IBI Volume 4 Issue 3

Volume 4 Issue 3

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E&L Standards Empowering Confident Labware Choices Residual Impurities in Biopharmaceutical Products CRISPR-Cas9 Knockout Screening Using Primary Human Cells in Drug Discovery How to Bring Your Oncolytic Virus Innovation to Market The Secret to Successful Development

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II INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Autumn 2021 Volume 4 Issue 3

Contents 04 Foreword WATCH PAGES 06 Shoring up Diagnostics Manufacturing for The Next Pandemic DIRECTOR: Mark A. Barker BUSINESS DEVELOPMENT: Ty Eastman ty@senglobalcoms.com EDITORIAL: Beatriz Romao beatriz@senglobalcoms.com DESIGN DIRECTOR: Jana Sukenikova www.fanahshapeless.com

As the global response to COVID-19 continues to move from a state of national lockdowns to the re-opening of societies, diagnostic assay developers are confronting two major challenges. Firstly, how to handle manufacturing volumes when level of demand is so uncertain. Secondly, where to invest in non-COVID diagnostics innovation and for which disease areas. Emmanuel Abate at Cytiva talks more about these two challenges.

REGULATORY & COMPLIANCE 08 4 Drugs Facing Key Patent Expirations and Potential Generic Entry From September 2021 – November 2021

RESEARCH & CIRCULATION: Jessica Dean- Hill jessica@senglobalcoms.com

A challenge in anticipating generic entry is elucidating which patents and regulatory protections constrain generic entry. Yali Friedman at DrugPatentWatch presents a set of estimated loss of exclusivity dates for four drugs, from September through November 2021. These estimated drug patent expiration dates and generic entry opportunity dates are calculated from analysis of known patents and US regulatory protections covering drugs.

COVER IMAGE: iStockphoto ©

10 Divided by a Common Language: PV Differences Between the UK and the US – Notes for Ambitious Biotechs

PUBLISHED BY: Senglobal ltd.

46 Plover Way, London SE16 7TT, United Kingdom

In the modern market, small and nimble biotechs possess many great advantages in terms of their ability to bring new innovation to market quickly, unhindered by legacy ways of operating. Yet the smallest start-ups share the same obligations as large pharma companies when it comes to safety and pharmacovigilance, which means they are likely to face a steep learning curve. Eric Caugant and Judi Sills at Arriello outline key differences in requirements on either side of the Atlantic and provide some practical tips for biotechs looking to optimise their PV budgets.

FINANCE DEPARTMENT: Akash Sharma accounts@senglobal.co.uk

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The next issue of IBI will be published in Winter 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 Senglobal ltd.

Volume 4 Issue 3 – Autumn 2021

12 Regulatory Challenges Associated with The Development of Cell and Gene Therapies The development of safe and effective cell and gene therapies (CGTs) relies on regulatory compliance and critical selection and qualification of raw materials. Regulatory classifications for advanced therapy medicinal products (ATMPs) vary globally and it is imperative for both manufacturers and suppliers to understand the requirements and obligations from each party, to achieve international compliance. Marlin Frechette at FUJIFILM Irvine Scientific discusses the regulatory environment surrounding bio manufacturing of CGTs and the significance of compliance with appropriate regulations to ensure safety, quality, and efficacy of the final product.

RESEARCH / INNOVATION / DEVELOPMENT 16 How to Bring Your Oncolytic Virus Innovation to Market: The Secret to Successful Development Cancer continues to be a major burden on healthcare systems across the world, with global spending on therapies and supportive care drugs expected to reach $200 billion by 2022.

The number of deaths worldwide has increased every year for decades, from 5.7 million in 1990 to 8.8 million in 2017. Kai

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 1

Contents Lipinski at Vibalogics explains how to bring oncolytic virus to market.

18 CRISPR-Cas9 Knockout Screening using Primary Human Cells in Drug Discovery While significant progress has been made carrying out CRISPR screens in immortalised cell lines, a more physiological and clinically relevant alternative are human primary cells, such as T cells, regulatory B cells, or natural killer (NK) cells. The use of phenotypically relevant primary cells, though, is not without biological and technical challenges, especially when their intended use includes gene editing and scale-up of these specialised cell types for screening. Verena Brucklacher-Walder at Horizon Discovery analyses the significant strides towards overcoming challenges associated with editing and screening primary human cells.

24 Are We Nearly There Yet? The Ongoing Journey of CAR-T Cell Therapies CAR-T cells have a genetically engineered T-cell receptor (TCR) that directs their binding to cancer cells. In first generation CARs, the TCR was engineered to express a new, antigen binding domain, usually the single chain variable fragment (scFv) of an antibody. Sophie Lutter at Oxgene discusses more about the ongoing journey of CART-T cell therapies.

PRE-CLINICAL & CLINICAL RESEARCH 28 Viral Vector Engineering to Improved Clinical Performance and Accelerate Timeline to Success for Novel Gene

reviews the most common types of residual impurities and identifies appropriate monitoring methods for a successful adaptive control strategy.

THERAPEUTICS 40 Innovations in Cell Line Development to Optimise Biotherapeutic Development Developing stable and high-producing cell lines to manufacture biologics is complex, multi-stage, and time-consuming, often resulting in development timelines that exceed six months when using classical techniques. This represents a frustrating bottleneck in biotherapeutic development, a market where speed-to-clinic is a priority. Louis Boon and Olivia Hughes at Sphere Fluidics present some of the latest strategies to optimise product concentrations and productivities, and high-throughput automated methods to accelerate screening, while reducing manufacturing costs.

TECHNOLOGY PLATFORMS 44 Accelerating Single-Cell Genomic Sequencing with Big Memory When fighting the global spread of a pandemic or working to fight cancer, faster time-to-discovery saves lives. For this critical bioscience work, scientists have employed singlecell RNA sequencing to assemble entire genomes and reveal genetic variants. Modern gene sequencing technology has grown exponentially in the last decade from studying hundreds of cells to millions of cells. Yong Tian at MemVerge explains how Big Memory technology helps accelerating single-cell genomic sequencing.

Viruses are known to be dangerous pathogens which invade host cells and hi-jack their cellular machinery to direct the replication and transcription of their own genome. They are also known as being ideal couriers to transport new DNA into a host cell and ensure that it’s transcribed. Sophie Lutter at Oxgene looks into three aspects of viral biology that make them efficient delivery vehicles for gene therapies, and some of the ways that biologists have taken advantage of these properties to improve clinical performance.

32 E&L Standards: Empowering Confident Labware Choices All labware has some extractable and leachable content. It is important to recognize that extractables and leachables are two different things. Extractables are organic compounds or metals removed from a material under extreme conditions, such as exposure to high heat or very high/low pH, whereas leachables are those that are removed under normal conditions of use. Sarina Bellows at Thermo Fisher Scientific examines the importance of E&L standards and their application for risk assessment during product selection and offers insight into relevant regulatory guidance and available E&Ltesting methods.

MANUFACTURING 36 Residual Impurities in Biopharmaceutical Products Process-related impurities or residual impurities are formed at any time during upstream or downstream processes. They are compounds that are present at very low concentrations in complex biomanufactured products and can vary from large proteins to small chemicals. Luc-Alain at SGS Health Sciences 2 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Autumn 2021 Volume 4 Issue 3

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Foreword Monitoring of residual impurities seen in bioprocessing can be quite challenging. Because of the range of potential impurities, many different analytical approaches may need to be used. After being developed, these methods must be qualified or validated for the intended use.

Although some impurities are related to the drug product, others are added during synthesis, processing, and manufacturing. Because residuals typically are present at low levels in difficult sample matrices, development and validation of assays and ongoing testing can be quite challenging.

Biomanufacturing is a complex process involving many steps from upstream fermentation, cell lysis, and solubilization to downstream refolding, purification, polishing, and formulation.

The sample matrix types can vary greatly because sampling at a variety of process steps is required to accurately monitor the target throughout the production process Process-related impurities or residual impurities are formed at any time during upstream or downstream processes. They are compounds that are present at very low concentrations in complex biomanufactured products and can vary from large proteins to small chemicals. Luc-Alain at SGS Health Sciences reviews the most common types of residual impurities and identifies appropriate monitoring methods for a successful adaptive control strategy.

Scientists across every discipline strive to design and conduct experiments that will deliver clear and meaningful results. Doing so demands meticulous attention to detail in assessing all aspects of the experimental setup, understandingthe variables involved, and anticipating and minimising potential unknowns. One critically essential, but easy to overlook factor is the influence ofthe labware used.

leachables are those that are removed under normal conditions of use. Sarina Bellows at Thermo Fisher Scientific examines the importance of E&L standards and their application for risk assessment during product selection and offers insight into relevant regulatory guidance and available E&L testing methods.

In this journal, we will also explore more about regulatory challenges associated with the development of cell and gene therapies. The development of safe and effective cell and gene therapies (CGTs) relies on regulatory compliance and critical selection and qualification of raw materials. Regulatory classifications for advanced therapy medicinal products (ATMPs) vary globally and it is imperative for both manufacturers and suppliers to understand the requirements and obligations from each party, to achieve international compliance. Marlin Frechette at FUJIFILM Irvine Scientific discusses the regulatory environment surrounding bio manufacturing of CGTs and the significance of compliance with appropriate regulations to ensure safety, quality, and efficacy of the final product.

CAR-T cells have a genetically engineered T-cell receptor (TCR) that directs their binding to cancer cells. In first generation CARs, the TCR was engineered to express a new, antigen binding domain, usually the single chain variable fragment (scFv) of an antibody. Sophie Lutter at Oxgene discusses more about the ongoing journey of CART-T cell therapies.

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

All labware has some extractableand leachable content. It is important to recognise that extractables and leachables are two different things. Extractables are organic compounds or metals removed from a material under extreme conditions, such as exposure to high heat or very high/low pH, where as

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

4 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Autumn 2021 Volume 4 Issue 3

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 5

Watch Pages

Shoring up Diagnostics Manufacturing for the Next Pandemic As the global response to COVID-19 continues to move from a state of national lockdowns to the re-opening of societies, diagnostic assay developers are confronting two major challenges. Firstly, how to handle manufacturing volumes when level of demand is so uncertain. Secondly, where to invest in non-COVID diagnostics innovation and for which disease areas. Both essentially come back to the same question: “What does a world now somewhat accustomed to being in a pandemic need from diagnostic testing next?”

While we at Cytiva cannot answer this broad question with certainty, I do believe it is vital that assay developers reduce their risk in relation to these two central challenges.

Advancing secure supply As a supplier to the industry, we have seen uncertainty from our customers as to how best to manage their own supply chains.

A year ago, the rapid spike in demand for COVID-19 assays led to intensive raw material purchasing, which in turn led to raw material constraints in a variety of areas. In the same way as grocery store shelves were left bare of toilet paper, so too were suppliers of assay components. Demand for COVID-19 assays has settled for the moment but uncertainty as to how to manage the supply chain remains. Should material stock levels be increased to de-risk future demand spikes? Should raw materials on the shelf be drawn down due to a testing plateau or even a decline?

The answer, as it usually is in our industry, is to hedge the risk.

Our customers, as they should be, are pushing their pressures further back in the supply chain. We are seeing a high demand for critical components like nitrocellulose and magnetic beads.

Even more importantly is the need for transparency and communication. We regularly review demand forecasts with our customers and discuss how and when we can best meet their needs within the context of the overall demand we are seeing. Finally, we are helping our customers structure contracts that give them flexibility to take product when they need it and scale down when they don’t.

Accelerating Development

By March 2020 assay developers had diverted their focus towards developing fast, scalable, reliable COVID-19 testing.

Now, developers are at a crossroads. They can continue developing better, faster assays or go back to the cutting room floor and pick up one of their pre-COVID projects. I believe this is a false binary. Successful developers will find ways to continue both COVID-19 and non-COVID programs and it is imperative that their suppliers step up as partners to support this way of working.

By providing expertise, access to materials and, most importantly, the increased pace of work that comes with iterating ideas quickly together, we are seeing that our customers can 6 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

work on more projects and achieve developmental milestones faster. Moreover, these projects can move to market faster because manufacturing and supply strategy can progress while the prototyping is still ongoing. That’s the power of co-development.

Conclusions This is a tough environment for an assay developer. The frenzied pace of the past 18 months is starting to ebb and is being replaced with an uncertain short-term future. I firmly believe that COVID-19 has created a broad understanding of the power of diagnostic assays in society and as such we are going to see more innovation and assay use in our everyday lives. However, resource allocation, stock levels, and new product development decisions all have to be balanced between caution and ambition. It is critical that component providers and developers work together in these areas to ensure mutual success.

Emmanuel Abate Emmanuel Abate, Vice President Genomics & Cellular Research and Head of Corporate Social Responsibility, works to bring out the best in his team so they can better serve the scientific community working on the next medical breakthrough. Genomics and Cellular Research comprises lab filtration products, genomics and diagnostics solutions, biomolecular imaging, western blotting consumables and cellular research tools. Emmanuel also leads the companywide Corporate Social Responsibility strategy.

Emmanuel’s career began in 1999 with GE Healthcare, where he worked in business development and eBusiness.

He earned his MBA in 2004 and joined the Boston Consulting Group from 2004–2007, based in Paris. When he returned to GE Healthcare in 2007, he served in product and commercial leadership positions for Interventional Imaging, Women’s Health Imaging, and Radiology.

In 2018, Emmanuel became the General Manager of Genomics and Cellular Research, when the business was part of GE Healthcare Life Sciences, now Cytiva. Emmanuel, known for his humble leadership, is inspired by the hundreds of scientists, specialists, and technicians working in on the instruments and consumables used to speed up the development of future diagnostics and therapies.

Autumn 2021 Volume 4 Issue 3

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 7

Regulatory & Compliance

4 Drugs Facing Key Patent Expirations and Potential Generic Entry From September 2021–November 2021 A challenge in anticipating generic entry is elucidating which patents and regulatory protections constrain generic entry.

Presented here is a set of estimated loss of exclusivity dates for four drugs, from September through November 2021. These estimated drug patent expiration dates and generic entry opportunity dates are calculated from analysis of known patents and US regulatory protections covering drugs.1 This methodology can be extended to ex-US jurisdictions by leveraging these estimates and tracking patent family members in other patent offices.

ADASUVE (loxapine) Estimated US Loss of Exclusivity Date: 26 October 20212* Generic Entry Controlled by: US Patent 8173107 Title: Delivery of antipsychotics through an inhalation route Abstract: "The present invention relates to the delivery of antipsychotics through an inhalation route. Specifically, it relates to aerosols containing antipsychotics that are used in inhalation therapy. In a method aspect of the present invention, an antipsychotic is delivered to a patient through an inhalation route. The method comprises: a) heating a composition, wherein the composition comprises an antipsychotic, to form a vapor; and, b) allowing the vapor to cool, thereby forming a condensation aerosol comprising particles with less than 5% antipsychotic drug degradation products. In a kit aspect of the present invention, a kit for delivering an antipsychotic through an inhalation route is provided which comprises: a) a thin coating of an antipsychotic composition and b) a device for dispensing said thin coating as a condensation aerosol."3 ADASUVE is marketed by Alexza Pharms and is included in one NDA. There are eighteen US patents protecting this drug, and two hundred and twenty-seven patent family members in twenty-one other countries/regional patent offices.

By analysing the patents and regulatory protections it appears that the earliest date for generic entry in the US will be October 26th, 2021, when US Patent 8,173,107 expires.

Patent 8,173,107 describesDelivery of antipsychotics through an inhalation route, and is assigned to Alexza Pharmaceuticals, Inc. (Mountain View, CA).2,3 EOVIST (gadoxetate disodium) Estimated US Loss of Exclusivity Date: 13 November 20214 * Generic Entry Controlled by: US Patent 6039931 Title: Derivatized DTPA complexes, pharmaceutical agents containing these compounds, their use, and processes for their production Abstract: "Compounds of general Formula I wherein Z.sup.1 and Z.sup.2 in each case independently mean the residue wherein m and n means the numbers 0–20, k, l, q and r means the numbers 8 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

0 and 1, and R means a hydrogen atom, an optionally OR.sup.1 -substituted C.sub.1 -C.sub.6 -alkyl residue, or a CH.sub.2 COOR.sup.1 group with R.sup.1 meaning it hydrogen atom, a C.sub.1 -C.sub.6 -alkyl residue, or a benzyl group, X means a hydrogen atom and/or a metal ion equivalent of an element of atomic number 21-29, 42, 44 or 57-83, with the provisos that at least two the substituents X stand for a metal ion equivalent; that one of the substituents Z.sup.1 and Z.sup.2 stands for a hydrogen and the other is not H; that – if n and l each mean the number 0 – k and r do not simultaneously mean the number 1; that – (O).sub.r – R is not – OH; and that Z.sup.1 and Z.sup.2 are not – CH.sub.2 – C.sub.6 H.sub.4 – O – CH.sub.2 – COOCH. sub.2 C.sub.6 H.sub.5 or – CH.sub.2 – C.sub.6 H.sub.4 – O – (CH. sub.2).sub.5 – COOCH.sub.2 C.sub.6 H.sub.5, as well as their salts with inorganic and/or organic bases, amino acids or amino acid amides, are valuable pharmaceutical agents, e.g., for NMR."5 EOVIST is marketed by Bayer Healthcare and is included in one NDA. There is one US patent protecting this drug, and thirtythree patent family members in twenty-four other countries/ regional patent offices.

By analysing the patents and regulatory protections it appears that the earliest date for generic entry in the US will be November 13th, 2021, when US Patent 6,039,931 expires.

