IBI 2021 Winter

Volume 4 Issue 4

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SLIM: A Gateway to the Adoption of High-Resolution Ion Mobility for Biotherapeutic Peptide Mapping Predicting Drug Bioavailability With the Modern-Day Toolkit CGT CDMO Partnering More than just Manufacturing Understanding the Role of Regulatory T Cells in Breast Cancer Metastasis

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II INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2021 Volume 4 Issue 4

Contents 04 Foreword WATCH PAGES 06 How Involved Should Biopharma CEOs Be In Outsourcing Activities?

DIRECTOR: Mark A. Barker INTERNATIONAL MEDIA DIRECTOR: Anthony Stewart anthony@senglobalcoms.com EDITORIAL: Beatriz Romao beatriz@senglobalcoms.com DESIGN DIRECTOR: Jana Sukenikova www.fanahshapeless.com FINANCE DEPARTMENT: Akash Sharma accounts@senglobal.co.uk RESEARCH & CIRCULATION: Jessica Dean- Hill jessica@senglobalcoms.com COVER IMAGE: iStockphoto © PUBLISHED BY: Senglobal ltd.

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Volume 4 Issue 4 – Winter 2021

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When outsourcing drug development and manufacturing, how involved should the CEOs of emerging biopharma be in the requisite day-to-day activities and processes? Louis Garguilo interviews James Mackay, CEO of Aristea Therapeutics to answer this question within his article How Involved Should Biopharma CEOs Be In Outsourcing Activities?

REGULATORY & COMPLIANCE 08 5 Drugs Facing Key Patent Expirations and Potential Generic Entry From December 2021 – February 2022 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 five drugs, from December 2021 through February 2022. These estimated drug patent expiration dates and generic entry opportunity dates are calculated from analysis of known patents and US regulatory protections covering drugs.

10 Contamination Control strategy Regulatory bodies expect that pharmaceutical companies have a Contamination Control Strategy (CCS) in place that outlines the control of contamination of utilities, manufacturing systems and environment and ultimately the pharmaceutical product itself. Patrick Nieuwenhuizen at Pharmalex explains more about contamination control strategy.

12 Digitisation versus Digitalisation – Understanding the Difference and the Role of LIMS in Achieving Both The system infrastructure for the creation, storage and management of electronic data has to be in place. If this isn’t the case, then completing a digitisation project using well accepted and proven solutions such as LIMS will provide considerable process benefits and form the foundations on which the concepts of digitalisation can be built. Simon Wood at Autoscribe Informatics shows that those two letters do make a difference, that it is important to understand that difference, and that once the difference is understood significant benefits can be gained through implementing these initiatives.

RESEARCH / INNOVATION / DEVELOPMENT 14 Predicting Drug Bioavailability with the Modern-day Toolkit Bioavailability is defined as the fraction of drug reaching systemic circulation following absorption in the gut and first pass metabolism in the liver. Accurately predicting the bioavailability of orally administered medicines in humans, during pre-clinical development is crucial as it forms the basis for setting safe and efficacious doses in the clinic. This significant parameter is therefore of interest to drug developers and regulatory agencies.

Dr. David Hughes and Dr. Yassen Abbas at CN Bio look into predicting drug bioavailability with modern-day toolkit.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 1

Contents 18 SLIM: A Gateway to the Adoption of High-Resolution Ion Mobility for Biotherapeutic Peptide Mapping

38 Maximising mAbs Purification Efficiency: Focus Areas for Reducing Bottlenecks in Downstream Processing

The demand for protein therapeutics, such as recombinant human proteins, monoclonal antibodies (mAbs), and fusion proteins, has grown significantly over the past decade. Often developed to treat previously unmet clinical needs, these biotherapeutics require increasingly advanced analytical technology for detection, identification, quantitation, and quality control (QC)/monitoring of molecular attributes. Melissa Sherman at MOBILion Systems Inc and Jared Auclair, Associate Dean, look over the adoption of high-resolution ion mobility for biotherapeutic peptide mapping.

Finding ways to remove bottlenecks and improve yields in downstream processing for monoclonal antibodies (mAbs) continues to be a key focus area for biopharma manufacturers.

In downstream processing, the goal is to improve recovery and reduce the cost per gram of protein produced. Nandu Deorkar, Jungmin Oh, Pranav Vengsarkar and Jonathan Fura at Avantor focus on areas where streamlining downstream processing steps can help improve overall mAbs processing efficiencies and help downstream productivity match the improved efficiencies achieved in upstream processing.

24 An End-to-End Solution to Accelerate CAR-T Cell Development from Concept to Clinic

THERAPEUTICS

CAR-T cell therapies have demonstrated tremendous success in relapsed or refractory haematological malignancies. Now therapeutics companies and academics alike are racing to break through the barrier of solid tumours, taking imaginative approaches to overcome the numerous obstacles in the way of turning a hugely variable starting material into an armed and precisely targeted weapon against cancer. Sophie Lutter at OXGENE analyses an end-to-end solution to accelerate CAR-T cell development from concept to clinic.

26 Benefits & Key Considerations for the Use of Human iPSC-Derived Disease Models in Drug Discovery The process of drug discovery, development and commercialisation is long and associated with high costs. In addition, it is estimated that only 1% of the initially tested compounds make it to the market. Noelia Muñoz-Martín and Elena Matsa at Ncardia describe how human iPSC-derived disease models improve drug discovery and what the main challenges and solutions are for the successful generation and application of these models.

42 Bringing Novel Therapies to Market: 5 Strategies for Success Advanced therapy medicinal products (ATMPs) were making headlines long before SARS-CoV-2, the virus that causes COVID-19, emerged onto the scene. The successful development of COVID-19 vaccines, including mRNA vaccines, has brought next-generation therapies into the spotlight and boosted the long-term projections for growth in this sector. Christian K. Schneider at Biopharma Excellence outlines 5 strategies for success while avoiding common pitfalls.

46 Understanding the Role of Regulatory T Cells in Breast Cancer Metastasis Breast cancer is the most common cancer amongst women accounting for 24 percent of new cancer cases worldwide and 15 percent of cancer deaths in 2018. While survival rates for breast cancer patients are improving with the help of early detection, the incurable metastatic stage of the disease has a poor prognosis, resulting in most breast cancer deaths. Christine Evans-Pughe explains more about the role of regulatory T cells in breast cancer metastasis.

PRE-CLINICAL & CLINICAL RESEARCH 30 Could Standardised Metadata be the Key to Optimising and Expediting Clinical Trials?

Clinical trials still offer the most effective way of testing the safety and efficacy of new drug treatments, and as more drugs are brought through development each year, the number of trials conducted grows. Gilbert Hunter at Formedix, explores how the use of next-generation, all-inone, cloud-based clinical metadata repositories (CMDRs) can provide the automation, standardisation and control needed to optimise clinical trials.

MANUFACTURING 34 CGT CDMO Partnering: More than just Manufacturing The biotech footprint has been exponentially expanding around the globe and no division has increased more feverishly in recent years than the Cell and Gene Therapy (CGT) space. This is where partnering with an experienced CDMO can de-risk your path to said critical milestones and provide that seamless transition from development to clinic and into both a regional and global commercial supply. Joe Garrity at Lonza Cell and Gene Technologies outlines the importance of partnering with an experienced CDMO. 2 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2021 Volume 4 Issue 4

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TO GO

Foreword The festive season is drawing ever closer and here at IBI, we are thrilled to bring you our fourth and final issue of the year!

Throughout human history, the morbidity and mortality associated with human disease has driven medical science into an ever-expanding quest for treatment and cure. Over the past century, a therapeutic approach complementing chemical drugs has been developing which uses proteins and peptides in the treatment of disease.

Many innovative protein therapeutic platforms are currently being employed and continue to be developed to attain cures in areas of unmet medical need; these include direct copies of natural protein structure and function as well as proteins with completely novel functionality. Today, protein therapeutics represents the fastest growing sector in the pharmaceutical industry and comprises 16% of prescription drug sales in 2011.

The demand for protein therapeutics, such as recombinant human proteins, monoclonal antibodies (mAbs), and fusion proteins, has grown significantly over the past decade. Often developed to treat previously unmet clinical needs, these biotherapeutics require increasingly advanced analytical technology for detection, identification, quantitation, and quality control (QC)/monitoring of molecular attributes. Melissa Sherman at MOBILion Systems Inc and Jared Auclair, Associate Dean, look over the adoption of high-resolution ion mobility for biotherapeutic peptide mapping.

Finding ways to remove bottlenecks and improve yields in downstream processing for monoclonal antibodies (mAbs) continues to be a key focus area for biopharma manufacturers.

In downstream processing, the goal is to improve recovery and reduce the cost per gram of protein produced. Nandu Deorkar, Jungmin Oh, Pranav Vengsarkar and Jonathan Fura at Avantor focus on areas where streamlining downstream processing steps can help improve overall mAbs processing efficiencies and help downstream productivity match the improved efficiencies achieved in upstream processing.

The process of drug discovery, development and commercialisation is long and associated with high costs. In addition, it is estimated that only 1% of the initially tested compounds make it to the market. Noelia Muñoz-Martín and Elena Matsa at Ncardia describe how human iPSC-derived disease models improve drug discovery and what the main challenges and solutions are for the successful generation and application of these models.

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 you all enjoy your festive season and I look forward to welcoming you back in the new year, with more enthralling articles to be included in IBI. Beatriz Romao, Editorial Manager

In this journal, we will also explore more about biotech footprint. The biotech footprint has been exponentially expanding around the globe and no division has increased more feverishly in recent years than the Cell and Gene Therapy (CGT) space. This is where partnering with an experienced CDMO can de-risk your path to said critical milestones and provide that seamless transition from development to clinic and into both a regional and global commercial supply. Joe Garrity at Lonza Cell and Gene Technologies outlines the importance of partnering with an experienced CDMO.

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

Winter 2021 Volume 4 Issue 4

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UK: oxgene.com

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USA: ctdmo.com

China: wuxiatu.com INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 5

Watch Pages

How Involved Should Biopharma CEOs Be In Outsourcing Activities?

When outsourcing drug development and manufacturing, how involved should the CEOs of emerging biopharma be in the requisite day-to-day activities and processes?

Now that’s a good question.

James Mackay, CEO of Aristea Therapeutics, and independent board member to emerging biopharma, says when he’s advising new CEOs, he answers by first informing them “at some point along the way, normally something in your CMC activities becomes problematic.”

And thus: “The CEO is likely to be the ultimate decisionmaker on more activities related to outsourcing than they might expect.”

Mackay, who earlier in his career at AstraZeneca helped guide six drugs to commercial approval, outlined the role of an independent board member for emerging biopharma in our earlier conversation.

Here he’ll turn specifically to our leading question.

CEO In The Weeds For an example of “normal” but “problematic CMC challenges” that can pop up for a CEO, Mackay uses his own situation at Aristea, where he is “living and breathing this right now.”

He describes the attempted nano-milling of an intermediate that “works beautifully on small scale, but not so well moving to large scale.”

CEO-level concern? Mackay thinks so.

But first, he says, it’s important – even at “virtual” start-ups – the CEO has at least a core team in place. That team can include an independent advisor suggesting approaches to the challenge; consultants and SMEs, who in this case, for example, can help the CEO better understand particlesize distribution, impacts of adjusting speeds, or the time of milling.

Next, as Mackay puts it, the CEO must recognise the people at the CDMO know their equipment and technology better than anybody. “They’ve seen more projects than we have,” he says.

“Working with them as part of your team is critical.”

“Nonetheless,” he continues, “in the middle of all these resources, as CEO I must be highly engaged.”

So, while CEOs will have other activities on their minds – and calendar – they should also serve as Chief Outsourcing Officer.

How an outsourcing impasse is resolved is a vital leadership decision.

6 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

In fact, insists Mackay, that leadership starts much before there’s a challenge with something like a spray-drying scale-up at a CDMO. It starts at company formation.

It requires a nuanced conception of the style and substance of the new company’s outsourcing model – and the resultant search for enabling partners.

The emerging biopharma CEO starts his decision tree with: “How will we approach outsourcing?”

“Which service providers will best enable that strategy?”

Says Mackay: “When as an independent board member I am advising the CEO, I relate my own experience by making it clear I’ve always talked one-to-one with my counterpart at the provider we're considering, so they understand the way we want to work together, and to create that ability to communicate at the top of the two organisations.

“Sometimes you will have to get up to that level to get some decisions made or resources released.”

But Keep Your Head Up The above, though, comes with a caution: All this outsourcing preparation from the outset, and day-today involvement, is done in service of the ultimate goal of commercial success.

“Although I'm a development guy by training, I've always been in that commercial interface,” says Mackay. “My colleagues in AstraZeneca used to describe me as the most commercial development leader we ever had!”

Mackay learned that as you design your program, if you are not thinking about that end goal, chances are you will not design the program appropriately.

“You should be thinking about regulatory approval; the market, even what the payer’s going to want; then how to build that into your clinical program,” he says.

“This is an area I’ve found I can add the most value in San Diego,” he adds. “Although we now have companies who have gone commercial – Acadia, Neurocrine, and GW, for example – historically San Diego companies don’t take their drugs through to market. So, helping CEOs focus on that future point, and then think back about what needs to be in the program, is valuable.”

Outsourcing Advice Wrapped Up Here’s our CEO checklist, as provided by an independent board professional: Winter 2021 Volume 4 Issue 4

Watch Pages

Start with a vision of ultimate commercial success. Work backwards to what you’ll need at each step of the way to attain it.

Define your outsourcing needs, style, and method. Select service providers that best meet those parameters.

Stay involved in the day-to-day outsourcing process and decision-making.

Mackay says the mindset is to take your program through to commercialisation, even if that ultimately is never going to happen, for example due to an acquisition by a strategic pharma. “It's still the way to think about it.”

This ensures the organisation makes the right decisions at the right time “for the development of the product.”

It also positions you for a deal with a strategic partner should that materialise.

“A larger pharma company will look at your program and say, ‘These guys have been thinking about this in the right way.’

“When we started up Aristea,” explains Mackay, “the first meeting we ever had with our CMC consultants was to talk about the ultimate commercialisation of the product. What would that look like? What are the indications, the size of the supply chain we’ll need? www.international-biopharma.com

“We worked back from that. It doesn't mean your initial CDMO has to be the one that's going to commercially supply the product. In our case, we didn't do that. However, we knew how we were going to transition the development program.

“We went with a more niche CDMO to begin with here in San Diego, with the ability to respond more rapidly than the bigger players. It was part of the overall plan, piece by piece.”

And day to day, with an eye towards the ultimate goal of commercialisation. The role of a CEO at a biopharma in 2021.

Louis Garguillo Louis Garguilo is chief editor and conference chair for Outsourced Pharma and a contributing editor to Life Science Leader magazine. He studied public relations and journalism at Syracuse University. Among other positions, Garguilo spent a decade at a global pharmaceutical contract research, development, and manufacturing organisation, and served under the governor of New York in the state's economic development agency, as liaison to the pharmaceutical and biotechnology industries.

Email: louis.garguilo@lifescienceconnect.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 7

Regulatory & Compliance

5 Drugs Facing Key Patent Expirations and Potential Generic Entry from December 2021 – February 2022 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 five drugs, from December 2021 through February 2022.

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.

LICART (diclofenac epolamine) Estimated US Loss of Exclusivity Date: 19 December 20212* Generic Entry Controlled by: FDA Regulatory Exclusivity LICART is marketed by Ibsa Inst Bio and is included in one NDA. By analysing the patents and regulatory protections it appears that the earliest date for generic entry in the US will be December 19th, 2021, when FDA Regulatory Exclusivity expires.

VAZALORE (aspirin) Estimated US Loss of Exclusivity Date: 19 December 20213* Generic Entry Controlled by: US Patent 9351984 Title: Compositions comprising lecithin oils and NSAIDs for protecting the gastrointestinal tract and providing enhanced therapeutic activity Abstract: "A novel pharmaceutical composition is provided by which nonsteroidal anti-inflammatory drugs (NSAIDs) are added directly to phospholipid-containing oil such as lecithin oils or to a bio-compatible oil to which a phospholipid has been added to make a NSAID-containing formulation that possess low gastrointestinal (GI) toxicity and enhanced therapeutic activity to treat or prevent inflammation, pain, fever, platelet aggregation, tissue ulcerations and/or other tissue disorders.

The composition of the invention is in the form of a non-aqueous solution, paste, suspension, dispersion, colloidal suspension or in the form of an aqueous emulsion or microemulsion for internal, oral, direct or topical administration."4 VAZALORE is marketed by Plx Pharma and is included in one NDA. There are five US patents protecting this drug, and forty-two patent family members in eighteen 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 December 19th, 2021, when US Patent 9,351,984 expires.

Patent 9,351,984 describes‘compositions comprising lecithin oils and NSAIDs for protecting the gastrointestinal tract and providing enhanced therapeutic activity’, and is assigned to The Board of Regents of the University of Texas System (Austin, TX).3,4 8 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

FEMTRACE (estradiol acetate) Estimated US Loss of Exclusivity Date: 21 December 20215* Generic Entry Controlled by: US Patent 6962908 Title: Oral pharmaceutical products containing 17 .beta.

