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Volume 4 Issue 2

International Pharmaceutical Industry

Supporting the industry through communication

Peer reviewed

Predictive Dissolution Testing Concepts and Challenges. A Robust, Scalable Platform For Recombinant Protein Expression Conducting Cystic Fibrosis Clinical Trials Methodological, Practical and Ethical Considerations UN 3373 Packaging Secure your Sample Shipments

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PUBLISHER: Mark A. Barker EDITORIAL MANAGER Jaypreet Dhillon jay@ipimedia.com BOOK MANAGER: Anthony Stewart anthony@ipimedia.com BUSINESS DEVELOPMENT: Farah Jamali farah@ipimedia.com DESIGN DIRECTOR: Ricky Elizabeth design@ipimedia.com CIRCULATION MANAGER: Cecilia Stroe cecilia@pharmapubs.com FINANCE DEPARTMENT: Martin Wright martin@ipimedia.com RESEARCH & CIRCULATION: Jean Baptiste Marty jean@pharmapubs.om COVER IMAGE: iStockphoto © PRINTED BY: SW TWO UK print@ipimedia.com PUBLISHED BY: Pharma Publications Unit J413, The Biscuit Factory Tower Bridge business complex 100 clements road, London SE16 4DG Tel: +44 (0)20 7237 2036 Fax: +44 (0)01 480 247 5316 Email: info@ipimedia.com www.ipimedia.com All rights reserved. No part of this publication may be reproduced, duplicated, stored in any retrieval system or transmitted in any form by any means without prior written permission of the Publishers. The next issue of IPI will be published in August 2012. ISSN No. International Pharmaceutical Industry ISSN 1755-4578. The opinions and views expressed by the authors in this magazine are not necessarily those of the Editor or the Publisher. Please note that although care is taken in preparation of this publication, the Editor and the Publisher are not responsible for opinions, views and inaccuracies in the articles. Great care is taken with regards to artwork supplied, the Publisher cannot be held responsible for any loss or damage incurred. This publication is protected by copyright.

STRY IN U D

DIRECTORS: Martin Wright Mark A. Barker

Contents 6

Publisher’s letter

Regulatory & Marketplace

International Pharmaceutical Industry

Supporting the industry through communication

UTICAL IN CE

Supporting the industry through communication

PHARMA

International Pharmaceutical Industry

AL

RNATION E T

8 What about ‘Melon’ Patents Recent approval by the European patent of a number of patent applications on food products, including the patent EP1587933 awarded to Syngenta on melons “with a pleasant taste”, and the patent EP1962578 rewarded to Monsanto on “closterovirusresistant” melon plants, reopened the debate on patents on foodstuffs, and why not on animal varieties. Bart Laenen at LC Patents discusses the appraisal of non-governmental organisations objecting to the practice of the EPO in awarding patents to companies. 12 The Proposed New European Unitary Patent and Unified Court Simplifying the European patent system should be something which all users would welcome. There should be benefits for all. However, political fudge means that insufficient attention has been paid to the detail, and representations by those seeking to avoid pitfalls are dismissed in the rush to do a deal. The Unitary European Patent is now closer than it has ever been in the 40+ years during which it has been under discussion. Vicki Salmon at IP asset LLP questions whether the system that is being proposed makes life easier or more difficult for technology companies, whether the political deal is a good one, and how, if it comes about, it should affect your current patenting and business strategies. 16 BIO-Europe Spring® 2012, Amsterdam Sets New Benchmark in Partnering For a small country, the Netherlands is definitely not a small player in life sciences and health. The Dutch sector owes its position to a strong foundation of excellent science, a vast clinical research infrastructure, and a culture of entrepreneurship and open innovation. Sandra Migchielsen at Amsterdam Innovation Motor provides an insight into the successfully hosted BIOEurope Spring® 2012. 22 Pennsylvania: the State of Innovation Pennsylvania is a vibrant business community. Last year alone, Pennsylvania had 23 foreign-based companies make the move with a total investment of more than $371 million. Recently, Site Selection, a national publication specialising in the corporate real estate and economic development field, ranked Pennsylvania third in the United States, with 453 new or expanded corporate facilities in 2011. Steve Kratz at Department of Community and Economic Development, and Nadine Späth at Pennsylvania’s Authorized Trade Representative explain why, and what sets Pennsylvania apart from the rest. 26 Interview Feature with BioWin BioWin federates all the stakeholders who are involved in the value chain process from various fields: biopharmacy, radiopharmacy, cell therapy, diagnostics, imaging, biotech products, medical devices and medical equipment, and CRO/CMO services. Jaypreet Dhillon from IPI speaks with Jean Stéphenne at GSK Vaccines about the milestones that marked the cluster from its very beginning and their vision of the future.

2012 PHARMA PUBLICATIONS Volume 4 issue 2 Spring 2012

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DRUG DISCOVERY, DEVELOPMENT & DELIVERY 28 Predictive Dissolution Testing – Concepts and Challenges To date, the question of the equivalence and the pharmacokinetic reliability of solid oral dosage forms could in most cases be answered only through clinical trials. Grzegorz Garbacz at the University of Greifswald, Werner Weitschies at University of Greifswald, and Sandra Klein at the University of Greifswald investigate how developing predictive in vitro test systems for solid oral dosage forms which are capable of a realistic simulation of gastrointestinal transit conditions is still a challenge. 36 A Robust, Scalable Platform for Recombinant Protein Expression As well as being an important tool for the advancement of science, recombinant proteins are the active molecules of many of the last 30 years’ therapeutic breakthroughs. Dr. Charlotte Dyring, Dr. Wian de Jongh at ExpreS2ion Biotechnologies and Dr. Ali Salanti at the university of Copenhagen explain why development and optimisation of alternatives, such as stable insect cell-based platforms, remains of interest for the advancement of scientific and medical research. 42 The Eradicable Cancer One out of every 145 women has cervical cancer, yet cervical cancer is the most preventable cancer and the only one that can realistically be eradicated in our lifetime. This is a fact supported by science and innovation. Jo Parrish and Rachel Griffith at SWHR look into how cervical cancer begins in the cervix and gradually grows more invasive.

58 Strategic Overview of Personalised Medicine Personalised medicine is not yet fully implemented in healthcare developments, though the concept is several years old. The reasons might be scientific, technological, financial, regulatory or ethical. The switch to personalised medicine cannot be achieved by merely considering one of those aspects isolated from the others, because they are all interleaved. It takes a global strategic analysis of how the healthcare industry should address this challenge. In this article, Thibault Helleputte at DNAlytics outlines recent trends regarding them and discusses the observation that relates to data science - or the lack of it - in current practice. 64 Validation of a Bioanalytical ICP-MS Method for Quantification of Potassium in Human Urine Potassium chloride is used to treat patients with hypokalemia and/or prevent hypokalemia for patients who are at risk of developing it. Potassium chloride has been approved by the United States Food and Drug Administration (US FDA) as a treatment for hypokalemia. A non-binding draft guidance on potassium chloride as recommended by the FDA was issued in 2011 for drug submission purposes. The objective of this study by Jennifer Ammerman, Chaoyang Huang, Daniel Wright at MPI Research was to validate a bioanalytical method for the quantitation of potassium in human urine samples obtained from clinical studies; with patients dosed with potassium chloride in support of required bioequivalence studies. LABS/LOGISTICS & COLD CHAIN SUPPLY

44 Three-Dimensional Scaffolds for Tissue Culture and Regenerative Medicine Tissue engineering is a relatively new tool available to scientists, first appearing in the early 1990s. It was originally conceived as a potential solution to the lack of availability of donor organs, and is now also additionally considered as an alternative strategy to animal testing for drug and chemical safety models. A threedimensional (3D) scaffold provides a support for cellular growth in such a way that specific cells form a 3D matrix of tissue, which in turn can be transplanted into a patient to allow regeneration of the specified region. Dr. Donald A. Wellings and Andrew Gallagher of Spheritech analyse a new novel biopolymer that has been developed, which has found many applications, especially in the formation of self-assembled macroporous biodegradable constructs specifically designed to support 3D cell culture. CLINICAL RESEARCH 48 Robust Medical Information Practices for Better Risk Mitigation Many companies are grappling with their post-marketing risk management strategies, determining whether their safety surveillance and risk management procedures are sufficiently robust to identify new safety signals and positively impact upon already identified risks. Companies need to evaluate if they have the core competencies to comply with the subsequent requirements for the implementation and measurement of risk management strategies. Dr. Ian Kovacs of PAREXEL International explains why ccompanies need to consider integrated approaches to risk management, ensuring that such activities commence early in clinical development.

70 Quality Assessment of Primary Human Keratinocyte Cultures with Online Measurement of Oxygen and pH The SDR SensorDish Reader allows precise, non-invasive analysis of the process parameters dissolved oxygen (DO) and pH in cell culture. A correlation between cell proliferation analysed with light microscopy, increase of oxygen consumption, and a decrease in pH value in the culture medium, is clearly evident. Harry Abts at Merz Pharmaceuticals GmbH provides an overview of why the oxygen and pH kinetics give valuable information about culture quality and efficiency. 76 UN 3373 Packaging: Secure Your Sample Shipments Thousands of samples of human or animal origin are collected and sent around the world every day by pharmaceutical companies, nursing homes, diagnostic laboratories, research centres and others. They are collected as part of medical tests, clinical trials, surveillance studies and various other tests. Abbes Kacimi at Sofrigam explores why samples must be transported in accordance with IATA regulations for the air transport of dangerous goods, and with the European agreement concerning the International Carriage of Dangerous Goods by Road. 80 Put Safety First in Pharma Logistics Taking a look at how a modern logistics system operates and the high-value investment in creating a system that works safely and efficiently suggests more consideration should be given to the humble pallet. Jim Hardisty at Go Plastic Pallets discusses how poorly chosen pallets can seriously compromise safety in pharmaceutical supply chains and can lead to unnecessary costs.

52 Conducting Cystic Fibrosis Clinical Trials – Methodological, Practical and Ethical Considerations Cystic Fibrosis (CF) is a chronic, progressive, life-threatening genetic disease that primarily affects the pulmonary, gastrointestinal, endocrine and reproductive systems. It is an autosomal recessive disorder that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Vikki Brandi at Quintiles discusses key methodological, practical and ethical issues in the conduct of CF clinical trials with a focus on site identification, feasibility and clinical research studies. 2 INTERNATIONAL PHARMACEUTICAL INDUSTRY

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MANUFACTURING 84 Peptides as Drugs Peptide research has seen progressive growth over the past few decades, in particular with respect to ‘Peptide Therapeutics’. Many companies specialising in their manufacture, along with companies developing peptide-based products such as new drug candidates, medical diagnostic devices, and cosmetics and food technologies, have come to the forefront of pharmaceutical acquisitions and venture capital groups. Dr Kamal Badiani at Pepceuticals provides an overview of why peptides have long been regarded as crucial to offering solutions to mounting and increasingly difficult world health issues, and the possibility of patient-specific therapy. 92 Looking After Your AHU Vibration sensing has been employed for some time now; indeed we can be certain that it has been in use for almost two thousand years. The oldest vibration sensor known to historians was a seismograph invented by Chinese astronomer Chang Heng in the second century AD. Chris Hansford at Hansford Sensors Ltd explains why vibration sensing is therefore one of the oldest solutions in the engineering workplace, with a lineage that outstrips other technologies by centuries. 96 Recent Advances in Raman and Surface-Enhanced Raman Spectroscopy: Pharmaceutical, Forensic and Biomedical Applications Iqbal T Shadi from Imperial College London NHLI and Roy Goodacre, Professor of Biological Chemistry at University of Manchester, explore how Raman spectroscopy has advanced in recent years, and how its use in both industry and academia has increased significantly and is redrawing the landscape in many fields, such as biomedical and pharmaceutical R&D. PACKAGING 102 Child-resistant Packaging Laws are becoming More Restrictive Throughout the world, we are getting more and more used to the “push and turn” and “squeeze and turn” functions of many household products. Drug manufacturers and packagers in both Europe and in the US over time have been met with far-reaching regulations with regard to ensuring that drugs are packed in a manner that effectively protects the welfare and safety of children. Rikke Lindhardt at Gerresheimer explains how constant focus on children safety and prevention of serious accidents seems to play a crucial role when choosing primary plastic pharmaceutical packaging. 104 EXHIBITIONS PREVIEWS & REVIEWS 110 News

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Spring 2012 Volume 4 Issue 2


Publisher’s letter IT WAS once only drug firms that developed drugs. But this is changing. Take the case of the Michael J. Fox Foundation, a Parkinson’s disease charity. On April 19th it announced that it would pay for a clinical trial of a drug developed by Sanofi, a French pharmaceutical giant, that might treat the mental symptoms of the disease. Some blame stringent regulators. Others grumble that big pharma firms are too bureaucratic. All agree that developing a new drug takes money (well over $1 billion) and time (over ten years in America). Whatever the cause, a shortfall in R&D has inspired a flood of new partnerships. Charities have been particularly bold. Companies, beholden to shareholders, are reluctant to do things that might lose them money. Charities, by contrast, exist to give the stuff away. The leading “venture philanthropist” is the Cystic Fibrosis Foundation. It has supported early research and clinical trials, small biotech firms and the biggest of big pharma companies, Pfizer. The foundation spent $75m on early research for Kalydeco, which in January became the first approved drug to target the mutated gene that causes cystic fibrosis. Kalydeco is owned by Vertex, a firm based in Massachusetts, but the foundation will get royalties from the drug’s sales, which will then support further research. Other charities have followed the Cystic Fibrosis Foundation’s lead. The Fox Foundation, for example, has spent $289m on research. This is all good news, which will see a more efficient and patient-centric

healthcare system evolving. I and my editorial team bring you a wide selection of interesting and informative series of articles in this issue. On the Regulatory & Marketplace front, Bart Laenan of LC Patents reviews recent approval by the European Patent Office (EPO) on a number of patent applications on food products, including the patent EP1587933 awarded to Syngenta on melons “with a pleasant taste”, and the patent EP1962578 awarded to Monsanto on “closterovirusresistant” melon plants, reopening the debate on patents on foodstuffs, as opposed to animal varieties. Meanwhile, Vicki Salmon of IP Asset LLP observes that the Unitary European Patent is now closer than it has ever been in the 40+ years during which it has been under discussion. But will the system that is being proposed make life easier or more difficult for technology companies? Is the political deal a good one? If it comes about, how should it affect your current patenting and business strategies? In the Drug Discovery / Development & Delivery section, Jo Parrish and Rachel Griffith of SWHR explain that one out of every 145 women has cervical cancer, yet cervical cancer is the most preventable cancer and the only one that can realistically be eradicated in our lifetime. Moving to Clinical Research, Vikki Brandi of Quintiles concentrates on conducting cystic fibrosis clinical trials, and takes into account the methodological, practical and ethical considerations. Experts from MPI Research concentrate on the validation of a bioanalytical ICP–MS method for

quantification of potassium in human urine. A detailed article by Harry Abts of Merz examines quality assessment of primary human keratinocyte cultures with online measurement of oxygen and pH. In the Labs, Logistics & Cold Chain Supply section, Abbes Kacimi, a Cold Chain Expert Engineer at Sofrigam examines issues relating to securing sample shipments with UN 3373 packaging. Always a strong section, Manufacturing focuses on peptides as drugs by Kamal Badiani of Pepceuticals, while Chris Hansford of Hansford Sensors Ltd concentrates on vibration sensing and air handling units. In IPI’s Packaging section, putting safety first in pharma logistics is a subject for Jim Hardisty, the founder and Managing Director of Go Plastic Pallets. We at IPI have been driving some incredible developments. IPI’s weekly newsletter has been newly updated. Packed with recent news sourced by our staff reporter, extracts of various articles we publish within IPI, and independent company news and banners, this weekly newsletter keeps you abreast of the current trends within this industry. So visit www.ipimedia.com and sign up now for the newsletter. You will also see that our website has been totally transformed, offering more opportunities and services to readers and clients.

Jeffrey Litwin, M.D., F.A.C.C. Executive Vice President and Chief Medical Officer of ERT

Rick Turner, Senior Scientific Director, Quintiles Cardiac Safety Services & Affiliate Clinical Associate Professor, University of Florida College of Pharmacy

IPI looks forward to meeting all readers at the DIA Annual Meeting in Philadelphia. I hope you all enjoy this edition, and see you all in August. Mark A. Barker Publisher

Editorial Advisory Board Bakhyt Sarymsakova, Head of Department of International Cooperation, National Research Center of MCH, Astana, Kazakhstan Catherine Lund, Vice Chairman, OnQ Consulting

Jeffrey W. Sherman, Chief Medical Officer and Senior Vice President, IDM Pharma

Deborah A. Komlos, Senior Medical & Regulatory Writer, Thomson Reuters

Jim James DeSantihas, Chief Executive Officer, PharmaVigilant

Robert Reekie, Snr. Executive Vice President Operations, Europe, Asia-Pacific at PharmaNet Development Group

Diana L. Anderson, Ph.D president and CEO of D. Anderson & Company

Mark Goldberg, Chief Operating Officer, PAREXEL International Corporation

Sanjiv Kanwar, Managing Director, Polaris BioPharma Consulting

Franz Buchholzer, Director Regulatory Operations worldwide, PharmaNet development Group

Maha Al-Farhan, Vice President, ClinArt International, Chair of the GCC Chapter of the ACRP

Stanley Tam, General Manager, Eurofins MEDINET (Singapore, Shanghai)

Francis Crawley. Executive Director of the Good Clinical Practice Alliance – Europe (GCPA) and a World Health Organization (WHO) Expert in ethics Georg Mathis Founder and Managing Director, Appletree AG Heinrich Klech, Professor of Medicine, CEO and Executive Vice President, Vienna School of Clinical Research 6 INTERNATIONAL PHARMACEUTICAL INDUSTRY

Nermeen Varawalla, President & CEO, ECCRO – The Pan Emerging Country Contract Research Organisation Patrice Hugo, Chief Scientific Officer, Clearstone Central Laboratories

Stefan Astrom, Founder and CEO of Astrom Research International HB Steve Heath, Head of EMEA Medidata Solutions, Inc T S Jaishankar, Managing Director, QUEST Life Sciences

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Regulatory & Market Place

What about ‘Melon’ Patents Recent approval by the European Patent Office (EPO) of a number of patent applications on food products, including the patent EP1587933 rewarded to Syngenta on melons “with a pleasant taste”, and the patent EP1962578 rewarded to Monsanto on “closterovirus-resistant” melon plants, reopened the debate of patents on foodstuff, and why not animal varieties. Once again we see the appraisal of non-governmental organisations (NGOs) objecting to the practice of the EPO of awarding companies like Syngenta and Monsanto with monopolies on plants as well as to the food products thereof, and accordingly depriving farmers and farmer organisations in developing countries from these innovations in agriculture. Where EP1962578 is still the object of an ongoing opposition procedure before the EPO, with a coalition of NGOs acting under the name ‘No Patents on Seeds’ as one of the opponents, the opposition to the ‘pleasant tasting’ melons (EP1587933) came recently to an end. In both cases one of the grounds used by the opponents has to do with the fact that the claimed subjectmatter would relate to plant varieties, or essentially biological processes for the production of plants, which are excluded from patentability by Article 53(b) EPC. Prior to addressing the grounds for the decision of the opposition division in maintaining the ‘pleasant-tasting’ melon patent, it seems appropriate to highlight the legal framework of the European Patent Convention (EPC) dealing with biotechnological inventions, and in particular inventions on plants and animals. To understand the reasoning of the EPO in allowing the foregoing melon patents, particular reference to Rule 26(4) is made setting out that a plant variety is any plant grouping within a single botanical taxon of the lowest known rank, which grouping, irrespective of whether the conditions for the grant of 8 INTERNATIONAL PHARMACEUTICAL INDUSTRY

a plant variety right are fully met, can be: (a) d e  fined by the expression of the characteristics that results from a given genotype or combination of genotypes, (b) d  istinguished from any other plant grouping by the expression of at least one of the said characteristics, and (c)considered as a unit with regard to its suitability for being propagated unchanged. But also Rule 26(5) should be taken into consideration when determining the patentability of the biological process involved. Within the context of Article 53(b) EPC, a process for the production of plants or animals is essentially biological if it consists entirely of natural phenomena such as crossing or selection. Please note in this respect the ‘or’ wording of article 53(b), meaning that a biological process within a patent specification may be excluded in that it entirely consists of natural phenomena,

which should not have an automatic bearing on the products (plants) thus obtained. As this appears to be a recurring debate, further interpretation of the basic framework of the European Patent Convention can be found in decisions of the EPO’s Enlarged Board of Appeal, with the recent ‘broccoli’ case (G2/07) and the ‘tomato’ case (G1/08). In making decisions, the Board is always guided by the general principle that patents can be granted in all technical fields, including biotechnology, in as far the application provides a technical solution to a technical problem. Only when this first hurdle on the technical character of the invention is taken, should the further requirements on novelty, inventiveness and industrial applicability be checked. As such, when allowing patents on plants, or processes for the production of plants, one should always seek to find whether the patent application concerns a technical development Spring 2012 Volume 4 Issue 2


Regulatory & Market Place which is new and industrially applicable and involves an inventive step. Evidently, this technical aspect of the invention must be reflected in the claims, through the technical characteristics of the product (plant) or process involved. By zooming in on the technical contribution of the invention over the art, one in principle tackles the fear of allowing patents claims to plant varieties per se from the start, as it is a priori to be expected that the technical feasibility of the invention is not going to be confined to a particular plant variety per se. Turning back to the general patent principles in the plant field, such technical development typically concerns characteristics such as improvements in yield, higher nutritional value or resistance to drought and pests. In the majority of patents on plants, this technical development is based on genetic modification of a plant by genetic engineering techniques, but also other technical means could form the basis for the technical development. One of such techniques is marker-assisted selection (MAS), wherein plant lines containing the desired substance / characteristic at a high level are selected by means of these markers and used in plant breeding. All of the foregoing elements can be found in the decision of the opposition division to maintain the ‘pleasanttasting’ melon patent EP1587933. In said patent, the problem addressed by the application relates to the fact that for certain fruits of the melon plant a major part of the total fruit fresh weight is not edible because of the low pH and low sugar content. It has accordingly been an object of the present invention to alter the taste of the fruits of the melon plant addressing the foregoing problem. In order to avoid claims directed to plant varieties per se, the applicant fully focused on the technical elements characterising the melon plant. As such, and confirmed in the decision of the opposition division, claims directed thereto are not excluded from patentability under Art 53(b) EPC even though they may embrace plant varieties. In the present case, the claim is drawn to a plant characterised by 10 INTERNATIONAL PHARMACEUTICAL INDUSTRY

parameters (e.g. sugar content, pH, citric acid content, and the ratio of citric acid to malic acid). The claim is not directed to plant varieties. While some of the exemplified melon plants may be varieties or may fulfil the requirements of Rule 26(4) EPC, the teaching of the patent at issue is not limited to these plants, since the patent shows the possibility to obtain plants with the foregoing features in different genetic backgrounds, and by using a variety of different melons as starting material. According to the opposition division it is also not evident that the plants having said characteristics are suitable to be propagated unchanged, as required by Rule 26(4) EPC. For those reasons, the opposition division came to the conclusion that the claims do not relate to plant varieties, and are not excluded from patentability by Art 53(b) EPC. The inventive activity underlying the presently claimed plants can be found in the combination of features characterising the fruits of the melon plant, as claimed with, in particular, the ratio of citric to malic acid. In the opinion of the opposition division, there was nothing in the cited prior art to suggest that the ratio of citric to malic acid, in combination with the other characteristics on pH and sugar content, would have such a favourable influence on the acid perceptions of the melons. For said reasons the opposition division concluded that the patent, and the invention to which it relates, meet all the requirements of the EPC. In other words the recent decision in the ‘pleasant-tasting’ melon patent confirms the practice of the EPO in allowing patents on plants in as far the claims directed thereto are based on the technical contribution of the invention over the art, excluding claims to plant varieties per se, even though they may embrace plant varieties. This brings us to my final question: are the facts about the “closterovirusresistant melon” patent that much different? The Monsanto patent (EP1962578) is directed to a melon plant resistant to the Cucurbit yellow stunting disorder virus (CYSDV) by the introgression of a CYSDV-resistance– conferring QTL or a CYSDV-resistanceconferring part thereof linked to a

genetic marker E11/M49-239. Where indeed the gene responsible for the resistance was already known in the art, previous efforts to introduce this genetic characteristic in commercial cultivars have been unsuccessful. It has accordingly been an object of the present invention to determine linkage of this genetic element with a marker that can be used in marker-assisted selection procedures to enable the introgression of the CYSDV resistance in commercial cultivars, and providing a marker that allows easy traceability of the new resistant plants. As such, in my opinion, this case is nothing more than a further MAS patent, and accordingly fits with the recent decisions on the broccoli and tomato patents. In said decisions, the EPO has hitherto held MAS to be a technical process and therefore patentable, because in addition to conventional breeding steps it also involves the use of marker genes to select particular characteristics. I accordingly do not expect to see a different outcome in the present opposition, and hope to get further confirmation that the patents will always be granted for technical solutions to specific technical problems. Bart Laenan graduated as Ph.D. in Biomedical Sciences at the University of Limburg (L.U.C.) in February 1999. He is a qualified European Patent Attorney with a rich experience in filing and world-wide prosecution of patent applications based on his former employment at Janssen Pharmaceutica NV. In March 2007 he started his own company, LC Patents, as an independent Patent Agent and IP Consultant mainly focusing on small / medium-sized life science companies in Belgium / The Netherlands. Bart lectures on different aspects of intellectual property at the University of Antwerp and the University of Hasselt and has been invited as speaker on seminars from C5 International and Leuven Inc. Email: bart@lcpatents.eu

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Regulatory & Market Place

The Proposed New European Unitary Patent and Unified Court Summary By the time you read this article, the outstanding issues surrounding the proposed Unitary European Patent and Unified Court system may have been settled, with the detailed timetable for implementation being just around the corner. Alternatively, issues relating to the Euro debt crisis may have pushed discussion of this package off the agenda for the meetings of European leaders in June 2012, putting the decision on this package back to a later date. The Unitary European Patent is now closer than it has ever been in the 40+ years under which it has been under discussion. But will the system that is being proposed make life easier or more difficult for technology companies? Is the political deal a good one? If it comes about, how should it affect your current patenting and business strategies? History The European Patent Office opened its doors for business in 1978. It has been a huge success and has grown from a handful of member countries to 38 member states, with extension possible to two other European states. However, there are two key points to remember about the European Patent Office:1 It is NOT an institution of the European Union; 2 Patents are still national rights. On grant, the single European patent application breaks apart into national patents for each member state. In order to keep that protection, the patent owner will have to take steps to validate and maintain that patent in selected member states. When a patent is infringed, the patent owner must bring legal proceedings to protect its rights: most of the time that means suing competitors on a country by country basis. There are some exceptions within the European Union, when one country 12 INTERNATIONAL PHARMACEUTICAL INDUSTRY

will adjudicate on patent infringement happening elsewhere in Europe. However, this is not the rule. In the pharmaceutical market, generic manufacturers frequently seek to invalidate patents before launching their own products. These actions must also be brought country by country. Ranbaxy’s attempts to have the Pfizer patents relating to Lipitor® overturned was one example of this. Different European courts came to different conclusions. In contrast to Europe, the entire US market can be protected by a single patent, and there is only a need for a single US court case. The continued fragmentation of the European Union for IP rights does not make commercial sense. But how to modify the European system, and how to make that work in a way which is better than the system we have now, are questions which remain to be answered. The Current Proposals The current proposals have two main components: • A Unitary Patent • A Unified Patents Court The Unitary Patent A Unitary Patent should be a nobrainer. To obtain one patent covering the whole of the EU must be better than up to 27 separate patents. That would be one validation, one set of renewal fees and one patent against which to register ownership changes. However, there are wrinkles. Extent of coverage – The Unitary Patent will not cover all countries for which the EPO grants patent protection. So there will continue to be fragmentation in Europe. Further,

Spain and Italy, although within the EU, have opted out of the Unitary Patent. So the Unitary Patent will only cover 25 countries. Obtaining the Unified Patent will follow the existing system – file an application with the EPO, wait for the EPO to examine and grant it. On grant, the patent owner will then have a choice. It can continue to validate the patent in selected member states, as now. Or it can elect to take a Unitary Patent for 25 EU states and validate selected countries in the other 13 EPO member states, as now. If key countries of interest include Switzerland, Turkey, Spain or Italy, then there will be no change to the current process. Third parties who object to the patent being granted can still oppose the Unitary Patent, as now, by filing an opposition with the EPO within nine months of grant. If that is successful, then revocation will affect all states for which the patent was validated. However, this time limit may expire before competitors find out whether a patented product is going to be successful. With the Unitary Patent there may be less pressure to file an early opposition, as a single later attack can be brought centrally against the Unitary Patent. So although it may be attractive to have a Unitary Patent, some companies would prefer not to put too many eggs in one basket. Validation costs – Currently, few patent owners validate in all possible member states. The costs of obtaining a Unitary Patent may be more than the costs which a business currently pays for validation in selected countries. The EU is currently touting figures for cost savings. But this would only affect those who routinely validate in all 25 Unitary Patent states. For everyone else, will any Spring 2012 Volume 4 Issue 2


LifeScience Krakow Klaster (LSK) is the network of institutions from Krakow and Małopolska Region (Poland) which agreed to collaborate in order to develop and deliver to the market innovations in the field of life sciences. These organizations represent six areas of interests: biotechnology and life science business, research & development, science and education, healthcare, business support and local government actively guiding and facilitating the cooperation at the regional level. As such the LSK is the perfect exemplification of a so called Triple Helix cooperation. Established in October 2006, LSK has been the concept of Jagiellonian University developed in conjunction with another bold decision taken in 2004 — to build first technology park in Poland be devoted to life sciences. Since the very beginning the LSK has been considered as the research driven collaborative network aiming to pursue following mission: • to create and sustain a life science network in order to enable effective global connectivity and the optimization of existing potential of individuals and organizations • to support innovation and to encourage effective commercialization of research results in the life science field • to develop the resources and competences in order to effectively explore existing and future opportunities related to development of a knowledge-based economy. Development of biotechnology and life science sectors has been directly addressed as the priority field in the Regional Innovation Strategy, overall aim of which is to build knowledge-based economy. This focus of the Region has been strongly supported by the complementarity of the local science and business activities, which include healthcare and medicine, pharmacology and drug development, food and agriculture, biology and zoology as well as informatics, nuclear physics or chemistry and environmental services — to name the most important ones. Important aspect of the life science strategic intent in Krakow and Małopolska Region is to associate “technologies and products improving quality of individual life”. This would involve drug discovery & research services, medical diagnostics, e-health and telemedicine, advanced-non-drug therapies as well as complementary food, cosmetics and environment technologies. What distinguishes Life Science Klaster in Krakow from others in Central and Eastern Europe is advancements in life science an biotechnology fields achieved due to interplay of several factors: - access to knowledge and skilled labor: Krakow is in fact the number one city in Poland with regards to quality and availability of labor force; take life science field only and you will meet more than 1000 scientists and 16000 students;

