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Volume 2 Issue 1

Peer Reviewed

Non-clinical Evaluations for Anti-cancer Drugs with ICH S9 Guideline What the Industry Needs to Know

The Effect of Insulin

On Cell Growth and Virus Production

LIMS and ISO/IEC 17025

An Opportunity not a Burden

Clinical and Investigator Initiated Trials’ Effect on MSL Responsibilities

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Contents 04 Foreword WATCH PAGES 06 Non-clinical Evaluations for Anti-cancer Drugs with ICH S9 Guideline: What the Industry Needs to Know DIRECTORS: Martin Wright Mark A. Barker BUSINESS DEVELOPMENT: Mark Sen mark@ibijournal.com EDITORIAL: Virginia Toteva virginia@pharmapubs.com DESIGN DIRECTOR: Jana Sukenikova www.fanahshapeless.com FINANCE DEPARTMENT: Martin Wright martin@ipimedia.com RESEARCH & CIRCULATION: Freya Gavaghan freya@pharmapubs.com COVER IMAGE: iStockphoto © PUBLISHED BY: Pharma Publications 50 D, City Business Centre London, SE16 2XB Tel: +44 (0)20 7237 2036 Fax: +44 (0)01 480 247 5316 Email: info@ibijournal.com www.biopharmaceuticalmedia.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 IBI will be published in Summer 2019. ISSN No.International Biopharmaceutical 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. 2019 PHARMA PUBLICATIONS / Volume 2 issue 1 – Spring 2019

The ICH S9 Guideline Q&A document – published in June 2018 – is designed to clarify some of the most common challenges experienced across industry when implementing ICH S9. Here, Robert Turcan at Envigo highlights a few key areas selected based on the advice sought by their customers during development of their anti-cancer pharmaceuticals. He concludes with a consideration of the outlook for oncology programmes, as well as recent FDA approvals. 08 Raising Organisational Fluency in Real-world Evidence The planet is currently experiencing global healthcare crises that affect huge populations. From diabetes to Alzheimer’s to mental health conditions, we still lack real-world data (RWD) on treatment outcomes that would enable us to more effectively manage complex diseases and fulfill the promise of personalised medicine. David Thompson and Yvonne Ash of Syneos Health examine how, regardless of the disease area or treatment option, RWE that is effectively planned for, collected and analysed outside of traditional clinical trial settings is emerging as integral to the future of global health. RESEARCH / INNOVATION / DEVELOPMENT 10 Applying Standardised NMR Spectroscopy to Research Human Diseases In this article Professor Matthias Nauck of Universitätsmedizin Greifswald and Roland Leiminger of Bruker BioSpin examine the importance of Biobanks and how they are continuously growing across the globe, with each one containing hundreds of thousands of samples that allow researchers to advance their knowledge on a range of diseases, including which individuals might be most at risk and how they will respond to certain treatments. This growing pool of knowledge contributes to the overall goal of safeguarding the future generation’s health. 14 Alzheimer’s Disease: The Way Forward Emily Burke at Biotech Primer examines Alzheimer’s disease (AD) and how it ranks as one of the toughest nuts to crack within drug discovery and development. Current treatments merely manage symptoms, so finding a better solution becomes more and more urgent as the aging population grows. With more hard work and investment, perhaps one of the many introduced in this article will lead to a cure – or perhaps the winning therapy may include a combination of these approaches. 18 Artificial Intelligence Expands the Destiny of DNA Analysis DNA is the code that has defined humanity for millennia. Until more recently, DNA has determined destiny. There was no escaping its design and code for individuals. It was the code that says how long people will live and what they will likely die from, at least in terms of propensity and predisposition towards certain conditions, from heart disease to diabetes to

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Contents colon cancer. Ian Jenkins at Frelii discusses the true epigenetic correlations that impact the destiny of DNA and how it relates to individual health and wellbeing to emerge in ways never before imagined.

REGULATORY / QUALITY COMPLIANCE

CLINICAL RESEARCH

The last two years have been some of the most remarkable years in pharmaceutical history. There were 46 FDA approvals in 2017, and this year we have received a record 59 approvals. Orhan Caglayan at CPhI Worlwide and bioLIVE explores how we might now be entering a golden age of innovation for pharmaceutical R&D, but what is most encouraging is that this new era of increased productivity is translating into drug delivery devices and packaging as well. Many of the experts believe that this new age of innovation is a golden opportunity for drug delivery and device manufacturers to bring new technologies to patients.

22 Diagnosis Still Poses the Biggest Challenge in the AIDS Battle Significant progress has been made in the AIDS response since 1988, however ensuring a swift and accurate diagnosis accessible to everyone is still a significant barrier which needs to be tackled in our battle to combat the disease. David Fraser at BBI Solutions identifies how AIDS diagnostics can be improved. He concludes that enabling mass access to swift, accurate, presymptomatic diagnosis requires collaboration and a deep-seated commitment to removing the stigma around HIV and AIDS. 26 Clinical and Investigator Initiated Trials’ Effect on MSL Responsibilities Among the many responsibilities assigned to medical science liaisons (MSLs) is facilitating company collaboration with thought leaders on clinical trial work. According to MSL teams surveyed by Cutting Edge Information, seemingly little time and ostensibly minor goals are assigned to this activity. But when considering the many tasks MSLs already perform and their headcount per region, even a minor focus on facilitating investigator-initiated trials (IITs) and other study types may be more than enough to meet company goals. Todd Middleton at Cutting Edge Information discusses further. MANUFACTURING / TECHNOLOGY PLATFORMS 30 Antibodies Exploration Thanks to Label-free Surface Plasmon Resonance Imaging Technology Surface plasmon resonance imaging allows the study of molecules binding in label-free and real-time conditions. The power of the technique comes from its singularities, which are multiplexing and imaging, leading to a considerable speed-up of the processes’ analyses. This will be illustrated for the study of antibody molecules by three applications, which Yannick Nizet at Synabs, and Chiraz Frydman and Karen Mercier of HORIBA reveal in this article. By experimenting with the surface plasmon resonance imaging technology via these three applications, they highlight the key advantages of this platform: sensitivity for the detection of small molecules, screening potential, and rapid experiment optimisation thanks to the label-free, real-time and multiplex capacities.

42 The Drug Delivery Innovation League table – Germany and France Lead the Way in Europe

46 LIMS and ISO/IEC 17025 – An Opportunity, Not a Burden Laboratory information management systems (LIMS) have wide-ranging usage within pharmaceutical organisations, wherever samples need to be taken, tested and the results evaluated, including QA/QC, stability studies, pre-clinical pathology, clinical trials, biobanking and environmental monitoring. The successful implementation of a LIMS provides an opportunity to improve the operation, performance and efficiency of any laboratory with the added benefit that it supports ISO 17025 compliance, as Simon Wood at Autoscribe Informatics explains. 50 Understanding Critical Quality Attributes With the increasing prominence of biopharmaceutical products in the modern therapeutic landscape, it has become vitally important to ensure the manufacture of these products is carried out reliably and efficiently. Therefore, the FDA introduced the concepts of quality by design (QbD) into the cGMP regulations in 2004. These guidelines provide the framework by which pharmaceutical companies can design their products, processes, and control procedures to ensure that a product has all the attributes considered paramount to its safety and efficacy – the critical quality attributes (CQAs). Omar Musleh at SGS Life Sciences explores how to use these to develop a life-saving biologic.

34 The Effect of Insulin on Cell Growth and Virus Production This article by Aziza Manceur from National Research Council Canada describes a project whose goal was to test insulin as a booster for cell growth and virus production. Insulin was chosen for many reasons. It is already used in cell culture, and it is approved by regulatory agencies. So this is in line with our mission to look for strategies that can be implemented quickly in industry. Insulin is also known for its anti-apoptotic and mitogenic characteristics. We expected that insulin would improve the growth profile of the cells that we work with, but its effect on virus production was unknown. 2 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Spring 2019 Volume 2 Issue 1


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Foreword Epigenetics delves into the way we can potentially affect our health by changing how our genes are expressed, and more specifically, how that expression can be altered through mechanisms that are independent of our innate DNA sequences.

The Clinical research section contains another interesting piece by David Fraser at BBI Solutions about the significant progress in AIDS since 1988, however David says that ensuring a swift and accurate diagnosis accessible to everyone is still a significant barrier which needs to be tackled in the battle to combat the disease.

As a brief overview, consider the following: the genes in our body are coded in such a way as to be responsible for deciding just about everything from eye colour to height to preponderance toward colour-blindness. There is a caveat though: not all our genes are always being expressed, and only the ones that are expressed are affecting us daily. As a matter of fact, the genome (complete set of genes) of every cell in the human body is the same. The structure and function of each cell is determined by their epigenome, or the set of genes that are expressed. A clear example of how this manifest itself is in the development of some cancers. According to a widely-accepted theory, one of the key steps in cancer formation is the activation, or “turning on”, of certain genes (known as oncogenes) and the deactivation, or “turning off”, of other genes (tumour suppressor genes). These genes control many of the proteins involved in cell growth and proliferation. When oncogenes are activated or tumour suppressor genes are deactivated, the normal cell processes that prevent malignancy are unable to occur. As it turns out, most of our genes work in a similar manner, affecting every aspect of our physiology.

One of the articles within the MANUFACTURING & TECHNOLOGY PLATFORMS section by Aziza Manceur from National Research Council Canada describes a project whose goal was to test insulin as a booster for cell growth and virus production. Aziza reveals that the insulin was chosen for many reasons – it is already used in cell culture, and it is approved by regulatory agencies. Among all interesting topics under the REGULATORY & QUALITY COMPLIANCE section Simon Wood at Autoscribe Informatics on a LIMS (Laboratory information management systems). Simon explains that successful implementation of LIMS provides an opportunity to improve the operation, performance and efficiency of any laboratory with the added benefit that it supports ISO 17025 compliance. I wish you all a successful 2019 and I look forward to bringing you more interesting articles in the next issue. Virginia Toteva Editorial Manager

By providing us with the first detailed map of the genome-wide DNA methylation pattern in human fat tissue, we are now able to definitively link exercise to altered adipose tissue DNA methylation, which may facilitate our understanding of how the human body stores and metabolizes fat cells. I am delighted to welcome you to the first edition of the IBI journal for 2019, where you will find hot topics within the biopharmaceutical industry from leading industry experts worldwide. One of these topics within the RESEARCH, INNOVATION & DEVELOPMENT section is the article by Professor Matthias Nauck of Universitätsmedizin Greifswald and Roland Leiminger of Bruker BioSpin who examines the importance of Biobanks and how they are continuously growing across the globe.

IBI – Editorial Advisory Board • Ashok K. Ghone, PhD, VP, Global Services MakroCare, USA

• Jim James DeSantihas, Chief Executive Officer, PharmaVigilant

• Bakhyt Sarymsakova – Head of Department of International

• Lorna. M. Graham, BSc Hons, MSc, Director, Project Management,

Cooperation, National Research Center of MCH, Astana, Kazakhstan

• Catherine Lund, Vice Chairman, OnQ Consulting

Worldwide Clinical Trials

• Mark Goldberg, Chief Operating Officer, PAREXEL International Corporation

• Cellia K. Habita, President & CEO, Arianne Corporation

• Maha Al-Farhan, Chair of the GCC Chapter of the ACRP

• Chris Tait, Life Science Account Manager, CHUBB Insurance

• Rick Turner, Senior Scientific Director, Quintiles Cardiac Safety

Company of Europe

• Deborah A. Komlos, Senior Medical & Regulatory Writer, Clarivate Analytics

• • Elizabeth Moench, President and CEO of Bioclinica – Patient Recruitment & Retention

• Francis Crawley, Executive Director of the Good Clinical Practice

Alliance – Europe (GCPA) and a World Health Organization (WHO) Expert in ethics

• Hermann Schulz, MD, Founder, PresseKontext • Jeffrey W. Sherman, Chief Medical Officer and Senior Vice President, IDM Pharma.

4 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Services & Affiliate Clinical Associate Professor, University of Florida College of Pharmacy

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

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

• 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 Spring 2019 Volume 2 Issue 1


www.biopharmaceuticalmedia.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 5


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Non-clinical Evaluations for Anti-cancer Drugs with ICH S9 Guideline Q&A – What the Industry Needs to Know The ICH S9 Guideline Q&A document – published in June 2018 – is designed to clarify some of the most common challenges experienced across industry when implementing ICH S9. Here, I highlight a few key areas selected on the basis of the advice sought by our customers during development of their anticancer pharmaceuticals. The finalisation of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) S9 Guideline – Non-clinical Evaluation for Anticancer Pharmaceuticals – in October 2009 was an important milestone in understanding anticancer therapy development. The tripartite harmonised ICH guideline provides information for pharmaceuticals that are intended to treat cancer in patients with late-stage or advanced disease, including both small-molecule and biotechnology-derived pharmaceuticals, regardless of the route of administration. It gives us the type and timing of non-clinical studies in relation to the development of anticancer pharmaceuticals and references other guidance as appropriate. Following a decade of questions and interactions with industry, a Q&A document was published in June 2018, designed to clarify some of the most common challenges experienced across industry when implementing ICH S9. It represents the current thinking of the US Food and Drug Administration (FDA) and, as such, provides non-binding recommendations.

6 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

The Q&A document contains 41 questions that cover the full scope of ICH S9: studies to support non-clinical evaluation, non-clinical data to support clinical trial design and marketing, and other considerations. Here we highlight a few key areas selected on the basis of the advice sought by our customers during development of their anticancer pharmaceutical. Are all initial development plans for anticancer pharmaceuticals covered under ICH S9? Right from the start, the document provides clarification on a commonly raised question – are all initial development plans for anticancer pharmaceuticals covered under ICH S9? ICH S9 makes clear that the intended treatment population is patients with advanced disease and limited therapeutic options. Question 1.1 of the Q&A document provides the further insight you need: ‘As most initial development programs are performed in patients (adult and pediatric) whose disease is resistant and refractory to available therapy, the nonclinical program described in ICH S9 is applicable. See also the answer to Question 2 (1.2). For other initial development programs in cancer that is not resistant and refractory, ICH S9 should be used as a starting point, and other studies added as appropriate with reference to ICH M3(R2) and S6(R1). In some situations where the development pathway is not clear, regulatory agencies should be consulted.’ In addition, while the guideline did not make reference to life expectancy, this has now been clarified – ‘… application of the guideline should not be based on an expectation of survival as

Spring 2019 Volume 2 Issue 1


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measured in years’. Finally, the Q&A document also makes clear that non-oncology therapeutics, even those for life-threatening disease with limited therapeutic options, are outside the scope of ICH9. Which studies are required? The requirement for planning additional toxicology studies (beyond "general" toxicology) is complicated, and there is often a temptation to cover a wide range of possibilities in the pre-clinical phase in order to avoid missing something. Now, the Q&A document clarifies, for example, that inclusion of supportive care drugs such as antibiotics during toxicology studies can be considered appropriate when secondary infection due to immunosuppression is observed in the study; however, giving such care to all animals prophylactically is not recommended. In addition, the document suggests that when the anticancer pharmaceutical is shown to extend survival of patients, additional general toxicology studies are usually not warranted; when such studies are required, they could still be submitted post approval. These two points allow you to reduce the studies required prior to approval, by judicious application of the knowledge of their product to the development program. Hidden under ‘Other considerations’ are a number of clarifications related to antibody-drug conjugates (ADCs), which you need to take into account. These clarifications cover requirements for pilot studies on the different elements of the conjugated material, tissue distribution studies, plasma stability, dosing in pre-clinical studies and the approach to setting a first-in-human starting dose. Given that nearly 250 ADCs are in the pipeline for the treatment of cancer, we anticipate that the points covered under "Other considerations" may prove the most useful clarifications over the next decade. www.biopharmaceuticalmedia.com

What's the outlook for oncology programmes? The ICH S9 Guideline Q&A is well timed: Targeted therapies account for around 87% of oncology drugs in development, and these small molecules, monoclonal antibodies and ADCs should reduce harmful side-effects for healthy cells and achieve a better clinical effect. And 2017 was the year that the Food and Drug Administration (FDA) first approved a cancer treatment based on a common biomarker, rather than the location in the body where the tumour originated. The clarification offered in the new Q&A help us all plan, design, execute and submit non-clinical studies to the FDA and other regulatory bodies around the world.

Robert Turcan Robert Turcan is Head of Regulatory Affairs and Program Management at Envigo. He and his team work directly with customers to provide wide-ranging scientific and regulatory support throughout product development, and provide advice and support in the design and execution of non-clinical development programmes. He is passionate about helping drug developers progress novel therapeutics from the lab to first-in-human clinical trials and beyond to market adoption. His many years of drug development experience in both small and large molecules enables him to design programmes that balance regulatory needs, scientific robustness and safety assessment efficiency, to deliver solutions that help customers achieve their development goals. Robert has a PhD in drug metabolism from the University of Surrey. He has been with Envigo for the last 3.5 years, having previously worked other in large pharma and CRO organisations.

Web: www.envigo.com

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Raising Organisational Fluency in Real-world Evidence The planet is currently experiencing global healthcare crises that affect huge populations. From diabetes to Alzheimer’s to mental health conditions, we still lack real-world data (RWD) on treatment outcomes that would enable us to more effectively manage complex diseases and fulfill the promise of personalised medicine. At the same time, underserved populations with rare diseases struggle to receive treatment and obtain the real-world evidence (RWE) that could provide guidance about living and thriving with their conditions. Regardless of the disease area or treatment option, RWE that is effectively planned for, collected and analysed outside of traditional clinical trial settings is emerging as integral to the future of global health. For biopharma companies, RWE is also becoming a critical success factor in achieving access to and adoption of new therapies. An expanding array of healthcare decision-makers – including payers, providers, patient advocacy groups, clinical guideline developers and regulators – now require, beyond traditional safety and efficacy measures, RWE based on real-world outcomes data. It isn’t easy, however, to get everyone in a company on the same page when talking about RWE. This fundamental problem can compromise a company’s ability to formulate a robust RWE research agenda and advance it within the organisation, which in turn can seriously impede the successful introduction of new therapies.