-estradiol-3-lower alkanoate, method of administering the same and process of preparation Abstract: "A pharmaceutical dosage unit for oral administration to a human female comprising a therapeutically effective amount of 17.beta.-estradiol-3-lower alkanoate, most preferably 17.beta.-estradiol-3-acetate, and a pharmaceutically acceptable carrier is disclosed. Also disclosed is a method for treating a human female in need of 17.beta.-estradiol and a contraceptive method by oral administration of the pharmaceutical dosage unit and a method of preparing a pharmaceutical composition that may be used to form the pharmaceutical dosage unit of the invention."6 FEMTRACE is marketed by Apil and is included in one NDA. There are three US patents protecting this drug, and eleven patent family members in ten 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 December 21st, 2021, when US Patent 6,962,908 expires.

Patent 6,962,908 describes Oral pharmaceutical products containing 17 .beta.-estradiol-3-lower alkanoate, method of administering the same and process of preparation, and is assigned to Warner Chilcott Company Inc. (Fajardo, PR).5,6 PHOXILLUM B22K 4/0 IN PLASTIC CONTAINER (calcium chloride; magnesium chloride; potassium chloride; sodium bicarbonate; sodium chloride; sodium phosphate) Estimated US Loss of Exclusivity Date: 13 January 20227* Generic Entry Controlled by: FDA Regulatory Exclusivity PHOXILLUM B22K 4/0 IN PLASTIC CONTAINER is marketed by Baxter Hlthcare Corp and is included in one NDA. By analysing the patents and regulatory protections it appears that the earliest date for generic entry in the US will be January 13th, 2022, when FDA Regulatory Exclusivity expires.

SPIRIVA RESPIMAT (tiotropium bromide) Estimated US Loss of Exclusivity Date: 4 February 20228* Generic Entry Controlled by: US Patent 7988001 Title: Container provided with a pressure equalization opening Abstract: "A process for producing a container comprising an outer container, an inner bag disposed therein and a pressure equalisation opening disposed in the outer container, and a container produced according to this process, is described, wherein a pre-moulding, comprising two coaxial tubes, is first produced by co-extrusion with the help of a blow mould and with an outwardly-projecting base seam being formed. The process forms a pressure equalisation opening in the outer container without endangering the integrity of the container, wherein a lower wastage rate and higher productivity are achieved. To do this, the base seam is partially cut off and a force Winter 2021 Volume 4 Issue 4

Regulatory & Compliance

which acts in the direction of the seam is introduced into the pre-moulding, which has a temperature of 40°C to 70°C, which force breaks open and plastically deforms the base seam so that a pressure equalisation opening is formed in the base area."9 SPIRIVA RESPIMAT is marketed by Boehringer Ingelheim and is included in one NDA. There are seven US patents protecting this drug, and one hundred and sixty-nine patent family members in forty-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 February 4th, 2022, when US Patent 7,988,001 expires.

Patent 7,988,001 describes‘Container provided with a pressure equalisation opening,’ and is assigned to Boehringer Ingelheim Pharma GmbH & Co. KG (Ingelheim am Rhein, DE).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 for information purposes only.

There is no warranty that the data contained herein is error free.

Financial & competing interests’ disclosure Y Friedman is the CEO of DrugPatentWatch. The author has no other relevant affiliations or financial involvement www.international-biopharma.com

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.

REFERENCES 1.

2.

3.

4.

5.

6.

7.

8.

9.

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, "LICART Drug Profile" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/p/tradename/LICART. Friedman, Yali, "VAZALORE Drug Profile" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/p/tradename/VAZALORE. The Board of Regents of the University of Texas System US9351984 Friedman, Yali, "FEMTRACE Drug Profile" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/p/tradename/FEMTRACE. Warner Chilcott Company Inc. US6962908 Friedman, Yali, "PHOXILLUM B22K 4/0 IN PLASTIC CONTAINER Drug Profile" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/ p/tradename/PHOXILLUM+B22K+4%2F0+IN+PLASTIC+CONTAINER. Friedman, Yali, "SPIRIVA RESPIMAT Drug Profile" DrugPatentWatch.com, 2021.https://www.drugpatentwatch.com/p/tradename/SPIRIVA+ RESPIMAT. Boehringer Ingelheim Pharma GmbH & Co. KG US7988001

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

Contamination Control Strategy

Regulatory bodies expect that pharmaceutical companies have a Contamination Control Strategy (CCS) in place that outlines the control of contamination of utilities, manufacturing systems and environment and ultimately the pharmaceutical product itself. The question is what are the key elements to be considered in order to meet the quality system requirements detailed in ICHQ10 in addition to ICHQ9 and EudraLex Volume 4 Annex 1 for a CCS? What points need to be considered to support the implementation of such a program within any manufacturing facility? What makes it robust? Should it be multifaceted to ensure its effectiveness?

These are all valid questions and key to ensure your CCS is robust. One important facet is that the strategy is to be built on a risk-based foundation. From the start, it requires a multidisciplinary team with a good understanding of the process, the utilities and equipment that serve the process. Cross-functional expertise allows scientific justified assessments to be made of potential microbiological, particulate, chemical and cross-product risk to your product.

A solid CCS also provides an estimation for the likelihood of a risk occurring and severity of the impact to the patient if the risk occurred. This allows you to identify the key areas of focus in the CCS and provides rationale for implementation of control or detection measures.

Development of a robust plan is critical in all manufacturing facilities – aseptic, terminal sterilisation and non-sterile. This is not futility as pharmaceutical manufacturing generally comprises a complex, multi-step processing system in which significant risks from various types of contamination are presented by different sources. Clear regulatory guidance is available on the need for exclusion and control of contamination at all stages during manufacture, however, addressing contamination control in a non-sterile manufacturing environment is less clear cut. Under all circumstances, good knowledge of your own process is paramount.

A CCS is a not a stand-alone plan but more a summary of practices and measures that are interlinked. The sum of all these individual aspects and how well these interact determines the effectiveness of the CCS from the selection and management of raw materials to final packaging of the product. For example, how the material and personnel flows are designed, is there cleaning, sanitisation, and sporicidal treatment and how is the robustness of the controls monitored through the environmental monitoring programme and in process or finished product testing.

When controlling contamination of pharmaceutical raw materials, the primary aim is to exclude any contamination which may subsequently result in deterioration of the 10 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

product or may harm the patient. If raw materials are not of the desired quality, they can be sources of contamination for your (intermediate) product. The origin and composition of the raw materials gives a good indication of whether the ingredient has the potential to be a source of contamination or can cause proliferation of microbial growth. Besides product contamination, raw materials have the potential to contaminate equipment and the manufacturing facility. This can lead to long term issues that are very difficult to eliminate and result in repeat contamination of product or cross contamination to other products. In particular, microbiological contamination poses a risk of biofilm formation or spore contamination that can be very difficult to remove.

Water is the most common used “raw material” within a pharmaceutical manufacturing process and different grades of pharmaceutical water are typically used for different processes.

As water is used in many different applications, there is not only the direct risk of contamination of the product, but also indirectly as water can act as a vector for contamination causing transmission from one system to the other. The design of your company’s water system augmented with a good preventive maintenance and sanitisation regime in combination with a sound monitoring program significantly contributes to microbial risk reduction.

One indispensable part of the CCS is environmental control in the cleanroom. Physical parameters such as relative humidity, temperature and differential pressures give an indication of the HVAC performance. In aseptic manufacturing environments air flow patterns are of significant importance in terms of transfer of contamination between areas. Your Environmental Monitoring (EM) programme needs to determine the types and level of microbial and non-viable particulate contamination present in the cleanroom. Sample locations must be selected through risk assessment. With an effective EM programme in place, analysis of the data collected can quickly verify that the cleaning and disinfection processes in place are effective and allow adverse trends to be quickly identified. EM is an important tool for determining the state of control of the facility and therefore, is an important part of the monitoring programme for all types of manufacturers.

As the number one source of contamination in controlled areas is personnel, training is key! The CCS must address personnel barriers to contamination whether the manufacturing process is designed for aseptic or non-sterile manufacturing facilities. The CCS should describe the rationale for the level of gowning chosen, the frequency of gown cleaning, behaviour of personnel in the controlled rooms and the acceptability of the gown materials for the type of manufacturing process.

Personnel working in the area must rigidly adhere to the gowning procedures in place. Training should involve a practical element; it should be continuous and ongoing monitoring is an essential part of an effective CCS. Training should include Winter 2021 Volume 4 Issue 4

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an initial and periodic assessment of gowning and until fully qualified, personnel access to the cleanroom should be restricted. A risk-based approach should be undertaken to assess frequency of retraining.

After personnel, the second highest risk of contamination is from materials and equipment brought into the controlled environment from the outside. For material transfer airlocks, it is essential that decontamination practices are in use prior to entry into the controlled environment. Items to be considered include the use of interlocking airlocks between entry points for classified areas of different grades, restricted and controlled access to aseptic areas through use of card readers etc., disinfection of materials to include use of a sporicidal where appropriate, sufficiently detailed material transfer procedures in an SOP and assessment of transfer techniques by an SME to validate compliance with your SOPs. The frequency of your cleaning and disinfection programmes should be risk-based and similar to the training programme, should be regularly reviewed. Two disinfectants should be used in rotation, one being a broad-spectrum disinfectant rotated with periodic use of a sporicidal agent. Have you validated your disinfectant prior to use? This is also key to an effective CCS and should include surface challenge testing to calculate the log reduction of each microorganism tested with the disinfectant. The selected surfaces must be representative of those that are present in your facility. Besides the obligatory ATCC strains the microbial challenge panel must also include environmental isolates to demonstrate effectiveness. Other www.international-biopharma.com

items to be considered under the cleaning and disinfection programme include the frequency and method for residue removal, performance of studies to demonstrate the ability to recover from loss of aseptic control and disinfectant efficacy studies to include contact times and expiry dating.

Consideration should also be given to identifying and defining your product contact surfaces and ensuring that in an aseptic process, these surfaces are sterilised and protected from contamination before, during and after processing. All wrapping materials in use must be of the appropriate quality to ensure a microbial barrier and low particle generation. The considerations in a non-sterile facility are similar as there needs to be proof that the sanitisation program used is effective and the frequency of use of sporicidal agents is appropriate and commensurate with the risk to the patient.

Patrick Nieuwenhuizen Quality professional with a Microbiology & Sterile Manufacturing background with over 25 years’ experience in the Pharmaceutical Industry. Worked for several global Pharmaceutical and Biotechnology companies across a variety of platforms including Biologics, Sterile Fill Finish and Solid Oral Dose. Qualified lead auditor and SME in Quality Control, Sterility Assurance, New Facility Design / Upgrade and risk facilitator for Quality Risk Management.

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Regulatory & Compliance

Digitisation versus Digitalisation – Understanding the Difference and the Role of LIMS in Achieving Both Communication can be a problem. A few misplaced letters in a word can have a huge impact on the meaning. Tony Abbott, the former Australian Prime Minister, is reported to have said that no one “is the suppository of all knowledge”,1 while Bertie Ahern a former Prime Minister (Taoiseach) of Ireland is supposed to have warned against ‘upsetting the apple tart’.2 Combine this with an industry like the IT industry, that revels in three letter acronyms, and where the use of jargon* is compounded by imprecise definition of the jargon itself, and you have a recipe for confusion. Within the context of laboratory informatics this can be frustrating to those for whom IT is a means to an end and not the end in itself. It obscures and confuses the benefits of what can be worthwhile technology driven initiatives.

Such is the case with a current hot topic in laboratory informatics, namely laboratory digitisation or digitalisation.

This article will show that those two letters do make a difference, that it is important to understand that difference, and that once the difference is understood significant benefits can be gained through implementing these initiatives. The role of a Laboratory Information Management System (LIMS) in supporting this will be covered.

Digitisation in the Laboratory Environment Starting with digitisation; this is the transformation of currently manual or paper-based operations to a digital format, generally enabled by one or more database applications. Within the laboratory there are many examples of how digitisation improves laboratory efficiency and helps ensure data integrity.

Looking at a simplified non-digitised sample workflow, samples arrive at a laboratory reception area accompanied by a handwritten test request form. Sample details and testing requirements are written into a laboratory notebook or logbook based on the submitted form. Once testing is to start a hard copy worklist may be created for a specific analyst or instrument. Before the test can be run the instrument logbook must be checked to ensure that it is in service and not awaiting maintenance or calibration. Instrument results are entered manually onto the work list or lab notebook and may require approval by another analyst or supervisor. In addition, they may require checking against applicable specifications or limits. Once the testing has been completed, the sample may need approval before the results are collated and a report created and sent to the sample submitter. During this process the submitter has been calling the lab to find out where their results are, while their colleague grumbles about late delivery of results for a sample the lab has no record of and threatens to formally complain about poor laboratory service.

This simplified example shows how digitisation through the use of a LIMS provides substantial benefits. Allowing test 12 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

requests to be submitted electronically through a LIMS portal removes potential issues with unreadable test requests, ensures only relevant tests are available and allows the requestor to print a barcoded label for the sample. Reading the barcode when the sample arrives allows the date and time of receipt to be automatically recorded. Samples that have been requested but not received are easily identified. Worklists can be automatically created based on samples and tests waiting to be tested. If QA/ QC run-sheets are required these can be built automatically based on predefined rules for the placement of the QC samples. If instruments allow bidirectional communication run-sheets can be automatically uploaded. Even if this is not possible most modern instruments can be integrated unidirectionally with a LIMS to allow automatic result capture. Transcription errors are therefore eliminated, as may be the need for result verification by another analyst. Results can also be automatically checked against specifications and limits. As LIMS can manage both instrument calibration and maintenance, and analyst competencies, it can prevent tests being run on instruments that are not in service, or by analysts not competent in the technique.

Once testing has been completed sample approval can be recorded in the LIMS and reports automatically generated and sent to the submitter. The lab hasn’t been called by the submitter to check progress because this can be done through the LIMS portal. Their complaining colleague has been told that the sample has never been delivered and that the key performance indicators available from the LIMS show the laboratory achieving their agreed targets for sample turnaround times.

Digitisation of the laboratory in this way through the use of computerised database systems such as LIMS has been taking place for many years. Experience shows that many organisations have gone at least some way down this path, however, few have implemented it fully and a surprising number still use paper-based systems. In addition, beware a feeling of complacency if you are running your laboratory using electronic spreadsheets, this is not an electronic database system (try running the question ‘Is Excel a database’ through a search engine).

Digitalisation, the Next Step Digitalisation is perhaps more difficult to define precisely, partly because it is as much a concept as anything else. However, we can look at it as how the digital environment, as enabled by the process of digitisation, will impact how we work and how we do business. One relatively simple example can be seen in laboratory stock control. Electronic stock records combined with defined reorder levels and the modelling of projected usage can support just in time ordering of laboratory supplies and reagents, while automated reordering and Business to Business (B2B) ecommerce removes the needs for time consuming manual purchase order and invoicing tasks. In effect digitalisation is the process of using the data we have, or could generate, to change, and hopefully improve our business.

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This is not necessarily a new idea but has been given added impetus lately by concepts such as industry 4.0 and laboratory 4.0, and specific tools including business analytics and business intelligence software. Increased connectivity throughout the laboratory (unfortunately sometimes referred to as the Internet of Laboratory Things or IoLT)) can provide more of the data needed. Increased automation and the use of robotics also have their part to play. Getting it right opens the way to potentially game changing technology such as machine learning and artificial intelligence. To some extent what can be achieved through digitalisation and how an organisation will benefit depends on the corporate imagination. However, it is also dependant on having the right data available in a suitable electronic format.

Once again to take a simple example, certain laboratories have always been concerned with how many samples they can process and what the impact of having an increased workload would be.

These may be high throughput laboratories wondering what the impact of a new screening project would be, or commercial testing laboratories needing to know if they can cope with a new contract. This has gained increased importance during the pandemic where testing laboratories that may already be handling hundreds of thousands of samples a week need to know if they can cope with increased throughput based on changing infection levels. This is dependent on many different variables within the laboratory, from the time it takes to unpack and receive samples through the capacity of liquid handling robots and other equipment, to how samples are approved, and results released.

With data driven tools and applications, together with increased access to relevant data, modelling the process and changes to the process becomes easier. The concept of Digital Twin software has grown up around the ability to model a physical process in order to optimise it and predict the impact of changes on the process.

This is a prime example of digitalisation. However, the ability to do this effectively is dependent on having the right digital data available, and in the laboratory setting, much of this will come from digitisation and a well implemented LIMS. www.international-biopharma.com

Getting the foundations right The aim of this article has been to differentiate digitisation from digitalisation in what is hoped is a clear and reasonably jargon freeway. The aim is to prevent laboratory organisations being led down a blind alley when it comes to process improvement.

