IBI Volume 4 Issue 1

Volume 4 Issue 1

Peer Reviewed

Automated Identification of Cardiac Hypertrophy Modulators Using a Hipsc-Derived Disease Model Maximizing mabs Purification Efficiency: Focus Areas for Reducing Bottlenecks in Downstream Processing High-Throughput Sequencing Technologies are Revolutionizing Antibody Discovery Live Cell Imaging: Non-invasive Kinetic Data to Better Understand Cell

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II INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Spring 2021 Volume 4 Issue 1

Contents 04 Foreword REGULATORY & COMPLIANCE 06 The Science is Stacking up for Regenerative Medicine DIRECTORS: Martin Wright Mark A. Barker BUSINESS DEVELOPMENT: Ty Eastman ty @pharmapubs.com EDITORIAL: Beatriz Romao beatriz@pharmapubs.com DESIGN DIRECTOR: Jana Sukenikova www.fanahshapeless.com FINANCE DEPARTMENT: Martin Wright martin@ipimedia.com RESEARCH & CIRCULATION: Virginia Toteva virginia@pharmapubs.com COVER IMAGE: iStockphoto © PUBLISHED BY: Pharma Publications J101 Tower Bridge Business Complex London, SE16 4DG Tel: +44 (0)20 7237 2036 Fax: +44 (0)01 480 247 5316 Email: info@ibijournal.com www.biopharmaceuticalmedia.com All rights reserved. No part of this publication may be reproduced, duplicated, stored in any retrieval system or transmitted in any form by any means without prior written permission of the Publishers.

The next issue of IBI will be published in Spring 2021.

ISSN No.International Biopharmaceutical Industry ISSN 1755-4578.

The opinions and views expressed by the authors in this magazine are not necessarily those of the Editor or the Publisher. Please note that although care is taken in preparation of this publication, the Editor and the Publisher are not responsible for opinions, views and inaccuracies in the articles. Great care is taken with regards to artwork supplied, the Publisher cannot be held responsible for any loss or damage incurred. This publication is protected by copyright.

2021 PHARMA PUBLICATIONS / Volume 4 Issue 1 – Spring 2021

A new peer-reviewed paper published in a special issue of Stem Cells International evidences the effectiveness of micro-fragmented adipose tissue (MFAT) in treating osteoarthritis of the knee. The special issue, entitled Mesenchymal Stem Cells and Regenerative Medicine 2020, was published in 2020. Simon Checkley at The Regenerative Clinic analyses the new data that shows positive potential for osteoarthritis treatment in knees.

10 Biosimilars – Increasing Regulatory Focus on Orthogonal Analytical Characterisation The COVID-19 pandemic has focused attention upon global pharmaceutical industries, and will continue to do so for some time to come. However, despite the initial disruption, some positive outcomes may be emerging from 2020 for the future of biotherapeutics in 2021 and beyond. One area which may be becoming more transparent is in the biosimilar regulatory sphere. Fiona Greer, Independent Consultant and Richard Easton at BioPharmaSpec look at proposed changes of regulatory emphasis from a global perspective and consider any potential impact on our analytical strategies for biosimilar testing.

RESEARCH / INNOVATION / DEVELOPMENT 14 Exploiting Epigenetics to Systematically Optimise Culture Conditions for Cellular Therapies One of the main challenges of cell therapies is the maintenance and/or expansion of the required therapeutic phenotype in vitro.

Moreover, to meet both the traceability and safety requirements for a clinical-grade therapeutic, cells need to be cultured in chemically defined conditions. Over the last two decades, scientists have sequenced the genome and epigenome of all known cell types in the human body, in addition to mapping hundreds of possible protein-protein interactions. Joachim Luginbühl and Rodrigo Santos at Mogrify explore epigenetics to systematically optimise culture conditions for cellular therapies.

18 Developments in Non-invasive Pre-clinical Lung Imaging to Support Drug Discovery Growing demand for safe, effective medicines for pulmonary disorders and diseases has increased the research into their treatment. This is key to discovering how to effectively manage pathological lung conditions including asthma, chronic obstructive pulmonary disease (COPD), pneumonia and lung cancer. Nicolau Beckmann at Novartis Institutes and Sarah Rebecca Herrmann at Bruker BioSpin show the developments in non-invasive pre-clinical lung imaging to support drug discovery.

PRE-CLINICAL & CLINICAL RESEARCH 20 Why Patient Diversity in Research Must Become the Norm While 2020 was not the year anyone hoped it would be, it did succeed in showing us what medical research can achieve when the life sciences sector works together to overcome a challenge. Three COVID-19 vaccines developed and licensed in less than a year, with more still in development, is certainly

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Contents extraordinary. But what is also remarkable is that, even in the rush to find and develop effective treatments and vaccines for a completely new virus, the life sciences sector achieved what it has struggled with for years: diverse patient involvement in clinical trials that truly represents the global population. Kate Shaw at Innovative Trials discusses why patient diversity in research must become the norm.

other diseases. The application of high-throughput sequencing (HTS) in monoclonal Ab (mAb) screening and selection are rapidly transforming the discovery process. Nestor Bernat, Josine Lohuis and Henk-Jan at ENPICOM discuss the impact of HTS and immuno-informatics on the accuracy and speed of Ab discovery workflows, and how these technologies are revolutionising the Ab development field.

22 Automated Identification of Cardiac Hypertrophy Modulators Using a Hipsc-derived Disease Model

40 Battling Biohazardous Liquid Laboratory Waste

Effective drug discovery and development rely heavily on the availability of predictive pre-clinical models. For decades, target-based drug discovery has focused on immortalised cells to identify and optimise inhibitory or activating molecules.

Elena Matsa at Discovery Technology examines automated identification of cardiac hypertrophy modulators using a hipsc-derived disease model.

28 Live Cell Imaging: Non-invasive Kinetic Data to Better Understand Cell Viability The current assessment of cell viability depends on endpoint measurements. Unfortunately, this ignores the levels and rates of in-between measurements. However, advances in technology and image analysis now allow cell viability to be observed more frequently and analysed more quickly.

Alternatively, live-cell imaging allows for kinetic monitoring in optimal conditions, helping researchers understand how the cells are reacting over time. Kendra Majewski at CytoSMART Technologies explores how researchers have studied cell viability and how live-cell imaging can provide a better understanding of cell viability.

30 Unlocking the Biomarker Revolution in Clinical Trials Completion of the first human genome sequence exactly 20 years ago opened up the tantalising prospect of a new era of precision treatments, with accurate genetic diagnosis enabling therapies to be targeted to smaller subsets of patients, increasing the chances of success and reducing side-effects.

Barnaby Balmforth at BiofidelityLtd explains the real value of diagnostics and their absolutely critical role in delivering the benefits of biomarkers in healthcare.

MANUFACTURING/TECHNOLOGY PLATFORMS

Treating biologically hazardous waste in the UK using a combination of chemical sterilants and a well-developed system of sewerage and treatment plants is not uncommon.

It is a system which can handle a wide range of biologically active substances, but it is not failsafe. Gareth West at Astell Scientific Limited will review alternatives with the aim of better understanding which method is more suited to the modern laboratory.

REGULATURY/QUALITY COMPLIANCE 44 Using Electronic Pipetting and Automated Dispensing Technologies to Accelerate Sample Preparation for RT-PCR Applications RT-PCR (real-time, reverse transcriptase-polymerase chain reaction) is a basic laboratory tool with a wide range of applications. Scientists use RT-PCR to quantify changes in gene expression, validate results from array analysis and for drug discovery. More recently, RT-PCR became the most accurate way to test samples for the SARS-CoV-2 virus that is behind COVID-19.

Tommy Bui at Thermo Fisher Scientific explains how to accelerate sample preparation for RT-PCR applications by using electronic pipetting and automated dispensing technologies.

TALKING POINT 46 An Interview with Dr. Mark Kotter, CEO and Founder at bit.bio on Transforming Drug Discovery and Medicine Research The identity and function of every cell is defined by which ‘genetic programs’ are active at any particular time. Dr. Mark Kotter, CEO and Founder at bit.bio, explains the breakthrough technology which, put simply, enables consistent reprogramming of cells based on the reliable activation of specific genes and these genetic programs.

32 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. 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.

36 High-throughput Sequencing Technologies are Revolutionising Antibody Discovery Therapeutics based on antibodies (Abs) are at the forefront of revolutionary treatments for cancer, autoimmunity and many 2 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Spring 2021 Volume 4 Issue 1

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Foreword We have been on a long and painful journey since COVID-19 was initially detected in Scotland approximately this time a year ago. Though we have experienced tragic losses in lives and livelihoods over these long months, our combined effort, resilience and sacrifice have also saved thousands of lives. We have pulled together in a spirit of solidarity and adapted to new ways of living to fight this destructive virus and to keep our country going.

We began the rapid roll-out of our vaccination programme, which we believe will diminish illness and deaths from COVID and also, ultimately, when a high proportion of our population has been vaccinated, let us return to a more normal way of living.

We have always known that vaccines would be our best way out of this pandemic and towards a more normal way of life. It is why we moved fast and early, supporting ground-breaking research from January last year. As a result of this work, we are the first country in the world to authorise a vaccine against COVID-19.

Today we have three authorised vaccines for COVID-19; more than any other country in the world. Our priority is to save as many lives as possible, as quickly as possible, while also reducing the hospitalisations that are creating such pressure on the NHS. Pharmaceutical companies, transportation providers, clinical customers and packaging vendors are increasingly focusing on their environmental impacts. Much biologically hazardous waste in the UK is treated with a combination of chemical sterilants and a well-developed system of sewerage and treatment plants: it is a system which can handle a wide range of biologically active substances, but it is not failsafe. Gareth West at Astell Scientific Limited will review alternatives with the aim of better understanding which method is more suited to the modern laboratory.

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.

In this journal, you will find some articles that will evaluate the biosimilar regulatory sphere. Although regulators have been heavily involved in pandemic activities, important milestones in biosimilar regulation in both Europe and the US were announced last year. Fiona Greer, Independent Consultant, and Richard Easton at BioPharmaSpec look at proposed changes of regulatory emphasis from a global perspective, and consider any potential impact on our analytical strategies for biosimilar testing.

Finally, the current assessment of cell viability depends on endpoint measurements. Unfortunately, this ignores the levels and rates of in-between measurements. However, advances in technology and image analysis now allow cell viability to be observed more frequently and analysed more quickly.

Alternatively, live-cell imaging allows for kinetic monitoring in optimal conditions, helping researchers understand how the cells are reacting over time. Kendra Majewski at CytoSMART Technologies explores how researchers have studied cell viability and how live-cell imaging can provide a better understanding of cell viability.

This journal will also include an interview with Dr. Mark Kotter, CEO and Founder at Drug Discovery and Medicine Research explains the breakthrough technology which, put simply, enables consistent reprogramming of cells based on the reliable activation of specific genes and these genetic programs.

I would like to thank all our authors and contributors for making this issue an exciting one. We are working relentlessly to bring you the most exciting and relevant topics through our journals. I hope that you enjoy reading this edition of the journal and keep well.

Beatriz Romao, Editorial Manager

IBI – Editorial Advisory Board •

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

•

Jim James DeSantihas, Chief Executive Officer, PharmaVigilant

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

•

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

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Catherine Lund, Vice Chairman, OnQ Consulting

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Mark Goldberg, Chief Operating Officer, PAREXEL International Corporation

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Cellia K. Habita, President & CEO, Arianne Corporation

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

•

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

•

•

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

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

•

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

•

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

•

Stefan Astrom, Founder and CEO of Astrom Research International HB

•

Steve Heath, Head of EMEA – Medidata Solutions, Inc

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T S Jaishankar, Managing Director, QUEST Life Sciences

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

•

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

•

Hermann Schulz, MD, Founder, PresseKontext

•

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

4 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

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The Science is Stacking Up for Regenerative Medicine

New Data Shows Positive Potential for Osteoarthritis Treatment in Knees

A new peer-reviewed paper published in a Special Issue of Stem Cells International evidences the effectiveness of micro-fragmented adipose tissue (MFAT) in treating osteoarthritis of the knee. The special issue, entitled Mesenchymal Stem Cells and Regenerative Medicine 2020, was published in 2020.

It shows that, out of 110 knees assessed for patient-centred outcomes of pain, functionality and quality of life, 81% of people responded positively and had an improvement in their arthritis and a significant decrease in their experience of pain.

Conducted over 12 months on patients at The Regenerative Clinic in London, the results show that more than four in five patients have responded well to their Lipogems® treatment for arthritic knees. Their response is extremely promising and equivalent to those who opt for a total knee replacement.

Simon Checkley, CEO, The Regenerative Clinic, says; “The potential is clear and we must now fully document the positive effects of using Mesenchymal stem cells, derived from fat, as an alternative for many orthopaedic conditions. This is a minimally invasive procedure that can be an alternative to major surgery. It can even aid post-surgery recovery. There are no major incisions or cuts. It can help in injury or with a long-term condition that limits daily activity and as a minimally invasive alternative to pain relief. There is much more work to do in proving the value of this treatment and we now need to validate findings with a long-term randomised controlled trial.”

The MFAT treatment that people undertook was Lipogems at The Regenerative Clinic in London, where people have been having treatment since 2017. Patients who opted for Lipogems to treat their knees saw positive results without the complications and risks of having operative surgery.

The Regenerative Clinic have an expert research team that measured patient responses to the MFAT Lipogems treatment in two distinct ways: •

• •

•

Using a visual analogue scale (VAS) and the Oxford Knee Score (OKS). These are both validated ways of objectively measuring the outcomes of treatments The VAS measures the level of the patient’s pain between 0–100; 0 = no pain and 100 = max imaginable pain The OKS measures the functionality of the joint between 0–48; 0 = no function at all and 48 = best possible functionality Results were measured at three months, six months and 12 months post-procedure

The results of the VAS of 110 knees treated show that 81% have responded to treatment. This means that, by the 12-month mark after the procedure, their degree of pain is less than the pain that they had prior to the treatment. On average, 6 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

patients come in with a pain score of 75. This has improved to a score in the region of 20–30, equating to an improvement of over 60%.

The individuals who have not responded to the Lipogems® treatment may have had an initial reduction in pain in the first three to six months, but this was not maintained at the one-year mark. The reasons for this are not entirely clear at this stage and further evidence-gathering is required.

The OKS results presented a similar rate of response as the VAS results. 79% responded positively. Two people did not complete the OKS scores, but these two were responders on the VAS data. On average, patients came to us with an OKS below 20 and by the one-year mark this increased to approximately 35. These results are comparable with having a total knee replacement, but without any complications that are associated with surgery.

Seven of the participants’ OKS scores remained constant throughout (i.e. their mean OKS remained between 20–25, where it started out from), thus no change in symptoms was witnessed in this group.

The deterioration that can be seen on the graph is presumably due to the natural progression of the arthritis disease and has no correlation to the Lipogems treatment.

The severity of arthritis on x-rays can be graded using the Kellgren-Lawrence system of radiological classification. Most patients presented with the most severe grading of arthritis on their x-rays. The response witnessed in these patients has shown to be similar to those who have less severe forms of arthritis. It can be inferred from this information that the vast majority of patients have actually responded to the treatment.

For example, in our grade IV group, which concerns the patients presenting with the most severe arthritis, 25 individuals have been treated. Of these, only two have not responded to Lipogems®.

Science and Evidence The Regenerative Clinic is the UK’s largest centre for regenerative medicine based in Harley Street. Established in 2017, there are more than 40 surgeons involved in the clinic, and each one is a specialist in their traditional discipline.

While clinicians at The Regenerative Clinic are clear that more research and more data must now be done, initial results show impressive potential for the treatment that is as effective in knees as a knee replacement; but is non-invasive; with a one- to two-day recovery time; rarely incurring post-treatment infection; and with the potential to be carried out at a significantly lower cost than traditional joint replacement.

Platelet-rich Plasma(PRP) Where appropriate, the clinic also offers PRP as a therapy. PRP is a concentrate of platelet-rich plasma protein derived from whole blood, centrifuged to remove red blood cells. It has a greater concentration ofgrowth factorsthan whole blood, and has been used to encourage a brisk healing response. Plateletrich plasma (PRP) has been established for 20 years and is used Spring 2021 Volume 4 Issue 1

Regulatory & Compliance by elite athletes and their coaches. There is now an abundance of high-quality evidence that supports the use of PRP injection for lateral epicondylitis and PRP for osteoarthritis of the knee.

There is moderate high-quality evidence which supports the use of PRP injection for patellar tendinopathy and of PRP injection for plantar fasciitis and donor site pain in patellar tendon graft BTB ACL reconstruction.

There is insufficient evidence to routinely recommend PRP for rotator cuff tendinopathy, osteoarthritis of the hip, or high ankle sprains. Current evidence demonstrates a lack of efficacy of PRP for Achilles tendinopathy, muscle injuries, acute fracture or non-union, surgical augmentation in rotator cuff repair, Achilles tendon repair, and ACL reconstruction.

Further data is urgently needed for ongoing study of mesenchymal stem cells (MSCs). Mesenchymal stem cellsare potentstromal cellsthat can differentiate into a variety ofcelltypes, including osteoblasts (bonecells), chondrocytes (cartilagecells), myocytes (musclecells) and adipocytes (fatcellswhich give rise to marrow adipose tissue). In regenerative therapy, MSCs are extracted from a person’s own fat for usage with Lipogems therapy or activated mesenchymal pericyte plasmainjections (AMPP).

AMPP® is a day case procedure performed in approximately one hour with minimal recovery time.

A paper funded by the Italian Ministry of Health and published in Knee Surgery, Sports Traumatology, Arthroscopy (2019) looked at all major studies conducted to date on Lipogems (MSCs derived from a patient’s own fat). The largest study records the results from 681 patients (840 individual knees) which showed that after 12 months, overall pain had decreased and knee function had increased.

The first conducted study* by Damian Hudetz published October 2017 was carried out on 17 patients (and 32 knees) with an average age of 69 with osteoarthritis classified as grade three to four (arthritis is graded one to four, with four representing the most advanced cases with little remaining cartilage and often bone-on-bone symptoms). Following the treatment, patients were tested at three, six and 12 months using a number of indicators including MRI scans and measurement of synovial fluid. In almost all cases, there was an improvement seen, with more than half (53.57%) of joints recorded seeing an increase in cartilage regrowth of +15% or more. The images of the following patient recorded an increase of 83% in cartilage after 12 months.

The treatment induces host chondrocytes to make rejuvenating structural and biochemical changes in the cartilage. It was also noted that it was not just the thickness but rather the quality of the regrown cartilage which was remarkable. Significant pain reduction was also noted across all patients after three, six and 12 months.

A separate study** conducted by Arcangelo Russo at the Sacre Cuore hospital in Calabria, Italy proves the effectiveness of reducing pain. The study is peer-reviewed and has just been published in the Journal of Experimental Orthopaedics.

87% of patients recorded a significant reduction in pain after 12 months (based on the VAS pain scale and Tegner Lysholm Knee). In a sample of 30 patients with a median age of 43, there were no major complications recorded. Russo found the treatment to be “sustainable, quick, one-step, minimally invasive, and with very low percentage of complications”.

Potential Advanced natural regenerative treatments help the body to www.biopharmaceuticalmedia.com

self-heal and may delay, or avoid altogether, the need for surgery.

Already a leading global expert in orthopaedic surgery, Professor Adrian Wilson was inspired by the work of Dr. Carlo Tremolada in Italy. Working closely with him, he began to understand the benefits of a new technology called Lipogems for healing, pain relief and the rejuvenation of joints to restore mobility.

