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Volume 2 Issue 4

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Cell Therapy

Challenges and Perspectives

Is Liver Biopsy a Gold or an Old Standard In NAFL and NASH

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Winter 2019 Volume 2 Issue 4

Contents 04 Foreword RESEARCH / INNOVATION / DEVELOPMENT 06 Cell Therapy: Challenges and Perspectives 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: Ana de Jesus ana@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 Spring 2020. 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 4 – Winter 2019

The achievements of cell-based therapeutics over the last decades have bolstered efforts in recent years to bring more of these products to market and across an ever more diverse range of applications. These advanced therapeutics offer promising potential to treat conditions which, to date, have defied traditional treatment modalities. Anna Gregson and Dean Houston at Mathys & Squire provide an overview of some of the key applications of cell therapies, as well as looking more closely at the challenges facing the evolution of this field. 10 Modelling Cancer for Immuno-oncology Applications: Past, Present & Future Cancer therapeutics have come a long way in a short time span, leading to significant paradigm shifts in drug discovery. The emergence of checkpoint inhibitors that target T lymphocytes has dramatically altered cancer treatment, availing a plethora of new opportunities that leverage the immune system either as a monotherapeutic, or increasingly within combinatorial treatments, in the clinical management of patients. Ivan Gladwyn-Ng at Taconic Biosciences reveals how a review of the past, present, and future of humanised models to support immuno-oncology demonstrates how these essential tools are helping researchers in their quest to advance cancer therapies. CLINICAL RESEARCH 14 Congenital Birth Defects Following Use of Dydrogesterone Versus Micronised Vaginal Progesterone as Luteal Phase Support: A Retrospective, Observational Study Incidence of congenital anomalies is reported to be higher in children born to couples with infertility compared to their fertile counterparts. Parental (chromosomal, genetic) and environmental factors are major contributory factors. In this study, Dr. Baidyanath Chakravarty et al. compare the incidence of congenital anomalies in babies born to infertile women with in-utero exposure to either dydrogesterone or micronised vaginal progesterone (MVP) following assisted reproductive technology (ART) and non-ART treatment, and in infertile women conceiving normally and not receiving any drug for luteal phase support (LPS). 20 Is Liver Biopsy a Gold or an Old Standard in NAFL and NASH? When defining the term gold standard, the most appropriate definition is “a benchmark test that is the best available under reasonable conditions”. Rafal Ziecina at Worldwide Clinical Trials demonstrates how when considering this definition, it becomes clear that non-invasive imaging is replacing liver biopsy as the gold standard for evaluation of fibrosis in non-alcoholic fatty liver disease (NAFLD). MANUFACTURING / TECHNOLOGY PLATFORMS 24 Split Butterfly Valve Technology and Eradicating the Risk of Contamination in Biologics Manufacturing As new biological therapies continue to be developed, the market has seen an increased need for innovation. Demand



Contents has grown for improved operational efficiency and achieving greater quality and cost reductions in manufacturing processes. Christian Dunne at ChargePoint Technology shows how one of the key challenges facing biopharma manufacturers is eliminating the risk of contamination, which requires effective containment strategies and monitoring of critical manufacturing processes. REGULATORY / QUALITY COMPLIANCE 28 Meeting the Standards Required for Effective Biorepository Management Biorepositories are a key asset for many organisations including those in the biotechnology, pharmaceutical and medical research areas. As they may contain human tissue and other material, possibly together with personally identifying information (PII), security is key and they can be subject to stringent regulations. Simon Wood at Autoscribe Informatics explores how the use of an appropriately configured laboratory information management system (LIMS) can contribute significantly to the efficient management of any biobank facility.

explains that it is essential that pharmaceuticals are protected throughout the supply chain end-to-end, as temperature excursions during transportation can even cause them to become toxic. SPECIAL FEATURE 44 Marine Extracts for Novel Medicines Developments: A Natural Solution to Antibiotic Resistance and Biofilmderived Infections In order to decrease the selection and spread of antibiotic resistance and to develop new therapies to fight against chronic infections, pharmaceutical companies are constantly looking for new resources to develop effective and safe medicines. Maxence de Villemeur at Lallemand Pharma discusses how for centuries, natural products have been used to develop medicines for the management and treatment of various diseases, as 70% of the planet is covered by water and is today one of the most promising sources of health ingredients for the future.

30 Is the Absence of Data Integrity Software Affecting the Assurance of Gel Clot Assays In Bacterial Endotoxin Testing? Data integrity remains an important topic in pharmaceutical and biopharmaceutical manufacturing science. The purpose of data integrity is to ensure that the accurate process of production and quality of the products are shown through the documentation that is associated with them. It is essential that facilities report what is occurring in their labs’ processes to ensure that good laboratory ethics are continuously practised throughout the production of their products. Although this is nothing new to the community, LaToya Mayfield at FUJIFILM Wako Chemicals U.S.A. Corporation states that every manufacturer around the world must ensure that the data related to their products has not been affected or compromised. 34 Best Practices of IoT Implementation for Smart Drug Research Drug discovery takes years to complete. Testing a single compound can cost more than £1.5 billion. Experiments on animals often fail to predict human behaviours and responses, because traditional animal anatomy often does not accurately mimic the human body. For these reasons, there is a vast need for other ways to emulate human diseases in vitro in order to accelerate the research and development of new drugs. Anindya Mookerjea, founder of S-Cube Technologies, explains why, to get the best of drug research using organ-on-a-chip (OOC) methods, connecting it to an IoT sensor would be the most effective. 40 Managing Global Supply Chains to Mitigate Risks in Pharma Logistics As temperature-sensitive pharmaceutical products are increasingly being shipped globally to more remote regions, there is an even greater demand for the effective and efficient management of global supply chains. Whether shipping finished products, transporting clinical trials materials or delivering sample drugs, Adam Tetz at Peli BioThermal 2 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

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Foreword Following the approval, in recent years, of the first immune checkpoint inhibitor, there has been an explosion in the development of immuno-modulating pharmacological modalities for the treatment of various cancers. From the discovery phase to late-stage clinical testing and regulatory approval, challenges in the development of immuno-oncology (IO) drugs are multi-fold and complex. In the preclinical setting, the multiplicity of potential drug targets around immune checkpoints, the growing list of immuno-modulatory molecular and cellular forces in the tumour microenvironment – with additional opportunities for IO drug targets, the emergence of exploratory biomarkers, and the unleashed potential of modality combinations all have necessitated the development of quantitative, mechanistically-oriented systems models which incorporate key biology and patho-physiology aspects of immuno-oncology and the pharmacokinetics of IO-modulating agents. In the clinical setting, the qualification of surrogate biomarkers predictive of IO treatment efficacy or outcome, and the corresponding optimization of IO trial design have become major challenges. In this issue of IBI Ivan Gladwyn-Ng at Taconic Biosciences reveals how a review of the past, present, and future of humanised models to support immuno-oncology

demonstrates these essential tools are helping researchers in their quest to advance cancer therapies. Within the Clinical Research Section, Dr. Baidyanath Chakravarty, a renowned IVF Phisician and his team, compares the incidence of congenital anomalies in babies born to infertile women with in-utero exposure to either dydrogesterone or micronised vaginal progesterone (MVP) following assisted reproductive technology (ART) and non-ART treatment. Within the Regulatory Section, Simon Wood at Autoscribe Informatics explores how the use of an appropriately configured laboratory information management system (LIMS) can contribute significantly to the efficient management of any biobank facility, and Anindya Mookerjea, founder of S-Cube Technologies, explains why, to get the best of drug research using organ-on-a-chip (OOC) methods, connecting it to an IoT sensor would be the most effective. The IBI Team wishes all our partners, a wonderful start to the year 2020, my team and I look forward to bringing more exciting articles and features in the next issue of IBI. Virginia Toteva, Editorial Manager

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.

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

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Research / Innovation / Development


Cell Therapy: Challenges and Perspectives

The achievements of cell-based therapeutics over the last decades have bolstered efforts in recent years to bring more of these products to market and across an ever more diverse range of applications. These advanced therapeutics offer promising potential to treat conditions which, to date, have defied traditional treatment modalities. Interest and investment in this sector are at an all-time high and whilst many are hopeful of a boom in the number of approved therapies in the coming years, the industry still faces significant challenges, particularly with regard to the manufacture and regulation of these cell-based products. In this article, we will provide an overview of some of the key applications of cell therapies as well as look more closely at the challenges facing the evolution of this field. To date, the applications of cell therapies have largely fallen into two broad categories; tissue regeneration and immunomodulation. With regard to the former, cell therapy has been viewed as one of the most promising techniques for the repair of damaged tissue, with applications in cardiovascular disease, neurodegenerative disease (for example, Parkinson’s and Alzheimer’s), musculoskeletal injury or degeneration and endocrine dysfunction (for example, type I diabetes). Cell therapies have proven particularly effective in the repair of articular cartilage, for which the intrinsic capacity for repair is low. The most established of these therapies have employed the patient’s own cells, i.e. autologous cells. In brief, harvested chondrocytes are expanded ex vivo, seeded into a collagen matrix and then re-implanted into cartilage defects in joints. Such products have been available for around a decade now (ChondroCelect, developed by TiGenix was first approved in the EU in 2009) and have shown considerable efficacy, although use of these advanced options is still low when compared to traditional treatment modalities (for example, joint replacement and analgesics). Whilst cartilage repair applications have tended to employ the terminally differentiated chondrocyte, bone repair applications have made use of the regenerative capacity of stem and progenitor cells. Bone marrow-derived mesenchymal stem cells (MSCs) have been proven in a range of orthopaedic applications over recent decades, including in the treatment of infants with osteogenesis imperfecta and in the repair of non-union fractures. Unfortunately, obtaining sufficient yields of pure MSC populations from bone marrow has proven difficult and there has been a switch in recent years to utilise MSCs derived from other sources, such as adipose tissue. Autologous cell therapies like those discussed above all depend on obtaining sufficient cell numbers from the donor patient and the ability to expand functional cells ex vivo. Off-theshelf cell therapies, which clinicians can employ for a range of patients, as and when needed, without concerns over yield or 6 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

expansion protocols, are likely to represent the future of cell therapy. UK-based biotech, ReNeuron, is one such company forging ahead with allogeneic cell therapies. Interestingly, ReNeuron’s neural stem cell line for the treatment of the disabling effects of stroke were cryopreserved prior to utilisation in the PISCES I (Phase I) clinical trial. Cryopreservation is just one of a number of advancements which will be necessary to bring off-theshelf cell products to reality. Whilst the regenerative applications of cell therapies have, at the very least, been researched for some time now, the immunomodulatory applications of cell therapy, in particular, chimeric antigen receptor (CAR) T cells, is a more recent development. Indeed, it was only in the early 90s when first-generation CAR T cells (which contained an antibody/T cell receptor fusion molecule) were developed and around the same time researchers were investigating adoptive transfer of patientderived virus-specific T cells. Since these early days, significant leaps forward have been made. In 2017, Novartis’ Kymriah (tisagenlecleucel) became the first CAR T cell therapy to be approved by the FDA, with Kite Pharma’s Yescarta (axicabtagene ciloleucel) following shortly thereafter. Data from the UK’s Cell and Gene Therapy Catapult clinical trials database indicates that there were around 22 clinical trials investigating the safety and efficacy of CAR T cells in the UK alone in 2018. The success of CAR T cells to date has largely been shown for haematological malignancies (indeed, Kymriah and Yescarta are approved for the treatment of acute lymphoblastic leukaemia and large B-cell lymphoma respectively). In contrast, despite extensive research, CAR T cell therapy for solid tumours hasn’t had the same impact, not least because of the challenges of targeting solid tumours including identifying a suitable target antigen and homing the cells to the hostile, tumour microenvironment. Nonetheless, strides are being made by combining CAR T cell therapy with other biologic agents, specifically checkpoint inhibitors such as pembrolizumab and nivolumab which target programmed cell death protein 1 (PD-1) a key regulatory protein found on T cells. The University of Pennsylvania, for example, is recruiting for a Phase I clinical trial assessing the safety of a CAR T cell/pembrolizumab combination therapy for the treatment of glioblastoma. This follows preliminary evidence from the Memorial Sloan Kettering Cancer Center that showed both safety and efficacy of a mesothelin targeting CAR T cell and pembrolizumab combination therapy in patients with malignant pleural disease. Thus, the use of CAR T cells for the treatment of solid tumours appears to be progressing. Immuno-modulatory cell therapies other than CAR T cells are also being investigated in the clinics. By way of example, Fate Therapeutics is currently assessing the safety of its off-the-shelf Natural Killer (NK) cell therapy. Unlike traditional CAR T cells, Fate’s NK cell therapies are derived from an induced pluripotent stem cell (iPSC) line allowing the production of large numbers of well-defined cells without relying on a patient’s own immune cells (which are often depleted in many cancers). Preclinical studies showed the efficacy of these cells in the treatment of checkpoint Winter 2019 Volume 2 Issue 4


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Research / Innovation / Development

inhibitor resistance tumours. At present, Fate has a pipeline of at least five different NK cell therapies. As well as the immuno-oncology applications, cell therapies are also being trialled for immuno-regulatory applications such as in the treatment of autoimmune disease and graft versus host disease. These trials have largely involved the use of autologous, expanded, regulatory T cells (Treg cells) which, through a range of mechanisms, are able to suppress a variety of immune cells. Treg cells used in studies to date have been isolated from both umbilical-cord blood and peripheral blood. A variety of Phase I studies have been completed or are in the process of assessing the safety of Treg cells for the treatment of type I diabetes. Although in the early stages of development, data to date is showing that Treg cells are well tolerated in patients and the ex vivo expansion methods are capable of generating sufficient numbers of stable and functional Treg cells. Future Phase II/III trials will of course be needed to reveal the true potential of these cells. Global investment in cell-based therapies increased to US$7.6 billion in 2018, a 64% increase from the previous year. In spite of this, the sector still faces a number of significant challenges before these advanced therapeutics become widely used. Research and development in this sector is undeniably booming, though difficulties in expanding, manufacturing and transporting cell products may be hampering the commercial viability and ultimate availability of these products. Achieving the quantity of cells needed with current production methods, especially if uptake of these therapies becomes more widespread, is one of the major hurdles facing the industry. By way of example, the recommended dose of ChondroCelect is 1 million cells/cm2 of cartilage defect. CAR T cell therapy Yescarta is dosed at a 8 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

staggering 2 million cells per kg (around 140 million cells for an average adult male). The issue is magnified somewhat by the focus of today’s research on the cell product per se; emerging biotech companies with innovative cell therapies should, at an early stage, consider the processes that will be necessary to achieve the desired cell numbers for later Phase II/III trials and beyond. These challenges also bring opportunities however, and there are now a number of innovative companies seeking to develop solutions for the industry, to simplify, accelerate and improve cell therapy manufacturing and supply. Automation of the manufacturing processes is currently of significant interest to the community. At present, the manufacturing processes employed in the generation of cell therapies largely resemble those utilised in other biopharmaceutical areas (for example therapeutic antibodies). Unlike therapeutic antibodies production, however, cell therapies (especially those relying on patient or donor cells) vary significantly from batch to batch, requiring complex and adaptive processes to generate consistent products within the regulatory confines. Through the implementation and training of a variety of mechanisms, e.g. sensors, robotics and image acquisition as well as processing software, researchers believe variability and reliability of current manufacturing processes can be improved. Whilst improvements in the manufacturing processes will hopefully lead to a reduction in the costs associated with the production of cell therapies, it should be noted that, unlike traditional therapeutic modalities, cell therapies are often a one-off treatment option for patients. Biotech companies must bear this in mind when attempting to recoup their research and development costs and, as such, costs are always likely to Winter 2019 Volume 2 Issue 4

Research / Innovation / Development be higher than traditional biologics. As it stands, the high costs associated with these therapies is proving challenging for healthcare providers to justify. The cost of these therapies is at least in part due to the convoluted path from bench to bedside. Cell therapies are considered differently to the conventional biopharmaceutical agents and have to undergo even more rigorous regulatory and quality assessments. This of course ensures public safety, but has also put the brakes on the number of cell therapies actually being approved (despite the ample number of trials). As is so often the case, the regulatory frameworks in place have not been able to keep up with the unprecedented scientific advances in this field. What’s more, the absence of harmonisation across jurisdictions has placed undue burden on the smaller players in this field. The lengthy timescales involved in obtaining regulatory approval (even after showing clinical efficacy) are exemplified by Holclar, an autologous cell therapy (comprising human corneal epithelial cells and limbal stem cells) for the repair of damaged cornea, which despite having shown clinical efficacy as early as 1997, only obtained regulatory approval in 2015. Regulation is of course paramount to ensure the safety of patients receiving advanced therapeutics (including cell and gene therapies) which have long been shrouded in safety concerns. These concerns are not without basis. Indeed, safety has been a major sticking point for stem cell therapies. The primary concern regarding stem cell therapies is unwanted differentiation, as has been shown in the cardiovascular setting, where calcifications have been identified in the myocardium of patients treated with MSCs following infarction (MSCs, of course, give rise to cells of bone and cartilage as well as muscle). Tumorigenesis has also been a concern for stem cell therapies, although this appears to have been unwarranted based on current data. In the immuno-oncology field, CAR T cells have also been associated with safety concerns including the development of cytokine release syndrome in patients receiving CAR T cell therapies, the engagement of target antigens on non-pathogenic tissues and host immune response to the specific recombinant proteins found in these cells. Pleasingly, the industry is seeking solutions to these problems and research is ongoing to improve the safety profile of these therapies. In the CAR T cell space, the incorporation of suicide or elimination genes into delivered cells is being investigated as a means to selectively deplete these cells in the body when necessary. The approved cell therapies are largely still in their infancy and data from future Phase IV clinical trials will be indispensable in assessing the long-term safety of these therapies. The number of cell therapies actually approved for clinical use remains small. This highlights that, despite the significant scientific advances and investment, the sector is largely still at the research and development stage. Having said that, the industry appears to have reached a critical mass and with the number of clinical trials in this field growing steadily, we can only assume that we will be seeing more and more of these therapies in the clinics. The industry seems to have clicked and more emphasis is now being placed on the challenges of efficiently, yet safely, manufacturing these products. Improvements in this key area could pave the way for wider implementation and access to these therapies. A multidisciplinary approach will be essential in the coming years to increase the number of approved therapies whilst still ensuring affordability and, importantly, patient safety. www.biopharmaceuticalmedia.com

Anna Gregson Anna is a partner in Mathys & Squire’s biotech team. She has over 10 years’ experience working with a diverse client base; from university technology transfer organisations to international corporations. Anna’s expertise covers a wide range of biotechnology and life sciences subject matter, with a particular emphasis on the cell and gene therapy space, including CAR T cells, iPSCs, neural regenerative medicine, cell culture technologies, and viral/non-viral gene therapy vectors. Email: algregson@mathys-squire.com

Dean Houston Dean is a trainee patent attorney in Mathys & Squire’s biotech team. He has a strong background in the cell and gene therapy sector, having completed his PhD at The Roslin Institute in Edinburgh. Dean has experience in the global prosecution of patent families across a diverse range of technologies, including therapeutic toxins, antisense oligonucleotides, vaccines and methods of neuronal stem cell culture. Email: dahouston@mathys-squire.com