- great business climate and best economic potential: Krakow and Małopolska have been ranked 6th alone and 7th within larger Southern-Poland region as having “best economic potential” in Europe; - strong science base: a number of universities and science institutes located in Krakow is active in research and development; this includes not only the oldest and the most prestigious in Poland — Jagiellonian University (est. 1364) together with its Collegium Medicum — but also University of Agriculture or University of Science and Technology followed by independent Pharmacology, Nuclear Physics or Animal Production national institutes; - diversified life science economy: number of biotech and life science businesses active in manufacturing of generic drugs, vaccines, innovative drugs R&D, imaging or molecular diagnostic technologies and services, clinical research, innovative cosmetics, functional food, diet supplements and others; - investments in infrastructure and R&D programs: due to availability and access to EU funds, only in the two recent years more than 0,5 billion € have been put in research infrastructure and research programs solely by LifeScience Klaster members in order to build both capacity and opportunity for collaborative development; - established networks for cooperation and support for innovation: coming to Krakow cluster you will meet ready-tocollaborate networks of interdisciplinary dimensions; technology transfer, VC & seed capital, marketing and business development, special economic zone and investor relationship; - quality of life in Krakow: the magic of the city is created by a unique combination of architecture, art and culture. Over 25% of Polish works of art are collected in Krakow due to more than a millennium of history and tradition. Thanks to that heritage in 2000 the city was honored with the title of the European Capital of Culture. Today, Krakow — a former capital city of Poland — is one of the most important cultural and tourist centers of the country. Contact: Kazimierz Murzyn Managing Director LifeScience Krakow Klaster Ul, Bobrzynskiego 14 30-348 Krakow, Poland phone: +48 (012) 297 4 605 mobile: +48 504 106 466 fax: +48 (012) 297 4 646 e-mail: kazimierz.murzyn@jci.pl http://www.lifescience.pl/


Regulatory & Market Place extra protection obtained across other EU member states be worth the extra cost? This may seem odd, so let me explain the complexity of this further. The EPO only operates in English, French or German. Patent applications filed in any other European language must be translated into one of those official languages. Before grant, the patent claims will be translated into the other two EPO official languages. So an application in English will have its claims translated into French and German, thus ensuring that the patent claims are in all three languages. Following grant, each country has rules as to how that patent is validated. For example France, Germany, Liechtenstein, Luxembourg, Monaco, Switzerland and the United Kingdom do not require any further translations at grant. Some countries, such as Denmark and Hungary, only require the claims to be translated into the local language, provided that the patent is in English. If the patent is in French or German, then the entire text must be translated into a local language or English. However, there are still countries, including Greece and Portugal (as well as Spain and Italy) which require a full translation of the text and claims into their local language. When it starts, a Unitary Patent will have to be translated in its entirety. Where the language of prosecution was French or German, then the full text will have to be translated into English. Where the language of prosecution was English, then the full text has to be translated into the language of any other participating state. So if your company would normally file its patent applications in English and only validate in the UK, Ireland, France and Germany, then opting for the Unitary Patent could initially add significant translation costs. The requirement to translate the full text is expected to be phased out in time, as computer-generated translations improve and can be used in place of those created by qualified translators. 14 INTERNATIONAL PHARMACEUTICAL INDUSTRY

However, the Unitary Patent will still not solve the problem of the additional costs incurred where protection is also required in Spain, Italy or the other European countries which are not in the EU. Renewal costs – To remain in force, renewal fees have to be paid for each year of the patent’s life. Currently these are paid in each European country of interest. The fees increase as the patent gets older. This is to encourage companies not to maintain patents which cease to have commercial value for them. Part of a company’s current renewal strategy may be, over time, to reduce the number of countries in which the patent is maintained. If a Unitary Patent is obtained, then that reduction in territorial coverage to save cost will no longer be open to the patent owner. The Unitary Patent will either be renewed in its totality or dropped. We don’t yet know the level at which renewal fees will be set. There has been a suggestion that they would start low and rise more steeply than they do now towards the end of the patent life. So when deciding whether to opt for the Unitary Patent, a company should also look at the current schedule of renewal fees to determine likely lifetime costs of the Unitary Patent over the current system. Enforcement – Although very few of the thousands of patents which are granted each year end up in the courts, the cost of patent enforcement and track record of the courts is an important consideration. If enforcement becomes too difficult or expensive for small companies, then the value which they should obtain from their investment in the patent system is seriously eroded. On the other hand, as the market becomes more and more complex, the chances of operating completely clear of competitor patents also becomes more difficult, and so consideration has also to be given to the impact on companies of being caught up in patent litigation as a

defendant. The system should not therefore be tilted in favour of patent owners. The Unified Patents Court The intention is that nearly all litigation for patents granted by the EPO for EU member states will be decided in the Unified Patents Court – whether these are Unitary Patents or national patents validated following an EPO patent grant. Patents can also still be obtained from national patent offices and these will be outside this system. So there will still be parallel systems. Italy has indicated that it may join this part of the system, even though it will stay outside the Unitary Patent. Spain is likely to remain outside. So this system may cover 26 EU countries. The choice of location to start litigation is likely to be complex. In addition to having a central court, each country may opt to keep local patent courts, or to collaborate with other countries to set up regional courts. For example, Germany has said that it wants four local courts, whereas the UK, the Netherlands and Ireland might co-operate to share a court. Additionally, there will be a single appeal court to which all appeals from the local, regional or central courts will go. A mediation centre will also be set up to help parties reach settlement without a full trial. This leads to a complex web as to where different types of patent action are begun – whether they should be brought locally or centrally, and whether that will give rise to a different quality of decisions, or differences in the way in which procedures are implemented. Location of the central court – European officials are currently portraying this as the only issue left to be decided, although, in the UK, we do not agree with this assessment. Four countries have made formal bids for the location of the Central Division – Germany, France, the UK and the Netherlands. There is a strong bid from the UK. More patents are granted in English Spring 2012 Volume 4 Issue 2


Regulatory & Market Place than other languages. London has good connections for those coming from outside the EU to litigate. Also, crucially, the EPO does not have an office in the UK. As the central court will hear appeals from the EPO in relation to Unitary Patents, it is important that the central court is not seen as being too “cosy” with the EPO. Bifurcation – However nice it would be to assume that all patents granted by the EPO are valid, we know that that is not the case. Although the EPO does its best to search and examine applications, competitors may know of other documents, presentations or instances of public use which are not going to be found by the EPO during examination. In the telecoms industry, in particular, a large number of the disputed patents granted by the EPO are overturned. In some European jurisdictions, notably Germany and Austria, patent infringement is heard in one court and patent validity is heard in completely different court. Anglo-Saxon lawyers find this hard: we operate under a basic premise that you cannot infringe an invalid patent, and so the court should look at the scope of the patent claims and whether or not they are valid before granting injunctions or awarding damages. Also, we fear that by separating these issues to different courts, patent owners are likely to argue for a broad claim construction during infringement proceedings (so as to catch the competitor) and narrow claim construction during validity actions (to avoid prior art). This favours the patent owner to the detriment of the competitor. It is particularly felt to benefit “patent trolls” – those who acquire patents (many of dubious validity) for licensing purposes and then use the threat of injunctions to extract maximum licence fees. For a patent owner, a system which enables it to obtain a swift injunction covering the whole of Europe is enticing. Under the Unified Patent Court system, where there are grounds for starting an action in Germany, a patent owner may get www.ipimedia.com

an early injunction ahead of trial and with no consideration of validity. On the other hand, where there is a risk of being a defendant, the location of operations which might be affected by that injunction becomes an important consideration. In the telecoms space, Apple recently relocated a distribution hub from Germany to the Netherlands to remove it from the effect of these swift German proceedings. Once the Unified Court is working, then those injunctions granted by the German local court will take effect throughout the relevant European Union states. What will this mean for the location of factories and distribution hubs? Will these need to relocate to Spain? Or out of the EU?

Conclusions Simplifying the European patent system should be something which all users would welcome. There should be benefits for all. However, political fudge means that insufficient attention has been paid to the detail and representations by those seeking to avoid pitfalls are dismissed in the rush to do a deal. The UK Parliamentary European Scrutiny Committee published a report on these proposals on 3 May 2012. It was not complimentary. So we wait to see whether we will all have to live with the current proposals or whether sense will prevail and amendments will be allowed, in order to make a system which will really deliver benefits to its users.

Other wrinkles – there are other provisions in the legislation which are still causing issues. There is a committee which is trying to draft rules for the court. As it gets into the detail, it becomes apparent that there would be better ways of writing the agreement to set up the court. However, with some countries refusing to discuss any changes to the legislation, the system is likely to be more cumbersome than necessary. The rules about patent infringement are being written into the two different legal documents. The rules for infringement of the Unified Patent are in an EU Regulation. This means that there is potential for referring questions on infringement of Unitary Patents to the Court of Justice of the European Union (CJEU). This will lead to delays (usually at least two years – which is significant in a patent dispute) and added costs. The CJEU does not have patent expertise, and many of its judgements in the IP field are already much criticised. When it starts to rule on patent issues, many commentators expect patent law to become less clear and harder to implement. Infringement provisions for national patents granted by the EPO for EU countries are in the other piece of legislation – so there is possibility for patent law under the two legal documents to diverge.

Vicki Salmon is a partner at IP Asset LLP. She is one of a handful of dual qualified p a t e n t attorneys and solicitors in the UK. With over twenty five years’ experience, and a degree in biochemistry, she advises on a wide range of intellectual property issues and technologies, from obtaining patent, trade mark & design protection, through to commercialisation and enforcement. Email: vicki.salmon@ipasset.com

INTERNATIONAL PHARMACEUTICAL INDUSTRY 15


Regulatory & Market Place

BIO-Europe Spring® 2012, Amsterdam Sets New Benchmark in Partnering Life sciences & health ranks high on the Dutch economic and social agenda. The Netherlands offers a long-standing tradition of biomedical research, with over 300 innovative companies forming a fertile breeding ground for new medicines, diagnostics and medical technology. Having successfully hosted BIO-Europe Spring® 2012 in Amsterdam, setting a new benchmark in attendance and partnering productivity, the Dutch convincingly illustrated their potential and strength in the European bioscience industry. Amsterdam ranks 8th in the world’s most popular places to host a conference. No less than a quarter of conferences held in Amsterdam are in the field of medical sciences. When the Amsterdam BioMed Cluster was created in 2006, its management team naturally investigated opportunities to organise an international life sciences event. The Amsterdam BioMed Cluster, hosted by the Amsterdam Innovation Motor, was set up to gain further economical spin off from the bio- and medical technology sector in the Amsterdam Metropolitan Area. Hosting an international event would offer Dutch life sciences & health organisations an opportunity to position themselves internationally and to participate in international partnering opportunities. As one of the premier partnering events in the world, BIO-Europe Spring ®, the springtime counterpart of BIO-Europe ®, was identified by Cluster partners as the event to target. BIO-Europe Spring®, produced by the EBD Group with the support of the Biotechnology Industry Organisation, has proven to be an important catalyst in forming the collaborations that drive the industry. When in 2008 Life Sciences Health 16 INTERNATIONAL PHARMACEUTICAL INDUSTRY

(LSH, the national cluster organisation of the Dutch life sciences and health sector) was created, the Amsterdam BioMed Cluster and LSH teamed up to host BIO-Europe Spring® 2012. Together they engaged the Dutch sector, consisting of circa 330 innovative companies employing 24,385 people 1, to benefit from this unique opportunity. Never before was a BIO-Europe Spring® meeting held in northern Europe. An All-time High Turnout BIO-Europe Spring® 2012 was held in Amsterdam from March 19-21, 2012 at the Amsterdam RAI Convention Center and turned out to be sunny and highly successful, setting a new benchmark in attendance and partnering productivity. The event grew more than 20% over the previous year, as shown under Highlights below. Also the host committee met its targets as the numbers of Dutch companies and attendees tripled compared to previous year(s). BIO-Europe Spring® 2012 Highlights • 11,172 one-to-one meetings • up 21% over 2011 • 2508 licensing opportunities posted - up 13% over 2011 • 2157 delegates - up 20% over 2011 • 1304 companies • up 15% over 2011 • 135 company presentations • 67 exhibitors • 44 countries represented • 18 workshops and panels • 300% growth in Dutch companies (139 compared to 46 in 2011) • 350% growth in Dutch attendees (259 compared to 72 in 2011) BIO-Europe Spring® 2012, through its online partnering system, enabled delegates to efficiently identify, meet, and get partnerships started with, companies across the biotech value

chain. High-level workshops, panel discussions, company presentations, and a lively exhibition together created a most productive event. Two pie charts show the distribution of the 2157 delegates in terms of industry and region, respectively [Fig 1a, Fig 1b]. Figure 1a: Distribution of the 2157 delegates in terms of industry

Fig 1.b, Distribution of the 2157 delegates in terms of region.

BIO-Europe Spring® 2012 attracted some of the best and brightest biotech companies in the sector, an array of pharmaceutical companies, and the venture capital community. Telling the Dutch Tale For a small country, the Netherlands is definitely not a small player in life sciences and health. The Dutch sector owes its position to a strong foundation of excellent science, a vast clinical research infrastructure and a culture of entrepreneurship and open innovation. Phillips, for one, is the world leader in medical imaging and patient monitoring. DSM is a leader in biomaterials, and MSD/Intervet innovates in veterinary life sciences. Innovative SMEs like Prosensa, Spring 2012 Volume 4 Issue 2


Regulatory & Market Place Galapagos and Agendia are thriving. No less than 10 of 15 European biotech partnerships and equity deals closed in 2008-2011 were with Dutch companies (total value exceeding EUR 4 billion). The wide range of dedicated life sciences service companies, as well as numerous specialised financial and business service providers, ensure that running a life sciences business in the Netherlands is efficient and effective. Sector figures show over 10% growth in number of companies and a 20% increase in number of products in development (2008-2010). Life sciences & health is one of the nine top sectors of the Dutch knowledge-based economy. Dutch Impressions The various elements of the BIOEurope Spring® 2012 programme were used to highlight the strength of the Dutch life sciences sector and put the Netherlands on the map of international companies operating in the field. (We have quoted freely from various press releases and social media snippets on the meeting.) Preconference Welcome Event In celebration of the opening, Life Sciences Center Amsterdam, Amsterdam inbusiness and Amsterdam BioMed Cluster held a preconference welcome event on Sunday, offering 80 guests the opportunity to meet, mingle and sample Amsterdam’s cultural offerings, including a chamber music concert by violinists of the Royal Concertgebouw Orchestra and an exclusive guided tour of the Van Gogh Museum. The event was highly successful and well received. After the welcome reception at the atmospheric Industrieele Groote Club at Dam Square kicked off BIOEurope Spring® 2012 in Amsterdam on Sunday March 18, the conference was officially opened by the mayor of Amsterdam on Monday morning, March 19. His elaborate speech was followed by a fantastic opening video about the Netherlands and the Dutch life sciences sector contributed by LSH2: Life is good in the Netherlands, through its continuing dedication to life sciences and health! 18 INTERNATIONAL PHARMACEUTICAL INDUSTRY

Prosensa’s CEO, Hans Schikan, gave an inspiring keynote speech followed by a plenary session on orphan indications and drug development. Also on Monday morning, the host committee organised a high-calibre workshop with well-known Dutch life sciences professionals, entitled “Close up and

personal: Recent deals examined from both sides”. Panellists of the workshop moderated by Roel Bulthuis, Head of Merck Serono Ventures, included Willem van Weperen of to-BBB technologies; Onno van de Stolpe of Galapagos; Rudy Mareel and Vincent de Groot of Synthon. Throughout the three days, many Spring 2012 Volume 4 Issue 2


Regulatory & Market Place Dutch companies, innovative startups, established biotech companies, large and mid-size pharma, presented information on products, partnering opportunities and corporate strategy. Informal matchmaking went on late during the evening receptions in the old stock exchange, ‘Beurs van Berlage’ and brand new ‘Muziekgebouw aan ’t IJ’ overlooking the IJ. The Dutch pavilion ‘Dutch masters in Life Sciences & Health’ formed the centrepiece of the exhibition floor. Whether it was the Drug Delivery Cluster-sponsored coffee, the DSM-sponsored ‘stroopwafels’, the delicious Dutch beer and cheese, or Mr C.P Buijink, Secretary-General of the Ministry of Economic Affairs, Agriculture and Innovation giving away the Rabobank-sponsored bike, traffic at the Dutch pavilion was excellent. Unique conference bags made from recycled denim and designed by regional artists brightened up serious suits and were much sought after. Young, local artists produced several art installations throughout the conference. The last day of BIO-Europe Spring 2012, Wednesday March 21, GlaxoSmithKline, Johnson & Johnson, and Index Ventures announced a EUR 150 million investment fund for earlystage biotechnology companies. Delivering Value for Growth On Thursday March 22, the day after the event, a number of Dutch life science clusters and parks welcomed interested conference delegates to their sites and displayed their complementary, regional offerings. Exciting visits were organised by Life Sciences Center Amsterdam, Maastricht Health Campus, Immuno Valley, and Medical Delta & Leiden Bio Science Park. Site Visit Alzheimer Center VU University Medical Center, Amsterdam Alzheimer’s disease presents challenges on many fronts; the complexity of diagnosis, clinical management, and the continuing unfulfilled search for effective therapy signal that we need to adopt new ways of cooperating to make progress. In this spirit, Life Sciences 20 INTERNATIONAL PHARMACEUTICAL INDUSTRY

Center Amsterdam proudly hosted a session entitled ‘Drug Development, Imaging, and Clinical Trials in Alzheimer Disease’ chaired by Dr Philip Scheltens. The session brought together experts from key elements of drug development and clinical trial value chain within AD, including: -V  Umc’s first-in-man trials capability in AD therapeutics, in conjunction with CHDR. -F  ull integration with Imaging Center VUmc, which offers radionuclide labelling, toxicology and pharmacokinetics, full GMP lab capabilities, PET tracer development, and combined PET/MRI. - Intimate linkage with EATRIS (European Advanced Translational Research InfraStructure in Medicine) headquartered at VUmc. -B  lood-brain barrier delivery companies. -A  D compound companies. -M  ultiple ongoing Phase I/II/III trials in AD. -A  large and very well characterised patient population of AD, especially early onset AD. -E  xtensive AD biology research, coupled with Neuroscience Campus Amsterdam and beyond. Site Visit Infectious Diseases and Vaccins, Immuno Valley, Utrecht Emerging infectious diseases such as SARS, Bluetongue or pandemic influenza, as well as widespread antibiotic resistance are a serious threat to our society by having immense socio-economic consequences. ‘Immuno Valley’ was founded in the Amsterdam-UtrechtFlevoland region of the Netherlands, to act as the network of excellence in infectious diseases detection and control. Immuno Valley organised a site visit, hosted by the Netherlands Proteomics Center, showing what the Netherlands has to offer in the field of infectious diseases and vaccines, from discovery to translational research. Renowned scientists and entrepreneurs addressed topics such as immunomodulation, discovery and bacterial and viral vaccine development. Also, there was focus

on important research facilities, business opportunities and public private partnerships. International partnering is expected to gain in importance for the future growth of Dutch life science companies. We thank the EBD Group, and all the companies, delegates and delegations, from as far as India and Australia, for coming to Amsterdam and making BIO-Europe Spring® 2012 such a successful event. As Onno van de Stolpe, CEO of Galapagos, already said, “The core value of Dutch companies is based on our strength to transform excellence in science into commercial success. Partnering is key to the sector.” We trust that by attending BIO-Europe Spring® 2012, more and more Dutch life sciences and health companies realise the benefits of partnering for growth. We shall have to wait till BIO-Europe Spring® 2013 in Barcelona to find out. References 1. L ife Sciences Health and The Decision Group, Dutch Life Sciences Outlook 2012, 2012 2. V  ideo on the Netherlands and Dutch Life Sciences sector www.youtube. com/watch?v=Nmo8Ua19sxY&feat ure=youtu.be

Sandra Migchielsen is senior project manager/ cluster manager for life sciences at Amsterdam Innovation Motor. After obtaining her PhD at Leiden University (1996), she managed and developed Elsevier’s (In)Organic Chemistry portfolio (19972006). Currently, together with industry, academia and the public sector, Sandra initiates projects that strengthen the knowledge based economy of the Amsterdam Metropolitan Area in the life sciences field. See also www.amsterdambiomed.nl. Email: sandra@aimsterdam.nl

Spring 2012 Volume 4 Issue 2


Regulatory & Market Place

Pennsylvania: The State of Innovation Great companies are built in Pennsylvania – including international ones. Major international companies including Shire Pharmaceuticals, Sanofi Pasteur, Siemens, Almac, Nestle, Toshiba and others have all found a place in Pennsylvania’s vibrant business community. Last year alone, Pennsylvania had 23 foreign-based companies make the move with a total investment of more than $371 million. Recently, Site Selection, a national publication specialising in the corporate real estate and economic development field, ranked Pennsylvania third in the United States with 453 new or expanded corporate facilities in 2011 – an increase of over 100 projects from 2010. What Sets Pennsylvania Apart from the Rest Located on the East Coast of the United States, within 800 kilometres of 40% of the US population and 60% of Canada’s population, Pennsylvania is a prime business location in the country. Only a few hours’ drive from Washington, DC and New York City, Pennsylvania sits between the nation’s seat of government and main financial

22 INTERNATIONAL PHARMACEUTICAL INDUSTRY

centre. With a gross domestic product of over $569 billion, Pennsylvania is the 19th largest economy in the world – larger than nations like Switzerland, Belgium and Saudi Arabia – and its continued success as an economic powerhouse has been ensured by the careful structuring of policies, legislation, and industry support. This area also represents 45 per cent of all US manufacturers and 41 per cent of the nation’s domestic trade and service industries. Reaching those customers and markets is easy and convenient because of Pennsylvania’s comprehensive transportation network, including: an outstanding network of interstate highways; modern freight railroads; six international airports; and three major ports providing access to the Atlantic Ocean, the Great Lakes, and the Gulf of Mexico. We also recognise that a quality workforce is a critical component of business competitiveness. Those companies with a presence in our state acknowledge our workforce as one of the best. The key to a quality workforce is a quality education. Pennsylvania is home to four of the nation’s top 50 universities, eight of

the top 50 liberal arts colleges, three of the top business schools and two of the top 50 law schools. The University of Pennsylvania’s Wharton School of Business is one of the nation’s top business schools, while Carnegie Mellon University is one of the country’s highest ranked graduate computer engineering schools. Pennsylvania State University’s Materials Science programme is ranked among the best in the world. Pennsylvania also offers an extensive range of customised job training programmes and partnerships, many of which specifically target the key industries in the state. Grants for workforce training are widely available for businesses choosing to locate within the state. Trade & Investment Support Programme Since the 1990s, Pennsylvania has been committed to breaking down barriers for businesses seeking to locate in the state, and supporting companies from Pennsylvania interested in doing business abroad. Led by the Pennsylvania Office of International Business Development (OIBD), the state has become a leader in cross-border projects and is one of the nation’s fastest growing exporters, thanks to its global network of 21 overseas offices which cover 62 markets worldwide. The Center of Trade Development (CTD) directs Pennsylvania’s Export Assistance Program consisting of a Regional Export Network (REN) and a global network of Authorized Trade Representatives (ATRs) offering assistance to Pennsylvania companies in markets abroad. Services and expertise for both new-to-export companies and experienced exporters seeking new markets include: market research, customised searches for qualified buyers, agents, distributors and partners, as well as in-country logistical support for business trips, trade missions and trade shows, Spring 2012 Volume 4 Issue 2


Regulatory & Market Place among others. A recent example of those services was the organisation and participation of companies from Pennsylvania at the 2012 BIO-Europe Spring in Amsterdam from March 19-21. The participation of the CTD together with five Pennsylvania companies has been a great success, thanks to the excellent preparation and close interaction between the CTD and our partners in the Netherlands and ´ Belgium. The participating companies had space to promote their products as well as the opportunity to take part in the numerous meetings that had been organised beforehand by Pennsylvania’s ATR. Participation in this conference has made it possible for companies from Pennsylvania to introduce their products to the Dutch / Benelux / European market and to form business partnerships in the international biotechnology community. In addition to working with Pennsylvania companies to expand trade opportunities, OIBD also provides assistance to foreign companies that are looking to establish a presence in the Northeast US – and there is no better place than in Pennsylvania. Working with the OIBD’s Center for Direct Investment (CDI) and its partners, international companies can easily learn what it takes to set up and operate a business in Pennsylvania, including information on taxes, immigration, site selection, real estate, and legal issues. CDI can also assist with gathering market intelligence and making initial introductions for companies to potential partners and others engaged in their industry across the state. These efforts have helped make Pennsylvania home to nearly 6000 foreign-owned businesses, employing more than 250,000 people. A Diverse Economy Pennsylvania’s diverse economy has a variety of strengths across five main industry sectors, including life sciences, advanced materials and manufacturing, business services, high technology and energy. Our world-class educational and research institutions, along with established 24 INTERNATIONAL PHARMACEUTICAL INDUSTRY

industry clusters in these sectors, and strategic support from the state government and local and regional partners, has helped Pennsylvania maintain stability and growth during challenging financial times. Pennsylvania’s advanced manufacturing and materials industry contributes over $64 billion to the gross state product and includes powdered metals manufacturing, agrofood processing, and prefabricated housing. Our business services industry ranges from network systems and software to finance and insurance, and is one of the most ambitious in the nation. High technology is a high priority for Pennsylvania. Our innovative programmes and initiatives are growing areas like next-generation technology and alternative energy. Growing Life Sciences in the Keystone State Pennsylvania is a national leader in the life science industry, and home to a cohesive community that unites biotechnology, medical device and diagnostics, pharmaceuticals and research institutions, and holds significant financial strength. Pennsylvania boasts 79,262 life science jobs across 2279 business establishments, ranking Pennsylvania among the top six US state employers in three of the four major life science subsectors. Known for being a state of Philadelphia, PA.

innovation, Pennsylvania ranks fourth in the United States in life sciencerelated patents, and fourth in total research awards from the National Institutes of Health (NIH) in FY2010. The evolution of a new model in the life science industry presents opportunities to link scientists, entrepreneurs, funding, programmes, and policy-makers in creating solutions and policies that will grow and sustain the innovation infrastructure; launching new companies and creating jobs that have a significant impact on human health globally. Pennsylvania is particularly unique in that it houses the entire continuum of the biosciences – world-class basic research, emerging companies, mature industry and global pharmaceuticals. These all interact to create a strong, vibrant community that is not found in many other states. We also have strengths that support the biosciences sector, including world-class academic/ research institutions, a location in the bio-pharma corridor providing access to a deep talent pool, an exceptional workforce, and partners for our region’s biotechnology companies. Despite recent economic recession, the life science industry continues to employ over 79,000 people in Pennsylvania, with wages of over $7.2 billion, and is a significant contributor to the state’s overall economy. The industry is poised for recovery and renewed growth as the economy rebounds. In fact, the number of firms representing the industry has actually increased from 2024 in 2008, to 2279 in 2011, a 13 per cent increase. This increase is driven by the addition of 300 firms (24%) in the category of research, testing and medical laboratory. The growth in the number of firms in this sector represents an opportunity for future industry growth as these companies grow and employ additional people. In addition to the research coming out of our universities and the recent growth of companies, Pennsylvania will continue to be a leader because of a supportive government with visionary leaders, at both the executive level with Governor Tom Corbett, and in the state legislature. Spring 2012 Volume 4 Issue 2


Regulatory & Market Place Pittsburgh, PA.

Open for Business A True US Leader Pennsylvania’s innovative programmes and initiatives are designed to enhance the state’s economy by providing the capital, infrastructure and talent necessary to create and retain high-paying, highly-skilled technology jobs. Our approach to economic development seeks to leverage our competitive business environment, manufacturing expertise, skilled labour force, world-class academic and research institutions, geographic proximity and advanced infrastructure to provide fertile ground for business growth. When considered together, these assets offer strategic and competitive advantages to businesses. Quality of life is also important for businesses looking to expand or relocate. From the urban vibe of our contemporary cities to the rustic and peaceful countryside, Pennsylvania has something for everyone. Pennsylvania is a great place to work, live, play and raise a family.

