IBI Volume 4 Issue 2 by Senglobal

Volume 4 Issue 2

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Antibody-Drug Conjugates: A Trojan Horse Story Standard vs. Hybrid FIH Trials: Advantages and Challenges Adapting Your Preclinical Animal Model Strategies for a New World Using Lentiviral Vectors to Advance the Development of Therapeutic Vaccines

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Summer 2021 Volume 4 Issue 2

Contents 04 Foreword TALKING POINT 06 A World Leader in Naturally Derived Products Discusses Commercial Success and New Innovations DIRECTORS: Martin Wright Mark A. Barker BUSINESS DEVELOPMENT: Ty Eastman ty @pharmapubs.com EDITORIAL: Beatriz Romao beatriz@pharmapubs.com DESIGN DIRECTOR: Jana Sukenikova www.fanahshapeless.com FINANCE DEPARTMENT: Martin Wright martin@ipimedia.com RESEARCH & CIRCULATION: Virginia Toteva virginia@pharmapubs.com COVER IMAGE: iStockphoto © PUBLISHED BY: Pharma Publications J101 Tower Bridge Business Complex London, SE16 4DG Tel: +44 (0)20 4541 7569 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 Autumn 2021. 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. 2021 PHARMA PUBLICATIONS / Volume 4 Issue 2 – Summer 2021

Mr. Darren Alkins CEO at SPL – Scientific Protein Laboratories, speaks with IBI about their continued development in their science and technical expertise to meet the demanding requirements for new applications and to realise commercial success in a timely manner. REGULATORY & COMPLIANCE 08 Supporting Mergers and Acquisitions in the Pharmaceutical/Biopharmaceutical Industry In recent years, we have seen pharmaceutical company megadeals that saw Takeda acquiring Shire for a total value of $81.7 billion, Bristol-Myers Squibb’s acquisition of Celgene for $74 billion, AbbVie’s $63 billion acquisition of Allergan and the proposed acquisition of Alexion by AstraZeneca for $39 billion. All of these acquisitions continue to have a lasting impact on the leadership and staff at these companies, which collectively employ hundreds of thousands of employees worldwide. In addition, there has been a plethora of product transfers between organisations with larger multi-national companies pruning portfolios, adding gene therapy and biotechnology divisions, and consolidating core assets. John Cahill at PharmaLex shows how mergers and acquisitions in the pharmaceutical / biopharmaceutical industry are critical for organisations to implement strategic changes to their business. RESEARCH / INNOVATION / DEVELOPMENT 12 Dealing with the Challenges of Post Translational Modifications (PTMs) During the structural analysis of recombinant protein biopharmaceutical products, a significant challenge is posed by the issue of Post Translational Modifications (PTMs). This is a very large area of investigation due to the number of PTMs that have been identified through the study of protein structures over the course of many years. Dr. Richard L. Easton at BioPharmaSpec explores more about the challenges of Post Translational Modifications. 16 Solving Critical Data Challenges for Chemicallymodified, Biologically-based Therapeutic Candidates The drug discovery research industry is seeing a growing prevalence of chemically-modified, biologically-based therapeutic candidates. This trend creates urgent and novel challenges for data management and analysis informatics software, necessary for supporting cross-functional teams of scientists bridging across biology and chemistry. Andrew LeBeau and Troy Humphreys at Domatics discuss key challenges and how to address them, such as rigour in scientific definition of candidate therapeutics and opportunities to automate and streamline data analysis workflows. 20 Using Lentiviral Vectors to Advance the Development of Therapeutic Vaccines Traditionally, vaccination has been used as a preventative strategy, aimed at curbing the impact of a wide range of

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 1

Contents infectious diseases. Over the past three decades, vaccine development has moved increasingly into the therapeutic area for the treatment of conditions such as cancer and chronic viral infections. To be successful, such vaccines must elicit a strong cellular as well as humoral immune response. Viral vectors, engineered to carry genes for the expression of a suitable immunogenic protein in the recipient, are widely used. Christian Bréchot at TheraVectys examines how a new generation of lentiviral vectors, capable of triggering effective, long-lasting T cell-mediated immunity, is demonstrating preclinical success in both therapeutic and prophylactic vaccine development. 24 Antibody-Drug Conjugates: A Trojan Horse Story According to the World Health Organization, cancer is responsible for 10 million deaths per year, and it is considered as the secondleading cause of death in the world. New anti-cancer drugs have entered the global market in recent years, but it is still not clear if they are sufficient to cure cancer patients. On the one hand, conventional therapies have severe side-effects and tumour cells become resistant. On the other hand, novel therapies have emerged, like immunotherapies, but so far, the clinical benefit is low. Panagiotis Parsonidis and Ioannis Papasotiriou at Research Genetic Cancer Centre analyse how to develop new approaches that could enhance the therapeutic efficacy. PRE-CLINICAL & CLINICAL RESEARCH 26 Adapting Your Preclinical Animal Model Strategies for a New World Investigators in the commercial and non-profit sectors have always faced pressure to accelerate their work, while balancing the need for quality and the realities of budget constraints. Pharmaceutical and biotech companies want to speed their time to market with novel, efficacious therapeutics. Non-profit institutions want to accelerate early-stage work that may lead to the identification of new drug targets or therapeutic approaches. Austin Jelcick at Taconic Biosciences looks at ways to adapt preclinical animal model strategies to achieve research and business objectives. 30 Standard vs. Hybrid FIH Trials: Advantages and Challenges First in human (FIH) studies are the key translational studies from preclinical to further clinical development processes. The goal of FIH studies is to investigate the pharmacokinetics (PK) and pharmacodynamics (PD), determine appropriate dosing, and document safety and tolerability. These studies serve as the basis for further efficacy studies in the targeted patient populations. Nariné Baririan at SGS examines the key considerations to consider when deciding whether to run a standard FIH in HVs or a hybrid clinical trial.

time and bringing therapies to commercialisation remains a hurdle. Vince Paolizzi at Pelican Bio analyses the intricacies and future considerations for the cell and gene therapy cold chain. 38 Four factors for a biopharmaceutical manufacturer to consider when developing and managing a single-use supply chain Single-use technologies are becoming widely utilised in the biopharmaceutical industry for the benefit of offering a more flexible, cost-effective approach to cGMP manufacturing. Single-use technologies are utilised in more than 45% of clinical mammalian cell culture processes and about 6% of commercial processes. In this report, Jay Harp and Timothy Korwan at Avantor will discuss four best practices that biopharma companies should consider in developing and managing their single-use supply chain. MANUFACTURING/TECHNOLOGY PLATFORMS 42 H96: Combining Freeze-drying and High-pressure Homogenisation for Ultra-fine Nanocrystal Production In recent years, nanomedicine has proven to be key in overcoming many of the challenges associated with poorly water-soluble drugs. Decreasing the size of drug particles can increase bioavailability and solubility can be observed, as a result of the increased active pharmaceutical ingredient (API) surface area. Due to the increased bioavailability, a lower amount of API is required, which in turn leads to a more cost-efficient product, with fewer risks and side-effects for the patient. Sebastian Prisacariu and Richard Lewis at BioPharma Group explore more about the process of combining freezedrying high-pressure homogenisation for ultra-fine nanocrystal production. 44 Data-rich Processing – The Growing Necessity of R&D to GMP Manufacture Improved diagnosis is driving precision in treatment, accompanied by increasing regulation stringencies from the likes of the MHRA and FDA. This ensures processes, as well as the products themselves, are fully understood during every step of the manufacturing process. The requirement for both the R&D and manufacturing processes to be ‘data-rich’ has become a prerequisite for drug product manufacturers and purchasers of drug products alike; a phenomenon driven not only when things go well, but where an irregularity might have occurred, even when confirmed to be within the validated ‘design space’ parameters. Richard Lewis at Biopharma Group explains the growing necessity of R&D to GMP manufacture. MARKET REPORT

SUPPLY CHAIN MANAGEMENT

46 Land use change and the global pandemic

34 Intricacies and Future Considerations for the Cell and Gene Therapy Cold Chain

Summer 2021 Volume 4 Issue 2

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Foreword Europe has traditionally been the major player in vaccine research, achieving a tremendous health impact by drastically reducing cases of devastating diseases, and providing new research strategies for combatting emerging diseases like EBOLA and now the COVID-19 virus. Many more infectious and non-infectious diseases could be eradicated by preventive and therapeutic vaccines. Vaccination has been used as a preventative strategy, aimed at curbing the impact of a wide range of infectious diseases. Over the past three decades, vaccine development has moved increasingly into the therapeutic area for the treatment of conditions such as cancer and chronic viral infections. To be successful, such vaccines must elicit a strong cellular as well as humoral immune response. Viral vectors, engineered to carry genes for the expression of a suitable immunogenic protein in the recipient, are widely used. Christian Bréchot at TheraVectys examines how a new generation of lentiviral vectors, capable of triggering effective, long-lasting T cell-mediated immunity, is demonstrating preclinical success in both therapeutic and prophylactic vaccine development. The drug discovery research industry is seeing a growing prevalence of chemically modified, biologically based therapeutic candidates. This trend creates urgent and novel challenges for data management and analysis informatics software, necessary for supporting cross-functional teams of scientists bridging biology and chemistry. Andrew LeBeau and Troy Humphreys at Domatics discuss key challenges and how to address them, such as rigour in the scientific definition of candidate therapeutics and opportunities to automate and streamline data analysis workflows.

In recent years, nanomedicine has proven to be key in overcoming many of the challenges associated with poorly water-soluble drugs. Decreasing the size of drug particles can increase bioavailability and solubility can be observed, because of the increased active pharmaceutical ingredient (API) surface area. Due to the increased bioavailability, a lower amount of API is required, which in turn leads to a more cost-efficient product, with fewer risks and side effects for the patient. Sebastian Prisacariu and Richard Lewis at BioPharma Group explore more about the process of combining freeze-drying high-pressure homogenisation for ultra-fine nanocrystal production. In this journal, we will also explore more about preclinical animal model strategies. Investigators in the commercial and non-profit sectors have always faced pressure to accelerate their work while balancing the need for quality and the realities of budget constraints. Pharmaceutical and biotech companies want to speed their time to market with a novel, efficacious therapeutics. Non-profit institutions want to accelerate early-stage work that may lead to the identification of new drug targets or therapeutic approaches. Austin Jelcick at Taconic Biosciences looks at ways to adapt preclinical animal model strategies to achieve research and business objectives. I would like to thank all our authors and contributors for making this issue an exciting one. We are working relentlessly to bring you the most exciting and relevant topics through our journals. I hope that you enjoy reading this edition of the journal and keep well. Beatriz Romao, Editorial Manager

The rapidly growing field of cell and gene therapy is both exciting and confusing to many, including those who work in the medical community and on the periphery. Advancements of therapies happen at a feverish pace. The number of clinical trials increases exponentially in a short time and bringing therapies to commercialisation remains a hurdle. Vince Paolizzi at Pelican Bio analyses the intricacies and future considerations for the cell and gene therapy cold chain.

IBI – Editorial Advisory Board •

Ashok K. Ghone, PhD, VP, Global Services MakroCare, USA

•

Bakhyt Sarymsakova – Head of Department of International Cooperation, National Research Center of MCH, Astana, Kazakhstan

•

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

•

Lorna. M. Graham, BSc Hons, MSc, Director, Project Management, Worldwide Clinical Trials

•

Mark Goldberg, Chief Operating Officer, PAREXEL International Corporation

•

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

•

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

•

Catherine Lund, Vice Chairman, OnQ Consulting

•

Cellia K. Habita, President & CEO, Arianne Corporation

•

Chris Tait, Life Science Account Manager, CHUBB Insurance Company of Europe

•

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

•

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

•

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

•

Francis Crawley, Executive Director of the Good Clinical Practice Alliance – Europe (GCPA) and a World Health Organization (WHO) Expert in ethics

•

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

•

Hermann Schulz, MD, Founder, PresseKontext

•

Stefan Astrom, Founder and CEO of Astrom Research International HB

•

Jim James DeSantihas, Chief Executive Officer, PharmaVigilant

•

Steve Heath, Head of EMEA – Medidata Solutions, Inc

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Summer 2021 Volume 4 Issue 2

LIFE SCIENCES

BIOPHARMACEUTICAL SOLUTIONS LIFE INSPIRED, QUALITY DRIVEN

Once a biologic has reached the first step of its development – R&D in vivo and in vitro activity proof of concept – it enters the more regulated chemistry, manufacturing and control (CMC) pathway. At this point, regulatory agencies require safety, identity, purity and integrity to be monitored. Our experts can support your requests with: Our Center of Excellence provides testing support for the biosafety and characterization of raw materials, cell bank and virus seeds for vaccines, cell and gene therapies, monoclonal antibodies and other recombinant protein. We provide established expertise for biopharmaceuticals characterization – from primary to tertiary structures, aggregation as well as physicochemical properties. Our solutions include antibody testing and batch release testing. We performs quality control (QC) testing to help you meet regulatory requirements at each stage of the supply chain. Our network enables one-stop full-panel cGMP biologics release and stability testing in North America, Europe, and Asia-Pacific

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www.sgs.com/lifescience

www.sgs.com/HealthCommunity

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Talking Point

A World Leader in Naturally Derived Products Discusses Commercial Success and New Innovations An interview with Mr. Darren Alkins, CEO at SPL Q: SPL – Scientific Protein Laboratories LLC (SPL) is a World Leader in Heparin and Pancreatic Enzymes. Can you tell our readers the brief history of your company, how the company got started and your growth so far?

A: SPL was started in 1976 as a branch of Oscar Mayer, a large meat producer located in Madison, the capitol of Wisconsin. Oscar Mayer launched SPL to take advantage of unused animal tissues containing pharmaceutical substances (Heparin & Pancreatin). SPL’s mission has always been to be the world’s leading manufacturer of high-quality Active Pharmaceutical Ingredients (APIs) derived from biological sources supplying the healthcare industries. Our growth has been driven by the increased demand for heparin and pancreatic enzyme products. As the uses for these invaluable, essential medicines has expanded, SPL has grown to keep pace with these innovations. SPL’s original focus was on Heparin, Pancreatin, and Blood Protein products. Now, 45 years later SPL’s portfolio includes Heparin and heparin derivatives, Pancreatic enzymes, Blood products, Thyroid, peptide hormones, collagen and related derivatives, molecular scaffolds, and other biologically derived products. SPL has expertise in extracting specific molecules/ substances of interest from all types of biological tissues. SPL’s products can meet the rigorous requirements for both pharmaceutical dosage forms, medical devices, and complex combination products.

derived from natural sources supplying the healthcare industries. Our expertise supports development, purification, and supply of active ingredient from a diverse range of biological sources. Because of our long history in handling, defining, and controlling these complex materials, we can produce high quality products that few others can manage. In some cases, we are the sole source. In all cases, our experience and skills managing the safety and compliance risks related to biological sources is unique and one of the best in the industry.

Q: You have placed great emphasis on expansion and diversification of your product pipeline, and corporate development programs. Can you tell our audience how you have achieved this and what are your vision for the future?

SPL has expanded its product portfolio in two primary ways. First, we have built the infrastructure and capabilities to meet and exceed our customer’s expectations. Second, we have formed strategic partnerships to bring new products to market. In both cases, the result is world class technology and quality. SPL has the scientific and engineering expertise to make any product derived from biological substances. Throughout the entire process, SPL applies the highest regulatory and quality standards to all aspects of the process. SPL is regularly audited by regional authorities and customers. We maintain a strong reputation for high technology, reliability and quality.

Q: How do you fit into the landscape of drug development?

Q: You offer more than 30 years’ experience in manufacturing APIs from biologically derived sources. What are your product pipelines, and how have you made a difference in the industry through your portfolio?

A: SPL, Scientific Protein Laboratories, is a world leader for manufacturer of high-quality Active Pharmaceutical Ingredients

A: SPL’s customer base includes some of the highest performing companies in the pharmaceutical industry. For decades SPL has

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Talking Point

been supporting new indications, regulations, patient safety concerns, and global supply chain changes. SPL has developed global experience with a wide variety of complex products and their related supply chains. The primary difference between SPL and our competitors is our science, quality, discipline, knowledge, and experience from the point of harvest, through purification of the product, up to and sometimes including the finished product. We offer high quality products, with excellent traceability and risk management controls. Our scientific, regulatory, and quality departments are best in class.

Q: Can you explain your Regulatory and Analytical Support Services? What are you offering, and how do you make a difference in the industry?

A: SPL has a world class fully operational QC, Micro, and qPCR laboratory. We specialize in bio-sourced materials but can work with a broad array of substances. Additionally, we have the capability to develop test methods for products, for new or existing method improvement. SPL’s analytical team is key to our success with manipulating biologically derived substances from a complex supply chain. We have special expertise with the complex matrices presented by bio-sourced products. Our regulatory group effectively supports drug filings across the globe. We also have significant experience in devices and other technologies that require biologically derived drug substances. We are best in class at bridging the gap between common pharmaceutical standards and non-drug industries.

Q: SPL offers a broad spectrum of technology capabilities in support of development and manufacturing services for the extraction, isolation and purification of naturally derived materials and fermentation, isolation, and recovery of therapeutic proteins. Can you give us a detailed market demand, and tell us your growth potential?

A: SPL’s capacity is often sold out well into the future. With each expansion and capacity increase, we establish close, long term customer relationships. We carefully select our partners to ensure we can support their needs and exceed expectations over the long term. www.biopharmaceuticalmedia.com

We judiciously manage the full supply chain, ensuring that everyone benefits and hold the finished product to the highest standards. As an example, the markets for porcine derived products (heparin and pancreatin in particular) are under pressure (globally) from the African Swine Fever (ASF) outbreak in 2019. In both cases demand for the products is at or above 2019 demand, but global raw material supply remains less than before. Our supplies and quality remain some of the highest in the world. Many of the new products we are working on are new markets or have limited competitors. In both cases, SPL is competitive and experienced great success.

Q: What major events have occurred in the industry and what has been its effects.

A: As the population in the developed nations has aged and as more people have access to modern medical care, the demand for SPL products has been growing robustly. We expect that these demographic trends will continue and will require SPL to continue innovating to meet this need. In addition, medical science continues to find new applications for bio sourced ingredients. SPL has continued to develop its science and technical expertise to meet the demanding requirements for these new applications.