Research / Innovation / Development

Modelling Cancer for Immuno-oncology Applications: Past, Present & Future Cancer therapeutics have come a long way in a short timespan, leading to significant paradigm shifts in drug discovery. The emergence of checkpoint inhibitors that target T lymphocytes have dramatically altered cancer treatment, availing a plethora of new opportunities that leverage the immune system either as a monotherapeutic, or increasingly within combinatorial treatments in the clinical management of patients. This successful targeting of T cells in oncotherapies has led researchers to explore the possibilities of harnessing other immune cell types that also contribute to cancer biology in immunotherapies. With the advancement of such treatment modalities, there has been a corresponding advancement in the animal models that serve as preclinical tools, most notably mouse models in which a human immune system is engrafted into an immunodeficient host, enabling the more faithful recapitulation of targets within the immune system. A review of the past, present, and future of humanised models to support immuno-oncology demonstrates how these essential tools are helping researchers in their quest to advance cancer therapies. The Past: T Cells Take Centre Stage There is no denying the substantial contribution of immune checkpoint inhibitors (ICIs), such as anti-PD-1, anti-PD-L1, and anti-CTLA-4, to the oncology field, as evidenced by the awarding of the 2018 Nobel Prize in Physiology or Medicine to James P. Allison and Tasuku Honjo – two immunologists whose work led to pioneering discoveries on the manipulation of immune checkpoints for cancer treatment. ICIs work by blocking immune checkpoint proteins from binding to their partner proteins, which prevent an “off-signal” from being sent to immune cells. The approved ICIs mentioned above prevent cancerous cells from evading attack from immune T lymphocytes, thereby improving the latter’s ability to mount an immune response against the tumour cells. Whilst the breakthrough in immuno-oncology is built upon a wealth of preclinical findings from in vitro and in vivo research – the latter comprising syngeneic and xenograft rodent models – the recent advancement in immunotherapies is increasingly facilitated by humanised mouse models. In greater detail, immuno-deficient mice are engrafted with either human hematopoietic stem cells (HSCs) or peripheral blood mononuclear cells (PBMCs) – or specific immune cells such as natural killer (NK) cells – in order to reconstitute the human immune system (or specific immune sub-compartments). The resulting models are known as humanised immune-system (HIS) rodent models. These HIS models are then implanted with human tumour cells derived from immortalised cell line- or patient-derived xenografts (CDX and PDX, respectively). In one early study, HIS mice were implanted with human leukocyte antigen (HLA) partially matched 10 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

tumours of various histological types from either PDXs or from a triple-negative breast cancer CDX. After treatment with the anti-PD1 pembrolizumab (Keytruda®), growth of the PDX and CDX tumours was significantly inhibited in the humanised immune system mice.1 The Present: A Broader Perspective With the increasing successes of preclinical investigations and clinical trials since the 2000s that led to the approval of blockbuster ICIs such as pembrolizumab, nivolumab (Opdivo®) and ipilimumab (Yervoy®) in the early 2010s, many subsequent preclinical studies are investigating the use of ICIs in combination with other therapeutic agents. The efficacy of pembrolizumab was explored in combination with ONCOS-102, an oncolytic adenovirus which the researchers hypothesised could serve as an immunosensitiser when used with an ICI. In HIS mice engrafted with human A2058 melanoma cells, treatment with pembrolizumab alone showed no therapeutic effect, whereas treatment with ONCOS-102 led to a significant reduction in tumour volume, and combinational co-treatment of pembrolizumab and ONCOS-102 reduced tumour volume to a greater degree.2 While checkpoint inhibitor therapy has become the treatment of choice for an increasing number of oncology indications – both as monotherapy and in combination – with greatly improved treatment outcomes, especially for a subset of cancer patients that were previously refractory to other treatment modalities, marked differences exist between responders and non-responders. Additionally, these T cell-based immunotherapies are associated with risks such as cytokine release syndrome and neurotoxicity, and they are not always feasible for patients who are already immuno-depressed after receiving a first-line cancer treatment. These limitations have led oncology researchers to venture far beyond the boundaries of T cells and ICIs. A natural evolution of the prior immunotherapies described above has been to enhance the efficacy of T cell-based therapies, which is being accomplished in several ways. Current immuno-oncology research is focusing on a wider range of therapeutic approaches and cell types. One such approach is chimeric antigen receptor (CAR)-T cell therapy, which involves genetically modifying a patient’s T cells to render them capable of recognising and killing cells that express the target protein. The first two CAR-T cell-based therapies were approved by the US Food & Drug Administration (FDA) for haematologic cancers (Kymriah® from Novartis and Yescarta® from Gilead Sciences) and reached the market in 2017, contingent on physicians completing adverse effects management training due to the known risks of cytokine release syndrome and neurotoxicity.3 In late 2019, Gilead released data that reported a three-year survival rate of 47% in patients with refractory large B cell lymphoma treated with its Yescarta® CAR-T cell therapy. Concurrently, Johnson & Johnson’s investigational CAR-T therapy (JNJ-4528) received the Breakthrough Therapy Designation from the FDA after a clinical Phase Ib trial in which patients with relapsed or refractory Winter 2019 Volume 2 Issue 4

Research / Innovation / Development

multiple myeloma showed a 100% response rate at a median six months follow-up post-treatment. Haematologic cancers have been the initial focus of adoptive cell transfer of T lymphocytes, which has proven efficacious in some tumour types. In a preclinical study of prostate cancer, adoptive transfer of naïve T lymphocytes (including CD4 T cells) from female mice were transplanted into irradiated male syngeneic mice that were subsequently implanted with TRAMP-C2 prostate tumour cells. The female lymphocytes slowed the growth of the implanted cancer cells, with no worsening of graft-versushost disease (GvHD) despite implanting cells across genders.4 Although CAR-T therapy has been approved for haematological cancers such as Non-Hodgkin’s lymphoma and acute lymphoblastic leukaemia, ongoing preclinical work is addressing its toxicity and efficacy, as well as the challenge of CAR-T cells migrating into solid tumours. In NOG mice expressing human IL-2 cytokine, CAR-T cell therapy was effective in targeting both xenografted uveal and cutaneous melanoma cells.5 These findings were consistent with the results of clinical trials, even in patients who had proven resistant to adoptive cell transfer of autologous tumour-infiltrating T lymphocytes (TILs). Nevertheless, the potential efficacy of CAR-T therapy must be balanced by the presence of similar risks as found in other T cell therapies, as well as a risk of cerebral oedema. Continued preclinical work aims to improve upon CAR-T therapy to overcome these drawbacks. Though these approaches have demonstrated good efficacy in some applications, T cell-based immunotherapy risks have prompted investigators to explore other immune cell types to develop newer therapeutic modalities. NK cells are one option www.biopharmaceuticalmedia.com

gaining traction because they can be mobilised to target tumour cells by using various germ line-encoded cell surface receptors, in order to circumvent the risks and limitations of T cell therapies. Whilst early efforts to engraft NK cells into immune-deficient models have been limited by poor cell uptake and a short survival window for the recipient mice, research at the Central Institute for Experimental Animals (CIEA) demonstrated improved NK cell engraftment and survival is feasible using a mouse model that transgenically expresses human interleukin 15 (IL-15). In comparison to the conventional NOG mouse, the engraftment of human PBMCs in this hIL-15 NOG mouse led to significantly higher levels of human NK cell reconstitution, with a longer duration of survival without indications of GvHD, all of which resulted in a feasible study window for assessing NK cell therapeutics.6 Recognising the role of cytokines in immune response, further characterisation of IL-15 and IL-2 is underway to determine their ability to stimulate NK cell anti-tumour immunogenicity, in order to overcome limitations in NK cells’ ability to zero in on tumours.7 Concurrently, clinical trials are investigating the subcutaneous administration of IL-2 in low doses in conjunction with NK cell therapy, based on preclinical studies demonstrating that low doses of IL-2, following chemotherapy-induced cytopenia, promoted anti-tumour response. One of the known challenges of modelling the interactions between the immune system and tumour cells is that the development of certain human immune cell lineages (including myeloid cells) is restricted in most HIS mouse models. In order to enable a more comprehensive reconstitution of the human immune system and improve the levels of human myeloid cells following HSC engraftment in mice, researchers are investigating INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 11

Research / Innovation / Development the efficient development of neutrophils, granulocytes, and macrophages in HIS mice. In turn, preclinical mouse models that express the human cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (CM-CSF) are becoming more widely used in immuno-oncology, as these cytokines are critical for myeloid development and differentiation. These models, including the NOG-EXL and huNOG-EXL, support expanded myeloid lineage development and improved T cell engraftment, making them especially suitable for studying ICIs in combination with other therapies. Using a breast tumour model with expanded development of lymphocytic and myeloid lineages, researchers investigated the effects of niraparib, a PARP-1/2 inhibitor, on the efficacy of an anti-PD-1 therapeutic and discovered the administration of these therapeutics together resulted in synergistic anti-tumour activities in both BRCA-proficient and BRCA-deficient tumours.8 Oncological Vaccines on the Rise While ICI therapy has become a standard of care in oncology, the relatively high percentage of non-responders continues to encourage researchers to explore new alternative treatments, such as increasing investigations into therapeutic cancer vaccines. Such vaccines are designed to elicit an immune response against tumour antigens, which are known to play a role in the development, progression and metastasis of cancers. Oncological vaccine research is yielding new insights and developments, in both target and antigen selection and in vaccine technology. Antigen selection can be especially problematic, as few antigens meet all the criteria considered necessary for cancer vaccine efficacy, including the need for the antigen to be expressed by cancer cells only, present on all cancer cells, essential for cancer cell survival, and highly immunogenic.9 The primary oncological vaccine targets are tumour-associated antigens (TAAs) and tumour-specific antigens (TSAs), including neoantigens. Many studies demonstrate that response to ICI therapy is often correlated with a high number of predicted neoantigens.9 Yet, most neoantigens are unique to the patient and their number varies by tumour type, necessitating a personalised approach that can prove cost-prohibitive. Nevertheless, preclinical proof-of-concept studies in mice have tested the feasibility and efďŹ cacy of employing vaccines targeting neoantigens.


Post-vaccination with synthetic long peptide neoantigens, three mouse models experienced noteworthy anti-tumour response,9 while a mouse model of Lynch syndrome (associated with a high risk of colorectal cancer and increased risk of other cancer types) was vaccinated with four tumour neoantigens and subsequently experienced significantly reduced tumour burden in the intestines and improved survival as compared to non-vaccinated mice.10 Nucleic acid vaccines, such as DNA-based vaccines, represent another avenue of promising immuno-oncology research. Most DNA vaccines are plasmids that deliver genes encoding tumour antigens, which in turn elicit an immune response against tumour cells bearing those antigens. While DNA vaccines have proven to be well tolerated without significant risk of major adverse effects, they tend to demonstrate limited therapeutic effects due to poor immunogenicity.11 A number of strategies are under investigation to improve DNA vaccine efficacy, including improving immunogenicity through selection of the optimal antigens for insertion into the DNA, or combining a DNA vaccine with other therapies in order to modulate tumour immunosuppression or improve immune cell volume and activity.12 A third approach to oncological vaccines is to adapt a patient’s dendritic cells to express tumour-associated antigens, thereby encouraging T cells to attack cancer cells. Currently, efficacy limitations have hampered the success of this approach in clinical trials, but several strategies may enhance efficacy, including the identification of subsets of dendritic cells that express high levels of targeted antigens and the improvement of vaccine delivery to lymph nodes.13 The Future: Modelling Pharmacokinetics and Pharmacodynamics Building on efforts to improve immunotherapy efficacy, future immuno-oncology research is likely to focus on enhancing the pharmacokinetic (PK) and pharmacodynamic (PD) profile of immuno-oncology therapeutics. A better understanding of the absorption, distribution, metabolism and excretion (ADME) of an immunotherapeutic, as well as its mechanistic action (including response magnitude and duration), is critical to improving the viability of these therapeutics, yet much remains unknown.

Winter 2019 Volume 2 Issue 4

Research / Innovation / Development A critical foundational tool for this work is preclinical models capable of modelling human pharmacokinetics and pharmacodynamics more faithfully, which remains a challenging endeavour. Syngeneic mouse models have been used to study the toxicity of immunotherapies, since such models represent the interactions between murine host cells and a competitive immune system.14 Hence, ongoing development of preclinical models is targeted towards improving their ability to accurately assess appropriate dosing levels that balance the efficacy and safety of immunotherapies, making this an essential area of emphasis in the evolution of model development. One of the most significant obstacles to gaining a better understanding of the PK and PD profiles of an immunotherapy is the potential for the human microbiome to impact drug activity, metabolism and toxicity. Enright et al reviewed the various ways in which human microbiota may increase a drug’s activity, render it inactive, or lead to the accumulation of toxic metabolites.15 In fact, preclinical and clinical studies have found that patients’ marked differences in response to ICI therapy may be a function of differences in their gut microbiome compositions. When patients with non-small cell lung, kidney or bladder cancer received antibiotics before or shortly after treatment with an anti-PD-1 therapeutic, they experienced shorter progression-free and overall survival than those who had not received antibiotics.16 When germ-free mice then received faecal microbiota transplantation (FMT) from patients, anti-tumour activity was evident in the mice that received FMT from responder patients and anti-PD-1, a response that was correlated with one bacterial strain.16 Using melanoma models, researchers at the University of Texas MD Anderson Cancer Center and the University of Chicago found similar correlations between the microbiome composition and response to anti-PD-1 or anti-PD-L1. The field of immuno-oncology has experienced both dramatic breakthroughs and steady improvements, all contributing to significant advances in how cancer is treated. Preclinical models have served and will continue to serve as effective tools for the study of immunotherapy efficacy and safety, across an expanding array of immune cell types and therapeutic approaches. As immuno-oncology investigators continually push the boundaries of their research, seeking the most efficacious treatments with the most desirable safety, PK and PD profiles, these models will evolve to stay in step with investigative trends.












1. Wang M., Yao L.C. et al. Humanized mice in studying efficacy and mechanisms of PD-1-targeted cancer immunotherapy. FASEB J. 2018 Mar;32(3):1537-1549. doi: 10.1096/fj.201700740R. Epub 2018 Jan 3. 2. Kuryk L., Møller A.W., Jaderberg M. Combination of immunogenic oncolytic adenovirus ONCOS-102 with anti-PD-1 pembrolizumab exhibits synergistic antitumor effect in humanized A2058 melanoma huNOG mouse model. Oncoimmunology. 2018 Oct 29;8(2):e1532763. doi: 10.1080/2162402X.2018.1532763. eCollection 2019. 3. June, C.H., O’Connor, R.S. et al. CAR T cell immunotherapy for human cancer. Science. 2018 March 23; 359, 1361–1365. doi: 10.1126/ science.aar6711. 4. Jenq R., Curran, M.A. et al. Repertoire enhancement with adoptively transferred female lymphocytes controls the growth of pre-implanted murine prostate cancer. PLoS One. 2012;7(4):e35222. doi: 10.1371/ journal.pone.0035222. Epub 2012 Apr 6. 5. Jespersen H., Lindberg M.F. et al. Clinical Responses to Adoptive



T-Cell Transfer Can Be Modeled in an Autologous Immune-Humanized Mouse Model. Nat. Commun. 2017, 8 (1), 707. doi: 10.1038/s41467017-00786-z. Volden, P. New PBMC-humanized Mice Support Efficient NK-cell Engraftment https://www.taconic.com/taconic-insights/oncologyimmuno-oncology/humanized-mice-nk-cell-engraftment.html (accessed Mar 11, 2019). Childs R., Carlsten M. Therapeutic approaches to enhance natural killer cell cytotoxicity against cancer: the force awakens. Nat Rev Drug Discov 14, 487–498 (2015) doi:10.1038/nrd4506. Wang Z., Sun K. et al. Niraparib activates interferon signaling and potentiates anti-PD-1 antibody efficacy in tumor models. Sci Rep. 2019 Feb 12;9(1):1853. doi: 10.1038/s41598-019-38534-6. Hollingsworth R.E., Jansen K. Turning the corner on therapeutic cancer vaccines. npj Vaccines 4, 7 (2019) doi:10.1038/s41541-0190103-y. Gelincik O., Ibrahim H. et al. Frameshift neoantigen vaccination prevent Lynch syndrome mouse model intestinal cancer. Proceedings of the AACR Annual Meeting 2019; 2019 Mar 29-Apr 3;: AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2723.  Yang B., Jeang J. et al. DNA vaccine for cancer immunotherapy. Hum Vaccin Immunother. 2014 Nov; 10(11): 3153–3164. doi: 10.4161/21645515.2014.980686. Lopes A., Vandermeulen G., Préat V. Cancer DNA vaccines: current preclinical and clinical developments and future perspectives. J Exp Clin Cancer Res. 2019; 38: 146.10.1186/s13046-019-1154-7. doi: 10.1186/s13046-019-1154-7. Riley R.S., June C.H., Langer R.  et al.  Delivery technologies for cancer immunotherapy. Nat Rev Drug Discov 18, 175–196 (2019) doi:10.1038/s41573-018-0006-z. Ochoa de Olza M., Oliva M. et al. Early-drug development in the era of immuno-oncology: are we ready to face the challenges? Annals of Oncology, Volume 29, Issue 8, August 2018, 1727–1740, https:// doi.org/10.1093/annonc/mdy225. Enright E., Gahan C.G.M. et al. The impact of the gut microbiota on drug metabolism and clinical outcome. Yale J. Biol. Med. (2016). Routy B. et al. Gut microbiome influences efficacy of PD-1-based immunotherapy against epithelial tumors. Science (80-. ). (2018). doi:10.1126/science.aan3706.

Ivan Gladwyn-Ng Ivan Gladwyn-Ng, PhD, is a Field Applications Scientist at Taconic Biosciences. He earned his PhD at the Australian Regenerative Medicine Institute at Monash University in Melbourne, Australia, where he studied cortical development, epilepsy and autism in children using transgenic mouse models. He completed postdoctoral fellowships at the GIGA-Neuroscience Institute and the Institut Pasteur.


Clinical Research


Congenital Birth Defects Following Use of Dydrogesterone Versus Micronised Vaginal Progesterone as Luteal Phase Support: A Retrospective, Observational Study Objectives: To compare the incidence of congenital anomalies in babies born to infertile women with in-utero exposure to either dydrogesterone or micronised vaginal progesterone (MVP) following assisted reproductive technology (ART) and non-ART treatment, and in infertile women conceiving normally and not receiving any drug for luteal phase support (LPS). Methods: This retrospective observational study was carried out at a tertiary level infertility centre, in Kolkata, India. Women (n=6549) in the age group of 21–45 years with a history of primary or secondary infertility were screened from January 2002 to June 2017, who had undergone ART or non-ART procedures, and had received either MVP or dydrogesterone as LPS. Infertile women with natural conception and not exposed to drugs for LPS served as control/s. Results: 6298 women met all criteria and were sub-stratified into women with natural conception not receiving LPS (n=2003; Group A), women who received MVP (n=2103; Group B) and those who received dydrogesterone (n=2192; Group C) as LPS. All demographic parameters were comparable amongst the three groups. The overall pregnancy rate was 32.01%, 29.15%, and 30.72% for Groups A, B, and C, respectively. Intrauterine foetal outcomes and neonatal characteristics were also comparable amongst the three groups. A total of 1851 children were born to 6298 women; 613 in Group A, 589 in Group B and 649 in Group C. Congenital (Group A: 2.45%, Group B: 2.89%, Group C: 3.08%), functional (Group A: 3.92%, Group B: 4.58%, Group C: 4.16%) and chromosomal (Group A: 0.49%, Group B: 0.68%, Group C: 0.31%) anomalies were comparable in all three groups. Conclusions: Dydrogesterone did not result in increased incidence of congenital malformations compared with MVP as LPS, and had similar incidence to those observed in infertile women who conceived naturally and did not receive LPS. Introduction Incidence of congenital anomalies is reported to be higher in children born to couples with infertility compared to their fertile counterpart/s1–3. Parental (chromosomal, genetic) and environmental factors (such as endocrine, infective-viral e.g. rubella, body mass index and smoking, etc.) are major contributory factors. Infertility treatments may have additional impact for higher incidence of congenital anomalies through various technologies involved, or from hormonal and non-hormonal drugs 14 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

administered 3,4,5 during the treatment procedure/s. Available data is, however, not conclusive on the actual risk involved from infertility treatments2,6–12. In fact, a longitudinal study of the Danish national birth cohort showed that congenital malformations in children born to couples with infertility were higher, irrespective of treatment1; and that the congenital malformations were partly due to the underlying infertility or its determinants. Hence, based on current evidence, there are no clear factors to distinguish congenital anomalies arising from the effect of parental sub-fertility or from those occurring as a consequence of infertility treatment3. Infertility treatments consist of two areas: procedural impact and use of drugs. A study by Pinborg et al. in 2013 indicates that overall risk of procedural impact is not increasing over time and there is a tendency for decline in the prevalence of congenital anomalies in the younger ART generation3. Procedural impact in ART or non-ART treatment drugs used in infertility management and their possible role on enhancing incidence of foetal congenital anomalies has not been specifically ascertained and highlighted in the majority of reviews published so far10. However, two recent reports indicate that amongst various drugs used in infertility management, progesterone may have an increased adverse impact on foetal congenital disease or deformities11,12. But a possible mechanism and large-scale evidence-based study has not been reported through either of the publications. Progesterone is a natural gestational support which has to be supplemented compulsorily as a luteal phase support (LPS) in pregnancies following assisted reproductive technologies (ART) 13,14 and possibly in other non-ART pregnancies achieved with or without treatment in women with infertility. The usefulness of progesterone14,15, especially in pregnancies following ART treatment and also in threatened and unexplained miscarriage, is well established16–18. The vaginal route is the preferred mode of administration for progesterone worldwide, and the pharmacological preparation commonly used is micronised vaginal progesterone (MVP). Dydrogesterone, an orally active progestogen with enhanced bioavailability, is similar to endogenous progesterone in its molecular structure and has a high affinity for progesterone receptors19. Dydrogesterone is approved in several countries for the treatment of progesterone deficiencies and has been used as a hormone replacement therapy since the 1960s. More recently, the non-inferiority of oral dydrogesterone to MVP (capsule or gel) for LPS in fresh-cycle IVF was demonstrated in the Phase III, randomised LOTUS I and LOTUS II studies20,21. Several other smaller studies including those by our team, and a meta-analysis have also shown that dydrogesterone is at least as effective as MPS for LPS22–28, with a favourable benefit-risk profile comparable with MVP20,22,26. Based on the LOTUS I and II studies, dydrogesterone has now been approved in several countries including India for use in LPS as part of ART treatment21. There are, however, conflicting reports on risk of congenital Winter 2019 Volume 2 Issue 4