For further information, please contact: David Briel, Executive Director Email: dbriel@pa.gov Center for Direct Investment (CDI) Peter C. O’Neill, Executive Director Email: peoneill@pa.gov Center for Trade Development (CTD) This article has been written in joint cooperation by Steve Kratz, Press Secretary Email: stkratz@pa.gov Department of Community and Economic Development Nadine Späth Email: spaeth@pspsconsultants.nl Pennsylvania’s Authorized Trade Representative (the Netherlands & Belgium) Pennsylvania Dept. of Community & Economic Development The mission of the Department of Community and Economic Development (DCED) is to foster opportunities for businesses to grow and for communities to succeed and thrive in a global economy. Office of International Business Development The Office of International Business Development (OIBD) develops economic and employment opportunity through its three Centers: Center for Direct Investment (CDI), Center for Trade Development (CTD) and the Center for Port Development. www.newpa.com twitter accounts: @investPA | @ tradePA

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INTERNATIONAL PHARMACEUTICAL INDUSTRY 25


Regulatory & Market Place

Interview with Jean Stéphenne (BioWin)

“The European leadership demonstrates that it is possible to convert scientific excellence into economic success in the [Health] sector” Created in 2006, BioWin saw an opportunity nestled among the framework of a major regional socioeconomic programme. Focusing on the health sector, the development of this cluster activities rely on four strategic initiatives: • Encouraging open innovation through private-public partnerships between companies, research centres and universities. • Contributing to innovation and training in the field of biotechnology and health in order to ensure the economic growth of regional big companies and SMEs. • Encouraging the creation of technological hubs able to meet the sector needs. • Promoting excellence in academic and industrial research in Wallonia in the field of biotechnology and health. With more than 100 innovative companies specialising in health biotechnology and medical technologies, 400 research units from three universities (ULB in Brussels, UCL in Louvain and ULg in Liège) and prestigious research institutes, BioWin federates all the

stakeholders who are involved in the value chain process (from basic research to commercialisation) and coming from various fields such as: biopharmacy, radiopharmacy, cell therapy, diagnostics, imaging, biotech products, medical devices and medical equipment, and CRO/ CMO services. After BioWin recently presented the results of its first five years of activity, the President of the cluster, Jean Stéphenne (GSK Vaccines), highlights the milestones that marked the cluster from its very beginning and shares his vision of the future. 1. What is your assessment of the BioWin cluster after six years of existence? What are its flagship projects and greatest achievements? BioWin’s success can be measured by the 25 R&D projects that have been selected and financed by the Walloon government since 2006, with a total investment budget (private and public) of 84 million Euros. In terms of employment, these projects have enabled the creation and safeguarding of more than 200 research posts and in the course of five years should contribute to

the creation of 1260 direct jobs in Wallonia. The first projects are now reaching their final stages, and three new spinoffs will soon get off the ground. In addition to encouraging innovation, the cluster has developed a strategy for the development of skills. The “BioPhare” programme, launched in 2011, is the result of BioWin’s ambition to provide solutions that are structured and adapted to the needs of the market for the development of technological and entrepreneurial skills. Almost 8300 people have already benefited from training and work placements offered by BioWin. I personally recently took part in a training session aimed at bioentrepreneurs, during which project leaders had the opportunity to pick the brains of experienced managers. For these young bio-entrepreneurs, these are golden opportunities which simply did not exist before the cluster. 2. What makes BioWin stand out on an international level? BioWin has reason to be proud of the strong support shown by the Walloon government towards innovation and research. It occupies a rather unique position in Europe, in particular regarding the level of funding for R&D. The establishment of WelBio, a virtual institute for life science research, completed the R&D value chain in the Walloon region. It funds scientific research which in turn feeds into the BioWin pipeline with applied research projects, aimed at developing and bringing to market new products and services. 3. What is your next big project (infrastructure, coordinated action, etc.)? In terms of collective infrastructure, together with the Mecatech cluster (which specialises in mechanical engineering) we have submitted a

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Regulatory & Market Place

plan for a proton therapy project that will involve the construction and use of a centre dedicated to the treatment of localised cancers that will also host research projects from universities. These universities may have links to other Belgian or foreign universities that are in partnership with industry. There is also the project for the Centre Innovations Médicales (CIM) the aim of which will be to develop activity in the field of translational medicine through partnerships between academics, industry and hospital doctors. These two platforms have just been added to “MastherCell”, a cell therapy production company that came out of BioWin and was launched in September 2011 as a private contract manufacturing organisation (CMO). 4. What are your key areas of action and your priorities for the next five years? Firstly, we wish to continue with the current strategy of the cluster and emphasise the four basic branches; namely open innovation, training, creation of technology platforms and internationalisation of stakeholders. Secondly, we must ensure that the leaders of R&D projects have economic ambition and a long-term entrepreneurial vision. We also want to consolidate support measures for live and future projects, by making sure that they are well run and that they develop in a direction that is compatible with the initially proposed plan. Finally, it is essential to sufficiently promote projects that are coming to an end in order to optimise their economic transformation (creation of spin-offs, licensing etc.). 5. What do you hope the recently appointed international “ambassadors” will achieve? It is essential that the cluster has an international outlook, as the market for “healthcare” biotechnologies and www.ipimedia.com

medical technologies is by its nature global. By participating in European projects and in various international partnerships, BioWin has over the years nurtured a vast network of contacts with other clusters in Europe, in Asia and also in the United States. The mission of the “ambassadors” representing BioWin abroad, from Shanghai to Boston, is to strengthen and perpetuate this network for the benefit of the Walloon businesses and organisations that are creating innovation.

demonstrates that it is possible to convert scientific excellence into economic success in this sector. Today, we have the means to position ourselves as market leaders in cutting edge technologies such as cell therapy, radio pharmacy, imaging and diagnostics. Our mission is to seize this opportunity and to ensure that innovations in medical therapies created in our region translate into economic benefits for the region.

6. What is your vision, as head of one of the industrial “big pharma” companies, for the relationship between pharmas and biotechs? What major changes do you foresee and how can BioWin support local SMEs in the quest for competitiveness? The cluster’s priority is to increase the number of SMEs working in the field of healthcare. In this context, the role of large companies is to enable SMEs to form groups with international scope in order, on the one hand, to ensure their technology complies with industrial quality standards and, on the other hand, to adopt good practices relating to the industrial development of their technology. This is the role played by, among others, GSK Vaccines, UCB, IBA and Eurogentec and more recently BAXTER which hosted around twenty members of BioWin (from businesses, university laboratories and research centres) at its European R&D centre located in Wallonia for a networking meeting to discuss their research topics.

Jean Stéphenne was born in Dinant (Belgium) in 1949. He joined SmithKline-Rit (the future GSK Biologicals) as a production development engineer in 1974. He became Director of vaccine research, development and production in 1984 before becoming Vice-President and General Manager of GSK Vaccines in 1991. From 1997 to 2000, he was President of the Walloon Companies Union (Union Wallonne des Entreprises). In 1998, he became Chief Executive Officer and General Manager of GSK. In 2000, in recognition of his commitment to research and development and as a reward for the industrial success that he had brought to the nation, King Albert II ennobled Jean Stéphenne and raised him to the rank of baron. He played an active role in the development of the Marshall Plan for Wallonia. In this context, he was appointed chair of Biowin, the Health Cluster of Wallonia (Belgium), in 2006. In 2009, Jean Stéphenne became a member of the GSK Group Corporate Executive Team (CET). In 2012, he remains very active at GSK but also in several companies & organizations boards. Email: jean.stephenne@gskbio.com

7. What do you think the future holds for biotechnologies, in Europe and internationally? Europe is home to several world leaders in the field of bio-pharmacy, including GSK in the field of vaccines. This European leadership

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Drug Discovery/ Development & Delivery

Predictive Dissolution Testing – Concepts and Challenges To date the question of the equivalence and the pharmacokinetic reliability of solid oral dosage forms can in most cases be answered only in clinical trials. However, with the increasing number of both in vitro test systems capable of mimicking selected aspects of human gastrointestinal (GI) physiology and various in silico tools and pharmacokinetic models applied to translate in vitro release data into plasma profiles, it is likely that at some point the equivalence and the pharmacokinetic safety of oral dosage forms might be answered without the need to use human subjects for bioequivalence testing, but using an appropriate in vitro test design. From our point of view, both questions may not be answered with a single in vitro test but a set of in vitro experiments with experimental settings that provide a realistic simulation of the complexity of the in vivo conditions. Developing predictive in vitro test systems for solid oral dosage forms which are capable of a realistic simulation of gastrointestinal transit conditions is still a challenge. Such predictive dissolution tests should be suitable to assess the robustness and reliability of the formulation, as well as to answer the question whether two oral drug formulations containing the same dose of the same drug will be bioequivalent. A prerequisite for developing predictive dissolution tests is detailed information on the physiological conditions a dosage form is confronted with during GI passage, as well as the implementation of such information in the rational development of both test systems and methodologies. The GI passage of dosage forms is characterised by a high variability of the physicochemical properties of the GI content, as well as by a discontinuous transit with changing dynamic conditions. The impact of GI fluid composition on drug release has been extensively investigated 28 INTERNATIONAL PHARMACEUTICAL INDUSTRY

within the last years1,2. It has been identified that there is a high interand intraindividual variability in the physicochemical properties (e.g. pH, ionic strength, surface tension), the solubilisation capacity and the digestive activity of GI fluids 3-5. It is also well known that food intake can result in a significant change in the composition and the properties of the GI fluids, which in turn (e.g. after intake of high caloric meal or ethanol consumption) can result in meaningful changes of the dissolution behaviour of oral dosage forms5. However, to date only a little attention has been paid to the impact of mechanical stresses like pressure and hydrodynamics on the drug delivery characteristics of solid oral dosage forms. However, such impacts are likely to be of great relevance for the pharmacokinetic safety and reliability of at least oral modified release (MR) formulations. In numerous human studies it has been shown that the GI passage of dosage forms is characterised by highly variable and discontinuous transit conditions. The transport of dosage forms along the GI tract is a result of alternating static phases of relatively long duration and events of dynamic transport6,7. During GI transport events, dosage forms are temporary handled with high velocities which can particularly be measured during gastric emptying and passage through the ileocecal junction. Recently, it has also been recognised that during GI transit, solid dosage forms are exposed to mechanical pressures resulting from typical GI motility events. Maximum pressures were registered in the regions of pyloric and ileocecal sphincters8-10. In addition to the transit conditions, the volume of fluid available in the GI lumen can have a significant impact on drug release. Data derived by magnetic resonance imaging (MRI) demonstrated that under fasting conditions liquids are not equally

distributed along the intestinal “tube” but limited to typically three to six discrete fluid pockets. In accordance with this observation, it could be shown that during GI transit, nondisintegrating solid dosage forms are not in continuous contact with GI fluids but are occasionally located in empty or gas-filled sections of the intestines11. With all this background information, it becomes clear that the design of standard dissolution methods does not provide for a realistic simulation of the GI-specific stress conditions. In the last decades the pharmacopoeial dissolution test apparatuses became powerful instruments for standardised dissolution methods used in quality control. Since these apparatuses fall short in simulating biorelevant stresses, their applicability in establishing test methods predictive of the in vivo performance of solid oral dosage forms is often limited. To better simulate the impact of in vivo hydrodynamics and mechanical stress on drug release, we developed a “biorelevant dissolution stress test device” capable of simulating the essential physiological stress parameters, such as the discontinuous movement of dosage forms in the GI tract, the variability of GI motility and pressure waves as well as the intermittent contact of the dosage form with GI fluids12. Dissolution Stress Test Device The dissolution stress test device was first introduced by G. Garbacz and W. Weitschies 12. In the meantime, the prototype of the apparatus has been technically upgraded and translated into a professional version manufactured by ERWEKA GmbH, Heusenstamm, Germany. The apparatus aims to simulate the dimensions of physiological mechanical stress that may occur during the GI passage of a solid dosage form. For this objective, the Spring 2012 Volume 4 Issue 2


Drug Discovery/ Development & Delivery dissolution stress test apparatus exposes a dosage form to sequences of agitation including movement and pressure fluctuations alternated with static phases as observed in vivo. Moreover, the device enables the simulation of an intermittent contact of the dosage form with the dissolution medium. The device is illustrated in Figure 1 and a more detailed description can be taken from references12-14.

are controlled by synchronised switching of valves, and the pressure can be regulated by a pressurereducing device. The central axis is driven by the stepping motor, which is computer-controlled and thus enables a programmable movement and positioning of the probe chambers in and outside of the dissolution vessel, resulting in immersion in the medium or in contact with air. This offers the possibility to simulate the interrupted

Figure 1: Biorelevant dissolution stress test device: schematic representation, construction detail, and photographic representation of the test setup with flow through UV Vis spectrophotometer (14).

Briefly, the dissolution stress test device consists of a central apparatus axle with seven spheres (probe chambers) made of a stainless steel wire netting in which the dosage forms are hosted throughout the test. Each sphere is divided into two parts. The bottom part is screwed into the central pipe-like axle by a bush and a nozzle. In the working mode the central pipe is placed on the deck plate of the device about 3mm above the top edges of a row of seven standard dissolution vessels in their symmetry plane. Consequently, each sphere operates in a separate vessel. On one end the central axis itself is coupled to a pressure regulation device, and on the other end a stepping motor is attached. Pressure waves are generated by periodic inflation and deflation of the balloons located inside the probe chambers. These balloons www.ipimedia.com

contact of the dosage form with GI fluid. All test parameters are controlled by custom-made software. The dissolution medium (1160-1200mL) is mixed by a separate paddle stirrer operated at 100rpm during the entire test. To date various experiments have been performed with this setup15. In these experiments the dissolution stress test apparatus was operated in simplified stress sequences as well as in more complex test programmes intended to simulate the mechanical forces acting on a dosage form during fasted GI passage. In the simplified experiments, the focus of the experiments was to examine the impact of a single parameter on drug release. Various stress sequences were applied. These were composed of stress events of maximal physiological fortitude applied in a frequency of one or three stress events per

hour. In the first set of experiments, dynamic stress with velocities of up to 100rpm and lasting over a period of one minute was generated by the rotational movement of the central axis. In the second set of experiments, pressure forces of maximal physiological fortitude were simulated by a sequence of three symmetrical pressure fluctuations with fortitudes of up to 300mbar and a duration of 6s each. Subsequent experiments were focussed on mimicking the variations of the mechanical parameters affecting orally applied dosage forms during their GI transit under fasting conditions. Several test programmes were developed to cover a whole range of such variations. These test programmes are composed of phases of agitation initiated by a rotational movement of the central axis intended to simulate events of transport which are followed by phases of pressure fluctuations mimicking motility events in the GI tract. During the rotational movement of the central axis, the dosage form is forced to move with velocities of up to 60cm/s which are in good agreement with those determined in an in vivo study examining transport events in humans with a magnetic marker monitoring technique17,12,16. Phases of high stress are composed of pressure events with pressures of up to 300mbar which are followed by one minute of rotation with a velocity of 100rpm. Such phases of high stress are intended to simulate the harsh conditions which may occur during gastric emptying and duodenal passage, as well as during passage of tablets through the ileocecal junction. The motility of the postprandial GI tract, particulary that of the stomach, has so far been simulated as a simple sequence of rotational movements and pressure fluctuations of biorelevant fortitude12,13,17. The test algorithms for simulating the dynamics and the variability of postprandial conditions are currently under development. Due to the known large variability of colonic motility, a realistic simulation of the conditions during colonic passage of dosage form is hardly possible. So far we simulated colonic transit by means of short phases of harsh agitations occurring in intervals of several hours (typically two to four hours). INTERNATIONAL PHARMACEUTICAL INDUSTRY 29


Drug Discovery/ Development & Delivery Figure 2: Results of the BE studies of Voltaren 100mg retard tablets; the in silico simulation of the drug plasma levels obtained from the measurement data; schematic representation of the test programmes 1 to 3 and dissolution profiles of Voltaren 100mg retard tablets obtained in the USP apparatus 2 at 100rpm, and in the dissolution stress test device under programmes 1 to 3. Dissolution medium applied was pH 6.8 phosphate buffer of the USP. Presented are means of n = 6; standard error is indicated by the error bars. Detailed description of the test setup is given elsewhere 12.

To date the device has been successfully applied for the identification of clinically undesired performance of various single-unit modified release (MR) formulations14. Results of drug release experiments of Voltaren 100mg retard performed with the novel apparatus clearly indicated that under physiological mechanical conditions, diclofenac release from the extended release (ER) tablets is highly variable, and strongly depends on the mechanical stress events of transport and motility 12. The study results helped to explain the irregular plasma peaks of diclofenac obtained in a fasted state in vivo study with Voltaren ER tablets. These irregularities, which could not be explained with any 30 INTERNATIONAL PHARMACEUTICAL INDUSTRY

of the pharmacopoeial dissolution methods, are most likely caused by the sensitivity of the tablet to events of mechanical stresses during GI passage, which can particularly be measured during gastric emptying and ileocecal passage. By simulating a range of different gastric residence times combined with varying pressures and transport events in the dissolution stress test device and subsequent translation of the resulting in vitro profiles into theoretical plasma profiles, a whole set of characteristic profiles was obtained. Similar to the plasma profiles obtained from the in vivo study, the theoretical profiles showed significant differences in both the maximum plasma concentration

and the time at which this concentration could be measured. The applicability of the novel device and the test protocol used in the Voltaren experiments was confirmed in further experiments where it was applied to screen for the interchangeability of generic ER formulations of diclofenac sodium 100mg17. The tested products were generic formulations of the original Voltaren retard formulation and regarded as bioequivalent. However, test results indicated pronounced differences in the release behaviour of the two generic formulations. As already observed for Voltaren retard, dissolution of the generic formulations was strongly dependent on the test conditions, and when applying mechanical stress of physiological intensity the different formulations were not affected in equal measure. From our point of view, such susceptibility of dosage forms to biorelevant stress might be the main reason for irregularities often seen in drug plasma profiles. However, since for these generic formulations corresponding in vivo data were not available, the clinical relevance of the results requires further investigation14. Another series of dissolution studies was performed with nifedipine ER formulations in order to clarify the reason for the dosage form-related food effects for Coral 60mg retard tablets reported in a previously performed human bioequivalence study13. Results from experiments with the dissolution stress test device indicated the lack of mechanical stability and a pH-dependent dissolution behaviour of Coral 60mg retard tablets. These factors are thus likely to be the explanation for the food effects observed in vivo. Overall, test results and experiences obtained to date suggest that the biorelevant dissolution stress test device is a useful tool for the identification of undesired biopharmaceutical properties of MR products like “dose dumping� and could therefore become an important tool for the successful identification of reliable and safe drug delivery characteristics13. To optimise the biorelevant dissolution stress test device with respect to better estimating the Spring 2012 Volume 4 Issue 2


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Drug Discovery/ Development & Delivery in vivo performance of MR after both fasted and fed state administration, further developmental steps aiming at the improvement of the test protocols are needed. Until now, little attention has been paid to the volume and composition of the dissolution medium. For many MR formulations one can assume that drug release should not be affected by the composition of GI fluids or the dissolution medium, respectively. For this reason, in early experiments performed with the dissolution test device, the main criterion for selecting dissolution media was to assure sink conditions rather than simulating the composition of the different GI fluids in detail. Thus, the composition of the media did not always take into account the whole set of fluid parameters, i.e. physiological pH, ionic strength, surface tension, solubilising capacity, osmolality, ionic strength etc. that might be relevant for drug release. For future experiments, these aspects also need to be considered where necessary. Whereas many MR formulations contain highly soluble drugs, and for such formulations drug release is often not that much affected by the volume of test medium applied in the experiments, the use of a non-physiological large volume is prohibitive for biorelevant dissolution testing of immediate release (IR) dosage forms, particularly when simulating fasting conditions. In order to extend the applicability of the biorelevant dissolution stress test device to IR dosage forms, we implemented modifications in order to reduce the media volumes towards volumes representative for physiological conditions. Moreover, recently we established test programmes that are intended to simulate the extremes of physiological conditions experienced by the dosage form after fasted intake, and reflect so-called edge parameters of physiological emptying patterns of liquids as well as for the mechanical stresses. We are convinced that with the biorelevant dissolution stress test setup or other methods capable of mimicking the most critical (stress) conditions occurring during gastro32 INTERNATIONAL PHARMACEUTICAL INDUSTRY

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Drug Discovery/ Development & Delivery intestinal passage, formulations with undesired drug delivery characteristics can be identified. Since the use of biorelevant test methods offers the opportunity to study drug dissolution under more realistic conditions, application of such methods in early formulation development will enable identification of formulations with unfavourable release characteristics without the need to perform Phase I clinical trials. Consequently, our methods might help to considerably reduce the failure risk of bioequivalence studies. They might also help to reduce the risks for the volunteers involved in studies of both original formulations and generics and will finally bring us a step closer to a predictive dissolution testing. References 1. K  alantzi, L., et al., Characterization of the human upper gastrointestinal contents under conditions simulating bioavailability/ bioequivalence studies. Pharm Res, 2006. 23(1): p. 165-76. 2. J antratid, E. and J. Dressman, Biorelevant Dissolution Media Simulating the Proximal Human Gastrointestinal Tract: An Update. Dissol Technol, 2009. 16(3): p. 2125. 3. V  ertzoni, M., et al., Estimation of intragastric solubility of drugs: in what medium? Pharm Res, 2007. 24(5): p. 909-17. 4. D  ressman, J.B., et al., Upper gastrointestinal (GI) pH in young, healthy men and women. Pharm Res, 1990. 7(7): p. 756-61. 5. D  ressman, J.B., et al., Estimating drug solubility in the gastrointestinal tract. Adv Drug Deliv Rev, 2007. 59(7): p. 591-602. 6. W  eitschies, W., H. Blume, and H. Monnikes, Magnetic marker monitoring: high resolution realtime tracking of oral solid dosage forms in the gastrointestinal tract. Eur J Pharm Biopharm, 2010. 74(1): p. 93-101. 7. W  eitschies, W., et al., Magnetic Marker Monitoring: An application of biomagnetic measurement instrumentation and principles for the determination of the gastrointestinal behavior of magnetically marked solid dosage 34 INTERNATIONAL PHARMACEUTICAL INDUSTRY

forms. Adv Drug Deliv Rev, 2005. 57(8): p. 1210-22. 8. C  assilly, D., et al., Gastric emptying of a non-digestible solid: assessment with simultaneous SmartPill pH and pressure capsule, antroduodenal manometry, gastric emptying scintigraphy. Neurogastroenterol Motil, 2008. 20(4): p. 311-9. 9. S  arosiek, I., et al., The assessment of regional gut transit times in healthy controls and patients with gastroparesis using wireless motility technology. Alimentary Pharmacology & Therapeutics, 2010. 31(2): p. 313-322. 10. Phillips, S.F., et al., Motility of the ileocolonic junction. Gut, 1988. 29(3): p. 390-406. 11. Schiller, C., et al., Intestinal fluid volumes and transit of dosage forms as assessed by magnetic resonance imaging. Aliment Pharmacol Ther, 2005. 22(10): p. 971-9. 12. Garbacz, G., et al., Irregular absorption profiles observed from diclofenac extended release tablets can be predicted using a dissolution test apparatus that mimics in vivo physical stresses. Eur J Pharm Biopharm, 2008. 70(2): p. 421-8. 13. Garbacz, G., et al., Comparison of dissolution profiles obtained from nifedipine extended release once a day products using different dissolution test apparatuses. Eur J Pharm Sci, 2009. 38(2): p. 14755. 14. Garbacz, G. and S. Klein, Dissolution testing of oral modifiedrelease dosage forms. Journal of Pharmacy and Pharmacology, 2012: p. no-no. 15. Garbacz, G., S. Klein, and W. Weitschies, A biorelevant dissolution stress test device background and experiences. Expert Opin Drug Deliv, 2010. 7(11): p. 1251-61. 16. Weitschies, W., et al., Bioavailability of amoxicillin and clavulanic acid from extended release tablets depends on intragastric tablet deposition and gastric emptying. Eur J Pharm Biopharm, 2008. 70(2): p. 641-8. 17. Garbacz, G. and W. Weitschies,

Investigation of dissolution behavior of diclofenac sodium extended release formulations under standard and biorelevant test conditions. Drug Dev Ind Pharm, 2010. 36(5): p. 518-30.

Werner Weitschies is a professor of biopharmacy at the University of Greifswald. His main research areas are the investigation of the in vivo behaviour of dosage forms and the development of nanoparticle-based imaging and therapy techniques. Email:werner.weitschies@ uni-greifswald.de

Grzegorz Garbacz joined the group of Professor Werner Weitschies in 2004 as a diploma student and started to work on the construction and optimisation of biorelevant test models for the simulation of mechanical parameters of the GI tract. Since 2010 he has been a postdoc at the University of Greifswald and the CEO of Physiolution GmbH. Email: ggarbacz@physiolution.eu

Sandra Klein is a professor of pharmaceutical technology in the University of Greifswald. Her research is focused on developing biorelevant in vitro models to predict drug bioavailability from oral and vaginal delivery systems, and on improving the bioavailability of poorly soluble drugs. Email: sandra.klein@uni-greifswald.de

Spring 2012 Volume 4 Issue 2


Biopeople, Innovation Across Disciplines Biopeople is Denmark’s Innovation Network for Health and Life Sciences, established by the Ministry of Science, Innovation and Higher Education. • We facilitate innovation and collaboration • For academia, industry, regulators and other stakeholders • Across borders, sectors and public-private boundaries. Biopeople is your point of entry for the entire Danish health and life sciences sector. Our Services Matchmaking: across professional disciplines, across regional and national borders & across public – private boundaries, around dynamic and inspiring formats. For example: • Science dating at scientific conferences • 1 to 1 matchmaking at international fairs • Taylor made partnering missions abroad Knowledge sharing: visionary and high quality conferences, symposia, workshops, and courses. Innovation and strategy development: Biopeople is a partner in several national and international innovation projects and can help your company to enter these networks. At BIO2012 in Washington Come and see Biopeople #2955, Hall B, at the Scandinavia Pavilion for • Partnering with Danish companies • Partnership search with Danish universities and research groups • General information about the Danish health and life sciences sector • Funding possibilities for cooperation with Danish companies or universities More information about our activities at: www.biopeople.dk


Drug Discovery/ Development & Delivery

A Robust, Scalable Platform for Recombinant Protein Expression As well as being important tools for the advancement of science, recombinant proteins are the active molecules of many of the last 30 years’ therapeutic breakthroughs. A multitude of platforms for expression of recombinant proteins has been developed, each with its specific advantages, but there is not one single expression platform that is suitable for all proteins. Therefore, development and optimisation of alternatives, such as stable insect cellbased platforms, remains of interest for the advancement of scientific and medical research. Drosophila Schneider-2 (S2) cells have been in use for several decades for generation of stable cell lines for recombinant protein production. S2 cells have been used for expression and analysis of a broad range of protein classes. They express heterologous proteins at high levels; the proteins are authentically processed and are biologically active. Over the last 10 years, insect cell systems have moved from the laboratory to cGMP manufacturing for clinical development and to the market. Recombinant proteins made in S2 cells have also been tested in clinical trials. In addition to the cell line itself, several other components are required to constitute a high-performing, recombinant protein production platform. These include the expression vectors, the transfection reagent and procedure, optimised culture media and knowhow on how to use these components for the generation of the stable, highproducing cell lines and for optimising production conditions. The strengths of S2 cell-based expression platforms reside in the relative speed of production, which allows fast access to the required proteins, excellent expression capability, scalability, applicability to high cell density perfusion cultivation, and regulatory friendliness. 36 INTERNATIONAL PHARMACEUTICAL INDUSTRY

For applications such as vaccines, where costs of manufacturing can weigh more than in other medical uses of recombinant proteins, a possible low-cost system such as S2 becomes very relevant. Selection of the appropriate expression system for recombinant or subunit vaccines is currently considered one of the most significant technical challenges for the vaccine industry. S2 cells have proved to be very effective for the production of a broad variety of protein classes, such as viral proteins, toxins, membrane proteins and enzymes (Table 1, 1,2), and insect cellbased expression platforms (S2 cells and Baculovirus-expression vector systems – BEVS) have now been established as versatile and robust

vaccine manufacturing platforms (Table 2). This is significant as thirteen of the viral vaccines recommended by the WHO for development are recombinant-based vaccines, and all but one (influenza) are in preclinical development3. A significant obstacle to the development of these promising and sorely needed vaccines is the development costs involved. An efficient manufacturing platform such as the Drosophila S2 cell-based system, which is robust and easily scalable, may become critical in enabling the development of these types of vaccines. An example of a Drosophila S2based protein expression system is the ExpreS2 platform, which consists of highly efficient vectors, a

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Drug Discovery/Development & Delivery proprietary and optimised expression host derived from Drosophila S2, a very effective transfection reagent to introduce the vector DNA into cells at high efficiency, and a cell culture medium developed to sustain increased growth and high titer production of recombinant proteins in Drosophila S2 cells. The platform allows for high success rates of recombinant protein expression, high yields, reproducibility, ease of scalability, time savings and compliance with regulatory requirements. The team that developed the platform has extensive experience in the use of stable transfected S2 cells for production of a broad range of recombinant proteins for research use and for clinical development. The team received approval and performed production of HER-2 AutoVacTM and RANKL AutoVacTM, used in human clinical trials Phases 1 and 2, and non-human primate studies, respectively. The effort of optimising each component of the production platform resulted in the ExpreS 2 system being easy to use, with a high success rate for expression of a broad range of protein classes (Figure 1), at consistently high yields, with excellent reproducibility, ease of scaling up, time/cost savings, and cGMP and regulatory compliance. Each optimised element of the ExpreS2 platform brings an added advantage:

Cloning vectors: compared to two of the most widely used commercially available vectors, the ExpreS2 vectors lead to a 20-fold higher expression level. Cell line: the proprietary ExpreS 2 cell line grows to 40% higher density than standard S2 cells. The ExpreS2 Insect-TR transfection 38 INTERNATIONAL PHARMACEUTICAL INDUSTRY

reagent results in more efficient introduction of DNA in both S2 and Sf9 insect cells; this is the basis for establishing good stable producing cell lines; Culture medium: ExpreS 2 –SFM (serum-free medium) allows a threefold increase in production yield of S2 cells (both ExpreS2 and ATCC S2 cells). Altogether, the ExpreS2 platform offers several important practical advantages to recombinant protein production and process development: transient expression can be attained in four days, allowing for fast screening and initial selection of variants for development. Generation of stable, high-yielding cell lines requires only three weeks after transfection. Thus, by using the S2 expression system, time from DNA to protein produced in stable polyclonal pools of transformed cells can be reduced from months to just weeks. The stability of the polyclonal pools, and the natural gene amplification that occurs (rather than the more time-consuming additional gene amplification needed for CHO cells), removes the time-consuming step of clonal isolation from the critical path in the discovery phase. This characteristic feature of the S2based system is particularly useful in screening many different proteins and/ or variants thereof for evaluation and characterisation of candidates, both in discovery and in R&D, in a short timeframe. Process development – including up and downstream and the establishment of analytical tools - can also benefit from the use of stable pools of S2 transfectants for protein production, as these steps can be optimised while screening and selection of a monoclonal producing cell line take place. This ability to have both activities in parallel dramatically speeds up the process, allowing for best use of resources. In production runs, cultures of S2 cells typically reach densities more than ten-fold higher than mammalian cell lines. Increased viable cell concentrations result in increased production yields and improved process economics. Furthermore, as S2 cells do not aggregate even at very high cell densities (>150 million cell/

ml), they are exceptionally well suited to perfusion processes. Perfusion processes deliver lower capital and start-up costs with equivalent or improved production yields. Another very important feature of the ExpreS 2 platform is the consistency and reproducibility of the protein coming from different production days and runs, which are vital parameters for pharmaceutical protein production. It is hard to control the variations in protein and glycosylation consistency when producing from lysing cells, such as in BEVS. In sharp contrast, consistency and reproducibility are very high in production from S2 cells (Figure 2). The ExpreS2 platform is now being used to produce the VAR2CSA protein, which is being investigated as the antigen for a placental malaria vaccine. This complex protein has been difficult to express in other platforms; only insect cell-based platforms such as Baculovirusbased and S2 cells have successfully expressed the correctly folded protein. The S2 system has been selected specifically for its potential to reduce cost-of-goods for this very cost-sensitive vaccine target. The most deadly form of malaria is caused by the parasite Plasmodium falciparum, carried by mosquitos and transmitted to humans by mosquito bites. Women, who, during childhood, developed immunity against malaria, are susceptible to malaria during first pregnancies4. Malaria in pregnant women leads to anemia, impaired fetal development, low birth weight, spontaneous abortion and possible death for the mother. Fortunately, women can acquire immunity against placental malaria (PM), the average birth rate becoming higher in second and third pregnancies. This relatively fast development of immunity raised the hope that a vaccine to protect against placental malaria can be developed. A possible strong antigen target is the product of the gene VAR2CSA, identified in 2003 by a team at the Center for Medical Parasitology of Copenhagen University4,5. The existing laboratory evidence supports the hypothesis that a VAR2CSAbased vaccine will raise antibodies that hinder parasite adhesion in the Spring 2012 Volume 4 Issue 2