Travelling back and forth to Italy, Wilson became determined to bring the treatment to the UK. He could clearly envisage the benefits as an alternative to costly and more intensive orthopaedic surgeries. Where surgery was required early in a person’s life for knee problems, for instance, Lipogems could give people additional time without surgery. In the UK alone there were more than 100,000 knee replacements last year, along with about 40,000 ligament reconstructions.

Wilson knew from his experience that some people, especially older people, may be less tolerant or suitable for traditional surgeries and the rigours of general anaesthetic and recovery time. One early patient with an advanced hip issue presented with severe kidney problems. A general anaesthetic (administered for a hip replacement) would have resulted in a near certainty of daily kidney dialysis forever thereafter. Results show improvement in this 75-year-old patient, whose mobility is restored, and his shoulder also received Lipogems for a less serious complaint. He continues to both work and act as a carer for his wife. He notes a significant decrease in pain for both hip and shoulder. Six months following treatment, results of increased mobility are present.

Professor Adrian Wilson says; “I have seen the remarkable potential of this treatment first hand. This is a new-to-the-UK procedure with potentially incredible results for patients. It is very exciting to be at the forefront of applying this new therapy but the real satisfaction comes in giving people back the joints that they used to have.”

How it Works The Lipogems technology works by harnessing the inherent rejuvenating properties of a person’s own fat cells. Fat contains lots of juicy blood vessels and natural repair cells (MSCs). The mesenchymal stem cells are extracted from fat, prepared using the Lipogems or AMPP technology and injected directly into the affected area (for example hip, knee or elbow).

These prepared cells trigger a natural healing response within the body where they detect injury and attach themselves to damage. At this point they react and begin to regrow tissue.

The day case procedure takes around one hour and is much less invasive compared to traditional methods of joint replacement.

Patients can return to normal activity after a day or so compared to the months of recovery associated with traditional joint replacement.

These pre-cursor stem cells have a pro-antibiotic effect that reduces pain and infection. Over 25,000 procedures have now been conducted worldwide with no post-procedural infections recorded, and a striking early success rate.

Lipogems is a quick, one-day procedure involving a multidisciplinary team including orthopaedic and plastic surgeons, an anaesthetist, a radiologist and specialist nurses.

With the patient sedated, the plastic surgeon injects a mixture of saline and local anaesthetic into tummy fat and it is left there for 15 to 20 minutes. The fat is extracted with a syringe from either side of the patient’s stomach in a process called lipoaspiration.

Unlike with liposuction, where more than three litres is removed, they take only 75ml (an espresso cupful).

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 7

Regulatory & Compliance It takes about 15 minutes to extract and is transferred to a Lipogems device consisting of a clear, airtight chamber with five marble-sized ball bearings and saline. This is shaken for 30 seconds to break down the fat globules. We add more saline and after six or more shakes, the solution becomes clear and the fat collects on the surface.

This process ‘washes’ blood and oil residue from the fat and exposes the pericytes. The fat solution containing them is then passed through a fine mesh to make the globules even smaller.

The radiologist injects about 10ml into the damaged area using a fine needle under ultrasound guidance. This takes about ten to 15 minutes per knee.

The pericytes start gradually releasing cells called cytokines, which inhibit and block pain and inflammation and promote healing. They block the same pain receptors as morphine and also recruit other cells involved in tissue healing, leading to the formation of new cells capable of forming new cartilage.

The patient can usually go home an hour later. There is often some minor discomfort for a couple of days. Most patients feel a slow release over the following weeks and gradually their pain eases.

A study by YouGov revealed that more than a third of Britons (38%) would prefer to have innovative stem cell treatments over a traditional joint replacement. The figure rose to 42% in the over-55 age group. Nearly half (45%) of Britons believe that stem cell procedures are the future of medicine, with 48% demanding to learn more about these new emerging techniques.

More men (49%) cite stem cell treatments as the future of medicine compared with 42% of women. This rises to 50% in 18- to 24-year-olds.

my knees felt better and soon no pain at all. I was astonished. I no longer limped and only felt occasional pain at night after a long day”.

A Success Story – John Wakefield John Wakefield, 57, from North Yorkshire, underwent the procedure. Eight weeks after the procedure, he could walk for 20 minutes without pain. For most of his life he had been active and played five-a-side football a few times a week for 16 years. But in August 2016, he suffered a sudden shooting pain inside the right knee while at work. This was followed by a feeling of heat deep inside the knee that did not go away.

He wrapped an ice pack round it, rested, then saw his GP a week later. He was referred for an MRI scan, which showed a tear in the cartilage lining the knee joint. An NHS knee specialist told him it probably happened during football. He said the flap of torn cartilage was catching on the joint whenever he moved his knee, causing the pain, and advised he could have microfracture treatment – tiny fractures are made on the surface of the bone in the knee, causing it to bleed. Once it scabs over, new cartilage forms.

Another consultant he saw privately for a second opinion said this might not work, as the new cartilage could break down in the future. Instead, he said it might heal on its own as it was quite a small tear. John stopped playing football but the pain affected other parts of his life. He limped and took painkillers.

The other knee started hurting. By October 2016 he was experiencing grinding pain whenever he walked, but an MRI scan couldn’t find any obvious cause. John saw Professor Adrian Wilson who diagnosed arthritis (cartilage damage in both knees) and recommended Lipogems. Done under local anaesthetic, John reports ‘not feeling a thing’. After 45 minutes he stood up and walked out of the theatre. He said, “The next day my knee joint felt inflamed. This sensation ebbed away after a week – though I was told it would keep happening for about three months. Gradually

REFERENCES

8 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Risks Independent consultants raise some risks. One leading specialist knee surgeon points out the possibility of bruising; “This treatment is interesting because there are no really obvious drawbacks as far as I can see. Infection is reported in very few cases. There seems to be a natural healing effect which protects from infection. This must be investigated further.

For a week or so there may be some mild bruising, with a risk of extensive bruising in one case in 80.

There is a small risk to the deeper structures of the abdominal cavity from the needle used when the fat is bring extracted. There are no research studies to determine the long-term benefits.”

AMPP In summer 2019, The Regenerative Clinic launched AMPP, a combination therapy for joint rejuvenation and preservation.

The new treatment was called activated mesenchymal pericyte plasma injections (AMPP®). AMPP® is a day case procedure performed in approximately one hour with minimal recovery time.

The combined treatment is thought to increase the effectiveness of the process at a cellular level, offering a bold treatment option for people with arthritis and other issues.

For more details about The Regenerative Clinic surgical and clinical team in the UK and globally please visit https://www. theregenerativeclinic.co.uk

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The paper, Patient-Centered Outcomes of Microfragmented Adipose Tissue Treatments of Knee Osteoarthritis: An Observational, Intentionto-Treat Study at Twelve Months, can be accessed in its entirety here: https://www.hindawi.com/journals/sci/2020/8881405/ *The Effect of Intra-articular injection of Autologous Micro-fragmented Fat Tissue on Proteoglycan Synthesis in Patients with Knee Osteoarthritis by Damian Hudetz Published October 2017 http://www.mdpi.com/2073-4425/8/ 10/270 **Autologous and micro-fragmented adipose tissue for the treatment of diffuse degenerative knee osteoarthritis by A. Russo, V. Condello, V. Madonna, M. Guerriero and C. Zorzi Published: October2017 https://jeo-esska.springeropen.com articles/10.1186/s40634-0170108-2 ***Injective mesenchymal stem cell-based treatments for knee osteoarthritis: from mechanisms of action to current clinical evidences https://www.researchgate.net/journal/0942-2056_Knee_Surgery_ Sports_Traumatology_Arthroscopy

Simon Checkley An experienced executive whose recent roles have been in the leadership of businesses with turnovers in excess of £100m and with employee numbers of more than 100.

A global commercial leader who has held senior roles across various geographies in sales, marketing and general management.

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Biosimilars – Increasing Regulatory Focus on Orthogonal Analytical Characterisation Introduction The COVID-19 pandemic has focused attention upon global pharmaceutical industries, and will continue to do so for some time to come. However, despite initial disruption, some positive outcomes may be emerging from 2020 for the future of biotherapeutics in 2021 and beyond. One area which may be becoming more transparent is in the biosimilar regulatory sphere.

Although regulators have been heavily involved in pandemic activities, important milestones in biosimilar regulation in both Europe and the US were announced last year.

For some time now, debates on the balance and appropriateness of the different types of data required to demonstrate biosimilarity – characterisation, biological activity, non-clinical and clinical – have swirled, with both FDA and EMA expressing their expectations of a future without large and expensive biosimilar comparative efficacy studies. A hot topic of discussion has been around the types of data required for demonstration of biosimilarity and the ability of analytical characterisation to provide a more accurate understanding of the originator and biosimilar molecules, in turn arguably reducing the need for large Phase III clinical trials. In April 2020, an international group of biosimilar industry experts published a paper “The Path Towards a Tailored Clinical Biosimilar Development”1 reviewing historical public information on both EU and US biosimilar approvals since 2006. The authors concluded that with current state-of-the-art analytical methods, a tailor-made development for biosimilars need not routinely require comparative efficacy trials. In September 2020, a position paper from experts working at the UK Medicines and Healthcare products Regulatory Agency (MHRA) also examined this issue2.

The big question being asked of analytical scientists is: what types of analytical, non-clinical data are most appropriate to support demonstration of biosimilarity? This article looks at proposed changes of regulatory emphasis from a global perspective and considers any potential impact on our analytical strategies for biosimilar testing, as it reinforces the importance of choosing orthogonal analytical techniques to interrogate quality attributes and assess biosimilarity in a step-wise fashion in early development.

New Proposed UK Biosimilar Regulatory Guidance Understandably, the regulatory emphasis during late 2020 was on the review of vaccine candidates to address the worldwide COVID-19 pandemic. Certainly, this task was a priority for the MHRA who performed it with speed and efficiency, culminating in the authorisation of the Pfizer/BioNTech vaccine product on 2 December and the Oxford University/AstraZeneca product on 29 December 2020. However, this groundbreaking news may have somewhat overshadowed an earlier announcement by the MHRA of a proposed new draft guidance for biosimilars on 7 October 20203.

With the transition period following the departure of the UK from the EU ending on 31 December 2020, the MHRA is once again responsible for evaluating biopharmaceutical products, including biosimilars, for marketing in the UK. It has published a short statement on the guidance to be used at present for Marketing Applications from 1 January 20214, based on current European Medicines Agency (EMA) guidance. However, in their October consultation document, they have taken the opportunity 10 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

to propose some changes, or perhaps it should be viewed as more of an evolution, to the approach of licensing biosimilars which diverges from the EMA’s CHMP guideline in some respects. Comments were invited during the consultation period of six weeks and we await the publication of these and the resulting new guidance.

The proposed new guidance derives from the cumulation of MHRA’s extensive experience accrued in the review of biosimilar applications since the first was approved in 2006; experience which is encapsulated in a September 2020 review paper of 20 EU biosimilar applications covering six complex reference products up to the end of 20192. This paper questions whether clinical efficacy trials are still necessary given their limitations as an effective discriminating tool and argues that, excluding exceptional cases, biosimilarity can be demonstrated by analytical testing and pharmacokinetic (PK) trials.

The proposed draft guidance is based on current EMA guidance, with additional details regarding UK reference standards, the lack of requirement for in vivo animal studies and changes in the requirements for a comparative efficacy trial. It is this last point which is of importance to analytical scientists working with biosimilars, as it signals the shift away from conducting biosimilarity assessments which include clinical comparability trials in favour of robust and extensive physicochemical and biological testing data, together with pharmacokinetic studies. In most cases, comparative efficacy trials will not be required.

In essence, the MHRA supports the use of in-depth scientific knowledge of the reference product (RP), gained using state-of-the-art analytical techniques to interrogate structure and biological activity.

These techniques are then utilised in comparative assessment of the biosimilar candidates, together with a pivotal comparative PK trial with no requirement for comparative efficacy/safety trials. The paper concludes that “extensive comparative analytical studies, together with an abbreviated clinical package…, is sufficient to assess biosimilarity in most cases”. The importance of performing detailed quality structural characterisation is discussed further below.

Understanding the Development Pathway for Biosimilars Comprehensive analytical testing of batches of RP followed by comparative testing alongside biosimilar candidates has always been a pivotal initial step in the development of biosimilars and will of course continue to be so, particularly as comprehensive data derived from orthogonal techniques are expected. A plethora of methods for primary and higher order (glyco) protein structure characterisation of the protein and carbohydrate moieties using both simple and highly sophisticated analytical techniques can be utilised to interrogate the structure of the RP and perform subsequent comparative testing. Regulatory authorities typically reference a “step-wise” or “step-by-step” approach. Thus, developers of biosimilars must demonstrate, through a comprehensive comparability exercise, similarity to the RP, in terms of quality characteristics and biological activity using appropriate physicochemical and in-vitro functional tests prior to undertaking clinical studies. Various guidelines require determination of physicochemical and biophysical properties, purity, impurities and quality according to ICH Topic Q6B. The skill of the analytical testing lab is in designing an appropriate programme of testing and this undoubtedly comes from a combination of experience, understanding of the regulatory requirements, and understanding of the strengths and weaknesses of the analytical methods themselves.

Importance of Quality and Biosimilarity Assessment An important central premise in any case for omitting a comparative efficacy trial is that extensive quality data should be generated, initially for the RP and then in comparison studies with appropriate numbers of Spring 2021 Volume 4 Issue 1

Regulatory & Compliance batches of biosimilar. This highlights the importance of detecting and defining the ranges of critical quality attributes of the molecule at both an analytical and in-vitro functional level.

For analytical scientists, the spotlight is once again turned towards the choice of analytical methods to perform both the detailed interrogation of the RP and the comparability exercise with the biosimilar. As regulatory authorities have extensive experience with modern analytical characterisation methods, they certainly expect the “big guns” to be used during this exercise. The draft MHRA guidance, for example, makes this clear, stating: “…comprehensive analysis of the proposed biosimilar and RP, using state-of-the-art methods with suitable sensitivity and orthogonal methods to determine not only similarities but also potential differences”.

Importance of Orthogonality The FDA, EMA and MHRA all state in their current and draft guidance documents that for biosimilar comparability studies, orthogonal methods should be used to give strength to the analytical data package3,5,6. There are a variety of methods that can be used to provide orthogonal data for various different aspects of structural characterisation; but the key,

however, is knowing how to apply these methods in the most meaningful way, so that conclusions from one particular technique can be matched (if possible) to data obtained from another technique. Table 1 below gives examples of orthogonal techniques and the areas of application to specific aspects of structure. Some approaches can be broadly applied, such as the assessment of deamidation and aggregation, since these will be common to all (glyco)proteins. However, different molecules will have their own unique aspects of molecular structure for which specific applications will be required, such as the degree to which the C-terminal lysine is present on the heavy chains of monoclonal antibodies.

An example of orthogonality for higher order structure (HOS) is shown in Figures 1 and 2 below, but examples of other orthogonal methods for glycan structure investigation have been illustrated in a previous article7. It should be noted that data generated will be based on the inherent properties of the technique being used. In this case, since CD and FT-IR employ different processes for determination of HOS, the outputs from the two methods will not be the same. This is widely understood and accepted within the industry and the regulatory bodies.

What matters from a biosimilarity perspective is how closely the data fit for each technique between biosimilar and innovator.

Figure 1a: CD Analysis of batches of Simponi (golimumab). Stacked far UV data (left) and stacked near UV data (right).

Figure 1b: Far UV CD secondary structure fitting data

Figure 2a: FT-IR ATR Spectral scans of the same Simponi (golimumab) batches as shown in Figure 1a. The full spectral range is shown on the left and the data normalised for the Amide I region is shown on the right. www.biopharmaceuticalmedia.com

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Regulatory & Compliance

Figure 2b: FT-IR secondary structure fitting data based on Amide I wavenumber region

Table 1: Examples of orthogonal techniques and areas of application to specific aspects of structure

Higher Order Structure and Aggregation Higher order structures (HOS) – secondary, tertiary and quaternary – of a biological molecule are unquestionably CQAs, reflecting not just the functional integrity of the molecule, but also the impact of manufacturing and processing.

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The conformation of the biologic is critical for its activity and is another important area of investigation when developing a fingerprint for biosimilarity. Again, many techniques – both qualitative and quantitative – can be applied to determine HOS. One of the most common quantitative Spring 2021 Volume 4 Issue 1

Regulatory & Compliance techniques utilised is circular dichroism (CD), which is most sensitive to helix content, providing information about both secondary and some tertiary structure. On the downside, the presence of buffers in the formulation can interfere with analysis.

Fourier transform infra-red spectroscopy (FT-IR) is an orthogonal quantitative method for secondary structure determination that is most sensitive to sheet content and less likely to be affected by buffers. Both intrinsic and extrinsic fluorescence techniques can also be used – the former for local tertiary structure, and the latter for surface hydrophobicity – but only give qualitative results. Other qualitative methods include differential scanning calorimetry (DSC), which looks at thermal stability, and UV-vis spectroscopy for local tertiary structure.

Hydrogen–deuterium exchange mass spectrometry (HDX-MS), a technique previously used in research applications, highlights details of dynamics, conformation and interactions and is now used to assess secondary and tertiary structures.

Another technique more normally applied in a research setting is two-dimensional protein nuclear magnetic resonance (2D NMR) which is an excellent way of probing 3D structure through assessment of the local environments of the functional groups in the molecule.

The way that biologics oligomerise and aggregate must also be studied. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) is an inexpensive but low-throughput tool for assessing aggregates, and dynamic light scattering (DLS) can be used to look for high-molecular weight aggregates.

Oligomers and aggregates in the dimer/trimer/tetramer range can be investigated using sedimentation velocity analytical ultracentrifugation (SV-AUC) and size-exclusion chromatography with multi-angle light scattering (SEC-MALS), both of which give quantitative results.

Differences in Analytical Results In real life, given the sensitivity of modern analytical techniques, some structural differences are bound to be observed between the biosimilar and the RP. For example, a frequently observed difference in glycosylated proteins (mAbs for instance) is the fucose, galactose and/or sialic acid content of the glycans. There are also often differences observed related to the age of batches used, e.g. increase in deamidation, oxidation, fragmentation etc., and/or differences in C/N terminal sequences. Understanding this, the MHRA guidelines state: “It is not expected that all quality attributes of the biosimilar are identical to the RP” but that “any differences detected in the quality attributes have to be appropriately justified with regard to their potential impact on safety and efficacy”.

A More Streamlined Path for Biosimilars?

The proposed new MHRA guidance, if finalised as written, has the potential to accelerate the speed and reduce the cost of licensing of biosimilar products for the UK market. However, as development is generally a global activity and clinical comparability studies are necessary elsewhere, it is unlikely to have a broader impact at present. Nevertheless, the emphasis on analytical characterisation highlights the importance of using the correct combination of analytical techniques, coupled with experienced interpretation of the findings to assess observed differences for further study using appropriate discriminating methods. www.biopharmaceuticalmedia.com

Over the past 40 years, advanced technologies have been developed for structural characterisation of biological molecules.

Indeed, the challenges of demonstrating biosimilarity have driven this development in part, with emerging novel techniques and improvements in older “classical” methods. There is also a greater appreciation of methods which can link structure with biological activity or predict how the molecule might interact within the biological system, for example, HDX-MS. Previous gold-standard research techniques such as protein NMR and X-Ray crystallography are also being utilised more. Essentially, regulators are looking for multiple orthogonal assessments to build a total profile of the molecule. This use of orthogonality in experimental design strengthens the value of the data beyond what each individual analytical technique could achieve. It is the strength of self-supportive analytical data that will weaken the call for extensive clinical comparability to be performed.

REFERENCES 1.

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Schiestl, M. et al. (2020). The Path Towards a Tailored Clinical Biosimilar Development. BioDrugs 34, 297-306.