Q: What role do SPL and your services play on their client’s journey to commercial success?

A: SPL’s quality, experience and expertise enable our clients to realize commercial success in a timely manner. We apply our experience to new products and processes, but always adapt to the specific requirements of the new innovations.

Darren Alkins Darren is the CEO of SPL and has 32 years experience in the pharmaceutical business. He started his career at Bristol- Myers Squib in sales, marketing and business development. He has been the president of three pharmaceutical companies. Darren is the former head of Teva US business development. Most recently, Darren was the General Manager of generics and dermatology at Sandoz US.

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Regulatory & Compliance

PEER REVIEWED

Supporting Mergers and Acquisitions in the Pharmaceutical/Biopharmaceutical Industry In recent years, we have seen pharmaceutical company megadeals that saw Takeda acquiring Shire for a total value of $81.7 billion, Bristol-Myers Squibb’s acquisition of Celgene for $74 billion, AbbVie’s $63 billion acquisition of Allergan and the proposed acquisition of Alexion by AstraZeneca for $39 billion. All of these acquisitions continue to have a lasting impact on the leadership and staff at these companies, which collectively employ hundreds of thousands of employees worldwide. In addition, there has been a plethora of product transfers between organisations, with larger multi-national companies pruning portfolios, adding gene therapy and biotechnology divisions, and consolidating core assets.

Mergers and acquisitions (M&As) in the pharmaceutical/ biopharmaceutical industry are critical for organisations to implement strategic changes to their business. Whether it be to (a) future-proof an organisation’s pipeline by accessing innovation, (b) obtain additional manufacturing capacity, or (c) to divest non-core assets (products, facilities, etc.), companies continue to grow, modernise and evolve to meet the targets set out in their strategic plans. When two or more organisations reach the ‘deal’ and it is announced that ‘A’ will take over ‘B’ or that A and B will share in ‘A-B’, or indeed that ‘A’ will sell part of their organisation to ‘B’, it is frequently followed by uncertainty and apprehension among internal stakeholders. This changing landscape tests an organisation’s ability to communicate the distinct ‘win-win’ elements of the deal. The Kübler-Ross change curve (see fig.1 below) is always worth bearing in mind during this transitional period of M&A, and never fails in tracking internal stakeholder mindset, albeit with differing levels of severity.

behind. It can take years before a post-M&A steady state is reached (sometimes never!), where full commitment to the change is obtained and all the anticipated ‘win-wins’ are realised. Some acquired organisations are left to their own devices (pardon the pun, medtech sector!) and they are run as true satellites whose contact with the corporate office is limited to communicating the positive financial results. In this scenario, the management team in-situ at the time of the M&A event are trusted to continue as-is and maintain the upward trajectory. Alternatively, and more commonly where there is a dominant merging partner, a strict cut-over timeline is applied for an acquired entity to morph into a fully incorporated affiliate. Typically, these sites implement corporate structures, policies and systems swiftly and assertively. Where M&A can become interesting is the cultural piece; everyone who has worked in an organisation through a merger or acquisition knows that there can be a seismic shift in the objectives of the new organisation… not so much what the objectives are as how the objectives are expected to be met. Post-M&A, organisations frequently change structure with new reporting lines, new titles, merged departments, increased/ reduced layers of management with revised spans of control. Systems of work can also change, where new policies are cascaded into procedures which are followed with varying degrees of success. Supporting systems, software tools and information flows are further material changes which tend to require extensive training and oversight in the early periods post-M&A.

Figure 1. Kübler-Ross Change Curve

The transition from pre-M&A to the post-M&A reality can be both fast and slow. The physical symbols of such transitions such as the company name, logo and headed paper can be changed in a matter of minutes, but the hearts and minds of management and employees can lag significantly further 8 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

When cultures collide in merging organisations, it has serious ramifications for business and its stakeholders. Industry is littered with mergers and takeovers that did not meet expectations simply because the cultural differences were too difficult to overcome. Naturally, organisations do not admit to failed mergers or acquisitions too often, but some of Summer 2021 Volume 4 Issue 2

Regulatory & Compliance

the more interesting ones are referenced below.1 Very often the differences in personal and collective discipline, personified in the leadership differences in the two organisations, is challenging for the organisations to reconcile. Where rigid, structured and conservative management methods meet innovative and unorthodox management, it can be a recipe for M&A difficulties.2 Specialist Manufacturing Sector Mergers and Acquisitions Increasingly, biopharma services companies, specifically specialist contract development and manufacturing organisations (CDMOs) are being seen by investors as important enablers of future personalised precision medicines innovation, in particular advanced therapy medicinal products (ATMPs). As a result, there is a growing ATMP ecosystem of targeted investment towards both intellectual property and innovation enablers. The bottleneck is in manufacturing sufficient clinical trial material, where the vast majority of ATMP innovation is in preclinical and early-stage clinical phases. Therefore, entry to GMP is a major value-enhancing milestone in the evolution of AMTP innovators, resulting in significant merger and acquisition activity at each of the value-add inflection points. CDMOs, and increasingly their investors, are realising the value that they can add to advanced therapy innovators, and are tooling up both in terms of scaling and global footprint,

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where many of the therapies require a combination of centralised and decentralised manufacturing responses, achieved largely through acquisition of different component capabilities to create end-to-end capability. The cell and gene therapy sector – specifically CDMO enablement – is explosive, putting it lightly, as a direct result of the manufacturing bottleneck in front of the rapidly growing pipeline of new advanced therapies being brought towards clinic. Gene therapy-enabling CDMO Brammer Bio, for example, was acquired in 2019 for $1.7Bn by Thermo Fisher, and Catalent acquired Paragon for $1.2Bn in the same time period. These are two examples of large CDMO pharma meets rapidly-growing specialist CDMO to expand their capabilities through acquisition and to enter into the cell and gene therapy sector as a strategic alignment measure. IPO/M&A remains the main exit route for sector investors, with transactions reaching multiples of 5–20 EBITDA not being unusual, revenue streams taking a back seat to potential growth trajectory and significant market opportunity, bringing the promise of transformational and even curative therapies that will revolutionalise healthcare systems in the near future. This is against the backdrop of increasingly complex regulatory and reimbursement uncertainty. $13.5Bn invested in 2020 in pharma IPOs was dominated by cell and gene therapy companies; for example, Sana’s IPO

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Regulatory & Compliance for a record $588M in 2021. Oxford BioMedica has grown in to an £840M market cap business since 2008 as a result of securing significant near- and long-term development and supply agreements to bring client cell and gene therapies to clinic and market. mRNA Manufacturing Capability Rapidly Growing as a Transformational Vaccine Innovation Similarly in the mRNA cancer and pandemic vaccine space, we are seeing significant merger and acquisition activity occurring already, with this technology bringing promise of new vaccines to market, transforming healthcare systems and pandemic strategies. The very recent Danaher acquisition of Precision Nanosciences in June, following its company Cytiva’s acquisition of Vanrx in January, positions parent company Danaher to provide end-to-end mRNA vaccine technological solutions for vaccine manufacturers globally. This will facilitate localised responses to pandemic vaccine development and manufacture, to give one example. Sartorius, Thermo Fisher and Terumo BCT are similarly significant players in this space, and are actively acquiring end-to-end capability through mergers and acquisitions.

parts of a merger or acquisition has been proven to be greater than one where the foundational compliance performance of the respective acquirer and acquiree are compatible. Preparing companies for a robust due diligence, leading to a transaction and successful merger or acquisition, is a core strength of PharmaLex’s global team of experts.

Canada’s ‘in-country’ self-sustainability programme is an example of one region’s response to the pandemic. In order to protect its citizens from future pandemics, it has cemented an investment of $164 million from the Government of Canada to modernise and expand Resilience’s Ontario biomanufacturing site. This develops pandemic preparedness for the current and future pandemics, and aligns well with mRNA vaccine development specifically, gearing Canada for future vaccine development dominance, potentially.

Small and Large Molecules Still Making a Dominant Healthcare Contribution Small and large molecule innovation remains by far the dominant route to market for new therapeutics, with a sustainably strong role to play for the foreseeable future. There are many examples of general acquisitions in the biopharma sector: Lonza’s acquisition in 2016 of Capsugel for $5.5Bn completed an acquisition of thousands of new customers who are developing new therapeutics, where the compliance effort leading up to the acquisition was a critical component of the buy. Lonza not only develops new therapeutics, but aids their development clients in the pathway to market; the acquisition of Capsugel was incremental in this journey and in extending their reach in both small and large molecular development. Recipharm acquisition of the Consort Group in 2020 similarly expands the company’s customers and service offerings, allowing it to improve its impact on the small and large molecule sector’s product development and commercialisation. Conclusion Compliance is fundamental to any successful pharma merger and acquisition closing, one that is increasingly forming an earlier part of the due diligence process, key to establishing whether or not there is a fitness for compatibility with respective synergistic incremental business. The sum of the 10 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

REFERENCES 1. 2. 3.

https://www.fiercepharma.com/special-report/top-15-m-amistakes https://hbr.org/2018/10/one-reason-mergers-fail-the-twocultures-arent-compatible Recipharm. 2021. Recipharm Offers to Acquire Consort Medical to Become a Leading Inhalation Company and Top Five Global CDMO Player. [online] Available at: <https://www.recipharm.com/ press/recipharm-offers-acquire-consort-medical-become-leadinginhalation-company-and-top-five> [Accessed 3 June 2021]. Catalent Biologics. 2021.Catalent Has Acquired Paragon Bioservices - Catalent Biologics. [online] Available at: <https://biologics. catalent.com/catalent-has-acquired-paragon-bioservices/> [Accessed 3 June 2021]. GEN - Genetic Engineering and Biotechnology News. 2021.Danaher Acquires Precision NanoSystems. [online] Available at: <https:// www.genengnews.com/topics/bioprocessing/danaher-acquiresprecision-nanosystems/> [Accessed 3 June 2021]. Size, F., Size, F. and WIRE, B. 2021.Resilience Receives USD $164 Million Investment from the Government of Canada to Modernize and Expand Its Ontario Biomanufacturing Site, Improving Pandemic Preparedness. [online] Businesswire.com. Available at: <https:// www.businesswire.com/news/home/20210518005956/en/ Resilience-Receives-USD-164-Million-Investment-from-theGovernment-of-Canada-to-Modernize-and-Expand-Its-OntarioBiomanufacturing-Site-Improving-Pandemic-Preparedness> [Accessed 3 June 2021]. Capsugel. 2021.Lonza to Acquire Capsugel. [online] Available at: <https://www.capsugel.com/news/lonza-to-acquire-capsugel-tocreate-leading-integrated-solutions-provider-t> [Accessed 3 June 2021].

John Cahill John Cahill is a Senior Consultant at PharmaLex with over 20 years’ experience in the pharmaceutical industry. John holds a PhD in Chemistry and a Diploma in Pharmaceutical Manufacturing Technology from the University of Dublin, Trinity College. John has extensive Pharmaceutical Operations experience working internationally in Manufacturing and Quality Leadership roles. He has extensive experience working with national and international regulators and preparing companies for GxP compliance inspections. John has provided consultancy support and audited numerous finished product manufacturers, active pharmaceutical ingredient (API) manufacturers and distributors of medicinal products across the globe. He is a lean practitioner and operational excellence exponent.

Summer 2021 Volume 4 Issue 2

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 11

Research / Innovation / Development

Dealing with the Challenges of Post-translational Modifications (PTMs) Introduction During the structural analysis of recombinant protein biopharmaceutical products, a significant challenge is posed by the issue of post-translational modifications (PTMs). This is a very large area of investigation due to the number of PTMs that have been identified through the study of protein structures over the course of many years. The requirement to assess PTMs cannot be ignored, as this is part of the expectation for structural characterisation as required by the ICH Q6B guidelines1. However, not all PTMs that have been identified need to be investigated for every protein, as many are very specific to a certain protein, cell type or cellular process. Nonetheless, analysing PTMs gives rise to many challenges, not the least of which is “what post-translational modifications am I even looking for?”

What are Post-translational Modifications? A PTM is defined as any chemical modification that occurs on the protein chain following translation. So “PTM” is an all-encompassing term for what could happen to a protein on its journey though the cell and the extracellular environment it finds itself in, be that culture media, purification process environments or drug product formulation media. PTMs of one form or another are therefore widely found on biopharmaceutical products. They can be part of the natural cellular machinery – modifications which are found on many proteins such as glycosylation and disulfide bridge formation would fall into this category2,3. Glycosylation, the attachment of carbohydrate to the protein, is arguably one of the most important PTMs as it could potentially affect efficacy and safety and, in some cases, result in immunogenicity. Furthermore,

Figure 1: Stylised representation of a protein chain showing some examples of post-translational modifications and the amino acids on which they are found. 12 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

the non-templated addition of sugars greatly adds to the heterogeneity of the biologic medicine. Other natural PTMs of the protein can also occur, but these may be restricted to a particular class of molecule (e.g. immunoglobulins) or even a specific molecule itself. Examples of these are proteolytic cleavage, e.g. for activation of an immature form of a protein or through the action of naturally occurring proteases and modification of the N- and C-termini such as the formation of pyroglutamate residues (as found at the N-terminus of some immunoglobulin light or heavy chains). Some PTMs are actually the result of controlled chemical modifications of the protein chains such as PEGylation (the introduction of a polyethylene glycol moiety to site within the protein). There are many other examples of PTMs that have been identified and a limited selection of the most commonly encountered types are shown in Figure 1 and summarised in Table 1. As well as the PTMs that are a natural part of protein processing, there are others that are the result of chemical modifications that take place during the protein production PTM

Amino acid(s) involved

Nature of the modification

N-glycosylation

Asparagine

Oligosaccharide attachment as part of biosynthesis of a glycoprotein

O-glycosylation

Serine, Threonine

Oligosaccharide attachment as part of biosynthesis of a glycoprotein

Disulfide bridges

Cysteine

Thiol cross-linking between two Cysteine residues. Note that disulfides and thioether linkages can also be found at low levels as a result of incorrect formation of the bridges

PyroGlutamination

Glutamine, Glutamic acid (only N-terminal)

Cyclisation of the N-terminus

Lysine removal

Lysine at the Cterminus of mAb heavy chains

Proteolytic cleavage of the C-terminal Lysine residue

Oxidation

Methionine, Tryptophan

Oxidation of the side chains of these amino acids as a result of exposure to an oxidizing chemical environment. Different degrees of oxidation can take place on the Methionine side chain sulfur atom producing sulfoxide (one oxygen atom added) and sulfone (two oxygen atoms added) forms.

Deamidation

Conversion of Asparagine to Aspartic acid Asparagine, Glutamine (Glutamine to Glutamic acid) as a result of the chemical environment

Glycation

Lysine

Non-enzymatic modification of the side chain amine of Lysine residues with Glucose or other Hexose sugars if present in sufficient concentrations in the protein buffer.

PEGylation

Cysteine, N-terminus or side chain primary amines (i.e. Lysine)

Attachment of a polyethylene glycol unit of usually between 5 and 20 kDa to the protein via specific chemistry

Phosphorylation

Tyrosine, Serine, Threonine

Specific modification of the hydroxy side chain of certain residues within the structure of the protein

Iodination

Tyrosine

Attachment of Iodine notably in the thyroid hormone thyroglobulin to produce the thyroid hormones

Table 1. Examples of more commonly encountered PTMs Summer 2021 Volume 4 Issue 2

Research / Innovation / Development process, purification scheme, or as a result of product formulation and storage. Since proteins, by their very nature, contain amino acids with different chemical side chains, it follows that a variety of PTMs can be produced through different chemical reactions during these stages of drug production and at least some of these PTMs will be frequently encountered as a result of side chain propensities for particular chemical reactions. The most commonly found PTMs that fall into this category are oxidation of the side chain of methionine (and tryptophan) and the deamidation of asparagine and glutamine. These PTMs may form over a period of time as a result of prolonged exposure of the drug product to a particular chemical environment, and thus can potentially build up in a product during storage. This formation of certain PTMs can be exacerbated by stress conditions such as the action of heat or light on the formulated sample.

spectrometry. A combination of intact mass analysis and, critically, peptide mapping will give significant information on both the nature and location of PTMs within any given product.

So, a “PTM” is effectively any modification that occurs on a protein and these modifications can happen during biosynthesis, drug product purification, shelf storage lifetime or as the result of directed chemical modifications of the drug during the manufacturing process (e.g. PEGylation). Importantly, these modifications cannot be predicted from the gene sequence and must be determined experimentally. Knowing that there are many PTMs that exist and can be formed at all stages of drug production, how does one go about assessing a product for these modifications?

Peptide mapping is a very powerful technique for assessing PTMs as the proteolytic cleavage of a protein into smaller peptides results in separation of PTMs and easier subsequent analysis. In a peptide mapping study, the sample is digested with a protease which cleaves the protein at defined sites (e.g. trypsin or endoproteinase GluC). The resultant peptides are separated by liquid chromatography and analysed by online mass spectrometry. Using a Q-TOF type of mass spectrometer for this work allows not only the intact mass of the peptide to be determined but also, through the use of the real-time higher energy fragmentation channel, data can be obtained giving confirmatory sequence ions from that peptide. For PTM assessment, peptide mapping in this way is very informative

Intact mass analysis as a first step allows the determination of the overall mass(es) of the molecule, which is necessary for identifying what the resultant overall mass or mass distribution of the product is, including any PTMs that may be present. The mass observed will be derived from the overall protein chain (the sequence and therefore mass of which can be predicted from the DNA sequence used) along with any subsequent post-translational processing of that chain. Intact mass, of course, is unlikely to allow a full determination of the PTM profile, as the mass observed is the product of the sum total of all PTMs present.