Embarking on a digitalisation project because it is the hot new concept is unlikely to be a success if the basic process of digitisation has not been completed. The system infrastructure for the creation, storage and management of electronic data has to be in place. If this isn’t the case, then completing a digitisation project using well accepted and proven solutions such as LIMS will provide considerable process benefits and form the foundations on which the concepts of digitalisation can be built. *Jargon – the technical terminology or characteristic idiom of a special activity or group OR obscure and often pretentious language marked bycircumlocutionsand long words.3 REFERENCES 1.

2.

3.

Liberals squirm as Abbott refers to ‘the suppository of wisdom – Sydney Morning herald August 12, 2013 https://en.wikipedia.org/wiki/Malapropism https://www.merriam-webster.com/dictionary/jargon

Simon Wood Simon Wood PhD, Product Manager at Autoscribe Informatics, has 30 years' experience in the commercial LIMS environment. He is an acknowledged expert in the field of scientific and laboratory informatics. With a degree in Plant Biology from Newcastle University and a PhD in Mycology from the University of Sheffield, Simon successfully moved into the field of laboratory informatics.

Email: swood@autoscribeinformatics.com

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Predicting Drug Bioavailability with the Modern-day Toolkit A central pillar in developing new medicines is understanding the pharmacokinetic/pharmacodynamic relationship (PK/PD). Can the drug reach the target site at a sufficient concentration, engage the target, and produce the desired pharmacological effect? For orally administered medications, prevalent in the pipelines of pharmaceutical companies, an important component of the PK/PD relationship is bioavailability. Bioavailability is defined as the fraction of drug reaching systemic circulation following absorption in the gut and first pass metabolism in the liver. Accurately predicting the bioavailability of orally administered medicines in humans, during pre-clinical development is crucial as it forms the basis for setting safe and efficacious doses in the clinic. This significant parameter is therefore of interest to drug developers and regulatory agencies. When oral bioavailability is insufficient, further optimisation of compound structure, changes to formulation or an alternative route of administration may be required, all of which are grounds to discontinue development.

The Need for Improved Bioavailability Predictions Studies report that PK/PD issues account for 5–10% of clinical failures. Whilst this percentage has fallen significantly over the past 30 years,1,2 it remains a concern. Interestingly, a 2014 AstraZeneca study2 reported that <10% of project teams had a high level of confidence that their drug exhibited “a combination of good drug properties and good pharmacological end points”, where good drug properties include bioavailability. So, which factors drove this fall in PK/PD issues and what can we learn going forward to improve bioavailability predictions?

Firstly, our understanding of the physiochemical properties of compounds with appropriate human PK profiles has improved, and this knowledge is being applied earlier in the discovery process.3 This has been aided by the derivation of absorption, distribution, metabolism, and excretion (ADME) parameters from improved in vitro models, such as the use of suspension cultures of primary human hepatocytes to evaluate liver clearance.

A second useful realisation has been the limitation of animal models. Traditionally, animal models have dominated the prediction of bioavailability and have been pivotal in establishing the PK/PD relationship. It is now well established that, while animal models maybe useful qualitative predictors of human bioavailability (i.e., low vs high), they are poor quantitative predictors (i.e., percentage oral bioavailability),4,5 with no absolute correlation for individual species or all species taken together. In part, this has driven the adoption of in silico models, particularly physiologically based pharmacokinetic (PBPK) models. Here, interactions between organs and physiological processes in the human body are described by sets of differential equations that enable animal and human PK and bioavailability to be predicted. In a learn, confirm and refine cycle, PBPK 14 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

models are first validated against animal data before making human predictions based upon parameters derived from in vitro experiments.6 PBPK models rely on a priori understanding of all the relevant processes, their description in the models and high-quality input data, without which the accuracy of in silico predictions is limited.

For bioavailability predictions to improve, more complex in vitro models capable of recapitulating tissue level function, such as organ-on-a-chip (OOC) or microphysiological systems (MPSs), are required to address the limitations of animal studies and better inform PKPB models. Importantly for the study of bioavailability, OOCs feature microfluidics to simulate blood flow, which in turn has enabled the interconnection of multiple organs to model systemic effects.7 Here we briefly review the state of the art in animal and in silico models and look at how the prediction of bioavailability can be enhanced by OOCs. Can Animal Models Accurately Predict Bioavailability?

Animal models dominate when predicting bioavailability, so it is important to question their correlation with humans and investigate the causes of divergence. One seminal study examined the literature and found 184 compounds with both reported human and animal oral bioavailability.4 Unsurprisingly, the overall correlation between animal and human bioavailability was found to be poor (R2 = 0.34). Mouse (R2 = 0.25), rat (R2 = 0.28), and dog (R2 = 0.37) are the most widely used animal models; however, these species are outperformed by non-human primates (NHP, R2 = 0.69). Despite this, using NHP remains unpopular, given the higher cost and more restrictive ethical considerations. The usefulness of animal models to assess bioavailability is perhaps best reserved for qualitative assessment, given the wide range of values compared to observed human data.

One of the fundamental challenges in drug discovery is extrapolating PK parameters from animals to humans and bioavailability is not unique in this. Differences in physiology and metabolic capacity between humans and rodents are the main contributors to the disparity. For example, the gut microbiome population differs substantially in rodents and rats do not have a gall bladder, one of the main constituents of the hepatobiliary system. Expression of enzymes that drive drug metabolism varies not only between rodents and humans but between mice and rat strains. Differences also manifest in absorption kinetics, expression of transporters in the gastrointestinal tract and extent of plasma protein binding, all important when determining PK parameters.5 Regardless, animals remain an important part of pre-clinical drug discovery. Unlike standard in vitro models, often cultured as a single cell type on plastic, animal physiology is akin to humans in many ways. It is complex with circulation, an immune system and organs that are interlinked. Therefore, the challenge for OOC technologies is to more accurately model the complexity of a human in vitro to improve pre-clinical drug discovery predictions.

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Figure 1 – Human oral bioavailability. Orally administered drugs are first solubilised by the stomach, then absorbed via the gut to be metabolised by the liver. Metabolites are then distributed through the body via transporters. Solubilised drug molecules that have not been absorbed through the gut are excreted out of the body.

Once achieved, these new technologies will likely reduce costs and experimental time versus animal studies, whilst also circumventing ethical constraints.

Despite their promise, it is unlikely that in vitro technologies such as OOC will completely replace animal studies in the short term, as regulators will still demand safety studies before clinical trials in humans. However, in 2021, the European Parliament passed a resolution to phase out animal testing in research, regulatory testing, and education. The immediate challenge therefore is to make OOC models as good – or better – at predicting PK parameters, bioavailability, and toxicology versus animals to reduce the numbers used. This goal is also in line with the commitment made by the Association of the British Pharmaceutical Industry in a 2015 report10 to the 3Rs principle of animal research: replacement, reduction, and refinement.

with time.6 As models become more complex, the number of compartments increase, with each compartment representing an organ or tissue. The compartments are interconnected by flow rates, which resemble circulating blood flow. These PBPK models use in vitro – in vivo extrapolation (IVIVE) techniques to predict drug plasma and tissue concentration, as well as bioavailability.

One of the advantages of PBPK is that models can be updated throughout drug development in a “learn, confirm, and refine” approach as experiments move from in vitro to in vivo then into clinical trials. Once validated in humans, the model can be used to investigate dosing regimens, likelihood of drug-induced liver injury and population effects such as age, race, and genetics.

The Integral Role of In Silico Models Over the past few decades, the use of in silico models has grown and they now form an integral part of the drug discovery development process. In submissions to regulators, in silico models are used to extrapolate PK parameters and predict PK profiles for a drug candidate. They also offer the possibility to explore diverse clinical questions, such as interactions with over-the-counter medications or the role genetics and aging play in the behaviour of a drug, which cannot easily be investigated in animals.

There is no doubt about the importance of in silico modelling in drug discovery, but there is also a need for continued improvement. The predictability of the model is dependent on the quality of the input parameters. Given PBPK relies heavily on data from early in vitro and animal studies, there is motivation to integrate models that better predict ADME, safety and efficacy profiles, with OOC likely to play an increasingly prevalent role.

For low clearance compounds, in vitro methods using liver microsomes lack the sensitivity to measure their metabolic rate and as drugs become more metabolically stable, transportermediated PK becomes more important. However, PBPK models often lack system parameters, such as transporters and abundance of enzyme in different organs, that make transportermediated drugs more challenging to predict.

The simplest in silico models used to predict PK profiles are built using compartments or “building blocks” that describe the relationship between the plasma concentration of a drug

As next-generation PBPK develops, it is likely to utilise emerging technologies to improve the in silico prediction of candidate drugs. OOC has the potential to considerably improve

www.international-biopharma.com

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Research / Innovation / Development

Figure 2 – Combining PBPK modelling and Organ-on-a-chip for human-relevant bioavailability

the quality of data inputted into PBPK, as its models meet the challenges of recreating the complexity of human physiology and disease. In parallel, computational tools such as artificial intelligence and machine learning have the capability to analyse large, complex datasets such as chemical properties, patient, and in vitro data. Given a “training dataset” for known drugs, these tools could be used in a complementary manner to predict the parameters that feed into PBPK models before in vitro experiments begin and, as technology advances, predictions of bioavailability and critical safety parameters (such as DILI and possible side effects) may be possible at the early, compound selection stage of drug discovery.

Organ-on-a-chip for Human-relevant Bioavailability Predictions Developments in tissue engineering and microfluidics have facilitated the design of OOC technologies which aim to recapitulate the functional unit of an organ or tissue at a smaller scale. OOC can be viewed as a bridge between traditional in vitro tissue culture on 2D plastic and in vivo animal models. There are several requirements that OOC should include: (1) fluid flow that mimics blood in vessels; (2) similar functionality to in vivo tissues; (3) use of human cells and where possible multiple cell types and (4) reproducible and long-term cell culture. The design of an OOC platform will depend on the end application and user.

For example, screening the effect of drug–drug interactions on bioavailability, which is a concern to regulators and can be impractical to undertake in vivo, will require OOC platforms with sufficient throughput to run multiple replicates and many combinations in parallel.

Liver-on-a-chip represents a great example of where OOC provides improvements over standard in vitro methods. Primary human hepatocytes, when cultured in suspension or 2D, have short-lived activity of cytochrome P450s, the group of enzymes that metabolise many drugs. The combination of fluid flow and formation of a 3D microtissue in the liver-on-a-chip from CN Bio (Cambridge, UK) has enabled improved functionality of hepatocytes in vitro, with measured metabolic activity for at least 28 days.11 Other OOC companies also provide models of the liver: for example, Emulate (Cambridge, US) embed cells of the liver into a classic chip-type device, about the size of a credit card.

The platform by TissUse GmbH (Berlin, Germany) is on a similar 16 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

microscale yet allows the interconnection of different organs into systems. In contrast, CN Bio use a larger scale, open-well design with embedded micropumps that also enable single and interconnected multi-organ studies. Providing easy access to media and cells, this latter multi-organ system is well suited to bioavailability assays.

For determining ADME parameters such as bioavailability in vitro, a multi-OOC model with fluidically linked gut and liver compartments is required. This enables an oral drug to be dosed into the gut compartment, where it passes through the intestinal barrier before entering the liver compartment for metabolism by hepatocytes.8,9 Medium samples taken over time are used to generate plots of drug concentration over time that resemble PK profiles, with an area-under-the-curve measurement made to estimate oral bioavailability. Multi-OOC also allows for the study of potential crosstalk between these organs; for example, the metabolites produced by liver metabolism of prodrugs used to treat cancer may have toxic effects on intestinal cells.

The translation of data generated by OOC to parameters relevant in PBPK modelling is a challenge that needs to be met to ensure relevance in pre-clinical drug development. To achieve this, mathematical modelling can be utilised, starting with differential equations that describe the concentration of a compound over time in an OOC system. In a multi-OOC, mechanistic models consider operational characteristics such as compartment volumes, flow rates and the scaling of hepatic cells that are seeded to that of a human adult liver.8 Once model analysis is performed, PK parameters such as hepatic and intestinal clearance rates as well as information on biodistribution can be obtained.

As OOC adoption increases in the pharmaceutical industry, there is a need for continued improvement. Where possible, OOC should utilise primary cells that better recapitulate the functionality of tissues and are more responsive to toxicological effects versus cell lines derived from cancer tumours. The inclusion of circulating and tissue-resident immune cells in OOC models would make disease models such as cancer more responsive to immunotherapies. For bioavailability, addition of the microbiome that is known to bioaccumulate and metabolise Winter 2021 Volume 4 Issue 4

Research / Innovation / Development certain drugs would provide an improved estimation of the amount absorbed through the small intestine and available for hepatic metabolism.

Conclusion Significant strides have been made in reducing clinical attrition due to PK/PD and bioavailability issues, but problems remain.

Animal models continue to be used in bioavailability research, but their limitations are serious and difficult to overcome.

The development and adoption of PBPK modelling and more latterly OOC, particularly multi-organ OOC, point the way forward. Improvements in prediction will be found using these technologies both separately and in concert. The three areas in which multi-organ OOC systems can offer benefits are (i) direct measurement of bioavailability, particularly those compounds and modalities that are not easily described in PBPK models, (ii) derivation of high-quality ADME parameters required by PBPK models and (iii) as a platform to validate PBPK models.

Now that expertise in PBPK modelling has expanded and commercially produced multi-organ OOC systems have been widely adopted, we should be questioning the use of animals in bioavailability testing. By providing scientists with more translationally relevant data that drives down failures due to PK/PD issues, will multi-organ OOC and PBPK models reduce and ultimately replace animals in this area? The early signs are promising, but all models – be they in vitro, in vivo or in silico – have limitations, and require a clear context of use to ensure successful application. As multi-organ OOCs are added to the toolkit, success will be achieved by defining the unique points at which they add value and placing them in a framework with other technologies to achieve a robust and well understood methodology for predicting bioavailability.

REFERENCES 1.

Kola, I. & Landis, J. Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov. 711–5 (2004).

2. Cook, D. et al. Lessons learned from the fate of AstraZeneca's drug pipeline: a five-dimensional framework. Nat Rev Drug Discov.

419–31 (2014).

3. Beaumont, K. & Smith, DA. Does human pharmacokinetic prediction add significant value to compound selection in drug discovery research? Curr Opin Drug Discov Devel. 61–71 (2009).

4. Musther, H. et al. Animal versus human oral drug bioavailability: do they correlate? Eur J Pharm Sci. 280–91 (2014).

5. Grass, GM. & Sinko, PJ. Physiologically-based pharmacokinetic simulation modelling. Adv Drug Deliv Rev. 433–51 (2002).

6. Jones, H. & Rowland-Yeo, K. Basic concepts in physiologically based pharmacokinetic modeling in drug discovery and development. CPT Pharmacometrics Syst Pharmacol. e63 (2013).

7. Edington, CD. et al. Interconnected Microphysiological Systems for Quantitative Biology and Pharmacology Studies. Sci Rep. 4530 (2018).

8. Tsamandouras, N. et al. Integrated Gut and Liver Microphysiological Systems for Quantitative In Vitro Pharmacokinetic Studies. AAPS J.1499–1512 (2017).

9. Abbas, Y. et al. Drug metabolism in a gut-liver microphysiological system. CN Bio Application Note. https://landing.cn-bio.com/ multi-organ-appnote 10. Animals and medicines research: Animals research for the discovery and development of new medicines. abpi. 2015: https://www.abpi. org.uk/media/1388/animals-medicines-research.pdf 11. Rubiano, A. et al. Characterizing the reproducibility in using a liver microphysiological system for assaying drug toxicity, metabolism, and accumulation. Clin Transl Sci. 1049–1061 (2021). www.international-biopharma.com

Dr. David Hughes Dr. David Hughes is CEO of CN Bio Innovations, a leading OOC company that has developed single and multi-organ microphysiological systems that improve the accuracy and efficiency of drug discovery, including the PhysioMimix™ lab-benchtop instruments. Dr Hughes is also Principal Investigator on a $26million US DARPA contract towards development of human in vitro multi-organ platforms and leads a €4million EU FP7 program aimed at developing stem cell derived models of human liver. Dr Hughes graduated from the University of Oxford with a Masters in Engineering Science and Doctorate in Chemical Engineering.

Email: david.hughes@cn-bio.com

Dr. Yassen Abbas Dr. Yassen Abbas is a Bioengineer at CN Bio Innovations. He completed an MEng in chemical engineering at The University of Edinburgh and joined the European Space Agency as a graduate engineer. He later received a PhD from the University of Cambridge and completed a postdoc fellowship, also at Cambridge on the development of a tissue engineered model of the human endometrium.He hasexperience withreal-time sensor technology,organoids and development of in vitro tissue models using human primary cells. Dr Abbas has published five peer-reviewed scientific articles, four as first author.

Email: yassen.abbas@cn-bio.com

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Research / Innovation / Development

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SLIM: A Gateway to the Adoption of High-Resolution Ion Mobility for Biotherapeutic Peptide Mapping The demand for protein therapeutics, such as recombinant human proteins, monoclonal antibodies (mAbs), and fusion proteins, has grown significantly over the past decade. Often developed to treat previously unmet clinical needs, these biotherapeutics require increasingly advanced analytical technology for detection, identification, quantitation, and quality control (QC)/monitoring of molecular attributes.