Bielsky, M-C. et al. (2020). Streamlined approval of biosimilars: moving on from the confirmatory efficacy trial. Drug Discovery Today 25, 11, 1910-1918. https://www.sciencedirect.com/science/article/ pii/S1359644620303433 Consultation document: MHRA guidance on the licensing of biosimilar products, 7 October 2020. https://www.gov.uk/government/ consultations/mhra-draft-guidance-on-the-licensing-of-biosimilarproducts/consultation-document-mhra-guidance-on-the-licensingof-biosimilar-products https://www.gov.uk/guidance/guidance-on-licensing-biosimilarsatmps-and-pmfs https://www.fda.gov/regulatory-information/search-fda-guidancedocuments/quality-considerations-demonstrating-biosimilaritytherapeutic-protein-product-reference-product https://www.ema.europa.eu/en/similar-biological-medicinalproducts-containing-biotechnology-derived-proteins-activesubstance Easton, R.L. and Reason, A.J (2020) The Benefits of Outsourcing for Earlystage Drug Development. IBI 3, 3,12-15

Dr. Fiona Greer Following a Ph.D. in Protein Biochemistry from Aberdeen University (1984) Fiona became a founding Director of M-Scan. Upon their acquisition in 2010 she became Global Director, Biopharma Services Development, SGS. With over 35 years experience in glycoprotein analysis she has been involved with a diverse range of biotechnology products, both novel and biosimilar and now consults to companies worldwide.

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

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Research / Innovation / Development

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Exploiting Epigenetics to Systematically Optimise Culture Conditions for Cellular Therapies One of the main challenges of cell therapies is the maintenance and/or expansion of the required therapeutic phenotype in vitro. Moreover, to meet both the traceability and safety requirements for a clinical-grade therapeutic, cells need to be cultured in chemically defined conditions.

Over the last two decades, scientists have sequenced the genome and epigenome of all known cell types in the human body, in addition to mapping hundreds of possible protein-protein interactions. The availability of biological data at this resolution and scale has enabled the development of computational tools capable of addressing the challenges associated with the development of scalable cell therapies.

One such platform, recently published in Cell Systems1, systematically predicts the best components for chemically defined culture conditions for cell conversion, maintenance, and expansion. This technology has been used to predict soluble factors that replace Matrigel in the derivation and maintenance of clinically relevant cell types, such as astrocytes and cardiomyocytes1. By allowing the integration of new datasets, platforms such as this could facilitate the systematic identification of culture conditions, differentiation and transdifferentiation stimuli for future cell therapy applications.

Consortia, Exponential Data Growth and the Rise of Epigenetics In February 2003, researchers working as part of an international

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research consortium, known as the Human Genome Project2, completed the 13-year-long hunt for a draft sequence of the human genome3,4. The consortium not only sequenced 94% of the human genome and generated a wealth of data about the architecture, development, and variation of the genomic landscape, but also established new standards for international research collaborations and the accessibility of the results.

However, elucidating the exact sequence of the 3.2 billion DNA base pairs encoded within the human genome, left researchers with more questions than answers.

The need to understand which of these base pairs formed functional units, either as templates for transcription or regulators of gene expression, subsequently sparked the foundation of several international research collaborations aiming to pick up where the Human Genome Project left off.

In September 2003, the Encyclopedia of DNA Elements or ENCODE project5, and its sub-project, the GENCODE consortium6, embarked on a quest to identify all functional elements in the human and mouse genomes across four consecutive phases.

The ENCODE project first used microarray-based methods to reveal basic organisational features in 1% of the genome (ENCODE 1), then switched to sequencing-based technologies that interrogated the whole human genome and transcriptome (ENCODE 2 and 3)7. ENCODE 3 introduced new types of assays to interrogate chromatin interaction and chromatin conformation, adding to the growing pool of data on epigenetic regulation of the human genome. The results of ENCODE 3, published in

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Research / Innovation / Development July last year8 offered the first version of an encyclopaedia of functional elements in the human genome, containing 20,225 protein-coding and 37,595 non-coding genes, 2,157,387 open chromatin regions, 750,392 regions with histone modifications, 1,224,154 regions bound by transcription factors and chromatin-associated proteins, 845,000 RNA subregions occupied by RNA-binding proteins, and more than 130,000 long-range interactions between chromatin loci.

Now in its fourth phase, the ENCODE project, amongst a multitude of other consortia and independent research, have greatly enriched and enhanced our view of the human genome from its original mapping in 2003, building on and rapidly expanding our understanding of the organisation and function of the human genome and epigenome. New consortia leveraging the latest technologies will continue to expand on this work; for example, the Human Cell Atlas9 uses single-cell RNA sequencing technology to create comprehensive reference maps of all human cells, which will add functional elements with high tissue- and/or cell-specificity to our understanding of the human genome.

Considerable growth in large-scale data is compelling scientists and bioinformaticians to develop novel methodologies for efficiently analysing and modelling the data generated.

The analysis of such datasets is also not limited to scientists within consortia such as the ENCODE sub-project, the Encode Data Analysis Center, but is also made available to myriad independent researchers and affiliated commercial entities,

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increasing the likelihood of their application in the development of cellular therapies. Two complementary examples, developed separately over more than a decade of multi-national research collaborations, utilise consortium and other data from the human genome and transcriptome to direct cellular conversion10 and the maintenance of cell identity1.

Can Epigenetics be Used to Predict Culture Conditions?

Understanding the development of cells and their conversion from one type to another is one of the great challenges in biology. Cellular conversion happens throughout development, from the first cell divisions of an embryo to the activation of cells in response to an immune stimulus. How can modern medicine draw on this knowledge to develop cell types for therapeutic applications? While the promise of cell therapies is enormous, most protocols do not make it to the clinic, fail in trials, or are not commercially viable, due to issues with efficacy, safety, and scalability. New solutions are required to improve these outcomes and address the challenges associated with the efficacy and manufacturability of cellular therapies.

Next-generation sequencing of the genome and epigenome, alongside high-throughput data approaches, are playing a key role in the identification of gene regulators (e.g. transcription factors and epigenetic modifiers), and soluble factors (e.g. cytokines and matrix proteins). These factors, once identified, can increase the generation, maturation, and maintenance of in vitro-derived therapeutic cellular products. Recent progress has seen novel technologies, such as Rackham et al. (2016)10

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Research / Innovation / Development and Kamaraj et al. (2020)1 deploy next-generation sequencing, gene regulatory and epigenetic network data to make unbiased predictions: using transcriptomics data to predict the key regulatory switches, such as an optimal combination of transcription factors, required for cell conversion10, and epigenetic data to rank the most important genes for each cell type, including soluble factors required for enhancing directed cell differentiation and maintenance of cell identity in chemically defined media1 (Figure 1).

type, called cell identity genes. The broader the breadth of the H3K4me3 peak, the more specific that gene is to a particular cell type (Figure 2). Such correlation is not replicated in gene expression levels; although cell identity genes are usually highly expressed in a particular cell type, as are housekeeping genes, whose functions are generally universal in all cell types. This striking correlation between H3K4me3 breadth and cell identity enables the prioritisation of proteins and signalling pathways that are critical for the survival of a given cell type.

Figure 1. Systematically predicting ligands, receptors and other factors to enhance cell identity, both for cell maintenance and conversion

Uncovering the ideal culture conditions for the derivation of cells in vitro, both from pluripotent stem cell differentiation and for direct cell transdifferentiation, is a demanding task.

Typically, achieving culture conditions in vitro that mimic the in vivo microenvironment relies on domain knowledge, and trial-and-error of several combinations of undefined culture medium and matrices containing different components (e.g.

Matrigel) and the addition of different sets of cell-typespecific factors, such as growth factors and extracellular matrices. Leveraging epigenetic data, the computational tool dubbed EpiMOGRIFY, published by Kamaraj et al. (2020)1, has been developed as a systematic means of identifying culture conditions that allow for both maintenance and conversion of any human cell type.

Kamaraj et al. describe an approach that incorporates data from over 100 human cell types available from the ENCODE and Roadmap Epigenomics consortia. Data from both projects were integrated into the same analysis pipeline to evaluate how different epigenetic marks correlate with gene expression.

Interestingly, the research observed that the breadth of the H3K4me3 peak at the gene promoter region correlates with the expression of genes that are specific to a particular cell

Following the discovery, the authors hypothesised that this could be leveraged to identify signalling molecules such as receptors and ligands that are essential for cell maintenance conditions. A third database was integrated: a receptor-ligand database previously described and recently updated11,12. With this integration, the authors were able to systematically rank receptorligand pairs, enabling prediction of the microenvironment required to maintain and support the conversion of target cell types in vitro.

To validate the approach, the predicted ligands required for maintenance of primary astrocytes and cardiomyocytes, and differentiation of these cells from pluripotent stem cells or neural progenitors, were tested. The predicted ligands for each cell type were shown to be sufficient to maintain the primary cells and differentiate pluripotent stem cells in the absence of Matrigel. In the case of astrocytes, one of the predicted ligands, LN1, was able to replace Matrigel alone, creating a simple chemically defined astrocyte differentiation and maintenance media. Of most interest, it was observed that a marker of mature cardiomyocytes, cardiac troponin T, was expressed in 45% of generated cardiomyocytes when the five predicted ligands were present, in comparison to 5% when Matrigel was used.

Further validation has revealed a similar trend, showing that the addition of predicted ligands increased the percentage of

Figure 2. Integrating epigenetic and protein-protein interaction data to predict factors for cell culture 16 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

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Research / Innovation / Development CD13/CD82-expressing cells five-fold by day 12. Even more impressively, the expression of maturation markers increased 80-fold after the addition of systematically predicted ligands by day 2113 (Figure 3).

6.

7.

8.

9.

www.gencodegenes.org, visited on 15 Jan 2021.

Abascal, F. et al. Expanded encyclopaedias of DNA elements in the human and mouse genomes. Nat. 583 (7818), 699–710 (2020).

Abascal, F. et al. Perspectives on ENCODE. Nat. 583 (7818), 693–698 (2020). https://www.humancellatlas.org/, visited on 15 Jan 2021.

Figure 3. Enhancement of cardiomyocyte differentiation from iPSC

Synergistic Input of Transcription Factors and Signalling Pathways for Cellular Reprogramming The use of computational power to analyse and model transcriptomic and epigenetic datasets is driving a revolution in how we view developmental biology and the production of in vitro cell therapies. The combinatorial use of computational tools predicting key transcriptomic switches, and chemically defined culture conditions, will enable the systematic generation and maintenance of clinically valuable cell types in vitro. Moreover, with the adoption of technologies enabling the sequencing of the transcriptome and epigenetic marks at a single-cell level, scientists are starting to gather information about defined sub-sets of cell types that were unknown or hard to capture.

Combined, it is possible to imagine a future where the generation and maintenance of cell types is no longer a black-box exercise, being instead a predictable and systematic process powered by computational methodologies.

Acknowledgements The authors would like to acknowledge Wei Wei, Senior Bioinformatician, and David Anderson, Principal Scientist, from Mogrify Limited, for the help in the manuscript and figure preparation.

REFERENCES 1.

2.

3.

4.

5.

Kamaraj, U. et al. EpiMogrify Models H3K4me3 Data to Identify Signaling Molecules that Improve Cell Fate Control and Maintenance, Cell Syst. 1–14 (2020). https://www.genome.gov/human-genome-project, visited on 15 Jan 2021.

Lander, E. S. et al. Initial sequencing and analysis of the human genome: International Human Genome Sequencing Consortium. Nat. 412 (6846), 565–566 (2001).

Collins, F.S. et al. International Human Genome Sequencing Consortium, Finishing the euchromatic sequence of the human genome. Nat. 431 (7011), 931–945 (2004). www.encodeproject.org, visited on 15 Jan 2021.

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10. Rackham, O. J. L. et al. A predictive computational framework for direct reprogramming between human cell types. Nat. Genet. 48 (3), 331–335 (2016).

11. Denisenko, E. et al. Predicting cell-to-cell communication networks. Nat. Commun. 1–11 (2020).

12. Ramilowski, J. A. et al. A draft network of ligand-receptor-mediated multicellular signalling in human. Nat. Commun. 6 (2015).

13. Skelton, R. J. P. et al. SIRPA, VCAM1 and CD34 identify discrete lineages during early human cardiovascular development. Stem Cell Res. 13 (1), 172–179 (2014).

Dr. Joachim Luginbühl Dr. Joachim Luginbühl is a Senior Scientist at Mogrify Limited. After completing his PhD at the children’s hospital in Zurich in a laboratory focused on tissue-engineered skin substitutes, he moved to Japan to join the laboratory of Jay W. Shin at RIKEN Yokohama and develop novel methods to study the direct reprogramming of human fibroblasts into different types of neurons.

Email: joachim.luginbuhl@mogrify.co.uk

Dr. Rodrigo Santos Dr. Rodrigo Santos is the Director of Cell Technologies at Mogrify Limited. He completed his PhD at the Stem Cell Institute, University of Cambridge, focused on the investigation of the biological mechanisms underlying the generation of induced pluripotent stem cells. Prior to Mogrify, Rodrigo was the Head of Technology at Bit Bio, and Principal Scientist at Horizon Discovery.

Email: rodrigo@mogrify.co.uk

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Research / Innovation / Development

Developments in Non-invasive Preclinical Lung Imaging to Support Drug Discovery Growing demand for safe, effective medicines for pulmonary disorders and diseases has increased the research into their treatment. This is key to discovering how to effectively manage pathological lung conditions including asthma, chronic obstructive pulmonary disease (COPD), pneumonia and lung cancer. As reported by the Respiratory Diseases Drugs Global Market Report, the market for respiratory drugs is forecast to grow at a CAGR of 42.5% in the next 10 years, due to the increased demand caused by COVID-19.1 As new drugs go through clinical trials, it will be important to ensure possible drug candidates have been through rigorous amounts of preclinical research to increase success rates.

Drug Discovery Process The full process of drug discovery and development is costly and time-consuming and often results in failure to win approval.

As such, finding new ways to evaluate drug candidates more effectively is an ongoing goal of the pharmaceutical industry. To support chances for successful drug development, researchers can improve their efforts through an earlier and deeper understanding of drug characteristics.

Research has been undertaken at the Novartis Institutes for BioMedical Research in Basel, Switzerland, on how the use of imaging techniques in preclinical studies can more effectively evaluate drugs and enable a higher chance of clinical success in humans. The work undertaken has shown that improving understanding of the molecular events that characterise lung disease and pathology enables researchers to better identify drug targets and the therapeutic candidates that interact with them.

Technology has driven forward data analysis, and increased instrument performance has supported the reproducibility efforts of experiments. More specifically, access to ultra-short echo time acquisition (UTE) for MRI experiments has significantly enhanced imaging techniques that now enable researchers to analyse tissues that would be difficult with more traditional techniques (Figure 1).

Improving the characterisation of compounds and their effects in early and relatively non-costly phases is one way to increase the chance of success in later phases of drug development. Research on pathophysiology, and its early diagnosis and characterisation, can also contribute to a better understanding of disease mechanisms, increasing the chances of finding a treatment that is successful.

Over recent decades, researchers have found value in using non-invasive bioanalytical technologies, such as imaging, in preclinical pulmonary disease drug discovery and development.

Imaging Techniques Imaging techniques including magnetic resonance imaging (MRI), computed tomography (CT), and positron emission tomography (PET) mean that researchers can non-invasively investigate, in vivo, animal biology and metabolism, disease models, and pharmacokinetics and pharmacodynamics of drugs.

They can detect early disease, meaning that therapeutics can be started earlier. Conventional approaches typically show advanced disease states that are often too late to treat.

These techniques and their combinations have become important in preclinical pharmaceutical research to examine lung tissue at high spatial and temporal resolution in small rodents at the anatomical, functional, and molecular or target levels. In addition to focusing on specific lung diseases, imaging enables the diagnosis and quantification of characteristics related to pathological conditions of the lung, which include inflammation, mucus secretion and clearance, perfusion, fibrosis and pulmonary arterial hypertension.

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Imaging the lung in small rodents increased significantly the sensitivity for detecting pathology in a shorter acquisition time. A bleomycin (BLM) model of pulmonary fibrosis illustrates this well. (a) Two-dimensional multislice gradient-echo and UTE acquisitions performed at 4.7 T on the same rat, at day 15 after BLM (4 mg/kg) administration. Image slices have the same geometrical parameters and positions for both acquisitions. It is clear that UTE allowed a more accurate detection of pathology reflecting tissue remodelling and fibrosis. (b) The same is valid for gradient-echo and UTE images at 4.7 T following administration of BLM (0.6 mg/kg) in mice. Of note, while the gradient-echo acquisitions lasted 22 min, UTE images were acquired in only 4 min in both rats and mice. Due to susceptibility effects, measuring the lung at a higher magnetic field is more demanding. Images c-e show representative images extracted from 3D UTE data sets acquired at 7 T with a TE of 0.02 ms. Acquisition times were 10 min for both species. (c) Images from a mouse at day 14 after BLM (0.6 mg/kg) administration. In addition to tissue remodelling/fibrosis along main airways (red arrows), the technique clearly allowed detection of parenchymal signal. (d) Rat images acquired on the same rat at days eight and 21 post-BLM (3 mg/kg) administration. A somatostatin analogue was administered on day nine. The tissue remodelling/fibrosis detected along main airways on day eight resolved on day 21. This shows that the compound had therapeutic effects on established fibrosis in rats. (e) UTE is also very useful for the detection of other disease conditions as well, as illustrated here with a mouse lung cancer model. The compound, an Alk inhibitor, essentially restored the lung to its pre-tumour state (tumours indicated in pre-Alk image by orange arrows).

All images shown here were acquired from isoflurane-anaesthetised, spontaneously breathing animals, without the use of any gating.

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Research / Innovation / Development Advances in developing imaging biomarkers for respiratory diseases have benefited drug development research, but what differentiates imaging biomarkers from others? Imaging readouts tend to be much more closely related to the disease phenotype, therefore facilitating direct associations between therapy and effect. After a lead compound has been validated and optimised, testing it in a relevant animal model of disease provides information concerning drug efficacy, absorption, distribution, metabolism, and elimination. This phase is where imaging holds remarkable benefits for the pharmaceutical industry.

Different Tools Provide Various Opportunities Imaging techniques clearly enhance research capabilities, but which type of instrument is best? Different tools each have their own individual strengths for various purposes, which are all equally important in preclinical research.

MRI Strengths of MRI are that it is non-invasive, and has high spatial resolution and exceptional soft tissue contracting capabilities.

The signal is governed by a number of parameters, and this wealth of information renders it a valuable tool for diagnosis, tissue characterisation and in vivo morphometry. Its use for the evaluation of animal disease and treatment models with high spatial resolution but without harmful radiation makes it particularly suited for longitudinal studies with repetitive measurements on the same subject. Both structural and functional MRI are also particularly valuable for non-invasive animal studies.

Micro-CT Computed tomography is considered the ‘gold standard’ for clinical lung imaging, providing 3-D X-ray imaging, often used in hospital scans. Micro-CT uses the same method, but on a smaller scale – providing high-resolution images and rapid data acquisition, key for detecting tissue structures, skeletal abnormalities, and tumours in small animals. It can also be used to supplement data from other molecular imaging techniques, such as PET, by providing images on a very fine scale.

PET PET detects radiation emitted from tracer substances injected into the body and labelled with positron emitting isotopes, with the isotopes bound to a target tracer. It is one of the most sensitive imaging approaches, being the method of choice for pharmacokinetic studies of biologically active compounds such as drugs or drug candidates.

The development of imaging strategies facilitates the translation from animal models to human subjects because they minimise changes in experimental paradigms even while the model organism is changed.

