COVID-19 Vaccine Urgency Throws Spotlight on Next-Gen Sequencing as Key Facilitator

Adventitious Agent Testing in Focus at CASSS CMC Forum Europe

The urgency of delivering a vaccine for COVID-19 in previously unattainable timeframes and volumes is throwing the spotlight on the expanded role next-generation sequencing (NGS) needs to play to avoid the time delays and other limitations of in vivo/animal testing.

The wave of COVID-19 vaccine development projects now underway is driving sponsors and regulators to further intensify their focus on how the full potential of NGS technology in assuring the safety and control of the vaccines can be realized.

Under review is how far the methodology has advanced over the past decade, how to best fill in and/or adjust to the knowledge and experience limitations that remain, and what needs to be communicated between sponsors and regulators to assure that the technology is performing its intended purpose.  

These pivotal issues were explored at a very timely session on “the use of next generation sequencing to characterize and detect adventitious viral agents in biological products” at the mid-May 2020 CASSS CMC Strategy Forum Europe.

The forum was originally scheduled to be held in Stockholm, Sweden, but was conducted virtually due to the pandemic. The conference agenda took shape in the latter part of 2019 before the pandemic was in view, making the choice of NGS as a topic of importance particularly prescient.

A session on “technologies related to viral safety” had been held at the December 2019 CASSS CMC strategy forum in Tokyo, Japan, in the wake of ICH’s release in November of a concept paper on updating its Q5A(R2) guideline on evaluating viral safety in biotech products (see Part IV).

The first session of the CASSS Europe forum, held on Monday May 11, included the traditional European Federation of Pharmaceutical Industry Associations (EFPIA) satellite session – formerly held under the “European Biopharmaceutical Enterprises” moniker. EBE has now been folded into EFPIA’s Manufacturing & Quality Experts Group (MQEG). The session covered the current CMC issues for polysorbates, antibody drug conjugates (ADCs), and adeno-associated viruses (AAVs).

On Tuesday, attention at the CASSS forum was on ICH developments regarding Q12, Q13, Q14, and Q2(R), before shifting later in the afternoon onto NGS. Wednesday sessions addressed bioassays for monoclonals and patient-centric quality standards, respectively.

Academic, Industry, and Regulator Perspectives Offered

In focus at the NGS session at the CASSS Europe forum was the expanding use of the methodology in qualifying vector and virus seed sequences and the absence of adventitious viral agents that may impact the quality and safety of cell-based products and vaccines.

Explored were the significant advantages NGS offers over the conventional in vivo methods, as well as the technical and regulatory challenges involved and the heightened urgency of addressing them in the COVID vaccine development context. Included was a review of the ongoing regulator/industry discussions regarding the validation of NGS assays, and what data should be submitted and in what format.

The session included insights on the NGS progress and technical/regulatory challenges from the academic, industry, and regulatory perspectives.

Introducing the speakers was EDQM European Pharmacopeia (Ph. Eur.) Division B Head Emmanuelle Charton, who co-chaired the session along with CBER Office of Vaccines Research and Review (OVVR) Division of Viral Products Deputy Director Robin Levis. Noting that falling under the “next generation” designation are “massive parallel,” “deep” and “high throughput” sequencing (HTS), Charton stressed that “if PCR revolutionized the world of molecular biology as first generation sequencing methods in the 20^th century, NGS revolutionized this world in the 21^st century.”

Throwing strong light on the technology’s potential, she explained, was its use in 2010 in detecting the unexpected presence of porcine circovirus (PCV) in a rotavirus vaccine. Since then, she pointed out, its development and use has expanded as a replacement for in vivo methods in identifying, characterizing, and controlling contaminants in biological raw materials and cell substrates.

The lead-off speaker was Ghent University researcher Sebastiaan Theuns, who explored the evolution in the sequencing technologies over the past decade and their expanded use by pharmaceutical manufacturers and veterinarians. Theuns heads a new Ghent University research spin-off company, PathoSense, that will serve industry in viral/bacterial detection with NGS.

Sanofi Pasteur Virology Analytical Expert Carine Logvinoff followed with a discussion of the evolving regulatory environment for HTS and the interactions Sanofi has had with health authorities – and particularly with FDA’s Center for Biologics Evaluation and Research (CBER) – regarding substituting HTS for in vivo tests in detecting adventitious viruses for its new viral vaccine candidates. [Logvinoff’s and Theuns’ presentations are reviewed in Part II.]