Clinical Research anomalies with dydrogesterone use, but reports on this topic are not univocal20,21,29,30,31. This discrepancy motivated us to study through retrospective analysis of our own data to evaluate the occurrence of congenital birth defects in children born to Indian women with infertility receiving dydrogesterone or MVP as LPS following ART and/ or non-ART treatment (ovulation induction [OI], intrauterine insemination [IUI]) and also with babies born to infertile women conceiving naturally and not receiving any LPS. Materials and Methods Study Design This retrospective observational study was conducted at the Institute of Reproductive Medicine, Salt Lake City, Kolkata, India, a tertiary level infertility centre. Medical records at the institute were screened to identify and follow-up babies born between January 2002 and June 2017 to women with infertility who received dydrogesterone or MVP as LPS following ART or non-ART procedures; additionally, women who conceived naturally during this period without LPS were included for comparison. The study protocol was approved by the Research Ethics Board of the institute prior to the commencement of the study. All women

consulting at the institute signed consent forms allowing the use of their health information except personal identification for the purpose of research or publication. Simultaneously, on admission for delivery, a consent form was received allowing the use of the newborn’s medical information for research purpose. As this was a retrospective study, data was obtained from the medical files of mothers and newborns. The trial is registered at http://www. ISRCTN.org (Trial ID: ISRCTN55659103). Patient Selection Women in the age group of 21–45 years with a history of primary and/or secondary infertility, e.g. fallopian tube obstruction, polycystic ovary syndrome, endometriosis and/or having partners with male factor infertility were included. Women with adenomyosis, hyperprolactinemia, hypothyroidism, congenital uterine anomalies, uterine synechiae, or baseline follicle stimulating hormone >12 international units, and those who were donor oocyte recipients or gestational surrogates were excluded from the study. All women were carefully scrutinised for the presence of the following risk factors for congenital malformation: history of congenital/chromosomal anomaly in the previous pregnancies; recurrent pregnancy loss; family history of birth defects; presence

Fig 1. Study design. OI: Ovulation induction, IUI: Intrauterine insemination, IVF: In-vitro fertilisation, LPS: Luteal phase support, IUGR: Intrauterine growth restriction, IUFD: Intrauterine foetal death, APGAR: Appearance, pulse, grimace, activity, respiration; NICU: Neonatal intensive care unit, RDS: Respiratory distress syndrome www.biopharmaceuticalmedia.com


Clinical Research of medical disorders; concomitant use of other drugs; smoking; alcohol consumption during pregnancy and occupational exposure to endocrine disruptors/ radiation. Exogenous Progesterone Supplementation for LPS Women undergoing ART or non-ART procedure/s received either MVP (Susten®, Sun Pharmaceutical Industries Ltd, India) 200 mg thrice daily, or oral dydrogesterone (Duphaston, Abbott India Ltd) 10 mg thrice daily as LPS for up to 12 weeks of gestation (Fig. 1).

Table 2: Causes of infertility categorized by type of treatment procedure in women receiving MVP or dydrogesterone

Table 1: Demographics and baseline characteristics of three groups with or without treatment of luteal phase support with micronised vaginal progesterone and/or dydrogesterone

LPS was initiated on day 16 and continued for 10 days in women undergoing OI, from day after insemination in women undergoing IUI, and from day of embryo transfer (ET) in women undergoing in-vitro fertilisation (IVF) or IVF/intra-cytoplasmic sperm injection (ICSI). All medicines were procured by patients. Assessments Serum β-human chorionic gonadotropin (β-hCG) level was estimated 13 days after OI / IUI / ET to confirm pregnancy. Clinical pregnancy was defined as the presence of a viable foetus in an ultrasound scan performed seven weeks after ET. The presence of at least one viable foetus at 12 weeks of gestation was classified as an ongoing pregnancy. Following successful conception, all women were followed up regularly for antenatal check-up at our institute. Pregnant women who could not come for regular follow-up were advised to visit at least thrice for antenatal check-up and at least once at six weeks post-conception. Foetal viability scan and detailed anatomy scans were performed at 6–7 weeks of gestation. Ultrasonography was performed at 12 weeks of gestation (completion of first trimester) and between 20 and 22 weeks of gestation to assess for congenital anomalies. The definition of congenital malformation, deformations and chromosomal abnormalities as stated in Chapter XVII, ICD-10 World Health Organization, International Statistical Classification of Diseases and Related Health Problems was used for the study purpose. According to the World Health Organization, congenital anomalies are also known as birth defects, congenital disorders or congenital malformations. Congenital anomalies were defined as structural or functional anomalies, including metabolic disorders, which are present at the time of birth. A Samsung SonoAce R7 (Samsung Medison; Seoul, South Korea) was used for the assessments, and the same trained sonologist carried out all assessments at every visit. The women were routinely examined by the obstetrician and the babies were thoroughly examined by a paediatrician during their postnatal visit. Electronic records till their postnatal check-up were maintained for all women. Demographic characteristics, pregnancy rate, miscarriage rate, congenital anomalies and foetal outcome were recorded. Information related to growth, learning 16 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

abilities and psychologic functioning was recorded by trained child psychologist, paediatrician and trained nurses (S1 File).The functional abnormalities in children were closely monitored. The follow-up reports were collected using various methods such as analysis of birth records, regular health check-up of children by the paediatricians and paediatric-psychologist during the annual baby get-together, and parents’ feedback. Statistical Analysis The proportion of congenital malformations between the study groups and treatment groups were compared by Yate’s corrected chi square test. Anomaly risk was quantified by univariate odds ratio (OR) with 95% confidence interval (CI). Unpaired t tests were used to compare the mean centiles between each group. Age and birth weight between different groups were compared by student t test. Data were expressed as mean ± SD and analysed using SPSS 20.0 statistical software (SPSS Inc., Chicago, Illinois, USA). A value of P<0·05 was considered significant. Statistical power of the study was obtained to be >95%, with a type I error of 5% based on null hypothesis and by G*Power software, available at http://www. gpower.hhu.de . Results Six thousand two hundred and ninety eight records were included after screening of 6549 history files of women reporting for infertility treatment between 2002 and 2017. The patients were stratified into three groups based on type of treatment received. Group A included women with natural conception and without any LPS (n=2003); women who underwent ART or non-ART treatment and received MVP as LPS (Group B; n=2103) and women who underwent ART and/or non-ART treatment with oral dydrogesterone as LPS formed Group C (n=2192). All demographic parameters were found to be comparable between the three groups (Table 1). The overall pregnancy (Group A: 32%; Group B: 29.15%; Group C: 30.7%) and the miscarriage rate/s (Group A: 10.61%; Group B: 14.19%; Group C: 13.52%) were comparable. No major pregnancy complications were observed in any of the intervened or control group/s.

Causes of Infertility Tubal factor, PCOS, endometriosis, and male factor in >800 women each were the reported causes for infertility; the cause was unexplained in the remaining women (Table 2). Since both Groups B and C were comprised of patients treated with a variety of regimens – oral clomiphene citrate, injectable gonadotropins, timed intercourse, IUI, IVF and IVF/ICSI and represent a heterogenous treatment regimen/s, chi square test was applied to evaluate the uniformity of patient distribution in each treatment arm. Winter 2019 Volume 2 Issue 4

Clinical Research Group B), and (n=649; Group C). The overall occurrence of congenital anomalies, functional and chromosomal abnormalities observed in these children (aged 0–5 years) were comparable between the three groups (Table 4). Congenital anomalies were observed in 2.45% of patients in Group A, 2.89% of patients in Group B, and 3.08% of patients in Group C (OR [95% CI]: A vs B: 1.18 [0.58–2.39]; A vs C: 1.26 [0.64–2.49]; B vs C: 0.93 [0.48–1.8]). Similar comparable findings were documented in both functional (OR [95% CI] A vs B: 1.17 [0.67–2.06]; A vs C: 1.06 [0.61–1.86]; and B vs C: 1.11 [0.64–1.91]) and chromosomal abnormalities (OR [95% CI] A vs B: 1.31 [0.61–6.23]; A vs C: 0.63 [0.11–3.77]; and B vs C: 1.03 [0.52–1.85]). No obvious differences were observed in congenital anomalies or functional abnormalities by type of treatment procedure in the groups treated with MVP or dydrogesterone (Table 5).

Table 3: Fetal and neonatal outcomes of three groups with or withouat treatment of luteal phase support with micronized vaginal progesterone and/or dydrogesterone

Table 4. Congenital anomalies and functional and chromosomal abnormalities observed in children in three groups with or without treatment of luteal phase support with micronised vaginal progesterone and/or dydrogesterone

Table 5. Congenital anomalies and functional and abnormalities observed in children born to women in three groups, by tretment procedure

Foetal and Neonatal Outcomes Intrauterine foetal outcomes such as gestational age, intrauterine growth restriction (IUGR) and intrauterine foetal death (IUFD), neonatal characteristics such as number of live births, body weight, and Appearance Pulse Grimace (reflex) Activity Respiration (APGAR) score; neonatal intensive care unit (NICU) admission; respiratory distress syndrome (RDS); neonatal jaundice; hypoglycemia; and hypocalcemia did not vary significantly between the three groups (Table 3). A total of 1851 children were born to 6298 women. The babies were sub-stratified into (n=613; Group A), (n=589; www.biopharmaceuticalmedia.com

Discussion Progesterone plays an important role in the establishment and maintenance of pregnancy33–35. It is well documented that stimulation of ovaries as part of infertility treatment procedures can cause luteal phase deficiency and a negative effect on implantation and maintenance of gestation36–38. Hence, LPS during these procedures is considered a standard practice to support implantation and improve pregnancy rates 39,40. The luteal phase, which from an embryologic point of view, ranges between the period of ‘dividing totipotent’ cells of cleaving embryo to the period of ‘differentiating pluripotent’ cells of expanding blastocyst, is the phase of embryonic development and organogenesis. While damage to the totipotent cells is more amenable to repair mechanisms 41–43, scope of repair slowly diminishes as the cleaving embryo progresses towards pluripotency, due to loss of unique transcriptome and/or modification in epigenetic and chromatin features, leading to a defective blastocyst44. This may be a possible explanation of teratogenic impact of the drug (progesterone) administered during the phase of embryonic organogenesis, i.e. during the luteal phase of an ovulatory cycle. However, in addition to several epidemiological studies which have shown no teratogenic effect of progesterone45, the ASRM committee in 2008 also suggested that there is no evidence which has been documented so far to indicate the maternal exposure to progesterone during pregnancy to increased risk for birth defects39. Dydrogesterone has been in use in pregnancy from the 1960s, with a total cumulative exposure of 26 million patient years up to 201846. While the extensive use theoretically rules out any substantial foetal risk, an overview of birth defects reported with dydrogesterone from 1977 to 2005, and a systematic review of all studies conducted until 2010, showed minimal risk of birth defects with dydrogesterone use.32,47 However, a cause-effect relationship may be presumed when a similar type of defect or disorder is detected in the offspring delivered following use of a specific drug or specific procedure used as part of infertility treatment. One retrospective study identified a positive association between dydrogesterone and congenital heart defects30. However, INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 17

Clinical Research counter reports have provided evidences to suggest that this study has several methodological constraints21,46. A recent patient database study has also suggested increased risk of teratogenic effect/s following exposure to dydrogesterone in pregnant women31. In the Phase III LOTUS I study, the incidence of congenital, familial and genetic disorders in the foetus or infants was limited and similar amongst dydrogesterone (1.0%) and MVP (1.2%) and in the group of women with no luteal support. A substratification of the LOTUS I trial in a subset of 216 Russian patients also demonstrated similar percentages of congenital anomalies in the two groups as those observed in the overall population; more importantly, no health issues were reported in the infants at six-month follow-up48. In the LOTUS II study, the incidence of congenital, familial and genetic disorders was 6.3% with dydrogesterone and 5.0% with MVP21. Consistent with findings in the LOTUS I and LOTUS II studies, we observed no notable differences in the incidence of congenital malformations and also no distinct pattern of defects in children born to mothers following dydrogesterone or MVP supplementation. Furthermore, the outcome for both the groups was comparable with that of spontaneously conceived cases, along with functional and chromosomal abnormalities in children of each of the three groups. Our findings are also in agreement with previous studies of dydrogesterone in other indications. El-Zibdeh and co-workers in 2005 reported no significant differences with respect to pregnancy complications or congenital abnormalities among women with a history of recurrent, unexplained spontaneous abortion treated with oral dydrogesterone, intramuscular human chorionic gonadotrophin or no additional treatment until 12 weeks of gestation17. Furthermore, Pandian et al., in 2009 stated absence of congenital anomalies after dydrogesterone supplementation for threatened miscarriage16. In a subsequent study in 200918, the authors again found no statistically significant differences in congenital abnormalities associated with dydrogesterone support in threatened miscarriage cases compared with women receiving no treatment. Despite our comparable findings, it is important to consider that infants born from singleton pregnancies after ICSI are at an increased risk of developing congenital malformations when compared with similar naturally conceived children49. This study is the first to our knowledge where congenital abnormalities were assessed in a real-life observational


setting in children born to women with infertility receiving dydrogesterone following ART or non-ART procedures. The limitations of the study are its retrospective nature, and it being a single-centre study in women with the same ethnicity. In conclusion, the study results demonstrate that dydrogesterone as LPS is associated with a similar rate of congenital anomalies. The proportion of children with congenital anomalies was similar to those observed with MVP as LPS and in women with spontaneous pregnancy achieved in infertile women and receiving no luteal support. Our findings also add to the existing evidence in support of oral dydrogesterone as an effective and well-tolerated drug for LPS in women with infertility. Acknowledgements The authors thank Sushanta Chakraborty and Sharmista Kundu Nag for assisting in collection of data. REFERENCES 1. Zhu JL, Basso O, Obel C, Bille C, Olsen J. Infertility, infertility treatment, and congenital malformations: Danish national birth cohort. Br Med J. 2006;333(7570):679. 2. Davies MJ, Moore VM, Willson KJ, Van Essen P, Priest K, Scott H, et al. Reproductive technologies and the risk of birth defects. N Engl J Med. 2012;366(19):1803-1813. 3. Pinborg A, Henningsen AK, Malchau SS, Loft A. Congenital anomalies after assisted reproductive technology. Fertil Steril. 2013;99(2):327332. 4. Levi Setti PE, Moioli M, Smeraldi A, Cesaratto E, Menduni F, Livio S, et al. Obstetric outcome and incidence of congenital anomalies in 2351 IVF/ICSI babies. J Assist Reprod Genet. 2016;33(6):711-717. 5. Elizur SE, Tulandi T. Drugs in infertility and fetal safety. FertilSteril. 2008;89(6):1595-1602. 6. Puumala SE, Ross JA, Feusner JH, Tomlinson GE, Malogolowkin MH, Krailo MD, et al. Parental infertility, infertility treatment and hepatoblastoma: a report from the Children's Oncology Group. Hum Reprod. 2012;27(6):1649-1656. 7. Kallen B, Finnstrom O, Lindam A, Nilsson E, Nygren KG, Otterblad PO. Congenital malformations in infants born after in vitro fertilization in Sweden. Birth Defects Research Part A: Clinical and Molecular Teratology. 2010;88(3):137-143. https://doi.org/10.1002/bdra.20645 8. Rimm AA, Katayama AC, Diaz M, Katayama KP. A meta-analysis of controlled studies comparing major malformation rates in IVF and ICSI infants with naturally conceived children. J Assist Reprod Genet. 2004;21(12):437-443. 9. Sharma S, Ghosh S, Singh S, Chakravarty A, Ganesh A, Rajani S, et al. Congenital malformations among babies born following letrozole or clomiphene for infertility treatment. PLoS One. 2014;9(10):e108219. 10. Olivennes F, Mannaerts B, Struijs M, Bonduelle M, Devroey P. Perinatal outcome of pregnancy after GnRH antagonist (ganirelix) treatment during ovarian stimulation for conventional IVF or ICSI: a preliminary report. Hum Reprod. 2001;16(8):1588-1591. 11. Liberman RF, Getz KD, Heinke D, Luke B, Stern JE, Declercq ER, et al. Assisted Reproductive Technology and Birth Defects: Effects of Subfertility and Multiple Births. Birth Defects Res. 2017;109(14):11441153. 12. Parazzini F, Cipriani S, Bulfoni G, Bulfoni C, Frigerio A, Somigliana E, et al. The risk of birth defects after assisted reproduction. J Assist Reprod Genet. 2015;32(3):379-385. 13. Soliman S, Daya S, Collins J, Hughes EG. The role of luteal phase support in infertility treatment: a meta-analysis of randomized trials. FertilSteril. 1994;61(6):1068-1076. 14. Pritts EA, Atwood AK. Luteal phase support in infertility treatment: a meta-analysis of the randomized trials. HumReprod. 2002;17(9):22872299. 15. Nosarka S, Kruger T, Siebert I, Grove D. Luteal phase support in in vitro Winter 2019 Volume 2 Issue 4

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16. 17.

18. 19.