Drug Discovery/Development & Delivery placenta, thereby keeping them in circulation and allowing for their destruction in the spleen6. The geographic distribution of malaria, affecting mainly populations in poor countries, has meant that the development of a vaccine to prevent PM, like for other tropical diseases, suffers from lack of economic incentives or political motivation. Public-private global health partnerships have been created to try to improve the development path for preventing and treating neglected diseases, but cost of development remains a critical parameter, and low cost manufacturing platforms will play a vital role in enabling the progress of the more modern recombinant protein-based products, necessary to address these diseases. Variants of ExpreS 2-produced VAR 2CSA protein are currently being screened for selection of the best vaccine candidate. As discussed above, a stable cell line system such as ExpreS2 allows for a broader variety of upstream process options, compared to the obligatory batch process of the BEVS. VAR2CSA variants have been produced in fedbatch and in perfusion, both methods leading to high production yields, with perfusion allowing for smaller footprint installations, a factor that may allow for more flexibility in early stages of production. In looking for more flexible, low capital cost investment alternatives to manufacturing, the ExpreS 2 platform has been tested in single-use reactors. S2 cells expressing a truncated variant of the VAR2CSA protein have been cultivated in a single-use batch system resulting in cell counts of 4050 E6 cells/ml and viabilities above 94%. The resulting protein was used to raise antibodies in animals, and these tested positively for opsonising and parasite adhesion blocking responses7. This work demonstrated how the ExpreS2 platform could be used to produce effective proteins in a single-use system, without the need for dissolved oxygen or pH control. In summary, ExpreS2ion Biotechnologies has developed over more than ten years the proprietary ExpreS2 protein expression platform, for production of recombinant proteins www.ipimedia.com

and recombinant vaccines. Unlike BEVS, ExpreS2 does not involve viral infection and is amenable to highly consistent and reproducible stable production, which is perhaps the most important feature distinguishing BEVS and ExpreS2. ExpreS2ion can facilitate cGMP production and scale-up of recombinant proteins and represents a potential solution for projects where flexibility and cost of manufacturing are limiting factors, such as malaria and other neglected diseases. References 1. Schetz, J.A. et al., Protein expression in the Drosophila Schneider 2 cell system. Current Protocols in Neuroscience 4(16) (2004) 2. Moraes, A.M. et al. Drosophila melanogaster S2 cells for expression of heterologous genes: From gene cloning to bioprocess development. Biotechnology Advances 30 (3):pp613-628 (2012) 3. Cox, M.J.M. Recombinant protein vaccines produced in insect cells. Vaccine 30 1759-1766 (2012) 4. Salanti, A. et al. Evidence for the involvement of VAR2CSA in pregnancy-associated malaria. Journal of Experimental Medicine 200 (9): pp 1197-1203 (2004)

Dr Charlotte Dyring, MSc, PhD Co-Founder, CSO and Managing Director at ExpreS2ion Biotechnologies Dr Dyring has an extensive track record in protein expression in eukaryotic systems, mastering a wide array of expression systems, tools and techniques, and having substantial practical experience with upstream process development according to industry standards, including process transfer to cGMP. Dr Dyring is a recognised worldleading expert on the Drosophila S2 expression technology, and her expertise was critical in the development of the technology to the current level of sophistication and robustness. Email: cd@expres2ionbio.com

5. T  uikue, N.G. et al. High level of VAR2CSA transcription by Plasmodium falciparum isolated from the placenta. Journal of Infectious Diseases 19(2): pp 331335 (2005) 6. Dahlback, M. et al., The chondroitin sulphate A-binding site of the VAR2CSA protein involves multiple N-terminal domains. Journal of Biological Chemistry 286(18):pp15908-15917 (2011) 7. De Jongh, W. et al. A plug-andplay disposable insect cell protein production system using the CELLtainer® and ExpreS2 expression system. Poster, 10th PEACe Conference (2011)

Dr Wian de Jongh, MSc, PhD VP Product Development and coFounder at ExpreS2ion Biotechnologies Dr de Jongh has a BEng and MSc in Chemical Engineering from the University of Stellenbosch in South Africa, and a PhD in Biotechnology from the Technical University in Denmark. Dr de Jongh has been instrumental in developing a Drosophila S2 protein expression system, and has more than six years of experience in applying the system to process development and clinical material manufacture. Email: wdj@expres2ionbio.com

Dr Ali Salanti, MA, PhD Associate P r o f e s s o r, University of Copenhagen Dr Salanti’s group of more than 25 scientists works on malaria vaccine development, with a particular focus on malaria during pregnancy. Dr Salanti’s group was the first to describe the VAR2CSA antigen, now the main focus for a malaria vaccine for pregnant women. Email: salanti@sund.ku.dk

INTERNATIONAL PHARMACEUTICAL INDUSTRY 39


Drug Discovery/Development & Delivery

The Eradicable Cancer One out of every 145 women has cervical cancer, yet cervical cancer is the most preventable cancer and the only one that can realistically be eradicated in our lifetime. This is a fact supported by science and innovation. Cervical cancer begins in the cervix and gradually grows more invasive. It is caused by persistent infection with the human papillomavirus or HPV, a group of common and contagious viruses. There are over 100 different types of HPV, and more than 30 types spread through sexual contact that can be categorised into oncogenic (cancer-causing) or non-oncogenic types. Cervical cancer includes squamous cell carcinomas which begin in epithelial cells, like the skin, and comprise about 80-90 per cent of all cervical cancers. Adenocarcinomas are cancer cells that begin in glandular tissue and comprise the remaining 10-20 per cent of cervical cancers. Most women will get HPV in their lifetime and most will clear high-risk HPV on their own, but clearing of the virus is variable. The high risk strains of HPV, which comprise more than 80% of all cervical cancer cases, are 16, 18, 31, 33, and 45. Most women acquire HPV within 12 months after their first instance of sexual intercourse, as it is highly commutable and incredibly common. 81% of women will resolve HPV infection within 18 months after diagnosis. Cervical pre-cancers and early cancers usually have no symptoms or signs but the most common symptom is abnormal vaginal bleeding. Additional symptoms include unusual discharge from the vagina that occurs separately from menstrual cycles, bleeding after sexual intercourse, and douching or a pelvic exam. After menopause, symptoms may include vaginal bleeding or increased vaginal discharge. Cervical Cancer’s Impact Worldwide, cervical cancer is the second most common cancer among women, resulting in an estimated 240,000 deaths each year. 80% of 42 INTERNATIONAL PHARMACEUTICAL INDUSTRY

all women will contract HPV at some time in their life before they are 50. Approximately 12,170 women will develop cervical cancer in the United States in 2012, and nearly 4220 will die from it. Cervical cancer affects some ethnicities more than others, especially Hispanic and African American women. 11.1% of all cervical cancer cases affect African American women, 12.7% Hispanic, 7.9% White, 7.6% Asian American/Pacific Islander, and 6.6% American Indian. The mortality rates also reflect similar patterns: 4.6% African American, 3.4% American Indian, 3.1% Hispanic, 2.2% White and Asian American. Cervical cancer is the most preventable cancer. Yet failure to screen (50% of all cases), large gaps between screenings (>5 years) (10% of all cases), and false-negative Pap smears (30% of all cases) are major factors in disease incidence. Screening Guidelines The US Preventive Services Task Force (USPSTF) makes evidencebased recommendations on clinical preventive services to empower healthcare professionals, healthcare systems, and the American people to make informed decisions about their health and healthcare, including cervical cancer screening guidelines. Established in 1984, USPSTF is made up of 16 experts in care, prevention and research methods — including physicians, nurses and health behaviour specialists. The work of this independent panel is supported by the Agency for Healthcare Research and Quality (AHRQ) and its initiative — the Evidence-based Practice Centers (EPC) Program. The most recent USPSTF cervical cancer screening guidelines, issued in 2011, suggest the first Pap smear should occur at 21 years of age, or three years after the woman’s first instance of sexual intercourse. Screening before then is unnecessary since cervical cancer in women under 20 years of age is almost nonexistent. For women 20-29 years old,

screenings are recommended every two years and for women over 30, screenings are recommended every three years if they have had three consecutive negative Pap results and are low-risk. Low-risk constitutes no history of Cervical Intraepithelial Neoplasia (CIN) 2 or 3 and being HIV negative. Cervical screening can be discontinued between 65-70 years of age but women should continue to receive annual exams. Women who have received the HPV vaccine still need to have regular screenings. Treatments There is no known cure for cervical cancer but it is preventable. A vaccine is available to protect women against cervical cancer, and was approved by the Food and Drug Administration (FDA) to be administered to women aged nine to 26. This vaccine prevents infection from HPV types 16 and 18, which are responsible for approximately 70 per cent of cervical cancer, and HPV types 6 and 11, which are responsible for approximately 90 per cent of genital warts. These vaccines are only used to prevent, not treat, cervical cancer. Because HPV is transmitted sexually, vaccinations for males are encouraged to help eliminate cervical cancer. Routine screenings also promote early detection. Of the women in the United States who develop cervical cancer, about half have never had a Pap test and an additional 10 per cent have not had a Pap test in the last five years. There are several different treatment options for patients with cervical cancer. The most common are surgery, chemotherapy and radiation. But vaccination is the key to prevention and ultimate eradication. Barriers to Treatment In the US, controversy about sexual behaviour and other barriers hinders the appropriate use of the HPV vaccine. Healthcare providers often do not offer the correct and complete explanation to patients about the HPV vaccine and the need for Pap Spring 2012 Volume 4 Issue 2


Drug Discovery/ Development & Delivery smears. Many parents are hesitant to vaccinate their young children for fear of promoting earlier sexual experiences. Universal screening is needed to detect and prevent cervical cancer and must be advocated for at the local level by trusted sources. Educational programmes in regions in the US with the most cervical cancer cases, such as North Carolina and Kentucky, have proven to be effective. Several vaccination barriers for rural populations include1 does the woman have male permission or support to get vaccinated?2 lack of transportation - public transportation in Kentucky is extremely limited, and3 cost factors including insurance coverage and high co-pays. Advocates suggest vaccinations be available at stores where women are likely to shop. For example, Wal-Marts in rural and convenient locations might encourage people who are already in the store to get vaccinated. Easy access may help women who have had the first HPV vaccination to continue with the second and third vaccinations to complete the set and be fully protected from cervical cancer. Partnership to End Cervical Cancer The Society for Women’s Health Research (SWHR), a national nonprofit dedicated to the study of sex differences, chairs the Partnership to End Cervical Cancer (PECC) and its 43 member groups dedicated to eliminating cervical cancer. PECC’s mission is to ensure universal access to education, screening and vaccination to prevent cervical cancer. For six years, PECC members have dedicated resources to reduce barriers to appropriate services, and to raise the potential for ending cervical cancer in the US with public and private stakeholders. PECC has produced public service announcements, produced web and print informational materials, held Capitol Hill and media briefings, and supported and shared programmes within the PECC member organisations on the importance of the HPV vaccine and the elimination of cervical cancer. PECC has also worked closely with www.ipimedia.com

Cervical Cancer Free America, which provides nationwide initiatives around education, screening and vaccination that have the potential to end cervical cancer in this lifetime. Great strides have been made over the past several decades to reduce the incidence of cervical cancer in the United States. The greater and largely unrecognised burden of cancer-causing HPV threatens the reproductive and emotional health of thousands of women, and costs the United States almost $4 billion annually. While eliminating cervical cancer in the United States may seem like an ambitious goal, experts agree that it is an achievable one. With organisations like SWHR, PECC, and Cervical Cancer Free America working towards universal vaccination, eradicating cervical cancer can quickly become a reality. References 1. Data presented by B. Casey in “Partnership to End Cervical Cancer meeting,” Washington, D.C., 17 Oct. 2011. 2. Data presented by T. Cox in “Partnership to End Cervical Cancer meeting,” Washington, D.C., 6 Apr. 2010. 3. www.cancer.org/Research CancerFactsFigures/ CancerFactsFigures/index, visited on 5 Apr. 2012. 4.h ttp://www.guttmacher.org/pubs/ gpr/15/1/gpr150108.html, visited on 6 Apr. 2012. 5. w w w . c a n c e r . o r g / C a n c e r / CervicalCancer/DetailedGuide/ c e r v i c a l - c a n c e r- k e y - s t a t i s t i c s , visited on 6 Apr. 2012. 6. w ww.womenshealthresearch.org/ site/PageServer?pagename=hs_ healthfacts_cervicalcancercf, visited 6 Apr. 2012. 7. w ww.womenshealthresearch.org/ site/PageServer?pagename=hs_ healthfacts_cervicalcancercf, visited 6 Apr. 2012. 8. www.uspreventiveservicestask force.org/about.htm, visited 12 Apr. 2012. 9. www.uspreventiveservices taskforce.org/uspstf/uspscerv. htm, visited 12 Apr. 2012. 10. Data presented by T. Cox in “Partnership to End Cervical Cancer Meeting,” Washington, D.C., 6 Apr. 2010. 11.w ww.womenshealthresearch.org/ site/PageServer?pagename=hs_ healthfacts_cervicalcancercf, visited

13 Apr. 2012. 12. D  ata presented by B. Casey in “Partnership to End Cervical Cancer meeting,” Washington, D.C., 17 Oct. 2011. 13. w ww.womenshealthresearch.org/ site/PageServer?pagename=hs_ healthfacts_cervicalcancercf, visited 13 Apr. 2012. 14. D  ata presented by B. Casey in “Partnership to End Cervical Cancer meeting,” Washington, D.C., 17 Oct. 2011. 15. D  ata presented by B. Casey in “Partnership to End Cervical Cancer meeting,” Washington, D.C., 17 Oct. 2011. 16. D  ata presented by B. Casey in “Partnership to End Cervical Cancer meeting,” Washington, D.C., 17 Oct. 2011. 17. w ww.cervicalcancerfreeamerica. org/, visited 13 Apr. 2012.

L. Jo Parrish, MA, MBA Jo Parrish oversees all aspects of a multi-faceted communications programme that includes public education, public and media relations, marketing, website, and publications. She also oversees all aspects of SWHR’s fundraising and development activities, including renewing support from current sources, expanding and diversifying SWHR’s funding base, and major fundraising events. Parrish joined SWHR in 1999. She has over 20 years of experience in organisations, associations, education, and cultural arts with special expertise in strategic and process planning in start-up or new programme operations. Email: jo@swhr.org

Rachel Griffith Rachel Griffith is the Communication and Event Manager at the Society for Women’s Health Research (SWHR). She manages and coordinates SWHR’s conferences, meetings and events. Griffith also manages outreach to SWHR constituents through the e-newsletter, social networks, research articles and media relations. She received the Aesculapius Award for Excellence in Health Communication in 2011. Griffith earned her BA in public relations from the University of Oklahoma.. Email: rachel@swhr.org

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Drug Discovery/ Development & Delivery

Three-Dimensional Scaffolds for Tissue Culture and Regenerative Medicine Introduction Tissue engineering is a relatively new tool available to scientists, first appearing in the early 1990s1. It was originally conceived as a potential solution to the lack of availability of donor organs, and is now also additionally considered as an alternative strategy to animal testing for drug and chemical safety models. A three-dimensional (3D) scaffold provides a support for cellular growth in such a way that specific cells form a 3D matrix of tissue which in turn can be transplanted into a patient to allow regeneration of the specified region. A new novel biopolymer has been developed which has found many applications, amongst which is the formation of self-assembled macroporous biodegradable constructs specifically designed to support 3D cell culture. The porosity of the biopolymer is controlled by self-assembly in such a manner as to accommodate cell and nutrient penetration within. The unique features of the 3D cell culture scaffold developed by the company include the fact that it is composed entirely of naturally occurring components. The main backbone of the polymer is polyƐ-lysine which is an edible, non-toxic material currently manufactured on a multi-ton scale and used predominantly as a preservative in foodstuffs 2. Poly-Ɛ-lysine can be readily crosslinked with any multi-functional carboxylic acid, and in particular it has been found that the naturally occurring bis-carboxylic acids such as sebacic acid and dodecanedioic acid impart advantageous properties on 3D scaffolds. Ultimately the polymer is susceptible to degradation by protease activity to produce lysine and fatty acids. Bi-functional fatty acids such as sebacic acid and dodecanedioic acid can be oxidised fully into CO 2 and H 2O by cells via the Krebs cycle 3. 44 INTERNATIONAL PHARMACEUTICAL INDUSTRY

Because of this dodecanedioic acid has been investigated as a substitute substrate to glucose in parenteral nutrition4. The biopolymer will therefore upon decomposition break down into natural components already found in vivo, providing a potential candidate for tissue regeneration and transplantation. In order to culture and differentiate cells in three dimensions, it is necessary to seed cells into an artificial structure capable of supporting tissue formation. These structures, usually referred to as scaffolds, are critical for allowing cells to influence their own microenvironments both ex vivo and in vivo. The scaffold must firstly allow cell attachment and migration, enable diffusion of cell nutrients and expressed waste and provide the requisite mechanical and biological properties. The porosity and properties under flow are crucial factors to consider when designing macroporous structures. The macroporous scaffolds utilised to date have poorly-defined pore structure, and the pores are generally either too small or have broad pore size distribution. Ideally a macroporous structure is required which allows efficient migration of nutrients and waste but importantly allows rapid and unhindered diffusion of cells. An optimum scaffold will have carefully engineered pores designed for cell type and rate of migration. Generally a workable pore size would be in the range of 40-200um, and the ability to create a specific pore size within this range is probably the ultimate goal for tissue engineers. A process has been invented for the manufacture of macroporous polymer scaffolds for cell culture. This novel process allows for a more precisely controlled pore structure, pore volume and pore dimension than

could previously be achieved. One example is a biomimetic peptide Figure 1 shows scanning electron micrographs (SEMs) of the new poly-Ɛ-lysine based 3D scaffold, SpheriGen TM, which is currently under investigation for applications in kidney regeneration, nerve regeneration, retinal repair and wound management.

motif (H-GGRGDG-NH2) containing the known cell adhesion tri-peptide ‘RGD’6. The ‘RGD’ motif is known to interact with transmembrane integrin receptors and facilitate not only cellular adhesion but also the initiation of intracellular signalling pathways. This results in the formation of growth factors and alterations in gene expression which in turn lead to the differentiation and proliferation of cells within their surrounding environment6.

Figure 2. It should be noted that SpheriGen TM can be manufactured in almost any shape or size including monoliths, tubes, sheets and membranes. Some examples are shown here.

Spring 2012 Volume 4 Issue 2


Drug Discovery/ Development & Delivery

Kidney Regeneration End-stage renal disease (ESRD) is, as the name suggests, terminal. ESRD progresses from long-term kidney diseases such as acute renal failure (ARF) and chronic kidney disease (CKD), resulting in total loss of kidney function7. Renal diseases are ever-increasing, especially in the western world, due to the rise in patients suffering from diabetes 8. Approximately one in every 500 of the population suffers from ARF or CKD, which has a mortality rate of 50% 7. When renal diseases progress to ESRD the only treatments presently available are dialysis or transplantation, both of which are expensive and inefficient treatments. Organ failure and rejection, coupled with limited donor numbers, have channelled investment and research into new alternative treatments, with the most promising being stem cell therapy9. Kidney stem cells or pre-cursors have recently been located in adult kidneys as well as in bone marrow 10. In the embryonic stages of life they are required for organogenesis, but in the adult body their role changes and becomes more maintenanceorientated through the mediation of tissue repair11. Adult kidney stem cells alone do not seem to have the ability, or be abundant enough, to repair the kidney after serious injury, and this usually leads to ESRD 10. For this reason, focus is turning towards the generation of tissue in vitro using kidney stem cells and a suitable 3D scaffold for in vivo transplantation. The product SpheriGenTM has been shown to support attachment and subsequent proliferation in true 3D. HEK293T:GFP cells supplied by the Stem Cell Group at the University of Liverpool, UK were seeded onto 1mm thick SpheriGenTM discs suspended by membrane supports in six-well tissue www.ipimedia.com

culture plates. Figure 3 shows a view of the cells proliferating on the surface of the discs at intervals over several days. Figure 4 shows micrographs of a section of the polymer across the depth of the 1mm-thick discs confirming proliferation in 3D. The cell count, determined using

Figure 5

Figure 3

Figure 4

the CCK-8 assay kit from Dojindo, confirms the anticipated rate of cell proliferation. The histogram shown in Figure 5 shows a comparison of SpheriGenTM with the ‘RGD’ coating, in parallel with a free carboxyl form of the polymer.

for nerve regeneration. Again two versions were tested in parallel, including a free carboxyl form and a version with a synthetic cell binding pentapeptide, IKVAV, derived from laminin covalently attached. Schwann cells were seeded onto each of the macroporous polymer scaffolds, and Alamar blue absorbance assay was used to determine cell proliferation. Alamar blue results show that Schwann cells initially attach and survive in both versions of SpheriGenTM after 24 hours. Initial Schwann cell growth and attachment is significantly better (P<0.001) on scaffolds functionalised with IKVAV compared to scaffolds in free carboxyl form. Furthermore, Schwann cell proliferation was significantly higher (P<0.001) over a four-day period on scaffolds functionalised with IKVAV (Figure 6).

Figure 6

Nerve Regeneration Early results from proliferation of Schwann cells within the product carried out at the University of Manchester also show promise INTERNATIONAL PHARMACEUTICAL INDUSTRY 45


Drug Discovery/ Development & Delivery Acknowledgements Dr Patricia Murray and Miss Chloe Williams, Institute of Translational Medicine, Faculty of Health and Life Sciences, University of Liverpool, UK. Professor Giorgio Terenghi and Dr Ben Minogue, Blond McIndoe Laboratories, School of Biomedicine, University of Manchester, UK. Co-author Andrew Gallagher Concluding remarks SpheriGenTM has been proven to support cell attachment and proliferation in 3D for kidney and nerve cells. The polymer scaffold has the potential to be finely tuned by incorporation of various copolymers including amino acids such as aspartic acid or cystine. Addition of these monomers will increase the flexibility with respect to protease activity and rate of biodegradation. Although SpheriGenTM is now commercially available in standard six-well plates supported by membrane inserts, the polymer can be provided in endless forms to suit customer requirements. By way of example, the company has recently supplied 50µm-thick membranes in 12-well plates for 2D retinal cell culture, which will ultimately lead to transplant for treatment of age-related macular degeneration. Ongoing developments include investigation of the polymer for wound care, in particular for applications such as corneal bandages and internal dressings to prevent post-operative adhesion. The physical properties of SpheriGenTM resemble those of natural collagen, with comparable elasticity and hydrophilicity. If necessary, the pore dimensions and polymer density can be carefully controlled to resist full cellular penetration of the matrix but still allow efficient fluid transfer so we also anticipate further developments for skincare, plus external and internal wound dressings.

46 INTERNATIONAL PHARMACEUTICAL INDUSTRY

References 1. H  ollister, S.J. (2005). Porous Scaffold Design for Tissue Engineering. Nature Materials. 4: 518-524 2. S  hih, I-L., Shen, M.H. and Van, Y-T. (2006). Microbial Synthesis of Poly(εlysine) and its Various Applications. Bioresource Technology. 97: 11481159 3. M  ingrone, G., De Gaetano, A., Greco, A.V., Capristo, E., Benedetti, G., Castagneto, M. and Gasbarinni, G. (1997). British Journal of Nutrition. 78: 723-735 4. G  reco, A.V., Mingrone, G., Capristo, E., Benedetti, G., De Gaetano, A. and Gasbarrini, G. (1998). The Metabolic Effect of Dodecanedioic Acid Infusion in Non-Insulin-Dependent Diabetic Patients. Applied Nutritional Investigation. 14: 351-357 5. R  uoslahti, E. and Pierschbacher, M.D. (1987). New Perspectives in Cell Adhesion: RGD and Integrins. Science. 238: 491-497 6. S  tevens, M.M. and George, J.H. (2005). Exploring and Engineering the Cell Surface Interface. Science. 310: 1135-1138 7. S  agrinati, C., Ronconi, E., Lazzeri, E., Lasagni, L. and Romagnani, P. (2008). Stem Cell Approaches for Kidney Repair: Choosing the Right Cells. Trends in Molecular Medicine. 14: 277-285 8. P  leniceanu, O., Harari-Steinberg, O. and Dekel, B. (2010). Concise Review: Kidney Stem/Progenitor Cells: Differentiate, Sort Out or Reprogram? Stem Cells. 28: 16491659 9. H  ayashi, M. (2006). Development of New Therapies, Including Regeneration of the Kidney, for Chronic Kidney Diseases. Clinical and Experimental Nephrology. 10: 99-101 10. Zerbini, G., Piemonti, L., Maestroni, A., Dell’Antonio, G. and Bianchi, G. (2006). Stem Cells and the Kidney: A New Therapeutic Tool. Journal of the American Society of Nephrology. 17: 123-126 11. Forbes, S.J., Vig, P., Poulsom, R., Wright, N.A. and Alison, M.R. (2002). Adult Stem Cell Plasticity: New Pathways of Tissue Regeneration Become Visible. Clinical Science. 103: 355-369

Dr Donald A Wellings CEO SpheriTech Ltd Donald Wellings is the owner and founder of SpheriTech Ltd based in Runcorn, Cheshire, UK. He has been performing laboratory to processscale peptide synthesis for almost 30 years and has a track record spanning careers in ICI, Zeneca and Avecia. He was responsible for the design, build, validation and commissioning of the cGMP facilities at Zeneca/Avecia for peptide synthesis. He is a regular speaker at international conferences and is the author of ‘A Practical Handbook of Preparative HPLC’, published by Elsevier in 2006. He is also the main inventor on 15 patents relating to solid phase synthesis, polymer chemistry and reactor design. SpheriTech was founded in 2009 to provide a consultancy-based business and laboratory-based resource for contract R&D and IP development. The company has a portfolio of IP related to the design, applications and manufacture of polymers and polymer particles for a range of applications including chromatography, solid phase synthesis, biocatalysis and cell culture. Email:don.wellings@spheritech.com

Spring 2012 Volume 4 Issue 2


Clinical Research

Robust Medical Information Practices for Better Risk Mitigation Many companies are grappling with their post-marketing risk management strategies, determining whether their safety surveillance and risk management procedures are sufficiently robust to identify new safety signals and positively impact upon already identified risks. Companies need to consider integrated approaches to risk management, ensuring that such activities commence early in clinical development. They also need to evaluate if they have the core competencies to comply with the subsequent requirements for the implementation and measurement of risk management strategies (e.g. REMS / RMPs), as required. Following changes to risk management regulations in recent years, it has become more commonly accepted that the concept of risk management needs to be integrated early into the clinical development process. Whilst in practice this is not always the case, the earlier risk management planning begins, the simpler it will be to establish an effective risk management strategy and develop the Risk Management Plan (RMP) / Risk Evaluation and Mitigation Strategy (REMS) necessary for an EU/US Marketing Authorization. Companies may consider enhancing the robustness of their data collection procedures. Bridging the link between safety surveillance procedures and case intake functions rarely happens in practice, currently, but is important to obtain specific information required for risk characterisation, particularly when such risks may potentially lead to product withdrawals. The scarcity of post-marketing follow-up information coupled with the current level of completeness of the spontaneous individual case safety reports (ICSR), makes challenging reporters with specific risk-characterisation questions at the point of case intake one of the most powerful signal 48 INTERNATIONAL PHARMACEUTICAL INDUSTRY

detection and tools available.

risk-characterisation

Introduction Pharmacovigilance requirements are more stringent now than ever before. Emergence of new legislation and methodologies, however, often follows the occurrence of major public health disasters such as that of thalidomide in the 1960s and rofecoxib in the 2000s. Consequently, legislative revisions have a tendency to be reactive rather than proactive. The result is a solution- rather than cause-focused approach to strengthening the pharmacovigilance system. Identification of risk is fundamental to protection of patient safety. All medicines have side-effects that is clear. It is how we detect, characterise and minimise these risks that ultimately affect both patients and companies alike. Many drug safety systems and processes were not designed to proactively manage risks now required by todayâ&#x20AC;&#x2122;s regulations. Typically companies have been challenged by a siloed culture, with R&D, drug safety and other stakeholders having varying levels of integration with respect to the reporting of adverse events. Clearly more needs to be done. Compliance per se is a necessary prerequisite, but it is not assured to demonstrate the safety of products and manage risks appropriately 1. By eliminating these silos, companies can benefit from a more proactive approach to risk management, allowing for better decision-making and greater strategy development. The primary focus over many years now has been firmly on risk management, with a particular emphasis more recently upon risk minimisation strategies. This is being considered a solution to prevent future public health disasters.