Italy National Center for the Control and Evaluation of Medicines Senior Researcher Domenico Genovese provided a European regulator’s perspective on the current regulatory landscape for evaluating the safety of vaccines and biological products and the challenges of balancing “regulation and innovation” in applying NGS to the task (see Part III) .

The Impact of the Wave of Coronavirus Vaccines Under Development

Session co-chair Levis then offered some highly valuable insights on CBER’s substantial engagement with NGS and the impact of the current pandemic.

She reviewed the research her Division of Viral Products has been doing under the leadership of Supervisory Microbiologist Arifa Kahn to expand virus reference resources – stressing the challenges and importance of the bioinformatics piece in particular – and highlighted the sponsor interactions with Sanofi and others through “technical working groups” that have led to the ability to use NGS in replacing in vivo testing.

She then brought the audience squarely into the present with the wave of coronavirus vaccines that her office is now involved with and the important discussions that are taking place on how to deploy NGS in assuring their safety in eclipsed development timelines where full validation is not an option.

 

Levis and Charton Stress the Harmonization Opportunity

Levis then moderated the panel discussion that followed (see Part II for a review of the full panel discussion). At its conclusion, she stressed that the introduction of NGS presents a “real opportunity” for harmonization between industry and regulators across the human and veterinary arenas and for sharing in the effort to maximize NGS’s effectiveness.

EDQM’s Charton agreed on the harmonization opportunity and benefits. Levis asked her fellow session moderator about the potential benefit of a pharmacopeial monograph for NGS.

Charton responded that in view of how quickly the technologies evolve, she did not envision having product-specific tests, but “maybe more guidance on how to validate the techniques” and how to demonstrate equivalence. She noted that how to further the use of these technologies was under active discussion at EDQM and that “there is lots to work on.”

She pointed to the appreciation for the NGS references in Ph. Eur. expressed by stakeholders at the major International Alliance for Biological Standardization (IABS) conference on “NGS for adventitious virus detection in human and veterinary biologics” held at the University of Ghent in November 2019.

“Of course, it is always a question of what to write and what not to write,” she commented, “because if we write too much, then you could be seen as preventing innovation. That is surely not what we want to do, but to continue to encourage the use of these techniques for the characterization or the identification of adventitious agents in biological medicines.”

[Highlights of the 2019 IABS meeting were provided by CBER’s Khan as part of her presentation on NGS in viral detection at the CASSS Japan Strategy Forum in December 2019, which is reviewed in Part III.]

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Sanofi Pasteur and Ghent University Experience with NGS

At the May CASSS CMC Strategy Forum Europe, Sanofi Pasteur Virology Analytical Expert Carine Logvinoff began a presentation on her company’s engagement with next-generation sequencing by highlighting the “noisy entry made by HTS [high-throughput sequencing] in the vaccine world” in 2010 with the discovery that the existing testing package could miss viral contaminants.

The impact on the regulatory environment, she explained, included vaccine manufacturers being requested to consider additional adventitious agent testing methods.

Sanofi Pasteur, in turn, made the decision to accelerate the exploration of new molecular technologies – finding HTS to be an “effective and valuable tool for both identification and reduction of unknown adventitious virus.”

Logvinoff outlined in detail the validation steps the company took to run the testing under GMP, with consideration of the new chapter 5.2.14 in the European Pharmacopoiea (Ph. Eur.).

The chapter recommends selection of a panel of representative, well-characterised model viruses to demonstrate that a proposed new method is equivalent in sensitivity to the in vivo methods. In view of the challenges in assessing the sensitivity of the broad in vivo methods, Logvinoff’s team took advantage of an NIH study published in 2014 to assess their test against the panel of 16 model viruses used in the research paper.

At the same time, the international regulatory environment was evolving, with publication of a series of technical reports by WHO and revision of the Ph. Eur. chapters on cell substrates and tests for extraneous agents in viral vaccines. Logvinoff explained that these revised texts supported Sanofi in changing its approach to the testing package – enabling a justification of the new technology based on the viral risk assessment.

She reviewed Sanofi’s extensive participation in international meetings and conferences – in particular, the International Alliance for Biological Standardization (IABS) meeting on next-generation sequencing (NGS) for adventitious virus detection in biologics in November 2019, held at the University of Ghent.  “I think it was a really important meeting” she said, “both for the technology aspect and the interface with the regulatory authorities.”