8 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Companies face three main challenges in this regard. The first challenge relates to the use of differing terminology and concepts; for example: What is RWD? How is that different from RWE? (Answer: RWD + analytics = RWE.) Is it the same as health economics and outcomes research (HEOR)? (Answer: No, it’s broader and encompasses much of HEOR.) In addition, confusion is widespread about the wide range of methodologic approaches to RWE study design, the selection of outcome measures and the real-world evidentiary needs of diverse health system stakeholders. The second major challenge involves RWE ownership within organisations. Typically pockets of RWE expertise are siloed within various teams – medical affairs, epidemiology, health economics, data science, market access – and among regional points of contact. Each may be looking to establish some level of ownership. Poor transparency makes it hard to see what data sources exist, who the subject matter experts are and where to find them. It also stifles best-practice sharing, which is essential in the evolving world of RWE generation and utilisation. The third challenge companies face arises out of the first two, and that is lack of planning. It is common to find that a company’s employees across functions and regions know what RWD and RWE are but that their definitions and experiences with the concepts are highly variable; they are often unsure how to utilise and optimise RWE in their research, strategies and customer engagements. Too often, RWE isn’t seen as a tool to be integrated into an overall set of evidence, and therefore also isn’t integrated into business and development processes, planning structures and templates, or budget and prioritisation decisions. Lack of planning for RWE leaves companies significantly behind and under-powered in their ability to demonstrate value and facilitate access and adoption at launch.

Spring 2019 Volume 2 Issue 1


Watch Page interactive learning experiences will help individuals engage with the material and practice and master the skills needed independently. Customised learning tracks can be designed to help all employees close knowledge and skill gaps, whatever their current level of experience with RWD and RWE. For example, it is important to raise awareness and educate about what RWE is at a basic level and why it is important to both individuals in various roles and to the organisation. With this baseline established, another learning track can facilitate adoption with a focus on RWE from an evidence generation perspective, so that anyone who works in some way with RWE, perhaps new in-role or learning to apply it, can navigate decisionmakers, defend why it is important to each major stakeholder in the healthcare landscape, or even speak and write with the confidence needed to apply common terminology and concepts. Taking this one step further would be a track on applying RWE in more proactive and seamless ways via frequent collaboration with colleagues and external partners, and by integrating RWE with other forms of evidence. RWE is a critical aspect of modern healthcare, providing evidence of real-world outcomes and disease insights that are increasingly a focus of healthcare policy-makers and other key stakeholders. However, biopharma companies are often challenged in building their RWE generation capabilities by inconsistency in the use of terminology and concepts, the silo-ing of expertise throughout their organisations, and lack of transparency – all of which contribute to an overall lack of planning for robust RWE generation. The ability to fully reflect the actual outcomes of medicines, technologies and other innovations requires a nascent skill set and knowledge base across multi-disciplinary and multi-functional teams. Biopharma companies will benefit from concerted efforts to more fully integrate their RWD and RWE expertise and to elevate organisational fluency in the concepts, utilisation and generation of RWE.

David Thompson, PhD Addressing these challenges requires not only a broad recognition of the imperative to establish and execute on a robust, rigorous RWE generation capability, but also a commitment to RWE awareness, education and training throughout the organisation. The “awareness” part is key. Employees across functions and geographies should understand – and be passionate about – how RWD and RWE can be used to improve the lives of patients. Once awareness is sufficiently raised, the stage is set to develop information resources and educational opportunities tailored to the needs of diverse audiences and different learning styles. That is also the time to facilitate the formation of collaborative networks of cross-functional experts who can share best practices from around the world, as well as their first-hand experiences with RWE challenges and solutions. An emerging best practice is for companies to create centralised “hubs” to help: • • • • •

Drive the creation of RWE expert networks within the organisation; Achieve broad consensus on RWE definitions and concepts; Clearly demonstrate the value of RWE evidence planning; Create a “one-stop-shop” for data sources; and Share case studies and lessons learned.

This hub can also serve as a platform for RWE awareness, education and training. Going sufficiently wide and deep to reach both new and experienced employees with varying levels of understanding about RWE will often require developing new communications channels and new content. Innovative, www.biopharmaceuticalmedia.com

David Thompson, PhD is Senior Vice President, Real World Evidence Research at Syneos Health, responsible for real-world research design consulting and thought leadership activities. David is a health economist with 25+ years of experience in HEOR & RWE generation. He also functions as Editor-in-Chief of Value & Outcomes Spotlight, a publication of the International Society for Pharmacoeconomics and Outcomes Research (ISPOR).

Email: david.thompson@syneoshealth.com

Yvonne Ash Yvonne Ash is Vice President of Solution Design at Syneos Health Learning Solutions. A recognised industry leader with 15+ years’ experience, Yvonne partners with major pharmaceutical, biotech and healthcare organisations to assess organisational effectiveness needs and development opportunities. She also serves as a strategic consultant to senior executives in sales and marketing to determine disease state education and product positioning needs across therapeutic areas.

Email: yvonne.ash@syneoshealth.com.

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Research / Innovation / Development

PEER REVIEWED

Applying Standardised NMR Spectroscopy to Research Human Diseases Biobanks are extremely useful tools for advancing the research of new therapies, and for the identification of disease biomarkers from large epidemiological cohorts. Biological material such as blood, urine, tissue and DNA is collected into large-scale repositories, which are invaluable to research institutes, hospitals, and biopharmaceutical and biotechnology companies. Individualised medicine, also known as personalised medicine, aims to provide optimal treatment for an individual patient, at a specific time, based on their specific genetic and molecular characteristics1. As an increasing number of biomarkers for diseases are becoming available, novel methods for implementing these in the context of individualised medicine are required. Biobanks are continuously growing across the globe, with each one containing hundreds of thousands of samples that allow researchers to advance their knowledge on a range of diseases, including which individuals might be most at risk and how they will respond to certain treatments. This growing pool of knowledge contributes to the overall goal of safeguarding the future generation’s health. One institute committed to developing biobanking studies is the University Medicine Greifswald (Universitätsmedzin Greifswald) in Germany, which uses state-ofthe-art analytical technology to measure samples and discover new biomarkers, to advance the field of individualised medicine. Biobanking Technology Collecting, preparing and analysing, storing and transporting biobank samples requires a method with high reproducibility, such as nuclear magnetic resonance (NMR) spectroscopy. Samples that are stored for long periods of time, such as those in biobanks, can be subject to change and develop artefacts. Although these can sometimes be of scientific interest, for longitudinal studies artefacts can be problematic and compromise the standardisation of results. NMR is useful for ensuring that samples remain unchanged over time. Scientists at the University of Medicine Greifswald are at the leading edge of biobanking research. Professor Matthias Nauck – Professor at the Institute of Clinical Chemistry and Laboratory Medicine – leads a laboratory that uses NMR spectroscopy for epidemiological research. Standardisation is crucial for these studies, and NMR as a technology provides the confidence needed that results obtained in 2018, for example, will be 100% comparable with results in 2028 taken from the same patient cohort. The laboratory combines this standardisation with a high degree of automation, to provide short turn-around times for analyses. Samples are transported from the emergency ward via a pneumatic tube system, covering a distance of 300 metres in 30 seconds. The sample is then immediately placed into the track system, which is connected to many analysers. The track system ensures that all steps, from sample preparation to centrifugation, are identical at every time point. Longitudinal Biobanking Studies If NMR is compared to other techniques, such as mass spectrometry (MS), which is far more sensitive, it is not possible to standardise MS measurements year-on-year. There would be systematic 10 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

shifts that would interfere with measuring the metabolome of individuals at several intervals across their lifetime. The outcome of long-term epidemiological studies can be translated into patient care, receiving additional information out of the metabolome, when these measurements are performed and rapidly calculated during the hospital stay of the patients. The consequences will be speeding up recovery times and improving overall health outcomes. The University Medicine Greifswald has initiated and been involved with a number of epidemiological studies over the past two decades. The longest-standing study began in 1997, the Study of Health in Pomerania (SHIP), which initially collected blood, urine and other biomaterial samples from 4400 individuals living in the Pomerania region2. The group is particularly interested in looking at biomarkers for diabetes risk. Probands are invited back every five years, so samples can be compared from 20 years, 15 years, 10 years and five years ago from the same patient cohort. The number of probands in the SHIP study is sufficient to assess common diseases, such as coronary heart disease and diabetes, but when looking at rarer diseases like cancer, higher numbers of probands are required. This was one of the driving forces behind the initiation of the German National Cohort, which is now the largest German epidemiological study, and aims to recruit 200,000 individuals in total across 18 centres across Germany. In this project, Prof Nauck is responsible for ensuring the quality of samples. To do this, he was engaged in the establishment of sample procedures at each study centre with identical equipment and standard operating procedures (SOPs). Two-thirds of the serum, plasma, urine and other biomaterials are stored at the Helmholtz Zentrum München, Munich, in the gas phase of liquid nitrogen at -180°C, which are the optimum conditions to keep the samples stable over long periods, whereas one-third is stored locally at the study centres. The aim of the German National Cohort is to look at individuals at various stages of disease development, and check for biomarkers from a clinical chemistry point of view. NMR spectroscopy is seen as the leading analytical method for such a prolonged study in order to detect sub-clinical deviations in metabolic state. The laboratory is focused on uncovering tiny sub-clinical variations that could potentially indicate a significant change in health status. A third project, known as GANI_MED (Greifswald Approach to Individualized Medicine)3, focuses on establishing an individualised medicine programme at the university hospital. The aim of this project is to improve the efficiency of treatment strategies, avoid adverse reactions to treatment, and to reduce healthcare expenditures, using sophisticated diagnostics and novel therapeutic interventions that account for the specific characteristics of an individual patient. Predicting Age-related Disease Progression Disease risk profiles depend on a number of factors, with aging being the most critical. Aging implicates a wide range of biochemical processes, and metabonomics has emerged as a new tool for characterising these biochemical changes. Metabonomics is a subset of metabolomics, and is the quantitative measurement of the metabolic response of an organism to physiological stimuli. Prof Nauck’s group used the SHIP probands to investigate the metabolic age of patients, adapted from the NMR spectra, using one urine spot sample4. The group showed that it is possible to capture biologically meaningful metabolomics information Spring 2019 Volume 2 Issue 1


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Research / Innovation / Development

within a single score on aging, derived from NMR-measured urine metabolites.

help individuals avoid developing specific diseases in the future.

It is thought that studies such as this will enable a more precise personalised medicine approach, and could form the basis of a precise estimator for the individual risk of age-related diseases. As urine is easily collectible, the metabolic age score based on this method could be a realistic tool for directly translating this approach into clinical practice.

REFERENCES

The Bridge to Routine Use Instead of providing clinicians with hundreds of pages of information, Prof Nauck and his team are now able to look at the analysis of the metabolomics data and provide a simple guide as to the condition of the patient’s organs. A traffic light system will be used with the NMR analysis, with green showing the organ is fine, yellow showing some limitations and red indicating a poor condition. The clinician then knows whether to look further at the test results to provide additional detail for clinical treatment. There are many techniques available for metabolomics studies, but NMR spectroscopy is preferred in Prof Nauck’s laboratory. One of the reasons for this is that sample preparation does not have to be carried out in advance. The reproducibility of NMR measurements over time is the key benefit of this technique however, as comparable data is crucial to detect small deviations between old and new results from a patient. The Future of Individualised Medicine Biomarkers that are able to classify the patients requiring medical intervention are required for optimal healthcare decision-making. Technology such as NMR spectroscopy is needed to measure reliable biomarkers that are critical for such decisions. Prof Nauck is now aiming to use NMR to detect minute changes from a ‘healthy state’ to a ‘disease state’, in order to inform preventative medicine approaches. Detecting individuals at risk of disease as early as possible is the overarching goal, and the University Medicine Greifswald is leading the way by measuring the metabolic status of individuals living in the local area, then re-measuring at subsequent time intervals to see if the development of the metabolic stage is normal, or if there are abnormal deviations. It is important to gain insights as early on as possible to 12 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

1. Grabe HJ et al. (2014) Cohort profile: Greifswald approach to individualized medicine (GANI_MED), Journal of Translational Medicine, 12:144, https://doi.org/10.1186/1479-5876-12-144 2. Volzke H et al. (2011) Cohort Profile: The Study of Health in Pomerania. International Journal of Epidemiology, 40:294–307. 3. http://www2.medizin.uni-greifswald.de/gani_med/ 4. Hertel et al. (2015) Measuring Biological Age via Metabonomics: The Metabolic Age Score, Journal of Proteome Research, 15:400-140, DOI: 10.1021/acs.jproteome.5b00561.

Matthias Nauck Matthias Nauck is a Professor at the Institute of Clinical Chemistry at University Medicine Greifswald (Universitätsmedizin Greifswald). His role is split between a focus on patient care and a range of scientific research projects. Prof. Nauck has played an integral role in introducing nuclear magnetic resonance (NMR) to the laboratory at Greifswald, for epidemiological research and biobanking studies. Email: matthias.nauck@uni-greifswald.de

Roland Leiminger Roland Leiminger is Executive Key Account Manager at Bruker BioSpin. Email: roland.leiminger@bruker.com

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Research / Innovation / Development

PEER REVIEWED

Alzheimer’s Disease: The Way Forward

Alzheimer’s disease (AD) ranks as one of the toughest nuts to crack within drug discovery and development. Current treatments merely manage symptoms, so finding a better solution becomes more and more urgent as the aging population grows. Approximately 70 per cent of dementia cases are caused by AD. It is a neurodegenerative disorder – neurons progressively lose structure and function. As the disease continues and more neurons are damaged and die, symptoms get worse. Neurons in the hippocampal region of the brain associated with memory formation are among the first affected. By 2025, the number of people age 65 and older with AD is projected to reach 7.1 million – a 40 per cent increase from the 5.1 million affected in 2015 (Alzheimer’s Association). A number of different companies are working to develop treatments, with several already in clinical trials. These drug candidates include a few that target the familiar amyloid-beta (Aβ) plaques and tau tangles, and several others that represent an entirely new approach to the disease. With no clear winner in sight, all comers are welcome in the attempt to defeat this devastating disease. Plaques and Tangles Plaques Alzheimer’s disease is associated with the build-up of amyloidbeta (Aβ) plaques in patients’ brains. Aβ plaques derive from the cleavage of the amyloid precursor protein, which is thought to play a role in the formation of synapses. Individual Aβ molecules clump together to form the plaques associated with Alzheimer’s. Until recently, the mechanism by which Aβ plaques might cause Alzheimer’s was not known. Research  from Stanford University suggests the plaques bind to a receptor on nerve cells, disrupting their function1. However, there is no absolute consensus on whether these clumps of protein are the origins of AD or a symptom of the underlying cause. Tangles Another brain protein associated with Alzheimer’s is tau, which is concentrated in the neurons and is primarily understood as a component in stabilising nerve cell structure. Abnormal aggregates of tau form “tangles” within nerve cells. These tangles are correlated with the onset of Alzheimer’s along with the characteristic plaque formations.

• •

Variant ε3 (APOE3) is neutral Variant ε4 (APOE4) is associated with a significantly increased risk of Alzheimer’s

The APOE proteins plays a role in clearing Aβ from the brain, with APOE4 carrying out this function less efficiently than the other variants2. There is also some evidence that APOE4 contributes to the breakdown of the blood-brain barrier seen in patients3,4, resulting in increased brain inflammation – another marker of Alzheimer’s. A better understanding of APOE4’s role in Alzheimer’s onset may lead to the development of a whole new class of drug. If At First You Don’t Succeed… A number of drug developers have attempted to use monoclonal antibodies  (mAbs) to disrupt the formation of the AD-associated Aβ plaques. Unfortunately, this approach has yet to experience clinical success. However, this doesn’t mean that the approach is not viable – different mAbs attach to different parts of Aβ, meaning that the outcome of one mAb trial does not necessarily predict the outcome of another. Biogen (Cambridge, MA) has an mAb therapy targeting AB plaques in Phase III clinical development. In earlier phase studies, this mAb showed the most promise in patients with less advanced forms of the disease. The prevention of Aβ plaques is also the focus of growing interest in creating an AD vaccine. Leading this effort is Novartis (Basel, Switzerland), whose CAD106 vaccine contains fragments of the Aβ protein and has been shown to be safe in Phase I trials. The goal of the vaccine is to activate an immune response against Aβ, thereby reducing its accumulation and potential to form plaques in the brain. CAD106 is currently in Phase II/III studies of efficacy, in which the vaccine is being tested in cognitively normal individuals between the ages of 60 and 70 who are at high risk of developing the disease based on their APOE4 status. Untangling Tau AbbVie (North Chicago, IL) is targeting tau, the other major protein associated with AD. The company currently has an anti-tau mAb therapy in Phase II clinical testing, and has also announced a partnership with Voyager Therapeutics (Cambridge, MA) to develop a gene therapy treatment that targets tau. The treatment will deliver a gene encoding anti-tau antibody directly to cells in patients’ brains – enabling those cells to make the antibody. If successful, this would bypass altogether the blood-brain barrier that makes it difficult for some drugs to even enter the brain.