32. 33.




fertilization: meta-analysis of randomized trials. Gynecol Obstet Invest. 2005;60(2):67-74. Pandian RU. Dydrogesterone in threatened miscarriage: a Malaysian experience. Maturitas. 2009;65 Suppl 1:S47-50. El-Zibdeh MY. Dydrogesterone in the reduction of recurrent spontaneous abortion. The J Steroid Biochem Mol Biol. 2005;97(5):431434. El-Zibdeh MY, Yousef LT. Dydrogesterone support in threatened miscarriage. Maturitas. 2009;65 Suppl 1:S43-46. Griesinger G, Blockeel C, Tournaye H. Oral dydrogesterone for luteal phase support in fresh in vitro fertilization cycles: a new standard? Fertil Steril. 2018;109(5):756-762. Tournaye H, Sukhikh GT, Kahler E, Griesinger G. A Phase III randomized controlled trial comparing the efficacy, safety and tolerability of oral dydrogesterone versus micronized vaginal progesterone for luteal support in in vitro fertilization. Hum Reprod. 2017;32(5):1019-1027. Griesinger G, Blockeel C, Sukhikh GT, Patki A, Dhorepatil B, Yang DZ, et al. Oral dydrogesterone versus intravaginal micronized progesterone gel for luteal phase support in IVF: a randomized clinical trial. Hum Reprod. 2018;33(12):2212-21. Chakravarty BN, Shirazee HH, Dam P, Goswami SK, Chatterjee R, Ghosh S. Oral dydrogesterone versus intravaginal micronised progesterone as luteal phase support in assisted reproductive technology (ART) cycles: results of a randomised study. J Steroid Biochem Mol Biol. 2005;97(5):416-420. Patki A, Pawar VC. Modulating fertility outcome in assisted reproductive technologies by the use of dydrogesterone. Gynecol Endocrinol. 2007;23:68–72. Ganesh A, Chakravorty N, Mukherjee R, Goswami S, Chaudhury K, Chakravarty B. Comparison of oral dydrogestrone with progesterone gel and micronized progesterone for luteal support in 1,373 women undergoing in vitro fertilization: a randomized clinical study. Fertil Steril. 2011;95(6):1961-1965. Salehpour S, Tamimi M, Saharkhiz N. Comparison of oral dydrogesterone with suppository vaginal progesterone for luteal phase support in in vitro fertilization (IVF): a randomized clinical trial. Iran J Reprod Med. 2013;11:913–918. Tomic V, Tomic J, Klaic DZ, Kasum M, Kuna K. Oral dydrogesterone versus vaginal progesterone gel in the luteal phase support: randomized controlled trial. Eur J Obstet Gynecol Reprod Biol. 2015;186:49–53. Barbosa MW, Silva LR, Navarro PA, Ferriani RA, Nastri CO, Martins WP. Dydrogesterone vs progesterone for luteal-phase support: systematic review and meta-analysis of randomized controlled trials. Ultrasound Obstet Gynecol. 2016;48:161–170. Saharkhiz N, Zamaniyan M, Salehpour S, Zadehmodarres S, Hoseini S, Cheraghi L. et al. A comparative study of dydrogesterone and micronized progesterone for luteal phase support during in vitro fertilization (IVF) cycles. Gynecol Endocrinol. 2016;32:213–217. Zargar MNS, Ejtahed M. Comparison the effectiveness of oral dydrogesterone, vaginal progesterone suppository and progesterone ampule for luteal phase support on pregnancy rate during ART cycles. Int J Pharma Res Allied Sci. 2016;5:229–236. Zaqout M, Aslem E, Abuqamar M, Abughazza O, Panzer J, De Wolf D. The impact of oral intake of dydrogesterone on fetal heart development during early pregnancy. Pediatr Cardiol. 2015;36(7):1483-1488. Koren G, Gilboa D, Rotem R, Levy A, Daniel S, Shalev V. Fetal outcome following dydrogesterone exposure in pregnancy. Arch Dis Child. 2019;104:e2. Queisser-Luft A. Dydrogesterone use during pregnancy: overview of birth defects reported since 1977. Early Hum Dev. 2009;85(6):375-377. Csapo AI, Pulkkinen M. Indispensability of the human corpus luteum in the maintenance of early pregnancy. Luteectomy evidence. Obstet Gynecol Surv. 1978;33:69–81. Couzinet B, Le Strat N, Ulmann A, Baulieu EE, Schaison G. Termination of early pregnancy by the progesterone antagonist RU 486 (Mifepristone). N Engl J Med. 1986; 315:1565–1570. Silvestre L, Dubois C, Renault M, Rezvani Y, Baulieu EE, Ulmann A. Voluntary interruption of pregnancy with mifepristone (RU 486) and a prostaglandin analogue. A large-scale French experience. N Engl J Med. 1990;322:645–648. Macklon NS, Fauser BC. Impact of ovarian hyperstimulation on the luteal


phase. J Reprod Fertil Suppl. 2000;55:101–108. 37. Beckers NG, Macklon NS, Eijkemans MJ, Ludwig M, Felberbaum RE, Diedrich K, Bustion S, Loumaye E, Fauser BC. Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropin, recombinant luteinizing hormone, or gonadotropin releasing hormone (GnRH) agonist to induce final oocyte maturation in in vitro fertilization patients after ovarian stimulation with recombinant follicle-stimulating hormone and GnRH antagonist cotreatment. J Clin Endocrinol Metab 2003;88:4186–4192. 38. Kolibianakis EM, Bourgain C, Platteau P, Albano C, Van Steirteghem AC, Devroey P. Abnormal endometrial development occurs during the luteal phase of nonsupplemented donor cycles treated with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonists. Fertil Steril 2003;80:464–466. 39. Practice Committee of the American Society for Reproductive Medicine. Progesterone supplementation during the luteal phase and in early pregnancy in the treatment of infertility: an educational bulletin. Fertil Steril. 2008;89:789–792.i. van der Linden M, Buckingham K, Farquhar C, Kremer JA, Metwally M. Luteal phase support for assisted reproduction cycles. Cochrane Database Syst Rev. 2011(10):CD009154. 40. Hansis C, Grifo JA, Krey LC. Candidate lineage marker genes in human preimplantation embryos. Reprod Biomed Online. 2004;8(5):577-583. 41. Jedrusik A, Parfitt DE, Guo G, Skamagki M, Grabarek JB, Johnson MH, et al. Role of Cdx2 and cell polarity in cell allocation and specification of trophectoderm and inner cell mass in the mouse embryo. Genes Dev. 2008;22(19):2692-2706. 42. Sun JH, Zhang Y, Yin BY, Li JX, Liu GS, Xu W, et al. Differential expression of Axin1, Cdc25c and Cdkn2d mRNA in 2-cell stage mouse blastomeres. Zygote. 2012;20(3):305-310. 43. Niakan KK, Han J, Pedersen RA, Simon C, Pera RA. Human pre-implantation embryo development. Development. 2012;139(5):829-841. 44. Posaci C, Smitz J, Camus M, Osmanagaoglu K, Devroey P. Progesterone for the luteal support of assisted reproductive technologies: clinical options. Hum Reprod. 2000;15 Suppl 1:129-148. 45. Kaur KK, Allahbadia G, Singh M. Luteal phase support using oral dydrogesterone-a prospective treatment for future replacing micronized vaginal progesterone. Open Acc J Repro & Sexual Disord. 2014;1(4). OAJRSD.MS.ID.000119. DOI: 10.32474/ OAJRSD.2018.01.000119 46. Carp H. A systematic review of dydrogesterone for the treatment of threatened miscarriage. Gynecol Endocrinol. 2012;28(12):983-90. 47. Sukhikh GT, Baranov II, Melnichenko GA, Bashmakova NV, Blockeel C, Griesinger G et al. Lotus I: A Phase III randomized controlled trial of oral dydrogesterone versus micronized vaginal progesterone for luteal support in in vitro fertilization, with focus on the Russian subpopulation. Akusherstvo i Ginekologiya/Obstetrics and Gynecology. 2017;7:75-95. (in Russian) 48. Lacamara C, Ortega C, Villa S, Pommer R, Schwarze JE. Are children born from singleton pregnancies conceived by ICSI at increased risk for congenital malformations when compared to children conceived naturally? A systematic review and meta-analysis. JBRA Assist Reprod. 2017;21(3):251-259.

Shovandeb Kalapahar, MS, DNB,1 Tushar K. Das, PhD,1 Sunita Sharma, MD, FNB,1 Sourav RoyChoudhury, PhD,1 Ratna Chattopadhyay, MBBS, PhD,1 Koel Chaudhury, PhD,2 Pratip Chakraborty, PhD,1 Baidyanath Chakravarty, MO, FRCOG, DSc.1 1. Institute of Reproductive Medicine, Salt Lake, Kolkata, West Bengal, India 2. School of Medical Science and Technology, Indian Institute of Technology, Kharagpur, West Bengal, India


Clinical Research

Is Liver Biopsy a Gold or an Old Standard in NAFL and NASH? When defining the term gold standard, the most appropriate definition is “a benchmark test that is the best available under reasonable conditions.” 1 When considering this definition, it becomes clear that non-invasive imaging is replacing liver biopsy as the gold standard for evaluation of fibrosis in non-alcoholic fatty liver disease (NAFLD).

Over the last 40 years, NAFLD has evolved from an unrecognised entity to a heterogeneous collection of overlapping liver diseases with a common phenotype of hepatic steatosis. Although NAFLD is quite common, affecting approximately 25% of the world’s adult population, it is increasingly clear that subjects with non-alcoholic steatohepatitis (NASH), and especially those with significant fibrosis, are at the greatest risks for excess mortality and adverse clinical outcomes, as well as impairment of patient reported outcomes and significant economic burden. Patients with NASH do not present with any obvious clinical signs until they are near liver failure. Despite the growing recognition of this important burden, there are significant challenges to accurately and non-invasively diagnose the progressive form of NAFLD. Although liver biopsy (LB) is still considered the current “gold” standard for diagnosing NASH and staging fibrosis, it is an invasive procedure with some variability in assessment of the key features of NASH. LBs require significant expertise both to perform and to interpret the results. Two clinicians are involved in obtaining and interpreting LB which represents a huge clinical process, especially in the settings of clinical trials. Given the significant number of patients with NAFLD, the limited number of hepatologists represents a serious bottleneck, often leading to delays in biopsy reads and confirmation of results. The accuracy of LB to assess fibrosis has also been questioned due to sampling errors and intra- and inter-observer variability that may lead to over- or under-staging, with even highly skilled and trained pathologists showing inter-observer concordance rates of less than 80%.2 The size of the biopsy specimen, which varies from 10 to 30 mm in length and from 1.2 to 2 mm in diameter, represents 1/50,000 of the total mass of the liver and is therefore subject to a significant risk of sampling error.3 Although LBs remain the gold standard for confirmation of liver fibrosis staging, they are costly, painful and pose risk of complications such as bleeding, damage to other organs, and potentially, although rare, death. Multiple LBs present a significant challenge in recruitment and retention of subjects in clinical trials due to above states risks and subject discomfort. Ideally, less invasive tests that are more widely available, less costly, and accurate can be confirmed and widely accepted by clinicians and regulatory bodies as the new gold standard. There are a number of such non-invasive tests such as radiographical 20 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

modalities, serum markers, and non-invasive predictive algorithms undergoing investigation for identifying those with increased risk of NAFLD and NASH and for confirmation of or staging of NASH fibrosis. The ideal test to discriminate advanced liver fibrosis due to NASH would be non-invasive, widely available, affordable, accurate, and reproducible. Imaging Modalities In the context of NAFLD, the first diagnostic challenge is to accurately show the presence of fat in the liver. Fat is thought to have its own chemical signature, which can be detected directly by magnetic resonance spectroscopy (MRS). MRS quantifies the proton density fat fraction (PDFF), a standardised measure of liver tissue.4 However, MRS has several limitations, including: limited availability, need for expertise in protocol prescription, data collection, and the requirement for spectral analysis. Furthermore, MRS is not available on routine scanners. Therefore, now, magnetic resonance imaging (MRI) based methods have been developed using MRI-PDFF to quantify liver fat without needing spectroscopy coils, using routinely available clinical MRI scanners.4,5 In contrast, fibrosis has no molecular signature that can be detected by current imaging techniques, so all imaging tests for fibrosis attempt to detect fibrosis indirectly using proposed biomarkers, which include: stiffness, diffusion, perfusion, metabolites, and image texture. Testing for the presence of advanced fibrosis is a primary concern when evaluating a patient with suspected NASH. It is known that fibrosis is the only independent predictor of associated morbidity and mortality. Confirmed presence of advanced fibrosis alters clinical management and consideration for treatment, potentially in the context of clinical trials. The leading biomarker of fibrosis is liver “stiffness” (or “elasticity”) and its family of related parameters.4 The most accurate non-invasive methods to assess the stiffness of the liver and to classify the patient into advanced versus non-advanced fibrosis include transient elastography (TE), magnetic resonance elastography (MRE), and emerging techniques such as shear wave elastography (SWE) and acoustic radial force imaging (ARFI). TE has been clinically useful as well, and has the means to replace LB as the gold standard. TE has high accuracy when identifying patients with F3-F4 fibrosis who are at greater risk for worse clinical outcomes.6 TE has been shown to have an area under the curve (AUROC) of 0.83 for advanced fibrosis when compared to blood tests. The most remarkable advantage of TE is that the procedure is non-invasive, without any of the complications associated with liver biopsy. In addition, its cost is one-fourth of that of liver biopsy, and it can be done in five minutes in the outpatient setting without any associated pain. MRE has the AUROC of 0.98 in identifying patients with F3–F4 disease, so at the very least is non-inferior to liver biopsy.7 Given Winter 2019 Volume 2 Issue 4


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Clinical Research its accuracy, MRE may also offer a good non-invasive tool to monitor changes in liver fibrosis. In a placebo-controlled trial of sitagliptin in NAFLD, MRE was shown to have robust correlation coefficient between baseline and 24 w  eeks.8 Longitudinal studies of contemporaneous MRE and liver biopsy are underway, and their results are eagerly awaited. Controlled attenuation parameter (CAP) is a novel ultrasound technique that measures steatosis simultaneously with liver stiffness during vibration-controlled transient elastography. Overall, CAP is a relatively simple and inexpensive method for steatosis assessment that is reasonably accurate for the diagnosis of steatosis. When combined with other clinical assessments, it is likely to help clinicians diagnose or exclude steatosis. Non-invasive Biomarkers in NASH In addition to the non-invasive tests based on the imaging modalities, there is an attempt to define non-invasive biomarkers using predictive models or serum biomarkers. These non-invasive markers include those that are based on alanine aminotransferase (ALT) levels, those that include components of metabolic syndrome, measuring circulating keratin18 fragment levels, as well as tests based on soluble markers such as FibroMeter, microRNA (miRNA) panels, and lipidomic panels, etc. The NASH test combines demographic characteristics (age, sex, and BMI), serum parameters (aminotransferases and lipids), and alpha-2 macroglobulin, apolipoprotein A1, and haptoglobin. Predictive Models for Advanced Fibrosis Serological markers of fibrosis evaluate alterations in hepatic function as well as collagen turnover. The severity and progression of liver fibrosis plays a key role for predicting outcomes and for making therapeutic decisions in NASH patients.


AST/ALT ratio: The aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio helps distinguish alcoholic hepatitis from NAFLD and NASH. Using these non-invasive tests to diagnose for NASH, current studies have found that the frequency of NASH in individuals with normal ALT (<35 U/L) was 11% whereas the frequency was 29% in those with elevated ALT ( 35 U/L); and if the ALT was two times the upper limit of normal (>70 U/L), predicting NASH was found to have a 50% sensitivity and 61% specificity for NASH.9Â However, another study found that individuals with NAFLD can have normal ALT levels as the disease progresses.10 Fibrosis-4 index: This scoring system combines age, AST, ALT, and platelet count. It has a negative predictive value of more than 90% for advanced liver fibrosis, according to experts. Results can be subject to rapid AST and ALT changes, though. BARD score: Calculated from body mass index, the ALT/AST ratio, and the presence of diabetes, this score, reported on a 0-4 scale, is routinely used to predict liver fibrosis in NAFLD patients. Scores less than 2 have a strong negative predictive value for advanced liver fibrosis associated with NAFLD. Enhanced liver fibrosis (ELF) test: Though not yet approved by the Food and Drug Administration and not sensitive to early-stage fibrosis, this panel is an algorithm comprised of three fibrosis markers â&#x20AC;&#x201C; amino-terminal propeptide of type III procollagen, hyaluronic acid, and tissue inhibitor of metalloproteinase. It detects advanced fibrosis with high accuracy in both adult and paediatric patients. Tailored Approach Significant progress has been made regarding the non-invasive

Winter 2019 Volume 2 Issue 4

Clinical Research assessment of liver disease in patients with NAFLD. Regarding detection and grading of steatosis, MRI-PDFF is the most accurate method but appears better suited for assessment and follow-up of selected patients in clinical trials, whereas conventional ultrasound, and if no steatosis is shown, CAP, as a point of care technique, could be used as triage in large unselected populations. As for the identification of advanced fibrosis, MRE, TE, as well as FIB-4 are the most accurate and validated methods. FIB-4 is best suited as a first-line tool in a primary healthcare setting to confidently exclude advanced fibrosis, whereas TE and MRE are more suited for referral centres. It is postulated that combinations of NITs in sequential algorithms can accurately detect advanced fibrosis while eliminating the risks associated with biopsy and reducing costs by minimising unnecessary testing.11 Regarding NASH, no highly sensitive and specific blood tests are available to differentiate NASH from simple steatosis. Neither imaging modality can reliably discriminate NASH from simple steatosis, although MR-based modalities are showing promise. Finally, there is increasing evidence that serum markers and liver stiffness, measured using TE, accurately identify the subgroup of patients with NAFLD at a higher risk to reach the outcome of liver-related complications and death/liver transplantation, especially when analysed together. Screening data from Phase 2 ATLAS study evaluating combinations of investigational cilofexor, firsocostat and selonsertib in advanced fibrosis due to NASH has been recently presented. This analysis demonstrates that the use of currently available NITs can accurately identify patients with advanced fibrosis due to NASH and potentially reduce the need for LB. When used in combination, the ELF test and FibroScan® (TE) accurately identified advanced fibrosis in >805 of patients (presented at The International Liver Congress 2019, Vienna). Looking Ahead Adopting new biomarkers in clinical trials requires substantial efforts and investment to validate the reliability of these biomarkers as surrogate endpoints. In an effort to address this challenge, developers are currently integrating exploratory markers as secondary endpoints in Phase II and Phase III studies. The field has also seen the formation of two multi-stakeholder consortia, LITMUS and NIMBLE, aimed at accelerating validation of non-invasive markers by sharing resources and patient samples. Histological diagnosis of NASH is still required in clinical trials, however non-invasive modalities can be used more frequently to follow at-risk patients over time, as well as be instituted for screening evaluations in the absence of the morbidity that unfortunately comes with LB. MRE has emerging data to support its non-inferiority to LB in terms of accuracy in fibrosis staging and, combined with the dramatic risk profile differences, should be soon considered a superior test. REFERENCES 1. Claassen JAHR. The gold standard: not a golden standard. BMJ 2005;330:1121 2. Bedossa P, Bioulac-Sage P, Callard P et al. Intraobserver and interobserver variations in liver biopsy interpretation in patients with chronic hepatitis C. Hepatology 1994;20:15-20 3. Bedossa P, Dargère D, Paradis V. Sampling variability of liver www.biopharmaceuticalmedia.com

fibrosis in chronic hepatitis C. Hepatology 2003;38:1449-1457 4. Dulai PS, Sirlin CB, Loomba R. MRI and MRE for noninvasive quantitative assessment of hepatic steatosis and fibrosis in NAFLD and NASH: clinical trials to clinical practice. J Hepatol 2016;65:1006–1016. 5. Park CC, Nguyen P, Hernandez C, Bettencourt R, Ramirez K, Fortney L et al. Magnetic resonance elastography vs transient elastography in detection of fibrosis and noninvasive measurement of steatosis in patients with biopsy-proven nonalcoholic fatty liver disease. Gastroenterology 2017;152:598– 607.e2 6. Boursier J, Vergniol J, Guillet A, Hiriart JB, Lannes A, Le Bail B et al. Diagnostic accuracy and prognostic significance of blood fibrosis tests and liver stiffness measurement by FibroScan in non-alcoholic fatty liver disease. J Hepatol 2016;65:570–578. 7. Loomba R, Wolfson T, Ang B et al. Magnetic resonance elastography predicts advanced fibrosis in patients with nonalcoholic fatty liver disease: a prospective study. Hepatology 2014;60:1920-1928. 8. Cui J, Philo L, Nguyen P, Hofflich H, Hernandez C, Bettencourt R et al. Sitagliptin vs. placebo for non-alcoholic fatty liver disease: a randomized controlled trial. J Hepatol. 2016;65(2):369–76. 9. Verma S, Jensen D, Hart J, Mohanty SR. Predictive value of ALT levels for non-alcoholic steatohepatitis (NASH) and advanced fibrosis in non-alcoholic fatty liver disease (NAFLD). Liver Int 2013;33:1398–1405. 10. Rinella ME, Loomba R, Caldwell SH, Kowdley K, Charlton M, Tetri B, Harrison SA. Controversies in the Diagnosis and Management of NAFLD and NASH. Gastroenterol Hepatol 2014;10:219–227. 11. Anstee QM, Lawitz EJ, Alkhouri N et al. Noninvasive tests accurately identify advanced fibrosis due to NASH: Baseline data from the STELLAR trials. Hepatology 2019;70(5):1521-1530

Rafal Ziecina Rafal Ziecina MD, PhD is Executive Director of Scientific Solutions at Worldwide Clinical Trials. Dr Ziecina is a board-certified pharmaceutical physician specialising in design and execution of cardiovascular and metabolic trials. He provides consultancy services around filing strategies, regulatory & safety strategies, regulatory agency interactions as well as protocol and drug development plans writing.