Analytical Challenges of Post-translational Modifications The most effective means of PTM analysis by far is mass 29.10

100

A 31.51 26.34

26.00

26.50

27.00

27.50

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28.50

29.00

29.50

30.00

30.50

31.00

31.50

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33.19

32.22

30.39 27.48

26.00

26.50

27.00

29.10

27.50

28.00

1297 1198

100

28.50

924 811

29.00

29.50

30.00

696 582 435 272

30.50

31.00

31.50

32.00

32.50

900.92

V-V-C-L-L-D-N-F-Y-P-R

901.42

355.06

371.09

372.09

205.05 241.06

200

259.08

250

330.99

300

350

491.25

391.27

400

450

701.36

584.81 601.25618.25

550

600

650

272.16

228.09 246.88 222.14

200

250

731.00

700

750

839.97

800

850

696.33

272.17

100

500

485.23

396.15 340.16 356.07

300

350

400

500

812.35 900.38

696.31

582.28

485.27

450

900

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979.84

1000

1052.01

1050

924.44

550

600

614.33

696.29

650

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1250.72

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925.42

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1198.55

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1326.36

1300

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435.23

299.13

901.45 901.91 901.93 900.29 937.78

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467.21

272.18

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1051.99 1015.72

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1200

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Figure 2: Online LC/ES-MS analysis of a tryptic digest of a protein. A: The upper two panels show a section of the total ion chromatogram and the extracted ion chromatogram for a deamidated peptide of interest. B: The lower two panels show the low energy mass spectrum demonstrating the presence of the deamidated peptide and the higher energy MSe data showing the fragmentation of that peptide. Signals observed are not only consistent with the peptide, confirming its detection but also demonstrate that deamidation has occurred exclusively on the first of the two adjacent asparagine residues. www.biopharmaceuticalmedia.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 13

Research / Innovation / Development A

7.20

100

2.00

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

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11.00

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12.00

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9.59

1.74

2.50

3.00

3.50

4.00

4.50

5.00

5.50

6.00

6.50

12.87

11.01

7.00

7.50

8.00

8.50

9.00

9.50

10.00

10.50

11.00

11.50

12.00

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120

140

160

180

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240

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728.35 279.09

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727.35

pE-V-Q-L-Q-E

100

12.12

6.98

5.08

2.00

11.83

8.42

7.19

260

280

371.10 390.63 355.06

296.06

300

320

340

360

380

400

470.04

420

440

460

480

729.36

506.02

500

520

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560

580

600

620

640

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700

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740

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780

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84.04

101.07

122.08 104.07

100

132.90

120

140

211.10 172.86 183.11 201.04

160

180

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220

240

258.10

260

276.11 279.09

280

340.16 311.16

300

320

350.99

340

452.24 378.78

360

389.19

380

400

420

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453.24

424.25

440

460

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540

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727.35 728.35 749.33 780.26

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Figure 3: Online LC/ES-MS analysis of an endoproteinase Glu-C digest of a monoclonal antibody. A: The upper two panels show a section of the total ion chromatogram and the extracted ion chromatogram for the predicted N-terminal heavy chain peptide if modified by pyroGlu (pE) at the N-terminal. B: The lower two panels show the low energy mass spectrum demonstrating the presence of the peptide and the higher energy MSe data showing the fragmentation of that peptide. Signals observed are consistent with the peptide bearing a pyroGlu residue at the N-terminus.

since not only can the peptide bearing the PTM be identified based on the intact peptide mass but, depending on the extent to which the PTM is present, it may be possible to generate fragment ions that not only confirm the identity of the modified peptide but also give the location of the PTM within the peptide. Two examples of this are shown in Figures 2 and 3 below, which represent the location of the site of deamidation within a peptide and the identification of the pyroGlu containing N-terminal peptide from an antibody heavy chain, respectively. Since modification of a peptide will virtually always result in mass and hydrophobicity changes to a peptide, a modified peptide will elute at a different position in a chromatogram compared to the native peptide. Furthermore, since PTMs are likely to result in partial modification of the peptide, a mixture of modified and unmodified forms of the peptide will exist in the sample. It is therefore possible in a peptide map to use extracted ion chromatograms (a chromatogram generated based on a search of the data for the predicted mass to charge ratio of the peptide) to search for the modified and unmodified forms of the peptides and determine the peak areas. The relative percentages of the modified and unmodified forms can then be determined from these peak areas. However, there are factors that must be borne in mind when performing this type of PTM assessment. Firstly, the ionisation efficiency of the modified and unmodified forms of the peptide may not be the same, meaning the relative signal intensity may not reflect the actual relative abundance. Rather it is a reflection of the amounts of material present and their ability to ionise. This does not in any way negate the assessments of the PTM abundances. For any comparability program, all samples are 14 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

handled in the same way such that any differences in ionization efficiencies are cancelled out. This means that comparisons can be fairly drawn. Secondly, depending on the nature of the PTM, there could be an effect on charge distribution (i.e. the most abundant charge state of the modified peptide may not be the same as the unmodified peptide). It is therefore important to ensure that the most abundant signal in the charge profile envelope is being assessed or XIC measurements are based on a summation of mass:charge values observed. Assessments of peptide mapping data can be performed either through manual interpretation or through the use of software-based data interrogation, such as Waters UNIFI, which often forms part of a general peptide mapping study. Software-driven data interrogation is a more rapid means of data interpretation, but care must be taken as false assignments can be made (through, for example, incorrect assignment of fragment ions) so a check of the output is essential to filter out false positives. Whilst mass spectrometry is an incredibly powerful tool for PTM analysis due to its ability to identify their nature and location within a protein chain, it is worth remembering that the use of other, orthogonal techniques can provide supportive data for their assignment. Importantly, multiple regulatory authorities expect orthogonal techniques and data to be presented in analytical data packages, as discussed in a previous article4. There are several techniques that can be used in an orthogonal manner to support the MS-based assessment of PTMs. One example is imaged capillary isoelectric focusing (icIEF) which provides a charge-based separation of proteins and can therefore give assessments of charge-based modifications such as variation in mAb C-terminal lysine and, more generally, deamidation events on Summer 2021 Volume 4 Issue 2

Research / Innovation / Development proteins. Ion exchange chromatography could also be used in a similar way to provide orthogonal data for charge-based PTMs. Other PTMs, such as PEGylation, glycosylation and proteolytic processing result in significant mass modification of the basic protein chain. Capillary gel electrophoresis (CE-SDS) can be used to give mass-based separation of species present in a sample to investigate these types of PTMs and support the MS-based investigations.

encountered PTMs in a general sense are oxidation and deamidation, since the chemical environments that proteins encounter during the manufacturing, purification process and storage can readily modify susceptible side chains. An assessment of these two PTMs not only provides information on the nature of the sample, but the demonstration of low levels of oxidation and deamidation is a good indication that the manufacturing process is well controlled.

Commonly Occurring PTMs So, given that PTMs are widespread, what types of modification are commonly observed and likely to need assessment irrespective of the type of protein? This is more easily addressed for modifications that are chemically derived, rather than as a result of biosynthetic processing. The most commonly

In terms of product-derived PTMs, disulfide bridges and glycosylation are the most common types of PTM encountered. For monoclonal antibodies, N-terminal pyroGlu formation (seen more frequently with glutamine than glutamic acid if present at the N-terminus of the heavy or light chain) and heavy chain C-terminal lysine need to be examined. It is also possible that directed chemical modifications of the drug as part of the production process may result in other unwanted PTMs occurring on the molecule, so it is very important to consider PTM assessments as different stages of production, where different PTMs may be created as a result of the varying chemical environments to which the protein is exposed. Conclusion In summary, an assessment of PTMs needs to be performed in order to fully investigate the structural characteristics of the protein as part of the requirements of the ICH Q6B guidelines. This is true both for the characterisation of new products but also for comparability studies of biosimilars, where PTMs need to be assessed between innovator and reference products as part of the comparability exercise. Mass spectrometry is a universally applicable technique in PTM investigations with its ability to obtain precise mass and fragment ion information, allowing detailed investigation of the potentially wide variety of PTMs that can be encountered on proteins. REFERENCES 1.

2. 3. 4.

https://www.ema.europa.eu/en/ich-q6b-specifications-testprocedures-acceptance-criteria-biotechnologicalbiologicalproducts https://biopharmaspec.com/blog/challenges-of-n-glycanstructures-in-complex-glycoproteins/ https://biopharmaspec.com/blog/dealing-with-the-challenges-ofdisulfide-bridge-analysis-in-biopharmaceuticals/ Greer, F.M. and Easton, R.L. (2021) IBI 4, 1, 10-13. https://biopharmaceuticalmedia.com/biosimilars-increasing-regulatoryfocus-on-orthogonal-analytical-characterisation/

Dr. Richard L. Easton Dr. Richard L. Easton, Technical Director of Structural Analysis at BioPharmaSpec, obtained his PhD in glycoprotein structural characterisation using mass spectrometry from Imperial College of Science, Technology and Medicine. Following postdoctoral research in the field of glycan elucidation, Richard worked at GlaxoSmithKline and then M-Scan Limited, where he held the position of Principal Scientist. At BioPharmaSpec, Richard manages all aspects of carbohydrate and glycoprotein characterisation.

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 15

Research / Innovation / Development

PEER REVIEWED

Solving Critical Data Challenges for Chemicallymodified, Biologically-based Therapeutic Candidates The drug discovery research industry is seeing a growing prevalence of chemically-modified, biologically-based therapeutic candidates1. This trend creates urgent and novel challenges for data management and analysis informatics software, necessary for supporting cross-functional teams of scientists bridging across biology and chemistry. We discuss key challenges and how to address them, such as rigour in scientific definition of candidate therapeutics and opportunities to automate and streamline data analysis workflows. The goal is to ensure scientists have the highest quality data to work with, and the time they need to drive drug discovery innovation.

Adoption of Informatics Software As the modern drug discovery industry emerged over the latter half of the 20th century, it was dominated by small molecule therapeutics. Vaccines and small peptide analogues were early biologics, and then in 1994 the first monoclonal antibody, Abciximab, was approved in the United States2. This was the landscape into which the widespread adoption of personal computers occurred in the final 15 or so years of the 20th century. This fostered and amplified the fields of computational chemistry and bioinformatics that had originally been developed on mainframe computers. The adoption of informatics software varied by scientific disciplines. In

general, research chemists appear to have adopted scientific informatics tools readily. Research biologists tended to focus on software applications for statistical analyses and for producing customisable, publication-quality scientific graphics. From this environment, scientific informatics vendors emerged, to provide personal productivity tools to these scientists. At that time, many drug discovery organisations maintained their own software development groups to serve their internal R&D groups with in-house developed solutions, though this is much less common now as the use of commercial informatics software has become the standard across the industry. Given this environment, decisions regarding informatics product features led naturally to the development of data capture systems, such as electronic lab notebooks (ELNs), that were oriented towards supporting medicinal and analytical chemistry, whereas biologists often favoured traditional paper notebooks. Chemical registration systems, used to define and enforce uniqueness of chemical molecules to ensure patentability of the new chemical entities (NCEs), were also developed. The digital capture of chemical structures encouraged the emergence of computational chemistry tools and rules such as Lipinski’s rule of five3, including the early adoption of QSAR and machine learning approaches, to predict activity and optimise drug characteristics. On the other hand, computational biology was the purview of bioinformaticians, who often came from the fields of mathematics, physics or

Figure 1. Examples of chemically-modified therapeutics. A: Antibody-drug conjugate. B: Bicyclic peptide. C: Stapled peptide. D: Peptide-drug conjugate. E: Antisense oligonucleotide 16 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Summer 2021 Volume 4 Issue 2

Research / Innovation / Development engineering, to analyse protein biochemistry and to solve large-scale challenges in sequence assembly and alignment. Bioinformaticians typically developed, and often shared, scripted algorithms, leading to a tradition of open-source software. Bench biologists typically did not use these tools themselves but engaged with bioinformaticians to solve their research questions. This description of the scientific informatics landscape for drug discovery is somewhat simplistic and a generalisation, but it has remained largely as described, until recently. The Emergence of Chemically-modified Therapeutics A broad class of novel therapeutic entity types has emerged. These are biologically-based molecules that contain some form of artificial chemical modification. While no widely recognised collective term for this class exists, this class can be referred to as “chemically-modified therapeutics” (CMTs), or “chemically-modified biologics” (Figure. 1). The latter term may be less desirable because a key goal of developing them is to avoid some of the challenges of biologic drugs. Such challenges include production challenges, dosing regime, and ADME (adsorption, distribution, metabolism, and excretion) profiles. This class of entities includes, but is not limited to, antibody-drug conjugates (ADCs), peptide-drug conjugates (PDCs), peptide mimetics, stapled peptides, cyclic and bicyclic peptides, and various RNA-based therapies, including antisense oligonucleotides (ASOs), small-interfering RNA (siRNA), and Modified RNA (ModRNA). A complete list is difficult to enumerate because it is such an active area of research that new entity types are emerging regularly. This is a diverse group of therapeutics, with significant differences in form and function. However, we find it useful to group them for the purposes of discussing the significant data management and informatics challenges they present given the current informatics landscape, as described earlier. It’s important to understand some of the key reasons why this class of therapeutics has emerged. Much has been made of the increasing cost of drug discovery4, and the decline on return on investment5. It has been suggested that much of the “low-hanging fruit” for small molecule therapeutics has been “plucked”, and fewer blockbuster drugs are coming to market6. On the biologic side, developability and delivery challenges, as mentioned earlier, have limited the breadth of therapeutic applications. Cyclic/bicyclic peptides and stapled peptides are perhaps the best examples of entities that bridge between chemistry and biology, meaning that they can address these challenges. They are small enough to avoid immunogenicity and other issues faced by antibodies, their chemical modifications protect them from ADME issues faced by analogues of natural peptides, and their larger size relative to traditional small molecules means that they can access pharmacological opportunities that aren’t open to small molecules. The various forms of RNA therapies primarily utilise their chemical modifications to avoid processing pathways faced by natural RNAs. Antibody and peptide conjugates typically act to deliver a cytotoxic small molecule using a biologically-based targeting system. Much of the innovation for ADCs is around the chemical linker technology attaching the small molecule payload to the antibody. From a scientific and research perspective, CMTs are very diverse in structure and in the modes by which they utilise the combination of www.biopharmaceuticalmedia.com

biology and chemistry. The diversity in molecular structure, mechanisms of action, targets and formulation adds to the challenges for scientific informatics systems. Accessing Data to Support Cross-functional Teams Considering data management challenges for CMT research, the number one issue is simply being able to access all the data that forms a research project. This is true for all drug discovery research but is even more acute for CMT research. Drug discovery research is a collaborative activity, often involving scientists within and across departments and sites, or even organisations (e.g., strategic partners or contract research organisations – CROs). The cross-functional nature of CMT science, drawing from both chemistry and biology, may mean that project data is distributed even more than usual across data capture systems. This is because many scientific informatics systems can handle either chemical or biological structures, data and workflows, but not both. If an early-stage organisation working on CMTs is in the process of selecting an informatics system, this is a key point to consider. For established organisations moving into CMT discovery, it raises the issue of how well the current informatics environment can support CMT data management and presentation. A key factor is the cross-functional teams working on CMTs. A single representation of project data, such as images of candidate therapeutics, is unlikely to satisfy all users. Chemists prefer to view molecular structure images whereas the lingua franca of biologists is typically sequence visualisations. However, sequence representations lack the detail that most chemists would seek. The richer molecular structure view also offers considerably better intellectual property (IP) protection than a traditional sequence view (see below). Biologists, by comparison, may lack the experience to perform effective structure-based queries. While different views and query mechanisms are available, there should only ever be one single source of truth. Therefore, CMT candidates should, wherever possible, be stored as a full chemical description, though it must be possible to find it through other search criteria, such as a sequence motif. This divergent capability extends also to the nature of the user interfaces provided for biologists and chemists. So, to summarise, data capture should be possible from both a traditional chemist (structure) and biologist (sequence) view, data storage should be with full chemistry, and data search should allow either structure- or sequence-based queries. Defining Uniqueness to Protect IP A second critical challenge for scientific informatics in CMT is the ability to rigorously define and capture compositional uniqueness of therapeutic candidates, to ensure the therapeutic design and associated IP protection needs are met. The bridging of CMTs across biology and chemistry brings key challenges in this respect. To be effective, scientific informatics systems must allow researchers to define entities in a format that is comfortable and familiar to them, while providing suitable rigour in the definition that ensures uniqueness. A situation where complications can occur is shown in Figure 2. In this example, we are using a conjugated peptide as our example entity. The entity consists of a peptide sequence, a chemical linker, and a chemical payload. The same entity can be represented as at least four meta-descriptions. In the first representation the INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 17

Research / Innovation / Development sequence, linker and payload are individually registered as standalone entities, and the complete entity is comprised of those three substituent (component) parts (Figure. 2A). This hierarchical representation affords the organisation flexibility to mix and match component units – a common use case – and therefore this representation is often the preferred choice. It is also consistent with de facto standards for information exchange such as via HELM (Hierarchical Editing Language for Macromolecules) notation7. As shown in Figure 2B, the same entity could also be represented as comprised of a joint payload–linker subunit, captured as a single molecule crosslinked to the sequence chain. Fig. 2C shows a joint linker–sequence subunit, attached to the payload. In this representation the linker is recorded as a modified N-terminal residue. Alternatively, the whole entity could be represented (and registered directly) as a single unit (Figure. 2D). All four representations are chemically identical; the distinct definitions simply reflect the preferred choice of a given scientist or their organisation. There are five distinct component entities that can be used in various combinations to represent the complete entity. The complication that may arise is that if the informatics registration system is unable to understand that all of these forms are identical, duplicate entities could be registered, undermining the smooth operation of the project and potentially creating IP issues. This can be avoided if the registration system generates a full chemical representation of the full entity. This not only prevents (or significantly reduces) the likelihood of duplicate registrations, it also provides a very robust definition of the entity.