Some recombinant therapeutics require post-translational modifications (PTMs) such as glycosylation for clinical efficacy, but erroneous PTMs can occur within the mammalian host expression systems that are typically used to produce these therapeutics.

In this context, PTMs are known as product-related impurities and can be broadly categorised as either enzymatic or chemical modifications. Enzymatic modifications most commonly include glycosylation, disulfide bond formation, and proteolytic cleavage of the protein backbone. Chemical modifications are often generated during downstream processing, formulation, and storage, and typically include oxidation, deamidation, glycation, and pyroglutamate formation. These impurities can impact the biological activity, half-life, and immunogenicity of protein therapeutic products, so must be characterised, controlled, and monitored throughout the development process to safeguard the drug’s stability, efficacy, and safety.

To achieve consistency in production, quality by design (QbD) is becoming a widely accepted strategy within the industry with the goal of enhancing pharmaceutical manufacture through design and control of processes.1 QbD systematically establishes the critical quality attributes (CQA) of a drug product, which are identified and closely monitored for efficient QC. A CQA is defined by the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) Q8 (R2) as a “physical, chemical, biological or microbiological property or characteristic that should be within an appropriate limit, range, or distribution to ensure the desired product quality.”2 The assays and instruments used to analyse CQAs are expected to be precise, reproducible, and robust, with commonly orthogonal techniques carried out to complement and provide confidence in results. Fast analytical methods that can produce large amounts of data have become vital to speed up bioprocessing timelines and the decision-making process.3 While conventional analytics such as chromatography and electrophoresis methods are important for early development and characterisation of therapeutic proteins, their sensitivity and specificity can be limited. Orthogonal mass spectrometry (MS) techniques are now recognised as an important tool, particularly for identifying and characterising PTM as CQAs in the biotechnology development pipeline. MS-based assays, such as peptide mapping, provide a means to directly measure PTMs at 18 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

the peptide and individual residue level. However, the analytical complexity of peptide mapping has, until now, limited its uptake in bioprocess monitoring and PTM characterisation.

Enhancing MS-based Peptide Mapping MS-based assays are providing ever-richer detail and a more granular perspective on PTM sites and are often coupled with liquid chromatography (LC) separations to overcome the challenge of ionisation variability, suppression, and difficulties in resolving isoforms with small mass differences. However, traditional LC separations have relatively low specificity and require long gradients to identify all components of the protein digest and resolve critical modifications for relative quantitation.

Routine implementation of downstream peptide mapping workflows using LC-MS has proven difficult due to the typical gradient times in the order of 90 minutes, with some as long as 190 minutes.4 In addition, considerable analytical expertise is required to interpret the data. The separation often requires these long analytical gradients to resolve critical near-isobaric or isobaric PTMs for quantitation.5 Incorporating ion mobility (IM) as an orthogonal separation that relies on peptide structure can reduce reliance on the LC separation by providing an additional differentiation filter to resolve isobaric peptides, reducing ambiguity in identification through mobility-aligned fragmentation. Established IM techniques suffer from limited resolution, meaning the condensed-phase separation is still necessary for relative quantitation of PTMs. In the IM separation, ions traverse a defined path through an inert buffer gas, such as nitrogen or helium, under the influence of an electric field. The arrival time of an ion at the end of the separation path increases with the ion’s collisional cross-section (CCS), a value that represents the rotationally averaged cross-sectional area of the ion in the gas phase.6 CCS values provide a fourth dimension of separation in addition to retention time, mass-to-charge (m/z) ratio, and spectral peak intensity, and have proven advantageous for complex separations and targeted peptide mapping workflows.7,8 Resolving power in IM typically scales with the square root of the separation path length, making the resolving power of IM separations with longer path lengths higher than that with shorter distances. Therefore, the path length of traditional IM technology (usually < 1m) can limit the power available to resolve critical isomeric species and necessitates long chromatographic methods for PTM quantitation.

Faster, More Detailed Analyses To overcome this challenge, researchers have developed a high-resolution ion mobility (HRIM) technique based on SLIM (Structures for Lossless Ion Manipulations) that can achieve 10 times higher resolution compared with traditional IM methods.

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 19

Research / Innovation / Development

Figure 1 – Glycoform quantitation on intact NIST RM 8671 using (A) a two-minute flow injection method on SLIM-MS (13m SLIM device, MOBILion Systems, 6545XT QToF MS, Agilent) and a five-minute LC-MS method, and (B) an eight-minute integrated LC-SLIM-MS method.14

SLIM – originally developed in the laboratory of Dr. Richard D. Smith at Pacific Northwest National Laboratory – transmits ions along a path between two parallel printed circuit boards (PCBs) with negligible ion loss, with a throughput of minutes, rather than hours. The electric fields that propel the ions also prevent them from striking surfaces while moving, therefore preventing any losses along their way. Ions are able to turn around the corners of SLIM’s serpentine path, removing the limitations set by linear paths and achieving previously impossible separations. SLIM has recently incorporated a multiple pass path for further enhanced separation of isomeric species.9–11 Similarly to other IM technologies, in HRIM separations, an ion’s migration time is determined by its mass-to-charge ratio (m/z) and its size and shape. As ions are driven along the separation path, the collision with an inert buffer gas slows them down to a degree proportional to their size. The serpentine path shape of the PCBs allows unprecedented ion path lengths to fit into a benchtop device (e.g., 13m SLIM device, MOBILion Systems), leading to resolutions high enough to separate the most challenging isomeric structures and collect data in the order of milliseconds to seconds. Compared with LC, HRIM set-up is relatively straightforward as there is no need to optimise columns, flow rates, or liquid components.

Enhancing Peptide Mapping Workflows Using SLIM-based HRIM coupled to a quadrupole time-of-flight (QToF) mass spectrometer, researchers can now achieve faster chromatographic separations for peptide mapping workflows. In a recent study, a NISTmAb was characterised by LC-HRIM-MS and LC-HRIM-MS with collision induced dissociation (HRIM-CID-MS) using only a 20-minute gradient – between 3–4.5x faster than traditional peptide mapping gradients.5 LC-HRIM-MS experiments achieved a sequence coverage of 96.5%, with LC-HRIM-CID-MS experiments providing additional confidence in sequence determination. HRIM-MS resolved critical oxidations, deamidations, and isomerisations that coelute with their native counterparts in the chromatographic dimension, demonstrating the ability of SLIM-based HRIM to maintain PTM quantitation capabilities and add isomeric resolution of critical coeluting PTMs. Glycoform Quantitation Protein glycosylation is a vitally important PTM in the functioning of a range of biological processes, such as molecular recognition, protein trafficking, regulation, inflammation, and abnormalities in protein glycosylation are associated with 20 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

several disease states such as cancer, inflammatory diseases, and congenial disorders. Careful monitoring of protein glycosylation is also crucial for the development of stable and effective drugs and for comparing biosimilar products to reference drugs, as required by regulatory agencies.12 Changes in manufacturing process conditions for biologics, such as process optimisation, scale-up production, and site changes, may impact glycosylation patterns of the resulting recombinant antibody.13 Glycans have many structural and functional roles and display enormous heterogeneity between glycoforms, so determining the profile of the glycans and glycosylation sites is crucial for producing safe, good quality, consistent therapeutic proteins. Although LC-MS can detect variations in glycoform species, peptide mapping for glycoform quantitation requires long gradients that hinder analysis in fast-paced bioprocessing environments.

Research has recently examined the utility of SLIM-MS and LC-SLIM-MS for faster and more in-depth characterisation of protein therapeutics.14 Glycoform quantitation on intact NIST RM 8671 was performed using a two-minute flow injection method on SLIM-MS and showed highly similar quantitation to a five-minute LC-MS method (Figure 1A). The study found that integrating SLIM to existing eight-minute subunit LC-MS workflows (LC-SLIM-MS) maintained quantitative capabilities of the method (Figure 1B).

The study also found that SLIM-integrated peptide mapping enables a five-minute analytical gradient compared to traditional 60–90 minutes using LC-MS alone, with the LC-SLIMMS method maintaining nearly complete sequence coverage.

Figure 2 shows coeluting critical quality peptide deamidations could be fully or partially resolved by SLIM separation using the five-minute gradient method.

Future of PTM Monitoring LC-MS has long been used for sensitive and accurate impurity analysis in the biopharmaceutical industry. However, developments in analytical technology over the last decade have accelerated the characterisation and monitoring of productrelated impurities such as PTMs beyond the capabilities of traditional LC-MS workflows. Advances in IM separations and the development of SLIM-enabled HRIM now combine higher resolving power with rapid speed of analysis, without compromising sequence coverage. The resulting increase in Winter 2021 Volume 4 Issue 4

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 21

Research / Innovation / Development Separations in SLIM. Analytical Chemistry, 2020; 92(7): 5004-5012.

12. Beck A and Reichert JM. Approval of the first biosimilar antibodies in Europe: a major landmark for the biopharmaceutical industry, mAbs, 2013; 5(5): 621–623.

13. Hahm YH, Hahm SH, Jo HY, et al. Comparative Glycopeptide Analysis for Protein Glycosylation by Liquid Chromatography and Tandem Mass Spectrometry: Variation in Glycosylation Patterns of Site-Directed Mutagenized Glycoprotein, International Journal of Analytical Chemistry, 2018; Article ID 8605021.

14. Arndt JR, Wormwood Moser KL, Deng L, et al. Biotherapeutic Characterization in Fifteen Minutes: Structures for Lossless Ion Manipulations (SLIM) Ion Mobility for Critical Quality Attribute Identification and Monitoring, ASMS 2020.

Jared Auclair Figure 2 – Coeluting critical quality peptide deamidations are fully or partially resolved

by SLIM separation in the five-minute gradient method shown here.

confidence of PTM characterisation and quantification leads to a more robust QbD approach to biopharmaceutical manufacturing and QC, with CQA monitoring that meets regulatory expectations in short time frames.

For more information about SLIM, please visit https://www. mobilionsystems.com/.

REFERENCES 1.

Finkler C and Krummen L, Introduction to the application of QbD principles for the development of monoclonal antibodies, Biologicals, 2016; 44: 282-290.

2. ICH, ICH Harmonised Tripartite Guidelines Pharmaceutical Development Q8 (R2) Step 4 version, 2009.

3. Oshinbolu S, Wilson LJ, et al. Measurement of impurities to support process development and manufacture of biopharmaceuticals, Trends in Analytical Chemistry, 2018; 101: 120-128.

4. Evans AR, Capaldi MT, Goparajuet G, et al. Using bispecific antibodies in forced degradation studies to analyze the structure–function relationships of symmetrically and asymmetrically modified antibodies. mAbs, 2019; 11(6): 1101-1112.

5. Arndt JR, Wormwood Moser KL, Van Aken G, et al. High Resolution Ion Mobility-enabled Peptide Mapping for High-Throughput Critical Quality Attribute Identification and Monitoring. Awaiting publication.

6. Kanu AB, Dwivedi P, Tam M, et al. Ion mobility–mass spectrometry.

Journal of Mass Spectrometry, 2008; 43(1): 1-22.

7. Ruotolo BT, McLean JA, Gillig KJ, et al. Peak capacity of ion mobility mass spectrometry: the utility of varying drift gas polarizability for the separation of tryptic peptides. Journal of Mass Spectrometry, 2004; 39(4): 361-367.

8. Olivova P, Chen W, Chakrabortyal AB, et al. Determination of N-glycosylation sites and site heterogeneity in a monoclonal antibody by electrospray quadrupole ion-mobility time-of-flight mass spectrometry. Rapid Communications in Mass Spectrometry, 2008; 22(1): 29-40.

9. Deng L, Webb IK, Garimella SVB, et al. Serpentine Ultralong Path with Extended Routing (SUPER) High Resolution Traveling Wave Ion Mobility-MS using Structures for Lossless Ion Manipulations.

Analytical Chemistry, 2017; 89(8): 4628-4634.

10. Nagy G, Attah IK, Garimella SVB, et al. Unraveling the isomeric heterogeneity of glycans: ion mobility separations in structures for lossless ion manipulations. Chemical Communications, 2018; 54(83): 11701-11704.

11. Nagy G, Attah IK, Conant CR, et al. Rapid and Simultaneous Characterization of Drug Conjugation in Heavy and Light Chains of a Monoclonal Antibody Revealed by High-Resolution Ion Mobility 22 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Jared R. Auclair is currently the Associate Dean of Professional Program and Graduate Affairs in the College of Science at Northeastern University and Associate Teaching Professor in the department of chemistry and chemical biology. In addition, Dr. Auclair is the Director of the Biopharmaceutical Analysis Laboratory (BATL), the Asia-Pacific Economic Cooperation Center of Regulatory Excellence in Biotherapeutics and Advanced Therapies and oversees the International Council for Harmonisation training. Lastly, Prof. Auclair serves as the Technical Supervisor for the Life Science Testing Center at Northeastern University, which is a state and CLIA-certified lab. Dr. Auclair collaborates with both academic researchers, industry, and government in the area of biopharmaceutical and cell/gene therapy development and analysis. He has expertise in mol. biology, protein biochemistry, analytical chemistry, protein crystallography, and biological mass spectrometry; and is interested in use inspired research for the biotechnology industry.

Melissa Sherman Dr. Melissa Sherman started her career as a research chemist with E.I. DuPont de Nemours and quickly transitioned from technical to marketing and business management positions in polymer fiber related industries. After DuPont, she worked for W.L. Gore as a product manager overseeing product development, manufacturing, regulatory, and sales for a variety of surgical product businesses.Melissabuilt an independent technology commercialization and strategy company, working with diverse clients in the regenerative medicine medical product sector. Melissa was hired by medical device companies, Kensey Nash and Aimago, to redefine and execute corporate growth strategies. Melissa was the Director of Technology and Business Development for IP Group Inc. where she managed early-stage technology investment at federal laboratories and was responsible for investment thesis development, deal origination, due diligence, and transaction execution. IP Group appointed Melissa to her current position as the CEO of MOBILion Systems Inc., an IP Group portfolio company. Melissahas a Ph.D. in Polymer Science from The University of Akron, and a B.S. in Chemistry from The University of Wisconsin – Eau Claire. Melissa, a Six Sigma Black Belt, will lead MOBILion into the future with her passion for building businesses, her visionary leadership, and her unmatched ability to execute tactically.

Winter 2021 Volume 4 Issue 4

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 23

Application Study

An End-to-End Solution to Accelerate CAR-T Cell Development from Concept to Clinic CAR-T cell therapies have demonstrated tremendous success in relapsed or refractory haematological malignancies. Now therapeutics companies and academics alike are racing to break through the barrier of solid tumours, taking imaginative approaches to overcome the numerous obstacles in the way of turning a hugely variable starting material into an armed and precisely targeted weapon against cancer. But building a biologic is a team effort, so while therapeutics companies focus on perfecting their scientific approach, partnering with a contract testing development and manufacturing organisation (CTDMO) can help streamline their preclinical and manufacturing processes, and accelerate their journey to the clinic.

Leveraging the Power of the Immune System to Treat Advanced Cancers TiCARos Therapeutics has leveraged the expertise of their team of immunologists from Seoul National University, College of Medicine to develop a promising pipeline of next generation immunotherapies for intractable haematologic malignancies and advanced solid tumours. TiCARos’ mission is to create medication based on science. TiCARos was founded by two leading T-cell immunologists, who have built a team with a deep passion for innovative science and immunology. Their strength is in leading-edge approaches to new CAR-T cell therapies. Since its foundation, TiCARos has established active collaborations with the renowned institutions Seoul National University College of Medicine and National Cancer Center, with which their founders have strong ties.

TiCARos’ cutting-edge technology platforms include CLIP CAR Technology, a strategy to stabilise the connection between CAR-T cells and antigen presenting cells, and CONVERTER CAR Technology, a strategy converting an inhibitory signal of CTLA-4 to an activating signal of CD28 in T cells by using

24 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

CTC28 chimera, which potentiates CAR-T activity without a corresponding increase in side effects.

Accelerating the Pre-Clinical Discovery Phase To accelerate the development of their CAR-T pipeline, TiCARos partnered with OXGENE, a WuXi Advanced Therapies company, for their expertise in construct design and engineering, and viral vector production.

DNA Engineering and Small-Scale Lentiviral Production In the first step of the project, OXGENE cloned four TiCARos constructs into OXGENE’s proprietary XLenti™ backbone.

OXGENE’s 3rd generation, self-inactivating (SIN) XLenti™ packaging plasmids are optimised for increased translation efficiency and have low homology between expression cassettes for an improved safety profile. This plasmid system outperforms other commercially available equivalents, and in this project, our team of expert molecular biologists’ custom engineered this system to optimise expression of TiCARos’ genes of interest.

In the second phase of the project, OXGENE generated concentrated and purified lentiviral vectors containing TiCARos’ custom constructs at shake flask scale, achieving good yields in each case.