All three of these imaging techniques can also work in collaboration with one another, as they provide different types of findings. For example, the data received from MRI or CT, with their high spatial resolution, can be used to provide a good anatomical reference for molecular data obtained with high-sensitivity PET or it may even provide complementary information. This could be achieved by post-processing of data obtained in different imaging sessions, or by utilising multimodality small-animal imaging instruments such as PET/MRI. www.biopharmaceuticalmedia.com

The Future of Drug Discovery with the Support of Preclinical Imaging New and improved imaging techniques enable greater advancements in research, meaning that preclinical work only proceeds to clinical trials once researchers have a greater degree of certainty that a drug candidate may be viable. Imaging maximises the available information at preclinical stage. Preclinical imaging techniques are central to evaluating the effectiveness and safety of new treatments and describing drug distribution patterns before clinical use, while preclinical small animal imaging has provided valuable insights into the mechanisms of many diseases, such as those affecting the lung, and the effects of treatments.

For more information about Bruker’s preclinical imaging solutions, please visit https://www.bruker.com/products/ preclinical-imaging.html REFERENCES 1.

Research and Markets, 2021.World Respiratory Diseases Drugs Market Analysis 2020 - Market Forecast to Grow to $92.6 Billion in 2020 at a CAGR of 42.5% Due to Increased Demand as a Result of COVID-19 Outbreak. [online] GlobeNewswire News Room. Available at: <https:// www.globenewswire.com/news-release/2020/06/11/2046856/0/ en/World-Respiratory-Diseases-Drugs-Market-Analysis-2020-MarketForecast-to-Grow-to-92-6-Billion-in-2020-at-a-CAGR-of-42-5-Dueto-Increased-Demand-as-a-Result-of-COVID-19-Outbreak.html> [Accessed 19 February 2021].

Dr. Nicolau Beckmann Nicolau Beckmann has an MSc in physics from the University of São Paulo in Brazil and a PhD in biophysics from the University of Basel in Switzerland. Currently he is the head of an imaging and histology group at the Novartis Institutes for BioMedical Research, Basel, and an assistant professor in Biophysics at the University of Basel. His research interests centre around the use of imaging techniques in pharmacological research, in the areas of musculoskeletal diseases, neurological disorders, respiratory diseases, arthritis, metabolism and transplantation. He has coauthored more than 100 publications and edited three books. Nicolau serves as reviewer for several scientific journals, research agencies and the European Commission, and is on the editorial board of the Journal of Magnetic Resonance Imaging and Frontiers in Pharmacology.

Sarah Rebecca Herrmann Sarah Herrmann has a degree of physics from the University of Heidelberg. After completing her PhD in magnetic resonance spectroscopy at the German Cancer Research Center, she joined Bruker, where she works in the Preclinical Imaging Market Product Management department.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 19

Pre-Clinical & Clinical Research

Why Patient Diversity in Research Must Become the Norm While 2020 was not the year anyone hoped it would be, it did succeed in showing us what medical research can achieve when the life sciences sector works together to overcome a challenge. Three COVID-19 vaccines developed and licensed in less than a year, with more still in development, is certainly extraordinary, but what is also remarkable is that even in the rush to find and develop effective treatments and vaccines for a completely new virus, the life sciences sector achieved what it has struggled with for years: diverse patient involvement in clinical trials that truly represents the global population.

Nearly half (42 per cent) of all global participants involved in Pfizer and BioNTech’s Phase III vaccine clinical trial had racially or ethnically diverse backgrounds. Meanwhile, Oxford University and AstraZeneca included more than 24,000 people from diverse racial and geographical groups in their Phase III vaccine trial. For Moderna’s COVID-19 vaccine, more than 11,000 people from racially and ethnically diverse backgrounds were enrolled into its Phase III clinical trial, representing 37 per cent of the study’s population.

This is important. In the UK, the Office for National Statistics (ONS) found that black men are around twice as likely to die from COVID-19 than white men and the risk of death for black women is around 1.4 times greater than for white women.

Men of Bangladeshi, Pakistani and Indian ethnicity are also at a significantly higher risk of dying from COVID-19 compared with their white male counterparts1. Meanwhile, a report by the Intensive Care National Audit and Research Centre (INARC) states that one in three critically-ill COVID-19 patients were either of BAME or mixed ethnicity2.

With people from Black, Asian and minority ethnic communities (BAME) disproportionately affected by COVID-19, this commitment to enrolling participants from diverse backgrounds is to be applauded. Unfortunately, the same cannot be said for every piece of clinical research. By June 2020, only six of the 1518 COVID-19 trials registered on ClinicalTrials.gov were collecting data on ethnicity3.

Pfizer, Moderna and AstraZeneca have shown us what is possible. We must all now follow them down the path they have started walking, in 2021 and beyond. Greater patient diversity is a ‘must’ in clinical research, not a ‘nice to have’.

Increasing Diversity in Clinical Research The issue of patient diversity within clinical trials is an important one: for any treatment to be developed it must be shown to be safe and effective. Clinical trials enrol people who are considered suitable for the particular treatment to help investigate how well tolerated the medicine or technology is and how well it can work.

However, when the patients that are enrolled into a trial are not representative of the population who are most in need of that particular treatment, we risk developing drugs that may not work so well for those populations.

20 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

This is widely acknowledged and is an issue that we, as a sector, have grappled with for years. For example, between 2008 and 2013, around one in five newly approved drugs demonstrated differences in treatment response across ethnic groups leading, in some cases, to doctors prescribing drugs differently according to ethnicity4. And in the US, it has been noted that AfricanAmericans need a higher dose of the blood thinning treatment warfarin than their white counterparts5.

Despite knowing this, however, people from BAME communities have continued to be traditionally underrepresented in research. Between 2008 and 2018, of the 230 clinical trials that led to cancer drug approvals by the Food and Drug Administration (FDA) in the US, ethnicity was reported in fewer than two in three (63 per cent)6. Meanwhile, a review conducted in 2017 of 12 Type 2 diabetes trials – a condition that disproportionately affects the South Asian community – found that participants of South Asian ethnicity in the UK were not proportionally representative of the country’s population. One in three of the studies did not report ethnicity data7.

According to another UK study, only around five per cent of people from BAME groups who had been surveyed had ever participated in medical research8, despite accounting for around 14 per cent of the UK population9. In the US, African-Americans account for 12 per cent of the population, but only around five per cent are involved in research10.

Reasons for this are complex, including issues such as cultural barriers and lack of knowledge of clinical trials11. Small steps forward have been taken over the years, but COVID-19 highlighted how seriously – and urgently – we must take the issue of patient diversity.

The FDA published new guidelines in November 2020, setting out a series of recommendations to increase the enrolment of under-represented groups and increase racial diversity in clinical trials within the US. It is an issue it takes seriously, launching campaigns and policies over the years to support the sector in tackling this issue, but uptake of these guidelines remains voluntary. In the UK there are no such guidelines.

We need to do better. We must go further, and together, if we are to truly tackle this issue. It must involve everyone involved in the drug development process – from funders and researchers to drug manufacturers – to take an integrated approach to tackle this inequality.

Promoting Diversity in Clinical Trials Trials must become fully inclusive so that medicines can be fully investigated within the patient populations that might need them most. Drug licensing and regulatory bodies are increasingly asking for evidence of drug safety and efficacy in diverse populations, which shows how important this issue is and is in itself another reason why we must take decisive action to end this inequality in healthcare.

Achieving this will require a different way of thinking – we know that traditional methods used to enrol participants into clinical research are not effective for BAME populations – but it Spring 2021 Volume 4 Issue 1

Pre-Clinical & Clinical Research is vital we adapt. Pfizer, Moderna and AstraZeneca have shown us it is possible.

A virtual event, which I chaired in November 2020, discussed whether a mandatory policy is needed to kickstart action in this area. While the response from the panel – which included a parliamentarian, pharmaceutical experts and BAME leaders – was mixed, it was clear that everyone involved thought change was required.

From my own perspective, I would like to see the research community and pharmaceutical companies work hand-in-hand with BAME communities to increase their understanding of research and participation in clinical trials, and design clinical trials that prioritise patients’ needs, placing this on an equal footing with the treatment being investigated.

Building relationships and trust is key to achieving this. This is not something that can happen overnight though. It will take time and effort and involve working in ways that we as a sector may not necessarily be accustomed to.

Visiting communities where people live and work and engaging with trusted community leaders and physicians, sharing relevant information in the most appropriate format and language, and giving individuals time to come to a decision about whether or not they want to participate in clinical research without applying pressure, are all vital elements. My team and I have used churches, grocery stores, hair salons and gyms as locations to talk to people about clinical research and found them to be much more receptive to trial information than they may have been otherwise.

More widely, the industry needs to consider how research is conducted internationally; is there potential to change how we share information or set up multiple sites, particularly in countries within Africa, Asia and Latin America, to ensure a more diverse representation of ethnicity?

And research funders could consider implementing patient diversity ratios where it is appropriate to do so, to ensure the studies they fund include a more equal representation of ethnicity.

It is important to acknowledge that one of the consequences of adopting these approaches is that the cost and time for delivering clinical trials will inevitably increase, but should this really be a reason not to better represent different populations in research? There is the argument that addressing this imbalance will generate savings elsewhere by targeting the right medicines to the righ populations, and fewer trials being abandoned because they do not meet the requirements of diversity set in the population being targeted.

In the UK, a National Taskforce is being established to create a blueprint for greater patient diversity in research and drive that forward to bring about real change. Spearheaded by Innovative Trials, COUCH Health and Egality, three organisations working in the patient engagement field, and recruiting members from across UK life sciences, the taskforce will focus on the critical issue of recording ethnicity in health research and clinical trials.

Without this data, there can be no benchmarks against which to measure improvement. Good data is also required for informed decision-making, which is itself crucial for long-term change.

Now is the time for all of us to come together to put the right processes, principles, resources, and monitoring in place. If we are going to find other effective treatments and vaccines for COVID-19 – or any other condition, for that matter – we must all www.biopharmaceuticalmedia.com

work together to make sure clinical trials represent those most at risk. If inclusivity is taken seriously, there is no doubt that this will result in better outcomes for all.

REFERENCES 1.

Office for National Statistics, Coronavirus (COVID-19) related deaths by ethnic group, England and Wales: 2 March 2020 to 15 May 2020, published 19 June 2020: https://www.ons.gov.uk/ peoplepopulationandcommunity/birthsdeathsandmarriages/deaths/ articles/ 2. These data derive from the ICNARC Case Mix Programme Database.

The Case Mix Programme is the national clinical audit of patient outcomes from adult critical care coordinated by the Intensive Care National Audit & Research Centre (ICNARC). For more information on the representativeness and quality of these data, please contact ICNARC: https://www.icnarc.org/DataServices/Attachments/Download/ af7be2d4-bdcd-ea11-9127-00505601089b 3. Pan D, Sze S, Minhas JS. The impact of ethnicity on clinical outcomes in COVID-19: a systematic review. EClinicalMedicine. 2020 doi: 10.1016/j. eclinm.2020.100404. published online June 3 https://doi.org/10.1016/j. eclinm.2020.100404 4. Ramamoorthy A, Pacanowski MA, Bull J, Zhang L (2014). Racial/ethnic differences in drug disposition and response: Review of recently approved drugs. Clinical Pharmacology & Therapeutics, doi: https://doi. org/10.1002/cpt.61 5. Akinboboye O. (2015). Use of oral anticoagulants in African-American and Caucasian patients with atrial fibrillation: is there a treatment disparity? Journal of multidisciplinary healthcare, 8, 217–228. https:// doi.org/10.2147/JMDH.S74529 6. Loree JM, Anand S, Dasari A et al. Disparity of Race Reporting and Representation in Clinical Trials Leading to Cancer Drug Approvals From 2008 to 2018. JAMA Oncol. 2019;5(10):e191870. doi:10.1001/ jamaoncol.2019.1870 7. Khunti K, Bellary S, Karamat MA, et al. (2017). Representation of people of South Asian origin in cardiovascular outcome trials of glucose-lowering therapies in Type 2 diabetes. Diabet Med. 2017; 34(1): 64-68. Doi: 10.1111/dme.13103 8. Harrison EK & Smart A (2016). The under-representation of minority ethnic groups in UK medical research. Ethnicity and Health, doi: 10.1080/13557858.2016.1182126 http://openaccess.city. ac.uk/14545/ 9. Office for National Statistics (2018). Population of England and Wales https://www.ethnicity-facts-figures.service.gov.uk/ uk-population-by-ethnicity/national-and-regional-populations/ population-of-england-and-wales/latest 10. Data presented by P. Sanders in "Dialogues on Diversifying Clinical Trials," Washington, D.C., 2011 Sept 22. http://www.womenshealthresearch.org/ site/PageServer?pagename=events_clinicaltrials 11. Symonds RP et al. Recruitment of ethnic minorities into cancer clinical trials: experience from the front lines. British Journal of Cancer. 2012.

107(7): 1017–1021. Doi: 10.1038/bjc.2012.240

Kate Shaw Kate Shaw is the founder and CEO of Innovative Trials, a UK-based clinical trials patient recruitment company, and has more than 20 years’ experience in patient recruitment support for clinical research. Her company has worked with pharmaceutical companies on nearly 200 clinical trials, recruited around 40,000 people into clinical research and played a role in six new treatments being approved for patients across the world. For more information, visit www.innovativetrials.co.uk

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 21

Pre-Clinical & Clinical Research

PEER REVIEWED

Automated Identification of Cardiac Hypertrophy Modulators Using a hipsc-derived Disease Model Effective drug discovery and development rely heavily on the availability of predictive preclinical models. For decades, target-based drug discovery has focused on immortalised cells to identify and optimise inhibitory or activating molecules. Testing on more complex biological systems takes place only during late stages in the drug development pipeline. Bringing the most relevant biology into the pipeline earlier would help to mitigate late-stage failures due to safety or efficacy concerns. However, complex biological systems are rarely available in the scale required for high throughput screening. Recent developments in human induced pluripotent stem cell (hiPSC) technologies hold great promise to overcome these limitations.

hiPSCs retain patient-specific genetic background information, differentiate into functional cell types, and closely mimic human pathophysiology. Ncardia has developed a system for scaled expansion and differentiation of hiPSCs, generating large batches of cells that can be cryopreserved until use. This enables the same batch of hiPSC-derived cells to be used for both hit identification and lead optimisation.

Automated Cell Culture Optimisation of hiPSC-derived Cardiomyocytes Fully automated cell culture protocols and assays are vital prerequisites for HTS campaigns. Ncardia chose the Fluent 780 workstation to enhance its drug discovery services. The first objective was to develop an infrastructure for the automated production of up to 42 pre-cultured 384-well microplates per day, with enhanced reproducibility versus manual culture, to enable screening of >3500 small molecules in a single study. Quality improvement was assessed by inter-well and inter-plate variation of the cell density (degree of confluence) and cell functionality (spontaneous beating quantified by a calcium flux assay). Three main scripts were developed for cell culture of hiPSC-derived cardiomyocytes in 384-well microplates: • • •

Assay plate coating, Cell seeding, Medium refreshment for a period of 10 days.

A schematic overview of the automated process optimisation can be found in Figure 2.

Successful drug efficacy screening and validation studies require not only a physiologically relevant cell model, but also validated high throughput screening (HTS) protocols to test the effects of drug candidates. To achieve this, Ncardia has automated its cell culture processes in a 384-well microplate format, as well as its assay readouts and data handling. Setting up such a complex workflow for highly sensitive and specialised cells is challenging. Optimal labware and workdeck configurations for automated liquid handling platforms are key to a robust and reliable process, requiring each step to be executed as quickly as possible to avoid variation across microplates. Tecan’s Fluent Automation Workstation offers the speed and flexibility required to meet such process-specific liquid handling needs.

This article outlines how the Fluent workstation was used to automate cell culture for Ncardia’s beating, hiPSC-derived cardiomyocytes. It also describes the use of these cultured cells to screen >3500 small molecules in a chemically-induced hypertrophy disease model, using a validated phenotypic assay (Figure 1).

Figure 2: Schematic overview of the optimisation process for automated cell culture of hiPSC-derived cardiomyocytes in 384-well microplates.

Figure 1: Schematic overview of the automated phenotypic screening workflow using hiPSC-derived cardiomyocytes.

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Spring 2021 Volume 4 Issue 1

Pre-Clinical & Clinical Research Results After several rounds of optimisation for labware teaching and liquid class refinement, a high degree of inter-well reproducibility was reached, based on measurements of monolayer confluence per well at 10 days post-seeding, using the Incucyte® Live-Cell Analysis System. The number of cells per well was also quantified using a CyQUANT™ DNA Count Kit and a Spark® 20M multimode plate reader. Before extensive process optimisation, assay plates cultured under fully automated conditions had a higher inter-well coefficient of variation (% CV) compared to assay plates handled manually. However, after process optimisation, assay plates under automated culture had comparable cell numbers with improved inter-well % CV relative to manually cultured plates (Figure 3).

Figure 4: Inter-well and inter-plate variability for assay plates generated using the automated scripts. Five 384-well plates were cultured using fully automated protocols for microplate coating, cell seeding and medium refreshment, and maintained in the automated incubator for 10 days. On day 10, calcium flux in the first and last plates was assessed using the FDSS/μCELL platform. Both plates showed similar beat rate and peak amplitude (top row). Moreover, inter-well and intra-well variation for each plate were within the acceptable ranges, represented by the dashed lines.

failure, arrhythmia and sudden cardiac death1,2. The natriuretic peptide fragment, NT-proBNP, becomes more abundant in HCM patients’ serum, and is an established clinical biomarker for the diagnosis of hypertrophy. Ncardia has developed a specialised NT-proBNP AlphaLISA assay to detect the secretion of this biomarker in cell culture supernatants from in vitro HCM models.

This assay was assessed and validated to ensure compatibility with HTS approaches, i.e., to demonstrate high reproducibility and a wide assay window.

Figure 3: Effect of labware teaching and liquid class optimisation on hiPSC-derived cardiomyocyte cell density, as determined by the CyQUANT viability/nuclear count assay. Two 384-well plates were cultured (one under fully automated conditions and the other manually). After 10 days of culture, the number of nuclei (DNA) per well was measured using the CyQUANT assay. The nuclear count per well, as well as the average nuclear count per plate (dotted line) and inter-well % CV, are shown for manual vs automated cultures, before and after optimisation of automation scripts.

The functionality of the automated hiPSC-derived cardiomyocyte cultures was also assessed at 10 days post-seeding by monitoring the spontaneous calcium flux properties in five assay plates. Figure 4 depicts the baseline measurements for calcium flux in the first and fifth (last) plates according to the handling order. Overall, there were no significant differences in the intra-well, inter-well or inter-plate measurements when comparing critical functional parameters. This data demonstrates that fully automated handling of assay plates for 10 days does not affect the morphology or functionality of hiPSC-derived cardiomyocytes. The automated scripts were therefore suitable for handling multiple assay plates per run in high throughput compound screening applications.

Development of a Hypertrophic Cardiomyopathy Phenotype Assay for HTS in hiPSC-derived Cardiomyocytes Hypertrophic cardiomyopathy (HCM) is an autosomal dominant disease of the cardiac sarcomere, associated with abnormal thickening of the left ventricular myocardium and resulting in elevated risk for clinical complications, such as progressive heart www.biopharmaceuticalmedia.com

Two hiPSC-based HCM models were used in these experiments: a patient-derived hiPSC line carrying an HCM-associated sarcomere mutation in the MYH7 protein, and a WT hiPSC line. Cardiomyocytes were derived from each line at large scale, using stirred tank bioreactors, and were treated with endothelin-1 (ET-1) – a known hypertrophy-inducing peptide typically produced by endothelial and smooth muscle cells. The chemically-induced HCM model in the WT hiPSC line was used for assay development and, later, for primary screening and hit confirmation, whereas the patient-derived model was used for hit confirmation only.