Capturing and Maintaining Data with High Fidelity A third essential need for scientific informatics in drug discovery relates to data quality considerations. A well-designed and implemented informatics system plays an essential role in capturing and maintaining data with high fidelity. This is true in general for drug discovery, but CMTs can create novel or exacerbated challenges with regard to data quality because of the cross-functional nature of the science. Researchers may be working outside their core expertise, where errors are more likely to occur, or less likely to be detected and solved. An example of this situation, and how informatics can alleviate the problem, is bioconjugation reactions for ADCs, PDCs, or for attaching reporter molecules to biological entities. A common method for doing this is “click chemistry”8. Several reaction schemes qualify as click chemistry, but one of the most common is the 1,3-dipolar cycloaddition reaction9. This reaction can be used with both peptides and nucleic acids, and there are commercially available reagents. Because the molecular structure of the reaction product (the conjugated substance) and the reactive portions of the input reagents (sequences and payloads) are clearly defined, creating these conjugates in a registration system can be automated. Properly implemented, an informatics system can implicitly draw connections between the components and create (in this case) the triazole crosslink without the explicit input of a scientist. This represents a hugely valuable capability that scientific informatics systems can provide, by identifying opportunities for automation. Such automation alleviates the need for scientists, who might be working at the limits of their experience and expertise

Figure 2. Variable representations of an identical peptide-drug conjugate. A: Each component is individually represented in a registration system, and the whole entity is comprised of the three sub-parts (as distinguished by the dashed lines). B: The molecule and linker are registered as a unit. C: The linker and peptide are registered as a unit. D: The entire entity is registered as a single unit. The informatics system must be able to recognise that all four representations of the whole entity are identical. 18 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

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Research / Innovation / Development higher quality data and allowing researchers to spend more time using that data for drug discovery innovation. REFERENCES 1.

6. 7.

Brown, D.G., Wobst, H.J. A decade of FDA-approved drugs (2010–2019): Trends and future directions. J. Med. Chem. 64, 2312–2338 (2021) Epic Investigators. Use of a monoclonal antibody directed against the platelet glycoprotein IIb/IIIa receptor in high-risk coronary angioplasty. The New England Journal of Medicine. 330, 956–961 (1994) Lipinski, C.A., Lombardo, F., Dominy, B.W., Feeney, P.J. Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 46, 3–26 (2001) DiMasi, J.A., Grabowski, H.G., Hansen, R.W. Innovation in the pharmaceutical industry: New estimates of R&D costs. J Health Economics 47, 20–33 (2016) Deloitte. Ten years on. Measuring the return from pharmaceutical innovation 2019. https://www2.deloitte.com/us/en/pages/ life-sciences-and-health-care/articles/measuring-return-frompharmaceutical-innovation.html. Visited March 2021. Li, J.J. Blockbuster Drugs: The Rise and Decline of the Pharmaceutical Industry. Oxford University Press (2014) Pistoia Alliance. HELM Hierarchical Editing Language for Macromolecules. https://www.pistoiaalliance.org/projects/ current-projects/helm/. Visited March 2021. Rostovtsev, V.V., Green, L.G., Fokin, Sharpless, K.B. A stepwise Huisgen cycloaddition process: Copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angewandte Chemie 41, 2596–2599 (2002) Haldon, E., Nicasio, M.C., Perez, P.J. Copper-catalyzed azide-alkyne cycloadditions (CuAAC): an update. Org Biomol Chem 13, 9528–9550 (2015)

Andrew LeBeau

Figure 3. Various forms of RNA-based therapies, each containing chemical modifications to either the nucleobase and/or backbone. A: Antisense oligonucleotide (ASO). B: small interfering RNA (siRNA), showing the RNA-Induced Silencing Complex (RISC). C: mRNA vaccine, showing coding mRNA.

due to the cross-functional nature of the work, from manual work, leading to improvements in data quality through a reduction in inadvertent errors. This is a critical area where scientific informatics can help to drive improvements in data quality. Ultimately, teams with a broad range of scientific domain expertise can now spend more time using their experience and knowledge on more productive tasks, working together with a common understanding, creating a win-win situation. Drug discovery research is challenging for many reasons and is characterised by many failures and few successes – but those successes are of monumental importance. Informatics systems that assist with managing, analysing and presenting data to researchers are crucially important in facilitating drug discovery research. They must evolve with developing trends in research, particularly the emerging importance of CMTs. These place additional challenges on informatics systems, such as ensuring a single source of truth for candidate entities, with the required level of scientific rigour. Opportunities to automate what would otherwise be manual, potentially error-prone tasks for scientists should be taken, resulting in www.biopharmaceuticalmedia.com

Andrew LeBeau, Ph.D., is Associate Vice President for Biologics Marketing at Dotmatics. He joined Dotmatics in 2017, bringing more than 15 years of experience in the life sciences industry. At Dotmatics, Andrew leads Dotmatics’ strategic product definition and marketing activities in support of the rapidly growing field of biologics drug discovery. Prior to Dotmatics, Andrew held Product Marketing and Product Management positions at Illumina and Accelrys / Biovia. Email: andrew.lebeau@dotmatics.com

Troy Humphreys Troy Humphreys, Ph.D., is a Principal Application Scientist at Dotmatics. He joined Dotmatics in 2017, bringing more than 20 years of biotech and academic research experience. As an application scientist he works with customers to implement Dotmatics solutions and also serves on cross-functional biologics strategy and product development teams designing the applications on the next horizon. Prior to Dotmatics, Troy worked at the lab bench making antibody products for Cell Signaling Technology. Email:troy.humphreys@dotmatics.com

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Research / Innovation / Development

Using Lentiviral Vectors to Advance the Development of Therapeutic Vaccines Introduction Traditionally, vaccination has been used as a preventative strategy, aimed at curbing the impact of a wide range of infectious diseases. Over the past three decades, however, vaccine development has moved increasingly into the therapeutic area for the treatment of conditions such as cancer and chronic viral infections. To be successful, such vaccines must elicit a strong cellular as well as humoral immune response. Viral vectors, engineered to carry genes for the expression of a suitable immunogenic protein in the recipient, are widely used. However, to date their success against agents such as HIV and against tumour cells has been limited. A major challenge in pre-clinical and clinical work is to ensure that the vectors used do not randomly integrate with host DNA and give rise to unwanted and potentially dangerous consequences. This article examines how a new generation of lentiviral vectors, capable of triggering effective, long-lasting T cell-mediated immunity, is demonstrating pre-clinical success in both therapeutic and prophylactic vaccine development.

The Challenge of Inducing Cellular Immunity Vaccines, including those based on viral vectors, stimulate two types of adaptive immune responses: humoral and cellular. A humoral, or antibody-mediated, response involves the activation of B cells, triggering a set of reactions that culminates in the production of specific circulating antibodies. This type of response is highly effective in dealing with an enormous range of infectious challenges and, to date, is the primary mode of operation for most successful vaccines. Antibody-mediated responses are, however, only very poorly protective against intracellular pathogens and tumour cells. Tackling these requires the activation of T cells and the development of a robust cell-mediated immune response. Cellular responses are both long-lasting and capable of destroying host cells infected by an intracellular pathogen or attacking a tumour cell. The process of activating naïve T cells to be able to recognise intracellular antigens requires the presentation of target antigens by mature dendritic cells within the secondary lymphoid organs. These antigen-presenting cells are the only ones capable of stimulating naïve T cells and they play a pivotal role in the cellular and molecular events that establish T cell responses. Dendritic cells trigger activation primarily through processing antigens, which they then present at their cell surface, making them accessible to the T cells. As well as being responsible for antigen presentation, dendritic cells give other signals involved in the induction, activation and proliferation of specific T cell clones. The crucial part that dendritic cells play in the development and regulation of cellular responses has led to them becoming a major target in both prophylactic and therapeutic vaccine development. They are, however, non-dividing cells, which makes their manipulation through 20 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

gene transfer particularly difficult. Lentiviral vectors are proving effective in overcoming this challenge.1,2,3,4 The Role of Viral Vectors Viral vectors have been studied for several decades now and a wide variety have been engineered as vehicles to transfer genetic material for gene therapy and, more recently, for vaccination.5,6,7 Among the most commonly used have been human and simian adenoviruses and the measles and yellow fever viruses. Retroviruses have also been the subject of intensive study and development as vectors, and in today’s gene therapy strategies lentiviruses sit alongside adenoviruses and adeno-associated viruses at the forefront of pre-clinical and clinical success.6 With respect to vaccine applications, the rapid and continuing development of vaccines effective against SARS-CoV-2 has put viral vectors firmly into the scientific and public spotlight, with emergency use listing now granted to adenoviral vector products. Viral vectors are exciting prospects for vaccine use as they have the capacity to introduce genetic material of interest and enable intracellular antigen expression that supports the induction of a cellular immune response.8 Generally, they also achieve a high level of immunogenicity without the use of an adjuvant. Despite continuing advances in the development and refinement of different types of vaccination against a wide range of infectious diseases, attempts to develop vaccines for cancer immunotherapy and for the treatment of chronic infections have been met with only limited success. Here a major challenge lies in being able to elicit a sufficient immune response to eliminate the virus or the cancer cells. Viral vector-based vaccines are a focus for these applications, but most viral vectors are unable to transduce non-proliferating dendritic cells. This means their action on dendritic cells, the keys to adaptive cellular immunity, is severely limited. Uniquely, lentiviral vectors do not suffer this drawback. Not only do they infect and transduce non-dividing cells they also have a natural in vivo tropism towards dendritic cells and have been engineered to provide a robust platform for vaccine development. The Practicalities of Using Lentiviral Vectors Lentiviruses are a subclass of retroviruses and can infect both dividing and, most importantly, non-dividing cells. The ability of lentiviral vectors to efficiently transduce non-dividing adult stem cells has already led to significant advances in gene therapy. Their effectiveness in delivering antigenic material to specific target cells, and their natural tropism for dendritic cells, has made them a major contender in vaccine development. Advanced engineering of lentiviral vectors now includes the application of DNA flap technology,9 a crucial breakthrough in enabling their more widespread use. Derived from the lentivirus genome, DNA flap technology consists of a DNA triple-stranded structure that enables the virus to cross the nucleus membrane and enter the cell nucleus itself. Engineered into lentiviral Summer 2021 Volume 4 Issue 2

Research / Innovation / Development vectors it enables the import of any gene into non-dividing cells. The inclusion of specific promoters for optimal expression of antigens ensures maximum expression at the right time and in the right place. With a strong proclivity to induce antigen presentation by dendritic cells, modern lentiviral vectors can therefore trigger sustained antigen expression throughout the endogenous pathway leading to efficient antigen presentation. This results in the induction of an intense and long-lasting T cell response. If the antigen is secreted or expressed at the surface of dendritic cells, a strong humoral immune response is also induced. The latest engineering also delivers vectors that contain a set of vesicular stomatitis virus (VSV)-G viral envelopes. Pseudotyping lentiviral vectors with VSV-G envelop glycoproteins have two major advantages. (i) Human populations have been barely exposed to VSV. Therefore, lentiviral vectors (LV) engineered to harbour VSV-G are not major targets of pre-existing immunity which could neutralise the vector and dampen its efficacy. (ii) Availability of diverse VSV-G variants allows the use of distinct viral envelopes for prime and boost immunisations. This will avoid the anti-vector immunity eventually induced by prime immunisation to impact the efficacy of the vector used in boost immunisation. Moreover, a high loading capacity allows these vectors to carry large (up to 8000 base pairs) antigens, or even multiple antigens, much more than is possible using adenovirus or similar vectors. Furthermore, they can be engineered to induce either antibody and T cell responses or T cell only, depending on their use. It is important to acknowledge past safety concerns with early generation retroviral vectors in gene therapy, where the occurrence of rare adverse events originally cast doubt on the technology.10 A challenge in using retroviruses for gene therapy is that the integrase enzyme can insert virus genetic material arbitrarily into the host genome, potentially disrupting or perturbating host gene function. If the disrupted gene is concerned with regulating cell division, then that division can become uncontrolled. Potential solutions involved changing the structural organisation of the lentiviral vector so as to modify the promoter that drives expression within the human genome, and/or generating a lentiviral vector that does not integrate but still retains the capacity to transfer genetic material into the human genome. Success in overcoming the issue of integration has been a major step forward in the safe use of lentiviral vectors for vaccine development. The latest-generation lentiviral vectors are genetically deficient in integrase and therefore do not integrate within the host DNA, but they do retain their original capacity to express foreign genes. Additionally, they are non-replicative, non-cytopathic and essentially non-inflammatory. Since safety is the overriding concern in vaccine development, the unique safety profile of today’s lentiviral vectors means they can be used with confidence. Extensive animal studies have demonstrated the absence of any serious safety concerns, and lentiviral vectors are already being used to insert CAR-T cell receptors and CRISPR-cas9 into human cells.11 Lentiviral vectors have also been used in a number of gene therapy trials without safety concerns. The safety of today’s lentiviral vectors contrasts with that of standard retroviral vectors, which are generally integrative, raising issues around genotoxicity. www.biopharmaceuticalmedia.com

Addressing the Question of Lentiviral Production Producing lentiviral vectors is a specialist area and previously there were challenges in producing volumes of the magnitude that would be required in a mass vaccination campaign. Great strides have been made in addressing the production question and processes can now be more readily scaled up to meet demand. Today these vectors are produced using a process similar to that for adenoviral vectors, involving a transient plasmid transfection of HEK293T cells. Production yields are inferior to those for adenoviral vectors, but this may be balanced by the predicted need for much smaller doses in humans – in the range of 108 particles versus 1010–11 for adenoviral vectors. The volumes needed to meet the demand for therapeutic vaccination are easily achieved and there is some expectation that stable packaging lines, similar to those used to produce other retroviral vectors, could be developed in the near future, with the additional benefit of reducing production costs. Demonstrating Pre-clinical Success The use of lentiviral vectors in vaccine development is maturing and there is a body of work that now covers 12 years of pre-clinical and clinical studies using advanced lentiviral vectors. Major goals have been to clearly demonstrate the safety, capacity and utility of non-integrating lentiviral vectors. Work to date has demonstrated both their efficacy and safety in a wide variety of diseases and different animal models, as the following example illustrates, and bodes well for success in humans. Protection of Macaques against SIV In a study of SIV, Chinese rhesus macaques were dosed with a lentiviral vector vaccine developed using a specific optimised antigen design. Following intramuscular injections of increased vaccine doses in a prime/boost/prime protocol, the macaques were given a very high dose intrarectal challenge with SIVMac251, the equivalent to a macaque model of HIV. All low-dose vaccinated animals showed: strongly reduced viremia during primo-infection; early control of SIVMac251 replication to undetectable viremia (under 37 RNA copies/mL); and stable control of SIVMac251 over time with no virus escape. Durable Protection against SARS-CoV-2 in Mice and Hamsters via Intranasal Route A new study has shown that a single intranasal administration of a lentiviral vector encoding a prefusion form of SARS-CoV-2 spike glycoprotein induces full protection of respiratory tracts and totally avoids pulmonary inflammation in a susceptible hamster model.12 The authors also generated a new transgenic mouse model with unprecedented permissibility of the brain to SARS-CoV-2 replication. In this highly stringent model, the authors demonstrated that an intranasal booster immunisation achieves full protection of both respiratory tracts and brain against SARS-CoV-2 and completely prevents lung and brain infiltration and pathogenic inflammation. The intranasal administration of a lentiviral vector, necessary to induce this efficient mucosal immunity, is possible thanks to the high safety of the vector, linked to its non-cytopathic and non-inflammatory properties. In Summary These and other studies together offer strong evidence that today’s lentiviral vectors enable a strong, long-lasting memory response. They have the potential for use in both prophylactic INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 21

Research / Innovation / Development

and therapeutic vaccination, and the effective dose is generally 1000 to 10,000 times lower than that required with adenoviral vectors. Future Promise There is clear evidence that non-integrative lentiviral vectors are effective in producing vaccines that rely heavily on eliciting a cellular immune response, as required for the treatment of cancer and chronic infectious diseases. Early safety concerns have been addressed and solved, and today’s lentiviral vectors have a unique and demonstrable safety profile. More than a decade of studies in various animal models provide pre-clinical evidence of their efficacy in cancer treatment and in the treatment of ongoing viral infections, pointing the way to success in humans. The unique ability of lentiviral vectors to target and transduce dendritic cells to stimulate the production of a strong and long-lasting T cell response may make them part of the best vectors capable of driving sufficient human cellular response to mitigate chronic viral infection. REFERENCES 1.

Firat H, Zennou V, Garcia-Pons F, Ginhoux F, Cochet M, Danos O, Lemonnier FA, Langlade-Demoyen P, Charneau P. Use of a lentiviral flap vector for induction of CTL immunity against melanoma. Perspectives for immunotherapy. J Gene Med. 4(1), 38-45 (2002). doi: 10.1002/jgm.243. PMID: 11828386 Esslinger C, Romero P, MacDonald HR. Efficient transduction of dendritic cells and induction of a T-cell response by third-generation lentivectors. Hum Gene Ther. 13(9), 1091-1100 (2002). He Y, Zhang J, Mi Z, Robbins P, Falo LD, Jr. Immunization with lentiviral vector-transduced dendritic cells induces strong and long-lasting T cell responses and therapeutic immunity. J Immunol. 174(6), 3808-3817 (2005). Ku MW, Authié P, Nevo F et al. Lentiviral vector-induced high-quality memory T cells via programmed Antigen expression in dendritic cells. Lundstrom K. Viral Vectors in Gene Therapy.Diseases. 6(2), 42 (2018). https://dx.. doi.org/10.3390%2Fdiseases6020042

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Bulcha JT, Wang Y, Ma H, Tai PWL, Gao G. Viral vector platforms within the gene therapy landscape. Signal Transduct Target Ther. 6(1), 53 (2021). https://doi.org/10.1038/s41392-021-00487-6 7. Lundstrom K. RNA Viruses as Tools in Gene Therapy and Vaccine Development. Genes. 10(3), 189 (2019). https://doi.org/10.3390/ genes10030189 8. Ura T, Okuda K, Shimada M. Developments in Viral Vector-Based Vaccines. Vaccines (Basel). 2(3), 624-641 (2014). https://dx.doi. org/10.3390%2Fvaccines2030624 9. Zennou V, Petit C, Guetard D, Nerhbass U, Montagnier L, Charneau P. HIV-1 Genome Nuclear Import Is Mediated by a Central DNA Flap. Cell. 101(2), 173–185 (2000). https://doi.org/10.1016/ S0092-8674(00)80828-4 10. Gore M. Adverse effects of gene therapy: Gene therapy can cause leukemia: no shock, mild horror but a probe. Gene Ther. 10, 4 (2003). https://doi.org/10.1038/sj.gt.3301946 11. Milone MC, O’Doherty U. Clinical use of lentiviral vectors. Leukemia. 32, 1529–1541 (2018). https://doi.org/10.1038/s41375-018-0106-0 12. Ku MW, Authié P, Bourgine M et al. Full Brain and Lung Prophylaxis against SARS-CoV-2 by Intranasal Lentiviral Vaccination in a New hACE2 Transgenic Mouse Model or Golden Hamsters. bioRxiv. (2021).02.03.429211; https://doi.org/10.1101/2021.02.03.429211

Christian Bréchot Christian Bréchot, MD, PhD, Senior Scientific Advisor to TheraVectys, joined the USF Health Morsani College of Medicine as Senior Associate Dean for Research in Global Affairs, Associate VP for International Partnerships and Innovation, Professor in the Division of Infectious Disease, Department of Internal Medicine, and heads USF’s Initiative on Microbiomes. He is also President of the Global Virus Network. In addition to his previous role as President of the Institut Pasteur, he has held senior positions at InstitutMeriux, Inserm (the French NIH) as well as Paris Descartes University. Dr. Bréchot’s research activities have focused on viral hepatitis, microbiomes, and viral infections.