Lentiviral Production and Process Development in Stirred Tank Bioreactors Following a positive outcome from the first project, TiCARos initiated a second project with OXGENE; this time to produce lentivirus containing the two best performing constructs in 1L bioreactors, followed by downstream concentration and purification. As well as measuring final infectious viral titre (through integrated viral copy number assay), OXGENE’s analytics team confirmed the total number of viral particles by p24 ELISA and quantity of viral genomes present in each

Autumn 2019 Volume 2 Issue 3

Application Study

Figure 1 – ?

sample by reverse transcription qPCR. The results were consistent across all assays.

Jae Won Lee, the CEO of TiCARos stated, “We’ve been extremely pleased with the work conducted by OXGENE to promote the pre-clinical development of our next generation CAR-T cell therapies. Working together with OXGENE in the UK, and later with WuXi Advanced Therapies in China has helped to accelerate the development of these promising clinical candidates.”

Producing GMP Grade Material for Clinical Studies TiCARos chose to work with WuXi Advanced Therapies for GMP plasmid and lentiviral vector manufacture. The process began with full characterisation of TiCARos’ plasmids; three lentiviral packaging plasmids and two transfer plasmids (from OXGENE).

WuXi Advanced Therapies then screened for the highest yielding clones to establish the research cell bank (RCB), and subsequently the master cell bank (MCB) and working cell bank (WCB) under GMP conditions. After establishment of the bacterial banks, WuXi Advanced Therapies conducted an upstream demo run to confirm and optimise the upstream fermentation process to meet target yield, as well as stability and long-term storage stability studies to ensure long-term plasmid quality. Next, WuXi Advanced Therapies carried out a downstream verification run to confirm and optimise the downstream purification process, before confirming the analytical assays to be used for quality control.

The final step before scheduling GMP plasmid production of the three lentiviral packaging plasmids and one of the transfer plasmids was for WuXi Advanced Therapies to produce process development and template batch record reports for TiCARos. Alongside GMP plasmid manufacture, WuXi Advanced Therapies also ran a full set of analytical and stability studies, as well as providing all the necessary documentation for their Investigational New Drug (IND) filing.

The last part of the project was to produce lentiviral vectors at GMP grade, using the plasmids OXGENE had created and WuXi Advanced Therapies had manufactured. Once again, WuXi Advanced Therapies conducted a demo run first to verify and optimise the upstream process and a verification run to confirm platform suitability and downstream processing conditions, making sure that optimal conditions were locked in for GMP production. www.biopharmaceuticalmedia.com

Figure 2 – ?

TiCARos is now using the GMP grade material produced at WuXi Advanced Therapies for pre-clinical studies and plans to begin clinical trials and submit an IND application next year. The biotech is committed to developing and commercializing novel CAR-T therapies in the near future for complete remission of both hematologic and solid tumours, and ultimately improve cancer patients’ survival.

TiCARos is seeking potential global partnerships for its CAR-T development.

OXGENE and WuXi Advanced Therapies: an End-to-End Solution to Accelerate the Development of Cell and Gene Therapies.

In March 2021, OXGENE was acquired by WuXi AppTec to become part of WuXi Advanced Therapies. This means that, like TiCARos, our customers now have access to OXGENE’s industry leading technologies and WuXi Advanced Therapies’ comprehensive manufacturing and testing capabilities. With sites in the UK, USA and China, OXGENE and WuXi Advanced Therapies’ combined capabilities support cell and gene therapy developers across the globe, offering technical support, manufacturing solutions and integrated testing, all the way from early discovery and pre-clinical research to GMP manufacture. Together, WuXi Advanced Therapies and OXGENE can accelerate this transition; aligned systems and processes allow for streamlined internal tech transfer between teams, accelerating development time and reducing the costs involved in bringing a cell or gene therapy to market.

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

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 25

Research / Innovation / Development

PEER REVIEWED

Benefits & Key Considerations of Using Human iPSC-Derived Disease Models in Drug Discovery The process of drug discovery, development and commercialisation is long and associated with high costs. In addition, it is estimated that only 1% of the initially tested compounds make it to the market.1 To decrease this high attrition rate, it is necessary to implement physiologically relevant disease models with higher predictability much earlier in of drug discovery. Disease models based on human induced pluripotent stem cell (iPSC) technology has the potential to revolutionise drug discovery. These models recapitulate, in vitro, many clinical features of human pathology and can be used for phenotypic screening with clinically relevant readouts. This article describes how human iPSC-derived disease models can improve drug discovery and what the main challenges and solutions are for their successful generation and application of these models.

Traditionally, target-based drug discovery has focused on biochemical assays or non-physiologically relevant cell-based assays. Biochemical assays are highly suitable for high-throughput screening (HTS), but it is difficult to predict the in vivo therapeutic potential of the hits found. Cell-based assays offer a more complex cellular environment and the possibility to evaluate the phenotypic effect of compounds.

However, traditional cellular models, such as immortalised cells, present several limitations regarding disease modelling and translatability to the clinic. As an alternative, primary human cells provide more representative responses, although they have a significant donor-to-donor variability and are rarely available in large-enough quantities for HTS, especially for less accessible organs such as the heart or brain.

Animal models do offer an in vivo complex environment, but are more expensive, have serious scalability constraints and the substantial inter-species differences hamper modelling of certain human diseases, such as cardiac arrhythmias or neurodegenerative diseases.

In the past decade, iPSC technology has emerged as a powerful tool to bring the human biological context earlier into the drug discovery funnel. Human iPSCs are obtained from patients’ or healthy donors’ somatic cells and reprogrammed to pluripotent stages. They have the ability to self-renew, while maintaining the potential to differentiate to nearly any functional cell type in the body, closely mimicking the human (patho)physiology. Human iPSCs are relatively easy to obtain from adult tissues and they retain patient-specific genetic backgrounds, making them a preferred system for disease modelling (Figure 1).2 BENEFITS OF HUMAN IPSC-BASED DISEASE MODELS The main benefits of using human iPSC technology for disease modelling are that iPSCs retain patient-specific genetic backgrounds and show clinically relevant phenotypes of 26 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Figure 1 – The process of disease modelling based on human iPSCs. Human iPSCs can be obtained from somatic cells with non- or low-invasive techniques and used to generate physiologically relevant disease models that retain patient-specific genetic backgrounds.

many human diseases. These features facilitate the study of disease development and complex pathological mechanisms.

In this section, we selected specific examples that highlight the advantages of human iPSC-based disease models.

Noonan syndrome with multiple lentigines, formerly known as LEOPARD syndrome, was one of the first diseases successfully modelled using iPSCs. Patients with LEOPARD syndrome develop cardiac hypertrophy in their late 40s.

However, iPSC-derived cardiomyocytes (CMs) from patients with this syndrome showed hypertrophic phenotype within 30 days of cell culture. This allowed elucidation of molecular signatures associated with the disease, in a relatively short timeframe.3 Winter 2021 Volume 4 Issue 4

Research / Innovation / Development Another advantage of using iPSCs in disease modelling was reflected by the derivation of CMs from patients’ iPSCs with Long QT syndrome (LQTS). LQTS is characterised by delayed repolarisation of the heart that can lead to a severe ventricular arrhythmia called Torsades de Pointes. Mouse models failed to mimic the disease because of the different electrophysiological properties. However, iPSC-derived CMs from LQTS patients manifested the electrophysiological signature of LQTS and proved to be a powerful system for pathogenesis studies and therapeutic compound testin.4 Schizophrenia is a multifactorial disease and patients can show a range of symptoms and responses to treatment.

The availability of iPSC-derived schizophrenia models from different patients can facilitate in vitro prediction of treatment responses and open the door for patient stratification and precision medicine.5,6 In a research study, iPSC-derived neurons from patients with Schizophrenia exhibited diminished neuronal connectivity and decreased neurite numbers, which are characteristic features of this disease, and responded to treatment with a clinically approved antipsychotic.

During the differentiation process of iPSCs, the stages of organ development are replicated in vitro, which is a great benefit for modelling congenital and developmental diseases.

Rett syndrome (RTT) is a genetic neurodevelopmental disorder that can manifest early after birth. RTT patientspecific iPSC lines have been used to investigate the phenotypic consequences of each specific mutation.7 At the other end of the spectrum, iPSCs can recapitulate disease progression, even for late onset disorders, which enables modelling Alzheimer’s disease (AD), Parkinson’s disease and other neurodegenerative diseases. A study with iPSC-derived neurons from different AD patients showed good data correlation with patients’ regimen-data, indicating the clinical translational power of these models.8 Moreover, more complex environments can be mimicked by deriving multiple cell lineages from the same iPSC line. For instance, iPSC-derived microglia and astrocytes from the same donor can be co-cultured with neurons for the study of cell-cell interactions.

An additional advantage of using iPSC-based models was brought by the breakthrough discovery of CRISPR. This genome editing technology is cost-effective, relatively fast, and efficient in iPSC. Using CRISPR, disease models can be derived from healthy human iPSC lines via knock-in, knock-out, or introduction of point mutations described in patients. For instance, an iPSC-derived model for hypochondrogenesis was generated by introducing a patient mutation (COL2A1 p.G1113C) in the collagen type II gene (COL2A1) with CRISPR/ Cas9.9 In addition, genome editing enables the generation of isogenic controls to minimise genetic-background related variability and identify the true impact of the genetic variants on cellular phenotypes.

HOW CAN IPSC-DERIVED DISEASE MODELS IMPROVE DRUG DISCOVERY? It has been demonstrated that human iPSC-derived disease models can successfully recapitulate many disease phenotypes that are clinically relevant and that cannot always be elucidated by traditional models. This feature makes iPSC technology an excellent tool to improve decisionmaking steps throughout the early phases of drug discovery (Figure 2).

TARGET IDENTIFICATION AND VALIDATION Target identification and validation stages benefit from the use of iPSC-based disease models because of their accurate representation of the disease and human biology, especially when the mechanistic landscape is not completely understood.

These models can make hypothesis generation more precise through the study of disease physiology and open the door for target validation based on phenotype rescue assays. As an example, human iPSCs derived from patients with a high ratio of mutant mitochondrial DNA were used to identify a potential therapeutic target for mitochondrial diseases. The patient-derived iPSC line exhibited defective differentiation into neuronal cells, and it was found that the compound tryptolinamide (TLAM) was able to rescue the phenotype.

Based on this approach, the protein inhibited by TLAM was identified, and could therefore be classified as potential therapeutic target.10

Figure 2 – Benefits of human iPSC-derived disease models along the different phases of drug discovery. The implementation of human iPSC-derived disease models in drug discovery can reduce costs, shorten the process, and decreases the attrition rate by bringing the true human biology earlier into the drug discovery and development process. www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 27

Research / Innovation / Development Hit Identification Phenotypic screening with physiologically relevant iPSC-based disease models facilitates the selection of relevant hits during drug efficacy testing. This type of screening also enables the description of novel therapeutic drug mechanisms of action and increases predictability. Since human iPSC-derived models can be used to study disease phenotypes, for example measured by changes in morphology, biomarker expression, metabolism or cellular function, they provide a more complete understanding of drug efficacy, compared to other screening platforms. Furthermore, the results of phenotypic screening with iPSC-based disease models are easier to extrapolate to the clinical situation because, in many cases, the selected readouts are equivalent to the clinical markers used for diagnosis. Recently, Ncardia developed a human iPSC-derived model of cardiac hypertrophy for the phenotypic screening of 3600 compounds. Human iPSC-derived cardiomyocytes were exposed to Endothelin-1 (ET-1) to induce hypertrophy and an AlphaLISA assay was developed to measure secretion of NT-proBNP, a clinical marker of hypertrophic cardiomyopathy.

341 hits were identified following this strategy and 192 confirmed with additional repetitions of AlphaLISA, high-content imaging of BNP protein expression and exclusion of false-positive hits.11 Hit to lead & lead Optimisation iPSC-derived disease models can be used to establish a structure-activity relationship (SAR) and measure the potency of newly synthesised compounds by looking at phenotypic changes, such as cellular functions, protein expression or metabolism. To facilitate a lead validation study with compounds that rescue Parkinson’s disease (PD) phenotype, Ncardia developed a PD model using Ncyte CNS Neurons. This co-culture of human iPSC-derived neurons and astrocytes was exposed to alpha-synuclein preformed fibrils (PFF) to induce neurodegeneration mimicking PD. Multi-electrode Array (MEA) analysis of the iPSC-derived PD model showed decreased neuronal firing rate in response to PFF treatment.

The same assay was used to calculate the PFF-induced toxicity and to successfully validate compounds that rescued PD’s phenotype.12 KEY CONSIDERATIONS Disease modelling based on human iPSC-derived cells has the potential to revolutionise drug discovery. Nonetheless, significant expertise is required to overcome some technical and conceptual challenges for the widespread implementation of these models in the pharmaceutical and biotechnology industry (Figure 3). Outsourcing can be a solution to achieve meaningful and actionable results in the shortest timeline possible. Collaboration with experts avoids common pitfalls, enables selection of risk mitigation strategies, enhances productivity, and ultimately reduces costs. hiPSC sourcing & reprogramming The first step to building a human iPSC-derived cell model is to obtain the most suitable iPSC line, taking into consideration patient genotype, tissue selection, age, gender, and availability of matched controls. This can be difficult due to informed consents not optimised for use in drug discovery.

In order to make iPSC sourcing less complex and save time with procurement, having a broad network of contacts and 28 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Figure 3 – Main considerations for the widespread use of human iPSC-derived cells in drug discovery. Significant expertise, in-house equipment and biological knowledge are required for the successful generation of disease models based on human iPSC technology.

agreements with multiple biobanks is beneficial. It is also essential to choose a reprogramming method that is efficient, has been validated in multiple somatic cell types, and has no genomic footprint.

Scale up differentiation The process of drug discovery involves testing thousands of compounds to identify the best hits that will potentially become a beneficial therapeutic. Ideally, the same batch of iPSC-derived target cell is used for the whole process to avoid additional variables impacting the screening campaign.

Therefore, the production of iPSC-derived cells must be of high quality, reproducible and in a large-enough scale for HTS applications. Automation, regular in-process monitoring, and multiple controls are needed to successfully scale-up the production of iPSCs to the required levels. Differentiation protocols in the public domain are typically established in 2D, with standard culture equipment and for a low number of cells. High understanding of cellular biology and high-level technology equipment are needed to set-up all the conditions and steps for an efficient iPSC differentiation in a large scale.

Assay Development Having the capabilities to measure phenotypic changes is essential for disease modelling and drug discovery. The assays used for phenotypic screening must be predictive, validated, and easy to perform in both high- and low-throughput. Finding the optimal assay conditions, in terms of cell density, number of replicates, type of cell culture media and coating matrices, volumes, washing steps, etc., requires time, experience and high knowledge of iPSCs and cell biology. During development, the assays are miniaturised and automated to avoid operatorrelated variability. However, the degree of compromise between throughput and assay complexity continues to be Winter 2021 Volume 4 Issue 4

Research / Innovation / Development a challenge. Another key aspect is the selection of the most suitable readouts for HTS, which must provide objective and clinically relevant data while being cost-effective.

CONCLUSIONS iPSC technology is a powerful tool to bring the human biological context earlier into the drug discovery funnel and mitigate late-stage failures due to safety or efficacy concerns.

Human iPSC-derived disease models are of great advantage because they retain patient-specific genetic backgrounds and recapitulate many clinical features of human pathology.

Nonetheless, significant expertise is required to overcome some technical and conceptual challenges for the widespread implementation of these models in the pharmaceutical industry. Working together with stem cell expert companies can a cost-effective and time-saving solution toeffectively implement the use of iPSC-derived disease models in pre-clinical testing and increase the success rate of drug discovery campaigns.

REFERENCES 1.

2.

3.

4.