However, whilst risk identification and quantification techniques are now almost unrecognisable compared to even a decade ago, failure to comply with legislation also brings far greater financial penalties than ever before. Despite this, legislation and industry guidance is, to some degree, still lacking. Smaller companies continue to struggle when it comes to understanding regulatory expectations regarding risk management. Until recently, many companies still used aggregate reports, such as the PSUR, as their primary safety surveillance tool. Clearly this is insufficient, and an effort placed by industry in understanding what is needed has to a degree overtaken the fundamental question of why it is important. The prime focus on risk management would suggest, perhaps, that both legislation and industry practices are overlooking the critical role of medical information. The Role of Medical Information in the Pharmacovigilance System Almost all analyses of risk will, at some point in time, require a manual qualitative review of the underlying data. Indeed, single case reports can be sufficient to warrant a labelling change if adequately detailed. In the post-marketing setting, however, it is all too common that adverse event reports are too poorly documented to even conduct a causality assessment, let alone meaningfully contribute to our understanding of the event concerned. Taking a step back from the complexity of data mining techniques, it is clear that the focus needs to be directed, in parallel, towards the point of adverse event intake to ensure that individual cases are not only detailed enough to enable the identification of potential issues, but contain the specific information required to rapidly characterise those issues. Spring 2012 Volume 4 Issue 2


Clinical Research Medical information as a fundamental component of the risk management system Medical information departments are commonly perceived to be primarily an information-providing service. However, they are most companies’ primary source of post-marketing adverse events. Given their potential influence on the amount of data obtained about spontaneously reported adverse events, this makes them one of the most important functions within the entire risk management system. Healthcare professionals and consumers alike commonly contact a company’s medical information department to request information. The scope of this information request is vast, but safety information is frequently requested because a patient has experienced an adverse event. Indeed from personal experience it is rare for safety information to be requested in the absence of an adverse event. Similarly, it is equally rare for an individual to inform a company of an adverse event without requesting information about what has happened. Thus the medical information department is one of the most important departments when it comes to obtaining safety information. Medical information departments exist, in part, because of legislative requirements. European Directive 2001/83/EC, as amended, requires Marketing Authorization Holders (MAH) to establish “a scientific service” in charge of information about the products that the MAH places on the market. This scientific service encompasses the company’s medical information department. However, legislation alone provides little information about how this service should be conducted and, in particular, the degree of overlap between this scientific service and the system. Similarly, the existing European pharmacovigilance legislative framework is equally unclear. Article 103 of Directive 2001/83/EC requires MAHs to have in place, “a system which ensures that information about all suspected adverse reactions…is collected and collated…” This implies www.ipimedia.com

that the scientific service referred to previously should also be considered as part of the pharmacovigilance system. Despite this, in the July 2009 to March 2010 Pharmacovigilance Inspection Metrics Report, the Medicines and Healthcare products Regulatory Agency in the UK reported that 7% of all critical findings related to non-compliance of medical information departments. It is therefore clear that medical information is firmly considered by regulatory authorities to be a key component of the pharmacovigilance system. Detailed guidance exists regarding requirements for processing of suspected ADRs, writing aggregate reports and performing risk management, all of which depend upon ICSRs. Yet such guidance provides little information about how suspected ADRs should be captured. Whilst the Council for International Organizations of Medical Sciences (CIOMS) V Working Group provides recommendations regarding how suspected ADRs should be followed up 2, it is well accepted that receipt of follow-up information, particularly from follow-up forms, is relatively low. Indeed, one recent study suggests that only 36% of follow-up attempts are actually successful in practice 3. The point of adverse event intake is, therefore, often the only opportunity by which information about adverse events can be obtained. Common adverse event intake strategies Figure 1 below demonstrates the typical route that an ICSR may take within the scope of many companies’ pharmacovigilance systems. Figure 1. The common approach to AE intake fails to provide formal linkage between safety surveillance and AE intake.

Adverse events are usually reported to the medical information department during an information request. To ensure regulatory compliance, any safety requests should be followed with a direct line of questioning to determine whether a patient has experienced an adverse event. Such questioning must be documented, along with the response. If it is determined that an adverse event has occurred, the call handler will usually capture all of the reported information about the event. Whilst they will usually be receptive to the information supplied, they are inevitably limited in their ability to request specific detail about the pathophysiology of the event. The captured adverse event is subsequently forwarded to the pharmacovigilance department and processed onto the global safety database. Regular reconciliation activities ensure that all safety information has been transferred from one department to the other. Depending upon the company’s safety surveillance procedures, it is likely that the report will be reviewed at some point in time, outside of routine medical review, to determine whether it contributes to either a new or existing safety signal. Regulators will always tend to take a conservative approach, and although many use sophisticated data mining algorithms themselves to aid in the identification of new safety signals, a large volume of reports of a particular drug-event combination will always trigger a potential signal alert, regardless of the data quality within individual cases. Depending upon the nature of the event, potential consequences range, at best, from a regulatory authority request to review the drug/event in question, to a suspension or withdrawal of the product from the market. The latter is ever more likely, as high profile cases hit the media and present a poor reflection of both regulatory authorities and companies alike.

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Clinical Research Strengthening the Medical Information Function to Increase Effectiveness of Risk Management Strategies It has long been accepted that the concept of risk management needs to be integrated early into the clinical development process. Whilst in practice this is not always the case, the earlier risk management planning begins, the simpler it will be to establish an effective risk management strategy and develop the Risk Management Plan (RMP) / Risk Evaluation and Mitigation Strategy (REMS) necessary for an EU/US Marketing Authorization. Yet it is rare during risk management planning for any great consideration to be given regarding the role that medical information – the department fundamentally responsible for how effective such strategies will be – will play in the overall risk management system. The essential component that is frequently lacking is a direct feedback mechanism between safety surveillance/risk management teams and the medical information function. It is rare that medical information teams are kept aware of safety issues under close monitoring, let alone what line of questioning to undertake in such circumstances. This lack of feedback occurs even more frequently when either medical information or pharmacovigilance services are outsourced, given that this is often not considered when compiling contractual agreements or Safety Data Exchange Agreements. Those conducting safety surveillance and risk management have a detailed understanding not only of what events are being closely monitored, but also what specific information is needed to characterise a particular ADR. To ensure that as much information as possible is captured at the point of intake, it is necessary to ensure that a formal feedback mechanism exists (see Figure 2). It could be argued that this should already occur as part of risk minimisation strategies within RMPs/REMs. However, such documents are usually based primarily upon clinical trial data, and although updated on an ongoing basis in the post-marketing phase, such 50 INTERNATIONAL PHARMACEUTICAL INDUSTRY

updates rarely extend to the role of medical information. At a predefined frequency, therefore, safety surveillance teams should provide the medical information department with a list of key events per product along with a small number of key questions per event. This is to ensure that when a particular drug-event combination is reported, the medical information advisor is prepared with a list of key questions that they can ask the reporter. At the time of adverse event reporting, the reporter is more often than not requesting information about that event. Therefore, the provision of the requested information can be provided in an almost negotiating manner. Reporters are often more forthcoming with information when they expect to receive something in return. Whilst it is in no way suggested that information is withheld subject to reporter compliance, in practice this is a highly effective method of obtaining information at the point of adverse event intake. Figure 2. Forging links between safety surveillance and medical information to strengthen the risk management system.

This approach also brings additional benefits. Rather than merely requesting the minimum information required to form a valid case, asking a set of key questions about the adverse event concerned often results in a much higher degree of reporter satisfaction, knowing that the company has demonstrated a high degree of diligence by truly attempting to obtain as much information about the event as possible. Summary Proactive risk management is fundamental to the identification of new risks and characterisation of

existing risks. It is key not only to protecting patient safety, which is of paramount importance, but an effective risk minimisation strategy can also help to prevent the subsequent financial repercussions that frequently follow public health disasters. Safety surveillance teams should provide the medical information department with better information of events and key questions per event ensuring that when a particular drug-event combination is reported, the medical information advisor is prepared so that companies can benefit from this highly effective method of obtaining information at the point of adverse event intake. References: 1. M aenni, U. “Pharmacovigilance: A Company-Wide Challenge” Applied Clinical Trials, February 2008 2. C ouncil for International Organizations of Medical Sciences (CIOMS) Report of CIOMS Working Group V. Current Challenges in pharmacovigilance: Pragmatic Approaches. (2001) 3. A nton, C., Cox, A.R., Ferner, R.E. “Improving follow-up rates in spontaneous adverse drug reaction reporting: effectiveness of a targeted letter used by a regional centre in the UK,” Drug Safety 32 (12) 1135-40 (2009) Ian Kovacs, PhD, is Director, Head of Pharmacovigilance Consultancy & Business Support at PAREXEL International Ltd where he oversees the pharmacovigilance consulting activities and provides subject matter expertise across the business. Ian has over 12 years’ experience in all areas of pharmacovigilance and medical information and has held a number of senior management positions. Ian graduated from the University of Southampton with a degree in pharmacology and a PhD in Pathology, during which he was shortlisted for a Young Investigators Award by the British Society for Haematology. Email: ian.kovacs@parexel.com

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Conducting Cystic Fibrosis Clinical Trials â&#x20AC;&#x201C; Methodological, Practical and Ethical Considerations Abstract Cystic fibrosis (CF) is a chronic, progressive, life-threatening genetic disease that primarily affects the pulmonary, gastrointestinal, endocrine and reproductive systems. It is an autosomal recessive disorder that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. To date, more than 1300 mutations have been identified in the CFTR gene. Approximately 70,000 individuals worldwide have CF, and although CF affects all racial and ethnic groups, it is more common in Caucasians of European descent. According to the Patient Registry Reports, the median age of survival for a CF patient in 2010 for the United States (US) is 38.3 years old and for 2009, 26.7 years old for Australians. Increased participation in clinical trials for CF patients is of paramount importance in order to further clinical and scientific knowledge and the development of novel medications and therapies. As with many other chronic diseases that are of orphan status, there are many challenges and considerations when conducting CF clinical trials. This article explores methodological, practical and ethical challenges frequently experienced by study participants and research staff. Key Words: Cystic fibrosis, CFTR, clinical trials, contract research organisation Background Over the past few decades, progress has been made to reduce morbidity and delay mortality for patients with cystic fibrosis (CF). The median predicted age of survival for CF patients in the US has dramatically increased over the past 20 years. In 1986, the median predicted age of survival was 27 years old compared 1 to 38.3 years in 2010 . A similar trend 52 INTERNATIONAL PHARMACEUTICAL INDUSTRY

for improvement in morbidity has 2 been observed in Europe . However in Australia, the median age of death for CF patients decreased in 2009 compared to 2008 and 2007 (26.7 compared to 30.3 and 30.7 years 3-4 old, respectively) . The reason for this decline is unclear. The overall global progress is due in part to neonatal screening which has allowed for earlier diagnosis and treatment, improvement in nutrition, consensus guidelines regarding the treatment of 5,6 CF patients and novel medications and therapies that treat not only the symptoms of the disease but also the underlying cause of the disease. Despite these advances, there is a continuous need for CF patients to become involved in clinical research, which will enable the development of new treatments and novel ways of managing the disease. However, as research subjects, CF patients are considered a vulnerable population and have special needs due to the relatively young age of study participants, likelihood for crossinfection, progressive nature of the disease, poly-medication use and complexity of disease due to exocrine gland dysfunction and multiple organ system involvement. As CF is considered a rare (â&#x20AC;&#x153;orphan statusâ&#x20AC;?) disease, there a limited patient population available for clinical research in addition to a limited number of CF centres. According to the clinicaltrials.gov website, there were a total of 143 clinical trials actively enrolling CF patients as of April 2012, as outlined below.

It has been estimated that currently less than 30% of all CF patients are potentially able to participate in clinical 7 trials . CF clinical trials are generally conducted at highly specialised CF centres, and therefore not only is there substantial competition for patients with this rare disease, but also for trained research sites able to conduct clinical trials to a high quality standard. It is extremely important for persons with CF to participate in clinical trials in order for new treatments to be developed. Patient advocacy organisations have done a good job to date of emphasising the need for participation in studies in addition to providing useful information on their websites about clinical trials. Development of clinical trial networks in the US and Europe has been beneficial for ensuring that CF patients have the opportunity to be involved in research. Development of similar networks in other regions such as Latin America is currently lacking, and would be advantageous. The global prevalence 8 of CF is displayed below .

The objective of this article is to outline key methodological, practical and ethical issues in the conduct of CF clinical trials with focus on site identification, feasibility and clinical research studies. Mitigation strategies for these challenges will also be discussed.

Spring 2012 Volume 4 Issue 2


Clinical Research CF Programme Considerations: Methodological Considerations There are several unique considerations when developing a CF programme. Although animal models for CF exist, such as the transgenic and CFTR-knockout mice, there are limitations with this species and it is unclear how relevant these findings are to humans. Furthermore, it is important to note that there are currently no animal models for CFrelated diabetes and hepatic disease. Due to these challenges, pig and ferrets as CF animal models have been generated, and there is hope that these novel CF models will lead 9 to new therapeutic approaches . When developing a CF clinical programme, it is important to review the regulatory guidance documents drafted by the Food and Drug Administration (FDA) and European 10,11 Medicines Agency (EMA) . When planning human CF studies, consideration needs to be given as to the strategy regarding age ranges of patients to include in the trials. Typically, an investigational product is initially studied in adolescents and adults (or only adults 18 years and older) followed by studies conducted in children. However, as CF is still mainly a paediatric disease generally diagnosed at birth, it may be advantageous to initiate trials in different age groups including children. Of course, the ability of children to successfully perform study assessments needs to considered, such as the lack of the ability of children to successfully perform spirometry. Additionally, for early phase studies involving a nebulised investigational product, it is important to ascertain whether the manufacturer intends to revise and/or discontinue manufacturing of the current nebuliser compressor system. In addition, the potential implications and consequences need to be considered regarding the availability of novel vibrating mesh nebuliser systems such as the Pari eFlowÂŽ and Source CFâ&#x201E;˘ nebulisers, should a patient use an unauthorised nebuliser system with the nebulised investigational product being studied. For early Phase 1 CF studies, it is important to determine 54 INTERNATIONAL PHARMACEUTICAL INDUSTRY

whether pharmacokinetic and pharmacodynamic data from healthy volunteers is applicable or not to CF patients. Additionally for trials involving children, unique issues to consider when designing a paediatric study include parent involvement, family decision-making, issues with phlebotomy, and the total volume of blood required. There are several additional key challenges that exist when designing pivotal Phase 2 and 3 CF studies. Randomised active-controlled trials are mandatory for confirmatory trials, even when a reference treatment exists. The decision whether to use a parallel group, crossover or cohort study design is important, due in part to the paucity of CF patients available to participate in clinical trials. Also essential is the timeframe required to obtain key data, potential for carry-over effects, potential seasonal effects, and the requirement for long-term exposure as required by regulatory authorities. For global studies, it is crucial to determine whether or not the CF patients are relatively homogenous across regions (including standard of care, survival rates and availability of medications including off-label usage). This can be difficult to determine, as scant data regarding CF exists for some regions such as Latin America and Central Eastern Europe. In these cases, feasibility is needed to best ascertain the risk/ benefit of conducting CF studies. Additional risks based on protocolspecific target populations also may be encountered e.g., if a study requires antibiotic-naĂŻve patients, they would be difficult to recruit. Another area that is particularly challenging is determination of appropriate endpoints. In contrast to oncology studies, survival is not a practical or ethical primary endpoint, therefore surrogate endpoints such as spirometry, pulmonary exacerbation and quality of life assessments are typically used and are deemed to be appropriate by regulatory authorities. Nonetheless, there can be a high degree in variability for endpoints such as forced expiratory volume in one second (FEV1) and typically only children older than five

or six years old are able to correctly perform spirometry. In addition, there is no global standard definition for pulmonary exacerbation, and typically studies use two to three different definitions. Standardisation of diets across study sites and geographical regions can also be difficult, and it is preferable to centralise such efforts with local input, especially because of the effect of nutrition on pulmonary function. To further complicate matters, the determination of a standardised clinically relevant effect has not been established for many endpoints, including changes in pulmonary function and nasal potential difference. Spring 2012 Volume 4 Issue 2


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There is not a universal solution to address all of the aforementioned study programme challenges, however, it is imperative to consider these key issues and make evidencebased decisions considering the context of CF patients and the variability in disease severity. Recent experience is important so that one is kept abreast of issues and lessons learned to effectively mitigate these challenges. Practical Considerations CF studies are unique in that many study sites are affiliated with patient advocacy groups such as the US CF Foundation (CFF) and European www.ipimedia.com

Cystic Fibrosis Society (ECFS). Both organisations have the ability to review the protocol, vet the protocol with their network of research sites, and provide the sponsor and/or contract research organisation (CRO) a shortlist of interested sites. These networks of sites include the premier CF centres that are very experienced in clinical research and generally have an extensive database of patients that can be utilised for pre-identifying patients for a study. When selecting sites, one must consider the mix of paediatric and adult centres to use. CF is distinctive in that many adults are still followed by paediatric CF centres, as patients can

be reluctant to change their healthcare providers. Therefore, even though a clinical study might involve adults only, it is likely that many paediatric CF centres would be able to recruit eligible patients either from their own patient database or by networking with their institution’s adult CF centre. In the US, approximately 67% of all CF patients are included in the 1 CFF’s National Patient Registry . This facilitates review of each site’s patient database in an effort to pre-identify patients who satisfy the protocol’s entry criteria. In Europe, the ECFS are planning to strengthen their registry in the near future. There are many study-specific challenges for CF studies, some of which are unique to CF studies. These include sputum induction, performing pulmonary function tests in infants, organising patient (and parent/caregiver) overnight visits, managing study visit impact on school or work schedules, and the hesitancy of CF patients to be involved in clinical trials, either due to misconceptions about research, and the burden of attending multiple study visits (and potential loss of income and/or identifying themselves to their employer as a CF patient). Compliance to the study schedule and patient follow-up is important for the study success. Telephone calls and text messages from sites to participants during the study would help in compliance with the study schedule. Other compliance-enhancing measures could be reimbursing patient travel for the follow-up visits as allowed by the local regulations, and flexible clinic schedule and visit windows to accommodate research patients around their school/work schedules. Training of all study staff is key towards ensuring quality data, as is standardising processes and procedures (i.e., sputum induction) across study sites. Educating patients about clinical research is vital, and both the CFF and ECFS provide useful brochures about clinical research to help in this effort. In general, use of advertising is not warranted as CF patients are already well known by highly specialised CF centres, and patients not treated by these centres may be under-treated. INTERNATIONAL PHARMACEUTICAL INDUSTRY 55


Clinical Research Ethical Considerations There are several ethical challenges facing CF clinical research, especially as the majority of the clinical trials involve children and adolescents, and/or utilise a placebo-control study design. Informed consent forms for CF studies can be extremely lengthy and difficult to comprehend, despite using readability statistics to ensure the document is no higher than a fifth-grade reading level. Ideally, both parents should sign the informed consent; however, some ethics committees (ECs) permit only one parent to sign the form. The precise age at which a child should be involved in the consent and assent process has not been fully elucidated. Ensuring that ECs and all study site personnel follow the tenets of good clinical practice (GCP) is key to protecting the safety of the patient. Other ethical considerations include conducting a clinical trial in a country in which the sponsor does not ultimately intend to market the medication. The intent of the sponsor to eventually market the drug in some regions can be questioned by regulatory authorities when submitting the clinical trial application. Additionally, non-Caucasians may be under-represented in CF clinical trials. Therefore, it is important to discuss this with study site personnel and provide support if needed to reach out to minority patients who might be eligible for a study. In addition, the need to provide patient-related documents in languages other than English would potentially help facilitate enrolment of non-Englishspeaking patients. Another interesting aspect is the length of time it takes for completion of a CF trial and publication of the study results in a peer-reviewed journal. It was determined that the median time is 3.25 years between completion of a CF trial and publication. Additionally, a large proportion of studies fail to report study results within five years 11 of study completion . This has potential negative consequences, especially as guidelines are based on evidence-based data, and patients who participated in these studies do not readily have access to study results and therefore cannot make 56 INTERNATIONAL PHARMACEUTICAL INDUSTRY

informed decisions about future study participation. Summary Although conducting clinical trials in CF is challenging, the roadblocks experienced (such as those summarised in this article) can be anticipated and mitigation strategies can be implemented when specific trigger points are experienced. The overall goal must be kept in mind – to provide novel medications and therapies which improve life expectancy and the quality of life of CF patients worldwide. Recent experience is key in order to understand the processes of CF advocacy organisations, and identify the most experienced research sites and develop mitigation strategies proactively to address considerations and challenges. References 1. Cystic Fibrosis Foundation patient registry annual report 2010. Bethesda, Maryland, USA: Cystic Fibrosis Foundation. 2. Meha G, Macek M, Mehat A. Cystic fibrosis across Europe: EuroCare CF analysis of demographic data from 35 countries. J Cyst Fibros 2010 Dec; Suppl 9; S5-S21. 3. Reid D, Blizzard C, Shugg D, et al. Changes in cystic fibrosis mortality in Australia, 1979-2005. Med J Aust 2011; 195(7): 392-395. 4. Cystic fibrosis in Australia 2009 – 12th annual report from the Australian cystic fibrosis data registry. Accessed online: http:// w w w. c y s t i c f i b r o s i s . o r g . a u / projects/dataregistry/. 5. Flume P, Mogayzel P, Robinson K, et al. Cystic fibrosis pulmonary guidelines – treatment of pulmonary exacerbation. Am J Respir Crit Care Med 2009; 180:802-808. 6. Stallings V, Stark L, Robinson K, et al. Evidence-based practice recommendation for nutrition-related management of cystic fibrosis and pancreatic insufficiency: results of a systematic review. J Am Diet Assoc 2008; 108:832-839. 7. Goss CH, Rubenfeld GD, Ramsey BW, Aitken ML. Clinical trial participants compared with

nonparticipants in cystic fibrosis. Am J Respir Crit Care Med 2006; 173:98-104. 8. T  he molecular genetic epidemiology of cystic fibrosis; Report of a joint meeting of WHO/ ECFTN, ICF(M)A/ECFS. Genoa, Italy, 19 June 2002. Accessed on 12 April 2011 at http://www.cfw. org/docs/ who/2002/who_hgn_cf_ wg.04d02.pdf. 9. F  isher JT, Zhang Y, Engelhardt JF. Comparative biology of cystic fibrosis animal models. Methods Mol Biol 2011; 742:311-334. 10. Guidance for Industry – Exocrine Pancreatic Insufficiency Drug Products – Submitting NDAs. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER); April 2006. 11. Guideline on the Clinical Development of Medicinal Products for the Treatment of Cystic Fibrosis. European Medicines Agency. London, 22 October 2009. 12. Hurley MN, Prayle AP, Smyth AR. Delayed publication of clinical trials in cystic fibrosis, J Cyst Fibros 2011;11(1): 14-17.

Ms. Brandi has more than 25 years experience in clinical research (Clinical Operations and Project Management) in addition to seven years practicing as a Physician Assistant. Ms. Brandi has worked for pharma, biotech and CROs and has managed Phase 1 to 4 studies domestically and globally. She has 20 years of experience in respiratory clinical research, including cystic fibrosis, asthma, COPD and allergy. Ms. Brandi is a Therapeutic Strategic Lead in the Allergy and Respiratory Center of Excellence at Quintiles. Email: vikki.brandi@quintiles.com

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Clinical Research

Strategic Overview of Personalised Medicine Personalised medicine is not yet fully implemented in healthcare developments, though the concept is several years old. The reasons might be scientific, technological, financial, regulatory, and ethical. The switch to personalised medicine cannot be achieved by merely considering one of those aspects isolated from the others, because they are all interleaved. It takes a global strategic analysis to determine how the healthcare industry should address this challenge. In this article, we outline recent trends in this regard. One observation relates to data science or the lack of it - in current practice. So far, two players have been working together: the biopharma industry, responsible for R&D, production and commercialisation of the treatments on one hand, and the in vitro diagnostic (IVD) industry, responsible for biological measurements production on the other. The latter are supposed to address the issues of the former. Our point is that there is a missing link: players who can extract useful knowledge from the information contained within the huge amounts of data produced, be it clinical, -omics, imaging, etc. But data scientists are traditionally not massively present in the healthcare business. The integration of this complementary component into the current landscape is in itself a challenge, but full of opportunities. 1. Personalised Medicine – What and Why? The model of blockbuster drugs aims at treating all patients with a given disease with the same treatment. This model has been the workhorse of the biopharmaceutical industry for decades. It allows for massive investments in R&D for long periods, which are amortised over a global 58 INTERNATIONAL PHARMACEUTICAL INDUSTRY

market and over several years through protection by patents. This model is gradually exhausted, both conceptually and economically 1,2. Conceptually, because the diseases that are difficult to handle nowadays are extremely heterogeneous, and therefore a single treatment is often ineffective - we speak of cancer when there are in fact many types of cancer, with very different specificities, while autoimmune diseases like lupus or arthritis may also have various forms. Economically, because investment in R&D and duration of treatment development are constantly increasing, while patent protection is limited to 20 years. Currently, a new drug development costs an average of 1.1 to 1.5 billion dollars according to estimates, and a clinical trial lasts about 13 years, leaving only seven years of patent coverage to recover this investment and realise the margin expected by investors. An extra protection of five years is possible in some cases (SPCs), but is not a sufficient extension. The ROI per Euro invested in R&D decreases, while at the same time the overall R&D budget increases exponentially. This is mainly due to failure to demonstrate sufficient efficacy of treatments in the final stages of validation. Medicine and healthcare then become progressively personalised and predictive by necessity, while putting the patient back at the heart of the business. New approaches are required, based on patienttailored therapies 3. There are in fact completely personalised medicine (a treatment per patient) and stratified medicine (a specific treatment for each subgroup of patients within the population with the disease) 4. Here, we adopt “personalised medicine” broadly to mean both. If there is a clear need for personalised healthcare,

how come there are not more such therapies available on the market? As we point out in this article, the causes are more due to a missing link in the value creation chain of the healthcare industry rather than to regulatory, political, economical or even ethical issues. 2. Ethical Issues Ethical issues do not really restrain advances in personalised medicine, even if its emergence is controversial. For example, what will happen if we do not identify treatments for all subgroups of patients with same disease? If the cost of developing a treatment is unchanged, but the population that it targets is narrower, won’t the price per dose explode? Conversely, must the community pay for the reimbursement of treatments when it can be known in advance they will be ineffective? Or for providing treatment to a patient if it can be predicted based on its profile that there will be harmful sideeffects (not to mention the indirect costs of treatments to deal with these effects)? Finally, some concerns are also discussed in terms of respect of privacy, especially in relation to the recent availability of affordable complete genome sequencing solutions. At some point, the DNA is the identity of the patient, more than his passport. These privacy issues are generally answered by freedom of information, and advances in secured communications. The next section shows that regulatory authorities even make some of these questions obsolete before they are answered. 3. Market Access and Reimbursement The Food and Drug Administration or the European Medicines Agency, although known for their strictness Spring 2012 Volume 4 Issue 2


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and the high impact of their regulations, are not conservative regarding personalised medicine. Recently, the FDA published many guidances (or drafts) that are consistent with rapid but intelligent deployment of personalised medicine strategies 5,6,7,8. Interestingly enough, the recommendations in this area involve skills outside the traditional scope of the biopharmaceutical industry, including a focus on data analysis. Some recommendations namely advocate for the systematic collection, and if possible analysis, of DNA samples from all patients involved in any clinical study. We discuss data analysis later in this text. Personalised medicine requires different treatments for different patient profiles, which in turn requires the design of diagnostic kits for determining these profiles. Currently, the activities of drug development and diagnostic kits conception are provided by different actors (biopharma on one hand and the IVD industry on the other hand), with huge problems of collaboration, allocation of financial margins, intellectual property, even of drug-kit adequacy, etc. 9. The FDA advocates in this area a closer collaboration for these activities, if not a merger: a personalised treatment and its companion diagnostic kit should be co-developed under the supervision of a single sponsor 10. As these recommendations are getting implemented, the industry has no choice but to comply. Thus, regulatory authorities are rather guiding personalised medicine development than preventing it. Aside from market access issues, new reimbursement strategies are also at stake. This question is generally not dealt with at a continental level, but more at a www.ipimedia.com

national level. It is thus not so easy to give a big picture. Just as an example, conditional reimbursement related to personalised medicine has been discussed by the Belgian parliament late in 2011 11. Most of those new policies are not operational yet, but go in the right direction. Anyway, at present, most of the few examples of already available personalised treatments and diagnostic kits are not reimbursed, even if they get market access. Rather surprisingly, studies have shown that even without reimbursement, some of these quite expensive products can be commercial successes 12. The same studies show that the two main factors for this success are i) the evidence of effectiveness of these therapies (patients are willing to pay more for a treatment whose efficacy is more likely), and ii) the views of professional associations. 4. Patientsâ&#x20AC;&#x2122; Empowerment After showing that neither ethical issues, nor market access and reimbursement are responsible for the lack of personalised medicine products on the market, let us consider the patient himself. Little has been said of patients so far, who are the beneficiaries of the entire healthcare industry. Could the patient be in disfavour of personalised medicine approaches? The patient is traditionally confident in his GP or the specialists he consults. However, with the advent of modern communication and information technology, many patients are keen to take control of their health. Today, even if the doctor is still the primary source of inside information taken by patients about a disease, 23% of patients look first on the internet, 25% go first to printed articles and books, and 43% of patients see medical websites

each month 13. Accordingly, we note the presence of actors recently offering B2C personalised medicine, such as 23andMe, Navigenics, and deCODEme. These online services offer to order a do-it-yourself DNA collection kit, which is then returned by mail. Hundreds of thousands of genetic traits are studied in a few days in an automated way, and the patient can then browse through his genetic peculiarities on the same web portal. These may be trivial in some cases (e.g., eye colour), but often have a prognostic value (x% higher than average risk of developing a disease) or include various information (the patient is genetically more of a long-distance runner than a sprinter, is lactose intolerant, etc.). These services are, in their current form, highly risky and questionable ethically, but this is beyond the scope of this work. The important point is that the patients want to participate in managing their health. Another evidence of that is the recent Genomera initiative, which enables any user to start a clinical study, in the web 2.0 spirit. Each user is free to participate or not, and to share his/her genomic data if available. Clearly, the patients, too, are enthusiastic about personalised medicine. 5. Diagnostics: Data Scientists at the Gate. Could the bottleneck of personalised medicine be the complexity of biology itself? Molecular biology and imaging technology have made dramatic progresses to enable us gain a better picture of living entities. We now have a hard time ahead of us to analyse this big picture, and extract useful knowledge from it. The IVD industryâ&#x20AC;&#x2122;s mission as a whole, as its name suggests, is to provide diagnostic INTERNATIONAL PHARMACEUTICAL INDUSTRY 59