Patent 6,039,931 describes Derivatized DTPA complexes, pharmaceutical agents containing these compounds, their use, and processes for their production, and is assigned to Schering Aktiengesellschaft (Berlin, DE).4,5 AMTURNIDE (aliskiren hemifumarate; amlodipine besylate; hydrochlorothiazide) Estimated US Loss of Exclusivity Date: 15 November 20216 * Generic Entry Controlled by: US Patent 8618174 Title: Synergistic combinations comprising a renin inhibitor for cardiovascular diseases Abstract: "The invention relates to a combination comprising the renin inhibitor of formula (I) ##STR00001## or a pharmaceutically acceptable salt thereof."7 AMTURNIDE is marketed by Novartis and is included in one NDA. There are two US patents protecting this drug, and one hundred and twenty-five patent family members in thirty-one other countries/regional patent offices.

By analysing the patents and regulatory protections it appears that the earliest date for generic entry in the US will be November 15th, 2021, when US Patent 8,618,174 expires. Patent 8,618,174 describesSynergistic combinations comprising a renin inhibitor for cardiovascular diseases, and is assigned to Novartis AG (Basel, CH).6,7 NAVSTEL (calcium chloride; dextrose; magnesium chloride; oxiglutatione; potassium chloride; sodium bicarbonate; sodium chloride; sodium phosphate) Autumn 2021 Volume 4 Issue 3

Regulatory & Compliance

Estimated US Loss of Exclusivity Date: 29 November 20218 * Generic Entry Controlled by: US Patent 7084130 Title: Intraocular irrigating solution having improved flow characteristics Abstract: "Improved intraocular irrigating solutions are described. The solutions have enhanced viscosities that reduce the risk of damage to intraocular surgical procedures by reducing the turbulence of the solutions and dampening the movement of tissue fragments and air bubbles. The solutions preferably also have modified surface tensions that more closely resemble the surface tension of the aqueous humour."9

for information purposes only. There is no warranty that the data contained herein is error free.

NAVSTEL is marketed by Alcon Pharms Ltd and is included in one NDA. There is one US patent protecting this drug, and twenty patent family members in sixteen other countries/ regional patent offices.

REFERENCES

By analysing the patents and regulatory protections it appears that the earliest date for generic entry in the US will be November 29th, 2021, when US Patent 7,084,130 expires.

Patent 7,084,130 describesIntraocular irrigating solution having improved flow characteristics, and is assigned to Alcon, Inc. (Hunenberg, CH).8,9 Disclaimer *Generic entry predictions are estimates. Drugs may be covered by multiple patents and regulatory protections. Although great care is taken in the proper and correct provision of this information, the author does not accept any responsibility for possible consequences of errors or omissions in the provided data. The data presented herein is for information purposes only. There is no warranty that the data contained herein is error free.

Acknowledgments Although great care is taken in the proper and correct provision of this information, the author does not accept any responsibility for possible consequences of errors or omissions in the provided data. The data presented herein is www.international-biopharma.com

Financial & competing interests disclosure Yali Friedman, Ph.D. is the CEO of DrugPatentWatch. The author has no other relevant affiliations or financial involvement with any organisation or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilised in the production of this manuscript.

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Friedman, Yali, "Generic Drug Launch Dates – A 1-step Solution to Navigate the Web of Drug Patents and Regulatory Protections" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/ blog/generic-drug-launch-dates-a-1-step-solution-to-navigate-theweb-of-drug-patents-and-regulatory-protections.

Friedman, Yali, "ADASUVE Drug Profile" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/p/tradename/ADASUVE. Alexza Pharmaceuticals, Inc. US8173107 Friedman, Yali, "EOVIST Drug Profile" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/p/tradename/EOVIST. Schering Aktiengesellschaft US6039931 Friedman, Yali, "AMTURNIDE Drug Profile" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/p/tradename/AMTURNIDE. Novartis AG US8618174 Friedman, Yali, "NAVSTEL Drug Profile" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/p/tradename/NAVSTEL. Alcon, Inc. US7084130

Yali Friedman Yali Friedman, Ph.D.,founder of DrugPatentWatch, a provider of global business intelligence on biologic and small-molecule drugs, dedicated to helping clients make better decisions.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 9

Regulatory & Compliance

Divided by a Common Language: PV Differences Between the UK and the US – Notes for Ambitious Biotechs Small biotechs come under the same scrutiny as large pharma when it comes to pharmacovigilance.

In this article non-executive advisors to Arriello, Eric Caugant in Paris and Judi Sills in New Jersey, outline key differences in requirements on either side of the Atlantic and provide some practical tips for biotechs looking to optimise their PV budgets.

In the modern market, small and nimble biotechs possess many great advantages in terms of their ability to bring new innovation to market quickly, unhindered by legacy ways of operating. Yet the smallest startups share the same obligations as large pharma companies when it comes to safety and pharmacovigilance, which means they are likely to face a steep learning curve.

It doesn’t help that requirements vary from region to region, from authority to authority, around the world. Relying on an individual CRO partner to manage all PV requirements internationally may be appealing, but is a risky strategy – unless there is experienced CRO oversight to ensure that the regulatory requirements are met in all countries where a compound is studied and/or marketed. A far better approach is for biotech companies to grasp for themselves the complexity of the task they face.

Before going to market Most pre-market PV requirements are the same across the European and US markets, in line with agreements and guidance on standardisation via the ICH – the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. But there are some small, noteworthy variations.

Companies tend to favour filing in the US first, because on top of the market’s vast size the US benefits from being one country, governed by one main agency – the United States Food and Drug Administration (FDA). In Europe, marketing authorisation can take much longer because beyond the central European Medicines Agency (EMA) each EU member state has its own unique requirements to navigate.

Even at a central level, EMA submissions have a different look and format to US dossiers so require different handling.

For instance the Summary of Product Characteristics (SmPC) and labelling in relation to side effects are not presented in the same way in Europe. Differences exist too between the risk management approaches – the FDA’s Risk Evaluation and Mitigation Strategies (REMS) and EMA’s Risk Management Plan (RMP) – one cannot be substituted for the other. Failure to factor in these differences could present an issue at the time of filing. In addition, national EU-specific requirements may be requested in certain countries, on 10 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

top of the RMP EU requirements, even for centralised procedures.

Get any of this wrong, and companies risk their dossiers being rejected, or authorisation being delayed as requests for amendments or additional information go back and forth.

Keep it together All of this means that biotechs need a clear strategy and timeline for how they will file to their target markets. Leaving Europe to one side until sales in the US are up and running is inadvisable, given the additional time that is likely to be required to prepare for EMA’s differing requirements – and those of each EU country beyond that. And of course the UK must now be treated as its own market, following Brexit which means it is no longer under the jurisdiction of EMA. It isn’t just European information and formatting requirements that differ and are more involved than in the US. Standard operating procedures (SOPs)/process requirements can be more complex in Europe too.

Real-world monitoring The post-marketing regulatory environment is highly regulated and inspection driven, and it is here that biotechs are likely to find the greatest challenges in managing their PV obligations.

Here, the differences between US and European requirements differ more significantly.

In the early 2000s, Europe revised its post-marketing PV requirements, making these very clear and prescriptive. In the US, equivalent post-marketing safety requirements are considerably older and quite vague in their language, leaving much to interpretation. For post-marketing safety studies, for instance, Europe has done quite a nice job of breaking down the requirements for interventional versus non-interventional studies and what needs to be reported – or not – for each. In the US, companies tend to tread a much more cautious path, interpreting the requirements more conservatively because precise guidance is lacking.).

If studies are used to support a product claim, and the right data hasn’t been collected in the right way for the given market, this could pose problems. So the different requirements do need to be well understood – and designed into post-marketing and market research studies – to avoid potential problems later.

Differences in definition A further variable in all of this is that categorisation and treatment of different products types by the authorities can differ between regions. A ‘device’ or ‘combination product’ (device plus medicine) may carry different definitions and requirements from one region to another, for instance. Being aware of this, and building this into PV processes and planning, is another international regulatory imperative then.

Autumn 2021 Volume 4 Issue 3

Regulatory & Compliance

Filling capability gaps The challenge for biotechs is that, while these companies have extensive product expertise, this is not typically matched in understanding and expertise in PV requirements and process rigour. To mitigate safety compliance related risk, they need to fill that gap – both with the right knowledge and experience, and with skills in writing SOPs and setting up PV systems which, in Europe, must be in place from the time of filing for marketing authorisation (checks for which could be made during filing/ pre-authorisation if the regulator feels in any doubt about a company’s PV provisions).

Relying on a third-party safety services provider to take on this burden without in-house oversight is not a practical or advisable solution. This is not least because the marketing authorisation holder retains ultimate responsibility for PV compliance: it is they rather than the CRO (contracted partner) that will be liable if anything goes awry.

So, irrespective of the biotech’s size and scale, the company will need to bring in someone experienced who understands PV and can keep a check on vendor quality – rather than simply send someone on a course.

Continuous tracking Where biotechs have entered into distribution partnerships/ relationships with other MAHs, there will be additional considerations - such as who will coordinate and be responsible for the PV requirements in a given market and how this will be written in any contracts. The MAH in the local country always is ultimately responsible for meeting PV requirements in that country. For PV, there is also the decision of who will be the global database holder (usually the company that developed the product and got it approved).

PV capabilities need to evolve, too – not just to keep pace with changes to regulatory requirements across the different markets, but also to stay on top of evolving channels and technologies when tracking safety signals. Where web sites and social media platforms create scope for market feedback, companies have an obligation to monitor and filter that content for potentially important real-world safety information, where www.international-biopharma.com

digital media is considered to be company-sponsored (if it is owned, paid for and/or controlled by the MAH).

Meanwhile, the increase combination treatments involving drugs and devices may drive new rules which clarify how responsibility for adverse drug reactions is calculated and apportioned between those relative components. So this situation needs to be tracked, too.

Ultimately, setting aside a PV budget to develop the right internal knowledge and connect with appropriate external guidance will be essential for any biotech navigating all of this international complexity. It may seem a lot for a small emerging biotech to take on board, but lay the right foundations early on and there will be a world to play for.

Dr. Judith M. Sills A non-executive director and advisor to Arriello, Dr. Judith M. Sills, Pharm.D. is president of JM Sills Consulting LLC, specialising in pharmacovigilance and benefit-risk management. She has over 30 years’ experience in pharmacovigilance in the pharmaceutical industry, FDA, and consulting. Prior to establishing a consulting practice in 2018, she was VP and head of global pharmacovigilance at The Medicines Company. Judi has also held senior positions at Novartis, Warner-Lambert and The Degge Group.

Dr. Eric Caugant Also, a non-executive director and advisor to Arriello, Dr. Eric Caugant has over 25 years’ experience in pharmacovigilance, drug safety, risk management, and more broadly pharmaceutical medicine. In 2017, he founded Pharmacovigilance Systems Consulting (PhVSC). Prior to this, Eric held senior positions in various pharmaceutical and biotech companies including Alexion, Bayer Lilly and Wyeth as well as the French Ministry of Health.

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Regulatory & Compliance

PEER REVIEWED

Regulatory Challenges Associated with the Development of Cell and Gene Therapies The development of safe and effective cell and gene therapies (CGTs) relies on regulatory compliance and critical selection and qualification of raw materials.

Regulatory classifications for advanced therapy medicinal products (ATMPs) vary globally and it is imperative for both manufacturers and suppliers to understand the requirements and obligations from each party, to achieve international compliance.

For example, raw materials of biological origin must undergo in-depth control procedures throughout every step of the manufacturing process. The risks associated with the use of biological raw materials must be understood, considered and mitigated with appropriate risk methods and quality control strategies. In this article we discuss the regulatory environment surrounding bio manufacturing of CGTs and the significance of compliance with appropriate regulations to ensure safety, quality, and efficacy of the final product.

Brief overview of ATMPs Advanced therapy medicinal products (ATMPs) are a rapidly growing field of novel CGTs and tissue-engineered products for complex diseases such as cancer and consist of products that contain modified genetic material or engineered cells and/or tissues.1 Their development is costly, highly complex, and lengthy due to stringent quality processes and ever-evolving regulatory requirements. The regulatory scope, definitions, and approval processes for development of ATMPs differ globally, particularly between the US and EU, increasing the time required to develop and bring these therapies to market. However, recent global events have demonstrated the urgency at which these medical solutions must progress, for example the effective yet prompt development of the COVID-19 vaccine for the SARS-CoV-2 virus.

Global international collaboration between manufacturers, researchers and regulators has been key in the rapid distribution of the COVID-19 vaccine, exhibiting that through the establishment of networks and exchange of information, the development and validation processes can be accelerated, for a faster route to market. A similar collaborative approach between manufacturers, raw material suppliers, and regulatory bodies should be followed in the development of CGTs to allow patients to more rapidly access and benefit from these innovative treatments. Manufacturers are required to have a thorough understanding of the entire supply chain and the processes expected by regulatory bodies. They should also work closely with certified suppliers to obtain high quality raw materials which will comply with the required quality standards, in order to maximise safety and chances of obtaining approvals.

Overview of the regulatory landscape To ensure the highest quality and safety standards of ATMPs, both 12 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

manufacturers and suppliers must comply with strict regulatory requirements. There is a concern surrounding the traceability of the biologic raw materials used to produce ATMPs, and the risks with the lack of traceability raw materials could pose to the safety and efficacy of the final product. The regulatory requirements set expectations and provide guidance for manufacturers so that ATMPs can be authorised and brought to market as swiftly as possible. As the field of ATMPs is still in its early days and the regulatory landscape surrounding their development is constantly evolving, collaboration between different suppliers, manufacturers, and regulators is especially important.

The regulations governing the development of ATMPs vary from country to country. Here we focus on the regulatory landscape of Europe and the US, where the majority of ATMPs are developed. In both regions, ATMPs are classed as biologics, however the sub classification groups vary across the two geographies. The European Medicines Agency (EMA) is the regulatory body responsible for reviewing and approving ATMPs across Europe. The EMA defines ATMPs as “medicines for human use that are based on genes, tissues or cells”.2 In the EU, ATMPs fall under four subgroups: gene therapy medicinal products (GTMPs), tissue engineered products (TEPs), somatic cell therapy medicinal products (sCTMPs), and combined advanced therapy medicinal products (cATMPs).

There are only two subgroups in the US: gene therapy and cellular therapy.1 In the USA, the Center for Biologics Evaluation and Research (CBER), a part for the FDA, regulates CGT products.

According to the CBER, cellular therapy products include “cellular immunotherapies, cancer vaccines, and other types of both autologous and allogeneic cells for certain therapeutic indications, including hematopoietic stem cells and adult and embryonic stem cells”.3 Currently there are 11 approved CGTs in Europe,4 with over 1000 ATMPs in ongoing clinical trials.5 In 2017 the FDA approved the first CGTs, with 20 approved on the US market today,6 and nearly 400 in development, as of 2020.7 It’s important to understand the differences in classification and requirements to generate the most appropriate data for regulatory submission. The differences in subgrouping must be well understood and considered to classify the product appropriately. An important difference between the US and EU in terms of regulatory scope, is that the FDA controls the clinical trials, whereas the EMA does not. Clinical trials in Europe fall under the jurisdiction of the country in which the trial is taking place and are overseen by national authorities. For example, in the UK, the Medicines and Healthcare Products Regulatory Agency (MHRA) is the competent authority for clinical trial authorisation. The review and approval process also differs significantly between the EU and US.1 Manufacturers should anticipate these variations in times and processes and adapt their development efforts accordingly.

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Despite these differences, the COVID-19 pandemic demonstrates how global scientific collaboration and research partnerships can overcome regulatory hurdles, to streamline vaccine development.

Achieved through facilitated information exchange, data sharing, and cross-border scientific and business support networks, these collaborative efforts aim to accelerate the development and manufacture of biologics. Agencies and governments have also contributed to this by fast-tracking the approval process and allowing large-scale manufacturing to meet increasing demand. Both the EU and US offer expedited approval processes for therapies for life-threatening diseases which offer greater therapeutic benefit than current treatment methods or meeting an unmet need. In the US, the FDA introduced the Breakthrough Therapy and Fast Track designation programs for serious disease therapeutics,8 while in the EU, the EMA introduced the PRIority Medicines (PRIME) designation scheme to support their development.9 In addition, working closely with trusted and experienced suppliers in the biologics field facilitates an efficient approval process by reducing risks associated with unqualified raw materials.

The varying global regulatory landscape (in EU and US) To develop successful ATMP products, the ATMPs must comply with stringent regulatory requirements. The raw materials used for their manufacture are being increasingly assessed in terms of quality, risk, and safety, and it is recommended that they are qualified and produced consistently in accordance with a recognised quality management system. In both the EU and US, Pharmacopoeia (Ph. Eur. and USP, respectively if available), a collection of quality standards for medicinal products, stipulate the required standards that the raw materials of biological origin must comply with.