Aldewachi, H., Al-Zidan, R. N., Conner, M. T., & Salman, M. M. High-throughput screening platforms in the discovery of novel drugs for neurodegenerative diseases. Bioengineering, 8(2), 30 (2021). https://doi.org/10.3390/bioengineering8020030 Halevy, T., & Urbach, A. Comparing ESC and iPSC— based models for human genetic disorders. Journal of clinical medicine, 3(4), 1146-1162 (2014). https://doi.org/10.3390/jcm3041146 Carvajal-Vergara, X., Sevilla, A., D’Souza, S. L., Ang, Y. S., Schaniel, C., Lee, D. F., ... & Lemischka, I. R. Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome. Nature, 465(7299), 808-812 (2010). https://doi.org/10.1038/nature09005 Itzhaki, I., Maizels, L., Huber, I., Zwi-Dantsis, L., Caspi, O., Winterstern, A., Feldman, O., Gepstein, A., Arbel, G., Hammerman, H., Boulos,

M., & Gepstein, L. Modelling the long QT syndrome with induced pluripotent stem cells. Nature, 471(7337), 225–229 (2011). https:// doi.org/10.1038/nature09747 5. Nakazawa, T. Modeling schizophrenia with iPS cell technology and disease mouse models. Neuroscience Research (2021). https://doi. org/10.1016/j.neures.2021.08.002 6. Brennand, K. J., Simone, A., Jou, J., Gelboin-Burkhart, C., Tran, N., Sangar, S., Li, Y., Mu, Y., Chen, G., Yu, D., McCarthy, S., Sebat, J., & Gage, F. H. Modelling schizophrenia using human induced pluripotent stem cells. Nature, 473(7346), 221–225 (2011). https://doi. org/10.1038/nature09915 7. Gomes, A. R., Fernandes, T. G., Cabral, J., & Diogo, M. M. Modeling Rett Syndrome with Human Pluripotent Stem Cells: Mechanistic Outcomes and Future Clinical Perspectives. International Journal of Molecular Sciences, 22(7), 3751 (2021). https://doi.org/10.3390/ ijms22073751 8. Kondo, T., Asai, M., Tsukita, K., Kutoku, Y., Ohsawa, Y., Sunada, Y., ... & Inoue, H. Modeling Alzheimer’s disease with iPSCs reveals stress phenotypes associated with intracellular Aβ and differential drug responsiveness. Cell stem cell, 12(4), 487-496 (2013). https://doi. org/10.1016/j.stem.2013.01.009 9. Lilianty, J., Bateman, J. F., & Lamandé, S. R. Generation of a heterozygous COL2A1 (p.G1113C) hypochondrogenesis mutation iPSC line, MCRIi019-A-7, using CRISPR/Cas9 gene editing. Stem cell research, 56, 102515. Advance online publication (2021). https:// doi.org/10.1016/j.scr.2021.102515 10. Kobayashi, H., Hatakeyama, H., Nishimura, H., Yokota, M., Suzuki, S., Tomabechi, Y., Shirouzu, M., Osada, H., Mimaki, M., Goto, Y. I., & Yoshida, M. Chemical reversal of abnormalities in cells carrying mitochondrial DNA mutations. Nature chemical biology, 17(3), 335–343 (2021). https://doi.org/10.1038/s41589-020-00676-4 11. Whitepaper: Automated cell culture and high-throughput screening of cardiomyocyte disease model. Ncardia Innovations Webpage (2021). https://www.ncardia.com/innovations/automated-cellculture-hts-cardiomyocytes 12. Case Study: Parkinson’s Disease Modeling. Ncardia Innovations Webpage (2021). https://www.ncardia.com/innovations/ case-study-parkinsons-disease-model

Noelia Muñoz-Martín Noelia obtained her PhD in biomedical research in 2019 at the Automous University of Madrid and worked as a postdoc at the University of Amsterdam. She has an extensive experience in cardiac arrhythmias and congenital heart disease research and has contributed to science communications of several organisations and companies from different angles: writing and editing peer-review articles and blogs, and creating website and social media content.

Elena Matsa Elena obtained her PhD in stem cell biology in 2010, and subsequently worked as a post-doctoral researcher at the University of Nottingham, and the Stanford University School of Medicine. She has extensive experience and high impact publications in modelling of human cardiac disease in iPSC-derived cardiomyocytes. Currently, Elena supervises the activities of Ncardia’s Discovery Technology Department, which runs disease modelling, and drug discovery and safety assessment projects in oncology, cardiovascular, skeletal, metabolic and neural disease areas.

www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 29

Pre-Clinical & Clinical Research

PEER REVIEWED

Could Standardised Metadata be the Key to Optimising and Expediting Clinical Trials?

Clinical trials still offer the most effective way of testing the safety and efficacy of new drug treatments, and as more drugs are brought through development each year, the number of trials conducted grows (see figure 1).

Indeed, at the time of writing, there are currently 390,644 registered studies live in 291 countries.1 Although clinical studies are tried and tested, they are by no means static; trials are constantly evolving in response to patient needs and changing technologies, incorporating more complex and adaptive designs, and allowing for more detailed data collection. Together with the need to comply with more stringent regulations and frequently updated standards, clinical trial managers are under increasing pressure to deliver optimised and streamlined trials that deliver safe and efficacious drugs faster and more cost-effectively.

Despite these pressures, many organisations still rely on manual processes, disparate systems and fragmented workflows to manage the metadata that supports and powers clinical trial design, data collection and regulatory submission. These traditional and out-of-date models can cause errors, inconsistencies and delays. Costs can soon escalate as trial managers struggle to find, use and manage metadata and, the issues don’t stop there, as when it comes to preparing metadata for regulatory submission, a longwinded programme of rework is often needed.

This article explores how the use of next-generation, all-inone, cloud-based clinical metadata repositories (CMDRs) can provide the automation, standardisation and control needed to optimise clinical trials.

Metadata: The Underlying Framework for Successful Trial Design and Execution Metadata underpins every clinical trial. It is the consistent framework that enables stakeholders to access, monitor, log and track data, and it is critical to every stage of a trial, from design to regulatory submission. Effectively managing metadata is critical to delivering fast, efficient and cost-effective trials that contain robust and reliable data. But to optimise the use of metadata, team members need to have access to the correct versions, in a readable format, when they need it.

When you think of metadata, regulatory submissions often spring to mind and, indeed, it plays a critical part in regulatory review. In fact, the US Food and Drug Administration (FDA) and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA), require metadata to be submitted in a standardised format: that defined by the Clinical Data Interchange Standards Consortium (CDISC). This stipulation is to ensure that results can be easily and consistently understood and processed during regulatory review.

However, CDISC standards aren’t merely intended as a tick box for regulatory submission. In fact, they’re a strategic tool to 30 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Figure 1 – Number of registered studies on the ClinicalTrials.gov website since its inception in 2000. In September 2005, the International Committee of Medical Journal Editors (ICMJE) started requiring trial registration as a condition of publication and in December 2007 registration was required in line with the Food and Drug Administration Amendments Act (FDAAA).

streamline the design and build of clinical studies, reduce the effort needed to build a trial from scratch, and automatically generate submission-ready data and metadata. Yet, in practice, metadata is often manually reworked to comply with the standards at the end of a trial, consuming considerable time and resources, and potentially resulting in errors. Despite the importance of easily accessible, accurate metadata to a clinical trial’s cost and success, many organisations don’t have the systems in place to maintain, control and access these valuable assets for all parts of a clinical trial’s lifecycle.

The Growing Challenge of Managing Metadata The real challenges of managing metadata come from its legacy and the complex networks that organisations make as they expand. The real-world practicalities of maintaining and using metadata are, therefore, complex and fraught with issues: •

Locating Metadata is Difficult – it is often held in different formats, folders and systems, and owned by different stakeholders.

•

Managing Metadata in Unconnected Files Can Introduce Errors – siloes form and metadata can be lost as it is

Figure 2 – CDISC standards model.

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Pre-Clinical & Clinical Research manually transferred, which can lead to transcription errors or deletion.

•

Change Control is Inadequate – approvals may be managed through multiple email chains, making it hard to maintain accurate change history and version control.

•

Impact Assessments are Lacking – when changes are requested, it’s difficult to see how they might impact other assets, standards or relationships with other metadata.

•

Metadata isn’t Standardised – when the planning and data collection parts of CDISC standards are not adequately embedded, the data tabulation and analysis metadata outputs are not automatically standardised. Because these standards are required for FDA and PMDA submission, metadata is often manually reworked for compliance at the end of the trial (see figure 2), causing delays and the potential for errors.

•

Metadata Can’t be Reused – it makes sense to reuse preapproved metadata, but without standardisation, version control and centralised storage, stakeholders can’t access this. Assets, such as electronic data capture (EDC) systems and electronic case report forms (eCRFs), need to be created afresh each time, often manually – a time-consuming process.

•

A Trial’s Data Can’t be Validated Until Completion – this means that errors or quality issues might not be caught or addressed early. Cohort-level trends, such as poor responses or adverse effects may also be missed, which will limit trial adaptability.

•

CDISC Standards Often Change – the constantly evolving nature of trials and their review means that standards must change. This means that experts are needed to keep abreast of developments and assess their impact.

Together, these issues mean that metadata isn’t carefully controlled, updated and accessible. In turn, this leads to the risk of quality issues creeping into trial designs and stifles the collaboration needed for a continuous improvement environment. Without collaboration and proper change control, changes could start to impact other projects and errors are more likely to occur.

Figure 3 – The latest all-in-one, cloud-based CMDRs provide a central infrastructure for all metadata relevant to an organisation’s clinical trials.

automation, standardisation, change control, in-trial validation and integration with other trial software.

CMDRs Provide the Means for End-to-End Standardisation and In-Trial Validation A critical attribute of next-generation CMDRs is that CDISC standards are built into their framework. Each stage of the CMDR is built with the next in mind, fostering seamless integration and inheritance between the stages. Since studies are designed and built with pre-approved, compliant metadata, they deliver submission-ready outputs, with no rework needed. By cutting out the manual tasks of aggregating and reworking metadata at the end of a trial, timelines and the risk of errors are greatly reduced.

Built-in CDISC standards offer another important function: templates can guide users to the correct versions for their submittable data, such as Study Data Tabulation Model (SDTM), Standard for Exchange of Nonclinical Data (SEND) and Analysis Data Model (ADaM). Since validation is integral to a CMDR, any compliance deviations are automatically flagged by the system, preventing late-stage validation issues.

Once CDISC-compliant metadata is approved, it can be stored and made readily accessible to other users. This pre-approved status forms the basis for the creation of organisational governing standards that stipulate how metadata is used.

Automation and Streamlining: The Driving Forces for Optimal Efficiency Standardised, pre-approved and centralised metadata can be used to automate and streamline tasks, unleashing the full power of the CMDR to optimise trials and increase efficiency.

CMDRs: Unleashing the Power of Metadata to Drive Effective Clinical Trials The key to unlocking the full power of metadata, and its unique ability to optimise clinical trials, is to build a single-source-oftruth; a place where metadata can be centralised, standardised and controlled. The latest, all-in-one, cloud based CMDRs provide this hub, with the capability to hold the huge volume of study metadata that exists as organisations run multiple, evermore complex clinical trials (see figure 3). With next-generation CMDRs, all stakeholders can access compliant, consistent and reusable metadata in a readable format, and monitor and track it for their specific purposes.

This single-source-of-truth also creates a space for governance, visibility and collaboration (see figure 4), which drive effective and efficient trials. CMDRs provide this unique capability through www.international-biopharma.com

Figure 4 – CMDRs enable visibility, governance and collaboration in one central single-source-of-truth.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 31

Pre-Clinical & Clinical Research A manual and time-consuming task in the creation of clinical trials is the building of the electronic data capture (EDC) system. Sometimes trials will use multiple EDCs, which requires manual transcription into different software systems.

Multiple EDCs can now be created directly from the trial brief within some of the latest CMDRs, saving a significant amount of time and reducing the errors that can arise from the manual translation of briefing files. Since the CMDR contains pre-approved CDISC-compliant metadata, the EDC is created to output compliant data, eliminating post-trial rework.

Some next-generation CMDRs can show electronic case report forms (eCRFs) before the more time-consuming EDC creation. This gives an early view of how a form will look, enabling review and approval to start sooner and progress quicker. Any underlying problems can be addressed early in the trial’s design, saving time later on when changes may take longer to implement.

Communication with EDC systems is just one function of the integration capability of some CMDRs. Using application programming interfaces (APIs), CMDRs can communicate with an array of trial software and safety programmes (see figure 5).

APIs provide a simple and accurate way to push and pull metadata between the parent system (the CMDR) and auxiliary systems; manual processing and transfer of data are, therefore, reduced, while the CMDR continues to control the single-sourceof-truth for all trial metadata.

it must have checks in place to prevent its use in live studies.

CMDRs provide the structure to set these lifecycle states as well as the checks and barriers that stop non-approved metadata from making its way into a study.

This structure is only the start. CMDRs create a deep understanding of the relationships that exist in and around metadata, and this underpins a change control protocol.

If a change is requested, such as adding, editing or retiring metadata, its impact on related assets can be evaluated before the change is made. The relational framework with the CMDR makes change control possible and gives the ability to track metadata. In a CMDR, user interactions create an audit trail.

Whenever a user works with metadata, or requests or approves a change, this is documented and evidenced within the system.

The automatically created trail further reduces the amount of work required to collate and validate metadata for regulatory submission.

CMDRs: Meeting the Multiple Challenges of the Modern Clinical Trial In response to growing and unmet health needs, pharmaceutical, biotechnology and contract research organisations are running greater numbers of clinical trials to bring life-saving treatments to patients. As trials increase in complexity and number, these organisations are under increased pressure to design and run faster, more efficient and more cost-effective studies.

CMDRs are fast becoming the tool of choice to help meet these challenges by providing the structure and tools to standardise and control metadata. By embedding CDISC standards in every part of their structure, CMDRs enable the creation of pre-approved, reusable metadata that meets regulatory standards from the outset. Furthermore, CMDRs power the automated tasks that reduce errors and time-consuming manual rework, helping teams build studies with efficiencies built-in as standard. Creating a single source of truth for all trial metadata enables teams to access a central hub for collaboration, governance and visibility. This, in turn, leads to higher quality outputs that meet regulatory requirements, and facilitates optimised, cost-effective trials.

REFERENCES

Figure 5 – Some CMDRs use APIs to transfer metadata and communicate with other clinical trial software.

A key benefit of a CMDR is that it provides the ability to reuse metadata. Considerable effort goes into defining and validating metadata and reusing it saves time, especially when trials are complex, involve large cohorts or run concurrently.

Mappings, annotations, controlled terminology and datasets can be reused, meaning EDCs and eCRFs don’t need to be created afresh each time, and data quality and consistency are improved.

Checks, Audits and Change Control A CMDR system will likely be accessed by multiple stakeholders all looking for metadata to inform their part of the clinical trial process. Lifecycle states must, therefore, be created for standards and studies to define how metadata is used and how it can be changed. When metadata is rejected or retired, then 32 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

1.

https://clinicaltrials.gov/ct2/resources/trends, site visited on 27 September 2021.

Gilbert Hunter Gilbert joined Formedix over seven years ago as a Technical Writer. Four years ago, the knowledge gained from content development together with his customer service skills marked him out for transition to the Professional Services Team. In his current role, Gilbert provides CDISC and software training, support and consultancy services to Pharmaceutical, Biotechnology and CRO organisations. He helps them save time and money by making their clinical trial design and regulatory submissions more efficient.

Winter 2021 Volume 4 Issue 4

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 33

Manufacturing

CGT CDMO Partnering: More than just Manufacturing

The biotech footprint has been exponentially expanding around the globe and no division has increased more feverishly in recent years than the Cell and Gene Therapy (CGT) space. With hundreds to thousands of products in the pre-clinical and clinical phases, the industry has improved in both a quantitative and qualitative way, but it has also exposed numerous pain points that have created critical bottlenecks affecting the ability for a smooth transition into clinic and subsequently to market. With the CGT space being at a critical inflection point, the current ways of working to develop, manufacture, and deliver products and treatments to customers (and subsequent patients) results in a greater need to access GMP manufacturing capacity for meeting clinical and commercial demand, particularly for small biotech firms developing cell andgene therapies. With continuing pressure coming from accelerated timelines and milestone deliveries without sacrificing quality, many of these firms lack the expertise, facilities, and supply networks needed to quickly scale up for clinical or commercial development. This is where partnering with an experienced CDMO can de-risk your path to said critical milestones and provide that seamless transition from development to clinic and into both a regional and global commercial supply.

While the cell therapy and gene therapy field has been around in some way, shape, or form since the 1950s and 1970s respectively, only in recent years has the industry really started to take off. Over those previous decades of predominately research and development, the landscape has morphed from viral vector to allogeneic to autologous and everywhere in between. Today’s landscape is a healthy mix of all three modalities and is expected to continue as such as depicted in Table 1.

Table 1: Sales Projections of CGT Modaltities1

Today’s CGT field has seen twenty products go to market2 and even despite the COVID-19 global pandemic, the market continues to rapidly expand. The expectation is that by 2025 34 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

there will be 10 to 20 Biological Licence Application (BLA) approvals per year for the Food and Drug Administration (FDA) alone. Of the 10 to 20 BLA approvals, there is expected to be nearly 10 times the amount of Investigational New Drug (IND) applications per year.3 This isn’t to suggest that the market is minimal and expected to ramp up, this is much the case of “the rich get richer” as the market continues to blossom more from prior blooms, and no modality has been greater affected than the autologous cell therapy modality.

With the successful commercial approvals of Kite’s Yescarta and Novartis’ Kymriah in 2017, the autologous market grew at a rate that few projected even a few years prior. Since then, other major autologous products have been approved consisting of Bristol Meyer Squibb’s Abecma and Breyanzi, and Kite’s Tecartus drug products.4 However, getting to the BLA or Marketing Authorisation Application (MAA) submission and approval stage is a steep hill to climb and the challenges of providing the market with a regional or global product begin much sooner.

Table 2: Cell Therapy Modality by Molecule Number5

Cell Therapy, specifically autologous cell therapy, has become the recent focus in cell therapy over previous years as depicted in Table 2, but faces, arguably, the most critical bottleneck in the industry, due to requiring the productreceiving patient to also be the donor of the product starting material that initiates the entire process: market reimbursement. When manufacturing an end-to-end cell therapy that results in a single dose or treatment, the costs tend to skyrocket, and the product is only as successful as the value the market will bear. But, despite this being one of the toughest challenges within the Product lifecycle, there are still numerous bottlenecks created upstream that add to the difficulties.