Clinical Research solutions. Based on a measurement of one or more biological parameters - whatever they are - these solutions are supposed to provide decisions (or at least decision support). Is the patient healthy or suffering from a given disease? And if he is, of which subtype, eventually? Among a given set of available treatments, which one will be more effective or safe? In practice, there is some discrepancy between this strategic objective and the reality of the solutions provided. Indeed, in many cases, the solutions simply provide IVD measures, and increasingly in large numbers, but do not really provide decision support based on these data. There is therefore a relatively low adoption of these solutions by medical practitioners, unable to interpret the results. Referring physicians are also inundated with information about the latest developments in medicine. These advances are increasingly complex, and are occurring at increasing frequency. Many physicians have therefore neither the time nor in some cases the skills to keep pace with new discoveries. These IVD solutions are also sold to medical research centres and biopharmaceutical companies where they are already more useful, mainly for clinical research. However even in this latter case, they do not deliver their full potential if analysis capabilities are not available to deal with the data they generate, in conjunction with clinical observations on patients. This chronic lack of data aggregators is pointed out by analysts 14, and is probably the main cause of the delay in personalised medicine successes. Real data scientists will not supersede biologists and medical doctors, but can help them make more of their tools. There are numerous examples of clinical research where data is planned to be produced “just in case” and then left without proper analysis 8. Data analysis is not about producing data. It is about generating knowledge from the information the data contain. A biomarker should not be named a biomarker just because 60 INTERNATIONAL PHARMACEUTICAL INDUSTRY

a clustering analysis has shown that several subgroups of patients seem “similar”. A biomarker should always be linked to a clinical outcome. But there comes a trickier issue: when many features are measured with respect to the number of samples available (as it typically is the case with modern IVD solutions), it is trivial to identify a biomarker that can perfectly split a cohort of patients into two groups. In fact, as soon as there are n-1 or more features that are measured on n patients, it is guaranteed that a perfect single biomarker can be found 15. Is it good news? Not at all. But it explains why analysing microarray data (several tens of thousands gene expressions) produced on a few tens of patients led to diagnostic, prognostic, or response prediction biomarkers that subsequently proved useless on new patients. Special care has to be taken when dealing with high dimensional data on few samples. Traditional statistics books even recommend not to analyse data when at least 10 samples are not available for each measured feature. Where will we find 30 billion human beings to get sequenced if we are about to analyse next-generation sequencing data? So, many analyses that pretend to be predictive are in fact simply descriptive of the patient cohort at hand, but will prove very poor on new, unseen patients, which is however the aim of biomarker identification, on which personalised medicine is supposed to rely. Designing analysis protocols that avoid selection bias (validating biomarkers on the same patients used to identify them) or overfitting (a similar issue for predictive models) in itself requires expertise in data analysis. And the distinction between the question of identifying biomarkers and identifying a good predictive model on those biomarkers is frequently simply overlooked. Another limitation is that many of the approaches for identifying biomarkers that are used today are univariate, meaning that they consider the predictive (or descriptive) power of each candidate marker individually. It can be shown that several markers that are totally useless alone, once combined,

can be very powerful at producing diagnostics. And is there a scientific rationale for univariate diagnostic solutions anyway? But how to deal with every possible combination of several biomarkers within a set of a few thousands of potential ones, when it amounts to evaluating more combinations than particles in the universe? Again, smarter approaches are available, but require specific expertise in data mining or machine learning 16. Those fields of research are related to statistics, applied mathematics/ optimisation and computer science, and are complementary to the set of expertises that are traditionally found in the healthcare industry. They will help personalised medicine perform major advances in the coming years. 6. A Vision of Open, Innovative and Data-Driven Healthcare Medicine and healthcare have become increasingly personalised and predictive, with the patients at the heart of their concerns. The model of blockbusters is exhausted, both economically and conceptually, and new approaches are required, based on more advanced diagnostics and treatments tailored according to patient characteristics. We outlined that most strategic aspects related to personalised medicine were good, but that the field required more attention to data analysis. The healthcare sector could then be seen as three axes working together on an open innovation mode. The first component is the biopharmaceutical industry, responsible for making available new treatments. Until recently, biopharmaceutical companies ensured three facets of their business: R&D, production, and marketing. While they still perform the last two, they tend to increasingly outsource R&D. At present, it is not uncommon that the very concept of a new treatment, or a new technology platform, has emerged in a small entity (biopharmaceutical, hospital, CRO or research centre) and is simply then bought by a larger biopharmaceutical company. Beyond the simple relation of outsourcing, there is an increasing Spring 2012 Volume 4 Issue 2


Clinical Research tendency to co-development. This is the open innovation model 17. Risks and investment are shared by a consortium of companies (and/ or academic partners) and the benefits are equally distributed. While this trend is commendable and promising, it is not easy to implement, mainly due to partner size asymmetries, and requires heavy prior legal work. Nevertheless, the open innovation tendency is likely to increase in the future. The second component is the IVD industry, allowing a range of measurements of biomarkers (genomic, proteomic, imaging ...) growing larger, more efficient and more affordable. The third component is the data mining industry, responsible for analysing the massive streams of data produced by IVD devices for the biopharmaceutical industry, but also for hospitals, physicians or patients directly. If applying real and fair open Innovation and incorporating data analysis solutions within the healthcare industry, all parties will benefit from personalised medicine. The biopharmaceutical industry will produce safer, more targeted and effective treatments, with reduced economic risk and development times. The IVD industry will meet its strategic objectives: producing real diagnostic tools, which are also useful to patients and doctors, rather than measurement devices. Patients will get better healthcare, with reduced, or at least more targeted, public health expenses. References 1. J ack, A. (21 November 2011). Big pharma counts the cost of R&D. Financial Times. 2. M aes, I. (2011). Recent developments on clinical trials in Belgium. PriceWaterhouseCoopers. 3. F rost & Sullivan (2010). Advances in personalized medicine. 4. Trusheim, M., Berndt, E., and Douglas, F. (2007). Stratified medicine: Economic implications of combining drugs and clinical biomarkers. Nature Reviews – Drug Discovery, 6, 287–293. 5. U.S. Department of Health and 62 INTERNATIONAL PHARMACEUTICAL INDUSTRY

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

Human Services – Food & Drug Administration (2005). Guidance for industry – pharmacogenomic data submissions. U.S. Department of Health and Human Services – Food & Drug Administration (2006). Draft guidance for industry clinical laboratories, and FDA staff – in vitro diagnostic multivariate index assays. U.S. Department of Health and Human Services – Food & Drug Administration (2011). Draft guidance for industry – clinical pharmacogenomics: Premarketing evaluation in early phase clinical studies. E uropean Medicines Agency (2011). Reflection paper on methodological issues associated with pharmacogenomic biomarkers in relation to clinical development and patient selection. F ierce Medical Device (2011). Companion Diagnostics: The Future of Medicine. U.S. Department of Health and Human Services – Food & Drug Administration (2011b). Draft guidance for industry and Food and Drug Administration staff – in vitro companion diagnostic devices. D e Block, M., Detiège, M., Somers, I., Rutten, G. and Van Cauter, C. (2011). Proposition de résolution relative à la médecine personnalisée et aux défis pour notre système de soins de santé. Chambre des Représentants de Belgique. M eckley, L. and Neumann, P. (2010). Personalized medicine: Factors influencing reimbursement. Health Policy, 94, 91–100. G eens, M. (2011). The e-healthcare age: New means of communication and information gathering in an era of healthcare consumerization & patient empowerment. InSites Consulting. P riceWaterhouseCoopers (2010). Biotech reinvented: Where do you go from here? Vapnik, V. (1995). The Nature of Statistical Learning Theory.

Springer. 16. H  elleputte, T. (2011). Stable Feature Selection in Empty Spaces: Applications to Gene Profiling and Diagnosis from DNA Microarrays. LAP Lambert Academic Publishing, ISBN 9783-8443-9014-8. 17. S hankar, B. (2010). Contract research outsourcing: Market trends – 2010 & beyond. Vital Signs.

Thibault Helleputte graduated as an Engineer in Computing S c i e n c e in 2006 at the Université Catholique de Louvain (UCL), where he obtained a PhD in 2010. He obtained a Masters degree in Management Sciences from the Louvain School of Management in 2012. For several years, he has been working on data mining solutions for high-throughput data analysis. In 2012, he co-founded DNAlytics, a UCL spinoff offering an innovative technology for data analysis enabling personalised medicine strategies for the healthcare industry. Email: thibault.helleputte@dnalytics.com

Spring 2012 Volume 4 Issue 2


Clinical Research

Validation of a Bioanalytical ICP – MS Method for Quantification of Potassium in Human Urine Synopsis Inductively Coupled Plasma Mass Spectrometry (ICP-MS) is being increasingly utilised to quantify the concentration of elements in biomatrices in support of nonclinical and clinical studies. These data support pharmacokinetic (PK), toxicokinetic (TK), and bioequivalency (BE) evaluations. The purpose of this paper is to present a bioanalytical ICP-MS method validation procedure applicable to the quantification of potassium in human urine in support of BE studies. This method was validated following applicable bioanalytical guidance documents, and includes assessments of sensitivity, selectivity, linearity, carryover, intra/inter-day accuracy and precision, matrix effect, ruggedness, and various stability parameters. The method objectives, materials, results, and conclusions are presented. Keywords Inductively Coupled Plasma Mass Spectrometry, ICP-MS, Good Laboratory Practice, Clinical, Bioequivalence, Bioanalytical Method, Method Validation, Potassium, Human Urine Introduction Potassium chloride is used to treat patients with hypokalemia and/or prevent hypokalemia for patients who are at risk of developing hypokalemia. Potassium chloride has been approved by the United States Food and Drug Administration (US FDA) as a treatment for hypokalemia. A nonbinding draft guidance on potassium chloride as recommended by the FDA was issued in 2011 for drug submission purposes1. The objective of this study was to validate a bioanalytical method for the quantitation of potassium in human urine samples obtained from clinical studies with patients dosed 64 INTERNATIONAL PHARMACEUTICAL INDUSTRY

with potassium chloride in support of required bioequivalence studies. Current methods for the determination of potassium in biological fluids include atomic absorption spectrophotometry2, ionselective electrode3, and inductively coupled plasma optical emission spectrophotometry4. These methods were used to support health screening purposes and were not intended for application to bioequivalence studies which require that the method be validated in compliance with requirements set forth by regulatory authorities such as the FDA or the European Medicine Agency (EMA). ICP-MS is a relatively new technique that has been widely applied for sensitive, accurate, and efficient quantitation of elements in environmental samples, drug articles, and biological samples. Herein we present a bioanalytical ICP-MS method for the determination of potassium in human urine. This method was validated in accordance with the US FDA Guidance for Industry for Bioanalytical Method Validation5, 21 Code of Federal Regulations (CFR) Part 58 GLP regulations6, industry conference bioanalytical method validation meeting reports7-10, and current MPI Research standard operating procedures (SOPs). Our approach was to apply the bioanalytical acceptance criteria and study design, typically regulated by the FDA bioanalytical method validation guidance document 5, to the ICP-MS analysis of potassium using a platform that is historically applied to environmental analyses.

and was utilised for the preparation of all quality control (QC) samples. NIST traceable aqueous potassium and scandium standards (10,000 µg/mL) were obtained from Ultra Scientific and used for the preparation of calibration standards and the internal standard, respectively. Ultrex, ultra-pure grade, nitric acid (HNO3) was obtained from J.T. Baker (Mallinckrodt Baker, Inc., Phillipsburg, New Jersey). Reagent water with a resistivity ≥18 MΩcm was obtained from a NanopureTM RO reversed osmosis system (Barnstead International, Dubuque, Iowa).

Materials and Methods Standards and Reagents: Potassium chloride standard reference material (SRM 999b) was obtained from the National Institute of Standards and Technology (NIST)

Calibration Standards With consideration to endogenous concentrations of potassium in human urine, calibration standards ranging from 500 to 10,000 µg/L were prepared in 1% HNO3 (diluent). The instrument

Control Matrix: Control matrices used for the preparation of QC samples (Bioreclamation, Inc., Westbury, New York) were prescreened for endogenous potassium concentrations. The control matrix with the lowest endogenous potassium concentration among screened lots was used to prepare QC samples. Replicates of the blank control matrix were analysed in every run to provide a baseline for subtraction from the fortified QC samples prior to determining the percent relative error. Preparation of Solutions and Samples: Internal Standard Working Solution (ISWS) and Calibration Stock Solutions The 10 µg/mL ISWS and 100 µg/mL potassium calibration stock solutions were prepared by dilution of the scandium and potassium standards in 1% HNO3 (diluent).

Spring 2012 Volume 4 Issue 2


Clinical Research concentrations in diluent standards are equivalent to 100 to 2,000 µg/mL relative to urine when multiplied by a dilution factor of 200, as reflected in the sample preparation steps.

Typical instrument parameters are presented in Table 2.

Linearity: The calibration standards were analysed in duplicate with one curve

Table 2 ICP-MS Instrument Settings

QC Fortification Solution (100,000 µg/mL) The QC fortification solution was prepared by dissolving potassium chloride reference standard (NIST SRM 999b) in reagent water. QC Samples QC samples at the low, mid, high, and dilution QC concentrations were prepared in unfiltered human urine. As calibration standard solutions were made in diluent, additional diluent QC samples were prepared to verify the validity of the calibration curve throughout an analytical run, and, more importantly, the ability of the curve to accurately quantify the analyte in both matrix and diluent QC samples. The concentrations of matrix and diluent QC samples are presented in Table 1. Sample Preparation Procedure: Table 1 QC Samples

Results and Discussion System Suitability Test: The instrument was tuned to optimise performance prior to each run. The typical evaluated performance parameters included: sensitivity, resolution, stability, oxide, and doublycharged ion ratios. The specifications were listed in Table 3.

each at the beginning and the end of each run. A linear regression with no weighting was used to calibrate the instrument response. The calibration curves had coefficients of variance (R2) of ≥0.998, which met the acceptance criterion (R2 ≥0.995). Figure 1 shows a representative calibration curve in the validation run.

Table 3 System Suitability Test Parameters

Standards, QCs, blank matrix, and unknown study samples were thawed unassisted, gently mixed by inversion, and the solids were then allowed to settle to the bottom of the tube. A 50 µL aliquot of the supernatant was transferred to a clean 15 mL tube. A 100 µL aliquot of ISWS and 9.85 mL of diluent was added to each tube. The tubes were capped, mixed, and an aliquot was analysed by ICP-MS. Equipment and ICP-MS Instrument Settings: A Thermo-Finnigan ELEMENT2 sector field ICP-MS and a Perkin-Elmer Elan 6000 quadrupole ICP-MS were used. 66 INTERNATIONAL PHARMACEUTICAL INDUSTRY

Selectivity: Selectivity was evaluated in six individual lots. Because of the endogenous nature of the analyte, whose concentrations vary across the entire calibration range among samples collected from the normal population, it is impossible to receive analyte-free blank urine for the evaluation of method selectivity. The analyte responses were reported as observed with no acceptance criterion. However, the scandium responses in these lots were compared to that obtained with a prepared calibration blank. The scandium intensity in all six lots did not exceed 5% of the signal intensity of the calibration blank.

Figure 1 Representative Calibration Curve

Carryover: Carryover was assessed by injection of one reagent double blank after each upper limit of quantitation (ULOQ) standard in each analytical run. The analyte and IS responses in the reagent double blanks met the Spring 2012 Volume 4 Issue 2


Clinical Research acceptance criteria that these should be below 20% and 5% of the respective responses in the preceding lowest limit of quantitation (LLOQ) sample. LLOQ: An assessment of the accuracy and precision at the LLOQ was conducted in two analytical runs (n=6 each). The intra-day accuracy results were 8.4% and 1.9% relative error (RE), and the intra-day precision results were 1.3 % and 0.4% relative standard deviation (RSD), respectively. Both met the ±20% RE and ≤20%RSD acceptance criteria. Accuracy and Precision: The intra day accuracy of the QC samples (n=6) was determined for each core validation run. The inter day accuracy of the matrix QC samples was determined across the three core validation runs. The acceptance criterion was a mean concentration within ±15.0% RE of the nominal concentration for each level. For the QC low samples, the intra day accuracy for the three core validation runs ranged from 10.1% to 6.0%, and the inter day accuracy was 0.9%. For the QC mid samples, the intra day accuracy ranged from 5.8% to 1.3%, and the inter day accuracy was 3.1%. For the QC high samples, the intra day accuracy ranged from 5.4% to 2.6%, and the inter day accuracy was 0.1%. All results met the acceptance criterion. The intra day precision of the QC samples (n=6) was determined for each validation run. The inter day precision of the QC samples was determined across the three core validation runs. The acceptance criterion was a RSD ≤15.0% for each level. For the QC low samples, the intra-day precision ranged from 1.2% to 3.4%, and the inter day precision was 4.3%. For the QC mid samples, the intra day precision ranged from 0.54% to 2.1%, and the inter day precision was 3.5%. For the QC high samples, the intra day precision ranged from 0.74% to 3.5%, and the inter day precision was 3.8%. All results met the acceptance criterion. Results from diluent QC samples in the three core validation runs also met the acceptance criteria (RE ±15.0%, www.ipimedia.com

RSD ≤15.0%). The intra-day accuracy ranged from -2.3% to 5.9% RE, and the intra-day precision ranged from 0.0 % to 1.3% RSD among the three concentrations. The inter-day accuracy ranged from -0.8% to 4.5% RE. The inter-day precision ranged from 1.5% to 2.0% RSD. Ruggedness: Ruggedness was conducted in a single validation run (n=6 at each concentration) using a different preparation analyst, a different instrument and on a different day. The intra-day accuracy ranged from 6.3% to 7.2% RE, and the intra-day precision ranged from 3.3 % to 3.9% RSD for matrix QC low, mid, and high samples. Dilution: As the highest potassium concentration in urine samples was anticipated to be below 4,000 µg/mL, the recovery for diluted samples was validated at 4,000 µg/mL (n=6) to accommodate analysing samples originally above the upper limit of the calibration range. Samples were diluted five fold with diluent to achieve a concentration within the calibration range. The average RE was 4.9%, and the RSD was 1.2%. Thus, both the accuracy and precision were demonstrated (RE ±15.0%, RSD ≤15.0%). Matrix Effects: Six different lots of human urine were fortified at 200 µg/mL and analysed. The responses of these samples, after the correction of endogenous contents, were compared to the response of fortified diluent solutions. The matrix factors (MF) ranged from 0.81 to 0.99 with an RSD of 7.8%. The matrix effect criterion was based on the precision (≤ 15%). The deviation of the analyte MF value from the

unity (1.0) was linearly related to the urine lot with a higher degree of endogenous analyte, indicating such a deviation in MF values was from instrument response to high endogenous concentration, instead of from the matrix effect. Stability Results: The stability for potassium in human urine was evaluated for freeze/ thaw, bench top storage, reinjection reproducibility (i.e., autosampler stability), and frozen storage, using QC high, mid (for autosampler stability only), low and the control with an acceptance of RE ±15.0%, RSD ≤15.0%. Stock solutions and calibration standard solutions were also evaluated for their ambient storage stability with acceptance criteria of relative difference (RD) ±10.0%, RSD ≤15.0%. Table 4 summarises the stability results in this validation study. Conclusion An ICP-MS assay for potassium in human urine was successfully validated by MPI Research. The method is accurate and precise for the determination of potassium in human urine over a calibration range of 100 to 2,000 µg/mL. The method was selective for the quantitation of potassium in human urine. Carryover does not impact the assay. Samples with a concentration up to 4,000 µg/ mL can be accurately quantitated after dilution. Matrix samples are stable for at least eight freeze/thaw cycles, up to 25 hours at ambient temperature, up to 72 hours at ambient temperature in injection solution, and up to 243 days at 20°C. The method has been successfully applied to the determination of potassium in human urine for several BE studies. This method is well suited

Table 4 Summary of the Validation Stability Results

INTERNATIONAL PHARMACEUTICAL INDUSTRY 67


Clinical Research for routine clinical urine testing, as it covers the typical range of potassium in human urine. A quick turnaround and high throughput are expected in this method due to the wide linear dynamic range feature of ICP-MS. Indeed, the high efficiency in the ionisation process and the multielemental capability of ICP-MS renders this method to be less prone to matrix effects and drifts that commonly occur in other atomic spectroscopic techniques. References 1. w w w . f d a . g o v / d o w n l o a d s / Drugs/GuidanceCompliance RegulatoryInformation/ Guidances/UCM270390.pdf 2. Grunbaum, B.W. and Pace, N. Microchemical Urinalysis: VI. Determination of sodium, potassium, calcium, and magnesium by atomic absorption. Spectrophotometry in the microliter range of urine. Microchemical Journal, 1970, 15 :666-672 3. Khalil, S.A.H, Moody, G.J. and Thomas, J.D.R. Ion-Selective Electrode Determination of Sodium and Potassium in Blood and Urine. Analytical Letters, 1986, 19: 17-18 4. Krejčová, A., Černohorský, T. and Čurdová, E. Determination of sodium, potassium, magnesium and calcium in urine by inductively coupled plasma atomic emission spectrometry. The study of matrix effects. J. Anal. At. Spectrom., 2001, 16: 1002-1005 5. F  ood and Drug Administration (2001) Guidance for Industry: Bioanalytical Method Validation. US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research 6. F  ood and Drug Administration (1978) 21CFR58 - Good Laboratory Practice for Nonclinical Laboratory Studies. Rockville, Maryland: US Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research. http:// www.accessdata.fda.gov/scripts/ cdrh/cfdocs/cfcfr/CFRSearch. cfm?CFRPart=58 68 INTERNATIONAL PHARMACEUTICAL INDUSTRY

7. Shah, V.P., Midha, K.K., Findlay, J.W., Hill, H.M., Hulse, J.D., et al. (2000) Bioanalytical method validation - a revisit with a decade of progress. Pharmaceutical Research 17: 1551-1557 8. Viswanathan, C.T., Bansal, S., Booth, B., DeStefano, A.J., Rose, M.J., et al. (2007) Workshop/ Conference Report - Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays. The American Association of Pharmaceutical Scientists Journal 9: E30-E42 9. Shah, V.P. (2007) The History of Bioanalytical Method Validation and Regulation: Evolution of a Guidance Document on Bioanalytical Methods Validation. The American Association of Pharmaceutical Scientists Journal 9: E43-E47 10. Shah, V.P., Midha, K.K., Dighe, S., McGilveray, I.J., Skelly, J.P., et al. (1991) Analytical method validation: bioavailability, bioequivalence and pharmacokinetic studies. Conference report. European Journal of Drug Metabolism and Pharmacokinetics. 16: 249-255G uidanceComplianceRegulatoryInf ormation/Guidances/UCM270390. pdf

Chaoyang Huang, PhD, is a Senior Analytical Scientist at MPI Research. In this role, Dr. Huang is responsible for method development and validation of inductively coupled plasma mass spectrometry based analytical methods supporting drug discovery, formulation, clinical and nonclinical research and other related studies. Dr. Huang received his PhD in chemistry from Queen’s University in Kingston, Canada, prior to joining MPI Research in 2006. He provides scientific expertise and solutions to analytical problems to meet our Sponsors’ various needs and is published in various peer-reviewed journals. Email info@mpiresearch.com

Jennifer Ammerman, BS, Analytical Study Director/ Manager at MPI Research, has nearly 10 years’ experience serving in the CRO industry. She received her BS in chemistry from Juniata College in Huntingdon, Pennsylvania. In her current role, she oversees management of the laboratory and study direction for bioanalytical studies in the ICP-MS and GC-MS analytical platforms. Jennifer has authored multiple publications related to ICP-MS and bioanalysis. Email info@mpiresearch.com

Daniel J. Wright, BS, joined MPI Research in 2007 and is the Director of Analytical Sciences at the MPI Research State College, PA facility. Mr. Wright has over 24 years of experience managing the operational and business aspects of contract analytical laboratories. He started as a bench chemist performing sample analysis by inductively coupled plasma atomic emission spectroscopy. He provides leadership and direction to the laboratory with an emphasis on laboratory automation, process improvement, and business development. Mr. Wright received his BS in biology from Manchester College in North Manchester, Indiana. Email info@mpiresearch.com

Spring 2012 Volume 4 Issue 2


Labs, Logistics & Cold Chain Supply

Quality Assessment of Primary Human Keratinocyte Cultures with Online Measurement of Oxygen and pH Abstract The SDR SensorDish Reader allows precise, non-invasive analysis of the process parameters dissolved oxygen (DO) and pH in cell culture. A correlation between cell proliferation analysed with light microscopy, increase of oxygen consumption, and a decrease in pH value in the culture medium is clearly evident. The oxygen and pH kinetics give valuable information about culture quality and efficiency. Future Potential for In Vitro Tests and Cell Therapy In vitro tests are standard screening processes in dermatological and cosmetical laboratories. New pharmacological substances, like e.g. against aging of skin, irritation or harmful UV light, can be tested on their efficiency, and quantitatively evaluated under defined conditions in culture vessels. The evaluation systems range from 2D fibroblast cell culture and keratinocyte monolayers up to complex 3D full skin models. In vitro cultivated epidermal keratinocytes have become increasingly important in medical research, especially in tissue engineering – e. g. in transplantation of cultivated autologous skin cells for the treatment of burns. The therapeutical reconstruction of destroyed skin after severe burns – in some cases a life-saving measure – shows remarkable success rates. Even successful treatment of non-healing chronic wounds was reported recently. The patient´s own keratinocytes are cultivated in vitro, and afterwards stabilised in a fibrin matrix, which is re-transplanted (Kopp et al., 2004). 70 INTERNATIONAL PHARMACEUTICAL INDUSTRY

In this context, it is obvious how important efficient and reliable in vitro cell propagation is. Modern monitoring and control systems are very advantageous to ensure reliable quality of cell cultures. They allow monitoring of the important metabolic process parameters – online and non-invasively. Now there is a noninvasive, optosensoric monitoring system for long-time measurement of DO and pH -SensorDish Reader (SDR, Fig. 1). Even minimal changes Figure 1. Online monitoring of DO and pH value in Kerationcyte cell culture with the SDR SensorDish® Reader by PreSens.

in any of the two important growth parameters can be detected – in close proximity to the cells. The outstanding performance of this system has already been demonstrated in preliminary studies at Celonic with metabolically active CHO suspension cells for fermentation culture 1. Different approaches for possible optimisation in biotechnological production of recombinant proteins had been investigated in those tests. This new study by Celonic now focuses mainly on two aspects: first, the sensitivity of the optosensoric SDR measurement system used with cells showing comparatively low metabolic activity; and second, the relation of cell proliferation and oxygen consumption rates or changes in pH values.