Email: rafal.ziecina@worldwide.com

Scott Beasley Mr. Beasley has more than 23 years clinical research experience and program leadership within the CRO and pharmaceutical industry. Mr. Beasley has successfully led a significant portfolio of several hepatology phase II and phase III programs in NAFLD/NASH as well as other metabolic disorders. Over the most recent years, Mr. Beasley has led the development of a NASH initiative training program to address the unique challenges associated with clinical trials in NASH/NAFLD. He has provided team expertise in managing complex NASH programs including development of operational strategies to proactively identify and mitigate risks to limit any potential impact. Mr. Beasley currently serves as Executive Director, Project Management and Franchise Area Lead for the NASH/NAFLD and Liver disease program at Worldwide Clinical Trials.


Manufacturing/Technology Platforms


Split Butterfly Valve Technology and Eradicating the Risk of Contamination in Biologics Manufacturing As new biological therapies continue to be developed, the market has seen an increased need for innovation. Demand has grown for improved operational efficiency and achieving greater quality and cost reductions in manufacturing processes. One of the key challenges facing biopharma manufacturers is eliminating the risk of contamination, which requires effective containment strategies and monitoring of critical manufacturing processes. Biopharmaceutical Market Landscape The biopharma sector has witnessed huge growth over recent years. Total annual revenue has increased from $4.4 billion in 1990 to around $275 billion at the end of 2019, an increase of 6,250%, and biopharmaceuticals now make up more than 25% of the total pharmaceutical market1. New technologies that can ensure vigorous and flexible containment strategies while also reducing a productâ&#x20AC;&#x2122;s time to market offer arguably the greatest benefits to manufacturers as the industry continues to evolve. Containment Strategies Manufacturing environments are open to many sources of potential contamination, which if not correctly controlled, pose possible hazards during the manufacture of biopharmaceutical products. Patient safety could ultimately be put at risk should microorganisms, particles or endotoxins enter a manufacturing environment. Potential sources of contamination include the equipment, materials used and the people within the manufacturing environment. Multiple technologies have been developed in response to the need to ensure the safe and sterile transfer of biologics, biosimilars and ingredients during aseptic processing. Restricted access barrier systems (RABS) and isolator technologies have become widely used in recent years. Isolators provide an airtight barrier around the aseptic processing line and can be employed in cleanroom environments to minimise the risk from contaminants. RABS provide a barrier between workers and processing lines, while offering operators the opportunity to interact with products as necessary. Despite this, both systems have disadvantages. Manufacturers using isolator technology may face difficulties when transferring materials in and out of the chamber, which can delay the shut-down and start-up process. The closed solution provided by RABS technology provides lower integrity chambers, and the technology relies on manual cleaning processes such as steaming in place (SIP) or sterilisation between uses which can be time-consuming and create delays. SBV Technology as a Solution SBVs are made up of an active half and passive half and enable the transfer of an active pharmaceutical ingredient (API) or product from a process vessel, container, isolator or RABS to another 24 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

without jeopardising sterility. When in use, the active half of the SBV is attached to the receiving container, while the passive half is attached to the flexible bag or discharging API or drug product container. When the two halves of the disc are brought together, a single plate is created, which allows the product to flow on the internal surface of each half. Thus, when the two halves are detached, the external faces remain clean and can be safely exposed to the process environment. When the valve of the SBV is sealed, an opening is created between the discs, which means decontaminating gas can be flushed through and decontamination can take place in a closed environment. Validation occurs using chemical indicators, which confirm full coverage of the enclosure has been attained. This is followed by biological indicators, to ensure a 99.9999% reduction (or 6-log reduction) in bacterial spores has been achieved. Case Study Sterile API Addition to a Mixing Vessel The CDMO is a full-service pharmaceutical company that leverages blow-fill-seal technology. Its capabilities extend well beyond manufacturing, with an in-house development team specialising in all aspects of bringing a product to market â&#x20AC;&#x201C; from lab scale batches, regulatory filings, scale-up, manufacturing and distribution. Challenge The CDMO was looking to solve the issue of charging sterile API into a mixing tank. This is a common problem with all aseptically prepared pharmaceutical products. Critical to the process was maintaining sterile conditions whilst docking a container to the vessel and then transferring solid API to form a liquid suspension. With a fully dissolved liquid, the product could be sterile filtered to ensure sterility as it was passed to the filler. Although in this case, the product being passed to the filler was a suspension and so this option was not possible. This required the whole process to be performed under aseptic conditions and as such would normally mean one of the following upgrades would be required. 1. Upgrade the whole room from a grade C cleanroom to grade A. 2. Upgrade the room to a grade B environment and additionally introduce an over-pressurised grade-A area around the point of fill. 3. Implement a laminar flow system around the point of fill, plus additional control due to the lack of a barrier. 4. Upgrade the room to a grade B environment and additionally introduce a RABS system at the point of fill or full vessel. 5. Maintain the grade C cleanroom but introduce isolator technology around the point of fill or full vessel. Traditionally RABs and isolator technology would have been favoured here due to the benefits they bring in terms of improved sterility assurance, employing the fundamental techniques of Winter 2019 Volume 2 Issue 4



Manufacturing/Technology Platforms separation and decontamination. That said, when considering some of the negative constraints which come with these technologies, such as high initial capital investment, space, ergonomics and ongoing cost and energy consumption, the company decided to look elsewhere to find a solution that was more suited to this critical task. Solution An aseptic bio-valve product was selected as an ideal solution to this problem, providing a sealed powder transfer in a small footprint mounted to the inlet port of the vessel. The valve can be pre-steam sterilised along with the vessel, unlike traditional SBVs or other conventional connections (see illustration 1a/b). On final connection, it also removed any room contamination from the mating faces of the transfer in a controlled and validated manner (see illustration 2a/b).

Dock SIP Cap illustration 1a

SIP through Cap – Pre-sterilising Active & Vessel illustration 1b

The AseptiSafe Bio valve works by creating a sealed chamber between the transfer container (passive section) and vessel (active section). When the two halves dock together, the sealed chamber is then bio decontaminated with vaporised hydrogen peroxide (VHP).

Illustration 2a

This removes any biological contamination to a validated 6-log reduction and leaves the space and mating faces clean and ready to fully dock together. Once fully mated, the disc 26 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

can be opened, which allows the product to be transferred from transfer container to vessel, free from the risk of contamination. Performing this transfer still within the grade C space provided enormous cost and production benefits, although the process needed to be fully validated to ensure the initial perceived benefits could be proven.

Illustration 2b

Validation The first step in microbiologically validating the process was to generate a validated decontamination cycle for the hydrogen peroxide gassing phase. This typically consists of four distinct phases, which the generator will run through to ensure a validated gassing cycle is performed each time. All the four phases are set on time. 1. Dehumidification – Where the chamber being gassed reduces the humidity within the chamber to provide ideal condition for biological kill. 2. Conditioning – Where VHP is introduced into the chamber to build up to levels to achieve good decontamination. 3. Decontamination – VHP concentration is maintained in order to deactivate any microbiological activity within the chamber. 4. Aeration – Where on completion of the biological decontamination, the VHP is removed from the system so that no harmful levels of residue are left. Normally the acceptance level is 1ppm, although in this instance 0.4ppm was used as the acceptance level. The client used a lower residue limit to ensure they had a really robust system and no chance of contamination of their product due to gas residue. The full decontamination cycle can be achieved in as little as four minutes, although 20 minutes is more typical. For this application the process was only being performed once a day and, to ensure a robust cycle was produced, additional time was added to each of the critical phases, ensuring that decontamination was confirmed, and gas was aerated from the system. This resulted in a 41-minute full cycle.

Initial cycles utilised chemical indicators (CIs) to determine H202 distribution. When satisfactory CI results were achieved, biological indicators (BIs) were introduced to the process to confirm the process was successfully achieved. Upon completion of each cycle, all BIs and CIs were collected. The CI strips were then checked for colour change to ensure uniform vapour distribution. The BIs were transferred to a suitable growth media, in this case Spordex culture media, and incubated at 55°C to 60°C for seven days. They were observed daily for any microbial growth. Winter 2019 Volume 2 Issue 4

Manufacturing/Technology Platforms

Acceptance criteria for the cycle included: A) All CI strips used in the cycle must have changed colour. B) The positive control BI must demonstrate growth. C) At least one BI from each location must not demonstrate growth. Once the cycle was developed it was then executed in triplicate to form the performance qualification (PQ) for this element of the process. In order to fully validate the system, the process was challenged with multiple media runs prior to validation. These successful media challenges were then carried forward with three media runs at PQ. The sterile hold was demonstrated at greater than 10 days with product transferred to the vessel and with the bio valve held in the closed interlocked position. The sterile hold period was demonstrated for the passive section (product in transfer container) for 48 hours, which was more than adequate as typically this would be at most half this time.

As an alternative to this process, the CDMO could choose a non-sterile API which is easier to handle, dispense this into pre-sterilised single use bags with the integral passive half of the valve which can be docked and product transferred. The whole package could then be sent away for gamma sterilisation, instead of having multiple individual sterilisation and aseptic assembly steps, again making the process more streamlined, easier to handle and more cost-effective. The benefits seen by adopting this method appear to be growing and, according to a report by ResearchAndMarkets, the global market for single use technology is estimated to be worth $7 billion by 2024. Final Thought As the requirements of biological products continue to evolve, it will become increasingly important for technologies in the sector to be agile in order to adapt to new demands. REFERENCES

Conclusion The installation is now operational and in full production. The original benefits seen at the outset of the project, such as low capital equipment cost, smaller footprint and ease of installation, have now been matched by improved sterility assurance, ease of use for operators, and low maintenance. The system is straightforward to use, easy to install / validate and has certainly improved the CDMOâ&#x20AC;&#x2122;s process. One learning from this project was at the dispensing stage. At the time of validation, the system installed was a fully rigid reusable solution where pre-sterilised API was supplied to the client in bags. These bags were opened and then subdivided and dispensed within an aseptic isolator to the pre-autoclaved transfer container and bio valve passive section. It would have been beneficial to sterilise the product, container and transfer connection in one step (gamma irradiation), although due to the constraints associated with gamma sterilising stainless steel and elastomeric assemblies as one item, this was not possible. www.biopharmaceuticalmedia.com

1. https://www.pharmamanufacturing.com/articles/2018/ biopharma-market-an-inside-look/

Christian Dunne Christian Dunne is the Global Head of Sterile Solutions at ChargePoint Technology for the AseptiSafeÂŽrange of products for sterile containment. He works on the advancement of ChargePoint Technology's split butterfly valve technology, designed to handle highly potent/sterile powders and small-scale components, where product and operator protection are paramount. While working on many aseptic applications, Christian integrated several different bio-decontamination systems and has an in-depth understanding of their performance and application.


Regulatory/Quality Compliance

Meeting the Standards Required for Effective Biorepository Management Biorepositories are a key asset for many organisations including those in the biotechnology, pharmaceutical and medical research areas. As they may contain human tissue and other material, possibly together with personally identifying information (PII), security is key and they can be subject to stringent regulations. With every aspect of specimen handling from collection through storage, processing, distribution, and eventual disposal to be considered, as well as the need to manage all the associated data and information, the use of an appropriately configured laboratory information management system (LIMS) can contribute significantly to the efficient management of any biobank facility. Guidance for managing biobanks is available from two highly respected sources. The ISBER Best Practices: Recommendations for Repositories Fourth Edition1, published by ISBER (International Society for Biological and Environmental Repositories) presents a set of recommendations for the  most effective practices for managing biological and environmental specimen collections and repositories. These are either evidencebased or consensus-based practices for collection, long-term storage, retrieval and distribution of specimens.  In addition, the recently published ISO 20387:2018(en)2 Biotechnology – Biobanking – General requirements for biobanking specifies general requirements for the competent, impartial and consistent operation of biobanks including quality control requirements for ensuring the quality of biological material and data collections. Creating a Robust Biobank Management System using LIMS By providing the functionality to control, manage, organise and document information within a dedicated database, a LIMS can meet many biobank management needs. Specifically, it can become a powerful component of an overall biobank quality management system. ISO 20387:2018(en) fully supports the use of computer software and hardware for data storage and tracking. However, a well-designed LIMS will also address issues around data security and access that help prevent loss or corruption of data. For example, commercially available LIMS provide functionality such as automatic data capture, username and password management, audit trails and database backup facilities that help ensure data integrity and prevent data loss. In addition, the data is held in a single place within an industry standard database for as long as required, with easy access using industry standard tools. From within the LIMS application, users have a user-friendly interface to search for, and easily filter, relevant data. However, the data they can access can be controlled to ensure they see only what they are authorised to see. Beyond just specimen management data, LIMS can have an important role to play in overall laboratory and organisational management, for example in monitoring equipment and instrument maintenance and calibration, managing staff training and competencies and monitoring corrective and preventative actions (CAPA). 28 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Specimen Handling Biobanks can contain a wide range of different specimen types with different storage requirements. The handling of these specimens, which may be defined by regulatory requirements, is of critical importance. LIMS provides a complete specimen management solution from sample collection and registration to sample disposition and eventual disposal through the allocation of unique IDs and tracking of actions performed on the specimens. The exact physical location of each specimen can be defined in terms of the storage hierarchy, for example facility, room, freezer, shelf, and rack. Container types, for example 96-well plates, can be specified as required and can include unique sample position information (Figure 1). All changes in location are recorded, giving a complete record of where a specimen has been. Aliquoting and sub-sampling can be managed with full history and chain-of-custody reporting, allowing each of these aliquots and sub-samples to be tracked and their relationship to the parent sample maintained. Randomised location auditing built into the LIMS provides evidence that specimens are where they should be.

Figure 1. Sample storage management

Handling Other Specimen-related Data In addition to data related to the type and location of the specimens, there is a significant amount of other specimen data and information to be managed. This can include patient consent data and potentially other patient-related data or specimenspecific information. The LIMS must be capable of recording this information (Figure 2). The biobank can define acceptance criteria for biological material and associated data, including biosafety, biosecurity and intellectual property rights. Collection procedures must be specified and documented. When samples are transferred out of the biobank, the minimum/maximum shipping temperatures and other key data should be recorded as required. Any processing or testing procedures required for any sample must be specified and documented. These various sample-related stages, such as collection, accession, acquisition, identification, preservation, long-term storage, quality control, transport, disposal, etc., must be described in a workflow (Figure 3). The issue with this is that all biobanks may work in different ways and record different information (indeed an individual biobank may have very different processes for different specimen types). Therefore, a LIMS must allow for these different working practices while maintaining control in terms of data integrity and security. Winter 2019 Volume 2 Issue 4

Regulatory/Quality Compliance

Figure 2. Sample registration

Figure 3. Biobank manager workflow

This is much easier to achieve in a truly configurable LIMS that can be set up to reflect the exact workflows and needs of the individual biobank. All procedures need to be specific to the biological material and associated data and should be fully documented. The LIMS can provide access to the specific documentation or standard operating procedures (SOPs) as required. Biobank Facilities and Competency Clearly, it is essential that equipment such as refrigerators and freezers within a biobank are kept fully operational and calibrated. In order to meet best practice requirements, the use of a LIMS allows the management of scheduled and unscheduled maintenance and calibration actions. Equipment can be flagged as out of service either automatically, for example if maintenance or calibration is past its due date, or manually in response to an unexpected event. Any equipment that is out of service can be prevented from being used. A permanent record of all actions can be maintained for regulatory and audit purposes. It is also necessary to check environmental conditions within areas of the biobank or indeed within specific equipment such as freezers, which could influence the quality of the biological material. A regime of spot checks may be set up to regularly monitor individual sampling points within locations throughout the biobank (Figure 4). These results can be recorded within the LIMS to ensure a permanent record of effective quality assurance. Failures and trends may lead to the raising of corrective actions that can also be tracked and managed through the LIMS. In fact, all non-conformances can be logged, tracked and corrected to provide management of QA preventive and corrective actions. It is also essential to ensure that personnel that carry out any action within a biobank have the appropriate level of training and expertise for the specific activity. A LIMS must allow the status of competency and training of biobank personnel to be recorded, with automated checks when specific actions are undertaken that prevent untrained or uncertified personnel carrying out the action. www.biopharmaceuticalmedia.com

Figure 4. Environmental monitoring

Reporting Given the amount of biobank data that can be managed by a LIMS, the ability to report on that data is essential. The LIMS must provide extensive reporting capabilities across all the available data, for example specimen reporting, management reporting and monitoring of key performance indicators. Typical reports may include the number of specimens accessioned over a period of time, specimens dispatched but not returned within the required time, or specimens due for disposal. Reports and searching functions can also be used to identify specimens that meet specific selection criteria for inclusion in projects or studies. Harnessing the Power of LIMS The ability of a LIMS to manage the varied nature and amount of data associated with a biobank means that it can make a powerful contribution to biobank best practice. However, the very fact that each biobank may be different and the data so diverse requires a flexible solution. A LIMS that is specifically designed for biobanks and biorepositories is clearly attractive, but it must also retain the flexibility to adjust the configuration to meet the exact needs of the particular organisation, rather than impose a particular workflow that is not appropriate. The ability to configure a LIMS without needing to change the underlying code offers significant benefits. It delivers a specific solution that does not compromise the underlying software and allows future changes to the system, for example in response to changing business needs or regulatory requirements, again without modification of the underlying software. This future-proofs the biobank solution and provides long-term protection for the investment made in the system. REFERENCES 1. 2.

https://www.isber.org/page/BPR https://www.iso.org/standard/67888.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. Visit www.autoscribeinformatics.com for more information. Email: simon.wood@autoscribe.co.uk


Regulatory/Quality Compliance

Is the Absence of Data Integrity Software Affecting the Assurance of Gel Clot Assays in Bacterial Endotoxin Testing? Data integrity remains an important topic in pharmaceutical and biopharmaceutical manufacturing science. The purpose of data integrity is to ensure that the accurate process of production and quality of the products are shown through the documentation that is associated with it. It is essential that facilities report what is occurring in their labs’ processes to ensure that good laboratory ethics are continuously practised throughout the production of their products. Although this is nothing new to the community, every manufacturer around the world must ensure that the data related to their products has not been affected or compromised.

understanding helps to reduce the chance of recalls and ensure that any potential failure in testing cannot be simply tested over without a trace. Data integrity forces companies to identify the cause of a potential failure rather than exchange information as if it never occurred. Regulators are strict about data integrity due to: 1. Alteration of raw data and manipulation of values 2. Changing or eliminating points without justification 3. Backdating results 4. Changing lot information or product information on the paperwork 5. Allowing technicians the ability to edit prior to approval 6. Security of the data is left unattended

Data Integrity Regulators have been cracking down on manufacturers to confirm that they have an adequate representation of their data. Quality assurance departments must enforce and have complete control over the data that is produced. This ensures that information is not manipulated during any stage of testing.

When these data integrity issues occur, facilities must become more stringent to ensure that the data generated have the integrity needed to be accepted by regulators. The system that is encouraged by regulators to be used for the integrity guidelines of facilities is to confirm that their data is Attributable, Legible, Contemporaneous, Original (true copy) and Accurate – the ALCOA Principle.

In FDA’s Data Integrity and Compliance with Drug cGMP Guidance for Industry it states that “data integrity is critical throughout the CGMP data life cycle, including in the creation, modification, processing, maintenance, archival, retrieval, transmission, and disposition of data after the record’s retention period ends. System design and controls should enable easy detection of errors, omissions, and aberrant results throughout the data’s life cycle.” The lack of information of what accurately occurred versus what is presented to regulatory agencies presents an issue of incompleteness which could be hard to determine when there is a product failure. Having the full

The Effect of Data Integrity on Bacterial Endotoxin Testing Endotoxin testing has been around to test pyrogens for many years. Bacterial endotoxin testing uses limulus amebocyte lysate (LAL) to detect pyrogens (specifically endotoxin) in pharmaceuticals, biologics and medical devices. Endotoxin is a toxin released from the lipid A portion of a lipopolysaccharide by a living or lysis gram negative bacteria. This release causes an adverse effect when it encounters the immune system of a living organism. The detection of this pyrogen is important to ensure the safety of end product testing of drugs prior to public release for human and veterinary use.