The hypertrophic phenotype of hiPSC-derived cardiomyocytes was assessed by measuring NT-proBNP secretion by AlphaLISA, in the presence of ET-1 and compounds with known anti-hypertrophic abilities. A Spark 20M multimode plate reader was used to detect AlphaLISA signals. The assay performance was evaluated using known quality control criteria, such as signal-tobackground ratio (S/B), Z-factor and % CV. This was necessary to ensure the robustness of high throughput compound screening for the novel cell-based platform.

Results The cardiac hypertrophy assay was robust (S/B >4) and reproducible (Z-factor >0.4 and % CV <20) for both ET-1 induced hypertrophy and inhibition of hypertrophy with the known anti-hypertrophic compound verapamil hydrochloride – used here as a positive control (Figure 5A). Validation of the assay for HTS by testing a larger set of pro- and anti-hypertrophic INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 23

Pre-Clinical & Clinical Research compounds in duplicate 384-well plates confirmed the reproducibility of the assay (correlation coefficient >0.9), and its suitability for screening (Figure 5B). This data confirms the successful development of a cardiac hypertrophy assay in hiPSC-derived cardiomyocytes that is appropriate for HTS.

Z-factor very close to the lowest accepted limit (0.32). This data shows that all plates met the predefined quality criteria and did not require rescreening. As a next step, the percent inhibition (PIN) of all compounds was calculated relative to the MAX effect (100 % inhibition). Based on distribution analysis of the compound effect at different cut-offs, the threshold for considering a compound as a hit was set at PIN >40 %.

According to this predefined cut-off, 341 compounds were identified as primary hits (Figure 6B).

Figure 6: Reproducibility and robustness assessment for primary screen. (A, Top left) S/B (MAX signal vs MIN signal) was assessed for 15 screening plates, with all 15 plates displaying an S/B >10. (A, Bottom left) 14 plates had Z-factor >0.4, and one plate had Z-factor of 0.32. (B) Each dot represents the effect of a single compound at 1 μM presented as percentage inhibition relative to MAX effect (100 % inhibition, red dots). The three libraries screened are shown in different colours. Blue dots depict MIN effect. Compounds with PIN >40 % (above red line) were considered as primary hits.

Following the primary screen, a hit confirmation analysis was performed for 341 compounds, using three distinct assays: Figure 5: Validation of NT-proBNP AlphaLISA for use with hiPSC-derived cardiomyocytes in HTS. Several replicates of four test conditions were used to assess assay performance in each 384-well plate. All groups showed low variation (% CV <20). (A) The assay was robust, as evidenced by an S/B of 6.3 and an assay window of 4.2. A Z-factor of 0.4 confirmed the reproducibility of the assay for high throughput compound screening. (B) For further validation, a selected panel of 96 anti- and pro-hypertrophic compounds at three concentrations (0.1, 1 and 10 μM) was screened in duplicate. The duplicate plates showed high reproducibility at all concentrations tested (correlation coefficient >0.9).

High Throughput Efficacy Screening for Identification of Anti-hypertrophy Drugs with Lead-like Properties After successful development of the ET-1 induced HCM model, and validation of the clinically relevant NT-proBNP AlphaLISA for HTS, a primary high throughput compound screen was performed using three chemical libraries: •

• •

the Prestwick library, consisting of 1180 FDA-approved, highly chemically and pharmacologically diverse small molecules with a high level of hit-like properties (screened at 0.1 and 1 μM), a library of 450 FDA-approved compounds (screened at 1 μM), a phenotypic diversity library of 2200 compounds consisting of diverse target classes and signallling pathway modulators (screened at 1 μM).

The entire screen consisted of 3830 compounds (5010 data points, as the Prestwick library was screened at two concentrations per compound) in fifteen 384-well microplates.

Each plate consisted of 24 replicates of 0.1 % DMSO plus ET-1 (MIN effect) and 0.1 μM verapamil hydrochloride plus ET-1 (MAX effect), enabling assessment of the quality of each plate throughout the screening.

Results As shown in Figure 6A, all the plates had a high S/B (>4) and robust Z-factor (>0.4), except for one plate which had a 24 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

• • •

NT-proBNP secretion AlphaLISA (technical duplicate), the AlphaScreen® TruHits™ assay to deselect false positives, high content imaging (HCI) to assess intracellular proBNP expression.

Based on the data obtained from the NT-proBNP AlphaLISA, 248 compounds showed a PIN >40 % (Figure 7A). To deselect false positive compounds (i.e., compounds that interfere with one of the components of the AlphaLISA technology, reducing the signal intensity), an AlphaLISA TruHits assay was performed. Two versions of the assay were used to exclude compounds interfering with either biotin (biotin mimics) or signal intensity (quenchers or scatterers) (Figure 7B). Based on the results, 192 of the hits with PIN >40 % in the NT-proBNP AlphaLISA had TruHits activity <20 % in both versions of the TruHits assay. This resulted in a final hit identification rate of 5%, which is a reasonable rate for a phenotypic cell-based screening using FDA-approved libraries.

Figure 7: Hit confirmation with three distinct assays. (A) Primary hits were first confirmed by repeating the NT-proBNP AlphaLISA at a 1 μM concentration for each compound. Compounds with PIN >40 % (red line) were confirmed as hits. (B) TruHits assays were performed to deselect false positives. The graph indicates compounds identified as colour quenchers, light scatterers (insoluble compounds), singlet oxygen quenchers and biotin mimetics (right of red line). Compounds with PIN >40 % in the NT-proBNP AlphaLISA, and TruHits activity <20 % (left of red line) were selected as confirmed hits. (C) The NT-proBNP AlphaLISA was multiplexed with HCI to confirm intracellular proBNP expression. The graph depicts the PIN effect of compounds in the NT-proBNP AlphaLISA (X axis) vs. the intracellular proBNP expression (arbitrary fluorescence units, AU) in proBNP+ cells (Y axis). Red dots represent the MAX effect reduction in proBNP levels, and blue dots represent the MIN effect.

Spring 2021 Volume 4 Issue 1

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BIOPHARMACEUTICAL SOLUTIONS LIFE INSPIRED, QUALITY DRIVEN

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Pre-Clinical & Clinical Research Additionally, the NT-proBNP AlphaLISA was multiplexed with HCI of proBNP and DAPI (nuclear DNA stain) to quantify intracellular proBNP expression (orthogonal assay) and evaluate any cytotoxic compound effects, respectively. As depicted in Figure 8C, the assay positive control (5 nM ET-1 + 0.1 μM verapamil) reduced intracellular proBNP expression levels compared to the negative control (5 nM ET-1 + 0.1 % DMSO). Furthermore, some hits reduced the proBNP expression in both assays (NT-proBNP AlphaLISA and HCI intracellular proBNP), while others only showed inhibitory effects in the NT-proBNP AlphaLISA. Finally, a potency analysis for the 192 confirmed hits was performed, using the NT-proBNP AlphaLISA. Dose-response curves were set up with eight concentrations per compound, ranging from 1 nM to 5 μM. The potency analysis was set up within eight plates that passed the predefined quality criteria (data not shown). This analysis revealed that hits had varying potencies, as shown in Figure 8. Some drugs showed low potency (estimated EC50 >10 μM), while others showed moderate potency (estimated EC50 <1 μM). Most compounds in the latter group had well-fitted ‘S curves’, enabling a precise calculation of EC50 values. Many confirmed hits had lead-like properties (sub-μM potency, favourable DMPK properties, good safety profiles, etc.) and are currently being further investigated as potential HCM therapeutics.

hiPSC-derivatives. As summarised in Figure 9, the phenotypic screen of >10,000 data points resulted in 341 initial hits. Hit confirmation assays narrowed this number down to 192 final hits. EC50 values were obtained for all 192 confirmed hits, and many of these have lead-like properties. Most importantly, these results hold great promise for the development of safer and more effective drugs for hypertrophic cardiomyopathy patients, and facilitate Ncardia’s mission to help get better drugs to patients faster.

Figure 9: Schematic summary of the phenotypic HTS campaign performed by Ncardia on the Fluent Automation Workstation to identify anti-hypertrophic compounds using hiPSC-derived cardiomyocytes (CRC = concentration-response curve).

REFERENCES 1.

2.

Maron, BJ et al. Images in cardiovascular medicine. Extreme left ventricular hypertrophy. Circulation, 1995, 92(9), 2748.

Kuwahara, K et al. The effects of the selective ROCK inhibitor, Y27632, on ET-1-induced hypertrophic response in neonatal rat cardiac myocytes – possible involvement of Rho/ROCK pathway in cardiac muscle cell hypertrophy. FEBS Letters, 1999, 452(3), 314-8.

Elena Matsa Figure 8: Potency analysis for the 192 confirmed hits. 8-concentration dose-response curves, ranging from 1 nM to 5 μM, were tested for each compound, identifying varying potencies. The top row depicts representative compounds with low potency (EC50 estimated at >10 μM). The middle row shows representative compounds with moderate potency (calculated EC50 from 1–10 μM). The bottom row depicts representative compounds with high potency (EC50 estimated at <1 μM).

Summary and Conclusions This study demonstrates how hiPSC-derived cells provide a physiologically relevant disease model that can be used in a high throughput screening campaign for assessment of drug efficacy.

Several hurdles were overcome to enable this hypertrophic cardiomyopathy phenotypic screen. First, Ncardia’s industryleading, scalable hiPSC-derived cell manufacturing enabled generation of sufficient quantities of cells from the same batch to execute a complete high throughput phenotypic screen, all the way from assay development to hit validation. The Fluent workstation allowed these hiPSC-derived cardiomyocytes to be cultured for a prolonged time period (≤14 days) in a fully automated way, with high well-to-well and plate-to-plate reproducibility.

The phenotypic assay described was robust and reproducible (S/B >4, Z-factor >0.4 and % CV <20), allowing 42 384-well microplates to be handled per run. This offers the speed and flexibility needed for the validation and execution of any phenotypic screening campaign using specialised 26 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Elena is the Director of Discovery Technology at Ncardia. She 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 modeling of human cardiac disease in iPSC-derived cardiomyocytes.

Email: elena.matsa@ncardia.com

Lucía Bruzzone Dr. Lucía Bruzzone is an application specialist at Tecan Switzerland. She studied molecular biology and biotechnology at the University of Buenos Aires in Argentina. During her PhD, she focused on cell biology and genetics at the Institut Jacques Monod in Paris, France. She joined Tecan in 2019 and focuses on the development and support of applications on Tecan’s liquid handling platforms in the cellomics and proteomics area.

Email: lucia.bruzzone@tecan.com

Spring 2021 Volume 4 Issue 1

YOUR BRIDGE BETWEEN LIFE-CHANGING THERAPIES AND PATIENTS

Introducing

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• Shorten timelines to proof of concept • Maximize dosage flexibility from cohort to cohort • Reduce API and drug waste significantly

Accelerate your Phase I / II studies with PCI services www.pciservices.com/speed-to-study

www.pci.com www.biopharmaceuticalmedia.com

talkfuture@pciservices.com INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 27

Pre-Clinical & Clinical Research

Live Cell Imaging: Non-invasive Kinetic Data to Better Understand Cell Viability Current assessment of cell viability depends on endpoint measurements. Unfortunately, this ignores the levels and rates in between measurements. However, advances in technology and image analysis now allow cell viability to be observed more frequently and analysed more quickly.

Alternatively, live cell imaging allows for kinetic monitoring in optimal conditions, helping researchers understand how the cells are reacting overtime. This article will explore how researchers have studied cell viability and how live cell imaging can provide a better understanding of cell viability.

Cell viability is a measure of the proportion of live/ metabolically active cells within a population. This is used as a baseline for cellular health and changes are monitored in response to various treatments and stimuli. Drug development, cancer research and regenerative medicine all utilise cell viability as a part of their research. Common methods to determine cell viability are cell counting, live/dead viability staining, metabolic activity assays, and colony formation assays to test continuous growth of cells1. A hallmark of dead and dying cells is the breakdown of cell and nuclear membranes.

Many viability assays use this characteristic of cell death to distinguish viable from non-viable cells.

Cell Viability Assays – An Overview Counting cells using dye exclusion methods (such as the Trypan Blue) is simple and quick but does require the cells to be in a single cell suspension before counting them2. These have been developed on the assumption that living cells have intact cell membranes, which will exclude the dye while dying/dead cells will uptake the dye due to impaired membrane integrity.

All the options that have been described so far are endpoint assays, where the cells need to be killed to obtain a readout.

Confluency from time-lapsed live cell imaging is a non-invasive alternative for these methods6. By imaging cells at several time points, the viability can be monitored over time without the use of toxic reagents or sacrificing samples, thus decreasing the overall amount of samples required. Another benefit of label-free methods over conventional cell viability assays is that it is quicker and cost-effective because it does not require several stages of sample preparation. It also allows cell cultures to have a controlled environment, where temperature, CO2 and O2 levels are controlled.

The most frequently used method is live cell fluorescent microscopy, which allows researchers to follow the location of different fluorescent proteins in cells7. A fluorescent reporter is introduced in the cell line and helps the research take mechanisms and areas of interest. However, the occurrence of phototoxicity is a common issue during fluorescent live cell imaging. Phototoxicity leads to cellular damage during illumination, and this affects sample physiology and possibly leads to cell death7. Reducing phototoxicity is vital to obtain reproducible qualitative and quantitative data, although it cannot be completely avoided.

Depending on the assay, fluorescence may not be required so the less phototoxic option of brightfield imaging can fit the researchers’ needs10. Monitoring cell viability with real-time live cell imaging not only provides quantitative data on confluency but also qualitative information about the cell morphology. Morphological changes present extra information about the impact of a certain compound on the cells that is

This invasive technique is an endpoint assay because the dye is extremely toxic for mammalian cells, making it unsuitable for long-term monitoring. A possible method to enable long-term viability monitoring would be to repeat counts with different samples at different time points, but it would add cost for required amount of cells, disposables and reagents. Moreover, this method is tedious since the samples need to be counted one at a time.

Colorimetric assays are centred on a biochemical marker demonstrating metabolic activity of the cells. The well-known MTT3 and resazurin (Alamar Blue) assays use metabolic activity as a measure for cell viability4. A compound of a specified colour is enzymatically transformed into a compound of a different colour by metabolically active cells. The level of absorbance of the converted compound is quantified to be used as a measure for cell viability. The advantage of metabolic activity assays over live/dead staining is that multiple samples can be analysed at once with the use of a plate reader. However, again most of the reagents are toxic, making it unfit for live cells5.

Furthermore, a plateau in the absorbance might be reached when the compound is completely transformed.

28 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Images of cell viability of C6 cells after 24 hours with different concentrations of Paclitaxel, a miotic spindle inhibitor.

Spring 2021 Volume 4 Issue 1

Pre-Clinical & Clinical Research

Phenotypes indicative of cell viability.

3.

Normalised cell confluency of C6 cells over time with different concentrations of Paclitaxel, a miotic spindle inhibitor.

missing when using metabolic activity assays or cell counting8.

Viability can be observed and quantified using image analysis algorithms and provide information not only on viability but also on proliferation and cell fate decisions. Cell death is always accompanied by specific morphological characteristics and they can be observed with a simple brightfield video system.

Cells can demonstrate abnormal morphology such as shrinking, blebbing, and detachment from the substrate20. A time-lapse approach can help to pinpoint not only if, but also when cells start to go into apoptosis.

Parameters like confluency, area infiltration, and colony growth can be used to generate quantitative data. This extra data and insight can also help researchers throughout the whole testing process. Cells that exhibit abnormal morphology can be signs of sickness or defects and should not be used for experiments10 It can also be critical for more difficult to culture cells such as stem cell cultures9.

Ansar Ahmed S, Gogal RM & Walsh JE, A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay.

Journal Immunology Methods, vol. 170, no. 2, 211–224, 1994.

4. Riss TL et al., Cell Viability Assays, in Assay Guidance Manual. vol. 114, no. 8, Sittampalam K, Coussens GS, Brimacombe NP, Ed. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences, 785–796, 2013.

5. Yang Fet al.Real-time, label-free monitoring of cell viability based on cell adhesion measurements with an atomic force microscope.Journal of Nanobiotechnology, vol.15, 23, 2017.

6. Pope I, Langbein W, Watson P & Borri P, Simultaneous hyperspectral differential CARS, TPF and SHG microscopy with a single 5fs Ti:Sa laser. Optics Express, 21, 7096-7106, 2013.

7. Icha J, Weber M, Waters JC & Norden C, Phototoxicity in live fluorescence microscopy, and how to avoid it. BioEssays, vol. 39, 1700003, 2017.

8. Aftab O, Fryknäs M, Hammerling U, Larsson R & Gustafsson MG, Detection of cell aggregation and altered cell viability by automated label-free video microscopy: a promising alternative to endpoint viability assays in high-throughput screening.Journal Biomolecular Screenvol. 20, 372-381, 2015.

9. Gómez-Villafuertes R et al. Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations. Journal of Visualized Experiments, 56291, 2017.

10. Jensen EC, Overview of live cell imaging: requirements and methods used. Anat. Rec.296(1), 1–8 (2013)

Cell viability assays for cell cultures are strong and trusted methods in life sciences. While compound-based assays such as the MTT assay are well known and trusted, these often lack kinetic insight. As researchers have discovered the acute effects compounds have on cell viability, a natural turn toward investigation of long-term effects is now of interest. Novel methods, such as live cell imaging, are presenting themselves as viable alternatives or enhancing results in tandem with traditional methods.

Over the last couple of decades, significant advances have been made in live cell imaging. The added data allows for a deeper understanding of cellular processes and behaviour.

Although many of the devices give the opportunity for kinetic monitoring, they have not overcome all the obstacles faced in maintaining an ideal environment for cell growth. As technology advances, the devices have become smaller and less disruptive to cell cultures. Continual advances in imaging techniques and design of fluorescent probes improve the power of this approach, ensuring that live-cell imaging will continue to be an important tool in biology.

REFERENCES 1.

2.

Stoddart MJ, Mammalian Cell Viability. vol. 740, pp. 1–6, 2011.

Mosmann T, Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays, Journal Immunology Methods, vol. 65, no. 1–2, 55–63, 1983.

www.biopharmaceuticalmedia.com

Kendra Majewski Kendra is a technical sales specialist for CytoSMART Technologies. She has obtained her BSc in zoology from Michigan State University and her MSc in water science from University Duisburg Essen.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 29

Pre-Clinical & Clinical Research

Unlocking the Biomarker Revolution in Clinical Trials Innovative, Fast and Cost-effective Diagnostics are Essential if the Long-awaited Era of Personalised Medicine is Finally to be Delivered.

Completion of the first human genome sequence exactly 20 years ago opened up the tantalising prospect of a new era of precision treatments, with accurate genetic diagnosis enabling therapies to be targeted to smaller subsets of patients, increasing the chances of success and reducing side-effects.

Although the concept was an exciting one, it met considerable scepticism in the pharmaceutical industry, which had built its success around one-size-fits-all blockbusters. The ability to identify the molecular pathways underlying a patient’s cancer, or the specific strains and resistance genes present in infectious disease, was both revolutionary and daunting.

Understanding of human molecular diversity was then relatively limited. Embracing the change would require a whole new approach to drug pricing, including charging more for drugs targeting smaller populations, and had enormous implications for the conduct of clinical trials. It also required a sea change in understanding the real value of diagnostics and their absolutely critical role in delivering the benefits of biomarkers in healthcare.