Genetics Plays a Role About 70% of Alzheimer’s cases are thought to have at least some genetic association, with different genes being implicated depending on the type of Alzheimer’s. A gene found on chromosome 19 called the apolipoprotein E gene (APOE) influences the development of late-onset Alzheimer’s. Individuals with different variants of the APOE gene have different risk profiles: •

Variant ε2 (APOE2) is rare and possibly lessens or delays Alzheimer’s onset

14 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

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Research / Innovation / Development

Amyloid-beta and tau remain tantalising targets for AD drug development because of their close association with the disease. However, new approaches are springing up as frustration over the lack of progress in treating this devastating disease grows.   Reviving the Brain? L  oss of neurons is Alzheimer’s signature, devastating effect. What if we could jump-start the development of new brain cells? Two companies are trying to do just that. Neuronascent  (Clarksville, MD) aims to develop small molecule activators of neurogenesis. By screening large chemical libraries, the company has identified compounds that show promise of sparking neurogenesis from adult neural stem cells in both tissue culture and mouse models. The company’s lead compound, NNI-362, promoted the growth of new hippocampal neurons in mice. The new cells migrated to the correct location and differentiated. Moreover, they survived long enough to reverse previously observed cognitive declines. The hippocampus is one of the first regions of the brain to show damage in AD and is thought to play a role in memory formation and spatial navigation. Neuronascent is preparing for Phase I trials of NNI-362. Neurotrope  Biosciences (  New York, NY) is developing bryostatin, a drug that activates protein kinase C epsilon (PKCꞓ). This protein plays a key role in forming memories. In animal models of stroke, traumatic brain injury, and Alzheimer’s disease, bryostatin appears to restore deficits in synapses (connections between brain cells) and decrease cell death. These results suggest that bryostatin could help to prevent the loss of neurons and restore synapses. Phase II clinical studies of late-stage Alzheimer’s patients demonstrated improved cognitive function as measured by the Severe Impairment Battery Scale (SIB), a standard tool for 16 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

evaluating treatment response in advanced Alzheimer’s. Their improvement was greater than that seen in patients given the placebo, but the difference was not statistically significant. A Neurotrope Biosciences spokesperson says that it considers the Phase II study exploratory, designed to determine correct dosing. The company is planning a larger confirmatory trial in hopes of demonstrating statistically significant efficacy. Neuroinflammation N euroinflammation is one of the drivers of neurodegeneration in Alzheimer’s disease, multiple sclerosis and other brain disorders. Research  suggests that the protein c1q is present at higher levels in people with Alzheimer’s disease5. C1q accumulates at neuronal synapses, the key points of communication between brain cells. This protein also signals other immune cells, such as macrophages – which then chomp up cellular debris present in affected brains. The accumulation of c1q could account for the loss of synapses and accompanying mental decline. South San Francisco-based  Annexon  is working on a promising therapy that centers on controlling inflammation in the brain. ANX005, now in preclinical development, is a monoclonal antibody that mops up excess c1q. Immunotherapy Immunotherapy – activating the patient’s immune system to fight disease – has taken the oncology world by storm. South San Francisco-based Alector is trying this approach on Alzheimer’s. Their lead Alzheimer’s candidate, ALOO2, targets and activates the TREM2 receptor, present on immune cells active in the brain. The idea is to activate those immune cells to clear out amyloidbeta and other potentially damaging proteins. The TREM2 target was identified based on genetic studies which showed that patients with a dysfunctional TREM2 gene had a significantly higher risk of developing AD, while those whose mutations led to an overexpression of the receptor had a lower risk. AL002 has begun Phase I clinical testing. Spring 2019 Volume 2 Issue 1


Research / Innovation / Development

Searching for a New Mechanism Rather than target Aβ plaques directly, Yumanity Therapeutics (Cambridge, MA) is trying to identify the problems they cause. Yumanity scientists have engineered yeast cells to overproduce the Aβ protein and monitor its detrimental effects, such as disrupting the action of other important cellular proteins. Because yeast share many molecular pathways with humans, researchers can use them to screen for potential drugs that address protein disruption. Promising candidates are then tested in Alzheimer’s patient-derived cells. By tackling a completely different disease mechanism, the new compounds may achieve greater success than seen so far with drugs that act directly on amyloid beta or tau. Yumanity is currently in the lead-optimisation phase of pre-clinical development. In partnership with  Biogen  (Cambridge, MA), Cambridgebased Proteostasis   Therapeutics  is targeting AD-associated protein aggregates by activating proteasomes. These cellular components get rid of damaged proteins and dysfunctional protein aggregates by dismantling their chemical bonds. The protein USP14 inhibits proteasomes. Proteostasis is working on the preclinical development of a USP14 inhibitor that allows proteasomes to fully activate in AD patients. This makes it more likely that the proteasomes will recognise and destroy amyloid plaques and tau tangles. Oryzon Genomics  (Barcelona, Spain) is taking an epigenetic approach to Alzheimer’s. Epigenetic modifications are chemical changes to gene sequences that don’t change the information content but instead affect how much that content is used – in other words, the amount of a particular protein that the body makes. Oryzon researchers identified an enzyme, lysinespecific histone demethylase 1 (LSD1), which makes epigenetic modifications to genes that results in “turning them down” so they produce less of the corresponding protein. LSD1 makes these changes to genes that support neuronal survival. Oryzon scientists have designed a drug, ORY-2001, that inhibits LSD1. Inhibiting LSD1 could mean that more neurons survive in AD patients, leading to improved cognitive function. ORY-2001 recently entered Phase II clinical trials. A Twist on  Tau Finally, the elusive AD treatment may lie in pursuing a wellestablished target after all – but at a new angle. That’s where  Ionis Pharmaceuticals  (Carlsbad, CA) is headed in the Phase I/IIA  clinical studies of their drug, IONIS-MAPT. This antisense drug targets the source of the tangles associated with AD. Like other antisense drugs, IONIS-MAPT destroys tau mRNA, thereby diminishing tau protein production. It’s encouraging to know how many therapies are in the Alzheimer’s treatment pipeline. With more hard work and investment, perhaps one of the many www.biopharmaceuticalmedia.com

introduced above will lead to a cure – or perhaps the winning therapy may include a combination of these approaches. The world awaits a winner in this all-important race. UPDATE: On Thursday, March 21st, Biogen announced that they are discontinuing Phase III testing of their mAb therapy targeting Aβ plaques after an independent data monitoring committee advised it was unlikely to meet primary endpoints. Reference: http://investors.biogen.com/news-releases/ news-release-details/biogen-and-eisai-discontinue-phase-3engage-and-emerge-trials REFERENCES 1.

2.

3.

4.

5.

6.

Kim T et al. (2013). Human LilrB2 Is a Beta-Amyloid Receptor and Its Murine Homolog PirB Regulates Synaptic Plasticity in an Alzheimer’s Model. Science 341:1399-1404. Wildsmith et al. (2013). Evidence for impaired amyloid β clearance in Alzheimer's disease. Alzheimer's Research & Therapy 5:33. Nishitsuji K et al. (2011). Apolipoprotein E regulates the integrity of tight junctions in an isoform-dependent manner in an in vitro blood-brain barrier model. J Biol Chem. 286(20):17536-42. Hafezi-Moghadam A et al. (2007). ApoE deficiency leads to a progressive age-dependent blood-brain barrier leakage. American Journal of Physiology, Cell Physiology 292(4): C1256-C1262. Hong S et al. (2016). Complement and microglia mediate early synapse loss in Alzheimer mouse models. Science 352(6286):712-716. Jonsson T et al. (2013). Variant of TREM2 Associated with the Risk of Alzheimer's Disease. N Engl J Med 368:107-116.

Emily Burke Dr. Burke is the Director of Curriculum for Biotech Primer, where she develops customized training for individuals and companies seeking a better understanding of the biotech industry and writes the Biotech Primer WEEKLY newsletter, a publication that explains the science behind the latest biotech innovations. Dr. Burke delivers training classes nationally and internationally, and has been an invited speaker at many events including the annual BIO International Convention.

Email: burke@biotechprimerinc.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 17


Research / Innovation / Development

PEER REVIEWED

Artificial Intelligence Expands the Destiny of DNA Analysis DNA is the code that has defined humanity for millennia. Until more recently, DNA has determined destiny. There was no escaping its design and code for individuals. It prescribes how a person looks, why they desire certain types of food and drink. It was the code that says how long they will live and what they will likely die from, at least in terms of propensity and predisposition towards certain conditions, from heart disease to diabetes to colon cancer. But there were also the “ghost in the machine” outcomes that didn’t make sense. Why identical twins could have completely different desires and personalities. If DNA were the primary factor, where did nurture come into play? Some like to refer to the “nurture” portion as epigenetic or above the genome – how genes are read, cut, folded and expressed. It incorporates the interplay between a person’s genes and their environment. Many in the medical genetics industry struggled for years trying to find a way to manually map the processes, to no avail. Then came a new type of machine learning that, in the past two years, has rapidly increased advancements in artificial intelligence (AI) learning of epigenetic interplay. The true epigenetic correlations impact the destiny of DNA and how it relates to individual health and wellbeing to emerge in ways never before imagined. AI and Precision Medicine One of the major challenges impacting effective patient outcomes has been the complex interaction between genes, drugs, food, and environment. Symptoms which may be affecting the patient can come from myriad potential causes, and cross multitudes of biochemical processes. With the use of AI in precision medicine, science can bridge the divide between complex interactions. What used to be a “trial and error” method, or what doctors refer to as the “art” of medicine, is becoming a precise and beautiful combination of understanding the patient as a whole. In the very near future, physicians may no longer need to wait for symptoms to present before interventions that treat the root cause are provided. This is an important approach within medical genomics. For example, there may be a patient who has a variant that is associated with an average of 32 per cent increase in aortic aneurism. Why 32 per cent for this individual? And where did that number come from? What would a doctor do for that patient? Without understanding how that number came to be and what other factors impact it directly, it becomes a nearly impossible task of prevention when it comes to clinical practice. Another example lies with the common variant in the MTHFR and or MTRR gene, which has been shown to correlate with symptoms such as over-sleeping, depression and lack of energy. However, not everyone with a mutation on MTHFR will have all of these symptoms and even if they do, correct methylation patterns with current treatment methods are not highly accurate. However, with precision medicine, the doctor can get to the root genetic cause and treat accordingly. They may need an increase of methylcobalamin version of B12, or it is possible that their body is struggling with methylation and they need increased 18 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

5-methyltetrahydrafolate supplementation. Or they may have other issues that need S-adenosyl methionine or even potentially intervention with thyroid metabolism. In many cases, doctors would treat the patient with anti-depressants but that may not correct the issue, when the body requires a methylated version of vitamin B12 or downstream methylation donors to get rid of the symptoms. Interestingly, these genes have interplay with many other variants across the genome and epigenetic patterns, so even with traditional MTHFR Sanger testing methods we were missing the whole story. Without knowing all of the parameters it is impossible for the doctor to make that kind of decision accurately. In previous generations, the healthcare community was focused on providing general care for the largest number of people. Doing the most good for the most people was the highest and noblest of causes. The problem with the traditional approach to drug trials, for instance, is that there has been a conceivably acceptable risk that a trial could cause adverse reactions or even death for three per cent of participants. This risk is arguably acceptable because for 97 per cent of trial study participants and eventual users, the drug would be safe and efficacious. Gathering generalised empirical evidence for the largest number of people was historically the best methodology and most practical approach based on the data and technology available to them. But with the advent of AI and an increasingly expansive knowledge of DNA gained on a daily basis, the risk of harming or even killing even three per cent of patients is unacceptable. AI technologies enable a change to the traditional approach, a change that is both fundamental and far-reaching. The EPMA Journal marks a shift in medicine by describing the paradigm change from the delayed interventional nature of traditional medicine to the concept of predictive, preventive and personalised medicine.1 The answer is precision medicine, using complete genetic and epigenetic analysis which allows medical professionals to specifically treat individuals with the drugs and treatment strategies that are specifically targeted to them. The National Institutes of Health (NIH) describes precision medicine as “An emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment and lifestyle for each person.”2 The NIH further states, “This approach will allow doctors and researchers to predict more accurately which treatment and prevention strategies for a particular disease will work in which groups of people. It is in contrast to a one-size-fits-all approach, in which disease treatment and prevention strategies are developed for the average person, with less consideration for the differences between individuals.” The evolution in approach is driven in part by the technical ability of medical professionals to leverage the predictive power of precision medicine and deliver targeted therapeutic interventions that not only promise more efficient treatment for patients and longer lives with improved quality, but also increases the role of personal responsibility for health, which is an understandable consequence of the shift from reactive disease-treatment oriented medicine towards the proactive approach of preventive medicine.3 Spring 2019 Volume 2 Issue 1


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Research / Innovation / Development

The key to this change is data. The practical ability to compile, analyse, cross-reference and coherently report on the data associated with the human genome is impossible without AI. The human genome contains approximately 3.2 billion base pairs, which reside in the 23 chromosomes. Each chromosome contains hundreds to many thousands of genes, which deliver the instructions for making proteins. Sequencing determines the exact order of the base pairs in a segment of DNA. This is no small feat. Human chromosomes range in size from about 50,000,000 to 300,000,000 base pairs.4 Sequencing typically produces roughly 3–17 gigabytes of txt data on a single human genome. AI is important for the analysis of human genome data because it then takes the world’s best genomics AI roughly 7,680,000,000 gigs of processing to analyse and cross-reference each data point against all other data points. This processing allows the AI to determine the true value of a single variant for the individual. Leveraging the Whole Genome When making medical recommendations about an individual’s body, a holistic perspective needs to be gained and every aspect of what makes up the person ideally should be taken into consideration. The whole genome needs to be sequenced and analysed. An analogy would be as if an astronomer were to take a single photograph of the night sky then attempt to explain the entire universe based on that snapshot. Most current solutions analyse less than 700,000 data points, generating less than 400 outcomes according to the International Society of Genetic Genealogy Wiki, such as ancestry, diet suggestions or identification of potential general health risks. Recent advancements in AI technology have dramatically increased the data points analysed to over 60 million, yielding more than 60 million outcomes.5 20 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

For example, a person may have a marker in the BRCA1 gene that denotes a risk for certain types of cancer. In looking at an incomplete genome, the fact that they have that risk factor may spur interventions that may or may not benefit the individual. A true analysis that includes the entire genome could show that the person has two or even three other markers that protect against that cancer and as such, other or no intervention is actually warranted. This is a rare case but it is still valid and should be considered to avoid unnecessary patient harm. The state of technology is such that there is still a great deal to learn regarding the human genome. There are more connections to understand; for instance, there is still much to learn about how epigenetics impacts the body well after birth and into adulthood. That said, AI can currently leverage the whole genome, and many of the interactions of the epigenome. When considering risk, the analysis has to take into account all known data and that requires participation by physicians, labs and patients. The industry will continue to add more data and connections. As this happens, the analysis will remain constant, but the recommendations will continue to improve over time. AI and Cannabis Questions One pressing application of AI-based DNA analysis technology applies to the rapidly growing global medical cannabis industry. The objective is to use DNA information to make medical cannabis use safer for the patient, inspire confidence in the prescribing physician, and create trust in the industry as a whole. Where medical cannabis is legal to prescribe, recommend or use, medical professionals are often left questioning how to best help their patients. The problem is, when compared to other interventions, there is a dearth of research and data regarding safety and efficacy of medical cannabis. As such, they are forced to take a trial-and-error approach or simply guess. Without data, Spring 2019 Volume 2 Issue 1


Research / Innovation / Development precisionmedicine/definition 3. Gefenas E, Cekanauskaite A, Tuzaite E, Dranseika V, Characiejus D. Does the "new philosophy" in predictive, preventive and personalised medicine require new ethics?. EPMA J. 2011;2(2):1417. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3405379/ 4. NIH: National Human Genome Research Institute. The Human Genome Project Completion: Frequently Asked Questions. https:// www.genome.gov/11006943/human-genome-project-completionfrequently-asked-questions/ 5. International Society of Genetic Genealogy. 23andMe https://isogg. org/wiki/23andMe

it is very difficult to prescribe it, dose it or use it with precision for medical applications. The reality is it can be an incredible intervention if executed correctly. When AI is leveraged to analyse the entire human genome and identify all known genetic markers, questions are turned into answers. With this information, a unique genetic profile can be created for each person with a customised recommendations report. This technology is particularly beneficial to the medical cannabis market due to the fundamentally varied manner in which medical cannabis has reached the market on account of patient demand, and not through sanctioned regulatory bodies and standards such as the FDA. Because medical cannabis is a fast-growing market with limited regulations, there are many “boutique or craft” cultivars who are growing strains and making unregulated medical claims with little to no research. While the industry is operating as if it exists within the “Wild West” it is creating a dark shadow on the true medical efficacy of the product. AI and genetic analysis of both the plant and the patient will allow for precise cultivar standards to guarantee efficacy of the plant while giving the prescribing physician confidence in the medication that he or she is prescribing. This will provide physicians much greater insight and decision-making perspective than what is currently available. The application of AI can also be used to help medical professionals determine the best or most appropriate plant genetics and dose to use with patients based on the patient’s genetic information. AI technology has the potential to greatly impact how medical cannabis is used, prescribed and recommended for medicinal purposes. In other words, where it is legal, medical professionals can know precisely how to move from recommendations to a true prescription of the correct dose (smoke, vape, edible, sublingual) of the correct compounds (CBD, THC, THCV, CBN, etc.) for the correct condition for the patient. The objective of applying AI to the genetics of cannabis and their impact on the human body is to enable a safer, faster and more targeted approach to prescribing medical cannabis. Genetic health data will allow doctors to feel more confident regarding the cannabis products they are prescribing. It will also allow the patient to trust that they are consuming a product that comes from a reputable source and will be safe for their body. REFERENCES 1. Costigliola V. Preface. EPMA J. 2010;1:1–2. doi: 10.1007/s13167010-0013-6 2. NIH. What is Precision Medicine? https://ghr.nlm.nih.gov/primer/ www.biopharmaceuticalmedia.com

Ian Jenkins Ian Jenkins is CEO of Frelii, Inc., which provides state-of-the-art DNA sequencing and analysis of the whole genome with powerful artificial intelligence (AI). Its unique technology and approach enables significant advancements in the areas of diagnostics, healthcare, pharmacology, telehealth, insurance and even personal health and wellness. Web: www.frelii.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 21


Clinical Research

PEER REVIEWED

Diagnosis Still Poses the Biggest Challenge in the AIDS Battle Significant progress has been made in the AIDS response since 1988; however, ensuring a swift and accurate diagnosis is accessible to everyone is still a significant barrier which needs to be tackled in our battle to combat the disease. According to the United Nations, only six countries have met the UN’s “90-90-90” goal (90% of all people with HIV know they are infected, with 90% of those taking sustained antiretroviral therapy, and 90% of those achieving suppression of the virus). Those countries are Botswana, Cambodia, Denmark, Iceland, Singapore, Sweden and Britain. The aim is that the world will achieve those marks by 2020 – but there is a long way to go and ensuring an accurate and rapid diagnosis is a crucial first step on the journey. Around the world, 37 million people are living with HIV, the highest number ever, yet a quarter do not know that they have the virus. However, since 2001, when the UN General Assembly hosted its first meeting on HIV/AIDS, the trajectory of the disease has shifted dramatically. Today, the UN estimates that some 22 million people living with HIV are receiving treatment.