There are two European committees responsible for evaluating ATMPs prior to granting marketing authorisation: the Committee for Advanced Therapies (CAT) and the Committee for Medicinal Products for Human Use (CHMP). ATMPs first undergo a safety and quality assessment by the CAT, which is then passed onto the www.international-biopharma.com

CHMP for a final authorisation decision. Manufacturers should refer to the Regulation (EC) No 1394/2007, which provides an overall regulatory framework for ATMPs in Europe. Since 2009, the CAT also provides specific scientific recommendations for ATMPs, in accordance with Article 17 of the aforementioned Regulation.10 Alliance for Regenerative Medicine (ARM), the international organisation promoting the development of ATMPs, supports greater harmonisation across the EU in response to the revision of the EU general pharmaceuticals legislation, and recently called for a centralised procedure around marketing authorisation and clinical trials.11 One of the aims of the revision is to “foster innovation, including in areas of unmet medical need” and simplify the access of medicines for patients.12 In the US, biologics are regulated under section 351 of the Public Health Services (PHS) Act and Title 21 of the US Code of Federal regulation.1,13 Biological products subject to the PHS Act also meet the definition of drugs under the Federal Food, Drug and Cosmetic Act (FDC Act), and therefore must be regulated accordingly.13 In 2016, the 21st Century Cures Act (Cures Act) was passed to accelerate the development of medical products and innovations, and to incorporate patients’ perspectives into the development of drugs, biological products, and devices in FDA's decision-making process. Another aim of the Cures Act is to modernise clinical trial designs and clinical outcome assessments, increasing the development and review of novel medical products. The Cures Act introduced the Regenerative Medicine Advanced Therapy (RMAT) designation to expediate the development of new therapies.14 Importance of regulation and Quality Attributes of raw materials of biological origin The risk due diligence and qualification of raw materials of biological origin used to produce ATMPs is imperative, due to their heterogeneity, which makes them difficult to control, and restricts the reproducibility and scalability for other uses. The use INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 13

Regulatory & Compliance of animal-derived materials also increases the risk of introducing contaminants to the final product. The FDA recommends using non-animal derived materials, such as serum-free cell culture media, to minimise risk of transmission and batch-to-batch variation.15 To avoid or minimise these risks, it is critical to understand the origin of the raw materials used to manufacture cell culture media as well as keeping tight controls on the quality and traceability of each component.

The FDA requires CGT manufacturers to provide information on chemistry, manufacturing, and control (CMC). A list of all materials used in manufacturing is required alongside the quality or grade of the materials used. The information should include the identity of the material, the supplier, the quality, the source (e.g., animal or human), country of origin and the stage at which the material is used in the manufacturing process. The manufacturer has a responsibility to establish a qualification program and provide necessary documentation to demonstrate the materials used to develop the final product meet the appropriate quality and regulatory standards. The documentation includes test results (such as adventitious agent, toxicity etc., testing) and certificates of analysis (CoAs), origin and transmissible spongiform encephalopathy/bovine spongiform encephalopathy (TSE/BSE) statements. All biological raw materials are required to undergo viral inactivation studies and confirm to be free of adventitious agents, including bacterial and fungal agents, cultivatable and non-cultivatable mycoplasmas, mycobacteria, and viruses.16 ATMPs that use non-graded and/or non-GMP materials without appropriate safety controls, testing and/or certifications, will require modification, such as substitution of the raw materials that are fit for clinical use, to gain regulatory approval. Many early clinical trials involved non-graded, unqualified raw materials; however, this is no longer accepted by the regulators.

Increasing number of ATMPs manufacturers are struggling to obtain regulatory approval if they use non-GMP and non-graded materials in their clinical studies. Many suppliers in the entire supply chain have expert knowledge in the regulatory field and can assist with the selection of the most appropriate raw materials and tools, which is why collaboration is key in the development, manufacture and scaling of ATMPs. In 2021, the FDA issued COVID-19-specific guidance for manufacturers of CGT products with risk-based recommendations to minimise the potential transmission of SARS-CoV-2 via the products. The guidelines highlight the need for compliance with cGMP requirements and ensuring quality-controlled evaluations of production controls. Although not specifically recommended by the FDA, manufacturers can include testing of raw materials, cell banks, intermediary and final products for SARS-CoV-2, in accordance with the manufacturer’s own quality control processes.6 How to ensure compliance and follow best practices Manufacturers of ATMPs should follow a risk-based approach throughout the development and manufacture of products. As part of the risk assessment, a robust quality by design (QBD) plan must be developed and should include a detailed list of identified risks with mitigation activities to minimise or remove these risks. Manufacturers should also work closely with raw material suppliers to ensure an in-depth risk assessment is performed prior to using the material in the final formulation.

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The raw material grade is very critical. Raw materials must comply with compendia requirements specified in appropriate pharmacopoeia, e.g., Ph. Eur., USP, BP, JP or multi-compendia.

If the compendia are unavailable, additional risk assessment and justification, or additional testing should be performed to establish the suitability of the material.

The selection of appropriate high-quality cell culture media is crucial. Chemically defined media are recommended as they minimise the risks of adventitious agents and remove the effects that undefined components could have on the cell. Reliable providers of high-quality cell culture media can optimise the media to support consistent cell growth and optimal performance to meet the manufacturers’ production needs while maintaining high quality standards.

It is important to understand each type of raw materials going into the product and how each material contribute to the intended use of the product. Then it is critical to qualify the raw materials to ensure safety and to demonstrate the qualification process in a well-documented traceable file. If the raw material is of animal origin, the supplier must demonstrate that all risk of viral transmissions has been removed either by viral inactivation studies or viral testing of the product. Presence of antibiotics should also be assessed to ensure that it will not present a threat to patient and/or understand the acceptable levels of such to be able to calculate the residual antibiotic quantity left in the material that is being qualified.

To obtain fully tested, highest quality raw materials, it’s important for manufacturers to partner and work closely with suppliers and gain appropriate certificates such as Certificate of Analysis (CoA), Certificate of Origin (CO) for both the country and the source material, such as chemical, plant, animal or microbial-derived. The definitions describing source materials are not harmonised, so it is important to make sure supplier definitions are in line with manufacturer’s definitions, e.g., animal component-free (ACF), animal-derived (AD), chemically defined (CD), xeno-free (XF), or serum-free (SF). However, all parties involved in the development and production of ATMPs such as raw material suppliers, media manufacturers, and cell processing centres must ensure safety of all materials used, this responsibility does not lie solely with the manufacturer of the final product.

Once the raw material is qualified, the final product needs to undergo further testing to ensure it meets all the safety and quality requirements before being approved and brought to the market. Furthermore, all medicinal products, including ATMPs, must comply with Good Manufacturing Practices (GMP). In doing so, GMP minimises batch-to-batch variability and ensures safety and purity of the final product, whilst confirming the supply chain has been audited for complete certification.

Summary & looking forward ATMPs are a novel field of therapeutics with a lot of potential, but also many challenges associated with their standardised development and manufacture. For effective and efficient scalability, the variability associated with raw materials specially those of biological origin must be minimised as much as possible by controlling the entire supply chain. Although it’s not possible to eliminate the variability associated with the patient’s own Autumn 2021 Volume 4 Issue 3

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cells, strict control of each and every step of the development process, including the qualification of raw materials, reduces the variability in the manufacturing process, and subsequently yields higher quality end products, with better patient outcomes.

Similarly, global alignment of the regulatory frameworks is required to facilitate approvals and speed up the process to bring these therapies to market. The rapid generation of the COVID-19 vaccine demonstrates the significant impact of collaboration between researchers, suppliers, pharmaceutical companies, and regulators, to reach patients at unprecedented speeds. Both the US and EU regulatory agencies have introduced fast-tracked programms to accelerate approvals of lifesaving and innovative therapies with significant public health benefit. Manufacturers need to continue to educate on the quality of the raw materials used for these development processes and understand the complex and continuously evolving regulatory environment, bring best industry practices to the processes to ultimately ensure the production of high-quality products. Cooperating with an experienced and trusted partner who can share their knowledge and provide support at every step of the development process can catalyse and minimise the challenges seen in the field and help drive innovation, to bring life-saving therapies to patients as quickly as possible.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

Iglesias-Lopez, C., Agusti, A., Obach, M., & Vallano, A. Regulatory Framework for Advanced Therapy Medicinal Products in Europe and United States. Front. Pharmacol. 10, 921 (2019). https://doi. org/10.3389/fphar.2019.00921 https://www.ema.europa.eu/en/human-regulatory/overview/ advanced-therapy-medicinal-products-overview, visited on 07 May 2021. https://www.fda.gov/vaccines-blood-biologics/cellular-genetherapy-products, visited on 14 May 2021. https://www.labiotech.eu/in-depth/atmp-cell-gene-therapy-ema/, visited on 07 May 2021. http://alliancerm.org/wp-content/uploads/2021/04/EU-PharmaLegislation.pdf, visited on 20 May 2021.

FDA, Manufacturing Considerations for Licensed and Investigational Cellular and Gene Therapy Products During COVID-19 Public Health Emergency. Guidance for Industry (2021). https://www.fda.gov/ media/145301/download, visited on 07 May 2021.

America’s Biopharmaceutical Companies. Medicines in Development

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8.

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2020 Report (2020). https://www.fda.gov/patients/ learn-about-drug-and-device-approvals/fast-track-breakthroughtherapy-accelerated-approval-priority-review, visited on 14 May 2021. https://www.ema.europa.eu/en/human-regulatory/researchdevelopment/prime-priority-medicines, visited on 14 May 2021. https://www.ema.europa.eu/en/human-regulatory/overview/ advanced-therapies/legal-framework-advanced-therapies, visited on 14 May 2021.

Alliance for Regenerative Medicine. Response to the revision of the EU general pharmaceuticals legislation (2021). https://ec.europa.eu/info/law/better-regulation/have-your-say/ initiatives/12963-Revision-of-the-EU-general-pharmaceuticalslegislation_en, visited on 14 May 2021. https://www.fda.gov/drugs/therapeutic-biologics-applications-bla/ frequently-asked-questions-about-therapeutic-biological-products, visited on 14 May 2021. https://www.fda.gov/regulatory-information/selected-amendmentsfdc-act/21st-century-cures-act, visited on 20 May 2021.

FDA, Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs).

Guidance for Industry (2020). https://www.fda.gov/media/113760/ download, visited on 14 May 2021.

FDA, Guidance for Industry - Characterization and Qualification of Cell Substrates and Other Biological Materials Used in the Production of Viral Vaccines for Infectious Disease Indications (2010). https://www. fda.gov/media/78428/download, visited on 14 May 2021.

Marlin Frechette Marlin Frechette has over 30 years of experience in the Medical Device industry, servicing Pharmaceutical and Biopharmaceutical customers. As Chief Quality & Compliance Officer at FUJIFILM Irvine Scientific, she has oversight and responsibility for the Company’s quality, regulatory, and compliance departments, including Quality Systems, Global Regulatory/Product Compliance, Corporate Compliance, and EHS. She holds a Bachelor’s of Science with a major in Business Administration & Personnel Management.

Email: marlin.frechette@fujifilm.com

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Research / Innovation / Development

How to Bring Your Oncolytic Virus Innovation to Market: the Secret to Successful Development Cancer continues to be a major burden on healthcare systems across the world, with global spending on therapies and supportive care drugs expected to reach $200 billion by 2022.1 The number of deaths worldwide has increased every year for decades, from 5.7 million in 1990 to 8.8 million in 2017.2

Nevertheless, despite all of this, there have been significant improvements in mortality rates for cancer sufferers over the last few decades. Since 1990 cancer mortality has declined from 142.5 (per 100,000 people) to 121.21 in 2017.3 The current therapeutics available to cancer patients, such as surgery, chemo- and radiotherapy, as well as hormone therapy, have all played a positive role in declining death rates. These treatments represent some of the most advanced healthcare on the market.

However, they all have critical drawbacks that make them less than ideal therapies. Notably, one or more of them have the following disadvantages: • •

Invasive: post-surgical complications, for example, can pose their own risks.

Toxic: radiation and chemotherapy, as well as hormone therapy are by their nature toxic, leading to harmful side effects.

A new approach is needed to treat cancer – by leveraging the close evolutional relationship between a human cell and its viral parasites it is possible to turn an old foe into a new friend.

Virologists are developing OVs as ‘Trojan horses’ to selectively kill cancer cells and deliver genetic payloads; the patient’s immune system is then educated to reject tumour cells as non-self-tissue.

The advantages of OVs Quite simply, an OV is a virus – either naturally occurring or genetically designed forms of common viruses, such as HSV, Adenovirus, Vaccinia Virus, or Reovirus – that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumour.

In addition to attacking the tumour themselves, OVs are unique among cancer treatments as they can be designed to stimulate the patient’s immune system to identify and target cancer cells itself. This is seen as a key goal in cancer therapy, because adaptive immune resistance of cancer cells often leads to a “cold” tumour microenvironment, where the tumour goes unnoticed by the body’s own immune system. By changing this immunological tolerance, a “hot” or active environment can be created around the tumour, soliciting an effective immune response, ultimately leading to systemic tumour rejection.

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This is not the only reason OVs are seen as a promising new entry into the cancer field. Compared with surgery, they are non-invasive, and relative to radio-, chemo- and hormone therapies, they are non-toxic too, minimising negative side effects.

With all of this in mind, it is clear that OVs can potentially add another effective modality to the treatment mix, in support of the traditional therapies utilised in the treatment of cancer.

It is no surprise, then, that the OV field is growing quickly – it is expected to be worth $962 million by 2030, with a compound annual growth rate (CAGR) of 26.28% through the forecast period from 2020 to 2030.4 Clinical trials in recent years have highlighted that they offer a high level of patient safety, with strong genetic stability, and show their effectiveness on a wide range of tumour types, from early to late-stage disease and even at metastasis.

The challenges for successful OV development While OVs are an exciting step forward in our fight against cancer, the treatment remains in its infancy, with just a handful at the clinical trial stage. OVs face a number of challenges that hinder their progress through the development process.

For example, the novelty of the technology, the current diversity of the OVs in development and their inherent complex structure, combined with the small number of projects that have been conducted so far, means that processes and best practice have yet to be firmly established or agreed by the industry. There are also production and product stability issues posed by the manufacturing of viruses that need to be addressed.

Compounding all of these issues is the relative lack of capacity and resource for scale-up. Few pharma companies have the specialist capability or the expertise in-house to develop their discoveries, navigate the global regulatory landscape and manufacture the finished product. Substantial investment would be required to bring OV processing capabilities in-house.

In addition to all of this, there is the more fundamental challenge of the sheer diversity of cancer profiles, as well as of patients’ own immune responses. All of this makes it even more of a challenge to find a promising OV candidate.

Centralising the solutions So, how can biopharma companies overcome these issues and maximise the chance of OV project success? There are several crucial measures that companies should take to ensure a return on their investment.

OV platform selection When selecting an OV platform making proper use of the strengths and weaknesses of platforms already in clinical trial or optimised for development can help streamline the development process and simplify regulatory approvals.

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Research / Innovation / Development and capacity for continued supply all need to be built into strategy early. This is crucial to ensure the Quality Target Product Profile (QTPP) can be defined and the product’s Critical Quality Attributes (CQAs) can be monitored and delivered upon effectively. With all of this in place, quality standards can then be successfully met with speed to first-in-human clinical trials and eventually market approval.

Finally, it is vital to have an expert strategy in place to navigate complex and diverse global regulatory approvals during the pre-commercial phases. This can go a long way towards mitigating risk, not just maximising the chance of the project succeeding, but also streamlining the therapy’s progress to the next phase of development. For example, your virotherapy CDMO will work closely with you on your approvalrelevant CMC package.

Collaboration is critical The pharmaceutical industry has only just begun to explore the full potential of OVs in revolutionising the fight against cancer.

There are many challenges holding back the development of the technology that must be overcome if OV-based therapies are to pass clinical trial and become a valuable commercial product.

Only through collaboration between industry stakeholders and making use of each partner’s core competencies, will OVs be able to reach their full potential in the near future, so they can transform patients’ lives for the better.

REFERENCES 1.

2.

Manufacturing platform Making use of already established virotherapy processes is a crucial step to creating a manufacturing platform for your selected OV candidate.

Partnering with a contract development and manufacturing organisation (CDMO) that specialises in virotherapy can really help. They have the subject matter experts and established manufacturing platforms where you can plug-in your new OV candidate. They will help you to avoid development and manufacturing pitfalls and reduce the risk of project failure.

In addition, such CDMOs understand the quality control and scale-up options open to biopharma companies. This means they can provide guidance and capacity, as well as recommend how to set-up your analytical release testing and stability programmes.

This helps to avoid issues that can occur later in the development timeline with product stability and quality.

Strategise for success OV treatments need to reach clinical trials and commercialisation rapidly, all while meeting the highest possible standards of quality and safety.

To achieve this goal, team expertise, product and risk understanding, operational experience, robust equipment www.international-biopharma.com

3.

4.

https://bisresearch.com/industry-report/cancer-immunotherapymarket.html https://ourworldindata.org/cancer#the-number-of-cancer-deathsis-increasing-as-the-world-population-is-growing-and-aging https://ourworldindata.org/grapher/cancer-death-rates https://www.benzinga.com/pressreleases/21/04/g20742197/ global-oncolytic-virus-therapies-market-report-2021-2030-focuson-commercialized-therapies-pipelin

Kai Lipinski Kai Lipinski, PhD, CSO joined Vibalogics in 2010 initially as Head of Cell Culture and Virus Production. Kai was named Head of Process Development in 2013, then promoted to Chief Scientific Officer (CSO) in 2020.

Kai has a wealth of experience in viral vector manufacturing from a variety of roles before he joined Vibalogics. He served as Principal Scientist at Cobra Biologics, focusing on upstream process development for virus and mammalian protein expression projects. Prior to that, Kai worked as Senior & Principal Scientist at ML Laboratories, where he was responsible for the development of targeted adenoviral vectors for cancer gene therapy approaches. At Vibalogics, Kai is central to the establishment of virus Process Development and Manufacturing capabilities, technical developments and the acquisition of many key clients.