One Size Does Not Fit All Two decades later, we can see that the industry, regulators and healthcare providers have clearly adapted to this new landscape. Already, more than 40 FDA-approved therapies require a companion diagnostic to gauge the suitability of an individual for a particular drug. However, there is still much more to do to unlock the full potential of biomarkers in driving precision therapies. While there have been enormous advances in our understanding of disease, the full benefits continue to be limited by the availability of the sensitive, rapid and cost-effective diagnostic testing required to take full advantage of the transformation in our understanding of biomarkers.

Research published recently by Trends in Molecular Medicine shows the scale of failure of the across-the-board approach to developing new therapies. It found on average that 50% of patients with arthritis did not respond to a particular drug in a class. This failure rate rose to 70% for Alzheimer’s patients, and 75% for cancer patients.

The dramatic impact of biomarkers on tailoring medicines to the people most likely to respond is seen with two of the most successful immuno-oncology drugs of recent years, pembrolizumab (Keytruda) and lenalidomide (Revlimid). When prescribed outside the guideline-recommended markers, they help only one in six people and one in three people, respectively. The opportunity that biomarkers offer to identify abnormal pathways that can be targeted with immunology 30 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

or molecular therapies before serious disease progression is therefore truly ground-breaking.

Rapid Growth Led By Oncology Biomarkers are now used at every stage of drug development.

This includes recruitment and stratification of patients in trials based on the presence of specific biomarkers, through to dose selection and the assessment of safety, efficacy and performance. Biomarkers enable companies to focus their time and resources on the most promising therapies with the best efficacy and safety profiles, reducing the risk of financial losses.

Oncology is leading the charge, with 21 new molecular entity approvals in the US in 2020, accounting for 37% of all drugs approved, up from 22% in 2014.

Identification of biomarkers has become critical to the process of developing precision drugs, making clinical trial design, execution and data analysis much more complex. The Personalized Medicine Coalition (PMC) found 55% of oncology clinical trials involved use of biomarkers in 2018 compared with just 15% in 2000 – a CAGR of 17%. Of these, breast (69%), lung (66%), leukaemia (64%), lymphoma (57%), melanoma (74%) and prostate (86%) were the most common tumours where biomarkers were being explored.

Trial strategy has also become more challenging, with more than 50% of biomarker trials now examining two or more biomarkers per trial. PD-1/PD-L1/2, ALK, TIL, CD4, MRD, and BRAF have all grown rapidly as a focus of interest in recent years.

Exploiting Biomarkers to Maximise Success in Drug Development Companies are understandably eager to use biomarkers to increase the chances of success and minimise failure.

Researchers at the London School of Economics estimated last year that the median cost of developing a new drug was $985 million. The LSE found oncology and immuno-modulatory drugs were the most expensive to develop of all, coming in at a median of $2.8 billion.

Not surprisingly, analysis by GlobalData found the most significant use of biomarkers (63%) in newly initiated oncology trials was to monitor treatment response and therapeutic efficacy to minimise risk of failure. Nearly 23% of trials used biomarkers in the trial inclusion process to identify trial participants who were most likely to respond. Biomarkers also allowed researchers to monitor treatment safety, enabling dosing to be adjusted or treatment halted to prevent toxicity becoming problematic.

Choosing one example, treatment for lung cancer has changed dramatically over the past 30 years, moving from platinum-based chemotherapy as standard of care, regardless of histology, in the early 1990s to the current age of biomarkerSpring 2021 Volume 4 Issue 1

Pre-Clinical & Clinical Research driven therapy. Clinical trials have evolved in line with this shift, leading to novel approaches that shorten the development process and allow evaluation of multiple patient cohorts.

Huge progress in the last decade in the identification of genetic mutations associated with various histologic subtypes of NSCLC means lung cancer is no longer treated as a single disease, with treatment options targeting specific genetic aberrations. As a result, patients are classified in ever smaller subgroups and treatments customised to their specific mutation. Traditional standard Phase I to III studies are no longer appropriate in an era of adaptive trial designs, where new knowledge is continually incorporated into the study process.

Efficient and flexible clinical trial designs are essential to keep up with the fast pace of biomarker discovery and therapy development, especially as combination therapy becomes increasingly common. Study designs also need to take account of biomarkers which continuously adapt to treatment, and incorporate strategies to address resistance to therapy, where sequential screening of biomarkers can be built into the trial design to allow for different treatments in response to changes in the biomarkers.

The Need for Faster, More Cost-effective Diagnostics Technologies While biomarker inclusion criteria can substantially increase the chances of trial success, they can also result in significant additional costs. The use of genetic markers requires accurate diagnostic tests that can be applied to screen patients for trial eligibility. This is typically carried out through tissue biopsy, an invasive and expensive procedure which cannot be undertaken by all patients, thereby reducing the number of patients enrolled. Where tissue samples can be obtained, the time lag between pathological confirmation of disease and biomarker testing often results in patients being put on therapy prior to trial inclusion, meaning they may become ineligible or that a 'wash-out' period with no treatment for up to four weeks can be required.

Delayed and reduced enrolment adds significant time and cost to already lengthy trials. Liquid biopsy – the testing for biomarkers directly from a patient’s blood – has been shown to offer significant reductions in average enrolment time. A recent trial in gastrointestinal cancer, for example, demonstrated a reduction from 33 to 11 days, more than doubling enrolment rates with no reduction in treatment efficacy.

Improvements in trial enrolment on this scale are compelling, but the diagnostic tests required to facilitate them can be expensive and technically challenging. Genetic markers are often a single base change in genomic sequence and may be present at fractions as low as 0.1% of the healthy background DNA in patient samples, making detection difficult. PCR-based diagnostics typically have a limit of detection of 2-5%, while the error rates of standard next generation sequencing (NGS) limit sensitivity to around 1%.

More complex NGS workflows and bioinformatics are now available as commercial diagnostics, and have successfully demonstrated limits of detection of 0.1% or less. However, the complexity and associated costs are prohibitively high for use in regular patient monitoring, and tests are only offered at a few specialist laboratories with the necessary infrastructure www.biopharmaceuticalmedia.com

and highly trained staff. The result is a cost per test of as much as $3–5,000. With many patients needing to be tested for each successful enrolment, this is a substantial burden to bear, and the cost of repeat testing for acquired genetic changes during treatment can be prohibitive.

NGS has proven to be a powerful tool in translational oncology, enabling the discovery of molecular mechanisms across a host of diseases. However, once these mechanisms and markers are established, using NGS as a routine test is like delivering a few Amazon parcels in a private jet. Solutions are needed which enable further reductions in enrolment time while dramatically lowering the cost and complexity of screening and monitoring of patients.

Substantial efforts are underway to make this vision a reality, with multiple approaches in development. Many of these require significant investment in new instrumentation, which is likely to lead to slow adoption. Genuinely broad and cost-effective access to precision genetic testing for trial enrolment for the critical biomarkers of interest will require further advances in molecular diagnostics that enable ultra-sensitive detection across large panels of markers to be delivered directly at trial enrolment sites using existing instrumentation and IT infrastructure.

The Future of Clinical Trial Enrolment The genomics era has caused a scientific upheaval which is transforming our understanding of the molecular mechanisms of disease. It’s leading to extraordinary advances in new therapies and improvements in patient care. The challenge now is that as therapies become increasingly targeted, the number of patients for which they are effective decreases, significantly driving up costs and extending the timescales of clinical trials.

To maximise the speed and success of clinical trials for targeted therapies, researchers need new approaches which enable the rapid and precise enrolment of patients without drastically increasing costs. Fortunately, the biomarker revolution is inspiring the development of new techniques and technologies that promise fast, accurate and on-the-spot testing of patients at clinical trial enrolment sites, from both liquid and tissue samples, at costs similar to traditional PCR-based tests.

This is the next great breakthrough that will accelerate the translation of the biomarker revolution into new, precisiontargeted therapies, bringing real benefits for patients and finally ushering in the long-awaited era of personalised medicine.

Dr. Barnaby Balmforth Dr. Barnaby Balmforth is CEO of Biofidelity Ltd, a private cancer diagnostics company based in Cambridge, UK. Biofidelity has developed ASPYRE, a revolutionary approach to genetic testing which enables ultra-sensitive, rapid diagnosis from tissue or plasma to be carried out in all laboratories using existing instrumentation and IT. Barnaby has more than 10 years’ experience in DNA sequencing technology development, including as Chief Operating Officer of Base4 before leading the spinoff of Biofidelity in 2019. He holds a PhD in Quantum Optics from the University of Cambridge and a Masters in Physics from the University of Oxford.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 31

Manufacturing/Technology Platforms

PEER REVIEWED

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, 32 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 Ready-to-use, cGMP 1x buffers Spring 2021 Volume 4 Issue 1

Manufacturing/Technology Platforms 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

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.biopharmaceuticalmedia.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 33

Manufacturing/Technology Platforms 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.

5.

6.

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.

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.

34 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Spring 2021 Volume 4 Issue 1

INSIGHT / KNOWLEDGE / FORESIGHT

SUPER PUBLICATIONS FOR SUPER PHARMACEUTICALS IPI

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

www.ipimediaworld.com

JCS

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

PHARMA’S DNA

www.jforcs.com

Listen to industry experts on the latest in drug discovery, development, research, industry regulations and much more at Pharma,s DNA, the podcast channel by Pharma Publications, available on Sound Cloud, Spotify, iTunes and YouTube.

IAHJ

IBI

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

www.animalhealthmedia.com

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

www.biopharmaceuticalmedia.com www.biopharmaceuticalmedia.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 35

Manufacturing/Technology Platforms

PEER REVIEWED

High-throughput Sequencing Technologies are Revolutionising Antibody Discovery Therapeutics based on antibodies (Abs) are at the forefront of revolutionary treatments for cancer, autoimmunity and many other diseases. The application of high-throughput sequencing (HTS) in monoclonal Ab (mAb) screening and selection is rapidly transforming the discovery process. By tightly integrating sequences with lab measurements, mAb candidates can be analysed in greater numbers and greater detail than ever before. Methods such as phylogenetic analysis allow researchers to better understand the differences between Abs based on their sequences and to rationally select the most promising candidates. Advanced modelling strategies are in their infancy but hold great potential for avoidance of developmental liabilities. In this article, we discuss the impact of HTS and immunoinformatics on the accuracy and speed of Ab discovery workflows, and how these technologies are revolutionising the Ab development field.

Antibody Therapeutics on the Rise mAbs have emerged as a major class of therapeutics for immunological infectious diseases and cancer. mAb development has proven to yield effective drug molecules, with currently over 500 clinical studies ongoing. The success rate for these studies between Phase I and approval is 17–25%1, which is double the general average (9.6% between 2006 and 20152). Overall, the number of available therapeutic mAbs for clinical use has steadily increased since the first approval in 1986 (Figure. 1), and had already managed to seize almost half of the Top 20 U.S. therapeutic biologicals sales back in 20073.

The success of mAbs on the therapeutic market has driven scientific and technological discovery. Recently, two Nobel prizes were awarded to research directly involving Ab discovery; one covers expansion of the discovery toolbox with phage display4;

the other highlights immune checkpoints, its most prominent therapeutic application5. Our improved understanding of the immune system and the use of novel technologies allow better Abs to be developed in a shorter amount of time. In this article, we will describe how HTS technologies are revolutionising the Ab discovery workflows and enable researchers to efficiently harness Ab characteristics for the development of sensitive and specific clinical impactful therapeutics.

Why Antibodies?

Diversity – Abs are incredibly diverse molecules. While most cellular receptors are hard-coded in the genome, Ab chains are generated by a semi-random recombination of gene segments and the addition of non-templated nucleotides, creating a huge variety of Ab. Mammal Abs are often formed by a dimer of two recombined Ab chains encoded in separate chromosomes, which together form the antigen-binding site. This creates truly unique binding sites, but also means that the full receptor cannot be sequenced together without creative molecular biology steps to link different mRNA products. The whole process has the capability to generate 1011 Abs with distinct specificities. This diversity contains Abs for an extremely wide range of targets.

Finding a candidate among this large diversity thus becomes the objective and can be performed by screening for functionality (binding) and Ab sequence analysis.

Specificity – Abs can accumulate mutations that increase their binding affinity. Upon antigen binding, the B cell will become activated and will undergo clonal expansion to form a group of cells, called a clonotype. These cells can acquire mutations through somatic hypermutation (SHM), which further improves the affinity for the target (affinity maturation), leading to a class of extremely selective binders for any given antigen.

Versatility – Natural Ab molecules possess various relevant effector functions such as engaging the complement system

Figure 1. FDA-approved therapeutical mAbs per year, grouped by the number of approvals per time period (blue bars) and cumulative including that period (orange bars).

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Manufacturing/Technology Platforms (ADCC) or Fc receptors, which takes their applications beyond mere target blocking. These effector functions can be further modified and refined by genetic engineering. Abs from different animal species have different properties or functionality advantages (e.g., nanobodies, IgNAR), and through bioengineering, Abs with multiple specificities (bispecific Ab) or linked to drugs (Ab-drug conjugates) can be constructed.

All-in-all, natural Abs are selected by the immune system to optimally bind to any antigen with strong affinity and high specificity and possess versatile effector functions. Our ability to select and engineer Abs with the right characteristics makes the B cell repertoire a perfect pool to fish for high-potential candidates to construct improved therapeutics.

The Different Roads to Rome A variety of different Ab discovery platforms are in use today, but all include a selection process from thousands or even millions of distinct antibodies, to the one that is best for the job. Ab discovery workflows still rely heavily on in vitro (biochemical) screenings of Ab candidates6. Unfortunately, although considerable improvements have been made in the throughput of these techniques, the rate of discovery is limited by the biochemical or binding phase of the workflow. Moreover, due to inevitable technical limitations, good candidates can be lost along the way and flawed candidates may only be dropped at a late stage.

Sequencing information captured throughout the process has shown to mitigate some of these issues and is therefore playing an increasingly important role in all discovery workflows6.

Sequence Data is Key There are several sequencing technologies available to elucidate adaptive immune receptors (Table 1). Sanger sequencing is often standardly performed in Ab discovery workflows for candidate validation purposes: the experiment start-up costs are low, and the read length is sufficient for almost any therapeutic construct (up to 1000 bp). Unfortunately, the throughput of this technology is very low compared to the natural Ab diversity. HTS technologies provide an unparalleled level of depth that, for the first time, allows for the interrogation of millions or receptor sequences.

This broad candidate space, which can now be captured and interrogated, is revolutionising the Ab discovery workflows and paves the way for in silico prediction and prioritisation, reducing the amount of necessary time-consuming binding selection. Each discovery platform faces its own technical hurdles and leverages HTS data in a unique way to improve the discovery process (Figure 2).

Hybridoma – By fusing plasma cells with myeloma cells,

Throughput Display libraries

B-cells

10

6

10

5

10

4

10

3

10

2

Single-Cell HTS

Bulk HTS

Hybridomas

Sanger

Developable candidates

Enrichment Selection

Figure 2. Schematic overview of the Ab discovery ‘funnelling’ process, starting from a throughput (y-axis) of millions, and progressively selecting candidates to thousands (single B-cell) or hundreds (hybridoma) of different Ab variants. After the validation of selected candidates, large-scale NGS data can be mined to expand the candidate pool (dotted flows upward), either to enrich the set of potential candidates, or to find Abs with better characteristics or without known sequence liabilities.

immortalised B cell lines (hybridomas) can be created to stably produce mAbs7. This workflow is labour-intensive and typically only hundreds of distinct receptor variants can be tested for binding, leaving a large part of the candidate space unexplored8,9.

HTS is increasingly applied to expand the candidate output (so-called ‘hit expansion’). Millions of receptor sequences can be identified through HTS and clustered together with the small set of validated binders (typically Sanger sequences). This enlarged and diversified candidate scope not only increases the chance of success but can even improve the quality of the final selected candidate, leveraging naturally occurring affinity maturation.

Display – The discovery of phage display technologies10 revolutionised the way in which we can functionally screen candidates. The display of Ab fragments in phages and selection with panning rounds allows phenotypically screening up to 10 million distinct receptor variants in a single experiment. Display workflows can make use of HTS data for quality control (e.g., to ensure library diversity) and to analyse clone enrichment profiles throughout panning rounds. Analysis of the fold increase in clone abundance, rather than solely cloning abundant candidates from final panning output, expands the candidate pool with more diverse binding profiles.

TECHNOLOGY

CHAINS

THROUGHPUT

DEPTH

READ LENGTH*

RELATIVE COST**

Sanger

Single chain

Low/medium

Shallow (hundreds)

1000 bp

Low

Bulk

Single chain

High

Deep (millions)

600 bp

Medium

Single-cell

Paired chains

Low

Average (thousands)

250 bp

High

Table 1. Overview of sequencing technologies to characterise the Ab repertoire. A comparison of the three commonly applied sequencing technologies based on features that guide the decision process, including sequencing depth, chain information, read length, and cost. * Most common commercially available length for Rep-Seq. ** Cost per sample, not base pair. www.biopharmaceuticalmedia.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 37

Manufacturing/Technology Platforms Single-B cell – Single-B cell workflows are based on the direct amplification of VH- and VL-region-encoding genes from single human B cells and their subsequent expression in cell culture systems11. The identification of paired heavy and light chains is frequently done with HTS technologies, sometimes accompanied by transcriptome analysis, and typically yields thousands of distinct Ab variants12. As for hybridoma construction, HTS can be applied on the original (unsorted) B cell pool to enlarge the candidate scope and to estimate the clone abundance more accurately, indicative of antigen binding.

A Use Case: Finding Better Antibody Candidates in High-throughput Sequencing Data A major bottleneck in some Ab discovery platforms is the expression and experimental testing of candidates. Even with cutting-edge isolation, expression and characterisation methods, the process is lengthy and yields less than 500 candidates, which is the tip of the iceberg considering the extremely diverse Ab repertoire typically generated against any antigen. Paired to this, affinity-based sorting methods are not perfect and can fail to select the best Abs. HTS offers the possibility to expand the candidate list by finding clonal relatives of the characterised Abs in a larger repertoire pool. For example, Phad et al. applied HTS of Abs to identify clonal relatives of single B cell candidates in different B cell compartments after immunisation13. These cells had undergone affinity maturation and had accumulated lots of mutations, which strongly correlated with neutralisation potency.

This demonstrates how the discovery process can be improved with HTS by specifically identifying alternative Ab sequences with better characteristics, reducing time and cost and improving the Ab properties. This concept of ‘target enrichment’ is applied in all of the different discovery workflows, including hybridoma and phage display.

A Use Case: Expanding and Diversifying the Antibody Candidates in Phage Display Phage display panning is a fast way to phenotypically select Ab candidates. Unfortunately, the calibration of the panning process to obtain a constant number of reliable candidates can be quite complex and may give uneven results. Panning is usually performed in sequential rounds, with amplifications in between to select for the stronger binders in a stepwise manner.

Without sequencing candidates at intermediate steps, the panning process is a black box, the results of which can only be measured at the end. By using HTS in between the panning steps, the selected clones can be tracked and their expansion can be measured, which correlates with binding strength14. Moreover, the information obtained from HTS allows for a highly stringent final panning round to select the best candidate; if the final panning output is meagre, this information can be used to revisit previous panning rounds in a targeted manner.

Analytical Discovery Challenges The analysis of Ab sequences has proven to be highly effective at improving selection and production of mAbs. However, the correct annotation of immune receptors, the interpretation of large-scale repertoires and the integration of different datatypes can be challenging.

Correctly Identifying Ab Sequences The processing of Ab sequencing data requires specific techniques that are related to the peculiarities of Ab sequences.

38 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

In a blood sample, the large diversity of Abs means that, on average, each one is represented by a single sequence in the data. This sequence contains segments from two to three highly polymorphic genes, and can, additionally, have a high proportion (>30%) of mutations. Sequencing data processing needs to take these factors into account to obtain the correct Ab sequences.