Although there remains much to do, huge strides have been made in the battle against the disease and as a result, annual AIDS deaths have fallen by half, from 1.9 million in 2003 to 940,000 in 2017, while the rate of new infection has decreased by nearly half in several of the hardest-hit countries according to WHO partner organisation UNITAIDS. Central to this success has been the continual innovation of treatment and diagnostics to meet the changing needs of communities and an ongoing commit-ment to shape care and treatment to the specific requirements and preferences and requirements of community groups – particularly those in hard-to-reach areas. Such novel and targeted models of care and diagnosis have helped to relieve the burden of large numbers of patients on health facilities and health workers and begun to ensure a more comprehensive response to communities affected. For example, in several countries, stable patients who prefer to visit their healthcare provider less often receive multi-month 22 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

supplies of medications. Other innovations include prescriptions which can be refilled at pharmacist-managed vending machines, and at-home HIV testing for people who live in remote areas, while for those found to have HIV infection, community treatment is supported by local health workers in many countries. Know Your Status Knowing your HIV status is an essential entry point to HIV treatment, prevention, care and support services. This knowledge is also a vital tool in enabling people to make informed decisions about HIV prevention options, including services to prevent children from becoming infected with HIV, male and female condoms, harm reduction services for people who inject drugs, voluntary medical male circumcision and pre-exposure and post-exposure prophylaxis. Early diagnosis is particularly critical for children who might have been exposed to the virus; it is commonly accepted that without treatment, half of all children born with HIV will die by the age of two and the majority will die by the time they are five years old. According to the WHO’s UNITAID, in 2013, only 42 per cent of children exposed to HIV were tested for the virus within the recommended first two months.

Current early infant diagnosis testing requires complex laboratory technology that is often only available at central laboratories with weeks to months for return of results. Without knowing the HIV status of a child, it is impossible to provide life-saving treatment and with over 90 per cent of paediatric HIV infection occurring during pregnancy, delivery or breastfeeding, known as mother-to-child-transmission; prompt identification and treatment of infants who are infected is crucial. However, due to the persistence of maternal antibodies in infants under 18 months, the use of antibody tests such as HIV rapid disposable tests cannot be used to accurately screen infants for the virus. Instead, WHO guidelines recommend DNA or RNA testing as the best option for determining the HIV status of young infants and strongly recommend that all HIV-exposed infants have HIV virological testing at four to six weeks of age or at the earliest opportunity. A New Generation of POC Tests Recognising the complex nature of human disease, overlapping symptoms and states of co-infections, there is increasing demand for multiplexed molecular systems that can detect multiple Spring 2019 Volume 2 Issue 1


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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 23


Clinical Research biomarkers simultaneously with improved sensitivity/specificity at the point of care (POC). Polymerase chain reaction (PCR) is considered one of the gold standard molecular diagnostic tests due to the high accuracy of the technology, which accounts for its use in central and reference laboratories.

the ease of use associated with current lateral flow technology. For end users of POC assays, this provides the convenience and familiarity of traditional POC lateral flow assays combined with the superior sensitivity and specificity of laboratory-based PCR testing.

The most widely used test for early infant diagnosis has been the DNA PCR molecular test, which is a qualitative nucleic acid test for the presence of pro-viral HIV. PCR tests can detect disease with as few as several copies of a gene – so is ideal for instances of mother-to-child-transmission. However, the laboratory-based nucleic acid tests typically require relatively sophisticated instrumentation and a trained laboratory technician.

Developing new, improved diagnostics is an important step in the journey to tackle HIV. In order to improve the accessibility of high-quality antiretroviral therapy, there is a growing demand for simple, affordable, reliable and quality-assured point-ofcare (POC) diagnostics for use in resource-limited settings. With improved POC diagnostics, antiretroviral therapy will become more scalable and will allow anti-retroviral therapy service delivery to be significantly decentralised to the community level.

DNA PCR tests are used primarily for early infant diagnosis for specimens obtained from prevention of mother-to-child transmission centres, clinics and the like. The infant’s blood is collected on dried blood spot (DBS) filter paper, which is transferred via couriers to labs for testing; test results are then returned to the clinic or other collection site for dissemination to caregivers. This process can be extremely slow – especially the return of results from laboratories to collection sites – creating delays in providing children with accurate and much-needed urgent treatment. Due to complicated workflows, complex procedures, a requirement for cold chain storage, and time and economic costs, PCR  hasn’t been truly feasible in a POC setting. However, recent advances for DBS specimens and POC platforms are enabling the potential to introduce molecular diagnostics in more decentralised settings. Although isothermal platforms have entered the POC market for use in clinicians’ offices and clinics, they tend to require an expensive instrument and cold storage for reagents – creating cost and logistical barriers which limit a widespread roll-out across public health systems.

Although no point-of-care PCR tests for infants currently exist, as technology evolves we expect to see rapid, point-ofcare PCR tests which could be used to test infants coming to market eventually – which could significantly boost the numbers of young children who can be accurately tested for HIV at the point of care. Encouragingly, advances in technology may reduce the cost of diagnosing and monitoring treatment of people living with HIV without diminishing the quality of care. In order to understand the benefits that POC diagnostics may offer, it is necessary to understand the current diagnostic technology landscape. Overcoming Barriers Access to diagnostics testing is crucial in facilitating early detection and treatment of HIV. Accurate detection, staging and monitoring will maximise the preventive impact of ART, and help to ensure an appropriate and rapid response to drug resistance – a problem likely to grow substantially over the coming years. However, alongside the physical barriers, societal factors also play a significant role in preventing people from securing an accurate diagnosis and starting the journey to treatment. With many people only getting tested after developing symptoms of AIDS, tackling the barriers presented by societal stigma is key to ensuring systematic, presymptomatic testing on a mass scale to prevent the spread of the disease. In many countries, discrimination is a huge deterrent for people considering taking an HIV test and access to confidential HIV testing is still a significant concern for people who fear being ostracised from their communities. New diagnostic approaches which make the process more secure and accessible can have a valuable role to play in helping to address these concerns and normalising presymptomatic diagnosis across communities.

However, the development of new PCR amplification methods, microfluidics and integration with lateral flow assay technology is enabling the development of new diagnostic platforms – which could have a substantial positive impact on the numbers of people able to access rapid, highly specific, sensitive diagnostics at the point of care.

As the United Nations has highlighted, HIV testing programmes must be expanded, which requires political will and investment, as well as novel and innovative approaches to HIV testing that are fully leveraged and taken to scale.

These platforms are looking to address some of the key challenges of bringing molecular assays to POC, by minimising the complexity and cost of the platform required to run the assays, removing the requirement for cold storage of the reagents and utilising lateral flow assays as the visualisation method for the assay result. The current generation of new PCR tests coming to market are able to offer outstanding sensitivity combined with simplified workflows, room-temperature stable reagents, lower costs, and 24 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Spring 2019 Volume 2 Issue 1


Clinical Research

Continued growth and progress are needed and developing innovative diagnostics to create an effective first line of defence against HIV is crucial. Investment in diagnostics to reach resourcepoor areas in the point-of-care setting is vital, both in developing countries and across often-overlooked communities in developed countries. Diagnostics need to be accessible, inexpensive and consumer-friendly. Lateral flow tests offer a cost-effective and easy-to-use solution. However, targeting HIV effectively is challenging and requires consistent innovation. Unlike many viral infections, HIV patients often seek diagnosis long before the onset of symptoms, compromising the typical viral diagnostic method of measuring the body’s immune response in a serology capture assay. To meet that challenge, currently available fourth- and fifth-generation tests combine serology approaches for HIV-1 and HIV-2 with an antigen capture of the p24 capsid protein of HIV. A new generation of p24 antibodies are being developed that have been tested in lateral flow and offer sensitivity that provides people with a post-event diagnosis, generally within days of exposure. Enhancing the sensitivity of existing rapid lateral flow HIV tests and improving the limit of detection in these tests is also helping to ensure that suspected sufferers can be even more rapidly and accurately identified. Thus optimised, lateral flow tests can play an even more effective role in the battle to diagnose and combat the spread of infectious diseases. Technological Advances Driving Positive Change Other areas of technological innovation are also gaining increasing importance as we move forward in the battle to identify and tackle AIDS. Mhealth, particularly mobile enabled point-of-care testing (POCT), is playing a key role in enabling an early and accurate diagnosis at the point of care. From breakthrough schemes like South Africa’s Project Masiluleke, which used mobile phones to send millions of text messages to people to increase HIV/AIDS awareness, testing and treatment – to in-field care workers and clinicians using mobile phones to trace HIV positive patients; technology, particularly mobile, has been a key weapon in the battle against AIDS. www.biopharmaceuticalmedia.com

Mobile enabled point-of-care technology is particularly valuable in hard-to-reach, rural communities – but it also has an important role in enabling testing where the social stigma of HIV might prevent people going to clinics to get tested. It offers a valuable tool, which could be used in the privacy of the home – giving people the ability to rapidly and accurately self-test, with the results shared securely and privately with healthcare professionals online. These diagnostic advances will have a transformative effect on people’s ability to access and secure a rapid, accurate diagnosis, particularly in rural or isolated communities. This access to early detection is an important step in determining the right care pathway for people who test positive for HIV. Enabling mass access to swift, accurate, presymptomatic diagnosis requires collaboration and a deep-seated commitment to removing the stigma around HIV and AIDS – thus ensuring people are equipped with the tools they need to know their status and move forward accordingly.

David Fraser David Fraser, MBA BSc (Hons) Lateral Flow Product Manager, BBI Solutions. With 14 years’ experience in the development and manufacture of diagnostics in the clinical and environmental markets, David has worked within the BBI Group for over nine years in a variety of technical roles. His current role is Product Manager for BBI Solutions’ Lateral Flow Services and Detection product portfolios. BBI Solutions offers a total solution to its customer base, providing services for teams in early R&D phase, from antibody development and all the way through to diagnosis.

Email: davidfraser@bbisolutions.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 25


Clinical Research

PEER REVIEWED

Clinical and Investigator-initiated Trials’ Effect on MSL Responsibilities Among the many responsibilities assigned to medical science liaisons (MSLs) is facilitating company collaboration with thought leaders on clinical trial work. According to MSL teams surveyed by Cutting Edge Information, seemingly little time and ostensibly minor goals are assigned to this activity. But when considering the many tasks MSLs already perform and their headcount per region, even a minor focus on facilitating investigatorinitiated trials (IITs) and other study types may be more than enough to meet company goals. Thought Leaders and Clinical Trials MSLs help disseminate scientific information and act as a link between a life science company and various external stakeholders. These stakeholders are thought leaders and other healthcare providers (HCPs) of varying degrees of influence. Interactions between MSLs and thought leaders usually involve discussion of the effectiveness and patient usage of products. Communication can progress beyond branded products, covering a range of scientific topics and, in some cases, how the thought leader and company might work together on research.

up 7.6% of pre-launch time and 7.9% of post-launch time. Not shown are the two activities that receive the largest allocation of MSL time: travel and interacting with thought leaders.

Figure 1: Percentage of Pre-Launch Time Spent on MSL Activities, Pre-Launch

An MSL does not directly solicit thought leaders for clinical trial involvement. Although conversations between liaisons and HCPs can lead to clinical trial collaboration, MSLs normally wait for HCPs to ask about ongoing trials first. Most often, an HCP will bring up the topic of emerging clinical trial data, which can involve asking about off-label uses for marketed products or the timing of new drugs coming through the company pipeline. MSLs can usually answer these questions freely while keeping regulatory requirements about off-label communication in mind. Once started, these conversations may lead to a thought leader wanting to conduct their own clinical trial – in other words, an IIT. MSL Time Spent on Trial Activities MSLs can provide a wealth of knowledge concerning ongoing clinical research and serve as a key facilitator for IITs. Because of these abilities and the necessity of clinical trials in getting a drug to market, one might assume that MSL teams spend a significant amount of time on trial-related activities. But according to a 2019 Cutting Edge Information survey, that may not be the case. Figure 1 shows the average percentage of time MSLs spend performing certain pre-launch activities in 2019, and Figure 2 displays similar activities and the time spent on them after product launch. Yellow columns indicate activities that focus on clinical trials. The Cutting Edge Information survey tracks a number of other MSLs activities, but this selection provides a basic idea of how much time is spent working on trials in relation to other MSL tasks. Meetings with internal stakeholders takes up the greatest preand post-launch time percentage. The next most time-consuming activity shown is attendance at medical meetings, which makes 26 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Figure 2: Percentage of Post-Launch Time Spent on MSL Activities, Post-Launch

Activities related to clinical trials take up smaller percentages of time during both stages. Leading up to launch, evaluating trial sites and developing clinical trial protocols take up just 2.0% and 1.1% of an MSL’s time, respectively. IIT coordination doesn’t take up a huge portion of time either – under 4.0% in both stages1. MSLs allocate relatively little time to coordinating IITs, but according to one MSL manager interviewed for another Cutting Edge Information report, starting an IIT involves a number of steps. The MSL manager, who works in the EU for a Top 10 life sciences company, details the process: In the discussion or hospital meeting, clinicians could bring up some good ideas for local trials. What MSLs can do is bring this idea to the office and discuss it with Medical Affairs and Spring 2019 Volume 2 Issue 1


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Clinical Research the medical advisors in that particular therapeutic area. Then the MSL can support the clinician by helping to translate their idea into a concept sheet and submit it to the company expert commission to see if there is interest to support the trial. In some cases, they have to go to a global-level expert commission. But once they have their approval, they can go back to the clinician or investigator to tell them they can start the trial. While MSLs help in getting company approval, it is the clinician that is accountable for the design and protocol of the IIT2.

Overall, clinical trial activities take up small amounts of time before and after product launch. Some tasks, such as attending internal stakeholder meetings and interacting with thought leaders, take up considerably more time; others, such as delivering promotional presentations, take up less. On the surface, it might seem that MSL teams could spend more hours supporting and facilitating clinical trials via ongoing thought leader engagement. But this is likely not the case when considering MSL performance metrics associated with clinical trials and average MSL headcount in a given region.

The manager’s description shows that IIT coordination aligns with but also adds to time spent on other activities. For example, meeting with internal stakeholders is essentially what an MSL does when they bring in a concept for an IIT. And although they are considered separate processes in the Cutting Edge Information report, evaluating trial sites and developing trial protocol may also include discussions with investigators who are sponsoring their own studies.

Comparing MSL Target Metrics with Average Headcount Tracking MSL team performance helps teams prove the value of their operations to upper management and the wider organisation. It also allows them to handle challenges in a more calculated way. An MSL’s performance can be tracked with a wide variety of different indicators, but most teams focus on just a handful of metrics. These can include the number of publications facilitated, the number of scientific speeches that thought leaders give, or the number of new opinion leader relationships created. But two specific metrics give an indication of an MSL’s performance in relation to clinical trials: the number of interactions with opinion leaders and the number of IIT proposals submitted via MSLs. Figure 3 displays these two monthly target metrics for MSLs during pre-launch and post-launch stages. These metrics come from MSL groups of varying company sizes (pharma top 50 and non-top 50) as well as company types (pharma and biotech). There is a large gap between the number of target interactions with HCPs per month and the number of IITs submitted per month. That said, both metrics help to facilitate successful clinical trial collaboration. Unsurprisingly, the number of IIT submissions an MSL can bring in correlates with the number of trials a company will likely take on1. On the other hand, more interactions with thought leaders may also promote a greater number of IITs, albeit indirectly. It’s not always the case that MSLs will discuss clinical trials with thought leaders; the type and subject of interactions vary widely. But when the number of target IIT submissions via MSLs per month is correlated to the number of interactions per month, survey findings suggest that some portion of interactions will end up pushing forward discussion of clinical trial involvement.

Developing protocol and evaluating trial sites make up between 1% to 2% of MSL pre-launch time. Evaluating trial sites is somewhat self-explanatory: MSLs go out in the field to various locations and assess their feasibility as clinical trial sites. Conversely, developing protocol is a primarily internal activity in which liaisons meet with medical affairs and clinical development representatives to come up with procedures for upcoming clinical trials. MSLs use their experience interacting with thought leaders to bring doctors’ perspectives to protocol meetings1. Coordinating Phase IV research and evaluating trial sites take up similar amounts of time. Much like protocol development, this task involves meeting with internal stakeholders to discuss Phase IV trial design and execution1. Still, MSLs perform other activities related to supporting clinical trials that are not shown in the graphs. In some cases, MSLs assist clinical development departments by smoothing out issues on a site level. During the course of a trial, MSLs may travel to sites that encounter problems and determine whether they can provide an immediate solution. This situation primarily applies to scientific issues. For example, investigators may have questions about an experimental drug’s formula or mechanism of action that aren’t easily answered over the phone or through email. If an MSL cannot provide a solution, they forward the query to the clinical operations department or the contract research organisation (CRO) in charge of the trial. Other tasks displayed take up even less time in pre- and post-launch phases. Interacting with patient advocacy groups and delivering promotional presentations each take up less than one per cent of MSL time during pre-launch; they only change by .1 percentage points after launch. Although not shown in the graphs, many other tasks take up less than a whole per cent of time. This list includes contributing to health economics analyses and product label development1. 28 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Figure 3: Average Monthly Target per MSL Performance Metric, Pre-launch and Post-launch Spring 2019 Volume 2 Issue 1


Clinical Research Regardless of where a product is in development, MSLs aims to submit an average of 1.1 IIT proposals per month. This seemingly low number lines up with Figures 1 and 2, which confirm that time allocated to IIT coordination is low. However, looking only at these measurements doesn’t give an accurate picture of how many IIT submissions a company or its MSL teams receive. Figure 4 shows the target number of IIT submissions an EU/Canada MSL team receives monthly.