The current ways of working to deliver CGT products to customers and subsequent patients utilises a variety of product flow paths that utilise variable entities, systems, and materials at numerous critical process touch points.

This creates a variety of pain points throughout the Chain of Identity (CoI) and Chain of Custody (CoC) pathway that Winter 2021 Volume 4 Issue 4

Manufacturing makes the path to clinical, commercialisation, and subsequent scalability extremely difficult and quite frankly overwhelming.

With all of the challenges the industry imposes on young cell therapy products, there is much discussion around how a company can both deliver a safe product with a high efficacy that is commercially viable and deliver it efficiently as speed is often the main driving factor for many therapies (first in clinic and first to market). This is where a partnership with a reputable CDMO can give large and small biopharma firms access to development, external GMP manufacturing capacity, and valuable expertise needed to meet critical milestones and control both the CoC and CoI pathways.

When choosing to partner with an external manufacturing company, the benefits stretch well beyond simply the manufacturing. To best understand the right fit for your company, you need to understand what the core drivers for your product are. Product drivers could fall into a number of categories, but a majority of the driving forces behind what CMO or CDMO a company should partner with fall within the timeline, cost, or manufacturing capacity buckets. Each of these buckets has a multitude of subsets that contain variable flexibility and options that may or may not fit your product.

Regardless of what the core driver is, a company needs to adhere to the plan that was put in place whether it’s for budgeting purposes, milestone achievements, board reports, etc. Within the CDMO space, the mantra in response to this need should always be “right first time” and what this results in is that de-risking the product process becomes the core driver a majority of companies aren’t aware they need.

De-risking ensures that the Product moves through the Product lifecycles with minimal rework and ensures that targets remain on track – critical for accurately measuring actuals vs proposed post-milestone achievement.

Due to CGT products being dominated by early clinical stage programs, most products still require some sort of development on the process, analytical, material, or equipment end and are not ready for clinical/commercial approval right out of the gate. This results in the external manufacturing CGT space being dominated by CDMOs as compared to CMOs. A CDMO will have a wider array of established development, operations, quality, and support departments dedicated to providing services with a variety of options providing flexibility to their partner.

Often, flexibility is a critical behind the scenes core driver that often exceeds the more dominated drivers such as cost and timeframes. This is driven by the understanding that time and cost saved now, isn’t necessarily saved later on in the product lifecycle. Pushing through a process for early clinical may expedite your time into clinic, but often will slow your time to market due to process optimisations driven by compliance and scale requirements; also, typically at cost magnitudes higher than previously would have been required.

This is where partnering with a CDMO is recommended as the CDMO partner can leverage the existing expertise, experience, and resources to de-risk your product right out of the gate and allow the customer to possess any stage of a product whether it be a process as simple as a late-phase “drop-in” process that www.international-biopharma.com

needs minimal optimisation or an idea based off of a gene of interest (GOI) and be confident that the CDMO can accurately and efficiently move that Product from the developmental stage into the GMP readiness phase with a process that is not only phase-appropriate, but focused on the long-term goal as well.

In the CGT industry however, talk is cheap: costs and timelines can be promised and never delivered. In an industry where speed and cost are critical to patient centricity, more than time and money are sacrificed when CDMOs over-promise and under-deliver. This is where experience plays a major factor in how a CDMO can successfully support a customer’s product. When selecting a CDMO, past experiences and market domination are often more valuable than upfront speed or cost. When developing a process or analytical method for a CGT product, issues are bound to arise through the initial product stages. However, what this results in is an experience that can be learned upon and passed onto future partners to better streamline their own product process and expedite time to clinic or market thus, creating a base that can be continued to build upon as time goes on.

This can be achieved a number of ways. When bringing in a product process, a CDMO can evaluate the process and process inputs/outputs and compare to past experiences to better prepare for any pain points that may have been apparent in past experiences. As shown in Table 3, the market modalities are often dominated by a common cell type leading to increased efficiencies. However, at some leading CDMOs this is taken a step further by establishing platforms where inputs/outputs are pre-evaluated and reviewed to allow a customer to drop seamlessly into a platform that has already been evaluated for clinical and potentially commercial implementation. This can be as straight forward as defining a full chimeric antigen receptor t cell (CAR T) process inclusive of analytical methods, materials, and equipment or take a bracketed approach where individual unit operations can be added/removed to streamline a portion of the process if other areas require customisation. Of course, there is always the full customisable approach as well if a customer wishes to develop the full process from end-toend, as leading CDMOs offer. Each of these options allows the customer to pick a product pathway within the CoC pathway that best fits their core product driver(s).

By focusing on late-stage or commercial viability earlier on in the clinical phase, it allows a customer to move more fluidly through the commercial readiness portions of the product lifecycle where typically processes slow down as they are extensively analysed through both a quality and regulatory microscope. This would include, but are not limited to, PLE studies, L/E studies, method validation, shipping/stability studies, hold time studies, RM qualification, etc. A CDMO will be able to focus on the areas that have previously provided bottlenecks to better plan for a streamline path to commercial rather than merely running through a list and coming across bottlenecks real-time without warning. As with the earlier stages of the product lifecycle, an experienced CDMO will treat this as a partnership and not focus on one party driving, but focus on a “how we can help you” approach as getting to market and successfully pushing through an approved therapy is a win-win-win situation for the customer-CDMO-patient.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 35

Manufacturing Leading CDMOs should be agnostic to the approach a customer wishes to take and instead focus on the successful product implementation for patient centricity and the priorities of the customer; all while providing consultation throughout each stage listed above without sacrificing overall quality.

trials when the demand forces the conversation and processes to be initiated. While the initial focus is (rightly so) on safety and quickly followed by product efficacy, there needs to be a focus on the supply chain side of the product earlier on during the process.

There are few integrated service offerings available in the market today that can assist in industrialising and streamlining the supply chain logistics with full traceability throughout the CoI/CoC processes; let alone at the scale demanded by common target indications. To attack these pain points and establish a robust and reproducible end-to-end process, CDMOs have looked externally to de-risk the overall “vein-tovein” processes and the available services that currently support the industry.

Table 3: Autologous Cell Type per Module Number6

While so much of the focus is on successful manufacturing of the drug product, the supply chain aspect of the product process is often heavily overlooked. This is understandably so as the system has set up products to be developed in a staged approach focusing on specific deliverables as the product moves through the product lifecycle and/or clinical phases. Drug products that target common indications such as solid tumours or blood cancers do not procedurally require any automation for scalability until the later phases of the clinical

36 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

This allows for large CDMOs to supplement their capabilities by utilising clinical management companies (Be The Match Biotherapies) and logistics’ companies (Cryoport) that all have the capability to be linked by an orchestration platform (Vineti, TrakCell, Salesforce). The outcome of this approach is a resolution for the more common pain points of logistic delays, process variability, scheduling adherence, compliance issues, and scalability. Additionally, this allows companies that may lack the necessary bandwidth allow the CDMO to manage areas outside of the standard CoC pathway and integrate traceability throughout the entire CoI pathway at a stage much earlier than initially planned.

CGT is often not alone within a larger CDMO and companies typically involve additional business divisions

Winter 2021 Volume 4 Issue 4

Manufacturing

that are aimed at improving the likelihood of moving a CGT product through the lifecycle stages. For example, Lonza leverages a Bioscience division to focus on improved transfection methods for non-viral genetic modification via electroporation and improved mediums for more improved and cost-effective culture conditions. Additionally, the Personalized Medicine division focuses on scalability through a proprietary GMP-in-box system combined with both a centralised and de-centralised manufacturing approach in the event a more pointed, point-of-care manufacturing approach is required.

efficient with a partner who has a map and knows all of the bottleneck areas to avoid or refocus on. In the end, this allows the therapy to have the best chance of succeeding and treating the patients, the top priority and focus.

Additionally, when selecting a CDMO partner, security of supply is a critical regulatory requirement needed to show commercial viability of your product. CDMOs often offer global footprints to extend patient supply across multiple areas of the globe; autologous especially as the logistics pathway is a bit more limited than more traditional viral vector or allogeneic manufacturing and trial support. When selecting a CDMO, the regional and global footprint is to be considered based on the long-term supply of your product. The growth also needs to be considered to ensure that the selected CDMO has available capacity for not only the existing demand, but the demand that extends into the commercial launch and peak commercial years.

3.

Combining the expedited time into clinic, the commercial viability assessment in the earlier clinical phases, and the scalability through assistance of other business divisions, a CDMO gives a great chance of pushing forward not only a product that works, but a product that will bear the weight of the market.

With the CGT industry expanding at a rate never seen before combined with a variety of pressure coming from expedited timelines, security of supply, and robust processes, partnering with an experienced CDMO allows the customer to allow experience, resources, and facility networks to do the driving on a road previously paved. Speed bumps are inevitable throughout a product lifecycle, but getting from point A to point B on the road to clinic or market is always much more www.international-biopharma.com

REFERENCES 1.

2.

4.

5.

6.

EvaluatePharma. Export: June 28, 2021 Approved Cellular and Gene Therapy Products. Food and Drug Administration, 27 Mar. 2021, https://www.fda.gov/ vaccines-blood-biologics/cellular-gene-therapy-products/ approved-cellular-and-gene-therapy-products. Accessed 30 Apr. 2021 Hanover, Larry. The Untapped Potential of Cell and Gene Therapy.

AJMC, February 2021, Volume 27, Issue 2, Pages: SP51, 16 Feb. 2021, https://www.ajmc.com/view/the-untapped-potential-of-cell-andgene-therapy. Accessed 30 Apr. 2021.

Approved Cellular and Gene Therapy Products. Food and Drug Administration, 15 Jun. 2021, https://www.fda.gov/ vaccines-blood-biologics/cellular-gene-therapy-products/ approved-cellular-and-gene-therapy-products. Accessed 14 Sep. 2021 Citeline Pharmaprojects Pipeline Search, July 1, 2021; Lonza internal analysis Citeline Pharmaprojects Pipeline Search, July 1, 2021; Lonza internal analysis

Joe Garrity Joe Garrity is the Head of Autologous Cell Therapy, Commercial Development at Lonza Cell and Gene Technologies where he works within their global Cell and Gene business unit. He works within the business leadership team to enable strategic development and growth of the autologous modality in Lonza CGT, as well as works with operations, development, and quality teams to streamline the clinical and commercial manufacturing services and offers provided

by Lonza. Joe has an educational background in biochemistry and a professional background of nearly a decade within the cell and gene CDMO space that has covered positions within development, operations, and business functions.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 37

Manufacturing

Maximising mAbs Purification Efficiency: Focus Areas for Reducing Bottlenecks in Downstream Processing Finding ways to remove bottlenecks and improve yields in downstream processing for monoclonal antibodies (mAbs) continues to be a key focus area for biopharma manufacturers. In downstream processing, the goal is to improve recovery and reduce the cost per gram of protein produced. Today, over 60% of the cost to produce a new mAb relates back to downstream steps.1 Any percentage of improvement in downstream recovery can contribute to improving the ultimate process yield for drug product of the target biologic.

When compared to upstream processing, finding efficiencies and economies of scale in downstream steps involves more complex analysis and optimisation. Significant investments have already been made in the technologies and processes used in upstream processes. Improvements to raw material characterisation and the addition of single-use systems, perfusion systems and more precisely controlled bioreactors in upstream processing steps are all leading to measurable increases in upstream yields. However, improvements in downstream throughput have not kept a similar pace to those for upstream, leading to potential bottlenecks in the end-to-end process.

Expanding the use of mixed-mode and multimode chromatography resins – using resins to target ligands for increased selectivity can help to process targeted molecules more efficiently – and exploring ways to make chromatography buffers more effective – using new kinds of additives and prepackaged single-use buffer materials to streamline buffer preparation steps – are two potential areas for optimisation that could lead to significant downstream improvement.

How Resin Choice Impacts Overall Operations Downstream processing generally takes place over a period of a few weeks. Multiple chromatographic steps, filtration steps, buffers and cleaning solutions are used as part of the process. A capture step is the first purification step where protein A has become the most widely used resin due to its highly specific nature, ease of use as a standard purification process and proven regulatory record.2 The protein A step is one area where process efficiencies and cost savings may be gained by selecting a high-performance protein A resin and optimising buffer preparation.

When choosing a protein A resin, the resin dynamic binding capacity (DBC) is one factor that can impact overall process productivity. A resin with a higher DBC can improve the productivity of the capture step while keeping the column sizes the same. This in turn can minimise the need to modify facilities, specifically for high-titre cell culture processes.3 A simulation was performed with BioSolve software using three model resins having binding capacities ranging from 30g/L to 65g/L to calculate the number of bind/elute cycles, 38 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

process time and amount of buffers required for a 2000L bioreactor batch. Assumptions made for the calculations are summarised in Table 1, where the column size was kept consistent as 68.6L for a 2000L cell culture reactor with a 5g/L titre value. The process’s productivity was evaluated in terms of number of cycles required per batch and process time.

Table 1. Process parameters used for simulation

Resins having higher DBC significantly reduce the number of cycles and total downstream processing time, as illustrated in Table 2. In addition to increasing productivity, reducing the number of cycles can also reduce operational risk and lead to lower costs for labour and consumables for each cycle.

Table 2. Process output based on resin capacity*

Reducing the amount of buffer consumed does more than impact raw material cost; it can contribute to verifiable savings in buffer preparation time, buffer tank size and method of preparation. In this model, total buffer consumption was reduced by approximately 30% with the use of resin with high DBC (Resin C) when compared to Resin B, and reduced by approximately 40% when compared to Resin A. Improving Buffer Preparation Workflows Lower buffer solution requirements also provide flexibility to either make buffers in-house or utilise ready-to-use buffers.

Buffers for the purification process can be prepared in multiple ways: • Powder hydration in fixed stainless-steel tanks or singleuse buffer prep reactors • Multicomponent buffer concentrates with in-line dilution, or single component stocks with buffer stock blending Winter 2021 Volume 4 Issue 4

Manufacturing Choosing a Hybrid Buffer Preparation Approach Industry organisations, including BPOG, have offered insight into how buffer stock blending and in-line dilution enable overall improvements across unit operations.4,5,6 The decision to select one option over the other (or a hybrid approach) will usually be dependent upon an economic analysis of items such as scale, batches of drug produced per year, raw materials used and other site attributes. Workflow improvements that can be implemented for each of the buffer prep options are listed in Table 3.

Figure 1. Buffer consumption of three protein A resins with different dynamic binding capacity (DBC) for processing of one 2000L bioreactor batch

Ready-to-use, cGMP 1x buffers The most commonly used method for in-house buffer creation uses WFI (water for injection) grade water to hydrate powders in stainless-steel tanks. While this well-established method is ideal for large volumes, it requires significant and ongoing investment in infrastructure. For example, a biopharma manufacturer or its contract manufacturer (CMO) may need additional warehouse space for storing raw materials prior to their use, as well as a dedicated weighing and dispensing area – all of which need to be properly managed, kept clean and in accordance with cGMP practices. In addition, the footprint for the stainless-steel tanks within the facility must also be considered; in an existing facility, stainless-steel tanks take valuable space away from value-added operations and in a new facility, specifying additional square footage to a project could increase the size of the initial CAPEX request and construction time.

Additionally, new developments in single-use technology have added flexibility in buffer preparation methods, giving small- and medium-scale facilities the freedom to choose single-use tanks for buffer preparation. This can support faster changeovers and cleanouts in buffer preparation, saving both time and cost in manufacturing processes.4

A hybrid approach using both in-house systems and outsourced buffers can streamline downstream purification unit operations significantly. As noted, the use of a protein A resin with a high DBC can reduce buffer usage to a more manageable level and the use of in-line dilution (ILD) systems will make the production of critical buffer components more efficient. Below are suggested buffer preparation methods for each buffer used in protein A step.

•

The cleaning buffer, usually a fixed normality of NaOH, can be prepared in-house using concentrate or can be purchased as a 1X concentration due to the smaller volumes used to reduce safety concerns.

•

The storage buffer (example: 20% ethanol) can also be managed in-house in the same way as described above due to low, consistent volumes that are typically required in the process, irrespective of the resin DBC.

•

Volumes required of equilibration buffers and wash buffers (examples: 1X PBS or 50 mM Tris, pH 7) significantly decrease with an increase in resin DBC, as shown in Figure 1.

Preparing these buffers using either in-house or single-use systems causes several operation challenges at lower DBC values due to high volume. For such buffers, the use of in-line dilution (ILD) systems using multicomponent concentrates (ex. 10X PBS) can provide operational advantages including facility footprint reduction, reduction in raw material management and availability of buffer on demand.

•

Elution buffers (example: 0.1M acetate buffer, pH 3.4) usage can also be streamlined through the use of in-line dilution.

Table 3. Suggested workflow improvements for various buffer preparation methods www.international-biopharma.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 39

Manufacturing

Conclusion There are a number of areas where streamlining downstream processing steps can help improve overall mAbs processing efficiencies and help downstream productivity match the improved efficiencies achieved in upstream processing.