Interpreting Large-scale Ab Repertoires Ab repertoires provide a wealth of information such as gene usage, sequence diversity and mutational load. The analysis and visualisation of these large datasets with millions of sequences for Ab development purposes (i.e., the identification of Abs with desired characteristics) has been shown to be challenging.

A very common step in discovery workflows is the clustering of sequences in clonotypes or groups that (presumably) share properties such as binding affinity15. Given the volume of HTS data, this easily turns into an analysis bottleneck. Subsequently, the mutations in the clusters often are analysed to trace the sequence history from VDJ recombination to a set of Abs (‘phylogenetic analysis’ or ‘lineage tracing’). Among others, this allows researchers to select Ab variants with similar binding characteristics, but different developability traits. The technical expertise required to perform phylogenetic analysis is substantial, hampering its use in day-to-day discovery workflows.

Leveraging Sequence Data for Ab Engineering mAb development is partly about the selection of Ab candidates, but also involves Ab engineering to obtain or enhance desired properties. Traditionally, mAb optimisation is done by custom, experience, or (if all else fails) trial-and-error. Fortunately, the field is moving towards a more efficient strategy that is based on risk mitigation and rational design. Based solely on sequences, computational techniques can pinpoint likely origins of undesired properties (so-called sequence liabilities) and suggest engineering strategies for mitigation16. A great example is the use of public sequence databases to statistically assess the likelihood of liabilities associated with specific amino acid residues at certain positions in the Ab sequences. Using such models to analyse novel Ab candidates can prevent stability and developability issues. Moreover, promising work on sequencebased predictions of specific properties (e.g., ‘humanness’ of a sequence), has led to Ab sequences being altered ‘rationally’ in order to gain the most optimal sequences. This field is still moving rapidly and comes with some challenging tasks such as database mining and structural modelling.

Data Integration to Create a Complete Picture The final challenge of Ab development is to bring all this information together such that a high-throughput analysis approach is possible. Linking existing and new data (e.g., Sanger and HTS) with new algorithms and methods will enable new insights to be gained. As new measurements and assay data (affinity, specificity for target, etc.) are added to quantify various aspects of Ab suitability, data analysis solutions should grow with the data to enable new insights.

The Right Toolset for the Job Bioinformatics software solutions aim to reduce the time spent on data processing and simplify the access to valuable analysis methods and algorithms. This allows researchers without extensive bioinformatics expertise to independently Spring 2021 Volume 4 Issue 1

Manufacturing/Technology Platforms

perform their discovery analysis and keeps them focused on what matters the most – the science. Academic researchers have created scripts and pipelines for Ab analysis, but these frequently have down-sides such as command-line interfaces, limited scope and/or usability, and sometimes sub-standard maintenance and validation approaches. Several commercial solutions exist for HTS data management and analysis, but these solutions are often generic and fail to take into consideration specific pitfalls related to Ab sequences.

ENPICOM and others have developed data analysis solutions specifically tailored to the peculiarities of Ab sequence data.

These comprehensive platforms sometimes incorporate (academic) point-solutions, in this way combining the best of both worlds: cutting-edge scientific methods and end-to-end robust solutions with a user-friendly interface. The integration of high-throughput screening and sequencing has proven very powerful for the rapid discovery of novel therapeutic Abs. Companies that leverage the full potential of HTS technologies and innovative immuno-informatic solutions will be ahead in this fast-growing field of therapeutic applications.

Elsevier; 2011. p. 453–7.

12. Mcdaniel JR, DeKosky BJ, Tanno H, Ellington AD, Georgiou G. Ultra-highthroughput sequencing of the immune receptor repertoire from millions of lymphocytes. Nat Protoc. 2016 Mar 1;11(3):429–42.

13. Phad GE, Pushparaj P, Tran K, Dubrovskaya V, Àdori M, Martinez-Murillo P, et al. Extensive dissemination and intraclonal maturation of HIV Env vaccine-induced B cell responses. J Exp Med. 2020;217(2).

14. Yang W, Yoon A, Lee S, Kim S, Han J, Chung J. Next-generation sequencing enables the discovery of more diverse positive clones from a phage-displayed antibody library. Exp Mol Med [Internet]. 2017 Mar 3 [cited 2021 Jan 11];49(3):308. Available from: www.nature.com/emm 15. Galson JD, Trück J, Clutterbuck EA, Fowler A, Cerundolo V, Pollard AJ, et al. B-cell repertoire dynamics after sequential hepatitis B vaccination and evidence for cross-reactive B-cell activation. Genome Med [Internet]. 2016 Jun 16 [cited 2021 Jan 14];8(1):68. Available from: http://genomemedicine.biomedcentral.com/articles/10.1186/ s13073-016-0322-z 16. Seeliger D, Schulz P, Litzenburger T, Spitz J, Hoerer S, Blech M, et al. Boosting antibody developability through rational sequence optimization. MAbs [Internet]. 2015 Jan 1 [cited 2021 Jan 11];7(3):505–15. Available from: http://www.tandfonline.com/doi/ full/10.1080/19420862.2015.1017695

Néstor Vázquez Bernat

REFERENCES

As an Application Specialist at ENPICOM, Néstor focuses on analyzing customer requirements, and project setup, management, and execution. During his PhD in Immunology, he isolated and expressed monoclonal antibodies after vaccinations and developed high-throughput sequencing library preparation protocols for B cell repertoires in humans, non-human primates, and other animal models.

1.

Email: n.vazquez@enpicom.com

Kaplon H, Reichert JM. Antibodies to watch in 2019. MAbs [Internet].

2019 Feb 17 [cited 2020 Dec 27];11(2):219–38. Available from: /pmc/ articles/PMC6380461/?report=abstract 2. BIO, Biomedtracker, Amplion. Clinical Development Success Rates 2006-2015 - Pharma intelligence. 2016.

3. Scolnik PA. mAbs: a business perspective. MAbs [Internet]. 2009/03/21.

2009;1(2):179–84. Available from: https://pubmed.ncbi.nlm.nih. gov/20061824 4. The Nobel Prize in Chemistry 2018 - NobelPrize.org [Internet]. [cited 2020 Dec 27]. Available from: https://www.nobelprize.org/prizes/ chemistry/2018/summary/ 5. The Nobel Prize in Physiology or Medicine 2018 - NobelPrize. org [Internet]. [cited 2020 Aug 12]. Available from: https://www. nobelprize.org/prizes/medicine/2018/summary/ 6. Bailly M, Mieczkowski C, Juan V, Metwally E, Tomazela D, Baker J, et al. Predicting Antibody Developability Profiles Through Early Stage Discovery Screening. MAbs [Internet]. 2020 Jan 1 [cited 2021 Jan 11];12(1):1743053. Available from: https://www.tandfonline.com/ doi/full/10.1080/19420862.2020.1743053 7. Köhler G, Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature [Internet]. 1975 [cited 2020 Dec 27];256(5517):495–7. Available from: https://pubmed.ncbi.nlm. nih.gov/1172191/ 8. Mackenzie M, Cambrosio A, Keating P. The commercial application of a scientific discovery: The case of the hybridoma technique. Res Policy.

1988 Jun 1;17(3):155–70.

9. Juy D, Legrain P, Cazenave PA, Buttin G. A new rapid rosette-forming cell micromethod for the detection of antibody-synthesizing hybridomas.

J Immunol Methods. 1979 Nov 15;30(3):269–75.

10. McCafferty J, Griffiths AD, Winter G, Chiswell DJ. Phage antibodies: filamentous phage displaying antibody variable domains. Nature [Internet]. 1990 [cited 2020 Dec 27];348(6301):552–4. Available from: https://pubmed.ncbi.nlm.nih.gov/2247164/ 11. Tiller T. Single B cell antibody technologies. Vol. 28, New Biotechnology. www.biopharmaceuticalmedia.com

Josine oude Lohuis As a Product Manager at ENPICOM, Josine is leading the development of the new antibody discovery module (ADM). This includes conducting strategic field exploration studies, defining new specifications, and driving software development by leveraging her diverse bioinformatics background and deep knowledge of immune repertoire sequencing technologies.

Email: j.oudelohuis@enpicom.com

Henk-Jan van den Ham As a Senior Scientist at ENPICOM, Henk-Jan is part of the research team focusing on the latest developments in immunology & bioinformatics, with a focus on adaptive immune repertoire analysis. With a PhD in theoretical Immunology & bioinformatics and post-doc experience in virology, Henk-Jan leads academic & industry projects, collaborations and grant applications at the interface of immunology, bioinformatics and software engineering.

Email: h.j.vandenham@enpicom.com

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 39

Manufacturing/Technology Platforms

Battling Biohazardous Liquid Laboratory Waste “So, what happens with your biosafety level 3 lab waste?” I asked the laboratory manager. “Our safety assessment says to just mix it with sterilant wash it down the drain – there is worse stuff down there already.” “How about genetically modified material?” “Yeah, that goes the same way – It’ll die in the drains”.

volume of liquid waste has been amassed, it is mixed with a chemical sterilant in the kill tank and held until sterile7 (see Figure 1).

Treating biologically hazardous waste in such a way is not uncommon in the UK – the combination of chemical sterilants and a well-developed system of sewerage and treatment plants can handle a wide range of biologically active substances, but it is not a failsafe system.

The 2007 foot-and-mouth disease outbreak within the county of Surrey, UK, is believed to be the result of a damaged drainage pipe leading from a containment level 4 laboratory1.

The facility had been permitted to dispose of liquid waste containing small quantities of live foot-and-mouth virus. After chemical sterilisation in an effluent decontamination system (EDS), liquid waste from the laboratory’s experiments was dispatched to the drain. The laboratory decontamination showers – deemed less likely to contain the category 4 virus – emptied directly into the drain2.

The unsterilised wastewater from the decontamination showers may have been the point of egress for the virus. By following the UK’s Health and Safety Executive (HSE) guidelines, the laboratory would have taken a different approach: collecting all wastewater from sinks and showers and deactivating the biologically hazardous material contained within it before discharging to the sewer3. This process, recommended for containment level (CL) 4 facilities, is also in most instances a requirement, if not strongly recommended for CL3 environments across varied settings from animal research4 and genetically modified organism5 labs, to large-scale biotechnology plants6.

Wastewater from the decontamination showers could have been sterilised by handling it in the same way the laboratory’s liquid waste was; through sending it to the EDS. An EDS is a single-purpose device that sterilises wastewater and effluent. While designs vary, these systems usually fall into one of two categories: thermal EDS and chemical EDS. The thermal EDS uses heat to sterilise waste, while a chemical EDS – as used by the laboratory at the centre of the 2007 foot-and-mouth outbreak – employs chemical sterilants. The following article will review both methods with the aim of better understanding which method is more suited to the modern laboratory.

The Chemical Effluent Decontamination System The EDS used by the laboratory employed chemical sterilisation to destroy hazard group 4 pathogens. Typically, such forms of chemical EDS collect effluent either in a sterilisation tank (also known as a kill tank), or in a storage tank. Once an adequate 40 INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY

Figure 1. Schematic diagram of a chemical effluent decontamination system, (Adapted from “Figure 8. Schematic drawing of the effluent decontamination system.” – Vijayan, V. & Ng, B. (2016). Validating waste management equipment in an animal biosafety level 3 facility. Applied Biosafety, 21(4), 185-192. )

In a chemical EDS, composition of the effluent forms a key factor determining sterilisation time. Organic and particulate matter in the mixture can encapsulate biologically hazardous agents shielding them from chemical sterilants. Consequently, the larger the amalgamation of material, the lower the efficacy of the sterilisation. Mechanical maceration and blending of the effluent may reduce the shielding effect, but to be effective, the sterilant must come in contact with the pathogen8.

By contrasting the results of two studies, it is possible to see the effect of effluent composition upon sterilisation time in the chemical EDS. Using sodium hypochlorite (bleach) as a sterilant at a concentration of 700 parts per million (ppm), it was possible to sterilise a mixture of Bacillus subtilis and water in 30 minutes9.

However a two-hour sterilisation time was required when using a concentration of 5700ppm of bleach to deactivate bacillus spores in a mixture of animal effluent, humic acid, and foetal bovine serum10. While a more viscous and solid-rich effluent does provide a greater challenge for chemical sterilants, it is the organic material’s reaction with the sterilant that explains the requirement for a stronger concentration of sodium hypochlorite in the latter example. Chlorine from the bleach reacts with protein, forming iV-chloro compounds and reducing the amount of active sterilant available to deactivate microorganisms and viruses11,12. To counter this, increased quantities of sodium hypochlorite are required.

Concentration of sterilant and processing time is also dictated by the type of biologically hazardous agents in the wastewater. Higher resistance to chlorine-based disinfection is shown within some genera of bacteria, (including Mycobacterium, Bacillus, Legionella, Pseudomonas and Sphingomonas13) fungi (such as Aspergillus 14) and viruses. In the later instance, adaption to tolerate warmer waters been shown to result in a greater tolerance to chlorinebased disinfectants15.

Once the effluent is disinfected, the mixture requires pH neutralisation before it can be released into the sewer. This process sees acids and alkalis – for example hydrochloric acid Spring 2021 Volume 4 Issue 1

Manufacturing/Technology Platforms and sodium hydroxide – added incrementally to the effluent until a pH range acceptable for the specifications of the local water board is achieved9. Only then can the effluent be dispatched to the sewer system.

Once it has left the EDS, the effluent may contain residual chlorine which can be carried into the sewer system. In 2019 alone, UK water companies overflowed untreated sewage into rivers and streams in over 200,000 instances for a combined 1.5 million hours16. With as little as 100–300 μgL of residual chlorine17,18 shown to be toxic to aquatic life18,19, wastewater that has been chemically disinfected may create environmental damage even when free of pathogens.

While sodium hypochlorite is not the only chemical sterilant available, it is the most cost-effective – an important consideration for laboratories outputting large volumes of biohazardous liquids.

Yet all chemical sterilants come with drawbacks. By their nature they are toxic and reactive substances which require specialist and dedicated storage, especially when used on the scale of an EDS. They are also less effective against solid-rich effluent, have varying levels of efficacy on different pathogens, and can be difficult to validate7.

in their construction allows these units to handle increasingly complex effluent types. For instance, biohazardous liquids that are little more than water containing pathogens require the simplest of thermal batch EDS. Sterilisation of such effluent can be achieved with a sealable pressure vessel containing an internal heating element, which can heat the effluent to the correct temperature for long enough to ensure sterility.

Challenged with sterilising effluent containing more varied constituents including solids, a thermal batch EDS with a jacketed sterilisation tank provides an effective solution7 (See Figure 2).

The jacketed sterilisation tank is a vessel with hollow walls.

Effluent is either pumped into the tank, or flows into it from a source above using gravity. Once the tank is sufficiently full, valves isolate it, and high-temperature pressurised steam is passed through the cavity in the walls of the jacketed vessel. This raises the temperature of the effluence to sterilisation temperature and pressure for long enough to destroy all pathogens within the sterilisation tank7. Once sterilisation is completed, the tank is emptied through displacement with high-temperature pressurised steam.

The shortcomings of utilising chemical EDS were highlighted in the HSE’s Final Report after the 2007 foot-and-mouth outbreak, which recommended a review of the process, stating “It is our experience that chemical treatments, while reducing the amount of pathogen in the liquid, may not render the liquid completely pathogen-free”2.

The Thermal EDS Chemical sterilisation of biohazardous material is a process commonly used by laboratories, in scales ranging from a vial of liquid waste to a whole facility’s effluent. However, analysing the chemical EDS and how its use contributed to the 2007 foot-and-mouth outbreak highlights the problems inherent with the sterilisation method. This is not the only method of liquid waste sterilisation available to laboratories. A simpler, more comprehensive method that does not rely on hazardous consumables is available: heat sterilisation.

Heat sterilisation of biologically hazardous material is a commonplace process, with autoclaves becoming ubiquitous in laboratories since their invention in 187920. By heating material to between 121°C and 134°C in a pressurised environment for between three and fifteen minutes, autoclaves can destroy all biologically active material21. Adapting the autoclave process to the singular task of wastewater treatment has resulted in the thermal EDS, a device for sterilising effluent via heat.

The structure of a thermal EDS can be varied according to need. Facilities ceaselessly expelling wastewater free of solid material can accommodate a continuous flow EDS7 – a length of heated pipe hot enough for the effluent to maintain sterilisation temperature, and long enough so that the flowing liquid has sufficient time to sterilise. For laboratories with a more varied and variable output, a thermal batch EDS is more applicable.

All types of thermal batch EDS collect a specified quantity of effluent, then heat it to a sterilisation temperature for long enough to destroy any biologically hazardous materials. Differentiation www.biopharmaceuticalmedia.com

Figure 2: Schematic diagram of a thermal batch effluent decontamination system.

A thermal batch EDS with a jacket vessel offers certain advantages over other types of EDS. It is not hampered by effluent containing organic material, does not add additional contaminants to the wastewater, and doesn’t require any chemicals. It shares these attributes with all forms of thermal EDS. However, unlike continuous flow EDS and chemical EDS, the thermal batch EDS can sterilise solid material in effluent, is not prone to clogging, and can be easily validated during operation7.

With capacity to adjust sterilisation times and temperatures, treatment parameters can be varied, while variation in the number and type of tanks used can create systems capable of efficiently handling any effluent flow rate.

Would use of such a jacketed-vessel thermal batch EDS have mitigated the foot-and-mouth outbreak in 2007? Maybe so. The penetrative and highly effective capabilities of heat sterilisation would have greatly reduced the possibility of materials leaving the EDS unsterilised. With low running costs, running all liquid effluent from the lab – including the showers – to the thermal batch EDS would be a simple and cost-effective solution during the initial site build.

Furthermore, it’s worth noting that this approach would have provided a more environmentally-friendly solution for the laboratory that was also safer for staff. As mentioned, chemical sterilants and neutralisers are hazardous substances that require INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 41

Manufacturing/Technology Platforms specialist handling and storage. Not only does this increase site safety protocols to mitigate the potentially harmful effects of the sterilants on staff and the environment, but it also necessitates more infrastructure. Chemicals require frequent shipping to site by road or rail, alongside buildings in which to store them until required; both factors that give the chemical EDS a large carbon footprint.

8.

9.

In contrast, even the most advanced thermal EDS can receive all the resources it requires from national energy and water supply networks. With electricity flowing from the grid into the device, and water for steam generation plumbed in, no manual loading is required. Even rare examples of thermal EDS that use natural gas as an energy source can be supplied through pipelines. This reduction in manual interaction improves staff safety – a factor further enhanced as the only potentially harmful by-product of the thermal EDS is heat.

While not all laboratories may be working with CL4 pathogens, facilities handling biologically hazardous material of any grade can benefit from a thermal EDS. AstellBio, sister company to the autoclave manufacturer Astell Scientific, produce thermal EDS solutions for a variety of uses. These range from small-scale sink units able to provide mobile and packaged EDS functionality, to larger EDS units capable of handling a whole building’s effluent.

By installing an AstellBio Thermal EDS, you can be assured that all effluence leaving your facility is sterile – without the need for chemical sterilants or disinfectants.

10.

11.

12.

13.

14.

15.

16.

17.

18.

19.

Figure 3: A selection of thermal batch effluent decontamination systems.

20.

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2.

3.

4.

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6.

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National Research Council (2012). Biosecurity Challenges of the Global Expansion of High-Containment Biological Laboratories: Summary of a Workshop. Washington, DC: The National Academies Press. https://doi.org/10.17226/13315.