Figure 4: Monthly Target Number of IITs Received by an MSL Team: EU/Canada

According to the graph, the average MSL team operating in the EU/Canada has 12 members and each member aims to submit 0.58 IIT proposals per month. That means the MSL team should receive up to 6.9 IIT submissions per month and up to 84 IIT proposal submissions in a year. IIT submissions in the US are even greater on average given increased MSL headcount and similar monthly targets. With that in mind, it’s clear that target goals associated with IITs are robust and can make a significant impact on future clinical work1. Conclusion At a glance, MSLs and their teams don’t place a great emphasis on supporting clinical trials and facilitating IITs. The amount of time MSLs allocate to these activities is relatively small when compared to a number of their other activities. It should be noted, however, that trial activities do beat out a significant number of other activities in terms of the time allocated to them. Trial-related activities have between 1% and 4% of MSL time allocated to them; by comparison, other activities, such as promotional speaking, have less than one per cent of time dedicated to them. Performance indicators associated with MSL activity also may appear to de-emphasise clinical trials as a priority among activities. Indeed, the target number of IIT proposals submitted via MSLs looks very low compared to the target number of opinion leader interactions. But when analysed in the context of average MSL headcount for a particular region, the number of IIT proposals a single MSL team can receive is quite high – up to 84 in a year for surveyed EU/Canada teams. Clinical trials, while critical, don’t require much MSL time or focus, according to Figures 1 through 3. However, the amount of time and level of activity allocated to trials may already be sufficient for a company’s clinical development 29 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

goals. MSL teams can obviously commit more time and focus to clinical trial-related activities, but that could take away from other core duties. To remain effective and ensure future drug development, companies must find a balance between MSLs’ clinical trial and non-clinical trial responsibilities. REFERENCES 1. Cutting Edge Information. Survey of Medical Science Liaison Management to be published in (2019) 2. Cutting Edge Information. Capture and Communicate the Full Value of Medical Science Liaisons. (2015)

Todd Middleton Todd Middleton is a research analyst at Cutting Edge Information, LLC. Located in Research Triangle Park, North Carolina, the company is a life science consulting firm specialising in fair market value rates and comprehensive qualitative analysis. Todd has been an employee at Cutting Edge Information for almost two years. He attended the College of Charleston, where he received a Bachelor of Science in international business administration. Email: todd_middleton@cuttingedgeinfo.com

Spring 2019 Volume 2 Issue 1


Manufacturing/Technology Platforms

PEER REVIEWED

Antibodies Exploration Thanks to Label-free Surface Plasmon Resonance Imaging Technology Surface plasmon resonance imaging allows molecules binding study in label-free and real-time conditions. The power of the technique comes from its singularities, which are multiplexing and imaging, leading to a considerable speed-up of the processes’ analyses. This will be illustrated for antibody molecule study by three applications. The first application aims to validate the sensitivity of the SPRi technology for the detection of small molecules. To do so, a monoclonal antibody highly specific to a steroid hormone (undisclosed) of 290 Da was analysed. This monoclonal antibody was developed at SynAbs using a new hybridoma technology for guinea pig mAb and was rescued and produced by Diaclone. In the antibody production processes, the final step is the characterisation of antibodies. Determination of immunoglobulin isotype is one part of the characterisation procedure. This second application demonstrates SPRi technique can be used to determine immunoglobulin isotype. Different rat monoclonal antibodies specific to different isotypes, produced by SynAbs, were immobilised on a functionalised SPRi-Biochip™. In this specific application, we highlight the screening capability of the SPRi technique, where different antibodies at different conditions can be analysed simultaneously and in a single biochip.

immobilisation improves the spot homogeneity and gives a higher immobilisation level. For this experiment, the flow rate of the SPRi-CFM was set to 15 µL/min and the contact time to 30 minutes. The printed SPRi-Biochips™ were then loaded into the XelPleX system. The interactions were monitored using EzSuite software. The running buffer was 10 mM PBS pH 7.4 and the working temperature was set to 25°C. Then, 200 µL of the studied molecules were injected into the fluidic system at a flow rate of 50 µL/min. A regeneration cycle was performed between each analyte injection by flowing either a 0.1 M glycine-HCl pH2.0 solution or a 10 mM NaOH solution with contact times between 30 seconds and four minutes.

Finally, the third application focuses on the experiment conditions optimisation. It shows how single-domain antibodies can be easily studied with SPRi technology. QVQ develops custom made single-domain antibodies from camelids (VHH), which can be directed towards specific epitopes and have similar high affinity and specificity as compared to conventional Abs. Surface Plasmon Resonance Imaging Platform (Figure 1) In each application, antibodies or single-domain antibodies were first immobilised on SPRi-Biochips™-activated surface using contact spotting (SPRi-Arrayer™) and flow printing (SPRi-CFM). The SPRi-Arrayer™ is an automatic and compact system for immobilising ligands in a multiplex format onto a SPRi-Biochip™ or a SPRi-slide™. This versatile instrument uses direct contact spotting and is suitable for printing on bare or 2D-functionalised SPRi-Biochips™ or SPRi-Slides™. Contact spotting allows fast and flexible microarray printings. The diameter of the printing pin can be adapted to the number of required spots in the matrix. Here, the diameter of the printing pin was 500 µm. The SPRi-CFM uses continuous flow deposition to immobilise up to 48 molecules in a single printing run. Three printing runs can be performed on a single biochip (and up to 144 spots per chip can be generated). The microfluidic 30 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Figure 1: Surface plasmon resonance imaging platform

Application 1: Steroid Hormone Detection A monoclonal antibody (mAb) developed at SynAbs by a new hybridoma technology for guinea pig mAb was rescued and produced by Diaclone. That monoclonal antibody binds specifically to a 290 Daltons steroid hormone (undisclosed). The hormone was injected at five increasing concentrations following a three-fold dilution series from 1.5 to 125 nM. The SPRi kinetic curves were analysed using the EzFit software to determine the kinetic constants and to calculate the affinity. This software is suitable for processing multiplexed data intuitively. The SPRi signal obtained on reference spots (i.e. Diaclone negative control antibody) were used for referencing. Then, the data were fitted locally (i.e. Rmax “maximum of reflectivity” different for each curve) using a 1:1 interaction model (see Figure 2; orange curves correspond to the fits). Spring 2019 Volume 2 Issue 1


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Manufacturing/Technology Platforms

Figure 2: Kinetic analysis of the antibody / hormone interactions (local fits using a 1:1 interaction model)

The limit of detection of the hormone by the guinea pig antibody was determined to be around 1.5 nM (~ 0.45 ng/mL). This limit of detection was obtained with a flow printing of the antibody on a 3D chemistry (dextran-based chemistry). It’s the same limit of detection as that of a conventional ELISA technique. ELISA is the standard technique used in the validation process of Diaclone. The kinetic curves profile showed mass transport limited kinetics. An affinity of 0.2 nM for the hormone / antibody interaction was calculated with this mass transport effect taken into account in the EzFit software by the addition of a km constant. The specificity of the antibody was also verified by flowing an analogue hormone at the same concentrations. No binding response was observed with the antibody of interest while injecting the analogue hormone. Thanks to the array-based format of the SPRi sensor chips, it is easy to extend these results to multiple interactions and to quickly integrate the XelPleX system into biomolecule production processes such as Diaclone’s monoclonal antibodies production process at the validation step. Application 2: Determination of Immunoglobulin Isotype Antibody isotyping consists in determining a monoclonal antibody class and subclass identity. Isotyping is a common basic test done in any immunological research and clinical diagnostics lab that requires antibody production. Determining the class and subclass identity of an antibody is a critical and valuable characteristic for functional activity, purification strategies, use in immunoassays and long-term stability. SPRi technology was tested to show how it can extend isotyping to multiple monoclonal antibodies produced.

Figure 3: Global overview of raw kinetic curves: one curve for one spot, after successive injections of the six mouse mAbs at 1 µg/mL and corresponding difference images below each mouse mAb injection. 32 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

The test consisted in an analysis of six different mouse mAbs from SynAbs immobilising six different rat anti-mouse isotype mAbs from SynAbs. Figure 3 shows a global overview of raw kinetic curves per spot obtained during the SPRi experiment after successive injections of the six mouse mAbs (MADNP-1, MADNP-2, MADNP-3, FLOPC-21, ABPC-105 and MADNP-5), each prepared at a concentration of 1 µg/mL. Regeneration steps (R) between each mouse mAb injection are not shown (light grey strips R identified). Each injection kinetic is associated to a corresponding difference image saved during the dissociation phase. As each rat anti-isotype mAb was immobilised at three different pHs and in replicates (three or six according to immobilisation system), 81 different curves in all are represented for each mouse mAb injection. Isotypes of mouse mAbs injected can be easily determined by looking at the raw kinetic curves of Figure 3 as well as the difference images. Difference images especially give a quick yes/no binding answer. Thus, if we go into more detail: MADNP-1 is an IgG1 isotype; MADNP-2 is an IgG2a isotype; MADNP-3 is an IgG2b isotype; FLOPC-21 is an IgG3 isotype; ABPC-105 is an IgA isotype; MADNP-5 is an IgM isotype. The mouse mAb concentration of injection was set at 1 µg/mL for the SPRi experiment as it is the minimal standard concentration used in isotyping kits. This concentration was completely suitable for a SPRi experiment. Indeed, isotype determination was greatly reliable for each mouse mAb tested. Exploiting at its maximum the wide printing surface of SPRi-Biochips™ and the multiplexing particularity of SPRi systems, one can easily imagine an isotyping analysis at a higher throughput level. Application 3: Single-domain Antibody Study Single-domain antibodies from camelids (sdAbs, VHH also referred to as Nanobodies®) are considered as the “third generation” of antibodies after conventional monoclonal antibodies (mAbs) and antibody fragments (Fab and scFv)1. They consist of single monomeric variable antibody domains. Because of their small size, they differentiate with several advantages of interest in medicine (enhanced tissue penetration, rapid clearance) and in biotechnology such as biosensors areas (higher immobilisation potential and enhanced detection sensitivity, superior stability)2. Q17c, a commercial sdAb from QVQ, recognises specifically HER2 recombinant protein, one of the epidermal growth factor receptors expressed in breast cancer. Q17c protein was immobilised under nine different conditions, i.e. using the two different immobilisation systems, prepared at three different pH levels (4.0, 5.0 and 7.4) and at six different concentrations (167, 333 and 667 nM with fluidic printing and 1.75, 3.5 and 7 µM with contact spotting) on a single biochip. The large working area of the SPRi-Biochip™ and the multiplexing capabilities of SPRi systems allow for the Spring 2019 Volume 2 Issue 1


Manufacturing/Technology Platforms

Figure 4: Specific responses retained for Q17c protein immobilised using two different spotting systems, at three different pH levels and at six different concentrations after the injections of HER2 protein at 2, 6, 17 and 52 nM.

immobilisation of different molecules and/or testing of different immobilisation conditions on a single biochip.

REFERENCES 1.

Specific HER2 protein binding responses retained on the spots of Q17c are represented in Figure 4. HER2 protein was injected at four different concentrations following a three-fold dilution series: 2, 6, 17 and 52 nM. For each injected concentration, the specific binding responses were measured during the dissociation phase of HER2 protein injections at the same time point. Values are reference-subtracted and spot-averaged. Specific binding responses were observed while injecting HER2 protein at different concentrations for Q17c immobilised at: • •

7 µM, in 10 mM PBS pH7.4 using contact spotting and pH4.0, whatever the concentration, using flow printing.

No binding was observed for Q17c immobilised in the other conditions. Optimal binding responses of HER2 protein were obtained for Q17c immobilised at 7 µM in 10 mM PBS pH7.4 using contact spotting system. In this study, similar specificity and affinity as conventional antibodies were also observed for single-domain antibodies. We demonstrated here how optimisation of immobilisation conditions can be fast. These optimal conditions can then be applied to design a chip with multiple different VHH molecules. Indeed, VHH molecules have key advantages that perfectly fit to biosensors to which SPRi technology belongs. Conclusion Antibody production companies are always looking for new technologies that allow them to accelerate the validation of their production and processes. They are looking for a solution that has the fewest steps, a solution that uses the least volume of reagents and a solution that provides a real-time response. Antibodies are commonly studied through the gold standard technology. By experimenting with the surface plasmon resonance imaging technology via three applications presented in this article, we highlight the key advantages of this platform: sensitivity for the detection of small molecules, screening potential and rapid experiment optimisation thanks to the label-free, real-time and multiplex capacities. www.biopharmaceuticalmedia.com

2.

Arbabi-Ghahroudi M. (2017) Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook, Front. Immunol. 8:1589 Saerens D. et al. (2008) Antibody Fragments as Probe in Biosensor Development, Sensors 8:4669-4686

Dr Chiraz Frydman Dr Chiraz Frydman is currently Global Senior Product Manager for SPRi and life science instruments. She has an engineering diploma in biology and a PhD in enzymatic engineering, bioconversion and microbiology. She has more than 20 years’ expertise in biophotonics and more than 10 years’ experience in optical instrumentation for molecular interaction analysis. For more than 10 years, she has been involved in more than 10 national and European projects.

Email: chiraz.frydman@horiba.com

Karen Mercier Karen Mercier, MSc, graduated with a Master of Science in proteomics from the University of Lille (France). She has been an application engineer specialising in surface plasmon resonance imaging (SPRi) technology in HORIBA Scientific (Palaiseau, France) since 2005. She is in charge of important developments for SPRi technology, dealing with biochips chemical functionalisation and new biological models’ studies.

Yannick Nizet Yannick Nizet received a PhD in immunology at the University of Louvain (Belgium). Working with the professor Hervé Bazin (inventor of the rat monoclonal antibodies) he has developed new immunisation methods as well as many monoclonal antibodies (including therapeutic) in mouse and in rat. Cofounder and CSO of Synabs since 2015, he is now developing a guinea pig monoclonal antibody technology.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 33


Manufacturing/Technology Platforms

PEER REVIEWED

The Effect of Insulin on Cell Growth and Virus Production The goal of our project was to test insulin as a booster for cell growth and virus production. Insulin was chosen for many reasons. It is already used in cell culture, and it is approved by regulatory agencies. So this is in line with our mission to look for strategies that can be implemented quickly in industry. Insulin is also known for its anti-apoptotic and mitogenic characteristics. We expected that insulin would improve the growth profile of the cells that we work with, but its effect on virus production was unknown. Cell-line Growth Properties We first examined whether insulin can improve the growth properties of an industrially relevant cell line. We work with a HEK293 cell line that was developed at the NRC. This cell line is grown in suspension in serum-free medium, and a Good Manufacturing Practice (GMP) cell bank is available. Two types of insulin-free media were selected: The in-house medium (IHM), which is a serum-free medium developed at the NRC specifically for this cell line, and the CD293 medium, a chemically defined and a protein-free Gibco medium from Thermo Fisher Scientific. The grey lines in Figure 1 (top row) show conditions in the absence of insulin with CD293 medium: The cell density remains below 1.5 million cells/mL with a high cell viability of nearly 100%. But the cell density never reached a density higher than 1.5 million cells/mL. On the other hand, when we added insulin, we reached a viable cell density of about 6 million cells/mL. We didn’t see a significant difference between the two insulin concentrations. So in this scenario, adding insulin alleviated limitations that we had observed initially for the chemically defined medium.

Figure A 34 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

A similar experiment was performed in the IHM medium developed specifically for this cell line (Figure 1, bottom row). With or without insulin we can reach a cell density of nearly 6 million cells/mL. But we can reach that cell density three days sooner when we add insulin (either of the two concentrations tested), thereby accelerating the process. Speeding up the process by three days is a sure benefit in bioprocessing. Accelerating Influenza Production According to the World Health Organization (WHO), the influenza virus kills about half a million people worldwide every year. Currently, vaccinating against the disease is the most effective course of action. About 60% of influenza vaccines are produced by inoculating fertilised chicken eggs, but new production platforms are in development, including using mammalian cell lines. Cell-based vaccine production offers a number of advantages over egg-produced vaccines: It bypasses risks associated with avian flu that could decimate the egg supply; it allows a faster and more versatile production platform because most strains can be adapted to cells quickly, and it opens the door to vaccine productions free of animal components. Figure 2 shows what insulin’s viral lifecycle looks like in mammalian cells. The multistep process begins with absorption and entry of the virus. Then it releases its viral RNA, which enters the nucleus where more RNA is produced and then exported into the cytoplasm. Finally, new viral particles exit the cells through a budding process. During this replication, the virus takes advantage of several cellular signalling pathways using enzymes and kinases that are part of the cell.

Figure BZZ Spring 2019 Volume 2 Issue 1


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Manufacturing/Technology Platforms In the diagram, they are shown in red, with the P13K/Akt pathway being implicated in several of those steps. What’s interesting is that insulin is a strong activator of the Akt pathway. So when we started this series of experiments, we worked under the assumption that insulin might enhance influenza production by increasing activation of those pathways.