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Producing breakthrough treatments requires continual advances in bioprocessing productivity. That’s why Avantor® is your trusted partner in producing life-saving therapies. Our J.T.Baker® BAKERBOND® PROchievA™ affinity chromatography resin offers best-in-class purification performance in the critical protein A chromatography step of monoclonal antibody production, using a proprietary ligand to improve efficiency. We focus our solutions on your needs so that you can focus on science. See how we help advance monoclonal antibody manufacturing at avantorsciences.com/moves-science-forward/ibi/performance www.biopharmaceuticalmedia.com

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Research / Innovation / Development

Antibody-drug Conjugates: A Trojan Horse Story

According to World Health Organization, cancer is responsible for 10 million deaths per year and it is considered as the second leading cause of death in the world. New anti-cancer drugs have entered the global market in recent years, but it is still not clear if they are sufficient to cure cancer patients. On the one hand, conventional therapies have severe side-effects and tumour cells become resistant. On the other hand, novel therapies have emerged, like immunotherapies, but so far the clinical benefit is low. It is crucial to develop new approaches that could enhance the therapeutic efficacy.1–2

Strong cytotoxic effect of drugs can be combined with the specificity of antibodies against cancer cells. Briefly, it is a combination of chemotherapy and immunotherapy for a targeted therapy. After the growth of monoclonal antibody technology and the approval of approximately 30 antibodies by the US Food and Drug Administration (FDA), there has been an evolution in antibody technology. Antibody-drug conjugates (ADCs) are targeted agents that consist of a monoclonal antibody, which recognises a specific cell surface antigen of cancer cells, and it is linked to a cytotoxic drug. The goal is to improve chemotherapy efficacy and reduce toxicity on normal cells.3 German physician and scientist Paul Ehrlich presented the concept of ADCs 100 years ago. Many years later, according to his concept, methotrexate was conjugated to antibody against leukaemia cells. Almost 40 years ago, clinical trials were performed with ADCs that were based on mouse IgG molecules. A decade later, chimeric and humanised antibodies replaced the mouse molecules. Over more recent years, production of human monoclonal antibodies has opened new horizons. They have been used in ADCs in order to increase the specificity and targeted delivery of cytotoxic drugs against cancer cells.4 Components Antibodies One of the most fundamental parts of ADCs is the monoclonal antibody that will lead the drug payload to target cells; target antigen selection and production of the appropriate monoclonal antibody. Tumour cells have more than 300 unique antigens that have the potential to be selected but there are some restrictions. The selected antigen has to be highly expressed by tumour cells and low expressed or preferably not expressed by normal cells. Moreover, the location of the antigen is crucial as it has to be placed on the cellular surface in order to be accessible by the monoclonal antibody and it must have internalisation properties to transfer the drug into the target cell.5 Antibodies have to be highly specific to tumour antigen and to possess high binding affinity. Apart from the connection to the target antigen, they should bear low immunogenicity and cross-reactivity. Murine antibodies that have been used in the 24 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

first generation of ADCs exhibited strong immune responses, resulting in low therapeutic efficacy. Genetic engineering was used to solve that problem and to generate the second generation of ADCs. Murine antibody was replaced by mouse/ humanised chimeric antibody and immunogenicity was reduced. It has shown great efficacy in cancer treatment but in some cases there has been human anti-chimeric antibody response. Moving forward, mouse/humanised chimeric antibodies were replaced by humanised antibodies, but there was still the same problem as none of those antibodies were fully truly human. Fully human antibodies have been used in next-generation ADCs and they do not produce immune response. Linkers Linkers are crucial parts of ADCs as they need to have key requirements. They need to be stable so that there will not be any off-target release of the drug. Also, they must keep the drug inactive as long as it is conjugated with the antibody, and they need to unleash the drug upon internalisation. There are two categories, non-cleavable and cleavable linkers. Non-cleavable linkers provide stable bonds and release of drug is performed after internalisation and degradation of antibody in lysosomes. Decreased efficacy might be observed due to reduced membrane permeability.6 On the other hand, cleavable linkers are cleaved by the differences that are present to the extracellular and intracellular environment, like pH or enzymes. There are linkers stable in the alkaline environment of systemic circulation but sensitive in acidic environments. Lysosomal protease-sensitive linkers have been commonly used and Cathepsin B-sensitive peptide linkers are an example of lysosomal proteases that are highly expressed by tumour cells. There are also β-glucuronide linkers and glutathione-sensitive disulfide linkers that can be used. All those linkers have common properties that can be functional in tumour environments and stable in blood flow.7 Payload Cytotoxic drug is the third part of ADCs. All three parts have similar importance as they have to be carefully selected and designed in order to achieve a successful therapeutic effect. Payload has to be activated as soon as it enters the cytoplasm of tumour cells and be able to kill tumour cells even at low doses. Candidate drugs should have in vitro IC50 value for tumour cells, solubility in the environment of antibody, small molecular weight, long half-life and low immunogenicity. There are 11 small molecule-based and five protein-derived drug classes that have been used in clinical-stage ADCs. In Clinics In 2009, gemtuzumab Ozogamicin (Mylotarg®) was the only FDA-approved ADC while 12 other candidate products were in clinical trials. Today, there are eight more products that got FDA approval (Table 1), while more than 80 are in clinical studies. Kadcyla®, Padcev®, Enhertu® and Trodelvy® are directed Summer 2021 Volume 4 Issue 2

Research / Innovation / Development against solid tumours. The other five ADCs are directed against haematological cancers. Among the targets of novel ADCs that have been under evaluation in clinical studies, there are CEACAM5, c-MET, Folate Receptor Alpha, HER3, Mesothelin, LIV-1and Tissue Factor.8 ADC

Target

Trade Name

Company

Approval Date

gemtuzumab Ozogamicin

CD33

Mylotarg®

Pfizer

2000

brentuximab vedotin

CD30

Adcetris®

Seattle Genetics

2011

adotrastuzumab emtansine

HER2+

Kadcyla®

Genentech/Roche

2013

inotuzumab ozogamicin

CD22

Besponsa®

Pfizer

2017

Enfortumab vedotin

Nectin 4

Padcev™

Seattle Genetics

2019

Famtrastuzumab deruxtecan-nxki

HER2+

Enhertu®

Daiichi Sankyo & AstraZeneca

2019

Polatuzumab vedotin-piiq

CD79b

Polivy™

Genentech/Roche

2019

sacituzumab govitecan

TROP-2

Trodelvy™

Immunomedics

2020

belantamab mafodotin

BCMA

Blenrep®

GSK

2020

Table 1. Antibody-drug conjugates approved by the Food and Drug Administration (FDA).

Resistance to ADCs Knowledge of the mechanism of action of ADCs can reveal the potential mechanism of resistance of tumour cells against ADCs. The steps of action are binding to the antigen, internalisation, release of payload in the cytoplasm and induction of cell death by apoptosis.9 The resistance of tumour cells may be attributed to downregulation of the antigen, defects of antibody binding or internalisation, defective lysosomal function, and lower or no release of the payload.10 Challenges Ahead Mechanisms of resistance, limited solid tumour penetration and toxicity are the major limitations of efficient treatment with ADCs. Those obstacles can be overcome with thorough research to identify the key factors affecting the efficacy of the products and with novel design of new ADCs, including novel targets, antibody production, linkers and potential cytotoxic drugs. Hence, drugs that have been tested against specific diseases in the past can be used as payloads in new ADCs, for drug repurposing/ repositioning, and have better pharmacological profile and efficacy. REFERENCES 1.

3. 4.

Mansoori B, Mohammadi A, Davudian S, Shirjang S & Baradaran B. The Different Mechanisms of Cancer Drug Resistance: A Brief Review. Adv Pharm Bull. 2017;7(3):339-348. Waldman AD, Fritz JM & Lenardo MJ. A guide to cancer immunotherapy: from T cell basic science to clinical practice. Nat Rev Immunol. 2020; 20: 651–668. Hafeez U, Parakh S, Gan HK & Scott AM. Antibody-Drug Conjugates for Cancer Therapy. Molecules. 2020;25(20):4764. Khongorzul P, Ling CJ, Khan FU, Ihsan AU & Zhang J. Antibody–Drug Conjugates: A Comprehensive Review. Mol. Cancer Res. 2020; 18, 3–19. Ponziani S, Di Vittorio G, Pitari G, Cimini AM, Ardini M, Gentile R, Iacobelli S, Sala G, Capone E, Flavell DJ, Ippoliti R & Giansanti F. Antibody-Drug Conjugates: The New Frontier of Chemotherapy. Int J Mol Sci. 2020;21(15):5510.

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Jeffrey SC, de Brabander J, Miyamoto J & Senter PD. Expanded Utility of the β-Glucuronide Linker: ADCs That Deliver Phenolic Cytotoxic Agents. ACS Medicinal Chemistry Letters 2010; 1 (6): 277–280. 7. Alley SC, Okeley NM & Senter PD. Antibody–Drug Conjugates: Targeted Drug Delivery for Cancer. Current Opinion in Chemical Biology 2010; 14 (4): 529–537. 8. Criscitiello C, Morganti S & Curigliano G. Antibody–drug conjugates in solid tumors: a look into novel targets. J Hematol Oncol. 2021;14, 20. 9. Beck A, Goetsch L, Dumontet C & Corvaïa N. Strategies and Challenges for the next Generation of Antibody-Drug Conjugates. Nature Reviews Drug Discovery 2017; 16 (5): 315–337. 10. Joubert N, Beck A, Dumontet C & Denevault-Sabourin C. AntibodyDrug Conjugates: The Last Decade. Pharmaceuticals (Basel). 2020;13(9):245.

Panagiotis Parsonidis Panagiotis Parsonidis is a scientist at the Research and Development department of Research Genetic Cancer Centre (RGCC) S.A.. He has experience in cellular and molecular biology techniques that have application in Oncology and Immunology. His research interest is focusing on gene and protein expression of Circulating Tumor Cells and Cancer Stem Cells. Panagiotis is currently involved in the development of Adoptive Cellular Therapies for cancer treatment.

Ioannis Papasotiriou Dr. Ioannis Papasotiriou, male, born in Munich, Germany. He graduated from Medical School of Thessaloniki University in 1997. He made his first specialty in Human Genetic (University of Zurich), and his second specialty in Hematology Oncology (MLU/UKH/Halle/Saale). He obtained two Master degrees, one in molecular biology in medicine from the Westminster University and one in Oncology from the University of Nottingham. He completed his promotion (MD, PhD) in MLU University in the area of TKIs in human cancer cells lines. Between 2001 and 2004 he established Arzt Genetik Zentrum in Thessaloniki where he was a director. Since 2015, Dr. Papasotiriou is a certified cytometris (Class A), qualified person for German authorities (Bundes Pharmazeutika), and Responsible Person (RP) for Swissmedic. Dr. Papasotiriou is certified consultant for ICH-GCP (EU, FDA), since 2018. Since May 2004 Dr. Papasotiriou is the medical director of R.G.C.C. INTERNATIONAL GmbH where the major field of expertise is molecular oncology with main interest in the entity of Cancer Stem Cell (CTCs) like.

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Pre-Clinical & Clinical Research

Adapting Your Preclinical Animal Model Strategies for a New World Investigators in the commercial and non-profit sectors have always faced pressure to accelerate their work, while balancing the need for quality and the realities of budget constraints. Pharmaceutical and biotech companies want to speed their time-to-market with novel, efficacious therapeutics. Non-profit institutions want to accelerate early-stage work that may lead to the identification of new drug targets or therapeutic approaches. At extraordinary times like we’ve experienced in the wake of COVID-19, these pressures have only heightened. In turn, it’s more vital than ever for R&D leaders and investigators to leverage preclinical animal models as effectively as possible. Given the trends shaping drug discovery in a post-COVID world, adapting the organisation’s preclinical model strategies is critical to achieving its research and business objectives.

The Research Landscape: Changing, Yet Familiar For most organisations, time, cost, and quality are key factors that influence strategic decisions. For commercial and non-profit organisations engaged in drug discovery and development, the situation is no different. A successful research pipeline demands balancing all three levers – a task that’s become more critical, yet more difficult in the current business environment. As COVID-19 emerged across the globe, the research community faced unprecedented operational and financial challenges. Lockdowns kept researchers from their labs; supply chain disruptions halted or severely delayed vital shipments; virus infections took staff members out of service; budgets were placed on hold due to economic impacts; grant funding slowed; and uncertainty made planning difficult. While vaccine rollouts have eased the situation somewhat, the environment in which researchers operate still doesn’t fully resemble pre-COVID times – and it maybe never will. It’s more likely the trends that were beginning to reshape the drug discovery landscape will become more entrenched in this new normal. Speed will continue to be a major driver. Time-to-market with a new therapeutic can put a startup biotech on the map, making it an attractive acquisition target, or give a mature pharma company a clear entry point into a new therapeutic area, making it more competitive. Speed is equally important in non-profit institutions, where grant review cycles are accelerating and grant funding is stipulating faster timelines. And for any investigator working on COVID-19 or other infectious diseases, greater urgency for answers will be the norm for the foreseeable future. Cost constraints remain a reality. That’s true whether the research funds are derived from commercial revenues or grant funding. Competing priorities will continue to tax budgets, 26 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

especially as organisations balance a surge in infectious disease work with equally compelling needs in therapeutic areas like oncology and metabolic disease. Attaining the right level of quality is a balancing act. Research organisations will always be challenged to achieve the highest level of quality, within the constraints of speed and cost – a fact that the pandemic experience made abundantly clear. As researchers working on SARS-CoV-2 vaccines and therapeutics are acutely aware, if you can achieve 95 per cent of your goal, five times faster than it would take to get to 100 per cent, that tradeoff may be worth making, especially in times of crisis. Counterbalancing these pressures is one bright note: The pandemic has deepened the commitment to biomedical research, both from governments and for-profit entities. When interest in SARS declined in the mid-2000s, so did the investment in research on coronaviruses, despite the fact that these viruses are on the rise. That proved a major lesson learned when SARS-CoV-2 emerged. In the aftermath, a focus on infectious disease research is increasing on the vaccine and therapeutic fronts, aimed at both new and repurposed drugs. At the same time, research efforts that were paused in fields like oncology, metabolic disease (including non-alcoholic steatohepatitis, or NASH), and neurodevelopmental/ neurodegenerative disease are returning to the forefront. Animal Model Strategies for a New World In this new normal, research organisations are rethinking and pivoting their strategies to reflect a business landscape that is likely changed forever. One area seeing a shift is the use of animal models in preclinical research. Several strategies are helping ensure researchers approach model generation, breeding, and colony management in ways that meet the evolving requirements of a new world. The In-house vs. Outsourced Decision Researchers have always grappled with deciding which tasks are best done by internal staff vs. outsourced, and speed is often a key driver of the decision. That’s likely to continue, especially for labs having trouble staying fully staffed or equipped or those struggling to meet pent-up demand post-COVID. Additionally, with investigators in non-profit settings expecting grant funding to increase, doors will open to more research opportunities than internal staff may be able to handle on a reasonable timeline. At the same time, risk mitigation will likely play a more prominent role in the decision, especially in the wake of supply chain and staffing disruptions experienced during the pandemic. Which tasks are best kept in-house vs. outsourced tends to vary by organisation, research project, and even project phase. Though not a hard-and-fast rule, investigators are often more comfortable outsourcing early-stage work and prefer to bring the work in-house as the project nears the clinical trial Summer 2021 Volume 4 Issue 2

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Comparison of timeline and animal usage for the generation of a monthly cohort of 30 homozygous animals. Natural mating is compared to IVF-mediated rapid expansion.

phase. Some research institutions may have the capability to work with embryonic stem cells (ESCs), conduct pronuclear injections, develop gene targeting vectors, or conduct the specialised testing required by biologics and advanced therapy medicinal products (specific to the EU). But recruiting, training, and maintaining the highly skilled staff needed for this work has become more challenging than it already was, given an environment marked by remote work and social distancing.

gene-editing step. Additionally, this technique is not limited by background strain or rodent type. In about 24 weeks, a small cohort of heterozygote mice with the desired modification can be generated and used to scale up a colony. But despite perceptions to the contrary, CRISPR isn’t appropriate for every genetic modification. Due to its technical limitations, it’s best used for single point mutations, small genetic insertions, simpler gene knock-ins, and constitutive gene knockouts.

When determining which tasks to take on internally vs. outsource, animal breeding is often one of the biggest question marks. The most important factors in this decision include access to staff with the necessary skillsets and experience, scope and capacity of animal housing facilities, quality control processes, genotyping and molecular analysis capabilities, the ability to maintain models at different health statuses, and total cost. Even if in-house breeding is deemed the best choice, having a backup provider for breeding is advisable to protect against a business interruption (as discussed later).

Homologous recombination in ESCs is more suitable for complex genetic modifications and large genomic insertions, such as when generating a genetically humanised mouse model. It is known as the “gold standard” in genome editing and has a long track record of successful use with complex genetic modifications. Fortunately, any risks with this technique are both limited and well-documented. However, this approach requires the longest timeline of the various techniques, averaging 42 weeks to arrive at a small cohort of heterozygous offspring.

Choosing a Model Generation Technique Investigators increasingly use genetically engineered models (GEMs) in their work for purposes like modelling a disease state, validating a gene target, or testing a drug candidate within a biological system. However, generating a novel GEM is not always as straightforward as one might think. There are multiple techniques used to modify the mouse or rat genome, and each has advantages and limitations from both a speed and cost perspective that investigators should be aware of.