Kai has a PhD in Transcriptional Regulation by Adenoviral E1A Proteins, and a Post-Doc, also on Transcriptional Regulation, from the University of Duisburg-Essen.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 17

Research / Innovation / Development

PEER REVIEWED

CRISPR-Cas9 Knockout Screening Using Primary Human Cells in Drug Discovery While significant progress has been made carrying out CRISPR screens in immortalised cell lines, a more physiological and clinically relevant alternative are human primary cells, such as T cells, regulatory B cells, or natural killer (NK) cells. The use of phenotypically relevant primary cells, though, is not without biological and technical challenges, especially when their intended use includes gene editing and scale-up of these specialised cell types for screening. However, overcoming these challenges and utilising new detection technologies for assay readouts herald a new era of cellular screening. Thus, functional genomic screening (FGS) with gene-edited human primary cells offer unique opportunities to accurately identify relevant drug targets and more reliably introduce validated therapeutics to the clinic.

CRISPR-Cas9 Knockout Screening to Understand Gene Function A logical method to understand the role of a factor within a system is to remove it and assess the result. In drug discovery, this loss-of-function approach is often employed to identify new drug targets by knocking genes down or out and investigating the resulting biological characteristics or phenotype. If the phenotype is unaltered, the targeted gene likely does not contribute to the phenotype under investigation. However, if the phenotype is altered, then the gene, and its protein, might be a potential drug target. Ideally, these screens involve systematic knockdown or knockout of genes from small scale up to whole genome level and are conducted in a cellular system that resembles in vivo conditions. Harnessing innovative technologies that include gene editing, cell culture, automation, end-point acquisition, and robust analysis is no longer science fiction, but a fully established reality.

CRISPR-Cas9 Knockout Technology Until recently, FGS was performed using RNA interference (RNAi).

However, RNAi does not completely abrogate gene expression and can potentially contribute to off-target effects. Permanent silencing of genes at the DNA level with minimal off-target effects is achieved with the CRISPR-Cas system allowing knockout of gene function using both Cas9 nuclease and guide RNA (gRNA).

The genomic target can be any ~20 nucleotide DNA sequence provided the sequence is unique compared to the rest of the genome and is immediately adjacent to a protospacer adjacent motif (PAM). The gRNA guides the Cas9 protein to the target sequence while the PAM serves as a binding signal. Then, the Cas9 protein and the gRNA form a complex enabling the Cas9 to cleave the DNA creating a DNA double-strand break (DSB) within the target site. To preserve the integrity of the DNA, DSBs are swiftly repaired. The most active and efficient DNA repair mechanism is the non-homologous end joining pathway that ligates the broken ends together, frequently giving rise to small nucleic acid insertions and deletions (indels) at the break site. This imperfect repair causes frame shift mutations, where the messenger RNA no longer encodes a functional protein effectively producing gene knockout.

CRISPR-Cas9 FGS There are two screening formats: pooled screening (Figure 1) and arrayed screening (Figure 2) that often utilise CRISPR knockout (CRISPRko) technology. Pooled screening involves introducing a pool of single guide RNAs (sgRNAs) or a sgRNA library into a population of cells. This is achieved by transducing cells with lentiviral particles packaged with sgRNA and Cas9-containing vectors (one vector per particle). Expression of sgRNA and Cas9 lead to target gene knockout. As these knockouts occur in a one cell population, pooled screening is restricted to readouts that physically separate edited cells (e.g., fitness endpoint or

Figure 1. Workflow of pooled CRISPRko screening in primary cells. CRISPR-Cas9 editing of one cell population with either an all-in-one vector or a two-step approach (sgRNA lentiviral library, Cas9 mRNA/RNP electroporation), followed by assay set up and data analysis.

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Autumn 2021 Volume 4 Issue 3

Research / Innovation / Development

Figure 2. Workflow of arrayed CRISPRko screening in primary cells. CRISPR-Cas9 editing of cells dispensed into individual wells by electroporation, followed by assay set up (mono-cultures or multi-cultures) with multiplexed assay read-out and data analysis.

cell sorting) with a phenotype of interest from those that do not. To link a phenotype to an individual gene, the integrated sgRNAs are deep sequenced and data are deconvoluted. Recent studies have shown that CRISPR-Cas9 based screens using whole-genome sgRNA-libraries can quickly and efficiently identify genes involved in different biological processes.1,2 The other screening paradigm is arrayed screening and involves targeting one gene per well in a multi-well plate format. Delivery of Cas9 and sgRNA is accomplished by transient transfection (e.g., electroporation) or lentiviral transduction. Because targeted genes are physically separated, unlike pooled screening, arrayed screens are compatible with a wide variety of complex assays and multiparametric endpoint readouts. The choice between pooled or arrayed format is highly dependent on the biological question, cell type and laboratory equipment.

CRISPR-knockout Screening in Primary Human Cells: Challenges and Solutions Traditionally, immortalised cell lines have been used in FGS because they are stable, predictable, and easy to handle. However, their ability to proliferate indefinitely (due to accumulated or induced genetic mutations) may affect their original physiological properties and cause them to differentiate from the human tissue they model. Therefore, immortalised cell lines may not provide the most biologically predictive results to determine therapeutic effect. By contrast, human primary cells are more physiologically relevant and therefore, screens using such cells can identify more clinically relevant targets.

In the last few years publications on CRISPR-Cas9 editing in primary human cells, such as fibroblasts, pancreatic islet cells, megakaryocytic precursor, monocytes, NK cells, B cells and T cells have emerged (Table 1). Most of these studies are proof-of-concept experiments targeting a very limited set of genes because primary cells used for CRISPR-Cas9 FGS encounter multiple challenges (Table 2).

Primary Cells in CRISPR-Cas9 FGS: Challenges Low cell yield due to limited biological abundance Cell lines have been immortalised to allow continuous propagation, while primary cells derived from human tissue www.international-biopharma.com

have a limited capacity to propagate in vitro. In addition, some primary cell populations are rarely present in body fluids and tissues. This poses an issue as the number of cells required for screening increases with the size of the sgRNA library. A sufficient cell yield to conduct whole genome screens can be achieved by sourcing the appropriate blood product (e.g., 109 peripheral blood mononuclear cells per leukopak), expanding/polarising cells (e.g., naïve T cells to induced regulatory T cells), pooling cells from various donors (e.g., myeloid cells) and assay miniaturisation (e.g., optimising cell numbers).

Viability issues due to limited lifespan Primary cells are inherently more difficult to culture than immortalised cell lines. As primary cells are isolated from human tissue or blood, they are sensitive to changes in their environment and often require special nutrient medium for their survival and growth. While cell lines can be kept alive in vitro for numerous passages, the in vitro lifespan of primary cells highly depends on its cell type. Some immune cell types, such as B cells and unstimulated T cells, have a very limited lifespan, but can be kept alive and functional by using specialist cell culture consumables and reagents.

Editing efficiency due to limited susceptibility to CRISPR/Cas9 gene editing Pooled and arrayed screening both involve the introduction of sgRNA and Cas9 into cells. In pooled screens using cell lines, sgRNA libraries and Cas9 are delivered using viral transduction; in arrayed screens using cell lines, CRISPR components are delivered in the form of DNA plasmid, lentivirus and synthetic sgRNA. However, primary immune system cells, such as T cells and NK cells, have an innate mechanism to resist foreign genetic material as they are perceived as signs of infection resulting in low editing efficiencies of T cells3,4 and NK cells.5 To circumvent this mechanism, high editing efficiency is needed for a penetrating phenotype and to select for edited cells.

While edited cells in pooled screening could be selected by cell sorting and antibiotic selection, this is not feasible for arrayed screening at large scale. In addition, unstimulated lymphocytes are not efficiently transduced by lentiviral vectors due to low levels of low-density lipid receptor6 which serves as a major INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 19

Research / Innovation / Development entry port for lentiviral vectors in humans.7 The requirement to stimulate lymphocytes for lentiviral transduction excludes pooled screening as an approach to study the polarisation of T cells or B cells into subsets as stimulation of these cells will drive them towards a default phenotype before editing has occurred.

Instead of using an all-in-one vector with sgRNA and Cas9 in one plasmid, researchers developed a dual system, using both lentivirus (for the sgRNA library) and electroporation (for Cas9 ribonucleoprotein (RNP) or mRNA) to overcome these challenges.3,4 High knockout efficiencies are achieved using algorithm-designed gRNA8 and screening libraries that employ a multi-guide design strategy, in which several sgRNAs target one gene, inducing one or more fragment deletions. Because these deletions remove several amino acids, they are highly likely to cause a complete knockout. This multi-guide strategy results in higher and more consistent knockout efficiencies compared to editing with just one sgRNA. For electroporation, a thorough optimisation phase in which parameters such as different electroporators, electroporation buffers, CRISPR-Cas9 amounts are tested to find the best condition in terms of cell morphology, cell viability, genome editing efficiency. To monitor any effects introduced by the electroporation pulse alone, mock and non-electroporated controls are necessary.

The inclusion of appropriate controls is crucial for any screen.

As negative controls, non-targeting sgRNA or sgRNAs targeting genes not associated with the downstream phenotypic readout may be advisable in case the DSB, itself, or the DNA repair pathways influence the readout. As positive controls, sgRNAs could be added that have a predictable effect on the phenotype being assayed. Other controls that provide information on editing efficiency in a binary fitness readout (live/dead) include lethal sgRNA mixes or target genes essential for cell health.

Assay variability due to heterogenous cell populations and donor-to-donor variability Higher variability of results should be expected in screens with primary cells than those using cell lines due to intra-donor (heterogenicity of cell populations) and inter-donor variations (donor demography). Intra- and inter-donor variability can be controlled by increasing technical and biological replicates, respectively. Even though increased variability might make data interpretation challenging, it also provides an insight into the spectrum of patient responses that could be expected in clinic and should not be viewed as a major drawback.

Scale up due to restricted technology for automation, data acquisition and analysis While the scale of a screen with cell lines is largely determined by the size of the sgRNA library and technical replicates, screens with primary human immune cells can easily double or triple in scope depending on the number of donors. Harnessing state-ofthe art automation and robotics, liquid handling systems enable handling and dispensing of numerous assay plates and flasks for replicable assay set up.

Similar to assay set up, readouts should not only be customised to address the biological question at hand but also be scalable depending on the size of the gene library. Hence, 20 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

there are limitations to assess knockout efficiency depending on the size of the screen. On a genomic level, analysis such as sequencing, tracking of indels by decomposition (TIDE) or T7 endonuclease I mismatch assay (T7EI) are not feasible for large gRNA library screens. On a phenotypic level, analysis such as flow cytometry, qPCR and western blotting might not reflect the genomic knockout as CRISPR guides target small regions of protein that render them non-functional. As such, unless there is direct overlap between the guide and the epitope to which detection antibodies bind, or protein transcription/translation is impacted, it is conceivable that the antibodies will bind to a protein that is otherwise non-functional. Being appreciative of these limitations for large-scale screens, it is advisable to plan for a validation screen to narrow down a gene list for thorough gene efficiency analysis.

The timing for assay set up and data acquisition is crucial for FGS with human primary cells. A fine balance between lifespan/ viability and absence of target protein must be identified. While immortalised cell lines have a rapid protein turnover as they are metabolically very active due to continuous propagation, primary cells, especially unstimulated or non-dividing ones, have a very slow protein turnover. So, even when a target gene has been knocked out successfully its functional protein may persist because it is not yet degraded. For the gene knockout to have a functional effect, the protein must be absent or non-functional by the time of assay set-up.

FGS relies on readout approaches that allow qualitative or quantitative evaluation at large scale. While screens utilising cell lines are often monocultures with a binary readout deploying high-throughput-friendly methods using plate readers (e.g., ATPlite) or high-content imaging, these are not always suitable options for co-cultured primary cells with a multiplexed readout.

For example, to assess the effect of gene knockouts in primary T cells in response to cell stimulation various surface expressed markers, cytokines, proliferation, and mRNA expression is of interest. All could be answered by classical protein or mRNA detection techniques such as tube-based flow cytometry, ELISA or western blot. However, these traditional readout methods have their limitations when it comes to scale up. To acquire data from a screen conducted with multiple donors and a 20+ sgRNA library, technology and approaches designed for microwell sampling and miniaturisation, such as plate-based flow cytometry screener or homogenous time resolved fluorescent assays, are harnessed.

Ideally, these technologies are supported by auto-fill stations and robotic arms.

The latest addition to readout approaches is single-cell RNA sequencing (RNA-seq), which allows insight into mechanisms by which each gene perturbation mediates its effect. Single-cell RNA-seq analyses transcriptomes on a cell-by-cell basis through microfluidic partitioning by capturing and barcoding single cells to obtain a next-generation sequencing (NGS) cDNA library.

Although powerful, these applications of single-cell CRISPR screening have been limited by low throughput and complex analytical methods. However, continued innovations in single-cell sequencing technologies can increase the utility of single-cell CRISPR screening for scale up.

The larger the scope of the screen (number of target genes, donors, technical replicates, and readouts), the larger the volume Autumn 2021 Volume 4 Issue 3

Research / Innovation / Development

Primary cell type

Healthy Donors

Editing approach

Size of library

Functional assay

Readout

Authors

Lung Fibroblasts

2

crRNA:tracrRNA, Cas9 RNP electroporation

SMAD2, SMAD3, PI4K4

Scar in a Jar assay

Western blot, immunofluorescence

Martufi et al., CRISPR J 2019

PDX1, KCNJ11

Insulin and glucagon measurement, immune staining, transplantation into mice, patch clamp studies

Pancreatic islet cells

3

All-in-one lentivirus

Insulin and glucagon measurement, TIDE analysis, ddPCR, qRT-PCR, ELISA, immunostaining, Na+, Ca2+ and KATP currents Insulin and glucagon measurement, TIDE analysis, ddPCR, qRT-PCR, ELISA, immunostaining, Na+, Ca2+ and KATP currents

Bevacqua et al., Nat Commun 2021

Mega-karyocytic precursor (MkPs)

3

All-in-one lentivirus

60 genes highly expressed in MkPs

Colony forming unitmegakaryocyte functional assay

qRT-PCR, FACS

Zhu et al., PNAS 2018

Monocytes

3

crRNA:tracrRNA or sgRNA, Cas9 RNP electroporation

B2M, CD14, CD81

Differentiation to DC and macrophages, phagocytosis assay

FACS, ELISA, imaging, phagocytosis, Sanger sequencing

Freud et al., J Exp Med 2020

FACS, immunofluorescent imaging, Western blot, TIDE, Luminescence, RNA-seq

Hiatt et al., Cell Reports 2021

3

crRNA:tracrRNA, Cas9 RNP electroporation

NK cells

1

crRNA:tracrRNA, Cas9 RNP electroporation

CXCR4

Transwell migration assay

Plate reader-based fluorescence-based method for quantifying cells, Flow cytometry, qRT-PCR, ddPCR, PCR, Lambert et al., Methods in Sequencing, TIDEPlate reader-based fluorescence-based Molecular Biology 2020 method for quantifying cells, Flow cytometry, qRT-PCR, ddPCR, PCR, Sequencing, TIDE

NK cells

3

crRNA:tracrRNA, Cas9 RNP electroporation, all-in-one vector

CD45, PTPRC, NCR1, CISH

Cytotoxicity and proliferation assay

FACS, Western blot

Rautela et al., JLB 2020

NK cells

3

crRNA:tracrRNA, Cas9 RNP electroporation

TGFBR2, HPRT1

Cytotoxicity assay

RT-PCR, plate reader

Kararoudi et al., J Vis Exp 2018

B cell

3

crRNA:tracrRNA, Cas9 RNP electroporation

CD46 and CDKN2A

EBV infection, B cell proliferation, plasma cell differentiation

ELISA, FACS, NGS, metabolically labeled with 4sU

Akidil et al., PLOS Pathogens 2021

B cells

3

crRNA:tracrRNA, Cas9 RNP electroporation

CCR5, PRDM1

Naïve cell to plasma cell differentiation

T7EI assay, Illumina sequencing for percentages of ontarget indels, viability, ELISA, NSG mouse transplant

Hung et al., Mol Ther 2018

B cells

2

crRNA:tracrRNA, Cas9 RNP electroporation

CD19, B2M

-

FACS

Laoharawee et al., Methods Mol Biol 2020

CD4+ and CD8 + T cells

3

crRNA:tracrRNA, Cas9 RNP electroporation

Classical T cell markers

Various classical T cell assays including anti-tumour efficiency of CAR T cells, HIV infection

e.g. FACS, in vivo tumour clearance, deep sequencing

Schuhmann et al., PNAS 2015; Ren et al., Clin Cancer Res 2017; Rupp et al., Scientific Reports 2017; Hultquist et al., Nat Protoc 2019

CD3+ T cells

-

gRNA-expressing Cas9-GFP plasmid

TCR, CD52

-

IDAA, FACS

Kamali et al., BMC Biotechnology 2021

CD4+ T cells & CD34+ cells

multiple

gRNA lentiviral, Cas9 in complex with NTC gRNA

2,585 genes and 13,243 genes

Proliferation

FACS-based enrichment, NGS

Ting et al., Nature Methods 2018

CD4+ T cells

1-2

crRNA, fluorescently labelled tracrRNA, Cas9 RNP, electroporation enhancer

TRAC, ZC3H12D, TBX21

Proliferation

FACS, T7EI, intracellular cytokine staining

Leoni et al., PLOS One 2021

CD8+ T cells

2

crRNA lentivirus, Cas9 RNP

Whole genome (19,114 genes, 77,441 sgRNA) & 6 genes (2 guides per gene)

Proliferation, T cell cloning

FACS-enriched NGS analysis for whole genome library & RNA-seq for custom library of six genes

Shifrut et al., Cell 2018

CD8+ T cells

4

crRNA:tracrRNA, Cas9 RNP electroporation

12 genes identified in a WG pooled screen to impact to enhance T cell proliferation

Proliferation

FACS

Shifrut et al., Cell 2018

Monocytes

SAMHD1, CXCR4, b2M, PPIA, AAVS1

Differentiation to DC and macrophages, in vitro infection of MDMs by Mycobacterium tuberculosis, HIV infection Differentiation to DC and macrophages, in vitro infection of MDMs by Mycobacterium tuberculosis, HIV infection

Table 1. Overview of publications using CRISPR-Cas9 editing in primary human cells. crRNA:tracrRNA (crRNA and tracrRNA complexed together), ddPCR (droplet digital PCR), ELISA (enzyme-linked immunosorbent assay), FACS (fluorescence-activated cell sorting), GFP (green fluorescent protein), IDAA (indel detection by amplicon analysis), NTC (non-targeting control), and qRT-PCR (quantitative reverse transcription PCR).

of data is generated. In parallel, methodological advances are driving renewed development in statistical modelling, machine learning, and artificial intelligence. Data analysis can only be as good as the data provided. Therefore, before a screen is conducted, statistical modelling must address the number of controls required for a powerful statistical analysis. Data complexity increases further by combining data in pathway www.international-biopharma.com

analysis, for example. Another key challenge is the graphical presentation, where various software options are available to choose.