Precise SDR Measurement Technique for Sensitive Cultures A spontaneously immortalised human keratinocyte cell line (HaCaTcells; Boukamp et al., 1988) and primary human keratinocytes were used for these experiments. Primary keratinocytes were carefully extracted from biopsy material with dispase and trypsin. A cell preparation with high proliferative potential was chosen. Both cell types have a critical, minimal cell density for inoculation. If this minimal value of cell density is not reached, cell growth is prolonged, or there is no growth at all. Both cell types were cultivated in parallel with four different starting cell densities of 2 x 103, 1.9 x 104, 6.7 x 104, and 3.8 x 105 cells/well. For each cell density one well was analysed online with the SDR. Reference value measurement was performed in cell-free samples. 24-well Oxo- and HydroDishes® by PreSens (Regensburg, Germany) were used as cultivation vessels. The multidishes have integrated chemical optical sensors for DO and pH measurement respectively at the bottom of each well. These sensors are read out non-invasively from the bottom with the 24-channel SensorDish® Reader. The surface of the 24-well dishes was coated with collagen for the experiments. The culture period was 12 days. Cultivation was carried out at standard conditions in 5 % CO 2 atmosphere with defined, serum-free keratinocyte-medium (Gibco, Defined Kerationcyte-SFM). The medium was replaced every day with equilibrated, fresh medium. The measurement results were recorded with the SDR software. The system allows simultaneous monitoring of 10 SDRs in a total of 240 wells. Spring 2012 Volume 4 Issue 2


Labs, Logistics & Cold Chain Supply Oxygen Kinetics and pH Value Measurement in HaCaT-cell Culture Measurements in epidermal HaCaTcell culture were used as a reference for primary keratinocytes. These cells are comparatively robust and can be split in cultivation as long as desired. Calf serum was used for generating this cell line. However for experiments in this study the HaCaTcells were kept in serum-free media. Analysing the oxygen kinetics of HaCaT-cells cultured with different starting cell densities led to several conclusions (Fig. 2). The sensitivity of the SDR system allowed detecting Figure 2. Oxygen kinetics in long-term cultivation of HaCaT cells.

even smallest changes in the hardly metabolically active HaCaT-cell culture â&#x20AC;&#x201C; even with starting cell densities of only 1.9 x 104 cells/well. Lower starting cell densities showed no difference to values measured in the cell-free control wells. Starting cell densities of 2 x 103 cell/well are too low for HaCaT-cells to grow. Microscopic analysis (Fig. 3) verified Figure 3. Microscopic analysis of HaCaT cells seeded at different cell densities after 3 (above) and 12 (below) days

that even single cells that had been adherent at the beginning were gone by day 12 of cultivation time. It could be observed that with continuing cultivation time the minimum DO value evened out at approximately 22 â&#x20AC;&#x201C; 30 % air sat. in all cultures independently from starting cell density. Remarkably, in the culture www.ipimedia.com

with the highest cell density of 3.8 x 105 cells/well the metabolically available DO concentration dropped to 5 % air sat. at the beginning of cultivation, but decreased to only 25 % air sat. after medium change. This is probably caused by removing non-adherent cells during medium change that could not grow onto the surface after inoculation because of the high cell density. On the other hand a continually increasing oxygen consumption rate could be measured for the cell culture inoculated at 6.7 x 104 cells/well. On day 6, this culture reached the same DO values as the culture seeded with highest cell density. Because of its oxygen consumption rate and the correlation with microscopic analysis (Fig. 3), cultures seeded at this cell density lie within the optimal detection range of the testing system. The cells can proliferate optimally with maximum growth rate. Starting cell concentrations of 1.9 x 104 cells/well can be considered not very usable. Only after 5 days could a continual decrease of the DO level in the medium be detected. Therefore the ideal starting cell density for HaCaT culture is 6.7 x 104 cells/well. High proliferation potential and metabolic activity can be deduced from the rapidly decreasing DO level to minimum values within a few hours after media change. Similar results could be observed for the simultaneously recorded pH values (Fig. 4). The decrease in pH Figure 4. pH kinetic in long-term cultivation of HaCaT cells.

values correlated closely with the respective starting cell density. For the culture with highest inoculation density, relative pH decrease was highest after each medium change â&#x20AC;&#x201C; and linked to cell number and metabolic activity. Different from DO, the pH values continually decreased

over the whole culture period of 12 days. Cultures with highest starting cell density show a pH value of 6.8 after 12 days of cultivation. Contrary to DO kinetics, differences caused by starting cell density could still be clearly detected towards the end of the culture period. Surprising Difference: DO and pH Value Kinetics in Primary Keratinocytes Oxygen consumption in primary keratinocytes (Fig. 5) is similar to that of HaCaT-cells at the beginning of the Figure 5. Oxygen kinetics in long-term cultivation of primary human keratinocytes

cultivation. In long-term cultivation there are noticeable differences though. Cultures with high starting cell densities reach a minimum DO level of 22 % after only 3 days. After an initial minimum of 10 % an upward trend can be detected on day 1. Different from HaCaT-cell culture, this increase continues after day 5. DO values stabilise at a higher level after each medium change. Towards the end of the cultivation period DO reaches a value of 70 % air sat. There is a significant difference to the measured values in HaCaT-cell cultures. It can be concluded that primary keratinocytes gradually reduce their metabolic activity. However, the noticeable decrease in oxygen consumption is not related to reduced viable cell numbers (Fig. 6). Figure 6. Microscopic analysis of primary human keratinocytes seeded at different cell densities after 3 (above) and 12 (below) days

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Labs, Logistics & Cold Chain Supply Primary keratinocytes show distinct contact inhibition compared to the HaCaT cells, and after forming a monolayer cells stop proliferating. In this state of cultivation metabolism is reduced respectively. Another reason for reduced metabolic activity can be the limiting culture conditions caused by high cell density. Some of the cells might have been damaged by this, which could have caused the reduced oxygen consumption during the following days. Microscopic analysis shows an increased number of small, darkappearing cells after 12 days â&#x20AC;&#x201C; probably with reduced vitality. Comparing this to the HaCaT-cell cultures after 12 days, cell complexes

72 INTERNATIONAL PHARMACEUTICAL INDUSTRY

of the primary keratinocytes seem looser. This demonstrates that primary keratinocytes react more sensitively under limited conditions than HaCaT cells. Cultures with cell densities of 6.7 x 104 and 1.9 x 104 cells/well show similar kinetics and tendencies to the above described highest cell density. In both cases a decrease of the DO level could be recorded with a respective postponement of 3 days. Kinetics of cell growth and microscopic analysis suggest that initial cell concentrations of 6.7 x 104 cells/well seem to be optimal conditions for fast, unrestricted growth and high metabolic activity in the cultures. After an initial minimum

value the DO level reaches higher values with every medium change. The DO value for the sample with 6.7 x 104 cells/well is approximately 45 % air sat., and for the sample with 1.9 x 104 cells/well approximately 30 % air sat. respectively â&#x20AC;&#x201C; in both cases with an upward trend. The sample with lowest starting cell densities of 2 x 103 cells/well shows distinctive differences. Unlike the HaCaT cells, this culture reveals significant metabolic activity from day 7 on. Primary keratinocytes can tolerate low initial cell densities better than the HaCaT cells. The serum-free culture system was optimised for the cultivation of primary keratinocytes by Celonic. It is obvious that HaCaT

Spring 2012 Volume 4 Issue 2


Labs/ Logistics & Cold Chain Supply cells adapted to serum containing media cannot adapt to the serumfree environment as well as primary cells. Microscopic analysis during cultivation confirmed a significant increase of cell density in cultures with starting cell densities of 2 x 103 cells/well (Fig. 6). On day 12 the culture vessel surface was almost completely overgrown with cells. pH kinetics (Fig. 7) for different Figure 7. pH kinetics in primary keratinocyte cultivation

inoculation sizes show the same tendency as DO kinetics. In cultures with higher initial cell density, pH values decrease from 7.2 to 6.7 after each media change. This recurrent decrease lessens from day 8 on. Cultures with lower initial cell density also show a decrease of pH value between media changes in the beginning of the cultivation period. In cultures with the second and third highest cell density this decrease lessens after day 10 and 11 respectively. Even in the culture with lowest starting cell density a pH value decrease could be detected after day 7, which became larger till the end of the cultivation period. These effects are associated with the cell number and activity of the primary keratinocytes, and are most obvious toward the end of the cultivation period. In the same way as for the kinetics of cultures with highest cell density, the decrease in pH of the cultures with 6.7 x 104 and 1.9 x 104 cells/well with reduced oxygen consumption is lesser – which indicates reduced cell activity in the older cultures. Insight into the Milieu of Sensitive Keratinocyte Cultures Precise insights into metabolic conditions within the culture vessels allow for new and promising perspectives in optimisation of cell 74 INTERNATIONAL PHARMACEUTICAL INDUSTRY

culture processes. The optosensoric measurement system SDR does not require invasive intervention to measure inside the culture. This method is safe and reliable, especially in long-term cultivation experiments. The cultures can be kept under sterile and constant environmental conditions. This reduces risks as well as workload. Online monitoring allows for continuous recording even of unexpected or unwanted conditions inside the incubator. Optimal culture conditions can be realised by noninvasive measurement at any time. Any interferences or unwanted conditions can be detected in time and adapted. The recorded kinetics give proof that the SDR system is ideally suited for monitoring of sensitive, primary keratinocyte cultures. Even at low initial cell densities DO and pH kinetics can be clearly determined. With these graphs detailed statements about culture development can be deducted. Measurement data allows identifying the timespan – dependent on the initial cell density – in which cultures will reach maximum metabolic activity and highest proliferation rates. The data also reveal periods of static metabolism or even degenerative processes. This way defined cultivation times can be set, according to process objective, in which cultures show optimal physiological conditions, as for example for the further use in transplantation or for pharmacological screening tests. Future up-scaling processes may be based on the results gathered in the 24-well format. The SensorDish Reader is not only a monitoring system but also a very sensitive sensor system for characterisation and adjustment of ideal physiological culture conditions. These conditions can be optimised methodically by specific modification of the parameters, as e.g. the addition of growth regulators like cytokines. The whole research project profits from more economically designed processes and can be transferred into a commercial application faster.

References 1. K opp J, Jeschke MG, Bach AD, Kneser U and Horch RE. Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix. Cell and Tissue Banking 5: 89–96, 2004 2. A bts H & Arain S. Process Monitoring in Suspension-Adapted CHO Cell Cultures, BioProcess International, January 2008, S. 64 – 66 3. B oukamp P, Petrussevska RT, Breitkreutz D, Hornung J, Markham A, Fusenig NE. Normal Keratinization in a Spontaneously Immortalized Aneuploid Human Keratinocyte Cell Line (1988). J Cell Biology 106: 761-71.

Dr. Harry F. Abts, Manager Biopharmacy Derma Laboratory (Merz Pharmaceuticals GmbH, Frankfurt a.M.), studied molecular biology and obtained his Ph.D. at University of Cologne in molecular-biology/ translational medicine (identification and characterisation of diseaseassociated genes). Worked for more than 6 years as research fellow and group leader in the Dept. of Dermatology at University of Düsseldorf on functional differential gene expression analysis in normal and diseased skin. He gained more than 12 years experience in pharmaceutical/biotech industry RD. Covering experience in cell-based assay development for target-gene based rational–drug development, “Tissue Engineering” for generation of in vitro skin-models and new expression system for GLP/GMP compliant eukaryotic production of biopharmaceuticals. Responsible at Merz Pharmaceuticals GmbH for research in dermal biology and development of topical formulations. Email: harry.abts@Merz.de

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Labs/ Logistics & Cold Chain Supply

UN 3373 Packaging: Secure your Sample Shipments Clinical Trials Thousands of samples of human or animal origin are collected and sent around the world every day by pharmaceutical companies, nursing homes, diagnostic laboratories, research centres and others. They are collected as part of medical tests, clinical trials, surveillance studies and various other tests. The results of clinical trials are crucial for market authorisation dossiers submitted by pharmaceutical companies to the relevant authorities. Samples must be transported in accordance with IATA regulations for the air transport of dangerous goods and with the European agreement concerning the International Carriage of Dangerous Goods by Road (ADR). Temperature-sensitive samples must for their part be transported in full compliance with the cold chain, on which their effectiveness depends. Depending on their stability, they are transported between +2 and + 8°C (or between +2 and +4°C) or below -18°C. To guarantee the cold chain, laboratories, depositories and CRO units use cooling packaging. For home treatment, qualified insulated bags are used to transport heat-sensitive products. Samples (blood, skin, etc.) are transported in UN 3373 insulated packaging, from the collection site (hospitals or laboratories) to analysis laboratories. UN 3373 Packaging: Transport of Class B Biological Substances The ADR and IATA regulations for air and road transport of infectious substances which may contain pathogens are based on UN recommendations. Category A infectious substances, regulated by UN 2814 and UN 2900, are those which can cause permanent disability or fatal illness in humans or animals. Category B infectious substances are those which are not classified as category A. They are regulated by UN 3373, and their official transport designation is “BIOLOGICAL SUBSTANCE, CATEGORY B”. 76 INTERNATIONAL PHARMACEUTICAL INDUSTRY

UN 3373 packaging consists of three layers: - L eak-proof primary packaging (container) that contains the substance; - S econdary packaging, which is resistant and leak-proof, protecting one or more primary containers wrapped in an absorbent material, enough to absorb all liquid in case of leakage; - O uter packaging that protects its contents and which guarantees the cold chain if necessary. UN 3373 packaging must be of good quality, strong enough to withstand the shocks and loadings normally encountered throughout the logistics circuits (transport, and manual or mechanical handling). Packaging must be constructed and closed so as to prevent any loss of contents that might be caused under normal conditions of transport, by vibration, or by changes in temperature, humidity or pressure. Depending on the material transported, liquid or solid, the primary receptacle and secondary packaging must meet leak-proof and mechanical strength requirements.

For liquid substances: - T he primary receptacle(s) must be leak-proof; - T he secondary packaging must be leak-proof; - If multiple fragile primary receptacles are placed in a single secondary packaging, they must be either individually wrapped or separated to prevent contact between them; - A bsorbent material must be placed between the primary receptacle and the secondary packaging. The absorbent material must be in sufficient quantity to absorb the entire contents of the primary receptacle(s) so that any release of the liquid substance will not compromise the integrity of the cushioning material or of the outer packaging; - T he primary receptacle or the secondary packaging must be capable of withstanding, without leakage, an internal pressure of 95 kPa (0.95 bar). In addition to these requirements the IATA adds the following conditions: - T he primary receptacle(s) must not contain more than 1 L; - T he primary receptacle or the secondary packaging must Spring 2012 Volume 4 Issue 2


Labs/ Logistics & Cold Chain Supply be capable of withstanding a pressure of 95 kPa in the range of -40°C to 55°C (-40°F to 130°F). - T he outer packaging must not contain more than 4 L. This quantity excludes the cold source used to keep specimens in the required temperature range. For solid substances: - T he primary receptacle(s) must be sift-proof; - T he secondary packaging must be sift-proof; - If multiple fragile primary receptacles are placed in a single secondary packaging, they must be either individually wrapped or separated to prevent contact between them; - If there is any doubt as to whether or not residual liquid may be present in the primary receptacle during transport then a packaging suitable for liquids, including absorbent materials, must be used. In addition to these requirements the IATA adds the following conditions: - It must not exceed the outer packaging weight limit; - E xcept for packages containing body parts, organs or whole bodies, the outer packaging must not contain more than 4 kg. This quantity excludes the cold source used to keep specimens in the required temperature range. How does the Outer Packaging Comply with Regulations? Clinical product refrigerant packaging has to respect the IATA regulations. The outer packaging should respect constraints about dimensions, design and resistance. The package must be properly marked, labelled and accompanied by shipping documents. Dimension: At least one surface of the outer packaging must have a minimum dimension of 100 mm × 100 mm. Design: To transport, the marking below must appear on an external surface of the pack on a bottom surface of contrasting color, and 78 INTERNATIONAL PHARMACEUTICAL INDUSTRY

be clearly visible and legible. The marking shall be square in shape and disposed at an angle of 45° (diamond-shaped), each side measuring at least 50mm long. The line width must be at least 2mm, and the letters and numbers must be at least 6mm high. The proper shipping name should be “Biological substance category B”, printed in letters not less than 6mm high. It must appear on the pack next to the diamond shape. Resistance: The package must be able to pass the drop test specified in the IATA, Chapter 6.5.1.1 – the height of the fall must not be less than 1.2m. After the series of falls indicated, there shall be no leakage from a primary receptacle, which must remain protected by absorbent material, if required in the secondary packaging. Refrigerated or Frozen Specimens: - W hen dry ice or liquid nitrogen is used as a cold source to keep specimens in a required temperature range, all applicable requirements of these regulations must be met. In this case, the cold source must be placed outside the secondary packaging or in the outer packaging. Interior supports must be provided to secure the secondary packaging in the original position after the ice or dry ice has dissipated. If dry ice is used, the packaging must be designed and constructed to permit the release of carbon dioxide gas to prevent a build-up of pressure that could rupture the packaging. - T he primary receptacle and the secondary packaging must maintain their integrity at the temperature of the refrigerant used, as well as the temperatures and the pressures which could result during transport. Clinibox: New Range of Packaging for Clinical Trials A range of UN 3373 packaging for clinical trials was launched at the beginning of the year. This range of packaging - Clinibox - ensures secure, safe, temperature-controlled transport for clinical trials (clinical samples, diagnostic specimens or

category B biological substances). It guarantees both compliance with the regulations for the transport of dangerous goods (IATA and ADR) and the respect of the cold chain. These UN 3373 transport kits consist of VIAL 650 pouches (secondary packaging) and a refrigerated box (outer packaging). The VIAL 650 pouch is 95 kPa certified by an IATA approved laboratory. It is divided into several separate compartments pre-equipped with absorbent material. Tubes are directly placed in these compartments, thus complying with the requirements of separation and protection against spillage. The insulated boxes are made of rigid polyurethane panels and refrigerated, according to needs, by eutectic gel packs or dry ice. They are tested and qualified by our partner Ater Métrologie laboratory, according to ISTA 7E and NF S 99-700 standards for thermal performance. They have also successfully passed drop tests from a minimum height of 1.2m. The Clinibox packaging range covers temperatures between +2 and +8 °C or below -18 °C, and durations from 24 up to 96 hours. It may be expanded for other specific needs: other volumes, other durations and other temperature ranges. Abbes Kacimi, Cold Chain Expert Engineer, SOFRIGAM. Abbes Kacimi is an Engineer in Refrigeration and Air-Conditioning. After a role as the head of Sofrigam Engineering Department (2001), responsible for the development and qualification of cooling containers for pharmaceutical products, Abbes is now the Sofrigam Cold chain Expert Director (2012). He is also member of cold chain commissions (as lIR Commission D2), and Co-author of “Practical Guidelines, Cold Chain for Medicines”. He contributed to several projects in Europe and Emirates Arabic, and has published a variety of articles on the cold chain. Email: abbes.kacimi@sofrigam.com

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Labs/ Logistics & Cold Chain Supply

Put Safety First in Pharma Logistics Taking a look at how a modern logistics system operates and the high-value investment in creating a system that works safely and efficiently suggests more consideration should be given to the humble pallet. In the UK materials handling and logistics sector, there is an average of 43 injuries per week, 11 of them serious. There are lots of safety hazards employees need to be aware of in the pharmaceutical supply chain – handling and storing pallets is just one of them. From shipping products to moving goods internally, pallets are an essential component in every pharmaceutical logistics operation. In Europe there are approximately half a billion pallets in circulation every year, with 70 million of those in the UK alone. Yet, despite manufacturers relying heavily on pallets to store and transit their goods, what is surprising is how little thought goes into selecting the type of pallets they use. Poorly chosen pallets can seriously compromise safety in pharmaceutical supply chains and can lead to unnecessary costs. Taking a look at how a modern logistics system operates, and the high-value investment in creating a system that works safely and efficiently, suggests that more consideration should be given to the humble pallet. Investment in Logistics Let’s start with racking systems. Racking systems are widely used in warehouses, as they offer considerable space advantages over floor storage, and allow easy access for the retrieval of goods. To ensure a racking system is of good mechanical construction, sound material and adequate strength, and is installed and maintained correctly, requires not just time and financial investment but manpower too. Modern racking systems are 80 INTERNATIONAL PHARMACEUTICAL INDUSTRY

Choosing pallets that have stated loading capacities, like plastic pallets, offers companies the reassurance that their products are being stored and handled in the safest possible way.

becoming ever more complex. There are experts in this field who can work out how to get the maximum number of pallets in the smallest amount of space to achieve the most efficient storage with the help of advanced computer systems, which leads me to my next point. Technology in warehouses today includes the use of advanced computer systems that not only store pallets in random locations, but can instruct an operative or an AGV (automated guided vehicle) to locate and retrieve a particular pallet when it’s required. These systems can track the movement of each pallet and are able to cope with the most complicated stock rotation requirements. Materials handling equipment is another key component of modern logistics systems. With the ever

increasing variety of handling equipment on offer, a great deal of thought is given to the selection process. There are automated varieties, narrow aisle trucks, bendy trucks, or trucks that require drivers – all of whom are given extensive training to ensure they operate the vehicles safely. Great care and consideration also goes into choosing suitable protective clothing for staff – high visibility jackets, helmets, steelcapped boots – in fact, failure to do so could leave companies vulnerable to prosecution. Despite all the time and huge financial investment that goes into selecting the best racking, computer systems and handling equipment to get a logistics system up and running, some companies are compromising all this hard work and effort by using second-hand wooden pallets. Spring 2012 Volume 4 Issue 2


Labs/ Logistics & Cold Chain Supply These second-hand wooden pallets, which are likely to have been used several times before, could be carrying any number of contaminants. Ignoring the contamination issue for a moment, how can you be sure that your second-hand wooden pallet is capable of bearing the weight of your pharmaceutical goods? The truth is, you can’t. Careful Pallet Selection If you ask a technical operative in any modern warehouse what the capacity of the racking system is, he’ll be able to define it based on the position of the pallet, the span of the beams and the drive-in bays; he’ll know that the beams are designed to support a specific number of kilograms on so many pallets per bay, and that the end supports have been bolted down using a particular bolt with a defined length and thread. Ask the same technical operative what the capacity of the wooden pallet being used on the racking system is, and he won’t be able to tell you as, whether new or reused, wooden pallets do not have guaranteed loading capacities. The reason for this is that the strength of timber varies. Make 100 wooden pallets one month and test them and they might all have the same loading capacity, but make another 100 pallets a few months later and there is no telling how the capacity of these pallets might differ, and what consequence this might have on your pharmaceutical logistics operation. Choosing pallets that have stated loading capacities, like plastic pallets, not only offers companies the reassurance that their products are being stored and handled in the safest possible way, but can also avoid costly incidents. A major UK supermarket chain recently trialled some of our plastic pallets in their automated storage system after a wooden pallet racked 10-racks high broke, causing a pyramid effect that destroyed a huge volume of product and cost £2.5m to resolve. Thankfully, no one was injured but, had staff been present in the warehouse at the time, the incident could have been lifethreatening. In July 2010, a pallet accident at 82 INTERNATIONAL PHARMACEUTICAL INDUSTRY

Plastic pallets are strong, durable and less vulnerable to damage than wooden pallets

work which caused the death of a Portuguese employee was the subject of an HSE prosecution at Manchester Crown Court. The accident occurred when a wooden pallet containing 55 bags of polypropylene collapsed, trapping the employee underneath the bags and crushing him to death. Another fatal pallet accident occurred in November 2010, when a man died from serious head injuries after he was reportedly struck by wooden pallets at a warehouse on the Bermuda Industrial Estate in Nuneaton, Warwickshire. Another man suffered serious arm injuries in the same incident. Recognising Pallet Safety The Health & Safety Executive’s Guide to Health and Safety in Warehousing and Storage (HSG76) was written to help reduce injuries and ill health in warehouses in order to achieve the Government’s injury reduction and occupational ill health targets for UK workplaces. The detailed guide includes advice on general health, safety and welfare, materials handling and storage systems, including the safe use of pallets. The guide states: “Flat timber pallets form an essential part of many mechanical handling systems in warehouses. Accidents directly attributable to these type of pallets usually arise from six main sources: poor design; poor construction; the use of a pallet which is unsuitable for a particular load; the continued use of a damaged pallet; bad handling; or the use of a pallet which is unsuitable for a particular racking system.”

By using plastic pallets, companies can eliminate virtually all of these safety hazards. Firstly, plastic pallets are moulded under extreme pressure in highly polished moulds. This process produces a high quality product – in design and construction – and ensures the consistent dimensional accuracy and loading capacity of each pallet. Secondly, plastic pallets have three stated loading capacities - static load, which is the maximum weight that a pallet can withstand when placed on a level surface; dynamic load, which is the maximum weight that a pallet can accommodate when being moved by a forklift, and racking load, which is the maximum weight that a pallet can accept when racked. These capacities help operatives choose the right plastic pallet for a particular load so there is little risk of getting it wrong, unlike with wooden pallets. Thirdly, plastic pallets are strong and durable, and less vulnerable to damage than wooden pallets. In fact a standard plastic pallet has a lifespan of up to ten years, offering an excellent return on investment. Once a plastic pallet reaches the end of its working life, the plastic still lives on, as the pallet can be recycled and the plastic can be reground to produce new plastic products. Finally, as plastic pallets have no nails, sharp edges or splinters and there is no risk of loose component parts breaking free under manual lifting conditions, they are far less likely to damage product packaging or cause injury to operatives. Safe from Harm A crucial consideration in the selection of pallets for use in the pharmaceutical supply chain that we have barely touched upon is hygiene. Those of you who read my last article in IPI magazine – ‘Good Pallet Practice – Product Recalls Highlight Need for Clearer Guidance’ - will be fully aware of a series of incidents in the last year where pharmaceutical manufacturers were forced to recall products due to contamination caused by a substance used to treat wooden pallets. Spring 2012 Volume 4 Issue 2


Labs/ Logistics & Cold Chain Supply

Goplasticpallets.com’s hygienic plastic pallet

Alarming as it is, it seems that manufacturers take great care packaging products to ensure product safety and avoid external influence, yet less importance is given to choosing the type of pallets the products are stored and transported on. Many countries have legislation that requires pharmaceutical and medical device companies to follow good distribution practice (GDP) guidelines to ensure the proper production and distribution of medicinal products for human use. In Europe, the EU GDP guidelines (94/C 63/03) offer companies guidance on personnel, documentation, premises and equipment, deliveries to customers, returns and selfinspections, but provide only vague advice on the topic of delivering medicinal products to wholesalers and pharmacies. Under the heading Deliveries to Customers, section 20 states: “Medicinal products should be transported in such a way that: they do not contaminate, and are not contaminated by, other products or materials.” Offering drug manufacturers clearer guidance on the distribution of medicinal products, in particular on the use and selection of pallets, will allow them to make an informed www.ipimedia.com

decision that could prevent palletrelated safety and contamination issues. All plastic pallets are hygienic when compared to traditional wooden pallets but, for transporting medicines and other pharmaceutical products, there are many advantages to using plastic pallets from our Hygienic range. Our hygienic pallets are made from virgin plastic or the highest foodgrade recycled materials, and comply with EU safety legislation. They have totally smooth, sealed surfaces, unlike wooden pallets, which are susceptible to cross-contamination issues caused by mould and dust. They can be easily cleaned manually or with an automated system, as they don’t absorb moisture – even under the most adverse conditions – and are tolerant of weak acids and alkalis. Next Steps Manufacturers give considerable thought, time and financial investment to create modern logistics systems that operate safely and efficiently, yet could be compromising the safety of their products and staff by using wooden pallets that have unqualified loading capacities and a recent history of causing product contamination.

As guidance on the use and selection of pallets has not been well documented in the pharmaceutical industry, it is down to the manufacturer to decide which type of pallet they should use to store and transit their goods. Comparing the safety and hygienic properties of different types of pallets will help pharmaceutical manufacturers choose the best type of pallet for their logistics operation.

Jim Hardisty is the founder and Managing Director of Goplasticpallets. com, the UK’s leading independent supplier of plastic pallets and containers. After 18 years working in the logistics sector and six years in the pallet industry, Jim set up Goplasticpallets.com in 2001 to facilitate the supply of plastic pallets to businesses in the UK. Ten years on, Jim has signed exclusive partnerships with a number of pallet manufacturers across Europe, allowing the company to establish a comprehensive and competitive product range. Email: jim@goplasticpallets.com Tel: +44 (0)1323 744057 Website: www.goplasticpallets.com

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Manufacturing

Peptides as Drugs Peptide research has seen progressive growth over the past few decades, in particular with respect to ‘peptide therapeutics’. Many companies specialising in their manufacture, along with companies developing peptide-based products ranging from new drug candidates to medical diagnostic devices, through to cosmetics and food technologies, have come to the forefront of pharmaceutical acquisitions and venture capital groups. What are Peptides? Peptides, typically classed as molecules containing between two and fifty amino acids bonded together, and proteins (larger peptide molecules containing over fifty amino acids, ‘polypeptides’) have long been regarded as crucial to offering solutions to mounting and increasingly difficult world health issues, and the possibility of patient-specific therapy. Peptides and proteins are found throughout biology, and possess a range of physiological and cellular functions. Their structure is often complex, capable of presenting in many different conformations, dependant upon their environment. It is perhaps in part due to the complexity of the tertiary and quaternary structures that the peptidedrug market stalled somewhat between the development of insulin in 1959 and the last decade. Other factors which have caused problems with development are the short halflife of peptide molecules due to their rapid metabolism in body, and also the delivery to specific organs. Acceptance over the last decade by the science community, that peptide/ protein drugs are viable options for therapy, has driven research organisations into screening libraries of peptides, looking for bio-interactions and potential lead candidates. The overall process of drug discovery can be summarised broadly as three separate components: the initial 84 INTERNATIONAL PHARMACEUTICAL INDUSTRY

stage of drug discovery, investigating protein behaviour and characteristics; the second stage of discovery, identifying potential candidates that can bind to the protein and adjust its behaviour; and the third stage, testing of a lead candidate’s effect on the native protein. For each of these stages, the use of peptide molecules assists research, and these reagents are seen as a key influence in future drug discovery.

total of sixty peptide drugs that had, at that point, been approved in the USA by the Food and Drug Administration (FDA). There were also 140 peptide drugs in clinical trials, and over 500 in pre-clinical development. With the majority of clinical trials targeting oncology, cardiovascular, metabolic and infectious diseases, there is a large opportunity to explore peptide therapeutics for other medical disorders.

With a clearer understanding of aspects of structural biology and drug metabolism pharmacokinetics, research into peptide/protein-based drugs has started to flourish, with the ability to deliver the drugs to specific sites, and the drugs offering high potency, and importantly, low toxicity. These key factors differentiate the peptide/protein therapeutics from more traditional ‘small molecule’ drugs, and leads science to ask “Why is this so?”

Of particular interest are the peptides targeting metabolic disorders, with the estimation of obesity being over 100 million people globally and increasing. This area of research has seen some significant breakthroughs, with the discovery of leptin, an adipose tissue peptide hormone intensifying studies. Leptin has been shown to act by decreasing appetite and increasing metabolic rate in studies in animals. Studies into this, and analogous peptides will continue to expand, and it is hoped that it could lead not only to treatments for obesity, but also to an antagonist of the mechanism, which could be employed in the treatment of anorexia. As obesity levels increase and concurrently generate more dependence upon treatment for diabetes, biomedical research into insulin-like peptides will continue.

At the molecular level, peptides influence the majority of physiological processes. They act by binding to specific cell surface receptors, and modulating the protein’s activity. The specificity obtained from using peptide therapeutics can be attributed to the complex structures of both the labile peptide, which may adopt a specific conformation allowing for interaction with only specific corresponding sites, and to the quaternary structure formed upon interaction with the protein. The generally lower toxicity of peptides can be attributed to both their lower instance of interaction with other molecules not of interest (which may be other drug entities or other proteins in the body), and also to their ease of metabolism into their component amino acid residues. Does the Indication Mean that Peptide Drugs have a Long and Secure Future? There has been a large growth over recent years in peptide drugs and drug candidates. In 2010, there were a

Insulin was the first peptide to be administered therapeutically, and has been used for over half a century in the treatment of diabetes. Despite research not offering suitable small molecule alternatives to insulin, there have been developments into novel analogues, such as lispro insulin. This is shorter-acting, allowing for a more convenient injection just prior to a meal compared with insulins that are injected thirty minutes prior to a meal. There have also been developments into non-injectable forms of insulin. Another peptide of interest is glucagonlike peptide-1 (GLP-1), which shows insulin-releasing properties and also suppresses glucagons levels, whilst Spring 2012 Volume 4 Issue 2


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Manufacturing delaying gastric emptying. The action of GLP-1 is glucose-dependant and as such it is more potent following a meal. Peptides drugs having multiple medical benefits have also reached the market, such as octreotide. This long-acting stable analogue of somatostatin has a number of therapeutic indications, having been used effectively in the treatments of acute pancreatitis, upper gastrointestinal bleeding, gastroenteropancreatic endocrine tumours and acromegaly amongst others. Further developments of this analogue have led to lanreotide and Sandostatin LAR, having longer-acting lifetimes and thus requiring less frequent injections. In 2003, Roche marketed the peptide enfuvirtide as Fuzeon for application in combination therapy towards the treatment of HIV-1 infection. The peptide, an HIV fusion inhibitor shown below, demonstrates the complexity of a peptide drugâ&#x20AC;&#x2122;s chemical structure as compared to more tradition small molecule drugs. Enfuvirtide was discovered at Duke University, by a small pharmaceutical company called Trimeris, formed by researchers at the University. After three years of development, Trimeris partnered with Hoffman-La Roche in 1999 to complete the development of the drug, and achieved FDA approval in March 2003 as the first HIV fusion inhibitor. It is estimated that the use of Fuzeon in therapy costs around $25,000 per person per year in the US.

for many years is that of antibiotic peptides. There have been two antibiotic peptides polymyxin B and polymyxin E (also known as colistin) that are already licensed for use. Colistin has been employed via injection, orally and by aerosol, and has been used to treat Pseudomona aeruginosa in cystic fibrosis patients. Polymyxin B has been used for the treatment of eye and ear infections and is employed topically. Research has identified that further classes of antibiotic peptides exist including magainins, defensins and cecropins amongst others. With the increasing ineffectiveness of traditional antibiotics, the research into peptidebased antibiotics will become essential for world health organisations. Increasingly, across the broad range of therapeutic areas the structures of these target compounds have become larger (more amino acids conjugated in the primary structure) and more complex. In order for these to be viable options for the healthcare industry, the technology to manufacture these compounds has had to develop considerably. Sustaining accessibility to the increasingly complex peptide species, the larger pharmaceutical companies, biotech SMEs and specialist CMOs must look at developing collaborations to ensure that manufacturing technologies can continue to achieve the drug

candidates and deliver the products on the scales required for the global demand. Short peptide sequences, typically less than 25-30 amino acids in length, are significantly more economical to manufacture than longer peptides. On a large scale, these sequences are typically prepared utilising solution phase chemistry, achieving a lower manufacturing cost as compared to solid phase peptide synthesis (SPPS) methods which allow access to much longer peptide sequences. The developments into SPPS using Fmoc chemistry have led to larger peptide molecules, and small proteins, being accessible by chemical synthesis on scales of multi-Kg and 100 Kg. The methodology of SPPS is based upon the first amino acid (at the C-terminus) being chemically bonded to an insoluble resin support. The amino acid residues are then chemically reacted sequentially on to the insoluble resin support, with all other reagents used in the synthesis being removed in solution away from the resin. The residues are added via a method of removing the Fmoc group that protects the amine function, and then adding a carboxyl-activated version of the next amino acid, forming the amide bond. Washing the insoluble support removes unbound molecules (reagents used in coupling chemistry).