Random integration transgenesis has broad applications in gene and protein level expression studies. It is faster than homologous recombination because it doesn’t require ESC manipulation and can quickly introduce a novel transgene for study. However, inserting the transgene into a random location creates some degree of risk of introducing unwanted mutations. When a researcher needs a model to study diseases caused by gene overexpression or copy number variations, random integration transgenesis may be a good choice for balancing speed and quality.

CRISPR/Cas9 is viewed as the fastest and most cost-effective of the available techniques. It eliminates the need to manipulate ESCs, greatly reducing the time and cost of the www.biopharmaceuticalmedia.com

Targeted transgenesis, as the name implies, involves integrating the transgene into one defined location in the INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 27

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genome, which reduces the risk of unwanted mutations. While this gene-editing approach can be suitable for more complex modifications than CRISPR, it requires ESCs, which lengthens the timeline. Developing the Optimal Breeding Plan Once a new model with the desired genetic modification is generated, or an existing model is selected, breeding can begin. The goal is to arrive at a cohort of animals with the desired age, sex, and genotype, in the quantity needed for the study, without wasting resources or incurring delays. Breeding is a biological process with limiting steps. There are many ways to approach it, each with an impact on the speed, cost, and quality of the outcome. That’s why each research project needs a customised, well-conceived, and well-coordinated breeding plan based on the study objectives and research priorities. When developing the optimal breeding plan, it’s essential to determine which lever is most important for the study. If speed is the top priority, the plan can exploit technologies such as rapid in vitro fertilisation (IVF) expansion combined with breeding strategies that accelerate this phase. If cost is a higher priority, the plan can include approaches that better leverage techniques and technologies to further reduce cage costs, for example. A well-designed breeding plan will enable the investigator to obtain a cohort that meets all the study criteria, on the right timeline, within budget, while reducing risks and minimising the odds of delays further downstream. 28 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Accelerating the Breeding Process Among the many ways to approach breeding, some are ideal for speeding the time to achieve the desired cohort size. Analysis of models generated using random integration transgenesis is a method for selecting transgenic founder (F0) animals most likely to transmit the desired genetic modification, avoiding wasted breeding cycles. It involves assessing the sperm of founder transgenic male mice to determine the presence of the transgene and the likelihood the germline will transmit to the next generation. Using endpoint tissue collection and expression analysis, breeding specialists can make this assessment and choose the best male breeders to move forward with – reducing wasted animals later in the process, avoiding unnecessary cage costs, and eliminating the risk of project delays. Rapid colony expansion is another method for driving time out of the breeding process by overcoming the limitations of natural breeding. This strategy was especially critical during early COVID-19 research, and it remains crucial for investigators who need to move fast in studying the impact of new variants or finding efficacious COVID-19 therapeutics. In vitro fertilisation (IVF) is one technique for rapidly scaling up a colony. Using only a few male donor mice, IVF makes it possible to produce hundreds of animals with the same week of birth, cutting the breeding timeline by three to six months and reducing total animal use. Together, the researcher and breeding provider can assess the reproduction potential of Summer 2021 Volume 4 Issue 2

Pre-Clinical & Clinical Research the desired mouse strain, along with known acceptance rates for IVF, to determine if this approach is viable for the specific project. If the investigator wishes to intercross two different models, that step can be integrated into the IVF process, further accelerating breeding. Successive IVF is another way to accelerate colony expansion. Female mice that are generated via IVF can become donors for a second round of IVF as early as three weeks of age, rather than waiting to do natural mating when they reach sexual maturity (about eight weeks of age). Rotational mating – rotating male mice with the desired genotype through different groups of female mice – also may be an option for speeding the time to achieve a study-sized cohort. Finally, integrating cryopreservation into the initial generation of a model can speed delivery of subsequent study cohorts by jump-starting the breeding process. Generating sufficient stocks of cryopreserved material early on enables the subsequent generation of 40-100+ animal colonies in ~10 weeks – far less than would be required when utilising traditional breeding. While care must be taken to replenish stocks of cryomaterial as they are used, early preparation and investment can provide for significant long-term benefits. Planning for Disaster Recovery Once a research organisation has invested time and budget to generate and characterise a preclinical model, protecting that investment is vital. However, it’s not uncommon for researchers to maintain a single breeding colony in a single location, with no backup. The potential consequences of this approach came to light after multiple natural disasters, like Tropical Storm Allison in 2001, Hurricane Katrina in 2005, and Superstorm Sandy in 2012. In each case, vivarium sites were destroyed or unreachable, resulting in a substantial loss of investment in genetically engineered rodent lines. With the COVID-19 pandemic greatly limiting access to research facilities, some labs found themselves in similarly difficult situations. Cryopreservation can minimise such risks and provide a better path to disaster recovery – essentially making the world the investigator’s lab. Once a genetically modified mouse line is cryopreserved, it’s easy to re-establish a breeding colony and the appropriate health standard if the live production colony is lost (due to a flood, fire, or other natural disaster) or because staff can’t access the facility (as in the early days of the pandemic) or in the event of disease outbreak in the colony or loss of environmental controls. Cryopreservation also enables researchers to move fast if there’s renewed interest in a model no longer in active use. When the pandemic emerged, investigators took cryopreserved material from the humanised ACE2 transgenic mouse models used in the early 2000s for SARS research and leveraged them to advance their COVID-19 research. While cryopreserving a model adds cost to a breeding project, it’s far lower than the cost of maintaining a live colony in a maintenance mode. It also greatly reduces space requirements and labour – two costly resources that are often in short supply in research facilities. Outsourced breeding is another form of protection against disaster. If an in-house vivarium can’t be staffed or accessed www.biopharmaceuticalmedia.com

due to a natural disaster or other crisis, having parallel lines of suppliers enables the investigator to maintain the colony without interruption. Commercial breeders have the resources, contingency plans, and cross-training to maintain live production colonies even in difficult situations. If needed, the provider can quickly cryopreserve tissue or sperm as an additional form of backup. As the pressure to accelerate drug discovery remains a constant, balancing that demand with the equally important drivers of quality and cost will prove a challenge. Preclinical model strategies can help research organisations arrive at the right mix. Adapting the organisation’s model generation, breeding, and colony management approaches for a post-COVID world will help R&D leaders and investigators achieve their objectives.

Austin Jelcick Austin Jelcick, PhD, is director of product management, scientific services, at Taconic Biosciences. Austin previously studied the impact of genetic variations in the mouse on the development of retinal degeneration and has worked with animal models for over 11 years.

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Pre-Clinical & Clinical Research

Standard vs. Hybrid FIH Trials: Advantages and Challenges Introduction First in human (FIH) studies are the key translational studies from preclinical to further clinical development processes. The goal of FIH studies is to investigate the pharmacokinetics (PK) and pharmacodynamics (PD), determine appropriate dosing, and document safety and tolerability. These studies serve as the basis for further efficacy studies in the targeted patient populations. Except for genotoxic/cytotoxic oncology drugs, FIH clinical studies have traditionally been conducted in healthy volunteers (HVs), which seems ideal for this type of early clinical research. However, in light of the emergence of non-cytotoxic agents (such as molecular targeting agents and immunomodulatory drugs), a number of FIH studies have recently been conducted in HVs to provide oncology indications. This approach has been further strengthened thanks to an improved understanding of pathology, genetic alterations, animal models, and mechanisms of action. However, early signals of development are critical to the survival of many drug development programs. With HVs, this clinical/therapeutic activity is not always possible or feasible. FIH clinical trials have evolved from traditional dose- and toxicity-finding studies in HVs to innovative complex study designs, sometimes requiring patients’ involvement. Hybrid trials offer a potential solution, allowing clinical study teams to enjoy the benefits of using both HV and patients while simultaneously mitigating the potential downsides. This article examines the key considerations to take into account when deciding whether to run a standard FIH in HVs or a hybrid clinical trial.

FIH Trials in HVs: Pros and Cons Relying on HVs only in FIH trials brings with it a number of potential upsides, as well as potential risks. First, what are the benefits of conducting FIH trials only in HVs? Clinical trial sites can draw from a large pool of HVs in the wider population when recruiting study subjects. Having a simple, straightforward recruitment process greatly reduces the overall study timelines. The second relevant advantage is that HVs are generally in good health with no major comorbidities and can be controlled for intrinsic factors (like age, ethnicity, renal and hepatic functions) and extrinsic factors (such as concomitant medications and smoking status). 30 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Running the entire trial from one site keeps logistical and operational challenges to a minimum and will avoid site-to-site variations. Additionally, studies conducted in HVs can include more extensive confinement than those conducted in patients. This makes it far easier to conduct intensive assessments such as pharmacokinetic (PK) sampling. During a washout period, the study participants are taken off the administered drug. When testing on HVs, clinical study teams can test the effects of a washout period without any potential ethical or safety concerns. It’s also worth highlighting that the process of creating a placebo group from a set of HVs is straightforward and doesn’t carry any potential ethical constraints. There’s a strong likelihood that most HVs will be able to complete the entire study. This can be attributed to lower levels of concern regarding patient mortality and health/safety. Unfortunately, there are also a number of cons to consider. HVs won’t gain any particular health benefit from the administered drug as they don’t need it. However, there’s still the risk that they might suffer from harmful side-effects. Therefore, potential levels of risk must be minimised compared to levels of risk considered generally acceptable among patients. PD data might be limited in scope, or even of no use as the target pathways are often expressed on or in the pathology cells. There may also be challenges associated with predicting safety and efficacy in patients based on HV data, since there may be differences in PK and PD, making the extrapolations difficult. HVs are generally given a lower initial starting dose than trials that treat actual patients. Moreover, the dose escalation strategy and stopping rules are stricter. Additional preclinical studies may have to be provided in order to support the exposure of HVs to the drug in question in FIH studies. This might include assessing their cardiovascular, central nervous, and respiratory systems, as well as conducting in vitro genotoxicity studies. When sponsors only include patients, these studies are typically not required until the submission of a marketing application. Given the above-mentioned disadvantages and risk/benefit ratio of performing FIH studies in HV, regulatory agencies are quite cautious and usually need a strong scientifically- and clinically-based justification, regardless of the population chosen. FIH Trials in Patients: Pros and Cons Similarly, there is a range of potential benefits and risks to using patients in FIH studies. If a drug does indeed bring about the desired therapeutic results, patients may immediately benefit. This provides Summer 2021 Volume 4 Issue 2

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a competitive advantage given the current regulatory environment in the United States as well in Europe by the rationalisation and simplification of early development processes in some therapeutic indications. This is especially apparent in the wake of recent FDA and EMA regulatory developments.

However, there are also a number of potential drawbacks to consider. FIH studies conducted on patients may have to take place across multicentre sites. Unsurprisingly, this thereby increases the logistical load on clinical study teams and greatly affects the trial’s timelines.

Clinical trials have become greatly more complex in recent years. Nowadays, they generally include more endpoints than ever before and require translational assessments. For instance, receptor occupancy assessments and pharmacodynamic biomarkers are necessary, which are not always measurable in HVs due to the absence of target indicators.

Clinical study teams may have to contend with multiple adaptations/amendments to existing protocols. It’s been reported that the total number of endpoints in any one protocol has largely increased during last years. Indeed, it’s likely that this has further increased in the years since.

There's no need for translation of PK data as the targeted population already possesses the relevant PK. The difference in PK properties between patients with some pathologies and HVs may be of crucial interest for some new investigational compounds, such as biologicals. In some instances, a starting dose that is substantially lower than the human expected pharmacological dose may not be appropriate for patients in an FIH study. The starting dose should take into account the nature of disease and its severity in the patient population included in the FIH trial. When starting at relatively high doses, the overall number of patients being treated at subtherapeutic levels will be minimised. www.biopharmaceuticalmedia.com

It’s incredibly difficult to recruit patients in an FIH study if there are efficient comparators on the market or at the advanced stage of clinical development. For the same reason, creating the placebo arm is a considerably complex process. Unsurprisingly, some patients might feel incredibly anxious when being treated with a drug that is given to humans for the first time. This could potentially impact the drug’s efficacy. Moreover, these anxious patients are also generally more likely to under-report adverse events (AEs) as they fear being removed entirely from the trial or receiving lower doses moving forward. In oncological drug development, for example, some patients might not survive until the trial’s conclusion. Furthermore, INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 31

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intensive PK sampling may also adversely affect a patient’s long-term health, safety, and wellbeing.

FIH Hybrid Trials By definition, hybrid trials are Phase I or FIH trials combining HVs and patients with the target indication in the same protocol.

development timelines. In hybrid FIH trials there is no confirmatory efficacy analysis given that they’re still in the exploratory phase, but a signal on PD effect may provide some confidence in the following development stages. So far, the number of subjects is still limited for HVs as well as for patients, and there is a relatively limited duration of dosing (often limited to 28 days based on generally available 28-days animal toxicity data). However, with hybrid trials, overall drug development timelines should be shorter, minimising the time it takes to initiate Phase II and beyond.

Hybrid trials possess two major advantages: the early generation of PD data and the potential shortening of

That being said, hybrid trials should not be considered a one-size-fits-all approach.

Patients may well have existing comorbidities and several necessary concomitant treatments that affect the drug’s performance, as well as influencing the PK.

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Pre-Clinical & Clinical Research There are multiple different considerations to take into account prior to commencing a hybrid trial. Does the clinical pharmacology unit (CPU) or clinical research organisation (CRO) have the capacity to support a complex hybrid trial? There are a number of CPU-related challenges when conducting hybrid trials within a Phase I unit. However, if recruitment allows, performing the HVs and patient parts in the same unit would be of an extremely added value. No trial method will survive a poor study design; therefore, when conducting hybrid FIH trials with patient involvement, it’s crucial that the design is appropriate. It should be investigated whether addition of patients into the study is justified and efficient to observe response to treatment within a rather short study timeline. Another challenging aspect is regarding patient accessibility and recruitment, since early-phase studies in general are not of high therapeutic benefit for patients. Moreover, only a subpopulation of patients may be eligible for the early-phase hybrid trials with less comorbidities, so-called “healthy patients”. Regulatory requirements must also be taken on board when designing a hybrid FIH study and if necessary, scientific advice can be sought from the agency. Similarly, consultation with patients and patient associations should be taken into account.

REFERENCES 1.

It should also be considered that with inclusion of both types of population in the same study, multiple objectives are included in a single protocol, resulting in increased trial complexity. So, there is a need for a flexible protocol and robust regulatory basis.

Different companies have different goals. Small biotech companies working on one individual project will likely have a different clinical trial strategy than large-scale pharmaceutical companies. Unlike their smaller counterparts, large institutions will prioritise quickly moving drugs through the clinical trials process in order to outrun the competition.

So how should clinical study teams determine whether or not to run a hybrid trial? They basically need to ask one overarching question: does a hybrid trial add genuine value? This question should be considered as early as possible. It must be answered using a science-driven approach over hunches or mere assumptions. For instance, you might be testing rare or orphan diseases. These trials are particularly well-suited to hybrid models, especially for smaller companies. Strong PD/efficacy data from hybrid trials sometimes means that trials can jump straight into Phase IIb, instead of having to also complete Phase IIa – making the trial notably quicker and more cost-effective. If you’re unsure about whether or not to conduct a hybrid trial, consider seeking regulatory advice. Most importantly, determine whether or not the hybrid trial will enhance, detract from, or have little overall impact on the patient’s safety. Patient safety must always be the primary concern – regardless of the drug being tested or the trial method being used. www.biopharmaceuticalmedia.com

J Shen, B Swift, R Mamelok, S Pine, J Sinclair and M Attar; Design and Conduct Considerations for First-in-Human Trials. Clinical and Translational Science; Jan 2019; vol 12, 6-19 R Paluri, P Li, A Anderson, L Nandagopal, T McArdle, M Young, F Robert, G Naik and M Saleh; First-In-Human Phase 1 Clinical Trials – A SingleCenter Experience In The Era Of Modern Oncotherapeutics. Scientific Reports, Nature Research, (2020) 10:7935 R Dresser; First-in-Human Trial Participants: Not a Vulnerable Population, but Vulnerable Nonetheless; J Law Med Ethics. 2009 ; 37(1): 38–50. M Ahmed, C Patel, N Drezner, W Helms, W Tan and D Stypinski; Pivotal Considerations for Optimal Deployment of Healthy Volunteers in Oncology Drug Development. Citation: Clin Transl Sci (2020) 13, 31–40; R Parchment and J Doroshow; Pharmacodynamic Endpoints as Clinical Trial Objectives to Answer Important Questions in Oncology Drug Development; Semin Oncol. 2016 August; 43(4): 514–525 J Karakunnel, N Bui, L Palaniappan, K Schmidt, K Mahaffey, B Morrison, W Figg and S Kummar; Reviewing the role of healthy volunteer studies in drug development; J Transl Med (2018) 16:336 Guideline on strategies to identify and mitigate risks for first-in-human and early clinical trials with investigational medicinal products; 20 July 2017 EMEA/CHMP/SWP/28367/07 Rev. 1 Committee for Medicinal Products for Human Use (CHMP)

Nariné Baririan Nariné Baririan, Director Consultancy and Clinical Pharmacology at SGS. By education Nariné is pharmacist with Research Master and PhD in Pharmaceutical Sciences. She has 15 years’ experience in Clinical Research. In her role, Nariné is leading the consultancy projects at SGS to support pharma companies in development of study design, protocol, CDP, STA, IB. She assists the dose escalation meetings and reviews preclinical or clinical study reports. Nariné is author of several articles, webinars and courses in early clinical research area.