A successful CRISPR screen can be measured by the hit list of potential targets generated for a new therapeutic. Next steps require prioritising targets that might have an impact in the clinic.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 21

Research / Innovation / Development Challenge

Solution

Cell yield

Limited capacity to propagate; Rare cell subsets

Appropriate blood product; Expansion/polarisation; Pooling donors; Assay miniaturisation

Viability

Sensitivity to change

Specialist cell culture consumables and reagents

Editing efficiency

Susceptibility to CRISPR-Cas9 reagents; Knockout efficiency

Stimulation; Choose the right screening format: Pooled vs Arrayed; Dual lentivirus/electroporation protocol; Algorithm designed; Optimising transfection/transduction conditions; Positive, negative and fitness controls

Variability

Intra-donor variability; Inter-donor variability

Technical replicates; Biological replicates

Scale up

Handling of plates and flask; Readout (editing efficiency and endpoint); Data acquisition and analysis

Automation/robotics, liquid handling systems; Validation studies; HTPautomation friendly methods; Software tools, computer power and scripting languages

Table 2. Challenges and solutions for CRISPR/Cas9-knockout screening with primary human cells

While bioinformatics and data mining help, experiments such as validation screens or compound cell panel screens are integral to move to the next stage.

Conclusion Researchers have made significant strides towards overcoming challenges associated with editing and screening primary human cells. As shown in Table 1, many of these insights are from recent years and are limited to proof-of-concept studies. FGS pipelines are a key tool in drug discovery; by efficiently identifying specific genes that are relevant to a particular phenotype, researchers

can more quickly identify drug targets that will be successful in clinical trials. However, the development of a robust, large-scale screening platform, especially for arrayed screening, is costly and currently only feasible for major pharmaceutical companies and specialist CROs. This dynamic is reflected by the emergence of only very few scientific reports sharing data – not due to lack of feasibility but rather linked to intellectual property concerns, proprietary methods, and internal policies.

REFERENCES 1.

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Wang, T. et al. Genetic Screens in Human Cells Using the CRISPR-Cas9 System. Science. 80–84 (2014).

Shalem, O. et al. Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science. 84–87 (2014).

Shifrut, E. et al. Genome-wide CRISPR Screens in Primary Human T Cells Reveal Key Regulators of Immune Function. Cell. 1958–1971 (2018). https://www.nature.com/articles/d42473-019-00168-7, visited on 20 Jun 2021.

Rautela, J. et al. Drug target validation in primary human natural killer cells using CRISPR RNP. J. Leukoc. Biol. 1397–1408 (2020).

Amirache, F. et al. Mystery solved: VSV-G-LVs do not allow efficient gene transfer into unstimulated T cells, B cells, and HSCs because they lack the LDL receptor. Blood. 1422–1424 (2014).

Finkelshtein, D. et al. LDL receptor and its family members serve as the cellular receptors for vesicular stomatitis virus. PNAS. 7306–7311 (2013). https://horizondiscovery.com/en/resources/featured-articles/ top-5-reasons-to-use-algorithm-designed-guide-rna, visited on 20 Jun 2021.

Dr. Verena Brucklacher-Waldert Verena is a Manager in the Cell-based Screening Department at Horizon Discovery, a PerkinElmer company, Cambridge, UK. She joined Horizon Discovery as Principal Scientist after supporting the Immuno-Oncology drug discovery/development pipeline in the biotechnology industry, as a Senior Scientist. A postdoctoral research fellowship from the German Research Foundation enabled her to intensify her studies in Immunology in Cambridge, UK, after receiving her doctorate in Neuroimmunology from the University of Tübingen, Germany.

Email: Verena.Brucklacher-Waldert@horizondiscovery.com

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Research / Innovation / Development

Are We Nearly There Yet? The Ongoing Journey of CAR-T Cell Therapies Brentjens et al. published results from the first clinical trial treating five adults with relapsed B cell acute lymphoblastic leukemia (B-ALL) with CD19-directed CAR-T cells in 2013.

These patients had a “dismal” prognosis, but all five achieved rapid tumour eradication and complete molecular remission post treatment.1 These remarkable results set the stage for a flurry of CAR-T research and clinical trials culminating in FDA approvals of two CAR-T therapies, Kymriah (Novartis) and Yescarta (Kite Pharma), in 2017.

These approvals were followed by Tecartus (Kite Pharma) in 2020, and Breyanzi and Abecma (both Bristol Myers Squibb) in 2021.

Generational improvements in CAR-T design CAR-T cells have a genetically engineered T-cell receptor (TCR) that directs their binding to cancer cells. In first generation CARs, the TCR was engineered to express a new, antigen binding domain, usually the single chain variable fragment (scFv) of an antibody. These early CAR-T cells showed promise in vitro, but in vivo displayed insufficient persistence and tumour killing activity.2 Second and third generation CAR-T cells addressed this by incorporating one or two co-stimulatory domains respectively into a first-generation CAR-T backbone.

This mimics the co-stimulation normally provided by antigen presenting cells (APC), which is required for full T-cell activation, and therefore improves CAR-T cell expansion, cell-killing activity and persistance.3 Fourth generation CAR-T’s aim for even greater improvements by further engineering CAR-T cells to release pro-inflammatory cytokines, like Interleukin-2 (IL-2), into the tumour microenvironment. This stimulates the innate immune system to divert endogenous immune cells towards the tumour, further enhancing tumour killing activity.3 The latter strategy is particularly advantageous, because it raises the possibility of an effective anticancer immune response even in the absence of universal expression of the target antigen on cancer cells, which is important since many cancers rely on antigen heterogeneity and spontaneous downregulation to evade the immune response. This limits the effectiveness of traditional CAR-T cell therapies, particularly in solid tumours where heterogeneous antigen expression is common.4 Breaking through the roadblock of solid tumours Poor antigen targeting, an immunosuppressive tumour microenvironment and inadequate T-cell infiltration, proliferation and persistence all contribute to CAR-T cells’ notorious lack of success in treating solid tumours to date.

Researchers have travelled numerous roads to overcome these obstacles, including the development of bi-specific CAR-T cells that target two antigens at once, or engineering CAR-T cells specific for a heterogeneously expressed cancer cell antigen to produce a bispecific T cell engager (BiTE) molecule.5,6 BiTEs are 24 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

recombinant bispecific proteins, with a different scFv fragment at each end; one binding to a target antigen on the cancer cell, the other to CD3 on the T-cell surface. In BiTE expressing CAR-T cells, the BiTE can recruit endogenous T-cells to kill cancer cells that don’t express the CAR’s target antigen, but localised expression reduces damage to healthy tissue outside the tumour site.6 Another tactic is to build ‘universal’ CAR-T cells, where, the T-cell is engineered to express a ‘universal’ antigen, for example a biotin-binding receptor or a leucine zipper. This can then be administered alongside scFvs targeting a range of antigens coupled to biotin or a leucine zipper binder, allowing a single system to target multiple cancer antigens without needing to re-engineer the T cells.7,8 Other teams have concentrated on mechanisms to improve CAR-T cell infiltration to the tumour site and persistence within it using oncolytic viruses to aid CAR-T cell recruitment to the tumour. In one effort, engineering an oncolytic virus with truncated CD19 resulted in enhanced CD19 on the surface of cancer cells. These were then targeted by CD19-directed CAR-T cells; a ‘belt and braces’ approach to cancer cell killing. Upon cell death, the dying cancer cells released additional copies of the CD19 encoding virus into the tumour microenvironment, which then infected neighbouring cells and further propagated the effect.9 Another approach, currently being tested in a Phase 1 clinical trial, uses an oncolytic adenovirus modified to express checkpoint inhibitors and stimulatory cytokines to improve HER2-directed CAR-T cell persistence and performance in mouse models of head and neck cancer.10,11 Checkpoint inhibition is another promising avenue of exploration for enhancing CAR-T effectiveness in solid tumours.

Indeed, using CRISPR gene editing to disrupt programmed cell death protein 1 (PD-1) on the surface of CAR-T cells has been shown to enhance tumour control and relapse prevention in in vivo models of breast cancer, demonstrating the potential of this approach.12 Finally, Klichinsky et al. describe using an adenoviral vector to introduce a CAR construct into macrophages, which then demonstrated antigen specific phagocytosis and tumour clearance in vitro and decreased tumour burden and prolonged overall survival in solid tumour mouse models.13 Navigating around CAR-T induced toxicity Even in haematological cancers, where CAR-T successes have been notable, it hasn’t always been plain sailing; in some trials impressive results have come at the cost of severe adverse events, and even death.

A major cause of CAR-T cell toxicity is the high levels of cytokines released by activated CAR-T cells. Once again, T-cell engineering allows for the development of several promising Autumn 2021 Volume 4 Issue 3

Research / Innovation / Development solutions to this. A phase 1 study of CD19-directed CAR-T cells with modifications to the CD8α molecule retained potent cytolytic activity with reduced cytokine release. Similarly, engineering the T cell to disrupt secretion of cytokines like GM-CSF or IL-1, which recruit monocytes and/or macrophages to the tumour site may also reduce cytokine release syndrome (CRS).14 Other strategies focus on putting the emergency brake on CAR-T cell activity in the event of CRS occurring. For example, in 2019 Mestermann et al. identified that the FDA-approved tyrosine kinase inhibitor dasatinib interferes with downstream CD3-ζ signalling, effectively halting all CAR-T cell activity without impacting cell viability in a mouse model of CRS.15 Accelerating towards off-the-shelf CAR-T cells The sheer logistical complexity of manufacturing an autologous cell therapy is likely the biggest challenge to widespread uptake of CAR-T cell therapies. The time from initial leukapheresis to reintroduction of cells to the patient, so-called ‘vein to vein’ time, can be problematic in patients with rapidly progressing disease.

Autologous therapies are also highly dependent on the number and quality of the patients’ T-cells, which may vary significantly between patients, and be impacted by age, disease burden, and previous treatment regimens.16 Finally, the inclusion of even a single malignant cell in the leukapheresis product may lead to its transduction with the CAR protein, thus protecting it – and its progeny – from CAR-T cell mediated cell death.17 Many of these challenges could be overcome with the use of allogeneic CAR-T cells. Leukapheresis from a single healthy donor can supply enough T cells to produce hundreds of doses, and availability of the cells prior to treatment allows time for more complex gene editing protocols as well as more stringent quality control.16 However, while this is an attractive proposition, significant risks of both Graft v Host disease (GvHD) and rapid clearance of the transplanted cells by the host immune system need to be addressed.

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One way to minimise the risk of GvHD is to use gene editing to target the constant region of the T cell receptor alpha chain (TRAC) and therefore disrupt endogenous TCR signalling in allogeneic CAR-T cells. This has been demonstrated clinically in two infants treated with allogeneic CAR-Ts for B-ALL on compassionate grounds, and again in two subsequent Phase 1 trials of adults and children with the same disease, demonstrating the safety and feasibility of allogeneic CAR-T cell therapies.18,19 Alternatively, NK cells, which don’t contain a TCR, can be used as the starting material to avoid GvH complications.

CAR-NK cells have demonstrated marked efficacy in vitro and in vivo, without corresponding CRS or GvHD; suggesting a promising alternative form of immunotherapy with simpler and faster manufacturing requirements.20 Aside from avoiding GvHD, it’s also important to put strategies in place to prevent immediate host rejection of donor T-cells. In the first allogeneic T cell clinical trials, this was accomplished by reinforced lymphodepletion prior to treatment with an anti-CD52 antibody. The donor cells were edited to delete CD52, protecting them from immunosuppression, and giving them time to work.18,19 Another possibility is to build a bank of T cells from donors with different HLA alleles, to offer at least partial HLA matching to the majority of the patient population. However, partially matched T cells are still unlikely to persist for as long as their autologous equivalents.16 Continuing the journey Enthusiasm for CAR-T cell therapies remains undimmed, despite numerous scientific, logistical, and manufacturing challenges.

We haven’t yet reached the final destination – readily available, rapidly produced and cost-effective CAR-T cell therapies, but with so many ingenious and innovative approaches under investigation, and such collective determination to succeed, we can certainly expect to make rapid progress towards that goal over the next few years.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 25

Research / Innovation / Development 8.

9.

10.

11.

12.

13.

14.

15.

16.

17.

Advances in targeted gene editing are paving the way for precisely edited CAR-T cells, accelerating progress towards increasingly potent CAR-T cell therapies with reduced side effects, allogeneic CAR-T cells, and CAR-T cell therapies for solid tumours.

REFERENCES 1.

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Brentjens RJ, Davila ML, Riviere I, et al. CD19-targeted T cells rapidly induce molecular remissions in adults with chemotherapyrefractory acute lymphoblastic leukemia. Sci Transl Med. 2013 Mar 20;5(177):177ra38. doi: 10.1126/scitranslmed.3005930.

Brocker T, Karjalainen K. Signals through T cell receptor-zeta chain alone are insufficient to prime resting T lymphocytes.J Exp Med. 1995;181(5):1653-1659. doi:10.1084/jem.181.5.1653 Benmebarek, M.-R.; Karches, C.H.; Cadilha, B.L.; Lesch, S.; Endres, S.; Kobold, S. Killing Mechanisms of Chimeric Antigen Receptor (CAR) T Cells. Int. J. Mol. Sci. 2019, 20, 1283. https://doi.org/10.3390/ ijms20061283 Huang, R., Li X., He Y., et al. Recent advances in CAR-T cell engineering. Journal of Hematology & Oncology (2020) 13:86 https://doi.org/10.1186/s13045-020-00910-5 Dai H, Wu Z, Jia H, et al. Bispecific CAR-T cells targeting both CD19 and CD22 for therapy of adults with relapsed or refractory B cell acute lymphoblastic leukemia. J Hematol Oncol. 2020 Apr 3;13(1):30. doi: 10.1186/s13045-020-00856-8.

Choi BD, Yu X, Castano AP, Bouffard AA, Schmidts A, Larson RC, Bailey SR, Boroughs AC, Frigault MJ, Leick MB, Scarfò I, Cetrulo CL, Demehri S, Nahed BV, Cahill DP, Wakimoto H, Curry WT, Carter BS, Maus MV. CAR-T cells secreting BiTEs circumvent antigen escape without detectable toxicity. Nat Biotechnol. 2019 Sep;37(9):1049-1058. doi: 10.1038/s41587-019-0192-1.

Urbanska K, Lanitis E, Poussin M, Lynn RC, Gavin BP, Kelderman S, Yu J, Scholler N, Powell DJ Jr. A universal strategy for adoptive immunotherapy of cancer through use of a novel T-cell antigen receptor. Cancer Res. 2012 Apr 1;72(7):1844-52. doi:

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10.1158/0008-5472.CAN-11-3890.

Cho JH, Collins JJ, Wong WW. Universal Chimeric Antigen Receptors for Multiplexed and Logical Control of T Cell Responses. Cell.

2018;173(6):1426-1438.e11. doi:10.1016/j.cell.2018.03.038 Park AK., Fong Y., Kim S., et al. Effective combination immunotherapy using oncolytic viruses to deliver CAR targets to solid tumours.

Science Translational Medicine 02 Sep 2020:Vol. 12, Issue 559, eaaz1863 DOI: 10.1126/scitranslmed.aaz186 Wang D. Binary Oncolytic Adenovirus in Combination With HER2-Specific Autologous CAR VST, Advanced HER2 Positive Solid Tumors (VISTA). https://clinicaltrials.gov/ct2/show/NCT03740256 Rosewell Shaw A, Porter CE, Watanabe N, et al. Adenovirotherapy Delivering Cytokine and Checkpoint Inhibitor Augments CAR T Cells against Metastatic Head and Neck Cancer. Mol Ther.

2017;25(11):2440-2451. doi:10.1016/j.ymthe.2017.09.010 Hu W, Zi Z, Jin Y, Li G, Shao K, Cai Q, Ma X, Wei F. CRISPR/ Cas9-mediated PD-1 disruption enhances human mesothelintargeted CAR T cell effector functions. Cancer Immunol Immunother.