Solid-phase cGMP peptide synthesis at Pepceuticals Ltd., using a batch-wise process.

Enfuvirtide, marketed as Fuzeon, has the amino acid residue sequence: A c - Ty r - T h r - S e r - L e u - I l e - H i s - S e r - L e u I l e - G l u - G l u - S e r- G l n - A s n - G l n - G l n - G l u Lys-Asn-Glu-Gln-Glu-Leu-Leu-Glu-Leu-AspLys-Trp-Ala-Ser-Leu-Trp-Asn-Trp-Phe-NH2

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Manufacturing There are two approaches to using SPPS, either in a batch-wise fashion, or in a continuous-flow fashion. Each approach has its benefits, dependant upon the stage in the drug discovery programme. Batchwise synthesis, shown in the picture above, is more rapid for delivering a large number of peptides, such as for screening, however the quality of the peptide produced may be low, and lead to increased purification costs. Continuous flow presents higher purity peptides straight from the synthesis, and can reduce purification costs, however continuous flow can take longer to synthesise the peptide, and current automated equipment does not have the same capacity as that of batch-wise synthesisers. More modern purification processes allow for larger-scale purifications of increasingly complex synthetic peptides, thus reducing the traditional ‘bottleneck’ in peptide manufacture. Peptide Market In the US, annual sales of peptide Kilo-scale purification at Pepceuticals Ltd., under cGMP conditions.

drugs exceeds $13 billion, representing 1.5% of drug sales globally. In addition, protein drugs such as therapeutic antibodies represent a larger share, with the combined biopharmaceutical market valued at over $70 billion. In Europe, Germany and the UK account for 63% of the peptide therapeutic market, with France, Italy, Scandinavia and Spain making up the rest of the major markets. With increasing biomedical research emphasis directed towards using peptides, the market is set to grow and broaden considerably over the coming years. In addition to the use of peptides as therapeutics, they have also found use acting as ‘carrier88 INTERNATIONAL PHARMACEUTICAL INDUSTRY

species’ when conjugated to a small molecule or other drug substance. The uptake and high specificity of the conjugated peptide gives better delivery of the active substance, and often leads to a decrease in toxicity. This has brought renewed interest to established treatments, and there is significant opportunity to exploit the intellectual property which is still widely available. Nutraceuticals, providing health benefits and prevention/healing of disease, has seen a respectable growth over the past few years. Supported primarily in markets where there is disposable income and an effort to maintain healthy living, nutraceuticals has seen a growth in North America of more than 6% for the period 2007-2011, with the protein and peptide section of dietary supplements expected to have a continued global growth for the foreseeable future. In 2011, peptides and proteins held the largest share of the dietary supplement market in North America, Africa and across the Middle East, as compared to vitamins and minerals, and herbal supplements. Increasing education and evidence about the benefits of such dietary supplements will continue to support the market, and its use of peptides/proteins is set to become more regulated, allowing opportunities for traditionally more pharma-oriented CMOs to expand their customer bases. Finding a specialist CMO which can offer a range of pre-cGMP development techniques and methodologies is often a hard task, and then transferring this to a cGMP-capable facility with the right experience is time-consuming and costly. CMOs offering a complete

service, or a range of key steps to support a drug discovery programme, are able to save unnecessary timewastage and investments and give a peptide product which has been confidently transferred through the development stages. CMOs understand that a customer may expect their product, presented as a high quality material, at a low cost price, to be delivered shortly after a purchase order is issued. However, unfortunately this is not something that the CMO can completely achieve, and it is often that one or two of the demands must be sacrificed in order to present the product correctly. It can sometimes be easy to lose sight of the overall target for the drug discovery programme – to deliver a quality and reliable product, with a specified manufacturing process, in a good time-frame, and at an economically advantageous cost. Looking to the Future The employment of peptides in medical therapies and diagnostics, and as conjugates to other therapies, is set to increase, along with the range of disease areas that they look to combat. In parallel, the use of nutraceuticals, antibacterials, agricultural products, and cosmetics will continue to flourish. Peptides it seems have finally, over the millions of years of their existence, started to take their place at the forefront of research, and will continue to provide solutions to a wide range of applications for years to come. Dr Kamal Badiani is Managing Director of Pepceuticals Ltd., a CMO specialising in the active substance manufacture of peptides and small molecules, based in Leicestershire, UK.

Kilo-scale purification at Pepceuticals Ltd., under cGMP conditions.

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Manufacturing development, analysis and quality control. To complement the contract pharmaceutical drug development services, Pepceuticals Ltd offers a comprehensive bioanalysis service, utilising state-of-the-art equipment for detection, identification and quantification of biomarkers. Waters Synapt High Accurancy Mass Spectrometer, capable of biomarker identification and characterization, protein sequencing and rapid sample analysis.

Pepceuticals Ltd., modern active substance manufacturing facility in Leicestershire, UK. Pepceuticals Ltd., established in 1998, specialises in the manufacture of bioactive molecules for the life science industry, ranging from peptides to small molecules. The new manufacturing facilities based in Leicestershire offer custom manufacture of researchgrade materials through to cGMP, in 90 INTERNATIONAL PHARMACEUTICAL INDUSTRY

versatile laboratories recently audited by the MHRA (July 2011). Pepceuticals Ltd currently has the capacity to manufacture from mg up to multi-Kg of material. The facility comprises of eight state-of-the-art cGMP suites, covering the complete manufacturing process (Grade C, Class 10,000) through to fill/finish (Grade A, Class 100). The cGMP manufacturing is supported by laboratories dedicated to pre-cGMP process

Dr Kama Badiani graduated with a Doctorate in Organic Chemistry from the University of St. Andrews in 1996. Since leaving St. Andrews Kamal worked within the pharmaceutical industry as a research chemist for two years before he founded Pepceuticals, in 1998. Under his leadership Pepceuticals soon became established as a manufacturer of synthetic peptides. As an organic chemist, Kamal has developed the novel chemistry used today in Pepceuticals to produce/ manufacture peptides, as well as the innovating new products that are currently in development. Over the last 14 years, Pepceuticals has grown to become one of the largest peptide companies in Europe, having moved to a new modern facility in Leicestershire, comprising of over 10,000 sq. ft of laboratory space. He has cultivated a reputation for outstanding quality and timely delivery. He believes that his team is fundamental to Pepceuticalsâ&#x20AC;&#x2122; continued success. Email: kamal.badiani@ pepceuticals.co.uk

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Looking After Your AHU A Brief History of Vibration Sensing Vibration sensing has been employed for some time now; indeed, we can be certain that it has been in use for almost two thousand years. The oldest vibration sensor known to historians was a seismograph invented by Chinese astronomer Chang Heng in the second century AD. This device responded to distant disturbances by depositing a bronze ball from the mouth of one of eight dragons placed at intervals around a large urn, thus indicating the direction of a distant earthquake and potentially saving lives by enabling the government to send assistance to the affected area. Vibration sensing is therefore one of the oldest solutions in the engineering workplace, with a lineage that outstrips other technologies by centuries. However, todayâ&#x20AC;&#x2122;s vibration sensing systems are no longer sculpted in the forms of mythical beasts; the development of vibration sensing has been less concerned with visual impact and more concerned with enhancing performance. This is because the users of each application in which vibration sensors are used (and there are many) demand ever-greater productivity from each mechanical system. There has therefore been a corresponding growth in the need for sophisticated vibration sensors that can maximise the performance of many engineering processes. Todayâ&#x20AC;&#x2122;s designers and engineers of vibration sensors have not only succeeded in providing devices of exceptional reliability, but also in packaging that functionality in a variety of resilient enclosures to enable its use within a wide range of applications. Vibration Sensing and Air Handling Units Air control as a method of manufacturing medicine is probably older than even vibration sensing, though the drying of animal flesh and bone to produce powdered drugs no longer represents the height of pharmaceutical technology. Today, 92 INTERNATIONAL PHARMACEUTICAL INDUSTRY

successful and effective air handling in the pharmaceutical industry requires maintaining machinery that can stabilise air conditions within precise limits during the manufacture of supplies. Consistent indoor air quality must be maintained, with no sudden changes to air flow rate, temperature and humidity that could potentially compromise production quality. To ensure that these levels are carefully managed, machinery must be well specified, maintained and run, since a mechanical failure can make a degradation in air standards almost inevitable. It is vital to ensure that a quality product is produced on any pharmaceutical production line, so there is a requirement to maintain air flow rate, temperature and humidity. Costs generated by lost production can be especially high in the pharmaceutical industry, owing to such issues as penalties resulting from late orders, and customer dissatisfaction can eventually result in the greatest damage if valuable business is lost. For these and many other reasons, the mechanical failure of air handling units must be avoided.

Taking Advantage of Vibration Sensing Vibration sensing assists engineers in preventing air handling units from unnecessary failure, but it needs to be carefully specified. However, because vibration sensing has become established as a powerful predictive maintenance solution within modern engineering systems, it can provide the right advice and specification for a growing number of applications. In addition, training in the use of condition monitoring components such as vibration sensors is now provided by organisations such as BINDT (British Institute of Non-Destructive Testing). This is important because, despite the fact that a vibration sensor offers high levels of performance and reliability, the component is only as good as its installer. For example, when mounting a sensor there may be a choice between drilling, tapping or gluing, but engineers need to understand and consider how these methods may affect the warranties on their equipment. However, with the right advice and assistance, the rotating elements of air handling units Spring 2012 Volume 4 Issue 2


Manufacturing can be cost-effectively monitored to enable vibration engineers to pick up early signs of any degradation in the equipment so that maintenance can be planned and carried out with minimal expense and disruption to service. A Closer Look at Vibration Sensors Accelerometers contain a piezoelectric crystal element, which is bonded to a mass. When subjected to an accelerative force, the mass compresses the crystal, and this causes the crystal to produce an electrical signal that is proportional to the force applied. This output is then amplified and conditioned by inbuilt electronics to produce a signal that can be used by higher level data acquisition or control systems either ‘online’ or ‘offline’. An online system is one that measures and analyses the output from sensors that interface directly with a programmable logic controller (PLC). An offline system is created by mounting sensors onto machinery and connecting them to a switch box; engineers can then use a hand-held data collector to collect readings. There are two main categories of accelerometer: AC accelerometers, which are typically used with data collectors for monitoring the condition of higher value assets such as turbines, and 4-20mA accelerometers, which are commonly used with PLCs to measure lower value assets, such as motors, fans and pumps. Both AC and 4-20mA accelerometers can identify misalignment, bearing condition and imbalance, while AC versions offer the additional capability to detect gear defects, belt problems, looseness and cavitation. Inside the Air Handling Unit A typical air handling unit comprises a supply fan and an extractor fan, coils that circulate steam or hot water for heating and chilled water for cooling and air filters, encased within a large metal enclosure with removable panels. The complete fan and motor assembly are contained within the enclosure, so engineers can easily gain access to carry out any necessary maintenance www.ipimedia.com

procedures by removing the panels. The supply fan and extractor fan can either be direct-drive or non-direct drive. Direct-drive fans may require a vibration sensor on both the driveend and non-drive-end bearing, while non-direct drive fans may require the addition of two accelerometers on the journal bearings of the gear shaft. A well-established, and cost-effective, technique for identifying imbalance and misalignment in air handling units employs 4-20mA sensors mounted on to the bearings and shafts, with the velocity levels being fed back to a PLC, allowing overall vibration trends to be monitored. A dual output sensor can provide not only a 4-20mA output but also an AC output, allowing the engineer to take more-in-depth vibration analysis via a data collector. In an air handling unit, an M12 connector used in conjunction with separate M12 cable assemblies offers an effective option, as the M12 connector is of a smaller size than many alternatives, and the associated cable assemblies have a tight bend radius. A further option is to use fixed AC sensors hard-wired to switch boxes outside the air handling unit, allowing data to be collected safely from the same positions on a regular basis. The limitation of this option is that readings only apply to the moment in time that they were taken. If there is limited access, for example, near the belt guards, it is worth considering that side entry sensors can be used, as well as especially compact small footprint sensors for small air handling units.

Specification and Installation When it comes to switch boxes, stainless steel units are often compulsory in pharmaceutical applications. A well-established supplier will offer valuable advice on specification issues such as this, and offer important guidance on installation. For example, accelerometers should be mounted directly onto the machine surface. This surface should be flat, smooth, unpainted, free from grease and oil, and larger than the base of the accelerometer itself. The accelerometer should also be positioned as close as possible to the source of vibration. If conditions (and product warranties) allow, the preferred mounting technique is to drill and tap a thread directly onto the machine so that an accelerometer with an integral ¼-28UNF, M6 or M8 mounting thread can be screwed into place. This ‘drill and tap’ method can also be used to fix a mounting stud, to which an accelerometer can then be attached, and specialised installation kits are available for performing this task. It is important to take care and use the right tools for the job, as tightening the sensor outside the appropriate torque levels can damage equipment or reduce its effectiveness. For example, over-tightening can damage the sensor by stripping the thread, while under-tightening will lead to inaccurate readings due to poor contact with the vibrating surface. If drilling and tapping is not an option, the next best thing is to attach mounting studs using adhesive. The issue to be aware of here is that you may need to consider the temperature present within your application and choose an adhesive that is capable of coping with that temperature. For applications in temperatures up to 100°C, a metal-filled epoxy adhesive would be the appropriate substance to use. A good spot facing kit will give you all the necessary tools needed to accurately mount a vibration sensor onto the rotating machine, including a tapping drill, taps, tap wrench and a spot facing tool. These kits are now available to allow for different mounting threads; ¼, M6 and M8. Correct mounting of the sensor is vital to ensure true readings and, INTERNATIONAL PHARMACEUTICAL INDUSTRY 93


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where possible, mounting a sensor via a drilled and tapped hole directly to the machine housing will give the best results. However, if the housing is not flat, a spot facing installation kit allows creation of a flat surface. Having installed accelerometers as carefully as possible, ensure that the accelerometer cable is clamped to the body of the accelerometer itself with a cable tie. This will not only prevent strain, but also prevent the false readings that can be generated when loose cables make excessive movement. If all of these specification and installation issues are carefully considered, not only will the air handling unit be able to maintain air control consistently to the benefit of the plant as a whole but also extend the operating life of equipment beyond 94 INTERNATIONAL PHARMACEUTICAL INDUSTRY

recommended maintenance intervals, guarding against the expense generated when increases in vibration lead to machine failure, downtime and unwelcome reductions in production volume or quality. We have come a long way since Chang Heng’s ingenious seismograph, and vibrations far smaller than earthquakes can now be detected instantly with accelerometers. Chang applied an earthly, pragmatic approach to the challenges presented by earthquakes, contradicting the orthodox opinion that viewed such disturbances as representations of the gods’ displeasure. While his elders awaited their fate, Chang preferred to take a more hands-on approach, and today’s engineers can follow in his footsteps by protecting their air handling units using vibration

monitoring techniques, rather than crossing their fingers and hoping for the best.

Chris Hansford is a qualified electro-mechanical engineer with over 30 years’ experience in the vibration monitoring industry. In 1986, he was involved in the formation of a sensor manufacturing company and, as Managing Director for 20 years, successfully grew the business and gained a wealth of commercial experience within the UK market. In 2006, Chris moved on to set up Hansford Sensors Ltd, a manufacturer of accelerometers and ancillary equipment that has already become a global market leader.

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PACKAGING

Child-resistant Packaging Laws are becoming More Restrictive Throughout the world, we are getting more and more used to the “push and turn” and “squeeze and turn” functions of many household products. It has been noticed that drug manufacturers and packagers in both Europe and in the US over time are met with far-reaching regulations with regard to ensuring that drugs are packed in a manner that effectively protects the welfare and safety of children. Child-resistant (CR) packaging for many prescription drugs (PD) for both solid dosages and liquid formulations - has been mandatory for many years; however during the last years there has also been a trend moving towards CR packaging for over-the-counter (OTC) products. With the still increasing consumption of medicines, there is a growing market for OTC products. Today an ordinary household keeps many different drugs, and thereby the exposure towards children is increased significantly. Industry has shown that reasons for using CR packaging for OTC products are not only regulative. The constant focus on children’s safety and prevention of serious accidents seems to play a crucial role when choosing primary plastic pharmaceutical packaging. In general, statistics show that suicide attempts among children have decreased, but unfortunately it also shows that children overdose more on medication use, i.e. a product like vitamins is the cause of children dying of overdoses because they unintentionally eat too many vitamins. In addition to safer storage at home, the solution could be to use CR packaging for vitamins. However regulations, recommendations, and common practice differ from country to country and from customer to customer. It is difficult to ignore seniorfriendliness when talking about child102 INTERNATIONAL PHARMACEUTICAL INDUSTRY

resistance, as these do not go hand in hand. The difficulty has always been to balance proper protection and ease of use. Recognising that the older population will be high over the coming decades, and at the same time more and more comprehensive standards for drug packaging are formulated, I consider that the design of the CR function is a fit subject for discussion. In the future the wellknown and well-incorporated “push/ squeeze and turn” moves will, to a greater extent, be accompanied by other designs which allow seniors and people with limited dexterity to better open and close the packaging. Opposite, or in addition to, children’s safety organisations, the interests of the aging population are also represented. Associations promoting societies without age barriers and ageism are also

very active when it comes to pharmaceutical packaging. They argue that there are more pensioners than teenagers, so their plea is to focus on pharmaceutical packaging with opening procedure properties which offer good patient adherence. Different institutions, such as Swedish Apoteket, are recognising the development of innovative packaging solutions with the prize for “Best Drug Packaging”. In 2002 the development of the innovative Duma® Multi-Grip Cap, which is designed for people with disabilities and influences the capability of administering self-medication in various ways, was honoured. Drug manufacturers and packagers want patients to get best value from their products in terms of health outcomes and safe use. As the packaging design has a key Spring 2012 Volume 4 Issue 2


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contribution to this we, as a supplier of pharmaceutical plastic packaging, want to establish a co-creating process with our customers with the aim of creating preference for their products. Lately we have become aware of the recommendation from the United States Consumer Product Safety Commission (CPSC) to make it mandatory to use CR packaging for all OTC and PD products containing more than 0.08mg of imidazolinegroup substances in a single package. Imidazolines (tetrahydrozoline, naphazoline, oxymetazoline and xylometazoline) are a family of drugs that are vasoconstrictors indicated for ophthalmic irritation and/or nasal congestion. According to CPSC, imidazolines can cause serious adverse reactions, such as central nervous system (CNS) depression, decreased heart rate and depressed ventilation in children treated with these drugs, or who accidentally ingest them. The notice of proposed rulemaking requiring CR packaging for www.ipimedia.com

imidazolines is dated January this year, and it has already resulted in a lot of activity at the Gerresheimer site, which is manufacturing dropper bottle systems, nebulisers and nasal sprays. This action is taken under the Poison Prevention Packaging Act (PPPA) of 1970. Assuming the CPSC’s proposed rule is made final, shortly thereafter, drug manufacturers and packagers using more than 0.08mg of imidazolines are obliged to change their primary packaging. Although no decision has been made yet, some of our customers in the human and animal health industries have already decided to change to CR packaging. Regulations on this area are anticipated – “it is just a matter of time”, they predict. And time can become the big issue. CPSC proposes a one-year implementation period after the effective date of the final rule. On behalf of the pharmaceutical industry, the Consumer Healthcare Products Association (CHPA) requested

an extension to child-resistant imidazolines product packaging. The CHPA argues that drug manufacturers and packers need at least two years to design, develop and test child-resistant packaging to ensure continued consumer access to eye drops, nasal decongestants and other products with imidazoline ingredients. The marketplace for primary packaging is not well represented with standard child-resistant packaging solutions for this type of product, and therefore design development is most likely to be done. Few suppliers of ophthalmic primary plastic packaging have standard CR solutions or CR closures which can be adapted to the customers’ existing bottles. Despite the best case of adapting an existing standard CR closure to the bottles already used, our experience is that such a change still requires a relatively long implementation period. In this line of business, many aspects must be considered to meet the stringent market demands of today and tomorrow!

Christina Desirée Holder is Product Manager for the European sites of Gerresheimer Plastic Packaging covering the business areas of pharmaceutical plastic packaging within solid dosage, liquid formulations, ophthalmic applications and plastic bottles for personal care. Christina has worked for Gerresheimer Plastic Packaging for nine years and has also held managerial positions within Customer Care Management and Marketing. Email: c.holder@gerresheimer.com

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NEWS

Hope for resistant TB as Otsuka’s delamanid shows efficacy With multidrug resistant (MDR) strains of tuberculosis reaching alarming levels in some areas of the globe, positive results from a phase II trial of Otsuka’s delamanid have provided hope that a new treatment option may be on the way. A phase II trial of the new drug, published in the New England Journal of Medicine (June 7), found that 45 per cent of patients treated with delamanid achieved a TB cure rate - defined as negative sputum culture - compared to 29.6 per cent of the placebo group. The treatment was given on top of a background regimen of anti-tubercular drugs. There were no dose-limiting toxicities with delamanid, although some patients on the drug showed evidence of a heart effect known as QT interval prolongation, according to the authors. Delamanid is a new agent derived from the nitro-dihydro-imidazooxazole class of compounds that inhibits mycolic acid synthesis, and if approved would be the first new drug for tuberculosis in the last 40 years. “Existing TB treatment regimens are long and cumbersome, which can lead to incomplete treatment, resulting in an increased risk of relapse and developing drug resistance,” commented Manfred Danilovits of Tartu University Hospital in Estonia, the lead investigator in the trial. “This study shows that delamanid … may help achieve earlier sputum conversion thereby reducing infectiousness and enhancing overall treatment options for MDR-TB,” he added. Source: IPI – Staff Writer – Cecilia Stroe

potential initial public offering (IPO) of a minority ownership stake in the new company, putting to rest rumours that the company planned sell off the unit in a similar fashion to the sale of its nutrition business to Nestle, with Novartis and Bayer touted as potential buyers. The announcement comes a week after the departure of former head of Pfizer animal health Cavan Redmond to become CEO of online health information service WebMD. At the moment, it is unclear what will happen to the rest of Pfizer animal health’s 9,000 employees, spread across 120 countries, although Pfizer said Zoetis will “build on the leadership of Pfizer animal health”, including the discovery, development, manufacture and marketing of animal vaccines, medicines, biopharmaceuticals, diagnostics and genetic tests for both livestock and pets. “Pfizer Animal Health is a dynamic business with strong fundamentals, an expanding and loyal direct customer base and a proven management team,” said Ian Read, chairman and CEO at Pfizer. “We are on track to create a standalone animal health company by our previously stated target of July 2013.” The launch of Zoetis, the name of which is derived from ‘zoetic’ meaning ‘pertaining to life’, comes as Pfizer restructures its business to cope with patent expiries for big-selling drugs like Lipitor. The company is looking to become a more streamlined business, focused on developing a strong pharmaceutical pipeline and maximising value for shareholders through suitable divestments. Source: Pfizer Ltd.

Pfizer to spin off animal health unit as Zoetis Pfizer has announced plans to set up its animal health division as a standalone company which it will name Zoetis. The US pharma firm will file for a

Targacept CEO J Donald deBethizy steps down after pipeline failures The CEO of US biotech Targacept Dr J Donald deBethizy is to leave his position following a difficult period of poor trial results and failed partnerships.

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Until a replacement is found his responsibilities will be taken over by board chair Mark Skaletsky who will assume a new role as head of office of the chairman, with Dr deBethizy to serve as an advisor during the transitional period. Dr deBethizy had been CEO of the nervous system specialists since 2000, but the company has faced many challenges in the past 18 months. This included the termination of its partnership with GlaxoSmithKline to develop products targeting neuronal nicotinic receptors, with Targacept losing out on a potential $1.5bn in milestone payments. The company’s collaboration with AstraZeneca was also brought to an abrupt end in March, 2012 after poor results in phase III studies for what would have been a first-in-class antidepressant. “I am proud of what we have built at Targacept over the years and continue to believe strongly that the company will do great things,” said Dr deBethizy on his departure. In addition to Skaletsky’s new position, three other members of the office of the chairman were appointed: Jeffrey Brennan, SVP, business and commercial development and chief business officer; Alan Musso, SVP, finance and administration, chief financial officer and treasurer; and Peter Zorn. SVP legal affairs, general counsel and secretary. Source: IPI – Staff Writer – Cecilia Store Abbott presents phase 3 study results for investigational use of HUMIRA® (Adalimumab) Abbott (NYSE: ABT) announced results from the Phase 3 ABILITY-2 investigational study of HUMIRA® (adalimumab) in patients with active peripheral spondyloarthritis (PSpA) who have not been diagnosed with psoriatic arthritis (PsA) or ankylosing spondylitis (AS). At week 12, nearly twice as many HUMIRA patients compared to those receiving placebo achieved the primary endpoint, defined Spring 2012 Volume 4 Issue 2


NEWS

as at least a 40 percent improvement in the peripheral SpA response criteria (PSpARC 40). These results were presented at the European League Against Rheumatism (EULAR) 2012 Congress in Berlin, Germany. ABILITY-2 is the first randomized, controlled trial to use the new Assessment of SpondyloArthritis international Society (ASAS) peripheral SpA criteria to enroll PSpA patients not including patients with PsA or AS - as well as evaluate an anti-tumor necrosis factor medication (anti-TNF) in treating these patients. PSpA is characterized by peripheral arthritis (asymmetric, lower limb or both), enthesitis (painful inflammation where a tendon or ligament attaches to bone) or dactylitis (a painful and swollen digit), in addition to the presence of other features (family history of SpA, history of inflammation in the eye, past or current history of psoriasis or inflammatory bowel disease). “ABILITY-2 is a landmark trial which focuses on an important subset of spondyloarthritis patients whose disease often goes unrecognized and inadequately treated because they do not present with a more recognized condition such as psoriatic arthritis or ankylosing spondylitis,” said Philip Mease, M.D., Director of Rheumatology Research, Swedish Medical Center and Clinical Professor, University of Washington School of Medicine, Seattle. Source: IPI – Staff Writer – Cecilia Stroe New immune therapy shows promise in kidney cancer An antibody that helps a person’s own immune system battle cancer cells shows increasing promise in reducing tumors in patients with advanced kidney cancer, according to researchers at Beth Israel Deaconess Medical Center. The results of an expanded Phase 1 trial presented at the American Society of Clinical Oncology’s annual conference in Chicago, showed that some patients treated with a www.ipimedia.com

fully human monoclonal antibody developed by Bristol Myers Squibb had a positive response to the effort by the agent, BMS-936558, to prolong the immune system’s efforts to fight off renal cell carcinoma without some of the debilitating side effects common to earlier immunotherapies. The presentation by David F. McDermott, MD, Director of Biologic Therapy Program at the Beth Israel Deaconess Medical Center Cancer Center and an Assistant Professor of Medicine at Harvard Medical School, highlights one of two key efforts underway to use the body’s own disease-fighting tools against cancer. Separate work by David Avigan MD, Director of BIDMC’s Blood/Bone Marrow Transplant Program, focuses on developing a personalized vaccine, compromised of the patient’s tumor and immune system agents, to battle kidney cancer. Cancer cells have the ability to trick the immune system, the body’s self-defense mechanism, which is designed to ward off infections. Immune therapy such as antibody treatment and vaccines is designed to reeducate the body to recognized cancer as an invader. “We’ve known for a long time that in certain cases the immune system can be boosted in a way that can create remissions” of hematologic malignancies like leukemia and lymphoma, says McDermott. “We’ve been trying to create the same long term results in solid tumors, which is more difficult.” Source: IPI – Staff Writer – Cecilia Stroe Trastuzumab emtansine (T-DM1) reduced the risk of cancer worsening or death Roche (SIX: RO, ROG; OTCQX: RHHBY) announced that the Phase III EMILIA study of trastuzumab emtansine (T-DM1) met its co-primary endpoint, of a significant improvement in the time patients with HER2-positive metastatic breast cancer (mBC) lived without their disease getting worse

(progression-free survival; PFS). The study showed that the risk of disease worsening or death was reduced by 35 percent for people who received trastuzumab emtansine compared to those who received lapatinib plus Xeloda® (capecitabine) chemotherapy (HR=0.65, p-value <.0001). The EMILIA study is the first randomised Phase III trial of trastuzumab emtansine in patients with HER2-positive mBC who had previously received Herceptin® (trastuzumab) and a taxane chemotherapy. There was also a trend for patients who received trastuzumab emtansine to live longer (overall survival, or OS, the other co-primary endpoint of the study) than those who received lapatinib plus Xeloda, but these data are currently not mature. The safety profile of trastuzumab emtansine was consistent with that seen in previous studies, with fewer patients who received trastuzumab emtansine experiencing Grade 3 or higher (severe) adverse events (AEs) than those who received lapatinib plus Xeloda (40.8 percent compared to 57 percent). The EMILIA data will be presented in the plenary session at the 48th Annual Meeting of the American Society of Clinical Oncology (ASCO) in Chicago by Kim Blackwell, M.D., Duke University School of Medicine (Abstract #LBA1, Sunday 3 June at 1:45 pm CDT). The EMILIA data were also featured in the official ASCO press programme on Saturday 2 June. “The encouraging efficacy, safety profile and quality of life results from the EMILIA study support our belief that trastuzumab emtansine may have an important role for patients with HER2positive metastatic breast cancer,” said Hal Barron, M.D., Chief Medical Officer and Head, Global Product Development. “We are working with global regulatory authorities to submit these data as quickly as possible and hope that trastuzumab emtansine will soon be available to patients with this aggressive type of breast cancer.” Source: IPI – Staff Writer – Cecilia Stroe

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