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Supply Chain Management

Intricacies and Future Considerations for the Cell and Gene Therapy Cold Chain The rapidly growing field of cell and gene therapy is both exciting and confusing to many, including those who work in the medical community and on the periphery. Advancements of therapies happen at a feverish pace. The number of clinical trials increases exponentially in a short time. And bringing therapies to commercialisation remains a hurdle. As biotech companies navigate bringing cell and gene therapies from clinical trials to commercialisation, companies within the supply chain also work to understand and support this evolving field of medicine. In the case of cold chain, this means navigating variable temperature requirements and offering solutions that accommodate people unfamiliar with temperature-controlled packaging, as well as innovating ways to track and monitor irreplaceable raw material and completed therapies as they travel from and to the patient. As therapies reach commercialisation and advance beyond one-toone treatments, adaptability and standardisation become increasingly important. The Market Cell and gene therapy are overlapping fields of biomedical research and treatment. Both serve to treat, prevent and cure genetic and acquired diseases. But they work differently. According to the American Association of Blood Banks, cell therapy replaces or repairs damaged tissues or cells with transplanted human cells.1 The cells originate either from the patient (autologous) or a donor (allogeneic). The applications are expansive, as there are hundreds of cells that make up the human body. Most cell-based therapies are currently experimental, with few exceptions. For example, hematopoietic blood stem cell transplantation was first explored in humans in the 1950s and is currently a well-established treatment for blood diseases. Gene therapies, by contrast, replace genes to help the body fight or treat disease, turn off genes that cause problems or replace genes that cause problems with ones that work correctly. Once a new gene is created, a vector is used to deliver new genes directly into cells – either inside or outside the body.2 Gene therapy was first introduced in the late 1970s, and the first gene therapy trial in humans began in 1990.3 Arguably, the introduction of successful chimeric antigen receptor T-cell therapy (CAR-T) in the early 2000s was a pivotal tipping point in the evolution of the cell and gene therapy market. Market growth is exponential. In 2012, there were 12 CAR-T clinical trials. By 2019, the number surged to 514. And between 2017 and 2020, three CAR-T products reached commercialisation, with the number estimated to reach double-digits by 2024. In 2019, CAR-T therapy earned 34 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

more than $700 million, and with more products expected to reach commercialisation, this rapid-growth industry is poised to produce over $10.8 billion by 2027. Since Kymriah and Yescarta received historic approvals of CAR-T therapies in 2017, their products have been infused into an estimated 450,000 patients worldwide. With growing numbers of approvals anticipated, the patient population for CAR-T therapy alone will reach approximately 2 million within the next 10 years.4 Currently, most of the therapies use products derived from blood samples. But with growing momentum to develop treatments for solid tumours and other tissues, more products with different requirements are likely to emerge. The Supply Chain The supply chain required for cell and gene therapy is simple, but variables not seen in a traditional pharmaceutical supply chain make it especially challenging. Unlike most pharmaceutical products, the raw materials used to develop potentially life-saving cell and gene therapies are most often a patient’s own cells. A trained medical professional collects the cells, ships them to another facility to create the therapy and the therapy is shipped back to the patient’s medical facility for infusion. However, medical facilities vary in infrastructure, available equipment and technologies. This poses a challenge when temperature-sensitive cells require temperature control and specialised packaging to transport them safely to a biotech company. Facilities may not have equipment to refrigerate or freeze cells, space to store bulky temperature-controlled packaging or appropriate freezers to condition packaging components. Staff familiarity with and training on temperaturecontrolled packaging could also introduce risk in the form of temperature excursions. Since cell and gene therapies are generally a treatment of last resort, patients are often very sick. Many would not be able to donate a second blood sample if the first sample or therapy experienced damaging temperature excursions. Fool-proof temperature control is necessary, especially when shipping a patient’s cells to a biotech company. Companies specialising in temperature-controlled packaging now make single-use and reusable refrigerated shippers that are easier to use at these sites. These new refrigerated shipping systems require no conditioning, and they weigh significantly less than shippers that use traditional coolants like ice. Just one touch of a button activates the cooling process for refrigerated samples, bringing a room-temperature product slowly to 2–8 degrees Celsius. These systems use evaporative, reactive cooling technology that responds and adjusts to varying ambient temperatures. This ensures the payload is protected from external temperature fluctuations throughout the duration of the shipping process. Summer 2021 Volume 4 Issue 2

Supply Chain Management Therapies shipped back to a patient’s medical facility are most often shipped frozen using vapourised liquid nitrogen (LN2). This requires special training since LN2 can cause frostbite or skin burn if touched, and its gases can damage delicate eye tissue. This shipping method also proves costly because of the specialised training needed to use LN2 shippers. When moving from one-to-one therapies to commercialised, mass-produced therapies, LN2 costs become too much to sustain. Temperature and Location Monitoring Temperature excursions that cause a patient’s cells or therapy to deviate outside of the allowed temperature range could damage the container’s payload, resulting in a devastating impact on the patient’s health. Maintaining supply chain integrity is critical to mitigating risks and ensuring the safe and secure transportation of health-giving and life-saving cells and therapies. Stakeholders in pharmaceutical shipments demand increasing levels of transparency regarding the status of shipments, including location tracking and temperature history. Cold chain and courier companies are increasingly using advanced asset management software systems to monitor temperatures and ensure packages are shipped to the right place, at the right time and arrive in the right condition. A key development includes real-time monitoring of payloads via smart loggers and devices that are interconnected to the internet of things (IoT), making it possible to access and assess the condition of the payload in transit. Being alerted about a temperature excursion by the shipper before it reaches its destination allows preventive or corrective supply chain actions earlier than would have otherwise been possible, especially if hand-couriered to a final destination. These new, advanced software systems and the integration of information technologies (IT) within the pharma supply chain play an increasingly vital role in protecting pharmaceutical shipments from excursions during transport to their destinations. Issues can arise if the shipper is opened during a customs inspection or tampered with during transit. The IoT device can track or warn when a shipper is opened, for how long, and if there is a risk to the payload’s temperature requirements. GPS is currently the only technology that provides real-time tracking. While there is growing interest in using GPS-enabled/ IoT sensors for temperature tracking, they are not widely used. This is because GPS technology tends to be heavy, expensive and takes up a fair amount of payload space. Additionally, when loaded on an airplane, the tracking signals must be turned off so they do not interfere with airplane navigation. This technology is most often used with individualised CAR-T therapies, where the loss of a product could mean life or death for the patient. When exact location and condition is not needed, another class of smart devices can be used.These are just in time (JIT) devices, and they often use other methods of communication when passing through a physical IoT gate or provide a download of data via Bluetooth, QR code, barcode, USB connection or other method. This data includes logged temperature information or the number of times the payload was opened, which can be assessed at the end of the journey. www.biopharmaceuticalmedia.com

Depending on the location, condition and temperature data needed, real-time or JIT devices can be an ideal solution when weighing urgency of data need versus cost and reduction in payload size. Data retrieved and shared can help biotech companies make more informed choices on the most appropriate cold chain solutions to deploy depending on specific shipping lanes. The latest global tracking technologies to ensure the protection of pharmaceutical payloads offer a wide range of capabilities. These include options to set up automatic maintenance, next shipment alerts and produce customisable reports. Related to advances in tracking technologies is increased interest in developing better, more effective solutions to enable use and sharing of data between packaging providers, logistics providers and biotech companies. Future Considerations Biotech companies face several complex challenges, including protecting the integrity of temperature-sensitive, high-value payloads during transportation. This must be done while mitigating costs, managing and tracking assets within a complex supply chain, meeting stringent global regulatory standards, navigating complex global shipping lanes and circumventing unforeseen challenges. Current cell and gene therapies are primarily autologous – using a patient’s own cells – and result in a high treatment price per patient. High treatment costs are not sustainable. As the industry evolves, it is anticipated that more companies will work on allogeneic-based therapies that are more cost-effective and more aligned with treatment reimbursement. These therapies could also offer an opportunity to move beyond one-to-one therapies into mass-produced therapies. Reimbursement constraints for patient therapies will play a role when biotech companies look at mass-produced therapies. When mass-produced therapies become a reality, it would be costly and impractical to continue using expensive and more complex LN2 systems for these therapies. Biotech companies will need to standardise temperature requirements for their therapies and move toward temperature ranges that offer economy and scalability – like dry ice. Cold chain companies and biotech companies need to work together to standardise shipping solutions. With biotech companies developing ever more complex and temperature-sensitive therapies, there’s growing demand to integrate IT solutions within the supply chain balanced alongside the established requirements of providing improved packaging performance and efficiency within cold chain logistics. Innovation, IT integration and new technologies to enhance supply chain monitoring and remote tracking will become increasingly important with bulk shipments. These technologies are crucial in addition to the continuous evolution of smart temperature-controlled packaging protecting cell and gene therapy payloads globally. The industry trend to deploy reusable systems coupled with asset management SaaS (software as a service) may catch on for cell and gene therapies. These systems can automatically INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 35

Supply Chain Management

collect and analyse data from company smart data logger outputs. These monitoring devices are increasingly used in cold chain as they become more affordable and accessible to biotech and pharmaceutical companies.These stakeholders are excited about the prospect of offsetting the costs of these new technologies with savings, based on reduction of lost products, and using data from smart devices to add efficiencies and fine-tune distribution models. Alternatively, IoT 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). It’s predicted that advancements in GPS tracking options via an online SaaS system will soon become standard within the industry. Benefits to biotech companies include knowing where their shipment is throughout its complex logistics journey. If payloads are intercepted, lost or get delayed en route, the biotech company can take steps to intervene and recharge or replace coolants, so the package or the bulk system gets delivered before expected temperature duration is exhausted.This presents a strong case for using IoT devices and supporting software and technology to mitigate a temperature excursion caused by a delay. 36 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

The cell and gene therapy industry continues to grow and expand as new, life-saving and health-giving treatments, technologies and distribution strategies evolve. Biotech companies, cold chain companies and other stakeholders will need to work together to standardise product requirements, maximise efficiencies and optimise benefits for patients who depend on their products and services.

Vince Paolizzi Vince Paolizzi, Director of NanoCool Sales, Pelican BioThermal has over 15 years of experience in various sales leadership roles within the Pharmaceutical packaging industry. He is responsible for the global sales growth of the NanoCool line of products. Paolizzi’s experience includes key account management to maintain strong relationships with customers as well managing multi-disciplined project teams to secure new accounts. Prior to joining Pelican BioThermal, Paolizzi held various sales positions within the packaging, tooling and food industries. He received his degree in Business Management from West Chester University of Pennsylvania. Email: vince.paolizzi@pelican.com

Summer 2021 Volume 4 Issue 2

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Supply Chain Management

Four Factors for a Biopharmaceutical Manufacturer to Consider when Developing and Managing a Single-use Supply Chain Single-use technologies are becoming widely utilised in the biopharmaceutical industry for the benefit of offering a more flexible, cost-effective approach to cGMP manufacturing. Single-use technologies are utilised in more than 45% of clinical mammalian cell culture processes and about 6% of commercial processes. The manufacturing of single-use systems requires a wide range of components, including thermoset or thermoplastic elastomer tubing, filters, connectors and process sensors to build complete assemblies. Any of these components could be impacted by supply chain shortages, leading to potential risk that can result in delays in the production of life-altering therapies. As more biopharma companies implement single-use systems (SUS) and single-use technology (SUT), extra attention should be paid early in the design phase to implement a robust, dependable global supply chain for any materials, components and assemblies used in manufacturing workflows. Selecting the right single-use integrator or provider can ensure a successful, sustained implementation of single-use in critical cGMP manufacturing and other operations. This report will discuss four best practices that biopharma companies should consider in developing and managing their single-use supply chain. 1. Adopting a Dual- or Multiple-sourcing Strategy for Components Biopharma manufacturers that are adopting single-use products and systems have historically followed a dual- or multiplesourcing model. This means that the developer will provide the same design or an assembly or component and request bids from two or more single-use equipment providers. Choosing multiple single-use equipment providers adds first-step redundancy that can improve supply chain security, allowing the greater chance for guaranteed delivery of critical products or components no matter the circumstances. Although, even with the use of multiple single-use equipment providers, a supply chain still may not be fully redundant, and leave the biopharma manufacturer at risk of a supply chain disruption. For example, if the exact same single-use design is being provided by two or more single-use providers, they are both relying on the same raw material supplier(s) for critical subcomponents, such as a particular filter, tubing, or connector. If that critical raw material supplier becomes productionconstrained, both single-use providers would be at risk – along with biopharmaceutical manufacturers’ supply chain. A growing number of components, such as tubing, bags, filters and gaskets, may also inherently have limited sources of material supply, because of an increased demand as more manufacturers adopt single-use technology as their 38 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

A dual-sourcing model, where a customer provides drawings for an assembly or component and requests bids from multiple vendors to ensure redundancy for supply chain security, has limitations. For that reason, it should not be the only method used to ensure single-use supply chain security.

manufacturing platform. Additionally, a specific brand of component may be specified for use each time. This introduces more risk if the brand-name material provider runs into a shortage or cannot produce the material. A smarter, more robust supply chain management practice would be to establish a part specification that defines material and performance properties, rather than selection of a single source of raw material or component, to allow more flexible alternate sourcing to limit risk. 2. Evaluating the Opportunities for Standardisation The ease of customisation and flexibility in the design of single-use assemblies can also be a cause for a supply risk. Global manufacturers with multiple manufacturing sites have the greatest opportunity to develop standard assemblies for unit operations. By developing standard assemblies for unit operations, biopharma manufactures will benefit by reducing the number of potential designs and can consolidate assemblies across an organisation. Establishing a comprehensive supply chain strategy as early as possible is a proactive way that a biopharma manufacturer can mitigate its risk. A biopharma manufacturer should engage with its single-use vendor(s) to better understand their unit operations and design assemblies that identify critical materials and components and where specific components may be at risk, then take that into account in the design process. Risk can also be reduced by focusing on good industrial design principles and system requirements for the application at hand. Manufacturers can reduce the number of issues they face by thinking ahead and anticipating scale-up challenges Summer 2021 Volume 4 Issue 2

Supply Chain Management as they design their systems. Manufacturers should strive to design the system for full GMP production at the pilot scale to identify and eliminate a potential hurdle later. In doing this, possible manufacturing bottlenecks or sourcing and supply issues can be identified and worked around before components are specified, drawings are created and a design is locked-in to the manufacturing process. 3. Examining a Single-use Vendor’s Quality Standards Manufacturing disruptions are not confined to just unavailability of parts or components: If single-use systems are produced but cannot be safely or consistently implemented in cGMP manufacturing environments, then the impact on the drug manufacturer can be just as severe as having delays due to missing parts, or possibly even more so. Several criteria for regulatory and quality compliance are worth considering when choosing a vendor for single-use products and technology.

Biopharma manufacturers should conduct an ongoing evaluation of a single-use supplier’s capabilities, including their supply chain management and quality systems. Understanding and aligning these capabilities with the manufacturer’s own practices is important, as biopharma manufacturers design facilities with single-use technology.

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Lot release testing. Lot release testing has been driven by downstream applications after filtration, so it is also important for the supplier to test finished products to verify that the product meets certain requirements after sterilisation. Most requests for this testing include USP <85> for bacterial endotoxins and USP <788> for particulate contamination.

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Quality documentation system. Documentation is a common theme in several of these steps for a reason: It’s not enough to simply state that tests are conducted, there must be tangible, referenceable proof. A biopharma manufacturer should not only ensure its vendors document their quality processes and systems, but should see evidence that the documentation is being used on a regular basis. On-site audits at set intervals are another important tool for validation.

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Quality risk management (QRM) programme. It is critical for single-use suppliers to use a systematic, documented process for the assessment, control, review and communication of risks to the quality of manufactured products. The best single-use vendors will invest in this kind of programme just as biopharma manufacturers do.

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Cleanroom management. Typically, single-use systems will be manufactured in an ISO Class 7 (Class 10,000) cleanroom. The production facility should be able to supply documentation of annual cleanroom recertification and a validated process showing a rotational cleaning regimen. Cleanrooms should also be monitored for real-time temperature, humidity and differential pressure with alert criteria and defined actions in place to address issues. It should also be able to demonstrate its processes and scheduling of air/surface viable and non-viable particulate testing. Product sterility validation. A well-defined, recognised reference standard should be identified by regulations, such as ANSI/AAMI/ISO 11137 (commonly referred to as VDmax25). One factor to be cautious about is singlepoint validation, which is insufficient. The manufacturer should have documentation that revalidation is routinely performed according to the standard and the single-use supplier. In addition, sterile barrier packaging validation (different from sterility validation) can also be used to establish shelf-life for an irradiated product.

As a best practice, a good QRM programme should include risk-based audits of raw material suppliers in order to understand the quality management capabilities and processes of these companies, followed by a risk-based classification of each supplier. In this, routine audits of high-risk suppliers would be performed. A tool such as a risk register can be used to track, identify and review where the greatest risks may occur. Quality agreements and performance reports should be established to track suppliers in a measurable and actionable way. On-time delivery, turnaround time for documentation (like drawings or quality documents), and manufacturing defects should be among the metrics identified and tracked on a regular basis to drive ongoing improvement. Collaborating first with a single-use vendor and focusing on good design principles and the requirements of the system can help identify any problem areas, bottlenecks or potential supply or sourcing issues in the single-use supply chain. www.biopharmaceuticalmedia.com

Finally, an internal business continuity plan is an effective tool for reducing supply chain risk that should also be included INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 39

Supply Chain Management A four-step CPFR programme would include the following: 1. Understanding customer requirements, such as important product/order attributes of dating, documentation, order frequency, lot control and storage material handling requirements 2. Effectively transferring documented requirements to internal systems to operationalise items, such as customercare instructions, warehouse instructions and setup of customer-specific inventory reserves 3. Regularly taking part in customer-planning meetings to obtain updated forecasts 4. Engaging with customers and suppliers to manage changes in key factors, such as required components and lead times.