2019 Mar;68(3):365-377. doi: 10.1007/s00262-018-2281-2.

Klichinsky M, Ruella M, Shestova O., et al. Human chimeric antigen receptor macrophages for cancer immunotherapy. Nat Biotechnol.

2020 Aug;38(8):947-953. doi: 10.1038/s41587-020-0462-y.

Rafiq, S., Hackett, C.S. & Brentjens, R.J. Engineering strategies to overcome the current roadblocks in CAR T cell therapy. Nat Rev Clin Oncol 17, 147–167 (2020). https://doi.org/10.1038/s41571019-0297-y Mestermann K, Giavridis T, Weber J, Rydzek J, Frenz S, Nerreter T, Mades A, Sadelain M, Einsele H, Hudecek M. The tyrosine kinase inhibitor dasatinib acts as a pharmacologic on/off switch for CAR T cells. Sci Transl Med. 2019 Jul 3;11(499):eaau5907. doi: 10.1126/ scitranslmed.aau5907.

Aftab, B., Sasu, B., Krishnamurthy J., et al. Toward “off-the-shelf” allogeneic CAR T cells. Advances in Cell and Gene Therapy, 2020 Jul; 3(3)e86. https://doi.org/10.1002/acg2.86 Ruella M, Xu J, Barrett DM, et al. Induction of resistance to chimeric antigen receptor T cell therapy by transduction of a single leukemic B cell. Nat Med. 2018 Oct;24(10):1499-1503. doi: 10.1038/s41591018-0201-9.

Qasim W, Zhan H, Samarasinghe S, et al. Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells. Sci Transl Med. 2017 Jan 25;9(374):eaaj2013. doi: 10.1126/ scitranslmed.aaj2013 Benjamin R, Graham C, Yallop D, et al; UCART19 Group. Genomeedited, donor-derived allogeneic anti-CD19 chimeric antigen receptor T cells in paediatric and adult B-cell acute lymphoblastic leukaemia: results of two phase 1 studies. Lancet. 2020 Dec 12;396(10266):1885-1894. doi: 10.1016/S0140-6736(20)32334-5.

Xu X., Huang W., Heczey A. et al.NKT Cells Coexpressing a GD2-Specific Chimeric Antigen Receptor and IL15 show enhanced in vivo persistence and antitumor activity against neuroblastoma.

Clin Cancer Res. 2019; 25: 7126-7138

Dr. Sophie Lutter Dr. Sophie Lutter is Scientific Communications and Marketing Manager at OXGENE, a WuXi Advanced Therapies company. OXGENE provides end-to-end research services to cell and gene therapy companies seeking to discover, develop, manufacture and test innovative drug candidates at scale for global commercialization. OXGENE's proprietary technologies and automation platforms for molecular discovery are seamlessly integrated with a full suite of technologies for cell and gene therapy manufacturing.

Web: www.oxgene.com

Autumn 2021 Volume 4 Issue 3

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 27

Pre-Clinical & Clinical Research

Viral Vector Engineering to Improve Clinical Performance and Accelerate Timeline to Success for Novel Gene Therapies Viruses are known to be dangerous pathogens which invade host cells and hi-jack their cellular machinery to direct the replication and transcription of their own genome.

They are also known as being ideal couriers to transport new DNA into a host cell and ensure that it’s transcribed.

Advanced therapy manufacturers have taken the head start that nature’s given them and used advances in genetic engineering, computational biology, and bioinformatics to perfect the design of the viral particle as a vector for therapeutic DNA and hasten the clinical success of groundbreaking new treatments. This article will look into three aspects of viral biology that make them efficient delivery vehicles for gene therapies, and some of the ways that biologists have taken advantage of these properties to improve clinical performance.

When efficient viral infection is a good thing Maintaining open channels of communication between cells is essential for nutrient transport, signal transduction and other forms of cell-cell interaction, in normal physiological conditions. Viruses are adept at exploiting these channels to hitchhike into the cell. For example, a virus often used as a vector for gene replacement therapies such as adeno associated virus (AAV), enters the host cell by interacting with cell surface receptors, leading to endocytosis from clathrin coated pits. From there, a change in pH triggers the release of the virus from the endosome, where it is rapidly trafficked to the cell nucleus.1 Which cells the virus infects, and how efficiently it enters them, is defined by the specific cell surface receptors it interacts with. This is known as viral tropism. So far, nine different AAV serotypes have been discovered in human cells, and a further four in non-human primates, each of which have different tropisms.2 The first AAV to be discovered was AAV2, which binds to the near ubiquitously expressed heparin sulfate proteoglycan (HSPG), resulting in a broad tissue tropism.3 Other AAV serotypes have different cell surface receptors, and therefore different tropisms, which in turn informs the choice of viral vector most appropriate for each gene therapy.

Efficient delivery of gene therapy vectors remains a challenge despite the divergent tropisms offered by different AAV serotypes. Natural exposure to AAV means that large sections of the population have developed neutralising antibodies against certain viral serotypes. Delivery to the target tissue can be difficult; for example, systemic administration of a viral vector often results in trafficking to the liver, which may not be the intended target. Access to the target tissue may present another challenge, for example the virus may get ‘stuck’ on HSPGs and other proteoglycans on the extracellular matrix outside the target cells.4 Indeed, any given AAV capsid may have both advantages and disadvantages for a particular clinical use.

28 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

But where nature has set challenges for the use of viral vectors to deliver gene therapies, science is striving to overcome them, in this case by capsid engineering to improve transduction of the target cell type, improve specificity of transduction and/or to evade a host immune response.

The first approach to this, termed rational design, relies on understanding the mechanism of transduction, and identification of the areas of the viral genome responsible for encoding the capsid’s interaction with the cell surface receptor.5 This allows the insertion of the viral genome sequence conferring the desired tropism into the genome of another AAV serotype, which may have other clinical advantages.

This approach has already proved viable in a clinical trial for Duchenne Muscular Dystrophy, where the capsid region responsible for the efficient transduction of skeletal muscle

by AAV1 was engineered into AAV2, whose safety profile was better characterised and could be easily purified.6 However, the exact mechanism of viral transduction isn’t yet clear for all AAV serotypes, so a more random approach to capsid engineering, termed directed evolution, may sometimes be necessary. This involves using the wildtype cap genes as a starting point to create large genetic libraries either by error-prone PCR to introduce random point mutations into the cap gene or random insertion of peptide-encoding sequences (for example those encoding a known cell surface receptor binder). Alternatively, mutations can be targeted to hypervariable regions of the capsid, such as surface-exposed loops, which can then be replaced by bioinformatically designed peptide sequences.4 The resulting viral particles can then be selected for transduction of the desired cell type in vitro or in vivo.

When turning the dial on viral genome expression matters Once they’ve entered the cell, viruses are adept at wresting control of the cell’s DNA replication machinery to force the transcription of their own genome. When the genes that would enable viral replication are replaced inside the virus particle by a therapeutic transgene, it is this DNA that the cell is forced to transcribe.

Almost all gene expression is driven by a promoter; a core region of DNA approximately 100bp upstream of the regulated gene plus an upstream activating sequence or enhancer.

Both core promoter regions and enhancers contain clusters of transcription factor binding sites, and transcription factor binding in these areas determines the level of gene expression.

Generally, either promoters endogenous to the target cell/ tissue type or viral promoters are used to drive transgene expression. Tissue specific endogenous promoters have the advantage of limiting gene expression to the target cells; however, typically low promoter activity or large promoter size may lead to sub-optimal expression levels. Viral promoters on the other hand are constitutively active, resulting in high levels Autumn 2021 Volume 4 Issue 3

Pre-Clinical & Clinical Research of therapeutic transgene expression in all transduced cells.

This carries a risk of toxicity due to transgene over-expression or stimulation of an immune response due to transgene expression within an antigen presenting cell.7 Engineering the promoter driving transgene expression therefore represents another opportunity to improve the clinical performance of the viral vector.

Error-prone PCR can again be used to insert random point mutations into promoter sequences to change transcription factor binding sites and alter promoter strength. However, this approach is most likely to prevent transcription factor binding and therefore reduce the strength of the promoter8.

While this may reduce the risks associated with transgene overexpression from a viral promoter, it won’t stop non-specific gene expression. Instead, hybrid promoters built by screening libraries of different promoter and enhancer combinations are likely to present a better option for generating tissue specific promoters with increased transcriptional strength.8 When harnessing the power of viral genome replication can aid manufacture Gene therapy manufacture relies on being able to produce huge quantities of viral vectors, using cells as mini virus-production factories. However, one of the properties of AAV that make it ideal as a vector for gene therapies – its inability to replicate without the help of a second virus – adds challenges for its manufacture. In nature, this help is provided by adenovirus, but this isn’t a system that’s easily co-opted for clinical production, because the adenoviruses must be purified out from the AAV before they’re suitable for clinical use, a complex and expensive process. To date, the adenoviral help necessary for producing AAV destined for clinical use is provided by a plasmid encoding an adenoviral genome that’s been pared back to only those genes essential for AAV replication. However, this is a far from ideal process, and the complexities and costs associated with plasmid-based AAV production at scale have been widely reported.

Adenoviruses, on the other hand, are extremely efficient at replicating themselves once they’ve gained control of the host cell systems. Indeed, following adenovirus infection, almost 90% of the host cells’ mRNA is derived from the adenoviral

genome. Here, viral vector engineering offers an opportunity to harness the power of adenoviral protein production and use it to improve the manufacture of cell and gene therapies.

In this case, scientists have done this with a clever two-step manipulation of the adenoviral genome. The first step was to manipulate the genome in such a way that it could still provide help for AAV production, but no longer transcribe adenoviral structural proteins. This worked because the adenoviral genome is divided into two temporal phases with distinct purposes. All the genes responsible for helping AAV to replicate are transcribed in the early phase, while the adenoviral structural proteins are all transcribed from late phase genes, under the control of the major late promoter (MLP). Inserting a Tet repressor binding site into the MLP, and the Tet repressor gene itself into the late region of the genome, essentially cripples adenoviral production; the more the adenovirus tries to transcribe its structural proteins, the more Tet repressor it produces to inhibit MLP activity (figure 1).

The second manipulation involves inserting either the AAV rep and cap, or the ITR flanked therapeutic transgene, into the early phase of the adenoviral genome. Now, the adenoviral genome can no longer produce adenovirus particles, but infecting cells with two of these edited adenoviral vectors is sufficient to produce high quantities of fully functional AAV9 (figure 2). Wildtype adenovirus and AAV have co- evolved and fine-tuned the supported replication of AAV, so it’s perhaps unsurprising that plasmid-based AAV production cannot replicate the AAV yield or quality of wild-type adenovirus.10 However, this adenoviral genome engineering approach retains all the natural adenoviral helper functions for AAV production, suggesting a manufacturing system that would produce higher yields of more infectious AAV than can be produced by triple transfection.

Over a period of a few years, our understanding of viral biology and genetics has increased exponentially, as has the availability of the tools required to manipulate them. This has led to the creation of ever more powerful viral vectors that can be targeted to specific cell types, fine tune expression of the therapeutic transgene within them, and – importantly for the accessibility of these new biologics – the ability to manufacture in greater quantities, at better quality and cost-effectiveness, and with greater ease. Indeed, the FDA expects to be approving

Figure 1: Inserting a Tet repressor binding site into the MLP of the adenoviral genome with a TetR gene downstream introduces a negative feedback loop, where the adenoviral genome cannot transcribe any late phase genes, which encode the structural proteins, in the absence of doxycycline. www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 29

Pre-Clinical & Clinical Research

Figure 2: Infecting HEK293 cells with engineered adenoviruses encoding the AAV genome, and AAV rep and cap genes respectively, results in the production of AAV.

between 10–20 new cell and gene therapies annually by 2025, suggesting that we are entering a new golden era for genetic medicine.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

Bartlett JS, Wilcher R, Samulski RJ. Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors. J Virol.

2000 Mar;74(6):2777-85. doi: 10.1128/jvi.74.6.2777-2785.2000.

Srivastava A. In vivo tissue-tropism of adeno-associated viral vectors. Curr Opin Virol. 2016 Dec;21:75-80. doi: 10.1016/j. coviro.2016.08.003.

Summerford C, Samulski RJ. Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.

J Virol. 1998; 72(2):1438–1445.

Kotterman MA, Schaffer DV. Engineering adeno-associated viruses for clinical gene therapy. Nat Rev Genet. 2014 Jul;15(7):445-51. doi: 10.1038/nrg3742.

Li, C., Samulski, R.J. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet 21, 255–272 (2020). https://doi. org/10.1038/s41576-019-0205-4 Bowles DE, McPhee SW, Li C et al. Phase 1 gene therapy for Duchenne muscular dystrophy using a translational optimized AAV vector. Mol Ther. 2012 Feb;20(2):443-55. doi: 10.1038/ mt.2011.237.

Domenger C, Grimm D. Next-generation AAV vectors-do not judge a virus (only) by its cover. Hum Mol Genet. 2019 Oct 1;28(R1):R3-R14. doi: 10.1093/hmg/ddz148 Blazek J, Alper HS. Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol J. 2013 Jan;8(1):46-58. doi: 10.1002/biot.201200120.

Su W, Duffy M, Seymour L et al., A Complete “Self-Repressing” Adenovirus System Enables Efficient Manufacture of Adeno-

30 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Associated Viral Vectors without Contamination by Adenovirus or Small Drugs (2020), MOLECULAR THERAPY, 28, 240 – 240 (conference paper) 10. Zeltner, N., Kohlbrenner, E., Clément, N. et al. Near-perfect infectivity of wild-type AAV as benchmark for infectivity of recombinant AAV vectors. Gene Ther 17, 872–879 (2010). https://doi.org/10.1038/ gt.2010.27

Dr. Sophie Lutter Dr. Sophie Lutter is Scientific Communications and Marketing Manager at OXGENE, a WuXi Advanced Therapies company. OXGENE provides end-to-end research services to cell and gene therapy companies seeking to discover, develop, manufacture and test innovative drug candidates at scale for global commercialization. OXGENE's proprietary technologies and automation platforms for molecular discovery are seamlessly integrated with a full suite of technologies for cell and gene therapy manufacturing.

Web: www.oxgene.com

Autumn 2021 Volume 4 Issue 3

YOUR BRIDGE BETWEEN LIFE-CHANGING THERAPIES AND PATIENTS

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talkfuture@pciservices.com INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 31

Pre-Clinical & Clinical Research

PEER REVIEWED

E&L Standards: Empowering Confident Labware Choices Scientists across every discipline strive to design and conduct experiments that will deliver clear and meaningful results. Doing so demands meticulous attention to detail in assessing all aspects of the experimental setup, understanding the variables involved, and anticipating and minimising potential unknowns. One critical but easy-to-overlook factor is the influence of the labware used.

One possibility, in particular, is the interference from extractables and leachables (E&Ls) that may be present due to the materials used to manufacture the labware, including both glass and plastic labware. While these chemical stowaways are often inert, it is crucial to be aware of their presence and be mindful of their potential impact on the experimental process. Testing and validation of labware is essential in establishing confidence in the quality and reproducibility of the experimental results that will be generated. This article examines the importance of E&L standards and their application for risk assessment during product selection and offers insight into relevant regulatory guidance and available E&L testing methods.

The Key Issue All labware has some E&L content. It is important to recognize that extractables and leachables are two different things.

Extractables are organic compounds or metals removed from a material under extreme conditions, such asexposure to high heat or very high/low pH, whereas leachables are those that are removed under normal conditions of use. Extractables are potential leachables, but not all extractables will become a leachable under each set of specific operating conditions.

Everything from glass and plastic to packaging materials and even label inks can leach unwanted contaminants.

Many of the additives used in plastics manufacturing, including plasticisers, pigments, lubricants, stabilisers, antioxidants and slip agents, can be an extractable and, therefore, a leachable under certain conditions. It is important to note that nearly all products made from plastics, including food packaging, toys and medical supplies, require the use of additives to ensure they deliver the needed properties of structural integrity, product performance, sterility and protection against environmental factors that would otherwise lead to degradation. Different resins may need more additives than others. A good example is that polypropylene (PP) and high-density polyethylene (HDPE) typically contain more additives than polyethyleneterephthalate (PET) or polyethylene terephthalate glycol (PETG). PP and HDPE used in products stored outdoors will often include both antioxidants and anti-UV agents as protective agents.

Although additives are necessary components in plastic materials, they also pose a risk of becoming leachables under 32 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

normal use conditions, and their passive migration from a piece of labware into a sample may result in unintended consequences.

High levels of additives in a plastic filtration device, for example, could lead to contamination of the filtrate. Labware from which there might be leaching has the potential to affect not only short-term experimental work but also the integrity of laboratory solutions stored for extended periods. Therefore, it is important to understand the risks associated with specific labware and consider the impact of E&Ls on an experiment or to assess the consequences of their accumulation throughout a process. The optimum scenario for all labware is that there are no leachables under normal use conditions. With so many different variables to consider when designing experiments, any opportunity to minimise unknowns is welcome. One of the best resources for understanding potential leachable contaminants is the manufacturer, who should provide significant insight and support.

Making an informed choice about labware requires access to E&L testing to understand all the factors likely to impact an experiment. Labware suppliers, therefore, have a duty to provide extractables analysis on the raw resin to aid the identification of potential leachables. This data provides users the tools needed to assess potential riskto their process. Although most E&Ls are not problematic for end-users can make that assessment themselves and conduct a leachables study under normal in-use conditions, if deemed necessary. Several regulatory bodies have issued guidelines and best practices for E&L testing. Key among these for laboratory products is the widely accepted United States Pharmacopeia’s (USP) <661.1> Plastic Materials of Construction,1 which outlines testing guidelines and specifications for raw resins to ensure the material is well characterised and suited for its intended use.