Winter 2019 Volume 2 Issue 4



Regulatory/Quality Compliance Like the heat block and water bath, this incubator can evenly distribute the temperature of 37 ± 1ºC for heat stability and vibration control to avoid inaccurate determination of results. Unlike the traditional water bath and heat block, the tube reader has user-friendly software to provide a visual trend of what is occurring with the sample and the direction the test is going in real time. This benchtop instrumentation is specific for endotoxin data processing. It is based on the protocols complying with three pharmacopoeias (USP, EP, JP) for bacterial endotoxin testing capable of a successful output of very detailed reports.

Endotoxin testing can be performed by three different methods; gel clot, turbidimetric and chromogenic. The ability to test endotoxin by the change in turbidity or chromophore release, the turbidimetric and chromogenic methods can be tested by a microplate reader. This can only happen when the instrument works in conjunction with software to ensure that data integrity of the testing was performed. In contrast, the gel clot assay cannot be performed on the microplate reader; it is the only method that is unable to prove that the integrity of the data was held throughout the testing process with software. Is the Gel Clot Method Enough Without Data Integrity Software? The gel clot method is an acceptable end product test to release products. It is also known as the “traditional method” because it is the first method for LAL testing. The gel clot method can determine the presence of endotoxin both qualitatively and semi-quantitatively. The method is relatively simple: the sample is incubated at 37± 1°C for 60 ± 2 minutes using a water bath or heating block. After the incubation, each sample tube is removed and slowly inverted at 180° to determine if there is a firm gel formed at the bottom of the tube. If the integrity of the gel within the tube is intact with no deformation, the result is positive for endotoxin. If the gel does not retain its integrity and collapses, the result is negative for endotoxin. The result of the gel clot method concludes the decision for that sample. Although the testing of this method is based on a subjective interpretation of the user, many can question if the user is trained properly to be able to have an accurate interpretation of the results. Performing such a subjective test can potentially differ from person to person, due to the judgement of a gel after inverting the test tube to 180°. Some users of this method have a second person to confirm the results as well. This could still pose an issue as well. The gel clot method itself is risky because it may be difficult to determine if the testing was carried out properly. Other common concerns associated with gel clot lack of data integrity are shown below, which leave room for the ALCOA principle to not be fulfilled: 1. 2. 3. 4. 5. 6.

The software has audit trail capabilities to help to ease audits by regulators. The robustness of the reader’s data processing functions allows statistical processing of the mean of the activation times and the standard deviations of the samples. The software permits the user to define the sample types, such as standard sample, negative control, positive control, etc. The system collects the data and ensures that the integrity of the actions abide to the 21 CFR Part 11 compliant regulation by providing: • • • • • • •

Audit trails Electronic signatures Raw data identification Archiving Backing up data (user management and system) Showing revisions when information is edited Unique user identification and level of access

Confirmation of Data Integrity for the Tube Reader As stated, FDA 21 CFR Part 11 is a feature of most tubers. This allows the tube reader to be able to have the proper electronic data and signatures from all parties involved in the testing of the product. There is a function that will show warnings if the user is not abiding by the guidelines set. In this case, no error in data entry can occur, further ensuring that the facility performing the test has the needed data integrity for their gel-clot assay. Workflows are created to ensure the protocol has been reviewed prior to performing the testing. Another advantage of performing the gel clot method on the tube reader is being able to see what is occurring as the gel is forming. On the tube reader, an indication that the sample’s gel is not forming, in the process of forming, or that gelation has occurred, is the colour of the light adjacent to each well. A time course plot gives the user the ability to be provided with a graph of the behaviour representation of the results in order to determine the outcome of the results quantitatively. This can solidify the user’s testing ability and provide that needed

Ability to manipulate data Providing a minute amount of information Unavailability of a second person to confirm the testing results Misinterpretation of gel Lack of audit trail Lack of unique identifier of each user

Data Integrity with Tube Reader There is instrumentation that is capable of gel clot evaluation. 32 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2019 Volume 2 Issue 4

Regulatory/Quality Compliance the same instrumentation for both. This technology allows for testing to be done by every method. It also gives the customer flexibility when introducing a new product to be tested for endotoxin testing. Most samples require dilution prior to testing due to the samplesâ&#x20AC;&#x2122; ability to interfere with the endotoxin or the LAL. In response, interference can be eliminated by diluting with bacterial endotoxin testing (BET) water or by a buffer. Gel clot assays restrict the user from making larger dilutions due to the maximum valid dilution that can be set from the lysate sensitivity that the user uses to test the sample. On the contrary, the kinetic method has a higher sensitivity than the gel clot assay and allows for a greater maximum valid dilution for samples. The kinetic method allows for a wider range of standards rather than a set two-fold bracket of concentration to use as a standard for comparison. Kinetic assays are typically faster and more accurate results than the gel clot assay. This will permit the user to gradually change from one method to another.

additional confirmation of quantitative accuracy of the gel. As a result, an extensive report can be printed to use as backup information. This will show more evidence of the test added to the qualitative positive or negative results for confirmation of the gel clot method. Transitioning Gel Clot Method from the Water Bath/Heating Block to a Tube Reader Validation of products is always tedious and time-consuming, however, it is a necessary aspect of ensuring that the product testing is accurate and reliable. It also confirms the suitability under actual conditions of use. Since this is the case, most facilities object to going through the process of revalidating a product unless enforced by a regulator or bringing a new product online. The gel clot transition from traditional instrumentation to a tube reader has become easier because the two are very similar. Testing performed on the tube reader should resemble testing with the traditional gel clot instruments. This similarity should eliminate the stress of having to change protocol. Transitioning from Gel Clot Method to Kinetic Methods using the Tube Reader This instrumentation and software are great for clients who are using the gel clot method but would like to start measuring endotoxin by the chromogenic or turbidimetric method. This technology gives the user time to gradually convert from the gel clot method to the kinetic application over time, while still being able to carry out the protocol they have already established using www.biopharmaceuticalmedia.com

Should there be More Emphasis on Data Integrity Software for Gel Clot Assays? The answer to this question is that it depends on how necessary it is to have supported documentation, security and backup quantitative results on the gel clot analysis to confirm what was proven qualitatively. Data integrity will continue to be an important topic in this industry. Whether the information is all recorded by handwritten documentation or by software that can ensure the data is kept secure and free of manipulation, ensuring that the intact data integrity should be the number one goal. If the paper system is ever questionable or human subjection causes a difference in results for a gel clot assay, it is imperative that the proper measures are in place to ensure the data is not at risk. There is much success in the tube reader and software approach for the completion, confirmation and accuracy of the data integrity to support the qualitative results of the gel clot assay. REFERENCES 1. U.S. Food and Drug Administration. Data Integrity and Compliance With Drug CGMP Questions and Answers Guidance. Available at: https://www.fda.gov/regulatory-information/ search-fda-guidance-documents/data-integrity-and-compliancedrug-cgmp-questions-and-answers-guidance-industry. Last accessed October 2019.

LaToya Mayfield LaToya Mayfield is a technical specialist for the LAL Division at FUJIFILM Wako Chemicals U.S.A. Corporation. She has a background in quality control and quality assurance. She is knowledgeable in cGMP, ISO and CAP regulations. LaToya performs customer trainings to assist in achieving successful product validations that comply with regulations. She is the founder and president of GiSTEM, Inc., a non-profit organisation designed to encourage girls to pursue and thrive in STEM fields. Email: latoya.mayfield@fujifilm.com


Regulatory/Quality Compliance

Best Practices of IoT Implementation for Smart Drug Research Drug discovery takes years to complete. Testing a single compound can cost more than £1.5 billion. Experiments on animals often fail to predict human behaviours and responses because traditional animal anatomy often does not accurately mimic the human body. For these reasons, there is a vast need for other ways to emulate human diseases in vitro in order to accelerate the research and development of new drugs.

IoT technology can evolve the sensors’ integrated OOCs. To achieve this, the first step is to integrate sensors to monitor physical and biochemical parameters associated with the functionality of OOC models. What is IoT in the Drug Industry Artificial intelligence (AI) in pharmaceutical companies has the ability to emulate human learnings in the analysis of complicated drug data without direct human input. AI technology gains information and processes it to give a well-defined output to the end-user through machine learning algorithms. The Internet of Things (IoT) in pharmaceutical companies is an application of the IoT for drug- and health-related purposes, data collection and analysis for research, and monitoring. IoT in pharmaceutical companies can be interpreted as connecting the drug discovery devices to the network to allow the scientists and researchers who may be located in remote locations and can still do the exchange of data and information and be able to take intelligent decisions for better drug research.

Is There An Alternative? In this context, organs-on-a-chip (OOC) has emerged as a new tool for drug discovery. Organs-on-a-chip is an in vitro tissue culture platform w   hich provides better evaluation of the effects of various chemicals on human tissue. Organ-on-a-chip An organ-on-a-chip (OOC) is a multi-channel 3-D microfluidic cell culture chip that simulates the activities, mechanics and physiological response of entire organs and organ systems, a type of artificial organ. OOC use microfluidics to reproduce the way a tissue or part of an organ works. Organ-on-a-chip can partially simulate organ function including lung, intestine, kidney, skin, bone marrow, blood-brain barrier, etc.

Drug Life Cycle

Although the field of OOCs has been looed at over some time, there is still a need to digitise the OOCs that will provide real-time monitoring and capturing the continuous information about the metabolic activity of the tissue from remote locations. Benefits of Integrating IoT Sensors in OOCs


Winter 2019 Volume 2 Issue 4

Volume 9 Issue 1 - Spring - 2017

Volume 9 Issue 1

Peer Reviewed

International Pharmaceutical Industry

Supporting the industry through communication

IPI â&#x20AC;&#x201C; International Pharmaceutical Industry


MALDI Mass Spectrometry in Drug Discovery Gaining A Deeper Understanding

Three Ways to Mitigate the Risk of

Late-Stage Failure in CNS Drug Development


The Foundation of Clinical Trials www.ipimediaworld.com

Temperature Management Keep Your Cool




Peer Reviewed, IPI looks into the best practice in outsourcing management for the Pharmaceutical and Bio Pharmaceutical industry.



Peer Reviewed, JCS provides you with the best practice guidelines for conducting global Clinical Trials. JCS is the specialist journal providing you with relevant articles which will help you to navigate emerging markets.


Volume 4 Issue 1 Volume 4 - Issue 1 Supporting the Development of Veterinary Drugs, Veterinary Devices & Animal Feed


Applying Game Theory to One Health Modelling Veterinary Healthcare Delivery International Animal Health Journal - Supporting the Development of Veterinary Drugs, Veterinary Devices & Animal Feed

Mastitis due to Mycoplasma bovis Insights Pet Obesity Prevention is Better than Cure Leadership Skills of Extraordinarily Successful Executives


Official Supporting Associations -

Sponsor Companies -

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Peer Reviewed, IAHJ looks into the entire outsourcing management of the Veterinary Drug, Veterinary Devices & Animal Food Development Industry.

www.animalhealthmedia.com www.biopharmaceuticalmedia.com


Peer reviewed, IBI provides the biopharmaceutical industry with practical advice on managing bioprocessing and technology, upstream and downstream processing, manufacturing, regulations, formulation, scale-up/technology transfer, drug delivery, analytical testing and more.


Regulatory/Quality Compliance Working Principle of IoT in the Drug Industry Let us first understand the working principle for IoT: Collect – Connect – Process – Present 1) Collect: Drug discovery devices with enabled sensors collect live data from the live environment. 2) Connect: The sensor sends collected data to a cloud infrastructure using various mediums of communications, including mobile phones or satellite networks, Bluetooth, WI-FI, WAN, etc. 3) Process: Once data is collected, and it gets to the cloud, the IoT software processes data into valuable information. 4) Present: The information needs are then presented to be available drug researchers. Challenges of IoT Data Collection • Unstructured data • Security and privacy issues Device Connection • Internet outages • Power cuts • Security issues • Lack of device performance • Industrial IoT integration – data loss, ineffective connectivity and desynchronisation Data Processing • Analysis of unstructured data • Inaccurate analysis due to flaws in the data Displaying the Information • Unpredictable situations • Incorrect information The IoT technology is expanding into the entire pharmaceutical and drug space. However, due to the challenges mentioned above and the lack of resources to overcome those challenges, companies may struggle to integrate sensors to OOCs. Research estimates that almost three-quarters of these projects fail due to these reasons. A recent forecast suggested that £192 billion in pharmaceutical sales could be at risk by 2024. This article aims to discuss the best practices of IoT implementation to empower the world of drug discovery. This is done by integrating the IoT implementation framework in the drug development stages, for seamless integration of IoT sensors to OOCs.

• •

Phase 1 – Pre-clinical Research of the Drug The following can be achieved with the correct implementation of IoT technology (Stage 1 to Stage 4)

• •


Improved drug discovery through improved information Unmet need met with real-world data

Stage 1 – Inception – IoT Implementation This stage includes understanding users’ needs of the drug and the drug discovery devices required to identify and assess the scope and value of the IoT implementation. The vast and diverse data collected from large sets of real-world cases will increase the genuineness of user research. However, the data must be correct and complete and of high quality. If the data is not high quality, then data mining must be performed: Data mining: Most of the time, medical research users lack critical real-world information. It mostly uses leftovers, controlled environments and volunteers for medical examination. Data mining ensures availability of meaningful and valuable data. By generating more practical and reliable data, IoT yields better solutions and discovery of issues that were previously unknown. That’s why improving data quality for IoT applications should be one of the most important activity to get the best ROI through IoT implementation. The below are the suggested best practices: • • • •

Gather data from large sets of real-world cases. Prepare data for mining. Apply the mining algorithms to data. Display mined data to the end users (patients/HCPs).

At the end of this stage, an intended use and indication of use document (covering all the points) having a clear and exact understanding of why IoT is needed in the drug discovery device space should be produced. The below are the suggested best practices: • •

Best Practices to Address These Challenges and Successfully Implement IoT Solutions The below figure illustrates the integration of IoT life cycle stages into the drug life cycle phases.

Phase 1: Pre-clinical Research Stage 1: Inception Stage 2: Planning Stage 3: Design Stage 4: Prototyping and verification Phase 2: Clinical Approval and Launch Stage 5: Validation and implementation Phase 3: Commercials Stage 6: Maintenance of IoT framework

• •

Document clearly what is the purpose of the drug discovery device. Document why IoT should be implemented for this particular drug discovery device. Document what clinical solution IoT will bring for this drug. Document the short-term and long-term usage of using IoT technology. Winter 2019 Volume 2 Issue 4

Regulatory/Quality Compliance • • • • • •

Document the end users – usage environment, age group, gender. Document the frequency of the drug usage. Document the user interaction methods with the drug discovery device. Document the risks of the category of drug and how IoT technology control the risk. Document the frequency of the drug usage. Document the interoperability needs with other medical devices.

Review of Stage 1: • Does the drug manufacturing company have complete clarity of why IoT implementation is needed for their drug devices and exact understanding of the IoT system requirements? • Is the risk management framework (identification-assessmentevaluation-control) understood? Stage 2 – Planning – IoT Implementation Once the user needs are well understood, the next step is to plan for the IoT implementation, considering multiple aspects of the devices. Find below the suggested best practices: •

Design planning of the IoT implementation: Successful IoT solutions require a solid strategy and workflow, combined with risks and human factors considerations. At this stage a plan document should be produced as per the given guidelines: • • • • • • •

• •

Plan for development kit – IoT hardware device, IoT software device, drug discovery device. Plan for the infrastructural needs. Plan for the risk management – technical and clinical risks. Plan for human factors – formative and summative activities. Plan for end-to-end security and privacy aspects for the end users in the IoT ecosystem. Plan for cold and hot path analytics. Plan for standards to be followed enabling companies to accelerate time to market and maximise the market size. Plan for post-market clinical follow-up. Plan to communicate with regulatory body to comply with regulations.

Resource planning: Some of the experts should be planned early for the IoT team. • •

• • • • •

Computer Engineer: a computer engineer specialising in embedded systems. Software Engineer: t  o design and implement computer programs, perform unit and integration testing, perform code reviews. Electronic Engineer:  to design circuit boards to be used in the IoT systems. Mechanical Engineer:  to design the hardware IoT components. Mechatronic Engineer: specialising in sensor use. Automation Engineer: automation of production processes. IT Expert:  to implement and maintain the internet


• •

network. Manufacturing & Production Engineer: someone with knowledge of the production processes, needs, and limitations. Telecom specialist:  to deal with the variety of protocols and gateways for IoT data transfer. Security & Vulnerability Engineer: someone with end-to-end security and vulnerability knowledge and formulate the security strategy to protect the entire IoT ecosystem.

Planning for maintenance and upgrades of IoT infrastructure.

Review of Stage 2: • Has the drug manufacturing company done sufficient planning to accomplish all the work? • Has the risk management plan (identification-assessmentevaluation-control) been implemented? Stage 3 – Design – IoT Implementation By now, the user needs are well understood and the plan is in place to build a strong and reliable IoT system. At this stage, you have to start designing the IoT framework that comprises IoT hardware and IoT software. To have an effective design, below are the suggested best practices: • • • •

Choosing the IoT platform Choosing the IoT hardware Choosing the IoT software IoT security solutions

Choosing the IoT Platform The parameters for choosing the IoT platform could be on the basis of the below suggested best practices: • • • • • • • • • •

Security Performance Storage and retention Scalability Usability Interoperability Adaptability with the legacy architecture Message protocols Disaster recovery Maintenance & decommission support

Choosing the IoT Hardware The below are the suggested best practices to choose the IoT hardware: Device to be connected: Choose the drug discovery device as per the intended use. Data acquisition: This is the component that contains the sensors that acquire real-world signals such as temperature, pressure, density, motion, light, vibration, etc. Decide the type and number of sensors you will need on your application. IoT sensors are the most critical IoT hardware. These devices consist of a variety of modules such as energy modules, RF modules, power management modules, and sensing modules. INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 37

Regulatory/Quality Compliance Data processing: This is the main unit that processes the data and performs operations such as local analytics, and stores data locally. Data display: It enables communications with the third-party system locally or on the cloud. For example, wearable electronic devices are the devices that can be worn on the body to get real-time information. Choosing the IoT Software The below are the suggested best practices to choose the IoT software: The Internet of Things (IoT) is an entirely new platform for developers, but one thing which remains consistent in this new world is the programming language. Developers utilise the same languages for their projects, while also integrating some specific changes for IoT. What languages are best for IoT? Selecting a language for IoT projects could be as difficult as selecting an IoT hardware platform. IoT software comprises a wide range of software and programming languages. C/C++, Python and Java are the most popular I oT programming languages. •

C/C++ is great for writing hardware-specific code and it works really well in Linux operating system (OS), which is the most popular IoT  OS. C/C++ is made to handle the hardware and complex processing at the same time, making it ideal for running on embedded systems. This language was written for the hardware systems, which makes them so easy to use. C is considered the most useful for I  oT  devices because it doesn't  require  a lot of processing power. Java:  While C and C++ are hardware-specific, the code in JAVA is more portable. It is more like a write once and read anywhere language. Programming   with  Java  makes   IoT  devices more efficient in exchanging information and making proper use of the information when and where it is needed. So, the device becomes more integrated. Python: Python is a good choice for data analysis in IoT systems. The language is simple and can be easily deployed. Its large community helps in providing help and libraries as and when required, which makes it an ideal language for IoT systems, especially for data-intensive applications. JavaScript: JavaScript works best in a wide range of environments, and is best in gateways and the cloud. It is very efficient when it comes to sensors because of having an event-driven modality option. PHP: PHP is a good option to develop apps using the GPS data from IoT devices.