In Figure 3, the x axis shows time in hours post infection (HPI). The red line indicates titers measured at different time points. Notice that there is a first viral exit at about eight HPI followed by a second, more important release of viral particles at 16 HPI. That’s what we call the budding process. Sixteen HPI also is when we start seeing a drop in cell viability, indicated here in black. We therefore measured phosphorylation of Akt in our cells by flow cytometry after infection. The figure shows activation of Akt starting at 15 HPI and ending at 24 HPI, which corresponds to the times of the second viral exit. Experimental Design First, several conditions were screened using a 24-well microbioreactor. When cells are infected, trypsin also is added to cleave and activate influenza. Then samples are loaded into the different wells and incubated for 48 hours at 35°C. At the end of the incubation period, we sample the different wells and determine the viral titer.

Figure 3–1

Figure 3–2

36 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

We used a multiplicity of infection (MOI) of 0.01, which means that we had one viral particle for every hundred cells. As shown in the template on the left side of Figure 4, four different concentrations of insulin were tested, ranging from 5 mg/L to 100 mg/L. Insulin was added at two time points: At the time of infection (TOI) and at 16 HPI, the key time point at which the budding process occurs. We tested influenza production in the same two media types that we used for cell growth experiments: the in-house medium (IHM) and the CD293 medium. We have also performed the experiments with two different influenza strains, one that belongs to the H1N1 subtypes and a second strain that belongs to the H3N2 subtype.

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Consistency. Proven

Increase specific Influenza production with recombinant human insulin The vaccine industry is challenged to produce large quantities of vaccines in a rapid and cost-effective way. Major changes to current bioprocesses are both difficult and very expensive to implement. Using HEK293, it has been demonstrated that addition of recombinant human insulin to commercially available chemically defined media can be used as a supplement to increase VCD and specific viral yield.

To learn more visit www.novonordiskpharmatech.com

Influenza production in HEK293 Total cell count (E05 cells/mL)

HA concentration (ug/mL)

40

HEK293 cells in CD 293 media*

30 20 10 0 0

5

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*Data kindly supplied by Aziza Manceur, National Research Council Canada. Hemagglutinin (HA) assay is used for quantification of Influenza (H1N1). Insulin Human AF used in the experiments is www.biopharmaceuticalmedia.com supplied by Novo Nordisk Pharmatech. CD 293 media is trademark of Thermo Fisher Scientific.

0

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 37


Manufacturing/Technology Platforms

Figure 4

AudienCe Poll 1: Methods of Choice The “Audience Poll 1” box shows a wide range of responses, but the hemagglutination assay earned a majority of the votes for quantifying influenza viruses. The array of responses emphasises my earlier point that quantifying influenza is not a straightforward task. Looking again at Figure 2, you can see two main proteins that are expressed on the surface of an influenza virus: neuraminidase and hemagglutinin (HA). Those proteins are used to identify influenza strains. H3N2 refers to a strain that expresses hemagglutinin protein from subtype 3 and neuraminidase protein from subtype 2. HA is about four times more abundant than neuraminidase, which is why it is the protein used to quantify influenza. HA comprises two regions: a head region and a stalk region (which is closer to the viral membrane). Most mutations take place in the head region, where most antigenic shift occurs and gives rise to new strains. Each strain will require a specific antibody – again, showing the difficulties of quantification. The good news is that there is a peptide in the stalk region called the fusion peptide that is highly conserved across the subtypes and strains because it’s important for virus replication. Sean Li, a collaborator at Health Canada, looked at about 4000 different strains using bioinformatics tools and determined that this peptide has a very high level of homology among the different strains.

Figure 5

chose to use the dot-blot technique. It has a 96-well capacity and is easy to implement with minimal cost. The protocol is quite simple. First we denature the samples with a mild denaturation solution consisting of 4M urea for half an hour to expose the epitope, which tends to be hidden within the virus envelope in the stem region. The samples are then loaded into the wells of the dot-blot apparatus, and vacuum is applied. After blocking for one hour, the membrane is incubated with pan-HA antibodies either overnight at 4°C or at room temperature for two hours. Figure 6 shows typical results. The first two columns show calibrating antigens that are loaded in duplicate, with concentrations ranging from 160 ng/mL to 20 µg/mL. Next to that are 10 samples that were also loaded in duplicate, but in four different dilutions. Figure 6b shows the standard curve that was generated from the calibrating antigen. If you consider the linear region, the R2 is 0.98, showing a strong correlation. Then using that standard curve, we quantified the 10 samples in the membrane in 6c, which ranged from almost no HA to 40–50 µg/mL HA. Many of the samples were not purified, but simply centrifuged.

Therefore, for quantifying influenza in our laboratory, we have generated pan-HA antibodies that can recognise multiple influenza strains. We have synthesised a peptide-conjugate based on the highly conserved sequence, immunised mice to generate monoclonal antibodies (MAbs), and ended up with two lead candidates. In the Western blot results shown in Figure 5, each lane corresponds to a different HA subtype. A 70-kDa band corresponds to uncleaved HA whereas the band at 25 kDa corresponds to the stem region of HA after cleavage (HA2). All subtypes are recognised by one or both antibodies. After further analysis, 11H12 was shown to be better at recognising HAs belonging to Group 1, whereas the second antibody (10A9) seemed to prefer HAs from Group 2. The HA groupings are based on the influenza phylogenetic tree. Combining the two MAbs created a pan-HA cocktail that enabled detection of all the subtypes tested. So far we have tested about 40 different strains of influenza produced in eggs or cells and even virus-like particles in plants. For quantification, a Western blot can be used, but it is a low-throughput assay and difficult to optimise. Instead, we 38 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Figure 6 Spring 2019 Volume 2 Issue 1


Manufacturing/Technology Platforms This technique is reproducible and robust. Some samples were quantified three or four times on different days by different operators and showed a standard deviation below 5%. Figure 7 shows an H1N1 strain tested in CD293 medium in a microbioreactor. HA concentrations were measured by dot blot, with the blue bars corresponding to results obtained when insulin is added at the time of infection. The grey bars denote when insulin is added 16 HPI. We found a significant increase in HA titer when 25–100 mg/L insulin was added at TOI.

Figure 7

We find it interesting that there is no significant difference in total cell density between the different conditions. This indicates that the increase observed in viral yield is not simply due to an increase in cell density; some other mechanisms are taking place. The maximum increase in HA titer with insulin was about a 1.7-fold increase. Results with the in-house medium were similar (with the H1N1 strain, Figure 8). The difference was that the increase in yield is observed at lower insulin concentrations ranging from 5 to 25 mg/L both at TOI and 16 HPI, except for one of the conditions. We saw no difference in the cell counts at time of harvest, which argues against a simple increase in titer resulting from an increase in cell density. Next (Figure 9) we looked at an H3N2 strain in the CD293 medium using the HA assay. As with the H1N1 strain in that cell medium, the increase in yield is observed only when insulin is added at TOI and does not affect the total cell counts at the time of harvest.

Figure 8

that work through similar mechanisms – such as lenti- and retroviruses, which are also enveloped – could probably benefit from using insulin. Other viruses such as adenoviruses use a different mechanism to exit infected cells. Insulin might still assist in their production, but it would be through a different MOA than for influenza. Increasing Influenza Production To continue my thoughts about how insulin mediates an increase in influenza production, we looked at the phosphorylation of Akt and mTOR using flow cytometry. In this experiment, insulin was added at 16 HPI. The control consisted of cells treated with trypsin only, but not infected with influenza. We calculated the ratio of phosho-Akt and phosphor-mTOR from infected cells over that from non-infected cells. Both kinases were activated by H1N1 starting at 15 HPI. That activation is stronger in the presence of insulin, which makes sense because it is an activator of this pathway. The two kinases play different cellular roles. mTOR is involved in protein synthesis. So increased mTOR activity indicates an increased protein synthesis. We do see an increase starting at 15 HPI and up to 24 HPI, but that increase is the same with or without insulin. The difference is not statistically significant. So activation of mTOR is probably not the mechanism behind the effect of insulin on viral production. Akt, on the other hand, is strongly activated by insulin infection with a 10- to 20-fold increase in phosphorylation. But additional insulin further increases that activation, especially at 18 HPI. Akt activation is associated with several cellular activities. Akt has nearly 100 cell substrates, and it is also tightly associated with increased cell survival and reduced apoptosis. So this could be the mechanism by which insulin enhances viral yield. We did measure cell viability, but only at the time of harvest (48 HPI). The viability was not increased by insulin, but insulin possibly could increase the cell survival at critical points between 18 and 24 HPI, during the budding process. That would allow production of more viral vesicles and delay apoptosis. So that is one possibility: that insulin, by increasing Akt activity, also would delay apoptosis and therefore allow the release of more viral particles during the budding process. That theory is further supported by some literature showing that a protein produced by influenza (NS-1, a non-structural protein) played exactly that role in the cells. NS-1 leads to activation of Akt and a decrease in apoptosis. If you follow the theory, it means that insulin basically mimics the role of NS-1. Also interesting is that NS-1 is strain-specific. All influenza strains have different types of NS-1. We compared Akt activation in cells infected by the two different strains and found that Akt is more highly activated by the H1N1 strain than the H3N2 strain. So it seems that H1N1 expresses an NS-1 protein that leads to higher phosho-Akt: higher Akt activation, higher production, and less apoptosis.

Figure 9

If you pool all results from the two different media with the two different influenza strains, it seems that the best condition is to add 25 mg/L insulin at TOI. That appears to increase the viral yield in all the different conditions used. Audience Poll 2: Examining the Mechanism of Action Based on the results of the second audience poll (see the “Audience Poll 2” box), I should say a few things about the mechanism of action (MOA). My guess is that insulin should work with other cell lines, especially with similar viruses. Influenza is an enveloped virus that buds out of a cell. So other viruses www.biopharmaceuticalmedia.com

Scaling up Production The cell scale-up team wanted also to see whether what was observed in small-scale and microbioreactor format could be reproduced at larger scale. Team members compared production of influenza in a 7-L bioreactor with productions in 50-mL shake flasks. Two controls were used for this experiment. An internal control consisted of cells obtained from the bioreactor after infection (a 50-mL sample). An external control was of the same cells infected in parallel in a shake flask, so those cells had not been in contact with the bioreactor. The “Bioreactor Parameters box” shows conditions used to run the bioreactor (the same as in the shake flask.): 35°C with an MOI INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 39


Manufacturing/Technology Platforms

of 0.01 and a harvest time at 48.5 HPI. The results showed about 13 µg/mL HA in the reactor compared with 14.9 and 14.8 in the shake flasks. So the team could scale up the process and obtain the same results in the bioreactor as in the shake flask. The effect of insulin on influenza production in a large-scale bioreactor is currently being evaluated. The team also is interested in how insulin could affect production of other viruses, such as lentiviral vectors, retroviruses, adenoviruses and adeno-associated viruses (AAV). They are hopeful that the similar treatment and condition would also help increase the production of lentiviral vectors and retroviruses, especially because they go through a similar budding process as influenza. For adenoviruses and AAV, the mechanism is likely to be different because the viruses go through a lytic process in which they basically tear open cells to be released. This work is ongoing. Increasing yield, Accelerating Cell Growth During cell growth we were able to increase maximal cell density by adding insulin to the chemically defined medium. And even in medium that was well-suited for cells developed in-house, we could accelerate cell growth by adding insulin. Although we have worked mostly with influenza so far, we have found that regardless of the strain or medium type, adding insulin at the time of infection increases yield by about two-fold for the H1N1 and about 1.5-fold with the H3N2 strain. The implication for manufacturing is that if you double the amount of HA produced, you can also reduce the number of runs by two. That is a cost-effective way of manufacturing. Audience Questions and Answers Why was insulin added every 72 hours during the cell growth experiments? We picked that time because the half-life of insulin is about 72 hours. We didn’t test too many other scenarios, so it is quite possible that fewer additions or even lower concentrations of insulin also could be tested, and it might depend on the cell type. Did insulin significantly change glucose consumption in the cultures? Yes. When we started, the glucose was around 20 mM. Whenever it dropped lower than 8 mM, we added glucose. Without insulin, we added glucose only once, on day 10. But in the presence of insulin, we had to add insulin twice, at day five or six and day 10. Which parameters were controlled in the microbioreactor? We were able to control oxygen levels at 40% throughout the whole experiment. The temperature also was maintained at 35°C and the pH at 7.2, but only through the addition of CO2. With the apparatus we had, we were unable to add a base, such as NAOH. How does the dot-blot method compare with other quantification methods? When we perform the dot blot, we are measuring the total HA concentration. So the best technique to compare it with is the HA assay, the one with the red blood cells, because that also measures total HA concentration. 40 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

We analysed about 50 different samples and looked at the correlation between the two techniques. There is good correlation between the dot blot and the HA assay with an R2 of about 0.8. The greatest differences observed were at high HA concentrations. The advantage of using the dot-blot assay instead of the HA assay is that you don’t have to rely on the availability of red blood cells from chickens. With the dot-blot assay you can run your assay whenever you want to. I’ve also found that the dot-blot assay is more reproducible than the HA assay because in the latter you need to have a specific concentration of red blood cells to get hemagglutination. However, when I tried to make similar comparisons with the single radial immunodiffusion (SRID) assay, the correlation was not that strong. In that assay, you actually measure the trimers that form the precipitation ring. So the correlation is not that good because we are not measuring the same thing. In the SRID assay we measure the trimeric form of HA, and with the dot blot we measure the total amount of HA. Why would one antibody bind preferentially to Group 1 HA and the other one target Group 2? Although we have not looked specifically at this issue, the homology for 12 amino acids out of the 14–amino-acid peptide used to generate the antibodies is about 99.9%. For the two remaining amino acids, the homology level is only 60–70%. Usually an antibody will bind to a peptide of 8–10 amino acids. It is quite possible that one antibody was skewed toward one end of the peptide and the other skewed toward the other end, or that the difference is due to the two amino acids with less sequence homology. That might explain the different binding affinity of the antibodies to different HA subtypes. But actual binding studies are in progress, after which we’ll have a clearer answer. Glycosylation is also different between the subtypes. That might explain why one antibody is better for one group than the other. Is there a restriction in using insulin for a parenteral bioproduct? You need to check that with regulatory agencies. But insulin is animal- free and well characterised, and it can be produced in yeast or bacteria. As long as you document it and you know the source of your insulin, you should be fine. Did you look at viral titers in addition to the HA content? We ran TCID50 assays for that best condition of 25-mg/L insulin. The trend is that it does seem to increase the TCID50 titer, but that assay has so much variation that we ended up with high standard deviations. The TCID50 titer was increased when we added insulin, but that increase was not significant when we ran statistical assays because of the standard deviation between the different replicates. We need to rerun both assays to be sure, but we did see an increase in the HA content. Acknowledgments The speaker thanks her colleagues at NRC - Sven Ansorge, Sonia Tremblay, and Rhonda Kuo Lee – as well as Clare Medlow and Magnus Franzmann from Novo Nordisk Pharmatech for their scientific expertise and for providing the insulin. These results described do not represent an endorsement of any product by NRC.

Aziza Manceur, PhD Aziza Manceur, PhD, is a research associate at National Research Council Canada. Email: aziza.manceur@nrc.ca

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Regulatory/Quality Compliance

The Drug Delivery Innovation League Table – Germany and France Lead the Way in Europe The last two years have been some of the most remarkable years in pharmaceutical history. There were 46 FDA approvals in 2017, and this year we have received a record 59 approvals. We might now be entering a golden age of innovation for pharmaceutical R&D, but what is most encouraging is that this new era of increased productivity is translating into drug delivery devices and packaging as well. Many of the experts believe that this new age of innovation is a golden opportunity for drug delivery and device manufacturers to bring new technologies to patients. Perhaps more significantly, the FDA is actively encouraging manufacturers to innovate, which should enable more of these new technologies to make it to market. This article focuses on the implications for the European market, including the inaugural release of a European drug delivery innovation ranking – the data from which was extrapolated from the recent CPhI Annual Report 2018 and released at Pharmapack Europe 2019. On the European side, the injectables market will reach $207.3 billion by 2020, nearly doubling from $114.7 billion in 2015, showing a compound growth rate of nearly 13% over five years.1 The population in Europe is suffering from a similar disease burden as the US, and is increasingly switching to advanced drug delivery methods – for example, with diabetes patients switching to needle-free, painless injectable forms. These again are often disposable products that do not require any specialist skills for administration. “What we will see in Europe in 2019 is a continued rollout of drugs coming to market using auto injector technology. The ease of use and patient experience will become fundamental to their design.” David Braun, Global Head of Medical Device Business Solution at Merck Group Oncology, particularly with the rise of combination therapies, is also stimulating innovation with injectables that can deliver two actives at once being increasingly developed. The unifying trend across all drug delivery devices is the global preference to improve the patient’s user ability, compliance and experience. For example, smart dose injectors, multi dose delivery injectable caps and smart, dry powder inhalers are just a small part of recent approaches that have been designed to control and monitor appropriate use. Similarly, implants and transferred patches are another growing area, whereby the drug developer can more closely control dosing and delivery of drugs, particularly for drugs that require sustained and controlled release. Switzerland remains another key innovation market, led by the large number of big pharma companies based in the country, but also by specialist drug delivery innovation companies such as SHL Group and a number of key drug delivery research officers and world-class university research centres. In fact, the European market for new drug delivery systems is already the second largest in the world, according to Mordor Intelligence, sharing 30% of the market. The CPhI Annual Report 2018 shows a concurrent trend with biologics manufacturing capacity, which we expect to overtake the United States in 42 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

the next five years. This analysis, conducted by Dawn Ecker of BioProcess Technology Consultants, was completed by looking at predicted biological expansions and new investments. This is of particular interest to delivery device manufacturers because a large proportion of biologicals in the next few years will increasingly come with unique delivery mechanisms. This would suggest that if greater manufacturing is brought into the European market, there is also a parallel opportunity for substantial growth in drug delivery and packaging within Europe. Interestingly, perceptions of quality of innovation of drug delivery and devices within Europe remains high – behind only the US and Japan. David Braun agrees that “it has the potential to boost the European Industry,” which could see a gradual shift towards Europe as we have seen in biologics in the last 10 years. However, Braun remains unsure about how this may play out in the near future, as he believes that “with today’s globalisation, things could remain as is. This industry has a hard time to change partners or suppliers unless there is a very good reason for that.” The European data analysis from the CPhI Annual Report 2018, containing insight from 350 global pharma executives, has been compiled, creating the first European ranking of innovation across drug delivery devices. Results from outside of Europe were excluded to create a European ranking table. In the original research, the United States, Germany and Japan retained, for the second year running, the top spots in terms of perception of ‘API’, ‘final product’, and ‘biologics’ manufacturing. This research shows that mirroring the findings in both solid dose and biologics manufacturing, Germany remains the preeminent source of innovation within Europe. Interestingly, for drug delivery innovation, France scored only narrowly behind Germany – perhaps due to a number of leading device manufacturers being based there, notably Nemera, BioCorp and Aptar. Third within Europe was Switzerland, followed closely by the United Kingdom. A second tier of nations, in Spain and Italy, finished the bottom of the primary European markets. “No real surprises here; the rankings are probably related to government support of innovation/R&D and the facilitation and levels of support offered to start-up/incubator companies in each country.” Gerallt Williams, Director Scientific Affairs, Prescription Division, Aptar Pharma Unfortunately, due to the methodology of the research, there was no data available for Sweden, the Netherlands or Ireland, but one might have expected these nations to score highly, perhaps between the tier one and tier two nations. For example, the CPhI Annual Report does contain data on the rankings of biological manufacturers in Europe, and both Sweden and Ireland scored particularly strongly due to the presence of several international contract manufacturers based in their countries. We would anticipate that innovative drug delivery start-ups would begin to appear around these notable manufacturing hubs over the next few years. “We would suggest that in terms of worldwide rankings it would be interesting to include Canada because of the number of facilities that do contract dose manufacturing in that country. In European rankings, it would be beneficial to include Sweden in the future because of the number of facilities in the country that perform contract dose manufacturing.” Fiona Barry, Associate Editor, PharmSource, a GlobalData product. Spring 2019 Volume 2 Issue 1