Focusing on new approaches to the protein A step is one area where significant opportunity exists. The flexibility and productivity of the mAb capture process step can be improved by utilising high-capacity affinity resins, along with optimal buffer management. A high-capacity resin reduces the process time by allowing less numbers of cycles required per batch, resulting in reduced process and labour costs, as well as reduced risk. Moreover, the implementation of a high-DBC resin decreases the volume of process buffers significantly. This reduced buffer volume provides flexibility to adopt different buffer preparation processes based on the facility requirements.

Since each mAb production process may have its own requirements and bottlenecks, it is important to have flexible process optimisation options so that unique solutions can be applied to various mAb products. However, by investigating and investing in these types of new technologies and new approaches, the ability to create and deliver these valuable, in-demand biologics more cost-effectively can help make sure that patients and communities worldwide benefit from these therapies.

REFERENCES 1.

2.

3.

4.

Deorkar, N., Berron, C. (2019). Key challenges and potential solutions for optimizing downstream bioprocessing production. International Biopharmaceutical Industry 2(2), 10-12.

Pabst, T., Thai, J. & Hunter, A. (2018). Evaluation of recent protein A stationary phase innovations for capture of biotherapeutics. Journal of Chromatography A 1554, 45-60.

J.T.Baker BAKERBOND PROchievATM protein A resin. vwr.com, accessed October 2020.

Vengsarkar, P., Deorkar, N. (2020). Improving mAb manufacturing productivity by optimizing buffer and media prep process flow.

BioPharm International, July 30, 2020.

40 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

5.

6.

Gibson, K. et al (2019). An economic evaluation of buffer preparation philosophies for the biopharmaceutical industry. BioPhorum Operations Group, December 2019.

Schrott, C. et al (2019). Nimble-Biophorum buffer stock blending system: A more advanced concept for buffer manufacturing.

BioPhorum Operations Group, December 2019.

Nandu Deorkar Nandu Deorkar, PhD, MBA, is the Vice President of Research & Development for Avantor. His expertise in materials technology research & development includes chemical/polymer R&D, drug development, formulation, drug delivery technologies, process development and technology transfer. Dr. Deorkar earned his PhD in chemistry from the Indian Institute of Technology, Bombay, and his MBA from Fairleigh Dickinson University, New Jersey (USA).

Jungmin Oh Dr. Oh leads product and process development projects at Avantor, where she is responsible for solving customer-centric problems with multiple biopharmaceutical industry partners.

She holds M.S. and Ph.D. degrees in Chemical Engineering focusing on the optimization of a continuous chromatography system.

Pranav Vengsarkar Dr. Vengsarkar is focused on product and process development for new cGMP products and excipients, and development and design of single-use raw material delivery systems at Avantor. He holds a Bachelor’s in chemical engineering ICT Mumbai and a Ph.D. in Chemical Engineering from Auburn University.

Winter 2021 Volume 4 Issue 4

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Contact us today to find out more 01933 357953 | archives@qualogy.co.uk | www.qualogy.co.uk INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 41 The Archivist, Qualogy Ltd, Po Box 6255, Thrapston, Northamptonshire, NN14 4ZL

www.international-biopharma.com

Therapeutics

PEER REVIEWED

Bringing Novel Therapies to Market: 5 Strategies for Success Advanced therapy medicinal products (ATMPs) were making headlines long before SARS-CoV-2, the virus that causes COVID-19, emerged onto the scene. The successful development of COVID-19 vaccines, including mRNA vaccines, has brought next-generation therapies into the spotlight and boosted the long-term projections for growth in this sector.

Nevertheless, although ATMPs hold great promise, exploiting the technology has been challenging.

Organisations need to adopt multidisciplinary strategies that begin as early in the development process. Christian K. Schneider, head of Biopharma Excellence and Chief Medical Officer, outlines 5 strategies for success while avoiding common pitfalls.

Novel therapies, known as advanced therapy medicinal products (ATMPs) in Europe and as cell and gene therapy products (CGTPs) in the US, could be game changers for the treatment of severe conditions that today have only limited treatment options. Driven by scientific innovations, impressive clinical outcomes, and a succession of new product approvals, the market for advanced therapies is set to be worth almost $21.2 billion by 2028, according to analysts.1 The ATMP sector is now considered to be at an “adolescent stage” by many analysts, which means it holds great promise for making personalised medicine a reality and improving global health through wider accessibility to innovative and personalised medicines and devices.

Promises and Challenges With promises, however, come challenges. The model for innovative therapies is very different from that for conventional development, and more tailored approaches are needed. ATMPs typically deal with smaller patient populations; special requirements for manufacturing where patients’ lives can depend on the speed with which a therapy can move from bedside to manufacturing and back again; and pricing models that can make the therapy prohibitive for many payers.

developments when they draft their guidance documents and regulatory scientific guidance can sometimes be too general for a developer to know how exactly to apply it to a given novel product.

Regulatory agencies should be involved throughout a development programme so that they stay in lockstep, and so that organisations can incorporate their insights into the programme. Regulators are increasingly open to dialogue for immature and early programmes, and they see their roles as enablers in addition to their more traditional roles as gatekeepers.

Ultimately, the overall goal is to build an agile approach to planning that minimises delays or risks of failure.

5 Strategies to Reduce Risk Strategies should be designed, from the outset, to build bridges between quality, non-clinical and clinical disciplines.

Advanced therapies involve complexities that need to be considered in the commercialisation process. Patient populations are very often smaller and more targeted and even though that means that product quantities can be low, they also have very specific logistical requirements. For example, manufacturing considerations and patients’ lives can depend on the speed at which a product moves from the bedside to the facility and back again, especially in cases where shelf-life is very short.

Advanced therapies might be potentially transformative, but pricing for them may prove prohibitive for some payers.

And the underlying quality, regulatory and manufacturing guidelines that apply to traditional drug development still need to be considered. Organisations therefore need to do the following: 1.

Assess the Risks and Benefits: ATMPs come with significant known and unknown risks, many of which are unique to this product class. Therefore, risk needs to be considered from an early stage, with a primary focus on safeguarding the patient but also on minimising risks to healthcare professionals and caregivers. The risk/benefit assessment should be designed as a gate to go/no-go decisions at each stage of development. Sometimes, the “go” will require a change in direction, so the process should be agile, and this should apply not only to the biological activity of the ATMP, but also the quality attributes, the manufacturing process steps and the therapeutic administration procedures.

2.

Develop an Integrated Product Development Plan (IPDP): To create a holistic IPDP, all development disciplines such as manufacturing, nonclinical and clinical development as well as regulatory affairs need to be involved. Even for early-stage programmes, commercial aspects such

In order to reduce the risks along the way, companies should plan early, building bridges between quality (CMC; chemistry, manufacturing and controls), non-clinical and clinical disciplines. They should also develop a regulatory strategy as soon as drug development begins and analyse the healthcare landscape to determine the market access model that will provide the greatest appeal for decision-makers and payers.

It’s also crucial to involve the regulatory authorities early on. Because ATMPs are complex biological entities, current regulations around them are also complex – and evolving constantly. Regulators are unable to anticipate future 42 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

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Therapeutics as targeting specific countries for commercialisation, the competitive environment as well as pricing / reimbursement aspects should all be considered.

The IPDP is a living document that will be updated as development progresses, promoting organisational prioritisation and decreasing time-to-decision. Defining the patient population, and the target stage for a given disease, for example, are important considerations, and these could have an impact on the design of non-clinical studies etc.

3.

4.

5.

Consider Models to Scale Manufacturing: Moving a therapy from the lab to scaling it for supply to patients can be challenging. To ensure scalability without wasting money, organisations need to align manufacturing readiness with the regulatory pathway, the patient population and suggested dosing.

Accelerate commercialisation with an Effective Regulatory Strategy: Distinct aspects to the regulatory plan should evolve as development progresses: 1) documenting the goal, which can be visualised via the Target Product Profile (TPP); 2) keeping pace with competitive therapies; 3) maintaining regular checkpoints with regulatory agencies; and 4) considering regulatory pathways, depending on markets or regions, indication areas, and classification of the therapy. The regulatory strategy should evolve along with development, and as new information comes in.

Begin a Market Access Strategy: Developers must be able to demonstrate clinical and economic evidence to providers, healthcare decision-makers and payers. Given the complexities of the payment systems for healthcare, it’s crucial to understand who will finance the therapy and how care will be reimbursed. Developers must offer a strong value proposition for decision-makers and visualise this from the proof-of-concept phase onwards, so that later considerations on risk-benefit and cost-benefit

converge and can be derived from overlapping evidence generated throughout the development.

Strategic Planning Hurrying from research to development without an integrated product development plan is a dangerous proposition.

Organisations must go through the planning process with the understanding that this will be a starting point only and that the plan will need to adapt as the science evolves. More importantly, through upfront structured planning – even while acknowledging things will change – the company will avoid road bumps and move faster as it progresses toward commercialisation of the product.

Developers of innovative therapies are charting new waters, so navigating these complex considerations can be challenging. But with proper strategic planning, organisations can clear the obstacles that lie ahead and move closer to commercialising the ground-breaking, curative therapies that people need.

Big pharma gets in on the act Six large pharma companies (defined as in the top 25 by prescription drug sales) have made it into the top 20, up from five in 2020. Pfizer is the new large pharma entrant into the top 20 ranking, with forecasts for gene therapy programmes in Duchenne muscular dystrophy and haemophilia, and antisense programs in diabetes and cardiometabolic indications.

Source: Evaluate Pharma consensus forecasts1 REFERENCES 1.

Advanced Therapy Medicinal Products Market Worth $21.2 Billion By 2028: Grand View Research, Inc, May 2021: https://www. grandviewresearch.com/press-release/global-advanced-therapymedicinal-products-market

Christian K. Schneider Christian K. Schneider, M.D., is Head of Biopharma Excellence and Chief Medical Officer (Biopharma) at PharmaLex. He was previously interim Chief Scientific Officer at the UK’s MHRA, where he was also Director of the National Institute for Biological Standards and Control (NIBSC) for five years. He has also held leading positions at the Danish Medicines Agency and at the Paul-Ehrlich-Institut, Germany’s Federal Agency for Vaccines and Biomedicines. At EMA, he has chaired the Committee for Advanced Therapies (CAT) as well as the Biosimilar Medicinal Products Working Party (BMWP) and served as a member of the Committee for Medicinal Products for Human Use (CHMP). He is one of the key architects of EMA’s advanced therapies and biosimilars framework. As a regulatory scientist, Christian has published 50+ articles in international, peer-reviewed journals.

Email: christian.schneider@biopharma-excellence.com www.biopharma-excellence.com

44 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2021 Volume 4 Issue 4

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 45

Therapeutics

PEER REVIEWED

Understanding the Role of Regulatory T Cells in Breast Cancer Metastasis Breast cancer is the most common cancer amongst women accounting for 24 percent of new cancer cases worldwide and 15 percent of cancer deaths in 2018.1 And cases are expected to increase by more than 46 percent by 2040, according to a recent article in The Lancet.2

While survival rates for breast cancer patients are improving with the help of early detection, the incurable metastatic stage of the disease has a poor prognosis, resulting in most breast cancer deaths. Of the 1.7 million new cases of breast cancer diagnosed annually worldwide, around 30 percent of patients diagnosed with localised disease get metastases in distant organs.3 How the immune system helps or hinders metastases is a promising field of breast cancer research. In the microenvironment in the tumour, for example, tumour-associated macrophages (TAMs) are associated with invasion, metastasis, and a worse prognosis. But tumour-infiltrating lymphocytes (TILs) are associated with a better outcome.

Cancers host a plethora of other immune cell subsets too, such as lymphocytes, various myeloid cells, and innate lymphoid cells, some of which aid and abet the tumour, while others hinder its progress.

Kevin Kos, a molecular immunologist in Karin de Visser’s Inflammation and Cancer research group at the Netherlands Cancer Institute (NKI)- in Amsterdam, and researcher of Oncode Institute, has been using Qlucore’s Omics Explorer for his PhD research on the role of T regulatory cells (Tregs) in breast cancer metastasis.

“Tregs act like the military policemen among the white blood cell population. Whereas other white blood cells fight pathogens, Tregs regulate immune responses to ensure that nothing goes out of control, making sure there isn’t excessive inflammation (Kos and de Visser, 2021). When this goes wrong, such as in auto-immune diseases like diabetes, multiple sclerosis, and inflammatory bowel disease, Tregs are either dysfunctional or reduced,” Kos explains.

To track the complex process of metastasis development, Kos is using mouse models in a way that closely mimics cancer development in humans.

“We use mice that are genetically engineered to develop tumours in their mammary glands. When these mice are about 8 months old, the tumours start to become palpable. At that point, we can test treatments such as chemotherapies and different combinations of treatments,” he explains.

“Additionally, we can also put small tumour fragments into healthy mice. When these tumours grow, we can surgically remove them. But like in human patients, these tumours recur in distant organs in the form of metastases. Within this window of disease progression, we can test novel treatments or other forms of analyses to study metastasis development.”

Kos is using Qlucore’s Omics Explorer to perform bulk RNA sequencing analysis on isolated Treg cells from various tissues such as the blood and lungs from both tumour-bearing and healthy mice.

“By doing this, we can see what kinds of mRNA the Treg cells express, which is a surrogate for what kind proteins they are making. So, we’re finding out whether they are active, are they dividing, are they functional. We learn all this from sequencing data,” he explains.

“Tumours affect the immune system throughout the body.

In human cancer patients, their blood is very different from the blood of healthy patients,” says Kos. “Ultimately, we want to use these insights to develop new interventions that may halt tumour progression.”

Easy Analysis and Visualisation of Transcriptional Data The Qlucore tool is used to perform differential gene expression analysis, clustering analysis, PCA analysis, and to produce publication quality figures of the transcriptome analyses.

The Netherlands Cancer Institute, founded in 1913, is a comprehensive cancer centre with a hospital and research lab. The lab is staffed by 750 scientists and scientific support personnel, while the hospital has 230 medical specialists, 212 beds, and an out-patients clinic with around 140.000 visits annually.

“There are many different projects running in our group,” says Kos. “On the clinical side, for example, we analyse patient samples to understand how various immune parameters change in breast cancer patients. I’m focused on the pre-clinical work, looking at how cancer spreads throughout the body and how the immune system is involved.”

46 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Winter 2021 Volume 4 Issue 4

Therapeutics

Differential gene expression analysis (see above) has been particularly useful in this project. “You get a statistical value for the differences between big groups,” says Kos. “We are sequencing thousands of different transcripts and to make sense of that it’s vital to see which genes are truly different between the subjects we’re comparing, such as healthy versus tumour-bearing mice.”

As with many research institutes, the bioinformaticians at the NKI have their own projects and very little free time to assist on ones that aren’t related. “The main benefit of using Qlucore is that it bypasses this need, and allows for easy, do-it-yourself, in-depth analysis of complex RNAseq data,” says Kos. The first paper from this research is under review and due to be published soon. Initial findings suggest that in all tissues analysed, Tregs behave differently in tumour-bearing mice compared to healthy mice.

Kos says: “It probably happens for more immune cells, not just Tregs. What we didn’t expect was that cells would be affected in distant organs like the spleen when the primary tumour is in the mammary glands. It’s a bit of a shocker. It means that the tumour is influencing Tregs far beyond their local environment. This behaviour has already been described for myeloid cells, so it’s interesting to observe it in lymphocytes as well,” he adds.

The results of this research may help to better understand the interaction between the immune system and breast cancer.

“Ultimately, the idea is to stop the Treg action for a short time in a way that interferes with functions that might help tumours to grow,” summarises Kos. Ready to Handle More Complex Single Cell Sequencing Data Bioinformatics analyses are getting increasingly complex.

While this latest project is about bulk RNA sequencing of thousands of cells, the next step is to look at the gene www.international-biopharma.com

expression in individual cells. Other scientists in the lab are doing that with patient samples.

“This is the future for gaining a much deeper understanding.

You can see what is different between a specific patient and a healthy person,” says Kos. “These kinds of analysis can also be done with the Qlucore tool. It’s super-nice as these kinds of analysis can normally not be performed by researchers that do not have the experience of bioinformaticians.”

REFERENCES 1.

2.

3.

4.

Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries by H Sung et al; CA Cancer J Clin. 2021 Global burden and trends in premenopausal and postmenopausal breast cancer: a population-based study by Emily Heer et al; The Lancet August, 2020 The genomic landscape of metastasis in treatment-naïve breast cancer models by Christina Ross et al; PLoS Genet, May 28, 2020 Kos, K., de Visser, K.E., 2021. The Multifaceted Role of Regulatory T Cells in Breast Cancer. Annu. Rev. Cancer Biol. 5. https://doi. org/10.1146/annurev-cancerbio-042920-104912

Christine Evans-Pughe Christine Evans-Pughe is a freelance science and technology writer whose work has featured in The Economist, The Guardian, The Independent, and BBC Focus as well as a wide variety of specialist journals. She was a regular feature writer for the UK's Institution of Engineering & Technology's award-winning Engineering & Technology magazine for over 15 years, and is co-founder of the website www.howandwhy.com.

Email: qlucoreinfo@qlucore.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 47

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