Low-level programming languages: B#, a language built from the ground up for very low power devices. It is similar to C#, but fitted with real-time control functions. Assembler is probably among the low-level languages, capable of running on just about everything. The downside is there’s no hand-holding at all, so if your code doesn’t work, or if a new processor doesn’t accept Assembler code, then you are in a really difficult situation. Weave (Google) could become popular if it receives more support from developers. Apple offers its open source language, Swift, currently marketed at iOS and Mac OS developers. To interact with the iPhones and iPads with your home hub, Swift is a good choice. IoT Security Solutions IoT security is the technology area for protecting the connected drug devices and networks in the IoT system. 38 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Challenges Lack of awareness: Design is one of the most sensitive phases in building the IoT system. The majority of vulnerabilities discovered in software are due to lack of awareness towards security aspects during the design phase. Statistically, more than 80% of all complex and expensive software refactoring is due to this issue. Low in priority: Security is often considered as an “afterthought” and as a result, the cost of fixing security issues in live software is much higher than the cost of implementing the secured design techniques at the design stage (before even beginning to code). Obsolete technology: Often, the connected legacy drug devices may not be inherently designed for modern IoT systems; hence the modern threats cause vulnerabilities. Weak password: Hardcoded or default passwords can lead to security breaches. Security patches: Mostly IoT devices (drug device sensors) are placed on a machine and left until end of life. They hardly ever receive security updates or patches and hence often cannot handle advanced encryption or other modern security measures. Lack of industry-accepted standards: While many IoT security standard frameworks exist, there is no single agreed-upon framework which makes it difficult not only to secure systems, but also ensure interoperability between them. Effect Apart from the financial and reputational losses, the worst could be compromising the patient’s sensitive data from malicious attacks. Injecting security solution on the basis of the suggested best practices: • Incorporating security features at the design phase • Strong encryption protocols adapted for secured communication between devices • Strong passwords policy in the IoT system • Updated digital certificates and continuous software updates for connected drug devices and IoT systems • API security for data integrity to ensure secured data transfers from devices to IoT backend systems • Unique device identifier (UDI) providing unique identifier for each device • Hardware security • Protecting IoT network by ensuring security gateways, port security, firewalls • Inventory management of IoT devices • Awareness and training – keep staff up to date with new systems, architectures and programming languages so they are ready for new security challenges.

Review of Stage 3: • Has the drug manufacturing company done the effective design to meet all the user requirements? • Has the risk management framework (identificationassessment-evaluation-control) been implemented? Stage 4 – Prototyping & Verification – IoT Implementation Now that IoT hardware and software are chosen, a secured IoT system prototype for the drug discovery device can be built. It Winter 2019 Volume 2 Issue 4

Regulatory/Quality Compliance is the IoT hardware and devices enhanced with smart sensors and embedded systems using many off-the-shelf components like sensors, circuit boards, and microcontrollers. Verification of the prototype shall act as proof that the concept will work the way it was envisioned. Verification of the prototype on the basis of the below suggested best practices: • • • • • • •

Code reviews Unit testing – verifies that the code works Integration testing – verified that the coded items are integrated with no interface challenges System testing – testing the drug device in the prototype of the IoT system Formative testing – early end use feedback. This can lead to some design changes Cold path analytics – testing the batch processed data Security testing – data integrity testing

Review of Stage 4: • Has the drug manufacturing company built the IoT System prototype for the drug discovery devices, and done sufficient verification? • Has the risk management framework (identificationassessment-evaluation-control) been verified? Phase 2 – Clinical Approval & Launch The following can be achieved with the correct implementation of IoT technology (Stage 5) • •

Ensure effectiveness of drug Meet post-market expectations

Stage 5 – Validation & IoT Implementation Once the prototype is verified, the IoT system implementation for the drug discovery device can be accomplished. Before it gets implemented in the patients’ and HCPs’ world, a thorough validation must be performed. • •

• • • • • • • •

Validate end-user requirements Validate risk and usability factors of patients and HCPs – summative testing with the reference of intended use of the drug device in the real IoT system Perform a risk-benefit analysis Compatibility testing of drug device in IoT ecosystem Clinical evaluations of device in IoT system Summative testing using real users in the real IoT system Performance testing Validate the hot path analytics Interoperability testing Security testing.

Review of Stage 5: • Has the drug manufacturing company implemented the IoT system prototype for the drug discovery devices in the real-world environment, and has it done sufficient validation in terms of summative testing and clinical evaluation? • Has the risk management framework (identificationassessment-evaluation-control) been validated? www.biopharmaceuticalmedia.com

Phase 3 – Commercial Phase of the Drug The following can be achieved with the correct implementation of IoT technology (Stage 6) • •

Monitor outcomes against competitive products Monitor safety.

Stage 6 – Maintenance of IoT Framework • Performance monitoring of the IoT system and measure of its ROI • Post-market clinical follow-ups (PMCF) - the data derived from the post-market clinical follow-up study is used to provide clinical evidence to support the  post-market surveillance of the IoT system for the drug discovery device • Corrective action (CA) – rectify a task, process, product that has caused an issue • Preventive action (PA) – change implemented to address a weakness in the IoT system that is not yet responsible for causing an issue. Review of Stage 6: • Is the PMCF process effective? • Is the CAPA process in place? Conclusion To get the best of drug research using organ-on-a-chip (OOC) methods, connecting it to an IoT sensor would be the most effective. This article is an attempt to suggest the best practices of IoT implementation in the drug research method, based on my rich experience in digital therapeutics and converging digital technologies with pharmaceutical products. REFERENCES 1. 2. 3. 4.

https://en.wikipedia.org/wiki/Internet_of_things https://en.wikipedia.org/wiki/Digital_health https://en.wikipedia.org/wiki/Drug_development https://en.wikipedia.org/wiki/Organ-on-a-chip

Anindya Mookerjea Anindya, Founder & CEO at S-Cube Technologies, is an Entrepreneur, Thought Leader, Business Visionary, Strategist & Founder of S-Cube Technologies, an IT Digital Solutions Company using Internet of Things (iOT), Artificial Intelligence (AI)/ Machine Learning (ML) & Robotic Process Automation (RPA) with potential contribution towards bringing digital technology in Healthcare, Drug and Life Sciences industries. His core specialisations include Advanced Digital Technologies, Connected Devices & Cloud-based App Development. Email: anindya@scubetech.co.uk


Regulatory/Quality Compliance

Managing Global Supply Chains As temperature-sensitive pharmaceutical products are increasingly being shipped globally to more remote regions there is an even greater demand for the effective and efficient management of global supply chains.

Increasingly there is a rising requirement for innovative solutions being placed on the temperature-controlled packaging industry, coupled with a rising requirement to track and trace pharma payloads throughout transit. Whether shipping finished products, transporting clinical trials materials or delivering sample drugs, temperature excursions can mean the difference between success and failure, profit and loss. It is essential therefore, that pharmaceuticals are protected throughout the supply chain end-to-end as temperature excursions during transportation can even cause them to become toxic. There is increasing recognition within the supply chain of the vital role smart packaging plays and in response to the stringent regulatory requirements, the packaging industry is taking a proactive approach. An impetus for the latest generation of high-performing packaging products comes as the pharma industry continues to grow at a significant rate, with an estimated global worth of $400 billion by 2020 according to the World Health Organization. Maintaining end-to-end pharma supply chain integrity is critical to mitigate risks within the pharma-logistics cold chain and better ensure the safe and secure transportation of healthgiving and life-saving pharmaceutical products. The market for packaging to transport temperaturesensitive materials for the global life sciences industry, such as pharmaceuticals, blood, tissue and organs, is currently valued at $2 billion and expected to grow to approximately $5 billion by 2026. The global life sciences industry faces several complex challenges – protecting the integrity of their temperaturesensitive high-value payloads is an important challenge. But the industry also must concern itself with mitigating costs, managing and tracking the assets within a complex cold chain closed-loop logistics system, meeting stringent global regulatory standards and navigating complicated global shipping lanes and unforeseen challenges. Various pharmaceutical compounds, utilised within the sector, are developed under certain temperature control conditions or designed to be stored at specific temperatures to maintain their stability. 40 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Therefore, it is vital, when shipping pharmaceutical products between locations, they remain at their storage condition temperatures to maintain their effectiveness at the point of use. Within the industry there are standard temperature ranges such as deep-frozen (below -50° Celsius), frozen (-50° C to -20° C), refrigerated (4° C to 8° C) and room-temperature (15° C to 25° C). With the pharmaceutical companies developing ever more complex and temperature-sensitive drugs, there is a greater demand in the cold chain industry to meet the growing market demand for supply as well as improved packaging performance and efficiency. To help mitigate supply chain risks, the industry is seeing a greater demand for higher performing packaging products. Coupled with the rise in regulatory requirements, there is an even greater emphasis on supplying the global market with more advanced, smart packaging that does more than act as a container for precious, high-value payloads being transported to emerging markets via complex shipping lanes. The expertise of experienced engineers is a vital component within the packaging industry, with providers deploying increasingly high performing packaging systems that need to mitigate supply chain risks and minimise temperature excursions. Any spikes or deviations in temperature, beyond the range specific pharmaceutical products are required to be stored and shipped at, could have a devastatingly detrimental effect on the payload, damaging the container’s contents and impacting on the efficacy of the products being transported. This could have financial repercussions equating to losses of hundreds of thousands of pounds; however, more importantly, it could have catastrophic consequences for the end user/patients reliant on the drugs being delivered remaining intact. More global clinical trials are requiring stricter temperature regulations, which command compliant cold chain conditions and increasingly innovative packaging solutions. Winter 2019 Volume 2 Issue 4

Regulatory/Quality Compliance

Temperature restrictions when transporting these pharma payloads present their own challenges, coupled with the fact more are being shipped to emerging markets where there are also extreme temperature ranges to contend with. An impetus for the latest generation of high-performing packaging products are these advancements in drug developments, which includes the increase in more fragile and temperature-sensitive pharmaceutical products. A driver for a substantial proportion of this projected growth in the life sciences sector includes the rise in temperaturecontrolled biosimilars and biologics, which are biologically-based pharmaceuticals as opposed to chemical-based. It is predicted more than 50 per cent of approved new drugs are going to be biologics or biosimilars in the next few years. The evolution of this latest drug development presents its own supply chain challenges when it comes to safe storage and transportation of these temperature- and time-sensitive pharma products. With the rapid rise of biologics and biosimilars within the pharma development sector, the need for temperature control during transportation is ever increasing. Any temperature excursion, from minor to major, within the supply chain can have costly consequences for pharmaceutical companies. Particularly serious would be a temperature excursion which impacts patient health. More complex distribution lanes, with emerging markets, geographies and increasing regulatory compliance conditions are some of the challenges when transporting these temperaturesensitive biologics/biosimilars. Innovation and new technologies are proving pivotal to the emergence and evolution of smart temperature-controlled packaging protecting pharmaceutical payloads worldwide, and successful management of supply chains is essential to provide pharma payload protection. Packaging companies continue to incorporate innovative design features and are utilising more advanced technologies, to produce and manage pioneering products and ever more sophisticated systems, which help eliminate excursions in temperature in cold chain. www.biopharmaceuticalmedia.com

Blockchain is another technology finding its place in supporting pharmaceutical manufacturingâ&#x20AC;&#x2122;s cold chain logistics processes. Blockchain is having a real impact on pharmaceutical shipments, from prevention of theft and counterfeiting of pharmaceuticals, to tracking root cause of a dangerous event that sickens a patient, to government tracking of source of origin to properly assess duties and taxes for imports. The industry is seeing an increase in the introduction of information-centric capabilities to assist with the safe shipping of pharmaceuticals around the globe. Packaging companies are increasingly utilising advanced asset management software systems, which are in place specifically to ensure pharmaceuticals are shipped to the right location, at the right time and critically, that they arrive in the right condition. Companies deploying pharmaceutical shipments worldwide benefit from the introduction of new technological advancements including web-based asset management software solutions, designed to track individual shipments globally. Integrating these cloud-based systems offers a range of capabilities benefiting the industry, including options to set up automatic maintenance, next shipments alerts and produce customisable reports. The industry is also seeing a growing trend to deploy reusable systems coupled with asset management SaaS (software as a service) and reaping the associated benefits. These systems can automatically collect and analyse data from company data logger outputs. Currently operating in the market is a range of SaaS products providing collection and analysis of brand-agnostic sensor data, as itâ&#x20AC;&#x2122;s linked to a variety of smart packaging options allowing packaging vendors to track a diversity of data including vibration, light, humidity, temperature and more. These software platforms capture and monitor information throughout the course of the shipments trip. The data retrieved and shared can help pharmaceutical companies make more informed choices on the most appropriate packaging systems to deploy, depending on the specific shipping lanes and routes their payload will navigate. Utilising asset management software within the supply chain process help life science industry clients reduce payload risk, distribution costs and their environmental impact, ensuring INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 41

Regulatory/Quality Compliance temperature-sensitive, critical and high-value payloads reach their destination safely. Integrating the cloud-based system supports and enhances engineering expertise that is incorporated into the development and design of the increasingly sophisticated systems utilised by the life science industries. Increasingly passive and active bulk systems are incorporating data loggers to track the temperature throughout the course of the trip throughout the supply chain. Issues can arise because often a parcel shipment will need to be opened to access the temperature logger stored inside. Alternatively, data logger devices can be attached to a specialised container to ship a pallet of products providing an isolated monitoring option to pick up data, which can be saved to the cloud via Bluetooth or radio-frequency identification (RFID). The latest development within the packaging industry transporting pharma shipments globally is the move toward GPS tracking, which would need to be managed via Bluetooth, RFID or manually scanned barcodes whereby pharma companies can track packages, and their shipment progress, online. GPS is the latest development in response to ensuring the protection of high-value pharma payloads. It is predicted advancements in GPS tracking options via a SaaS system will be part of the industry in the near future. There are benefits to pharma companies, including knowing where their shipment is throughout its transportation trip. If payloads are lost or get delayed en route, the pharma company take steps to intervene and recharge or replace coolants so the package or the bulk system gets delivered before expected temperature duration is exhausted, and this would help mitigate a temperature excursion caused by a delay. What’s exciting about leveraging all of this smart technology is the promise of the capabilities to have data logger sensors and SaaS platforms communicate directly with each other. The latest developments will be something that interests the pharmaceutical companies; the availability of information through a SaaS platform will be a market differentiator for the companies that produce this type of smart packaging option. Currently the information being captured is primarily via barcode, which requires human intervention to manually collect and then input the information into a SaaS system. It’s predicted there will be a move to a system whereby the relevant information will be captured and then stored in a centralised database. That information could be updated frequently at check-in points or instantly via Bluetooth. Additionally, forensically these smart packaging systems would offer insight to find out at what point the systems failed and where that happened with a combination of GPS and temperature data. This would allow pharma companies to discover when and where a failure occurred and then diagnose if it’s a problem with the shipment, with logistics, or with the local customs office or similar. 42 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

On the horizon is the integration of GPS and temperature logging to a SaaS system in an effortless way that doesn’t involve human interaction to leverage the movement towards an Internet of Things (IoT), which allows all devices to be assigned an IP address, allowing each device to interact as a unique entity on the internet. Leveraging a movement to IoT will help packaging manufacturers with making future packaging truly smart, by even providing information on the shipment’s status, temperature, current location and more back to its owner in a central office. It also allows for mid-transit interventions if needed or examining efficiencies of scale identified, such as using different shipping lanes or different ways to palletise or bulk-ship smaller products. Specialised software systems can also aid reverse logistics within the industry and can provide real-time tracking and trading through the entire end-to-end distribution cycle. These optimisation tools can help ensure payload efficacy and efficient life cycling of reusable packaging inventory assets, providing a high return on investment. Often easy-to-learn and use, these superior software systems help the global life sciences industry manage its demanding and expanding cold chain logistics supply chain operations while critically meeting the stringent requirements of a highly regulated industry. All of these innovations based on clever uses of smart technology build upon the robust performance of advanced thermal shippers. Traditionally packaging vendors relied on more basic thermal control methods providing passive packaging products, where the key forms of technologies deployed were ones incorporating insulating material for the outer box of polystyrene or foams, providing protection and insulation. To maintain the temperature within the packaging, combinations of chilled and frozen water were utilised. Because of the requirement to carry a lot of water to control temperature and often bulky insulation, these systems proved heavy and their performance against highly variable external temperature challenges was not reliable. Winter 2019 Volume 2 Issue 4

Regulatory/Quality Compliance

Although useful for an anticipated and unchanging external environment, these antiquated systems are being replaced more regularly with technologically advanced packaging systems. These advanced temperature-controlled packaging systems were developed to meet the demand and regulatory requirements for pharmaceutical companies and their cold chain supply chain service providers. Most recently the technological advancements introduced to the market have seen the advent of better insulation options by incorporating vacuum insulated panels (VIPs), reducing the thickness of the insulation required and improving performance. The traditional water-based systems are rapidly being replaced with ones using phase change materials (PCMs) where the melting point of the coolants deployed is designed to the ideal temperature required, while holding that temperature for as long as a week without any additional, external thermal energy. These latest advancements mean temperature-controlled shipping systems using PCMs are far more reliable, providing thermal stability within the payload space at the desired temperature while using less weight and space. The payload efficiency of these newer systems can be more than twice that of the traditional water-based and foam-insulated shippers, proving more cost-effective when it comes to logistics services. Although these more sophisticated shippers can be more expensive, the trade-off is you are less likely to experience temperature excursions en route, which would lead to damaged products providing a costly consequence in the long run. In line with the increase in more complex shipping lanes and limited infrastructure in place in some developing destinations, the need for smarter, more secure, robust packaging has become more prevalent. The more sophisticated a shipper, the more the unit cost can be, so increasingly there is a requirement for reusable systems to provide better return on investment and which are more www.biopharmaceuticalmedia.com

cost-effective in the long run. Additionally, reusable systems provide considerable environmental benefits. Provided the infrastructure is in place to recapture and reuse higher performing systems, that contain higher-value components, reuse makes economic and environmental sense. The rise in reuse has trigged a corresponding increase in the global network of service centres being established to facilitate the reconditioning and repurposing of these smarter packaging options. It has also sparked the rise in SaaS systems within the packaging industry with cloud capabilities that better enable collaboration in the sometimes complex packaging supply chain and the ability to centralise data focuses the management of packaging services intelligently. Ultimately the aim always is to continue to reduce the supply chain costs and improve performance and reliability.

Adam Tetz Adam Tetz is the Director of Worldwide Marketing at Peli BioThermal and has more than 25 years of marketing experience. He is responsible for telling the story of Peli BioThermal to our worldwide audiences, including brand identity, product launch and communication strategy. Prior to Peli BioThermal, Tetz held positions in product management and marketing communication across a variety of industries, including medical software, financial software, information services and professional consulting services. H   e holds an MBA in marketing from the University of Saint Thomas, a BA in advertising from the University of Minnesota and is a veteran of the United States Coast Guard. Email: adam.tetz@pelican.com


Special Feature

Marine Extracts for Novel Medicines Developments: A Natural Solution to Antibiotic Resistance and Biofilm-derived Infections In order to decrease the selection and spread of antibiotic resistance and to develop new therapies to fight against chronic infections, pharmaceutical companies are constantly looking for new resources to develop effective and safe medicines. For centuries, natural products have been used to develop medicines for the management and treatment of various diseases. As 70% of the planet is covered by water, today one of the most promising sources of health ingredients for the future could be the oceans. In fact, marine extracts have attracted a great deal of attention due to their potential effects in promoting health and reducing disease. The research in this field is still at its premise; nevertheless, some pioneer pharmaceutical companies and academic institutions have decided to focus their research and development in marine-derived extracts to answer to one of the major concerns of the healthcare community. Novel Resources for Medicine: Encouraging Findings Despite the marine biosphere being the largest one on earth, it remains unexplored, as the majority of oceans, especially the deep stages, are still inaccessible to researchers. Nonetheless, marine environments harbour inestimable riches of microbial diversity and natural bioactive compounds (e.g. enzymes, biopolymers, biosurfactants, exopolysaccharides) that possess a wide variety of potential properties which differ from their terrestrial counterparts.1,2 Aquatic environments, especially oceans, are one of the most adverse. This is due to variations in temperature, pH, salinity, currents, precipitation regimes and wind patterns.

peculiar characteristics, such as water solubility, biodegradability, biocompatibility, bioadhesivity, swelling and gelling power.5 Shallow marine hydrothermal vents of Eolian Islands, Italy, in the Mediterranean Sea, provide an easily accessible extreme environment to study marine microbial diversity and several extreme bacteria. A specific bacteria, Bacillus licheniformis, part of Lallemandâ&#x20AC;&#x2122;s marine bacteria strains bank, has been recently isolated from the shallow hydrothermal vents in the sea surrounding the island of Panarea. The unique metabolites produced by this bacteria, also defined as postbiotics, have demonstrated unmet anti-biofilm activity, representing a potential to face the alarming resistance of bacterial infections caused by pathogenic biofilms development.6â&#x20AC;&#x201C;8 Biofilms Activities and their Involvement in Resistance to Antibiotics What is Microbial Biofilm Biofilm Formation The research into and interest in microbial slime is in fact quite ancient. In 1868, the German biologist Ernst Haeckel hypothesised that life on earth originated from primordial slime at the bottom of the oceans. His successor Henry Huxley observed and analysed mud on the Atlantic seafloor, sure to have discovered the source of primordial matter. In honour of Haeckel, he named this slime Bathybius haeckelii. Although Huxley wrongly thought that this slime was a new organic substance, his discovery enhanced several researches on microbial slime, to reach current knowledge about microbial biofilm, as described below.9 At their normal stage, planktonic (floating) bacteria live individually in suspension in a liquid environment. However, in response to a stressful environment, the planktonic pathogenic bacteria start to regroup themselves in a community to adhere to the mucosa, to adopt another lifestyle: the biofilm.