Standardised Testing of Plastic Materials No single test can identify all the compounds present in a sample. For example, detecting elemental impurities, such as metals, involves Inductively Coupled Plasma-Mass Spectrometry (ICP-MS). Volatile and semi-volatile compounds are typically detected using Gas Chromatography-Mass Spectrometry (GC–MS). Liquid Chromatography-Mass Spectrometry (LC-MS) is widely applied to the analysis of non-volatile organic compounds. Properly conducted E&L studies require the use of a range of analytical technologies if they are to deliver the complete picture.

In addition to USP guidelines, additional regulatory bodies issuing E&L testing guidelines include the US Food & Drug Administration (FDA), the International Standards Organization (ISO), and the Product Quality Research Institute (PQRI). Each standard will differ slightly and is typically based on the product's intended use – medical devices versus pharmaceutical packaging. If a manufacturer wishes to test a material whose intended use is not an exact fit with any of these guidelines, then either the best fit can be selected or Autumn 2021 Volume 4 Issue 3

Pre-Clinical & Clinical Research a new method may be developed and validated. It is widely accepted, however, that using a standard method enables clear comparisons and avoids a lengthy validation process.

then assess the impact they may have. This reinforces the need for labware suppliers to perform E&L testing on their products and provide scientists with the necessary information.

USP <661.1> provides guidance and specifications for testing raw resins. Resins that pass the guideline’s criteria are acceptable in packaging systems for pharmaceutical use.

It outlines the analyses necessary for a complete understanding of a material’s composition. Most resins must be tested for material identification, physicochemical properties and plastic additive composition. PET, PETG and polycarbonate (PC), however, typically contain few or no additives, so additive testing is not a requirement. PC guidelines do require additional tests for bisphenol A (BPA), a breakdown product of PC, and also for the presence of residual solvents from the manufacturing process.

How Different Can It Be? An examination of four different filtration receiver bottles illustrates the importance of careful selection when considering labware. In this study, extractables testing was carried out using methods based on USP <665> requirements. Triplicates from the same lot of polystyrene receiver bottles from three different manufacturers were tested with 100 ml of three extraction solutions: water, 50:50 ethanol/water, and 2% nitric acid in water. The receiver bottles containing the extraction solutions were incubated for 21 days at 50oC prior to analysis.2

Although the analyses outlined in USP <661.1> are essential in gaining a comprehensive understanding of a material's composition, it is the plastics’ additive and elemental analyses that are often of greatest importance to end-users. For example, when plastics are oxidised they can become brittle and break apart. Antioxidants are added to avoid this. With polyolefin materials, such as high-density polyethylene and low-density polyethylene (LDPE), and also for homopolymer and co-polymer polypropylenes, USP <661.1> defines specification limits on 13 common plastic additives that are characterised as phenolic antioxidants, non-phenolic antioxidants and slip agents. USP Plastic Additive 2 (pentaerythritol tetrakis (3-(3,5-di-tert-butyl-4hydroxyphenyl) propionate )) and USP Plastic Additive 5 (tris (2,4-di-tert-butylphenyl) phosphite) are phenolic antioxidants that are often used to work synergistically to react with oxygen, thereby protecting the plastic from oxidation.

However, in an application like cell culture, USP Plastic Additive 5 can oxidise and form breakdown products that are known to inhibit the growth of certain cell lines. Material for use in products for cell culture should, therefore, be free of this compound.

Three analyses were then run on each of the extracts. Total organic carbon (TOC) was measured from the water extracts using a laboratory TOC analyzer. Measurements of λmax absorbance in the 50:50 ethanol/water extracts were made by UV-Vis spectrophotometry. Finally, ICP-MS was applied for elemental analysis of the 2% nitric acid in water. Figures 1, 2 and 3 show comparisons at the two ends of the results scale with products from supplier A and supplier B.

Figure 1. TOC results from testing receiver bottles from two different suppliers.

This is one of the few examples of a known impact of a plastic additive on a specific application. It is often up to the end-user to test and validate labware, but suppliers can perform E&L testing per regulatory guidelines, such as USP <661.1>, to ensure confidence in labware selection.

USP <661.1> outlines testing of raw resins, but a separate guideline, USP <665>, outlines testing of molded products.

When plastic is molded, some of the antioxidants are oxidised, and trace metals from the molding equipment may transfer to the surface of the molded part. Therefore, the molded part's organic and elemental E&L profile will be slightly different from the raw resin. The USP <665> guideline is being used across the industry for standardised testing of leachables from plastic parts used in pharmaceutical manufacturing. The tests include E&L profiling of three separate solvent systems at high pH, low pH and in an organic solvent. There are no standard criteria for the results, and so they are often presented in the form of a report.

The actual impact of any leachables will be highly dependent on how and for what purpose a piece of labware is being used.

Here, the vendor can provide extensive support to the user by providing a full analysis of potential leachables. The user must www.international-biopharma.com

Figure 2. Absorbance (λmax) results from testing receiver bottles from two different suppliers.

Figure 3. Metal analysis results from testing receiver bottles from two different suppliers.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 33

Pre-Clinical & Clinical Research TOC results showed 996 parts per billion (ppb) from supplier A's bottle compared with 2074 ppb for supplier B. TOC is often the result of catalysts used during polymer synthesis, from the polymer itself, or slip agents used in molding. UV-Vis absorbance data at 245 nm shows results for supplier A to be 0.153 compared with 0.217 for supplier B. Increased absorbance is directly proportional to an increased concentration of extractables when comparing similar λmax values. Finally, there were no detectable metals in supplier A's bottle, whereas silicon (28Si), titanium (47Ti) and tantalum (181Ta) were present in supplier B's bottle.

These results illustrate the importance of knowing the properties of the materials being used in an experimental process. Lowering the levels of extractables provides less chance of those compounds leaching into the sample, and it is clear that not all materials are equal.

Knowledge is Power The sheer number of E&Ls can still be daunting. This is why testing must begin with a detailed review of the materials used in labware. E&L data has a crucial role in risk assessment during product selection, and enables the avoidance of materials that contain additives or elements with the potential to impact specific experiments. Resins that pass USP <661.1> criteria can be confidently used for pharmaceutical purposes, and the data can provide users with knowledge to select the best product for their application. This data can also help scientists identify potential leachables in their specific application.

The level of transparency provided by the supplier can be another key consideration. Materials, specifications and processes may periodically change from time to time for various reasons. It is critical that the supplier has a systematic approach in place toalert customers to changes so that they can assess any potential impact to their experimental work. Digging deeper into understanding your labware’s E&L profile will empower you and leave you feeling confident in your selection.

REFERENCES 1.

2.

3.

<661.1> Plastic Materials of Constructionhttps://www.uspnf.com/ sites/default/files/usp_pdf/EN/USPNF/revisions/661.1_rb_notice. pdf(Accessed 2 June 2021).

Application note. 'Are the extractables for Nalgene Rapid-Flow receiver bottles lower when compared to similar devices?'

ThermoFisherScientific.https://assets.thermofisher.com/TFS-Assets/ LPD/Application-Notes/SN_ELRapidFlow_final.pdf(Accesses 2 June 2021).

Extractables and leachables: Regulatory requirements for vaccine and biologic products. Thermo Fisher Scientific. http://tools. thermofisher.com/content/sfs/brochures/Extractables%20and%20 leachables_Whitepaper_W11.pdf (Accessed 16 June 2021).

Sarina Bellows Sarina Bellows, Ph.D., is an R&D Staff Scientist for Thermo Fisher Scientific’s plastic labware.

Sarina is also a chemist and subject matter expert in extractables and leachables, working closely on new product development within Thermo Fisher.

34 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Autumn 2021 Volume 4 Issue 3

LIFE SCIENCES

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 35

Manufacturing

Residual Impurities in Biopharmaceutical Products Process-related impurities or residual impurities are formed at any time during upstream or downstream processes. They are compounds that are present at very low concentrations in complex biomanufactured products and can vary from large proteins to small chemicals. Therefore, the proper detection and evaluation of residuals requires extensive knowledge of biotherapeutics manufacturing as well as solid expertise in current analytical methodology.

This article reviews the most common types of residual impurities and identifies appropriate monitoring methods for a successful adaptive control strategy.

Introduction Biopharmaceuticals represent a wide range of therapeutic drugs manufactured in living cells or organisms. The characterisation of these products is particularly difficult because biopharmaceuticals are composed of several different structures, more commonly referred to as variants. Variants of the biopharmaceutical product presenting different safety and/or efficacy profiles are defined as product-related impurities. On the other hand, process-related impurities or residual impurities are not structurally related to the intended biopharmaceutical drug and can occur at any time during the upstream or downstream process. This makes their detection and evaluation particularly difficult, requiring extensive knowledge of the biopharmaceutical drug manufacturing process.

Different impurity types can form at various stages during the manufacturing process. It should be noted that adventitious viruses, endotoxins and mycoplasma are considered as contaminants, not impurities, and therefore will not be discussed in this article.

Types of Impurities: •

The first impurity type to be considered are upstream impurities, which are split into two categories, cellsubstrate derived and cell culture-derived impurities.

Examples of cell-substrate derived impurities are host cell proteins (HCPs), DNA residues or virus-like particles.

Examples of cell culture-derived impurities include antibiotics, inducers, antifoam or media components, and virus inactivating agents.

•

The second type are downstream impurities, are common components such as purification reagents (chromatographic solvents, buffers), column and tubing leachates, or metals.

•

The third important type of residuals are raw or ancillary material related impurities. Compendial monographs often provide useful standardised tests and specifications for assessing the purity, presence and concentration of raw and ancillary materials.

36 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

•

The fourth and final category of impurities are formed during the storage phase of the product and may interact with the biotherapeutic itself. Hence, monitoring of key stability indicators such as critical Quality Attributes (or cQAs) of the drug substance is an essential step of the drug development pathway. The appearance of non-product related impurities during storage should always be part of the Control Strategy.

Biopharmaceutical Characteristics The concept of a well characterised biopharmaceutical product is usually acknowledged as the physico-chemical and biological characterisation of the drug substance itself. However, the identification and quantification of both product-related and process- related impurities is also an integral part of the definition of a well characterised product.

One of the goals of process control and monitoring during the manufacturing process is to ensure the safety and efficacy of the final product as well as to minimise any unwanted effects. Ensuring that a biopharmaceutical is safe can only be done by filing a complete and well-defined product profile. For well-known biologic modalities such as Monoclonal Antibodies (MAbs), proteins expressed in Chinese Hamster Ovarian (CHO) or Escherichia Coli cells, the permissible levels of the most common impurities (such as protein A, aggregates, or HCPs…) is well documented. Even the removal of these common impurities is part of established adaptive control strategies.

For other, less-common classes of biologic drugs, including Antibody Drug Conjugates (ADCs), Gene Therapy products, Oligonucleotides (DNA, RNA), Cell Therapy products, or Enzymes, in depth studies are required to establish efficient monitoring of process related impurities. This starts with defining the Quality Target Product Profile (QTPP), followed by identifying which of the possible process related impurities will be classified as cQAs. Residual impurities Examples of commonly encountered residual impurities are listed in the table below, together with suggested monitoring methods. The impurities can be segregated into two major groups, chemical-based and biological-based.

Most biological-based impurities such as residual DNA, host cell proteins, or fetal bovine serum, require specific monitoring methods such as PCR or Immunoassays. On the other hand, online LC-MS could address most of the commonly encountered chemical-based impurities. This is why real-time, direct analysis will play an increasingly important role in control of the manufacturing process of biopharmaceuticals. Because of its versatility, LC-MS is a perfect example of a real-time, direct analysis technique that can become an essential tool for In Process Control (IPC) monitoring. This will ultimately facilitate the implementation of Quality by Design (QbD) criteria in the manufacturing of Autumn 2021 Volume 4 Issue 3

Manufacturing biotherapeutics, which will in turn result in significant time and cost savings.

5.

However, the analysis of aggregates or particles in solution is particularly challenging, mainly due to their broad size range which can vary by up to six orders of magnitude1 from soluble dimers to large, insoluble aggregates. Once identified, these impurities should be clearly classified as either process related or product related, to allow for a better control strategy. Since no analytical technique is available to cover a six order of magnitude size range, a combination of analytical methods is needed to accurately characterise this type of impurity. Finally, the presence of trace metals can be monitored with Inductively Coupled Plasma (ICP) Spectroscopy.

The regular improvement of existing methodologies or the development of new ones, helps in continuously lowering the sensitivity levels for detection and measurement of process related impurities. These technological advancements, especially in Mass Spectrometry, make it possible to monitor various impurities and product quality attributes in parallel with the development of a Multiple Attribute Monitoring (MAM) concept.3 Moreover, if connected to a production stream, MAM may allow real-time, in-process control so that production parameters can be instantly adjusted.

IMPURITY TYPES Cell substrate derived: Host cell protein Host residual DNA and RNA Virus like particle

Upstream

Downstream

Cell culture derived: Antibiotic (Amoxicillin, Chloramphenicol, Kanamycin...)

Antifoam (Tween, Pluronic acid, PPG...)

Biological Ingredients (Insulin, FBS...)

Chelating agents such EDTA Inducers such IPTG Metal ions Process enhancing agents (DTT, Glutathione...)

Selective agent such Methotrexate Solubilizers (Guanidine, Urea...)

Buffer components (Citrate, Glycine, Imidazole, Sucrose, Tris...)

Leachates (columns, tubing) Protein A Residual solvents (Acetonitrile, DMF, DMSO...)

Visible particles Subvisible particles

MONITORING METHOD Immunoassays, MS PCR Nanoparticle tracking analysis, SEC-MALS HPLC, MS HPLC, MS HPLC, Immunoassays HPLC, MS HPLC, MS ICP HPLC, MS HPLC, MS HPLC, MS

HPLC, MS HPLC, MS HPLC, Immunoassays HPLC, MS Appearance Light obscuration, Liquid particle counter, Micro-flow imaging, Nanoparticle Tracking Analysis (NTA), Direct Light Scattering (DLS), Size Exclusion Chromatography-Multi Angle Light Scattering (SECMALS), Transmission Electron Microscopy (TEM)

any process related impurities to an acceptable level Set the acceptance criteria for the residual impurities in the DS and DP

Examples of In Process Control (IPC) During the second chromatographic step of the downstream processing of a Monoclonal Antibody (Mab), an aliquot of the eluent fraction from ion exchange chromatography was collected for residuals and product quality attribute analyses.

LC-MS monitoring data for the residual concentration of a polyethylene glycol antifoam and the level of oxidation of a specific Methionine residue are given below as two examples of IPC. The figure below shows 3 traces: the buffer blank, the buffer blank spiked at 0.5ug/mL of the antifoam and the sample.

Four parent ions were selected across the distribution of the antifoam mass spectrometric signals to be fragmented in the collision cell. A common fragment ion was monitored for each parent. The antifoam level in the sample was calculated at 0.13ug/mL.

Table 1: Examples of common families of residual impurities with suggested Table 1: Examples of common families of residual impurities with suggested monitoring monitoring methods methods

Control Strategy Plan In order to detect and quantify process related impurities, Control Strategy Plan In order to detect and quantify process related impurities, sensitive have toThis be sensitive methods have to be established and methods validated. established and validated. This provides the only means to effectively develop an provides the only means to effectively develop an efficient process with an associated control strategy plan, to guarantee the elimination of impurities, or at least their reduction to an acceptable safety level.2 The first step in assuring the purity of a biopharmaceutical product is to prepare a strategic plan, such as the one below: 1.

2.

3.

4.

Identify the nature and source of each process related impurity Assess the risk for patient safety of each residual impurity.

The risk should be weighed against the probability of occurrence of each impurity Develop a method sensitive enough to quantitate the residual impurities at various steps of the manufacturing process (for the Drug Substance (DS) and the Drug Product (DP)).

Develop a process capable of eliminating or at least reducing

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Figure1: LC-MS/MS chromatogram showing the TIC (total ion chromatogram) obtained from the sum of four transitions specific to the antifoam agent used. Top trace: standard sample spiked at 0.5ug/mL, middle trace collected sample, bottom trace blank buffer eluate.

The MS chromatogram below shows the MS profiles of a native tryptic peptide generated by enzymatic digestion of the MAb (top trace) and its oxidised variant (lower trace). The ratio of peak areas allows the oxidation level of a specific Methionine residue within the Mab sequence to be estimated at 4.9%. To evaluate Methionine oxidation, it is important that the oxidised peptide produced by the enzymatic treatment of the parent drug does not coelute with its corresponding native parent peptide, since oxidation can occur in the detector. Moreover, the sample preparation protocol should be carefully developed so as to avoid the occurrence of oxidation.

The increasing use of disposable manufacturing equipment coupled with the recent development of continuous or semiINTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 37

Manufacturing

technologies will need to be developed. MAM and therefore MS-based methodologies are extremely valuable, as they can provide “real-time”, in-process control monitoring of a wide range of impurity types. The recent development of continuous downstream processes can become the ideal testing ground for direct, real-time analysis, facilitating the adoption of QbD principles for biopharmaceutical manufacturing.

REFERENCES 1.

Figure 2: LC-MS chromatogram showing the SIR traces of the native Methionine containing peptide (top trace) and its oxidized variant (bottom trace). Respective peak areas are integrated.

continuous downstream processes makes the implementation of IPC with MAM methodologies much easier. When applied early, these developments allow a better understanding of the manufacturing operation and product attributes. This greatly facilitates the execution of a QbD approach4 for the production process, resulting in consistently shorter processes, an improved control strategy, and a reduction in the number of QC tests needed for batch release.