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Regulatory/Quality Compliance the issues around delivering these complex molecules. We’re going to see these trends affect drug delivery revenue growth, as many CMOs will not have the capability to provide the new technological capabilities required. So finished dose CMOs will have to improve their formulation and drug delivery technologies to compete for clients, with products that are increasingly complex and have poor solubility.” When looked at holistically, the combined trends of the drug delivery and packaging sector over the next few years demonstrate a clear desire from regulators, developers and patients to improve the overall patient experience and centralise treatment options around the patient. In Europe, this aspiration coupled with a growth in biologics manufacturing and a new age of innovation is redefining the art of the possible and reimagining delivery options and adherence options. In fact, we would predict that a number of new innovation hubs with a more diverse mixture of smaller, highly agile companies will become established in the next few years across France, the UK, Switzerland and Germany. These countries in particular have an established centre of innovative companies, coupled with a large and growing biologics industry that is driving many of the innovative drug delivery options.

What’s clear is that the industry is collectively trying to move the delivery of drugs closer to the patient – potentially reducing the overall burden of healthcare providers. For example, pre-formulated, pre-filled syringes, which reduce the need for dosing. Taken a step further, auto-injectors can also allow a patient to conduct the delivery in isolation at home, with potentially a smart tool such as an app that notes the time of administration and simultaneously informs the healthcare provider. In fact, we predict that over the next one to three years, the rate of innovation with drastically increase as an even wider array of new companies get funding to produce exploratory clinical products. “Smart devices and connectivity remain hot topics. The technology is rapidly becoming more established, more available, more compact and cheaper; a common pattern with electronics and IOT-related technology in other markets,” added Andy Fry. The challenge, of course, in such a heavily regulated space will be having the size and experience to transfer the promising innovations through the pipeline and into commercial solutions. The net result of this is likely to be an increase in licensing deals between small and larger firms, as well as numerous acquisitions and co-development deals. “The big challenge is and will remain in scaling up from prototyping into an approved product going through clinical trial, and the same is most definitely true of bringing the product to full commercialisation.” – David Braun, Merck Group Barry, summarising her perspectives collectively on what the recent spate of innovations we have seen and the rising number of FDA approval mean for the contract manufacturing sector, added: “Our trend report (PharmSource: Contract Dose Manufacturing Industry by the Numbers: Composition, Size, Market Share and Outlook – 2018 Edition, August 2018) shows the complexity of new APIs is increasing, and so in turn, are 44 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Such is the rate of innovation in Europe, the injectables market should double in size in just over five years, reaching some €200million by 2020. Auto-injectors and needle-free injectors are likely to grow particularly quickly. Connected technology and the internet of medical things are also bringing newer market entrants into the European drug delivery market and our experts forecast this will bring in established technologies from other areas that will have profound and transformative implications for patients. The only potential ‘brake’ to this growth and acceleration is a reluctance by global pharma to change partners and work with newer providers, as well as still some degree of uncertainty as to how many of these more innovative drug delivery devices will be successfully scaled-up and commercialised, whilst still meeting stringent regulatory requirements. It’s one thing to demonstrate proof of concept, but a good deal more complex to deliver an approved device. As a result, what we will likely see is the larger drug delivery manufacturers and pharma companies scouring these new innovation hubs for licensing partners and acquisitions. Ultimately, these new combinations of small and large companies should help accelerate and enable these new, ground-breaking technologies to reach the patient without becoming slowed by scale-up challenges or regulatory approvals. In fact, it is likely that VC funding – that is currently in a relatively buoyant period – will be required to support the early stages of innovation before pharma and larger companies take over the latter stages of development. Other potential factors slowing innovation, particularly in the United Kingdom, will be the long-term implications of any trade deals negotiated between the United Kingdom and the EU. For instance, there is a good deal of uncertainty around the advanced therapies market – which the UK is currently the European leader in – and how this will be affected and/or if manufacturing will be moved to other European centres. Serialisation, which is now being implemented across the EU, could also be the beginning of a new age of connected devices healthcare solutions reaching the market. Technologies that track Spring 2019 Volume 2 Issue 1


Regulatory/Quality Compliance is to be averted, they will need to rely on the ‘good grace and flexibility of the regulator’. What is most exciting about all of these changes, when looked at collectively, is that there is an increasing diversification of the types of companies, types of professionals, and collaborations in the industry driving innovation forward. Other industries have clearly shown that when an open-access approach to new technologies is taken, transformational changes are often quick to follow and in five years’ time, it is likely that the drivers for change will see a much larger and diversified drug delivery and packaging innovation hub in Europe. There will be a far larger injectables, biologicals and connected devices market, with a number of innovation hubs established, not only in the major advanced European pharma markets, but also potentially in newer and smaller centres such as Ireland, Sweden, the Netherlands, and Belgium. The last two years have been a golden period for FDA approvals, however, the coming five years are expected to be a golden period of innovation for drug delivery and packaging devices across Europe.

Orhan Caglayan

not only where a pharmaceutical product is in the supply chain, but also at what date and time the patient administered the drug, result in improved compliance, clinical trial data and reduced physician intervention. However, many analysts forewarn that despite a number of years of preparation, a large number of smaller companies across the EU remain inadequately prepared for full implementation of serialisation regulations and if a crisis

Orhan Caglayan is the brand director at CPhI Worlwide and bioLIVE – two events examining the intersections between business and biotech/pharma.

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Regulatory/Quality Compliance

PEER REVIEWED

LIMS and ISO/IEC 17025 – An Opportunity, Not a Burden Laboratory information management systems (LIMS) have wide-ranging usage within pharmaceutical organisations, wherever samples need to be taken and tested and the results evaluated, including QA/QC, stability studies, pre-clinical pathology, clinical trials, biobanking and environmental monitoring. ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories, is the international reference for testing and calibration laboratories wanting to demonstrate their capacity to deliver reliable results. Accreditation to the ISO/IEC 17025 standard confirms that laboratories have the management, quality, and technical systems in place to ensure accurate and reliable analyses and results. For pharmaceutical laboratories, the adoption of ISO/IEC 17025 provides a vehicle to achieve and improve the quality and reliability for test results demanded by the industry. However, all too often the adoption of systems such as ISO/IEC 17025 is seen as a necessary burden required to achieve compliance with a specific standard. However, the opposite is true, especially if the adoption of the standard is combined with the implementation of a suitable laboratory information management system (LIMS). The successful implementation of a LIMS provides an opportunity to improve the operation, performance and efficiency of any laboratory, with the added benefit that it supports ISO 17025 compliance. The LIMS can be viewed as an ‘enabling technology’; however given the rapid pace of change within the laboratory environment, it is a technology which must be able to evolve and adapt, ideally under direct control of the user, as business requirements change.

be valid from 30 November 2020. The standard recognises the use of both computerised and non-computerised laboratory management systems. However, since some of the requirements are better suited to computerised systems, a LIMS can greatly facilitate the accreditation process. Facilitating Laboratory Testing with LIMS The laboratory process involves more than just the registration of samples, entering of results and reporting of those results. Amongst many other items, laboratories must allocate resources to tasks, including suitably trained personnel and properly calibrated and maintained instruments and equipment, calculations need to be performed, results compared to defined limits and QA/QC runs and measurement of uncertainty managed. Commercially available LIMS are designed to provide the basic functionality of controlling, managing, organising and documenting information within a dedicated database. This streamlines the whole process, reduces the likelihood of process error, and can provide a complete audit trail that records when and by whom an action was completed. A LIMS can be used to: • •

• • • •

The Evolution of ISO/IEC 17025 The third edition of the standard, ISO/IEC 17025:2017(E)1, was published on 30th November 2017. This cancels and replaces the second edition (ISO/IEC 17025:2005), and has a stronger focus on information technologies. It takes into account the fact that hard-copy manuals, records and reports are slowly being phased out in favour of electronic versions, and incorporates the use of computer systems, electronic records and the production of electronic results and reports. In addition, the scope has been revised to cover all laboratory activities, including testing, calibration and the sampling associated with subsequent calibration and testing. 
The process approach now matches that of newer standards such as ISO 9001 (quality management), ISO 15189 (quality of medical laboratories) and the ISO/IEC 17000 series (standards for conformity assessment activities), putting the emphasis on the results of a process instead of the detailed description of its tasks and steps. 
For laboratories already accredited to the second edition of the standard (ISO/ IEC 17025:2005), it has been agreed that a three-year transition period (from date of publication) will be allowed for them to make the appropriate changes and transition to the new edition. Accreditations to ISO/IEC 17025:2005 will therefore cease to 46 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

• •

Log incoming samples, print and scan barcodes to avoid errors Keep a complete chain of custody, track samples from initial sampling location through the lab to disposal, recording location, custodians and other key data Facilitate and organise laboratory data Account for the entire quantity of sample received, used and disposed of Create certificates of analysis (CoA) to local regulatory requirements Incorporate all tests, instruments, sample information and result data (etc.) in one place Keep a record of all laboratory instruments and a history of calibration and maintenance events Keep a defendable audit trail of all sample and test result information

Laboratories not using a LIMS are generally using paper-based systems or non-specific electronic systems such as Excel. However, paper-based systems can be time-consuming and difficult to manage in a way acceptable to regulatory and auditing agencies. Every time data is written on a piece of paper or copied from one to another there is the potential for transcription errors. Non-specific electronic systems can help but are not designed for the task, making it difficult to both control the laboratory process correctly and efficiently access the required information. By moving to a LIMS, there can be significant improvements in laboratory operational efficiency through: • • •

Cumulative time saving on job processing Elimination of time-consuming paperwork Automatic allocation of tasks and tests to samples as they are registered 
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Elimination of transcription errors through automatic data capture Streamlining of final report production and delivery based on the lab’s, and the customer’s, particular needs

Thus a LIMS helps laboratories manage large volumes of data to strict standards, while at the same time improving efficiencies, automation and turnaround times. Facilitating ISO/IEC 17025 Accreditation Clearly a LIMS can bring many benefits to a laboratory, and the latest version of the standard recognises the use of validated commercial, off-the-shelf LIMS software for data management. However, it can offer much more than just data management for the laboratory process. Its use can be extended to cover many of the other management requirements for accreditation to ISO/IEC 17025:2017(E). These requirements include1: • • • • • • •

Control of management system documents Control of records Actions to address risks and opportunities Improvement Corrective actions (CAPA) Internal audits Management reviews

A reputable commercial LIMS will not only support these requirements by providing data needed to show adherence, but will provide the functionality to manage these requirements, for example CAPA management, document management and audit planning. Choosing a Suitable LIMS As mentioned earlier, a LIMS can be used in any application where samples need to be taken and tested and the results evaluated. However, not only will the requirements for various applications differ, but even laboratories in the same discipline can have different ways of achieving the same ends. Outside of the testing process there are a multitude of other ways in which the workflow of laboratories differ; starting from the way samples are submitted for testing, through the sample lifecycle to the point at which results are authorised and reported. A LIMS must be flexible enough, therefore, to support these different needs and working practices. Most commercial LIMS can be adapted to meet the requirements of the individual laboratory but, in many cases, this process means relying on the vendor to modify the

Figure 1. Laboratory Process Flow Overview www.biopharmaceuticalmedia.com

Figure 2. Monitoring Instrument Calibration & Maintenance

underlying code of the LIMS or the organisation employing its own IT staff and programmers to do so. This also means that if any further changes are required to the system during its lifetime, the code will have to be further modified. A more flexible approach is offered by LIMS products that provide a configuration ‘layer’ within the system. This provides configuration tools that allow the setup and modification of the LIMS without writing any new code. The system can be configured to look and behave according to the precise user requirements in terms of workflows, screen designs, menu designs, terminology, numbering schemes and report designs. In this way the system can be readily adapted to the needs of the laboratory and any future changes can also be simply incorporated in the LIMS. Most importantly, end users can also be trained to use these tools, enabling them to modify their configurations if they are authorised to do so. The ability to configure the LIMS takes on even greater importance by allowing additional functionality and specific needs to be incorporated into the LIMS, further helping towards ISO/IEC 17025 accreditation. Control of Management System Documents All of the processes and procedures established as part of ISO/ IEC 17025 need to be documented and provision made for access for everyone who needs them. Issues such as the appropriate indexing and monitoring of document updates also need to be addressed. It should be possible for LIMS to track, store and distribute all documents from a central location. Including document versioning provides a full audit trail, allowing the history of revised documents to be traced for internal and external auditing and reporting. Documents can never be lost as saved information is automatically backed up. Staff can be required to record the fact that they have read and understood controlled documents, therefore providing the required training record. They can be automatically notified when training becomes due and management reports can be generated to identify any staff that have not read the appropriate documents. Corrective and Preventive Actions (CAPA) for Laboratory Systems Laboratories need to be accountable for the analytical results that they produce, and this leads back to the procedures and processes they have in place. ISO/IEC 17025 requires them to have policies to deal with customer complaints, with records being kept not only of these complaints but any subsequent investigations and corrective actions. In addition, laboratories will INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 47


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Figure 3. Document Management

need to have policies to deal with any aspect of non-conformance in testing or calibration, even if it is not instigated by a customer complaint. Any changes that are necessary as a result of corrective action investigations must be documented as well as implemented. Since an evaluation of the significance and wider implications of any non-conforming work needs to be made, the recording and date-stamping of activities in a LIMS will help with this. Corrective action is, by definition, a reactive response to a problem. Laboratories are also required to have a preventive action policy, to allow the identification of improvements and potential sources of non-conformities. Any preventive action plans that are developed in this way must be documented and monitored. Using a configurable LIMS, screens can be configured to reflect and implement identified improvements. Customer comments and feedback can be logged directly into the system to help to drive continual service improvement. Issues and actions can be defined and allocated to appropriate staff for implementation with outcomes stored within the system. Managing CAPA through a LIMS allows users to:

Decrease the mean time to resolution of issues for the benefit of both internal staff and customers

Competency in the Laboratory ISO/IEC 17025 requires not only that all staff are suitably qualified for the tasks that they undertake in terms of appropriate education, training, experience and/or demonstrated skills, but also that the competence requirements for each function influencing the laboratory results are documented. A configurable LIMS should include functionality to record and manage personnel skills and training records. Training courses can be scheduled automatically and certificates of competency issued and stored to provide a historical record of skills acquired. Automatic renewal reminders can be issued for time-dependent qualifications. Having all personnel qualifications and competencies documented also allows easy matching of staff to the specific needs of the laboratory. For example, if a backlog builds up for a specific test or analytical method, additional suitably qualified staff can be identified from within the

Figure 5. Competency Tracking

• • • •

Capture the necessary QA information needed to investigate deviations and out of specification (OOS) results Provide a clear record and assignment system for corrective and preventive actions Reduce the cost and operational risk associated with addressing recurring issues 
 Improve operational performance of the laboratory with standardised processes 


organisation to reduce the backlog. Furthermore, personnel can be prevented from carrying out tests they are not qualified to do. Multiple security levels available for each system function, down to screen, field and button level, make it possible to: • • •

Implement data management strategies that increase security of data Allow highly controlled third-party access to the system if required Ensure that specific system functions are restricted to suitable personnel, for example the approval or authorisation of results and the issuing of final reports

Utilising the Data Not only can a LIMS be configured to manage a diverse range of data over and above the traditional testing of samples, but by providing access to this information from a single integrated source, many of the other requirements of ISO/IEC 17025, such as internal audits, risks and opportunities, improvement and management reviews can be addressed. A LIMS can maintain a clear, audit-trailed, searchable record of all samples and test results and reports issued. Complete and readable copies of all records (acquired data, audit trail, electronic signatures, etc.) can be produced in electronic format or paper format.