A single-use supplier’s supply chain expertise and ability to comply with regulations and quality standards can ensure that the drug manufacturer successfully migrates a single-use system from pilot scale to full, cGMP production.

as part of a QRM programme. This could include a business impact analysis (BIA) report of potential scenarios associated with business continuity and disaster recovery, as well as relevant plans of action. This way, the vendor has a method for responding to a serious event that could have the potential to interrupt operations. As biopharma manufacturers redesign facilities to make greater use of single-use systems, there will be benefits from understanding and aligning the manufacturer’s own quality practices with a supplier’s capabilities. A single-use supplier’s knowledge of industry regulations and quality standards can make a difference in how effectively a drug manufacturer can adopt single-use in their operations. Single-use suppliers that can demonstrate that they not only just comply with regulations and quality standards, but have deep understanding and documentation of the same, can be critical partners to biopharma manufacturers to help navigate potential roadblocks in production. 4. Collaborative Planning, Forecasting and Replenishment (CPFR) Additionally, lead times for finished goods can be another challenge to address. Although stocking finished goods is one strategy that can help address this challenge, it comes with related costs for proper storage of large volumes of products. Biopharma manufacturers with operations around the world may choose to find alternate solutions for local storage and quick delivery. Working with single-use suppliers who have a global network of distribution centres, backed by strong logistics support and access to an open-architecture product portfolio with components sourced from multiple suppliers, can further help mitigate this risk. SUS supply chain management can be enhanced when biopharma manufacturers are able to work with suppliers in collaborative planning, forecasting and replenishment (CPFR) programmes. This collaborative effort can effectively support manufacturing planning and ensure that orders are delivered on time. 40 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Additional procurement and supply management programmes offered by a single-use vendor might include onsite services and technology, custom kitting solutions and ancillary supplies for production to help customers streamline procurement, optimise inventory levels and spend less time managing supplies. Avoiding Supply Chain Disruption is the Goal Quality single-use systems and technologies can help biopharma manufacturers reduce contamination risk, improve resource efficiency and ultimately lead to breakthrough solutions for many of the world’s most challenging diseases and chronic conditions. Having dual suppliers for all critical components and materials isn’t enough to fully mitigate risk in a single-use supply chain. Instead, true redundancy must be built into a single-use component supply chain, along with careful attention to system design, choosing a reputable single-use vendor and fully understanding their supply chain strategy and quality systems, and collaborating with that vendor early on and throughout the single-use system design and implementation process. These four best practices can position a manufacturer to avoid a worst-case scenario: being dependent upon a supplier whose own sources of components or materials could be threatened.

Timothy Korwan Timothy Korwan is a director of new product introduction for single-use at Avantor. Tim has more than 20 years of experience as an engineer and in business development with PAW BioScience Products, VWR and Biopure Corporation where he has designed single-use products, components and systems for the global drug and vaccine manufacturing industry.

Jay Harp Jay Harp is director, single-use product management at Avantor. Jay has more than 15 years of experience engineering single-use solutions and systems utilised in upstream, downstream and filling applications used by global biopharmaceutical companies. Along with his current role within the Avantor organisation, Jay is also an active member of the ASME-BPE and BPSA industry groups.

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Manufacturing/Technology Platforms

PEER REVIEWED

H96: Combining Freeze-drying and High-pressure Homogenisation for Ultra-fine Nanocrystal Production In recent years, nanomedicine has proven to be key in overcoming many of the challenges associated with poorly water-soluble drugs. Decreasing the size of drug particles can increasebioavailability and solubility can be observed, as a result of the increased active pharmaceutical ingredient (API) surface area.

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Smaller achievable particle size. Improved in vivo.

Due to the increased bioavailability, a lower amount of API is required, which in turn leads to a more cost-efficient product, with fewer risks and side-effects for the patient1. High-pressure homogenisation has long been the favoured method of particle size reduction, which involves the forced passage of sample through a very narrow gap/nozzle. According to the Bernoulli equation, this leads to an increase in the dynamic pressure, and a reduction of the static pressure on the liquid. The static pressure falls below the vapour pressure of the dispersion medium water, leading to cavitation2.

The H96 process involves initial freeze-drying of an organic solution of the poorly water-soluble drug. The drying process consists of freezing the product and decreasing both pressure and temperature below the solvent triple point to start the sublimation of the solid solvent directly into vapour. The lyophilisate is then dispersed in an aqueous surfactant solution, and homogenised. Lyophilisation makes the drug material more brittle and fragile, which allows nanocrystals <100nm to be obtained by homogenisation. Homogenisation alone is often unable to achieve these particle sizes2.

Figure 1: Graph – Pharmaceutical drug emulsion A before processing

The decrease in particle size is a result of the pressure applied and the number of cycles/passes, until the lowest size reaches a minimum. At which cycle number this lowest size is reached depends on the physical properties of the drug (e.g. hardness, number of imperfections in the crystal, amorphous fraction)2. High-pressure homogenisation is known as a first-generation approach. More recently, second-generation approaches have been applied. These involve a combination of technologies (SmartCrystal processes). One of which is known as the H96 process: The combination of freeze-drying and high-pressure homogenisation (freeze-drying being the first step). The aim of this process is to modify the starting material in such a way that it can be better broken down by high-pressure homogenisation1. • • •

Benefits of combinative technology include: Higher physical stability. Faster production of the nanocrystal, by reducing the number of require homogenisation cycles/passes.

42 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Figure 2: Graph – Pharmaceutical emulsion A after processing at 18000psi

Another benefit of the H96 approach is a reduction in the required number of homogenisation cycles/passes to achieve the desired particle size and/or size distribution. At the pilot and production scale, this could bring about a significant cost-saving, as a result of reduced processing and labour time, reduced wear rates and reduced machine downtime, all of which improve the overall commercial viability of the process/project. Summer 2021 Volume 4 Issue 2

Manufacturing/Technology Platforms Premilling is often applied prior to homogenisation in the field of nanocrystals, to break down excessively large crystals. This prevents the homogenising valve gap from being blocked2. The premilling step can be done via the homogenisation system itself, simply by utilising a lower pressure. The below link describes a premilling step on an Avestin Inc. homogeniser (available for purchase at Biopharma Group):

Pharmaceutical, biotechnology, chemical and food industries have been supported with a wide range of freezedrying solutions for product characterisation, formulation & cycle development, cycle audit, troubleshooting, tech transfer, scale-up and production. For over 30 years, Biopharma Group’s experience has been established with more than 3000 projects developed for APIs, biopharmaceuticals, therapeutic delivery systems, PCR reagents, whole organisms, vaccines, blood components, food and nutraceuticals ensuring success in many industrial processes, globally.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845550/ The above link demonstrates how aprepitant drug particles were processed via homogenisation alone (with initial premilling), compared with the H96 (freeze-drying then homogenisation) process: • •

Via our in-house laboratory, and demonstrative freezedrying/high-pressure homogenisation equipment, we encourage anyone interested in trialling the H96 process, to get in contact Biopharma Group on today!

Homogenisation alone: Pre-mill (five cycles at 5000 psi) then 10 cycles at 15,000 psi H96: Freeze-dry, then homogenisation (five cycles at 15,000psi).

The results show how a smaller particle size was achieved via the H96 process, despite only involving <half of the amount of homogenisation cycles. Earlier studies have also seen similar benefit3, and in 2006, a patent was filed for an amphotericin nanosuspension with a 62 nm particle size, prepared by the H96 process4. Specifically designed and executed freeze-drying and homogenisation protocols will enhance the quality of the final products. In general, depending upon the nature of the API and its formulation, a gentle freeze-drying process optimises the retention of the drug activity and structure before the homogenisation step5. Avestin Inc. are the global market leader in the high-pressure homogenisation field, offering robust, scalable equipment, manufactured to deliver superior results, from the benchtop to production scale.

REFERENCES 1.

Dahiya, S. (2017). Drug Nanonization: An Overview of Industrially Feasible Top-down Technologies for Nanocrystal Production.Bulletin of Pharmaceutical Research. (2), 144. 2. Al Shaal, L., Muller, R.H., Shegokar, R. (2010). smartCrystal combination technology–scale up from lab to pilot scale and long term stability.Pharmazie. 65 (12), 877-884. 3. Salazar, J., Heinzerling, O., Muller, R.H., Moschwitzer, J.P. (2011). Process optimization of a novel production method for nanosuspensions using design of experiments (DoE).International Journal of Pharmaceutics. 420 (2), 395-403. 4.` Möschwitzer, J., Lemke, A. Method for carefully producing ultrafine particle suspensions and ultrafine particles and use thereof. WO/2006/108637. United States Patent US; 2006. 5. Ward, K.R., Matejtschuk, P. (2018). Lyophilization of Pharmaceuticals and Biologicals.Springer.

Sebastian Prisacariu Figure 3: Particle size reduction of pharmaceutical emulsions. Diameter before processing 435nm. One pass through the filter extruder with 80nm pore at 0psi following which initiated.

Biopharma Group supplies high-pressure homogenisers in the UK & Ireland, as well as the market-leading SP Scientific Freeze Dryers – all fully supported by our Technical Service Department. In addition, Biopharma Group provides worldwide assistance in the research and development in freezedrying. www.biopharmaceuticalmedia.com

Sebastian Prisacariu (Biopharma Group) -Capital Equipment Sales Executive – UK

Richard Lewis Richard Lewis (Biopharma Group) -Head of Equipment Sales UK & Ireland

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 43

Manufacturing/Technology Platforms

Data-rich Processing – The Growing Necessity of R&D to GMP Manufacture Improved diagnosis is driving precision in treatment, accompanied by increasing regulation stringencies from the likes of the MHRA and FDA. This, in turn, ensures processes, as well as the products themselves, are fully understood during every step of the manufacturing process.

The requirement for both the R&D and manufacturing processes to be ‘data-rich’ has become a prerequisite for drug product manufacturers, and purchasers of drug products, alike; a phenomenon driven not only when things go well, but where an irregularity might have occurred, even when confirmed to be within the validated ‘design space’ parameters.

From Theory to Practice US-based SP Scientific, has put data necessity at the forefront of its breakthrough suite of new lyo / freeze-drying technologies named Line of SightTM, supported in the UK, Ireland, and France, by Biopharma Group. Line of SightTM encompasses process analytical technologies (PAT), designed to assist pharmaceutical developers / manufacturers in achieving drug commercialisation objectives, whether for scale-up or scale-down. All aspects of the range are designed for use at R&D scale, but capable of use at full GMP production too. This allows scientists to know from the outset of an R&D programme that the technology can be scaled up successfully – it is imperative technology can work at the commercial scale, as well as R&D. Some of the most significant freeze-drying data interpretation tools for key data post-processing are: •

Where the products are usually very expensive to manufacture, manufacturers are always keen to use the data to prove, even if an irregularity took place, the batch can still be released rather than simply quarantined and / or rejected. From a regulatory perspective, the above method of data interpretation and auditing for corroborating a product is safe for release is also hugely beneficial.

44 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

AutoMTM/SMART™ Freeze-dryer Technology This is a primary drying cycle optimisation tool for in-depth product information to determine: • • •

The ice interface temperature Product resistance (Rp) Cycle optimisation

Summer 2021 Volume 4 Issue 2

Manufacturing/Technology Platforms appearance, reduced protein aggregation, skin formation, and vial cracking, watch this short video – https://youtu.be/JM00HTm8XaU • •

LyoFlux® TDLAS Sensors Available for use at any scale for the accurate measurement of vapour mass flow in the calculation of critical product attributes such as: • Primary and secondary endpoints • Average product temperature & resistance • Heat transfer coefficients, such as Kv • Assists with engineering freeze-dryer performance qualification

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Tempris® Wireless Sensors All Line of SightTM freeze-dryers are compatible for use with Tempris probes. Wireless probes allow the same probe type to be used for R&D and production, whereas wired probes used for R&D are not compatible at manufacturing scale.

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ControLyo® Nucleation Technology • Precise control of the freezing point in all vials simultaneously, including vials with thermocouples • Increasing nucleation temperature, lowering supercooling, saving time and money • Larger ice crystals – giving a reduced cycle time and faster reconstitution times • To discover more about the improvement in cake

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LyoCapsule™ This freeze-dryer enables accelerated freeze-drying cycle development with as few as seven vials, and has all of the functionality listed above, which is particularly beneficial when small-scale development is required due to: • High costs of the product being trialled • Low availability of the product itself – allowing R&D to occur with very small sample volumes, creating data that can be used efficiently at larger scale

As can be seen from the importance of PAT at the heart of Line of SightTM, whilst the science of freeze-drying remains king in the production of viable product, it is apparent that one of the most necessary / critical elements of processing is the availability of auditable data, which – no matter how good your product looks and performs post-process – generates both regulatory and consumer confidence in the product and subsequently the manufacturer.

Richard Lewis Richard Lewis from Biopharma Group for BPS Crowthorne

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 45

Market Report

Land Use Change and the Global Pandemic

While medical research has found the path to lead us slowly but steadily out of the current pandemic, the strategies to prevent such pandemics from happening in the first place remain under-researched, let alone applied. There is widespread speculation that some environmental conditions make zoonotic spillover (the transmission of a pathogen from one species to the other) more likely. When humans encroach into wildlife habitats, be it due to urban expansion, land demand or dietary shifts, the ecological interface between humans and wild species increases, together with the probability of contact between them, especially when livestock animals act as transmitters. The goal of our research was to transform this assumption into something measurable.

Most human diseases originate from animals, meaning there are several ways in which environmental change, through the altered interactions between animals, humans and the environment, can affect the development and transmission of infectious diseases. Environmental change may moreover contribute to the emergence of infectious diseases in wildlife, and to the flow of pathogens across species. The loss of biodiversity resulting from environmental degradation can also increase the prevalence of infectious diseases in their hosts. The encroachment of humans and their domestic animals into wildlife habitats can decrease the distance between people and pathogen hosts, exposing humans to new infections. Whilst the origin of SARS-CoV-2 is unclear, Asian horseshoe bats are known to host the greatest diversity of SARS-related coronaviruses. For this reason, we made a

46 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

systematic collection of published data on bat distributions and locations, within a domain of almost 30 million square kilometres. On this area, we mapped high resolution data (from 30m to 1000m) representing different land use patterns, including forest cover, croplands, human settlements, and data on the population density of livestock. From forest cover, we calculated forest fragmentation, the process by which human encroachment alters a once continuous forest into multiple disconnected and jagged patches. Our previous study on Ebola demonstrated that forest fragmentation was a particularly good indicator of the impact of environmental change on spillover risk because it better represents the increased interface between anthropised land and wildlife habitat. Therefore, we studied this indicator, along with human settlements and livestock density, using tools through which we could both identify the hot spots, looking for clusters of locations with a mix of high values of the indicators, and classify the locations into groups with similar land use patterns. Through this approach, we identified actual and potential coronavirus spillover hotspots from horseshoe bats. Actual hotspots are regions at risk because the high levels of forest fragmentation, human settlements and livestock density place wildlife and humans into closer contact, facilitating the transmission of pathogens. Whilst potential hotspots are areas not classified as hotspots, they could transition to a hotspot status if at least one of the indicators (forest fragmentation, human settlements, livestock density) increases. While in some hotspots it’s the concurrence of different factors making the difference, the impact of some factors may be greater than others in the creation of other hotspots.

Summer 2021 Volume 4 Issue 2

Market Report

The same happens for areas at risk of becoming a hotspot: some areas are already fragmented and may turn into a hotspot if human and livestock presence increases (or vice versa), and some other areas may become at risk as the result of the expansion of a nearby hotspot. This information could be useful for policy-makers, through the prioritisation of interventions towards the mitigation of one or the other risk factor, and the development of surveillance plans. Indeed, it is crucial for hotspot areas to increase biosecurity and disease surveillance, in order to monitor and decrease contact rates between humans, wildlife and livestock. Land planning should aim at reconstructing the ecological network in order to reduce the extension of exposed habitat edges. At the same time, areas close to the tipping point can identify their local risk factors and establish prevention strategies to keep them within sustainable limits. Whilst environmental degradation can increase the risk of zoonotic spillover, potential strategies to mitigate this risk do overlap, in some cases, with those typically aimed at containing the biodiversity crisis and reducing greenhouse gas emissions. Habitat restoration, sustainable agriculture and livestock production, together with the adoption of less meat-based diets, are some examples of policies with multisectoral potential positive outcomes. In the framework of the UN2030 Agenda, they would address Sustainable Development Goals #2 (“End hunger, achieve food security and improved nutrition, and promote sustainable agriculture”), #3 (“Ensure healthy lives and promote well-being for all at all ages”, including the fight against communicable diseases) and #15 (“Protect, restore and promote sustainable use of terrestrial ecosystems, sustainably manage forests, combat desertification, and halt and reverse land degradation and halt biodiversity loss”). This intersection of problems and solutions, cutting across the abiotic and biotic environment, and human society, supports the implementation of the One Health approach to mitigate risks associated to anthropised environments. www.biopharmaceuticalmedia.com

REFERENCES 1.

2. 3.

Rulli, M.C., D’Odorico, P., Galli, N. & Hayman, D. Land-use change and the livestock revolution increase the risk of zoonotic coronavirus transmission from rhinolophid bats. Nature Food 2, 409–416 (2021). https://doi.org/10.1038/s43016-021-00285-x Santini, M. The land use–food–coronavirus nexus. Nature Food 2, 390–391 (2021). https://doi.org/10.1038/s43016-021-00290-0 Rulli, M.C., Santini, M., Hayman, D. & D’Odorico, P. The nexus between forest fragmentation in Africa and Ebola virus disease outbreaks. Scientific Reports 7, 41613 (2017). https://doi. org/10.1038/srep41613

Maria Cristina Rulli Dr. Maria Cristina Rulli is full professor in hydrology and water and food security at the Politecnico di Milano, Department of Civil and Environmental Engineering since 2019. Previously she was associate professor at the Politecnico di Milano and researcher associate at the University of California at Berkeley. Email: cristina.rulli@polimi.it

Paolo D’Odorico

Paolo D’Odorico of Department of Environmental Science, Policy, and Management, University of California, Berkeley, Berkeley, CA, USA.

Nikolas Galli

Nikolas Galli of Department of Civil and Environmental Engineering, Politecnico di Milano, Milan, Italy.

David T. S. Hayman

David T. S. Hayman, of Molecular Epidemiology and Public Health Laboratory, School of Veterinary Science, Massey University, Palmerston North, New Zealand.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 47

Ad Index

Page 23

Avantor

Page 11

Biopharma Group

Page 45

BioPharmaSpec Ltd

Page 37

Cerba

Page 41

Richter-Helm Biologics GMBH & CO. KG

IBC Plasmid Factory

Page 3 Oxgene

IFC R.G.C.C. Group

SPL – Scientific Protein Laboratories

Page 5

SGS SA

I hope this journal guides you progressively, through the maze of activities and changes taking place in the biopharmaceutical industry

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Summer 2021 Volume 4 Issue 2

AAV

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Summer 2021 Volume 4 Issue 2 Document SPL1001 22 June 2021