The adaptation of marine organisms to a wide range of environmental conditions render them an enormous reservoir for biotechnological improvements, and findings are encouraging: marine microorganisms provide a tremendous biodiversity, offering great promise as a source of pharmaceuticals for the future.3 Bacteria from marine sources have the uncommon ability to grow in extreme environmental conditions, such as high temperature, salt water, and high concentrations of hydrogen sulfide and heavy metals; and the exopolysaccharides released by marine species were found to have unique physical properties and molecular structure.4 Marine bacterial exopolysaccharides have a wide range of biotechnological and pharmaceutical applications, thanks to their 44 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2019 Volume 2 Issue 4

Special Feature Microbial biofilm is an organised structure of microorganisms living in community within an extracellular matrix attached to a surface. It is composed of 97% water, 2% protein, 1–2% of polysaccharides and < 1% of eDNA and eRNA. Extracellular DNA (eDNA) represents an important component required for biofilm formation and maintenance10. Bacteria account for 5–35% of the biofilm volume, while 65–95% consists of the extracellular matrix. In fact, this biofilm is a kind of “building” embedding the microbial community. The crucial stage in biofilm formation is the initial adhesion of the microbial cells to the host epithelial cells. Non-specific adhesive mechanisms as well as adhesins, which are cell-surface components of bacteria, facilitate the adhesion of the microbial cells to the mucosa and to each other. It represents the first stage of biofilm formation, but is still a reversible adhesion. The second stage, also called the colonisation stage, results from the production of extracellular polymeric substances by the microbes leading to their irreversible attachment to the mucosa. This substance will also form the matrix that embeds the microbes. The third stage is the early development of the biofilm in three-dimensional architecture with protective scaffolding and water channels. The fourth stage corresponds to the maturation of the biofilm architecture, its size increasing while its architecture becomes more complex. During this stage, the bacteria are in near-dormancy state and grow slowly. They also communicate between themselves and exchange genetic material, and release enzymes and inflammatory molecules until the biofilm is destroyed. The last stage is the dispersion of single microbes, when the bacteria resume their planktonic state to spread and colonise new surfaces. Some of these bacteria have acquired new abilities such as resistance genes or biofilm development and can restart the cycle of biofilm development, which in the case of pathogenic bacteria lead to recurrences and relapses of infections.10 Microbial biofilm represents more than 80% of all bacterial infections. The pathologic mucosal biofilm can be viewed as an extension of the concept of “dysbiosis”, which, once formed, contribute to a stable pathogenic microbiome leading to disease persistence. The pathologic biofilm can act in two ways: as a “protective home” for further pathogens that can egress the biofilm in the planktonic phase and cause locally recurrent infection, or by disrupting the healthy local microbiome, promoting inflammation even in the absence of infection.11

communication, also called Quorum Sensing. Quorum Sensing is the regulation of the gene expression in response to the fluctuation of the microbe’s density inside the biofilm thanks to signalling molecules “autoinducers”, and of the availability of the nutrients. By this process, the microbe’s density is regulated.13 In conclusion, pathogenic bacterial biofilms are a real challenge for the healthcare community, as the microbes embedded in such a matrix can reproduce a real ecosystem, rendering them almost invincible. They are hidden in this protective matrix and can mislead our immune system and stay in a near-dormant state. They are also more resistant to antibiotics and biocides. In fact, they can be compared to “Trojan horses” in IT security, able to lure our system of defences, stay in a latent form, and then infect the whole system. It was then imperative to develop new approaches to prevent and disrupt the biofilm formation in infectious diseases. Biofilm and Application in Pharmaceutical Industry As we previously discussed, pathogenic biofilms can develop on different mucosa and epithelial cells, oral, nasal, urogenital but also on abiotic and hydrophobic non-polar surfaces (Teflon, and plastic) such as central venous and peritoneal dialysis catheters, pacemakers, mechanical heart valves, etc. The applications are then numerous, depending on the anatomical region of the body infected: urinary tract infections, catheter infections, middle-ear, oral, periodontal and dental infections, middle-ear infections, nasal infections, and many others. Taking the example of dental plaque and oral biofilms, these biofilms are responsible for the development of periodontal diseases, endodontic infections, and caries. The pathogens responsible for the development of such infections are Streptococcus mutans, and Actinomyces spp, and some aciduric bacteria lactobacilli, and Bifidobacterium spp.13 Speaking about chronic rhinosinusitis (CRS), methicillinresistant Staphylococcus aureus (MRSA), but also Pseudomonas aeruginosa have been isolated frequently in CRS patients. The bacterial colonisation and biofilm formation in the sinuses is involved in the recurrences and resistance mechanism.14 Ventilator-associated pneumonia (VAP) occur in intubated patients and is one of the most frequent hospital-acquired infections.15 The mortality rate is quite important and reached about 50%, with VAP caused by microbial biofilms formed on the inner surface of the endotracheal tubes (ETT). They are derived from various species: Staphylococcus aureus, Enterococci, Enterobacteriaceae, Pseudomonas aeruginosa, Acinetobacter baumannii and Candida species.16

The biofilm allows the bacteria to protect themselves from immune system, antibiotics and biocides; it is to note that bacteria embedded in biofilms can tolerate 10-1000 higher doses of antibiotics to be eradicated than their counterparts at planktonic level.12

The research on biofilm development allowed a better understanding to investigate different control strategies. In fact, acting at different steps of the development could avoid the spread of biofilm infections:

The promiscuity of the bacteria inside the biofilm allows them to adopt collective behaviour facilitating the cell-to-cell

• •


Inhibition of the adhesion of microbes forming biofilm Inhibition of the QS INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 45

Special Feature â&#x20AC;˘

Disintegration of the matrix: enzymes, improved cleaning and disinfection of surfaces.

Such approaches could also allow the reduction of antibiotic resistance spread and associated healthcare costs, as we discussed before about the transmission of antibiotic resistance genes between microbes embedded into a biofilm. Antibiotic Resistance The Emergence of Resistance In recent years, critical bacterial species have become a threat to a growing number of infections due to their resistance to the available antibiotics.17 Diffusion of antibiotics into the biofilm is limited and leads to bacterial exposure to suboptimal drug concentrations. To respond to this issue, practitioners tend to increase the duration and the doses of antibiotic prescription, a condition favouring the selection of antibioticresistant mutants, the so-called superbugs. The emergence of new bacterial resistance is no longer compensated by the discovery of new active antibacterials. The main factors leading to antibiotic resistance come from uncontrolled distribution of antibiotics, prescription of broad-spectrum antibiotics and use of antibiotics in industries (e.g. agriculture). In other words, it is found widely from agricultural settings to general practitioners as well as in hospitals. Prof. Giovanni Di Perri (Professor of Infectious Diseases, University of Turin, Italy) warned about the antimicrobial resistance emergency, and how there is a sharing of responsibilities at the different levels of care.18 The increasingly aged patient population with multiple comorbidities represents an additional factor contributing to antibiotic prescriptions increase. The inter-human passage of antibiotic-resistant bacteria through different environments has led to the nowadays common occurrence of difficult-to-treat severe bacterial infections. In the most critical settings (e.g. intensive care units) virtually untreatable infections occur at an increasing rate.

Methicillin-resistant Staphylococcus aureus (MRSA) infection also remains a global healthcare issue. S. aureus causes a wide range of diseases involving skin, soft tissue (mucosa), joints, and infections induced by catheters or prosthetic devices. It is responsible for almost all S. aureus bacteremia and is associated with poorer clinical outcomes. The prevalence of MRSA differs widely around the world. In Europe, while Netherlands displays a low rate at 0.9%, Romania reaches the highest rate with 56% of MRSA isolates. A higher proportion of MRSA isolates in the southern regions can also be noted. Despite a decrease of MRSA prevalence during the last decade, a review of 15 studies shows a worldwide prevalence ranging from 13 to 74%.22

Figure 1: Percentage of Streptococcus pneumoniae resistant to Macrolides

Figure 2: Percentage of Pseudomonas aeruginosa resistant to Carbapenems

A Universal Issue Despite measures taken in response to the threat of the global antibiotic-resistance crisis, researchers from the Center for Disease Dynamics, Economics and Policy (CDDEP),19 Princeton University, and the Princeton Environmental Institute (PEI), ETH Zurich and the University of Antwerp, found that the worldwide use of antibiotics in humans soared 39 per cent between 2000 and 2015, particularly in low- and middle-income nations. In 2016, 490,000 people developed multi-drug-resistance globally. Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas aeruginosa, and Mycobacterium tuberculosis frequently implicated in respiratory infections are strains with reduced susceptibility to multiple classes of antibiotics.20,21 (Figure 1 and 2) 46 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Reduction of Antibiotics Reducing the selection and spread of antibiotic resistance involves rationalisation of antibiotic use, especially in the outpatient setting. A reduced antibiotic use might favourably impact on bacteria sensitivity to antibiotics by decreasing the selective pressure on the resident flora. By reducing the selective pressure in the outpatient setting we might actually decrease the likelihood of bacteria evolving into complex resistance patterns, eventually circulating among humans. Some new therapeutic approaches shall be evaluated, such as prophylactic measures that could improve the overall profile of infection management. Moreover, the universal issue among biofilm-associated bacterial infections justifies the focus on biofilm. Dealing with this issue involves studying the specificity of every species responsible for biofilm and the options to interfere with, inhibit or control the critical steps of the biofilm formation, as for example the adhesion of biofilm forming bacteria. Winter 2019 Volume 2 Issue 4

Special Feature The Biological Activities of Exopolysaccharides Isolated from T14 Strain Extracellular substances secreted by bacteria may be both the constituents of the biofilm extracellular matrix as well as bacterial products able to inhibit the biofilm formation from other strains23–27. One of these bacterial strains, an extremophile thermophilic marine Bacillus licheniformis termed T14 is able to secrete new marine postbiotic: metabolites composed of exopolysaccharides. The peculiarities of this bacteria can be explained by its environmental habitat. T14 was isolated in the sea of the Eolian archipelago, where the submarine volcanic activity offers unique hydrothermal shallow vents hosting a treasure of microbial diversity. These exopolysaccharides showed the ability to display anti-biofilm activity, most likely by exerting anti-adhesive properties on several pathogenic bacteria species, such as Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus aureus. Those properties have not been previously found in polysaccharides from algae or plants.28 In a recent study, the biofilm formation with or without exopolysaccharides from T14 was measured spectrophotometrically. Experiments showed in fact that E. coli, K. pneumonia, P. aeruginosa and S. aureus biofilms were all significantly inhibited with the exopolysaccharides5. The exopolysaccharides also showed a surfactant activity by modifying the hydrophobicity of bacterial cells reducing the chances for the bacteria to adhere to the mucosa and the formation of an eventual biofilm. The exopolysaccharides do not modify the growth of the bacterial isolates, meaning that exopolysaccharides do not have any antibiotic activity. The high content of fructose and fucose contained in exopolysaccharides also participates in the inhibition of bacteria adherence, since it decreases the formation and activity of fimbriae and pili presents in bacteria.29 Further ex vivo and in vivo studies aim to assess the anti-biofilm effect of exopolysaccharides on several pathogens responsible for respiratory and oral infections. Lallemand Pharma is currently performing additional researches on the anti-biofilm activity of this postbiotic: An ex vivo study to evaluate the anti-biofilm activity of the exopolysaccharides on S. aureus and S. epidermidis using the nasal swabs from patients with chronic rhinosinusitis (CRS). Several other researches are ongoing to study the efficacy of this postbiotic to inhibit biofilm formation of other respiratory and oral pathogenic bacterial and also viral strains on epithelial nasal cell models. Conclusion Over the years, the concept of biofilm and its consequences has become better understood. A biofilm is a protection for bacteria building their own houses. The biofilm acts as a defence and adaptative mechanism for bacteria to gain better survival against external factors, which can lead to an antibiotic resistance pattern. Antibiotic resistance is a real concern in this time, whose selection and spread keep on going at an alarming pace. New directions in the prevention and reduction of risk factors of infectious diseases seem to be actually pursuable by taking advantage of mechanical action of the unique marine exopolysaccharides produced by Bacillus licheniformis T14 ability to inhibit biofilm formation, and its surfactant activity to improve cleaning and disinfection procedures. www.biopharmaceuticalmedia.com

The anti-biofilm activity, anti-adhesive properties, water solubility, surfactant potential, thermostability within a wide temperature range and full biodegradability of this unique postbiotic suggest a large potential for Lallemand Pharma’s new health applications in the prevention of biofilm-derived infectious diseases. REFERENCES 1. Jayadev, Ayona et al. Marine bacteria : a potential bioresource for multiple applications. International Journal of Scientific & Engineering Research. 6(9);2015. 2229-5518 2. Kang, H. K., Seo, C. H., & Park, Y. (2015). Marine peptides and their anti-infective activities. Marine drugs. 2015 Jan; 13(1): 618–654. 3. Wiese, Jutta & Imhoff, Johannes. Marine bacteria and fungi as promising source for new antibiotics. Drug Development Research. 2019 Feb;80(1):24-27. 4. Fitter L, Herrmann R, Dencher RA, Blume A, Hauss T. Activity and Stability of a Thermostable α-Amylase Compared to Its Mesophilic Homologue:  Mechanisms of Thermal Adaptation Biochemistry 2001; 40: 10723-10731. 5. Antonio Spano et al, “In vitro antibiofilm activity of an Exopolysaccharide from the Marine Thermophilic Bacillus licheniformis T14”, Curr Microbiol (2016) 72:518-528. 6. Raad, I. et al. Successful salvage of central venous catheters in patients with catheter-related or central line-associated bloodstream infections by using a catheter lock solution consisting of minocycline, EDTA, and 25% ethanol. Antimicrob. Agents Chemother. 60, 3426–3432 (2016). 7. Mistry, S. et al. A novel, multi-barrier, drug eluting calcium sulfate/ biphasic calcium phosphate biodegradable composite bone cement for treatment of experimental MRSA osteomyelitis in rabbit model. J. Control. Release 239, 169–181 (2016). 8. Lemire, J. A., Harrison, J. J. & Turner, R. J. Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat. Rev. Micro 11, 371–384 (2013). 9. Hans-Curt Flemming. EPS-then and Now. Microorganisms (2016), 4, 41. 10. Brandas SS, Vik A, Friedman L, Koiter R. Biofilm: the matrix revisited. Trends Microbiol 2005;13:20e6. 11. Daniel L Hamilos, Biofilm formations in Pediatric Respiratory Tract Infection, Current Infectious Disease Reports (2019) 21:6. 12. Ceri, H et al (1999). The Calgary Biofilm Device: new technology for rapid determination of antibiotic susceptibilities of bacterial biofilms. J. Clin. Microbiol. 37, 1771–1776. 13. Xiaoqing Hu et al. Antimicrobial photodynamic Therapy to Control Clinically Relevant Biofilm Infections. Frontiers in Microbiology (2018) 9:1299. 14. Li, H.,et al. (2012). Relationship between bacterial biofilm and clinical features of patients with chronic rhinosinusitis. Eur. Arch. Otorhinolaryngol. 269, 155–163. 15. Azoulay, E et al. (2006). Candida colonization of the respiratory tract and subsequent pseudomonas ventilator-associated pneumonia. Chest 129, 110–117. 16. Gil-Perotin, S., et al. (2012). Implications of endotracheal tube biofilm in ventilator associated pneumonia response: a state of concept. Crit. Care 16, R93. 17. European Centre for Disease Prevention and Control. Surveillance of antimicrobial resistance in Europe – Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net) 2017. Stockholm: ECDC; 2018. 18. Di Perri G., Ferlazzo G. Mucosal immunity in the infections of airways. Supplement of Clinical Perspectives in respiratory Medicine No.5. 2019 May. 19. De Beer, D. et al. (1997) Measurement of local diffusion coefficients in biofilms by micro-injection and confocal microscopy. Biotechnol. Bioeng. 53, 151–158. 20. Guitor AK, Wright GD. Antimicrobial Resistance and Respiratory Infections. 2018 Nov;154(5):1202-1212 21. Ranita Roy, Monalisa Tiwari, Gianfranco Donelli and Vishvanath INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 47

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Tiwaria. Strategies for combating bacterial biofilms: A focus on anti-biofilm agents and their mechanisms of action. Virulence. 2018; 9(1): 522–554 22. Hassoun A. et al, Incidence, prevalence, and management of MRSA bacteremia across patient populations – a review of recent developments in MRSA management and treatment Crit Care (2017) 21:211. 23. Jiang P, Li J, Han F, Duan G, Lu X, Gu Y, Yu W: Antibiofilm activity of an exopolysaccharide from marine bacterium Vibrio sp. QY101. PLoS ONE 2011, 6(4):e18514. 24. Joseph LA, Wright AC: Expression of Vibrio vulnificus capsular polysaccharide inhibits biofilm formation. J Bacteriol 2004, 186:889-893. 25. Davey ME, Duncan MJ: Enhanced biofilm formation and loss of capsule ynthesis: deletion of a putative glycosyltransferase in Porphyromonas gingivalis. J Bacteriol 2006, 188:5510-5523. 26. Valle J, Da Re S, Henry N, Fontaine T, Balestrino D, LatourLambert P, Ghigo JM: Broad-spectrum biofilm inhibition by a secreted bacterial polysaccharide. Proc Natl Acad Sci USA 2006, 103:12558-12563. 27. Qin Z, Yang L, Qu D, Molin S, Tolker-Nielsen T: Pseudomonas aeruginosa extracellular products inhibit staphylococcal growth, and disrupt established biofilms produced by Staphylococcus epidermidis. Microbiology 2009, 155:2148-2156. 28. Rendueles O, Kaplan JB, Ghigo JM. Antibiofilm polysaccharides. Environmental Microbiol 2013; 15:334-46. 48 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

29. Imberty A, Wimmerova M, Mitchell EP, Gilboa-Garber N (2004) Structures of the lectins from Pseudomonas aeruginosa: insight into the molecular basis for host glycan recognition. Microbes Infect 6:221–228.

Maxence de Villemeur Maxence de Villemeur has been Marketing and Sales Manager for Lallemand Pharma since October 2010 and is in charge of global business and marketing development of polyvalent mechanical bacterial lysates. Prior to this, she was Product Manager in the health division of Lallemand. Maxence has over 15 years of marketing and business experience in international healthcare and pharma markets. She holds an MBA in international management from ESC Clermont-Ferrand, France, and graduated in product development engineering from Cergy-Pontoise Industrial Biology Engineering School at EBI, France. Email: mdevillemeur@lallemand.com

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