Figure 4. CAPA Management 48 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

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areas for potential improvement and detect serious problems. For example, such trends could potentially identify the source of bottlenecks in a particular laboratory, allowing actions to be taken to adapt the workflow to minimise these. The LIMS can be used to model the effect of potential business changes. By using historical information on turnaround times and the number of trained analysts, for example, it is possible to model the impact of increasing expected sample throughput on the laboratory. Therefore, the impact of a new contract on training requirements, staffing levels and equipment utilisation can be assessed and managed before the samples start to arrive and backup in the sample reception area. Smoothing the Path to ISO/IEC 17025 Accreditation A LIMS offers so much more than just a repository of information regarding sampling, testing, and results. A properly implemented LIMS is a business-critical system that enables informed decisions to be made regarding a multitude of laboratory processes, as well as providing the foundation for ISO/IEC 17025 accreditation. When implementing ISO/IEC 17025, it must be looked at as an opportunity to improve laboratory performance and efficiency, www.biopharmaceuticalmedia.com

an opportunity that can be maximised with the use of a fully configurable LIMS. REFERENCES 1.

www.iso.org/standard/66912.html

Simon Wood Simon Wood PhD, Product Manager at Autoscribe Informatics, has 30 years’ experience in the commercial LIMS environment. He is an acknowledged expert in the field of scientific and laboratory informatics.   Autoscribe is a global supplier of LIMS to both the laboratory and the wider business markets, with distributors in every continent offering localised technical support.

Email: simon.wood@autoscribe.co.uk

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Regulatory/Quality Compliance

Understanding Critical Quality Attributes To develop a life-saving biologic, it is not enough to focus solely on what it can do. It is just as vital to know where and why it can fail. Introduction With the increasing prominence of biopharmaceutical products in the modern therapeutic landscape, it has become vitally important to ensure the manufacture of these products is carried out reliably and efficiently. To achieve this and address the unique challenges of scalable manufacturing of biologics and other pharmaceuticals, the FDA introduced the concepts of quality by design (QbD) into the cGMP regulations in 2004. Subsequently, ICH guidelines Q8 “Pharmaceutical Development”, Q9 “Quality Risk Management” and Q10 “Pharmaceutical Quality Management” have been issued to provide guidance to drug developers and manufacturers that would allow for the implementation of QbD principles into industrial processes. Taken together, these guidelines provide the framework by which pharmaceutical companies can design their products, processes, and control procedures to ensure that a product has all the attributes considered paramount to its safety and efficacy - the critical quality attributes (CQAs). CQAs are defined in ICH Q8(R2) as any physical, chemical, biological, or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality. Unfortunately, this statement contains little in the way of guidance for its application to biologics development. More practically, CQAs form the basis from which the entire chemistry, manufacturing and control (CMC) is built. A product’s chemistry may be modified in order to achieve greater potency or stability; the manufacturing process may be modified to reduce the risk of impurities or imperfections in the final product; quality control testing panels may be designed in order to assess batch-to-batch variability as well as the acceptable limits of the observed variability. Each of these elements of the CMC process are defined by the CQA profile of the product, and so it is a useful exercise to take a closer look at what a CQA is, and how one might go about determining those that are relevant.

to be a very daunting task. Fortunately, answering the much simpler first question, identifying the end goal or product, represents a major step towards identifying the relevant CQAs. From the perspective of biopharmaceuticals, the first task is to identify the broad classification of the biologic in question. Is it a monoclonal antibody, or an enzyme replacement therapy? Will it be administered by injection or infusion, or will it be formulated into a capsule? What excipients will be included in the final formulation, and at what concentrations? Simply assessing a range of tangible factors and inputs starts to put some constraints around that open-ended second question. These include the nature and mechanism of the active pharmaceutical ingredient, the characteristics of the product and how it is manufactured, and historical knowledge of similar products. Mechanistic Knowledge As an example, imagine an injectable monoclonal antibody with a benign liquid formulation. Its efficacy is, essentially, contingent on enough of the antibody performing the action for which it was designed. Thus, from a CQA perspective, we already know two traits we need to confirm: that there is enough of the antibody (i.e. concentration determination), and that it is doing what it should (in the case of an antibody, binding a target antigen). Existing knowledge about effective concentrations and binding activity allow UV absorbance assays and ELISA assessments to show that the product should, indeed, be efficacious. Armed with this confidence that it is likely to work, the antibody can be injected into a CQA practice mouse for confirmation. Product Characteristics However, what if the mouse’s condition appears to have deteriorated the next day? The results from a series of tests indicate that the drug’s efficacy is poor, despite the previous in vitro confirmation of its binding activity and the prior knowledge of its expected pharmacokinetics. Further investigation shows that the drug is distributed very abnormally within the mouse, and the root cause is a high concentration of an acidic isoform of the antibody. The next step is, therefore, to make another batch of the product, but this time adding charge heterogeneity analysis

There are two very general questions that should be asked when examining any product or process – whether developing and manufacturing a revolutionary new biopharmaceutical product, or just replicating grandma’s famous lasagne recipe. The first – ‘What is the end goal or product?’ – is usually fairly straightforward to answer. The second, however, is a far more difficult question: ‘What can go wrong?’ Providing answers to both of these questions represents the central challenge involved in identifying and understanding CQAs. Identifying CQAs Producing a list of every possible way that a process might go wrong to answer that second, open-ended question appears 50 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

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Regulatory/Quality Compliance those that are truly critical. How likely it is that the risk will be realised, and how much of an impact would it have? By scoring likelihood of occurrence against potential impact, a ‘risk index’ can be developed. Here, each index score must be above a certain threshold to be considered relevant. It should be noted that, as with most risk assessment, this approach is largely qualitative, as both the likelihood of occurrence and impact are, for the most part, scored subjectively. It is often prohibitively difficult, if not impossible, to obtain objective metrics.

to the CQA assessment panel before the drug is injected into a second practice mouse. Manufacturing Process Unfortunately in our example, the second mouse develops an immunogenic response to the new batch of product. There is a strong suspicion that this is a batch problem rather than an inherent flaw in the product, as the first mouse did not exhibit any such response. It can be surmised that another CQA must have been missed. Looking back at the manufacturing process, the resin from the protein A purification column looks oddly discoloured, leading to the conclusion that this purification did not work as expected, and there are likely to be host cell contaminants that led to inflammation in the mouse. Another CQA gets added to the list, and purity checks by SDS-PAGE electrophoresis and host-cell ELISA are added to the testing panel. Historical Knowledge It becomes clear that this trial-by-fire strategy for the determination of CQAs is not very efficient. There is a huge amount of experience in the manufacture of therapeutic antibodies in the industry, and therefore a wealth of information that could be mined from that past experience. And there are, indeed, many more CQAs that could – and should – be on the list. Running through a new, comprehensive, list of CQAs and associated quality control tests provided by external experts leads to the realisation that a high level of aggregates were present in the product because of an error during ultrafiltration. A new batch of product is manufactured carefully, and then tested against this longer list of CQAs. It is then injected into the third mouse, and this time cures its illness without the side-effects experienced by the two previous mice. Assessing Risk While the example above constructs the CQA identification process from the bottom up, it is common practice to start by taking a top-down approach. Rather than discovering each CQA one at a time, the starting point is to consider a large set of likely potential CQAs, and assess which are likely to pose the greatest risk to safety and efficacy. In order to reduce down this long list of CQAs, all four of the CQA identification strategies highlighted above should be applied – mechanistic knowledge, product characteristics, manufacturing process and historical knowledge. However, to keep it manageable, the process should be streamlined, with empirical assessments of risks focused on confirming only those CQAs that are likely to be relevant in each individual case. This strategy requires robust criteria to be developed to identify which should be considered relevant. Once the potential risks, and therefore CQAs, have been identified, two filters can be applied to create a shortlist of www.biopharmaceuticalmedia.com

Conclusions While it is easy to understand the basic definition of a CQA, the identification and assessment of these attributes is a much more uncertain, involved process that requires a deep scientific insight into the product and its manufacturing process. Even building the requisite knowledge is often insufficient, as new discoveries about characteristics that affect product quality are made regularly. Such knowledge must be combined with thorough pre-clinical studies to provide empirical support to the initial risk assessment strategy, which then further informs aspects of continued development, manufacturing, and control strategies. As such a fundamental part of CMC and QbD, the level of attention to detail when determining CQAs is essential if a safe and potent product is to make it to market. Further Reading This article is intended as an introduction to critical quality attributes. For more detail, the following is recommended: • ICH Q8(R2): Pharmaceutical Development • ICH Q9: Quality Risk Management • ICH Q10: Pharmaceutical Quality Management • Understanding Quality by Design • Quality by Design for ANDAs: An Example for ImmediateRelease Dosage Forms

Omar Musleh Omar Musleh is a client services manager at SGS Life Sciences Canada, working with partners across the biologics and biopharmaceutical industry to establish and implement their analytical quality control testing needs. With a specialisation in biomedical research, Omar brings a diverse scientific background to SGS, with research experience in structural biochemistry, biosensor engineering, and cGMP Method Development and Validation. Email: omar.musleh@sgs.com

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Application Study Automated Glucose Control Stuart Tindal, Sebastian Ruhl, Diana Dreiling

1. Introduction One of the goals of mammalian upstream bioprocess development is to handover a process to clinical manufacturing that is robust, safe and in the end produces enough material to meet the studies demand. These process development teams are increasingly being asked to do more and in less time. Thus, automated feeding strategies are becoming more and more popular as a way of reducing the development efforts and as a side benefit have shown to improve process performance and product attribute consistency. Cell cultivation’s primary feed component is glucose which is used as a starting point for all growth and energy pathways within the cells. Ensuring that the cells do not have too much or too little, enables them to grow fast and maximize the product secretion. Additionally, it has been noted that an excess in glucose concentration has an effect on the glycosylation rate of secreted soluble proteins. Thus, controlling the available glucose would improve the consistency of glycosylation patterns on final products and may improve the quality.

level enabling a user defined glucose setpoint within the BIOSTAT® B-DCU control software. The first two runs, insitu glucose concentration was maintained by discontinuous bolus feeds. Subsequently the third run utilized a glucose setpoint controller using defined PID settings and an internal, speed-controlled peristaltic pump.

Within this application note a stepwise example method on how to establish glucose feed control is documented. The materials and methods show how this was done using Sartorius Stedim Biotech hardware and equipment. An overview of the results and discussion of the data is given in order to highlight some of the key benefits of applying this method to other processes. Material & Methods A BIOSTAT® B-DCU was used as bioreactor control system. The BIOSTAT® B-DCU is a bioreactor for advanced process optimization and characterization featuring the option of a glucose concentration controller. The integration of the BioPAT® Trace allows a real-time monitoring of the glucose

The BioPAT® Trace was set up for dialysis mode glucose & lactate measurement at a 20 minutes sampling rate. It was set to a fully automated and self-calibrating protocol as part of the schedule tab function. Each 17 day run used 12 L of transport buffer, two calibration solutions (high: 10 g/L glucose; low: 1 g/L glucose) and a 10 L waste container attached to the tube set. The dialysis probe was prepared, filled and installed into the UniVessel® Glass 5 L prior to sterilization and connected to the BioPAT® Trace tube set and primed for analysis before inoculation. The UniVessel ® Glass 5 L was equipped with two 3-blade segment impellers for low shear stress and good homogenization of the cell broth. The blade angle was 30° and set to down pumping. A ring sparger with holes faced up was used for all trials. Additionally, the vessel was equipped with several ports for feeding, a classical pH sensor, pO2 sensor, dialysis probe, exhaust cooler and gas filters.

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Application Study To evaluate the BioPAT® Trace integration in the BIOSTAT® B-DCU a CHO fed-batch process was used. The 17 day cultivation comprises of a 3 day batch phase and a 14 day fed-batch phase. After the inoculation with 0.3 + 106 cells/mL the peak viable cell density (VCD) is typically reached at day 8 with 25 – 30 + 106 cells/mL and a viability of 99 %. After the following 9 day dying phase the VCD should be above 10 + 106 cells/mL with a viability of more than 50 % at the point of harvest. The bolus feeding from day 3 comprises feed medium A (FMA), feed medium B (FMB) and a highly concentrated glucose solution (400 g/L). The fed amount of FMA and FMB is constant throughout the complete fed-batch phase. Typically on day 7, additional glucose is needed to maintain a glucose concentration of at least 3 g/L in the cell broth. The feeding process is automated using balances and pumps connected to the digital control unit (DCU) and S88 recipe in the SCADA software BioPAT® MFCS. Analytics were an essential part of this evaluation. Among other things, offline glucose and lactate measurements were performed with the Radiometer ABL 800 basic. VCD and viability were analyzed with a Cedex HiRes.

Figure 2: Feeding scheme 3

In trial 3 the bolus feeding of FMA was modified to continuous feeding to remove daily peaks in the glucose concentration. After glucose reached a concentration below 6 g/L, a controlled feed of glucose solution (400 g/L) was initiated to maintain the glucose concentration at 6 g/L. Results & Discussion The batches ran sequentially on the same BIOSTAT® B-DCU and BioPAT® Trace systems. The results are shown in figure 3 and 4 to demonstrate the reproducibility and conformance of the process batches (compared to the historical golden batch). The glucose monitoring of the BioPAT® Trace and subsequent glucose controller are shown in figures 5–7.

A total of three different trials were performed using the following characteristics: 1. Monitoring Trace technology was solely used to monitor the online glucose trend throughout the cultivation. 2. Online value replaces offline measurement During the CHO culture a daily sample is taken. The offline glucose value is used to fill to a certain glucose concentration in the bioreactor. In trial 2 the glucose online value is used instead of the offline value for this feeding procedure. 3.

Glucose control During the third trial glucose control at 6 g/L was activated after 6 days. The usual bolus feed of FMA was modified to continuous feeding to reduce glucose concentration peaks.

Feeding scheme for trial 1 and 2:

Figure 3: VCD trend trial 1–3

Trial 1 and 2 fit the golden batch trend well. Due to a general process modification (continuous FMA feed) the VCD trend of trial 3 has a slightly reduced peak VCD. Final VCD and viability fits well of exceeds golden batch values for all trials.

Figure 1: Feeding scheme 1 and 2

For trials 1 and 2 the feeding scheme was identical. During the batch phase no medium was fed to the culture. On day 3 a daily bolus feed of FMA and FMB started. After glucose fell below 6 g/L an additional feed of glucose (400 g/L) started. www.biopharmaceuticalmedia.com

Figure 4: IgG trend INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 53


Application Study Comparable productivity in all three trials was demonstrated by daily measurements of the product concentration (IgG concentration, figure 4). Due to a lower cell growth in trial three a slightly lower product concentration was achieved in this run.

Figure 5: Glucose and lactate trend trial 1

The transition from glucose monitoring to glucose control requires a change of the process. The effects of tighter glucose control are known to be beneficial to the process performance and product quality1 (reference An Zhang et al). Considering this, changing a process using non tried and tested technology is undesired. Thus, learning the system and building confidence around the measurement output (comparing to offline sampling) and ensuring the device’s robustness is generally needed before making that change. The first two process runs show the high resolution monitoring capabilities of the integrated BIOSTAT® B-DCU and BioPAT® Trace whereas the third run engages the glucose controller. The overall user operational interactions with the bioreactor related to sampling glucose are removed. This means the normal hours and out of normal working hours needed to maintain the BIOSTAT® B-DCU system in a glucose controlled state are reduced and more manageable. In table 1 a summary of the time savings are presented, in addition all user interaction work with the system was done within the initial stages of the BIOSTAT® B-DCU setup. Table 1: FTE resource balance per batch when using the BioPAT® Trace for glucose

Despite of a small offset in the first 24 hours the online and offline measurements of glucose and lactate fit well during the cultivation.

Summary & Conclusion The stepwise integration of the glucose control technology to a CHO fed-batch process was successful and maintained an inprocess steady-state glucose concentration of 6 (+/- 0.25) g/L.

Figure 6: Glucose and lactate trend trial 2

For trial 2 the online and offline glucose and lactate measurements showed good comparability.

The direct integration of the BioPAT® Trace technology into the BIOSTAT® B-DCU is fully functional and the utilization was user friendly, easy to establish and dynamically variable when required. As the system package is coming from Sartorius Stedim Biotech completely, the lifecycle of the glucose controller is managed by us through all scales of development and commercial manufacturing. This ensures the benefits uncovered in development can be transferred to commercial production for the complete product lifecycle. Outlook Further trials with varying, automatically controlled glucose concentrations will be performed in order to improve product concentration and product quality attributes. Additionally, the operator time saving (7 hours per batch) and manageable working hours makes steps towards a walk away bioreactor. REFERENCE 1.

Figure 7: Glucose and lactate trend trial 3

Also in trial 3 the online and offline trends fit well for glucose and lactate. At day 13 and 15 an unnecessary recalibration was performed (operational mistake). 54 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Zhang, A.; Tsang, T. et al. Advanced process monitoring and feedback control to enhance cell culture process production and robustness. Biotech & Bioeng. 2015, 112(12), 2495–2504

Disclaimer This feature has also been published in the AMERICAN LABORATORY Journal SEPTEMBER 2017 Spring 2019 Volume 2 Issue 1


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Ad Index Page 13

Abzena Ltd

Page 27

BioGenes GmbH

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Biopharma Process Systems

Page 15

Expres2ion Biotechnologies Aps

BC Page 23

FUJIFILM Wako Chemicals U.S.A. Corp

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GenXPro GmbH

Nemera

Novo Nordisk Ltd

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PCI Pharma Services Company Pharmaceutical Cleanroom Technology Europe

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Pharma Publications

Page 3

PlasmidFactory GmbH & Co. KG

IBC

Quick International Courier

IFC R.G.C.C. Group Page 11

Richter – Helm Biologics GmbH & Co. KG

Page 52

Sartorius AG

Page 35

SGS

Page 41 Page 5

Source BioScience

Wickham Laboratories Ltd

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