Conclusion The identification and monitoring of residual impurities is an essential step in the development of biotherapeutic drugs. With new modalities of biologics emerging, such as oligonucleotides, RNAs, next-generation peptides, antibody drug conjugates, cell & gen therapies and others, new potential impurities will continue to emerge. These novel impurities will require new methods of monitoring and sensitive analytical 38 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

2.

3.

4.

Mirasol, F. When Assessing Protein Aggregation Grows Increasingly Challenging, Pharmaceutical Technology's In the Lab eNewsletter 10-07-2020,Volume 15,Issue 10 Challenger, C. A. Eliminating Residual Impurities Starts with a Strategic Plan. Biopharm International 33 (7) 22-25 (2020) Rogers, R.S., Abernathy, M., Richardson, D.D. et al. A View on the Importance of “Multi-Attribute Method” for Measuring Purity of Biopharmaceuticals and Improving Overall Control Strategy.AAPS J20,7 (2018) Quality by Design. European Medicines Agency. May 13, 2020. https://www.ema.europa.eu/en/human-regulatory/researchdevelopment/quality-design. Accessed June 23, 2021.

Luc-Alain Luc-Alain is the Global Head of Biologics at SGS Health Sciences, Drug Development.

In his current role, Luc-Alain leads the SGS development strategy for Biologics, including the positioning of activities in the Biologics marketplace. Prior to this role, he was the co-founder and managing director of M-Scan SA, a contract research organization specializing in high level bioproducts characterization, sold to SGS in 2010.

Autumn 2021 Volume 4 Issue 3

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Therapeutics

PEER REVIEWED

Innovations in Cell Line Development to Optimise Biotherapeutic Development Developing stable and high-producing cell lines to manufacture biologics is complex, multi-stage, and time-consuming, often resulting in development timelines that exceed six months when using classical techniques. This represents a frustrating bottleneck in biotherapeutic development, a market where speed-toclinic is a priority.

The challenge to overcome these limitations and meet the increasing demand for novel biologics encourages constant innovation in the manufacturing processes.

This article presents some of the latest strategies to optimise product concentrations and productivities, and high-throughput automated methods to accelerate screening, while reducing manufacturing costs.

Biologics represent the fastest growing sector of the pharmaceutical industry, and this constant growth has been complemented with significant improvements in upstream process development.1 Many biopharmaceutical companies are acting upon new insights and experimenting with ways to accelerate traditional cell line development timelines. Three novel advancements that have had a significant impact on these processes are as follows: 1) a novel method to influence the charge profiles of the biologic to improve cell-specific productivity, 2) a technique developed to precisely modify the critical quality attributes of the biologic to optimise effector functions, and 3) an automated, high-throughput approach to search and select the optimum high-yielding proven monoclonal cell line.

Harnessing Citrulline for Improved Cell Culture Productivity Advances in cell culture systems, focused on improving process efficiency by increasing volumetric productivity, can be applied to facilitate process optimisation. Conventional cell culture strategies generally focus on improving the viable cell density (VCD) during upstream processing to increase volumetric productivity. However, while a high VCD results in higher product titres, such changes often lead to difficulties downstream due to the increased complexity of clarification and the requirement for larger filter surfaces. A higher VCD is also associated with a higher concentration of host-cellrelated impurities, which researchers must remove during the downstream processing phase, thereby increasing the overall cost of production.

Recent studies to increase specific productivity show that the use of citrulline is a promising alternative.2 In contrast to conventional practices, the effectiveness of this technology is based on increasing the cellular volume, rather than increasing the VCD. The addition of citrulline to the culture medium increases the volume of mammalian cells by more than two-fold while simultaneously decreasing the volume of the culture medium by 60%, with no negative effects on 40 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

total biomass or productivity. Furthermore, citrulline has the potential to modulate the charged profile of a product by lowering the percentage of acidic species in the product's composition.

Citrulline has also been shown to reduce the protein content of host cells by 40% while simultaneously increasing the filterability for clarification by 40% per gram of product. This is due to the increase in cell size and, consequently, the increase in cell-specific productivity. The latter has significant implications for the good manufacturing practice footprint of the cell culture clarification step. With an increase in specific productivity, less cell debris needs to be removed during clarification, resulting in less product loss and, in turn, lower costs.

Enhanced Modulation Strategy for Improved Process Efficiency with In addition to increasing volumetric productivity, optimising product quality is critical in the production of biotherapeutic proteins. The majority of biologics are composed of recombinant N-glycosylated proteins that are produced in mammalian expression systems, such as Chinese hamster ovary cells. These biologics require human-like post-translational modifications and a cellular system to perform the complex protein folding and glycosylation steps crucial for therapeutic efficacy.

Post-translational modifications are dependent on many parameters, including the host cell line and culture conditions, which, coupled with the inherent variability of the cellular factories during bioproduction, can introduce significant issues.

Difficulties occur as recombinant biotherapeutic proteins have the potential to misfold and stress the secretory pathway, resulting in protein degradation or cell death.

Multi-chain biotherapeutic proteins may fail to pair correctly, and insufficient or incomplete glycosylation can occur, affecting the biological activity, stability, and immunogenicity of therapeutics. Furthermore, variation in N-glycan species can have an impact on effector functions, including antibody-dependent cell-mediated cytotoxicity and complement-mediated cytotoxicity, both of which are associated with drug efficacy.

Accordingly, engineering approaches to reliably generate complex proteins with the correct post-translational modifications are an area of continuous research and development. Recent findings show that researchers can achieve improved control over the production of biologics by leveraging an advanced process modulation toolbox that draws upon advances in pathway platform knowledge and the requirements for growth and production, integrated with an understanding of the cellular environment.2 Using the modulatory strategy, it is possible to specifically modify critical quality attributes of novel biologics, including terminal galactosylation, core-fucosylation, and mannosylation to enhance efficacy. In conjunction, it is possible to modulate the charge profile of the product by using Autumn 2021 Volume 4 Issue 3

Therapeutics

Figure 1. A high-throughput screening workflow to significantly reduce timelines and bring more molecules towards commercialisation.

molecules like citrulline, as described above. Another essential factor to consider is the optimisation of culture media.

Automating Screening and Selection of Production Cell Lines Several aspects are considered when choosing a production cell line. The optimum cell line will have high cell-specific productivity and titres, demonstrated scalability and stability, as well as well-documented proof of monoclonality to meet regulatory requirements. The subsequent selection process to identify the desired cell line is traditionally a lengthy process.

Major bottlenecks include heterogeneity in a clonal cell population, requiring extensive screening for desired traits, and the regulatory requirement for assurance of monoclonality, demanding multiple rounds of single-cell cloning.

Selecting single clones has conventionally been achieved using the classical, resource-intensive method of limiting dilution or semi-automated technologies, such as fluorescent activated cell sorting, colony picking, and single-cell printers.

Once isolated, clones are imaged using instruments such as cell-in-well imagers and then evaluated for productivity, before progressing into small-scale bioreactors for batch production.

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Overall, this is a costly, time-consuming, and resource-intensive process, requiring separate pieces of equipment for each technique, taking up valuable laboratory space and increasing the risk of sample contamination. As a result, the industry's research and development efforts to consolidate steps in the cell line development workflow and alleviate some of these limitations continue to be a major area of focus.

Utilising the combined benefits of picodroplet-based microfluidics and automation, researchers can now bypass the traditionally time-consuming and multi-step selection, isolation, and verification process with a fully automated, high-throughput screening solution. In doing so, optimum cell lines capable of delivering high productivity and antibody quality can be identified through more commercially competitive workflow timelines. Given their potential to reduce critical path timelines, picodroplet single-cell analysis systems have proven to be transformative in the field of cell line development (Figure 1). Studies show that high-throughput microfluidic technologies, such as this, significantly improve the accuracy and efficiency of screening and culture development, enabling large numbers of cell lines

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 41

Therapeutics

Figure 2. An integrated workflow packaging the screening, sorting, isolation, and verification of high-secreting clones into a fully automated process.

to be assessed early in the development process and providing high assurance and probability of monoclonality.3,4 Picodroplet-based microfluidic technologies encapsulate single cells into picolitre-volume aqueous droplets. These miniaturised picodroplets provide a highly controlled microenvironment for studying individual cells, chemical and biochemical reactions, and cell-cell interactions, while preserving cell viability. Picodroplet technologies are increasingly applied to optimise cell line development, offering several advantages over conventional processes, including high single-cell screening throughputs, rapid-yetgentle cell processing, highly sensitive quantitative assays, and reduced reagent costs.3,4 Within a picodroplet, encapsulated cell-secreted molecules quickly accumulate to a concentration that can be measured consistently and quantitatively utilising a Förster resonance energy transfer theory (FRET) assay. In a FRET experiment, two fluorescent probes, the donor and acceptor, will attach to the target secreted molecule. The binding event brings the probes together, resulting in energy transfer from donor to acceptor, seen as a reduction in the donor's fluorescence signal and a rise in the acceptor's fluorescent signal. Picodroplets containing desired cells can then be fluorescently sorted and distributed into individual wells on a microtitre plate for downstream analysis.

Fully integrated instruments, actuated through intuitive single-cell analysis software, also incorporate high-speed imaging into the dispensing step, allowing researchers to recover high value clones with clear visual evidence of monoclonality, without additional screening or instrumentation (Figure 2). During dispensing, as the picodroplet travels through the microfluidic channels, the encapsulated cells are imaged multiple times to provide visual verification of clonality.

Additionally, in requiring less equipment, reagents and hands-on time, the integrated instrumentation minimizes operating costs to enable more cost-efficient upstream processing, and enhanced biologics manufacturing capabilities.

Conclusion Novel developments have had a considerable influence on the cell line development process, helping to increase cell-specific productivity, improve process efficiency and establish high-throughput methods to advance the generation 42 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

of high-quality clonal cell lines with monoclonality assurance.

These innovations in host cell line optimisation, selection, and screening strategies can be combined to improve product consistency, quality, and robustness. In doing so, biologics development processes are streamlined and less labourintensive, to prioritise speed to clinic.

References 1.

2.

3.

4.

Papathanasiou, M. M., & Kontoravdi, C. Engineering challenges in therapeutic protein product and process design. Curr. Opin. Chem.

Eng. 27, 81–88 (2020). https://polpharmabiologics.com/en/knowledge-center/downloads, visited on 30 July 2021.

Josephides, D. et al. Cyto-Mine: An Integrated, Picodroplet System for High-Throughput Single-Cell Analysis, Sorting, Dispensing, and Monoclonality Assurance.SLAS TECHNOLOGY: Translating Life Sciences Innovation.25(2), 177–189 (2020). https://bioprocessintl.com/analytical/cell-line-development/ using-fret-based-microfluidic-screening-for-analysis-sortingimaging-and-dispensing-development-of-high-producing-clonalcell-lines/, visited on 30 July 2021.

Dr. Louis Boon Dr. Louis Boon is the Chief Scientific Officer at Polpharma Biologics. He has over 25 years of experience in the discovery and development of novel antibody therapeutics and is the inventor of over 20 patent applications for biologics across different disease areas. He is also a thought leader in molecular and cellular immunology, having co-authored over 320 publications in international scientific journals in the field of medical biotechnology.

Olivia Hughes Olivia Hughes is a life science writer and Digital Marketing Associate at Sphere Fluidics. Sphere Fluidics develops and manufactures single-cell analysis and monoclonality assurance systems to enable leading-edge research and accelerate biotherapeutic discovery and development.

Email: olivia.hughes@spherefluidics.com

Autumn 2021 Volume 4 Issue 3

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 43

Technology Platforms

PEER REVIEWED

Accelerating Single-Cell Genomic Sequencing with Big Memory The Extra-Exponential Growth of Cell Data When fighting the global spread of a pandemic or working to fight cancer, faster time-to-discovery saves lives. For this critical bioscience work, scientists have employed single-cell RNA sequencing to assemble entire genomes and reveal genetic variants. Modern gene sequencing technology has grown exponentially in the last decade from studying hundreds of cells to millions of cells.

At the same time, the modality of data has increased exponentially to improve the profiling of different aspects of a cell, including its genome, transcriptome and epigenome, and the spatial organisation of the above -nomes. The emergence of multi-modal studies of millions of cells has resulted in the extra-exponential growth of cell data resulting in DGM (data is greater than memory).

In figure 1 below, the charts on the top and bottom left show that in 2010, studies were based on 1 hundred cells, and by 2020 had increased 4 orders of magnitude to 1 million cells per study. On the top right are examples of how the modalities of data increased.

from storage into memory for each stage. With terabyte data sets, loading data from storage into memory takes a long time.

And when all the cell data doesn’t fit in memory, then code execution becomes IO-intensive as data is swapped from storage.

Even with high-performance solid-state disk, repetitively loading data from storage and executing application code with IO to storage is 1,000x slower than memory. The traditional model and storage IO has become a bottleneck in many types of multi-stage analytic jobs with massive data sets.

Big Memory Computing: The Future of IT Infrastructure for Genomics Research Technology has emerged that enables bioinformaticians to keep pace with the extra-exponential growth of cell data and the corresponding storage bottleneck in the singlecell sequencing pipelines. The new technology is called Big Memory Computing.

Big Memory was designed for large data sets (big data) that must be processed quickly (fast data) because time-to-

Figure 1 – Growth of data driven by the increase in number of cells/study and modalities of data

A Problem Emerges: Storage Becomes a Bottleneck When Data is Greater than Memory For the last 50 years, computing has been dominated by a model which uses storage as “virtual” memory for data that cannot fit into DRAM. The R error message in the lower right-hand corner of figure 1 is an example of how the extra-exponential growth of data is crushing IT infrastructure such as the current computing model and R. Single-cell sequencing jobs are multi-stage analytic pipelines using very large matrices that need to be loaded 44 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

insight is critical to applications such as genomics research.

To process this big and fast data, Big Memory Computing integrates DRAM, lower cost persistent memory, and memory virtualisation to provide a lower cost pool of memory that gives bioinformaticians fine-grained control over memory capacity, performance, availability, security, and mobility.

Real-Life Examples of Big Memory Accelerating Single-Cell Sequencing Below are two examples of Big Memory Computing at work in the Bio IT World. Analytical Biosciences was founded Autumn 2021 Volume 4 Issue 3

Technology Platforms their time to insight. In Figure 4 below, the chart on the left shows that pipeline execution time was reduced 58% to 2.4875 hours. The pie charts on the bottom show that after Big Memory was deployed, pipeline execution time was improved because storage IO was reduced to only 3% of total time.

Figure 2 – Traditional Computing Model

Figure 3 – Big Memory Computing Mode

to create and harness the human disease precision atlas through cutting-edge single-cell genomics and bioinformatics.

The Translational Genomics Research Institute (TGen) is a non-profit genomics research institute that employs genetic discoveries to improve disease outcomes by developing smarter diagnostics and targeted therapeutics.

Analytical Biosciences Reduces Pipeline Throughput by 61%, Storage IO by 97% As the growth of their cell data and pipeline throughput exploded, Analytical Biosciences deployed persistent memory and memory virtualisation software to accelerate

The diagram on the upper right shows why loading data at each stage is faster. The red diagram shows data is loaded from storage. The grey area shows that data is loaded from persistent memory at each stage. This is possible with another new technology, in-memory snapshots from DRAM to persistent memory. Once the cell data is snapshot, even a terabyte can be restored at each stage from persistent memory in a few seconds.

TGen Transforms a Single-Threaded Pipeline into a Multi-Threaded Pipeline TGen was recognised in Science Advances for the use case shown below in an article titled, “Single-cell RNA sequencing reveals profibrotic roles of distinct epithelial and mesenchymal lineages in pulmonary fibrosis.” Like Analytical Biosciences, bioinformaticians at TGen deployed persistent memory and memory virtualisation software to accelerate their time-todiscovery.

In figure 5 below, the diagram on the left shows their pipeline architecture before Big Memory. The diagram on the right shows their architecture after Big Memory, including the transformation to a multi-threaded application. Total pipeline throughput was reduced 36% from 6 hours, 31 minutes, to a little over 4 hours, 11 minutes. This was made possible by faster data load times, faster code execution with all data in-memory, and by using in-memory snapshots for parallel code execution.

Figure 4 – Analytical Biosciences reduces storage IO while creating parallelism in pipeline www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 45

Technology Platforms

Figure 5 – TGen RNA Sequencing Before & After Deploying Big Memory

Yong Tian

Big Memory for a Healthier World For the expanding universe of real-time applications such as genomics sequencing, Big Memory technology can change the game. Virtualising DRAM and persistent memory, and adding a layer of management intelligence, allows applications that must process Big Data to do so faster and more reliably without facing the storage I/O bottleneck. That means significantly more processing speed and efficiency for the critical work being done to find the cures we need for a healthier world.

46 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Yong Tian is director of product management forMemVerge, the pioneers of Big Memory Computing. He heads product strategy for the company’s Memory Machine software which helps accelerate time-to-insight in Big Data Analytics, AI and bioinformatics. Previously Yong was Co-Founder and CEO of Enlighten Robotics and Co-Founder and COO of UltraSee Corp. He holds a master’s degree in Electrical Engineering from the University of Illinois.

Email: yong.tian@memverge.com

Autumn 2021 Volume 4 Issue 3

INSIGHT / KNOWLEDGE / FORESIGHT

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 47

Ad Index

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

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Autumn 2021 Volume 4 Issue 3

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Autumn 2021 Volume 4 Issue 3 Document SPL1001 22 June 2021