Health and Safety Executive (2007). Final report on potential breaches of biosecurity at the Pirbright site 2007. Health and Safety Executive. http://news.bbc.co.uk/1/shared/bsp/hi/ pdfs/07_09_07finalreporthsefandm.pdf Health and Safety Executive (2006). Biological agents The principles, design and operation of Containment Level 4 facilities.

Health and Safety Executive. https://www.hse.gov.uk/pubns/ web09.pdf Health and Safety Executive (2010).Working safely with research animals: Management of infection risks. Health and Safety Executive.

Health and Safety Executive (2014). The Genetically Modified Organisms (Contained Use) Regulations 2014. Health and Safety Executive.

Health and Safety Executive (2010).The large-scale contained use of biological agents. Health and Safety Executive.

Trembalay, Gilles, Langer-Curry, Rebecca, Chris, Kiley and

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Cory, Ziegler (2010). Effluent Decontamination Systems: Addressing the Challenges of Planning, Designing, Testing, and Validation. Applied Biosafety. 15 (3): 119–129. doi:10.1177/ 153567601001500304. S2CID 114865675. Retrieved 12 October2020) Winward, G. P., Avery, L. M., Stephenson, T. and Jefferson, B. (2008).

Chlorine disinfection of grey water for reuse: effect of organics and particles.Water research,42(1-2), 483-491.

Vijayan, V. and Ng, B. (2016). Validating waste management equipment in an animal biosafety level 3 facility. Applied Biosafety,21(4), 185-192.

Cote, C. K., Weidner, J. M., Klimko, C., Piper, A. E., Miller, J. A., Hunter, M., ... and Glass, P. J. (2020). Biological Validation of a Chemical Effluent Decontamination System. Applied Biosafety, 1535676020937967.

Van Bueren, J., Simpson, R. A., Salmax, H., Farrelly, H. D. and Cookson, B. D. (1995). Inactivation of HIV-1 by chemical disinfectants: sodium hypochlorite.Epidemiology & Infection,115(3), 567-579.

National Research Council. (1995). Preventing HIV transmission: the role of sterile needles and bleach.

Luo, L. W., Wu, Y. H., Yu, T., Wang, Y. H., Chen, G. Q., Tong, X. ... and Hu, H. Y. (2020). Evaluating method and potential risks of chlorineresistant bacteria (CRB): A review.Water Research, 116474.

Mattei, A. S., Madrid, I. M., Santin, R., Schuch, L. F. D. and Meireles, M. C. A. (2013). In vitro activity of disinfectants against Aspergillus spp.Brazilian Journal of Microbiology,44(2), 481-484.

Carratalà, A., Bachmann, V., Julian, T. R. and Kohn, T. (2020).

Adaptation of Human Enterovirus to Warm Environments Leads to Resistance against Chlorine Disinfection.Environmental Science & Technology,54(18), 11292-11300.

Laville, S. and McIntyre, N. (2020). Exclusive: water firms discharged raw sewage into England's rivers 200,000 times in 2019. Retrieved 30 January 2021, from https://www.theguardian. com/environment/2020/jul/01/water-firms-raw-sewage-englandrivers United States Environmental Protection Agency. (1999). Wastewater Technology Fact Sheet: Chlorine Disinfection.

Hassaballah, A. H., Bhatt, T., Nyitrai, J., Dai, N. and Sassoubre, L. (2020). Inactivation of E. coli, Enterococcus spp., somatic coliphage, and Cryptosporidium parvum in wastewater by peracetic acid (PAA), sodium hypochlorite, and combined PAA-ultraviolet disinfection.

Environmental Science: Water Research & Technology,6(1), 197209.

Stewart, A. J., Hill, W. R., Ham, K. D., Christensen, S. W. and Beauchamp, J. J. (1996). Chlorine dynamics and ambient toxicity in receiving streams.Ecological Applications,6(2), 458-471.

Durkan, R. Ozel, M. B. Bagis, B. and Usanmaz, A. (2008). Chronological reference marks-Charles Chamberland (1851-1908) Chronological reference marks-Charles Chamberland (1851-1908), 2007.Dental materials journal,27(4), 640-642.

Rogers, W. J. (2012). Steam and dry heat sterilization of biomaterials and medical devices. InSterilisation of Biomaterials and Medical Devices(pp. 20-55). Woodhead Publishing.

First published in International Labmate, Volume 46, Issue 2.

Gareth West Gareth West is the Marketing Executive at Astell Scientific Limited – a leading cross-sector manufacturer and supplier of laboratory, pharmaceutical and hospital Autoclaves/ Sterilizers, Steam Generators and Effluent Decontamination systems (EDS), with UK-wide Calibration and Validation Services. Established in 1884, Astell products can be found in use in over 100 countries around the world.

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INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 43

Regulatory/Quality Compliance

Using Electronic Pipetting and Automated Dispensing Technologies to Accelerate Sample Preparation for RT-PCR Applications Introduction RT-PCR (real-time, reverse transcriptase polymerase chain reaction) is a basic laboratory tool with a wide range of applications. Scientists use RT-PCR to quantify changes in gene expression, validate results from array analysis, and for drug discovery. More recently, RT-PCR became the most accurate way to test samples for the SARS-CoV-2 virus that is behind COVID-19.

Although scientists can perform RT-PCR in more than one way, the general process includes five basic steps: select the approach; prepare the sample; design the primer; remove any genomic DNA; and perform RT-PCR. At some stages, the use of integrated commercial kits simplifies and streamlines the process.

Getting a sample ready for RT-PCR requires considerable liquid handling that many scientists still perform manually.

This work involves the precise and accurate delivery of varying volumes of different liquids with varying characteristics, such as differences in viscosity. So, multiple dispensing steps are performed to prepare multi-well plates. As a result, the steps upstream of the actual RT-PCR process can be very time-consuming, labour-intensive, error-prone and inconsistent when performed manually. In addition, extensive manual pipetting can cause repetitive strain injuries. Nevertheless, some scientists often underestimate these important challenges.

Fortunately, advanced electronic pipetting and automated dispensing technologies can replace the manual liquid handling for RT-PCR experiments. More than just making RT-PCR easier, such upgrades produce more reliable results, and improve a user’s experience in comfort, thus reducing the likelihood of an injury.

Tracing the Steps The exact RT-PCR workflow depends on the selected approach, but some commonalities exist. Before extracting the nucleic acids, the samples must be placed in the desired labware, such as 96-well plates. However, the samples can come in different kinds of containers, such as plates or tubes, and it is burdensome to move them precisely and accurately into a multi-well plate.

In some protocols, the nucleic-acid extraction can include three wash plates, an elution plate and a sample plate. Wells in the sample plate will include several liquids in various volumes. In one protocol, for example, preparing the sample plate requires five liquids in volumes of 5–275 microlitres. So, even before reaching RT-PCR, as many as five plates might need to be prepared using as many as nine different liquids. Beyond the physical challenge – particularly with manual pipetting – scientists need to get the right volume of the correct liquid in the intended wells, all while minimising cross-contamination. This can be performed manually with pipettes or with automated liquid-handling platforms.

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Next, a scientist combines the purified/extracted sample with the RT-PCR reagents. Here, researchers work with reagents such as the RT-PCR master mix, the assay and nuclease-free water to make the reaction mix. This cocktail of chemicals goes into each well with the extracted sample. In addition to dealing with various liquids, they must be dispensed in small volumes, often two microlitres or less.

Throughout the entire process of preparing samples and running them through a RT-PCR instrument, the samples and reagents must be handled properly and transferred accurately from plate to plate. The steps must be performed consistently and without errors, and it’s challenging to meet those objectives with manual pipetting.

Given so many steps and requirements, it’s not surprising that setting up plates is the most time-consuming part of the RT-PCR process.

Manual Pipetting Challenges Despite the challenge of manual pipetting, it continues to be the ubiquitous method of delivering liquids for many reasons. A testing laboratory for COVID-19, for instance, receives samples in a collection of labware, such as test tubes and microcentrifuge tubes, which makes automation too complicated.

Some laboratories also continue to use manual pipetting in RT-PCR to reduce costs. In small laboratories that work with small numbers of samples, managers might resist the investment in the equipment, training, and maintenance of electronic or automated equipment. The flexibility of manual pipetting also appeals to some scientists and some might be resistant to change.

Still, every laboratory aims to be more efficient – doing more with less, and manual pipetting can have many inefficiencies.

It requires training new laboratory personnel, and ensuring the same technique is implemented consistently across all experiments to facilitate the delivery of accurate, reliable and reproducible results.

Even where manual pipetting works out, it is a workout.

In addition to being physically and mentally exhausting, making the same movement over and over causes repetitive strain injuries in laboratory personnel. That adds another cost associated with manual pipetting, in the form of reduced personnel productivity and medical treatment expenses. Plus, errors in manual pipetting, resulting from poor ergonomics or injury, increase a laboratory’s costs through the need to redo experiments, which requires using more reagents, samples and supplies.

Importantly, some of the conventional thinking around pipetting has created a preconceived notion that electronic pipettes are only required for high-throughput applications. That Spring 2021 Volume 4 Issue 1

Regulatory/Quality Compliance is not the case. Electronic pipetting enables the most consistent results, which benefits any laboratory. It also provides ergonomic benefits. Human interaction, by far, has the biggest impact on pipetting performance. Using electronicpipettingor automated dispensing technologies removes variance in dispensing and aspirating liquids, because these processes are automated and not adversely affected by the user. Consequently, electronic or automated methods help to improve liquid-handling results, while also having a positive impact on speed as well as overall employee satisfaction and health.

Electronic and Automated Options Many scientists claim that the key pain point associated with RT-PCR is pipetting – at least, when it is performed manually.

After that, designing the experiments and then making the required calculations are the most common complaints. Clearly, electronic and automated technologies resolve the first pain point, and some technologies even include pipetting programs that can be downloaded to an electronic pipette to eliminate the need for manual calculations. Making protocols available for download eliminates the need to learn how to program a new electronic pipette or the several kinds being used in a laboratory.

With electronic and automated technologies, aspirating, dispensing and multi-dispensing are much more consistent than manual options. The electronic approach really adds efficiency when, for example, aspirating 1000 microlitres and then dispensing 100 microlitres 10 times. In short, an electric motor drives a pipette piston more accurately and consistently than a scientist’s thumb.

Electronic and automated technologies can also be combined.

For example, some laboratories use an automated dispenser to set up a series of plates and then an electronic pipette to distribute the samples.

Simplifying the Switch The comparisons of using manual pipetting versus electronic and automated techniques for RT-PCR experiments show the latter’s many benefits, from accuracy and ergonomics to repeatability and throughput. In laboratories that still rely on manual pipetting, changing to more advanced methods can require actually seeing the difference. By witnessing the benefits enabled within laboratories that have made the transition to advanced liquid-handling technologies, more laboratories might move to these more efficient methods.

To smooth the transition from manual pipetting to electronic and automated dispensing methods, scientists can take relevant training to learn how to program a specific device. The transition can be further simplified by using equipment that allows for cloud-based downloads of protocols for custom workflows, such as those mentioned above. Overall, the upfront investment time in training and the right technology will save time in the laboratory for the life of the devices and beyond.

Conclusion Although many scientists once thought of pipetting as a standalone technology, no matter what the application, that is changing. Today, an increasing number of scientists see pipetting more holistically. That means thinking of a pipette as part of a laboratory’s overall workflow. www.biopharmaceuticalmedia.com

In RT-PCR, scientists want to improve pipetting by gaining greater efficiency, less human involvement upstream and downstream, and cost savings. To achieve that collection of improvements, scientists must include the pipetting technology in the design of an experiment. So, from the beginning of an experiment through data analysis, scientists should think about what pipetting solutions will solve problems, like efficiency and human error. In this way, liquid handling becomes integrated in the entire workflow, which makes it easier to develop standard operating procedures and create an audit trail.

Selecting the best liquid-handling devices for a laboratory depends on several key elements: the pipette’s features, as well as the manufacturer’s commitment to quality and service.

In addition, such a holistic perspective is likely to reveal that a laboratory will not optimise its efficiency with just one kind of pipette. Instead, different kinds of liquid handlers – from manual and electronic pipettes through to automated dispensers – can be combined to create the most effective and efficient tools for a specific task.

Overall, advanced electronic pipetting and automated dispensing technologies accelerate sample preparation for RT-PCR experiments, as well as accuracy and reliability of the generated results – ultimately, increasing laboratory throughput and productivity, all while improving the comfort and performance of laboratory personnel.

Tommy Bui Tommy Bui is Senior Manager of Business Development at the laboratory products division of Thermo Fisher Scientific focused on manual liquid handling solutions. With a BSME from the University of California, San Diego and an MBA from the University of California, Los Angeles, Tommy has over 20 years of experience in life science equipment and automation.

INTERNATIONAL BIOPHARMACEUTICAL INDUSTRY 45

Talking Point

Transforming Drug Discovery and Medicine Research An interview with Dr. Mark Kotter, CEO and Founder at bit.bio Q: Let us start with an overview of bit.bio. What are your key capabilities?

A: bit.bio is a synthetic biology company focussed on human cells.

Cells are of fundamental importance – they are the building blocks of life. Virtually all diseases are disturbances at the cellular level; replacing or augmenting dysfunctional or lost cells, their function will enable us to re-establish health.

We are at a critical time in biology and healthcare. There's one huge bottleneck to the next generation of cures: a robust, consistent and scalable source of human cells. If you remove this bottleneck, you have a huge positive impact across the whole healthcare industry, enabling widespread implementation of cell therapies, and potentially increasing the efficiency of drug development, while reducing the cost and time. bit.bio develops capabilities to produce every human cell type at scale. We believe this will transform the biotech and medicine research landscape and enable a new generation of cell therapies. Synthetic biology for us is essentially the transition of biology as we know it to engineering and that’s the approach we are taking to cells.

Q: bit.bio’s flagship technology is called opti-ox. Please tell us about this technology and what it does?

A: The identity and function of every cell is defined by which ‘genetic programs’ are active at any particular time. opti-ox is bit.bio’s breakthrough technology which, put simply, enables consistent reprogramming of cells based on the reliable activation of specific genes and these genetic programs. This is not trivial, as cells are able to detect foreign DNA and they silence it. opti-ox is a gene targeting strategy that overcomes this silencing and enables the reprogramming of the cell. It reliably engineers in and then activates specific transcription factors within the cells’ DNA, without them being silenced.

Transcription factors are key regulators that determine the activity of genetic programs in a cell.

If we activate the correct program using opti-ox, this results in deterministic reprogramming of entire human iPSC cultures into the target cell type.

So it is a real departure from classical differentiation methods and overcomes the inherent limitations associated with them.

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Our approach allows reliable, reproducible and consistent production of human cells at scale, and within days versus weeks or months. We started working on the technology in 2012, in my research lab in Cambridge University. In 2017, my team showed that opti-ox works reliably in producing neurons and myocytes, sparking consideration of cell therapies for the diseases that affect these cell types, as well as an existing demand from the community that researches these diseases. In 2019, a year after we formed bit.bio to bring opti-ox to market, we commercialised our first product, ioGlutamatergic neurons, and launched our ioSkeletal Myocytes shortly afterwards.

You’ve spoken before about bit.bio’s mission to reveal the ‘operating system of life’ and the huge impact this could have across science and medicine. Could you tell our readers what you mean by this?

As mentioned above, the identity and function of all cells is determined by genetic programs. Every cell’s DNA contains the same information; the difference between cell types is determined by which of these programs are active. Together, you could view these programs as ‘the operating system’ of a human cell or the operating system of life.

Transcription factors are the ‘code words’ of the operating system – they control the programs (the gene regulatory networks) of the cell and hence, tell a cell what to do.

Q: How do you expect this merging of biology and engineering to influence medicine in the future?

A: Pharmaceutical companies have historically developed small-molecule drugs to treat a range of diseases, and this has had a huge impact on medicine. Then, biotechnology companies began developing larger molecules for the same purposes, such as proteins, and this has also greatly improved the field. Small molecules and biologics will continue to play an important role. To speed up their development and to increase the probability of clinical success, bit.bio has commercialised two cell types for the research & drug discovery markets: ioSkeletal Myocytes and ioGlutamatergic Neurons. These are the first human cell products that can be used for high-throughput screening of drug compounds – the starting point for drug development.

Then there is the next step where human cells are used as treatments and therapies. This has truly revolutionary potential.

Human diseases are dynamic and often unpredictable with the ability to mutate their own genetic coding, or even lie undetectable within the human body. I am convinced that as we move forward as scientists and clinicians, we must focus our efforts on therapies that are adaptable or ‘intelligent’ and can respond to, and work with, e.g. the immune system.

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Talking Point With the recent approvals of the world's first CAR-T cell therapies in haematological malignancies, we have a glimpse of a future where cancers and other diseases can be eradicated, and health restored to the patient. Cell-based therapies have widespread potential for reversing a terminal diagnosis from a devastating degenerative and autoimmune disease.

Unlocking the full potential of cell therapies will be possible once we have a reliable supply of cells.

So the future of medicine for me is very much about cell therapies becoming widely available at a cost that is affordable to everyone.

Q: Cell therapies are making huge promises of routes for treating many different diseases, but they are held back by the lack of availability of human cells. How can bit.bio help overcome these barriers and support the widespread use of cell therapies?

A: The two most used approaches for cell therapy are either autologous (patient’s own cells) or allogeneic (donor cells). With either approach, you are limited by the availability of the cells.

In some instances, you are limited by the number of available cells and in other cases, you are limited by what a donor is willing to donate.

Both approaches require the isolation and expansion of the cells in cell culture. However, not all cells can be harvested and those that can are restricted in their ability to grow.

Moreover, donor cells are never all the same – they are not consistent or reliable. This can lead to challenges with regards to characterising the final therapy product given back to the patient.

If we want to reach the full potential of cell therapies, we need a source of consistent, reliable, healthy cells to work with; and we need them at scale. opti-ox allows us to overcome these limitations.

Q: High-throughput screening (HTS) has long been seen as an effective way of identifying new potential drugs for further investigation, and boosting drug discovery.

How can bit.bio support the first effective HTS work?

A: HTS has long held promise as a method for fast-tracking drug development, but until now, has lacked biologically-relevant, reliable and consistent models to deliver on this promise.

Animal models do not necessarily accurately represent human characteristics and are not as effective as human cell models for investigating the effect of drugs for human disease.

The main reason that so many novel drugs fail is that they have been screened or tested on animal models, and do not have the desired impact on human cells. By moving to human cells, researchers will gain a better understanding of the mechanism of the disease, enabling more effective development of treatments. www.biopharmaceuticalmedia.com

bit.bio’s cells address this demand. These cells are physiologically relevant and phenotypically characterised, and for the first time, available at scale, offering predictive in vitro cells for drug screening.

HTS based on relevant, human and disease-specific cells could form the backbone of new drug discovery pipelines, rapidly testing the biological activity of large numbers of molecules such as drugs. This will reveal molecules that demonstrate biological activity in human model, lowering the threshold for clinical translation. Even a small increase in the probability of success would enable drugs to come to market quicker, at lower cost to pharmaceutical companies, and therefore lead to significant improvements in quality of life for patients.

It is anticipated, in the foreseeable future, that the use of bit.bio’s human cell types will markedly improve the current approximately 3% success rate of new drugs progressing through the clinical development stages. Ultimately, moving away from the over-reliance on both animal models and immortalised cell lines for relevant human cells throughout the drug discovery process will improve patient quality of life through a greater range of available medicines.

Dr. Mark Kotter Dr. Mark Kotter is a stem cell biologist and neurosurgeon at the University of Cambridge.

By combining synthetic and stem cell biology, his team has developed a benchmark technology for the efficient and consistent production of human cells for use in research, drug development, and cell therapy. He is the founder of bit.bio and co-founder of the cultured meat startup Meatable.

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

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