Sanofi’s collaborative engagement around NGS has included active participation in PDA’s Advanced Virus Detection Technologies Interest Group (AVDTIG). Sanofi participants are now co-leading the IG sub-groups on bioinformatics and follow-up investigations.

[Editor’s Note: See Part III for further discussion of the 2019 IABS meeting and AVDTIG by FDA’s Arifa Khan at the CASSS CMC Strategy Forum Japan in December 2019.]

Logvinoff concluded with a review of the valuable meetings Sanofi has had with FDA and other regulatory authorities on its use of HTS.

At the initial meeting with CBER in 2017 on Sanofi’s proposal to replace in vivo tests for a new vaccine, the company was invited to have “any type of technical meeting as we felt the need,” she said. 

A pre-IND meeting was held to discuss data submission requirements in 2019, followed by another at which the testing challenges were further addressed. These “open and extremely fruitful discussions” ensured that there was “mutual confidence” in the HTS test and that “CBER expectations” were being met. [A link to Logvinoff’s full remarks at the forum is provided below.]

Researcher Theuns Explores Fast Moving NGS Technology

In offering his extensive research experience with the rapidly evolving NGS technologies at the CASSS Europe session, Ghent University researcher and PathoSense leader Sebastiaan Theuns explored the wide range of analytical applications now in play and discussed the innovations of major sequencing companies such as Illumina, PacBio, ThermoFisher, Oxford Nanopore Technologies, and MGI (BGI).

Theuns was a member of the scientific planning committee for the November 2019 IABS conference. This second IABS meeting expanded the scope from a 2017 meeting to consider both human and veterinary biologicals. The planning committee for the 2019 conference included representatives from industry, European regulatory agencies, FDA and WHO.

In his presentation at the CASSS forum, Theuns noted the range of genome sizes being studied by NGS for adventitious agent testing, diagnosis of pathogens, and whole human genome sequencing, including: ● viruses at 1.5-150 kilobases ● bacteria in the 1-5 megabases range and ● human genomes at 3.2 gigabases. Depending on the sample source and purpose of the testing, amplification and both short- and long-read NGS technologies may be needed to investigate genome sequences of interest.

A common challenge for everyone working in the field of NGS, he stressed – whether in fundamental or applied research in academia, industry or regulatory agencies – is the rapid evolution of sequencing technologies, with weekly improvements in accuracy, the release of new equipment models, and the strong competition among manufacturers.

After discussing the technologies, Theuns moved on to the ‘major hurdle’ of the bioinformatics expertise and hardware needed for storage and real-time analysis of the data generated.

“In the past,” he explained, “we have mainly been using central processing units (CPUs), which are the typical chips in your computer.” But increasingly, graphic processing unit (GPU) hardware – the graphical cards in a computer that are used by gamers – is crucial to speed up analysis, and is “really the way forward.”

As a current example of the utility of NGS in diagnosis in real-time, Theuns spoke about use of the technique to diagnose and monitor the spread of Covid-19 in people crossing the border into the Netherlands, as well as at the local level in hospital departments. 

Questions Posed by Panel Moderators and Participants

During the latter part of the session, a panel discussion provided an opportunity for presenters and moderators to further explore some of the issues in deploying NGS technologies for virus control that had been raised in the presentations.

Joining Logvinoff and Theuns on the panel was Italy National Center for the Control and Evaluation of Medicines Senior Researcher Domenico Genovese, who presented on balancing regulation with innovation (see Part III for more on Genovese’s remarks). Moderating the discussion were CBER Division of Viral Products Deputy Director Robin Levis and EDQM Ph. Eur. Division B Head Emmanuelle Charton.

The NGS issues drawing discussion were:

● whether system cost and capabilities related to data quality

● the level of accuracy needed for adventitious testing and how to validate it

● how to maintain continuity of data quality in a GMP environment with rapidly evolving technology

● whether NGS could be used for quantifying bioburden as well as adventitious virus testing

● how to ensure complete DNA/RNA extraction by using two kits

● ease of use of different platforms and the need for appropriate expertise to interpret the big data generated

● the importance of virus standards in the early implementation phase of NGS with the many variables involved

● what discussions there were with other regulatory bodies besides FDA – in particular, European agencies

● product-specific validation using model viruses and during method transfer

● the opportunities to speed up coronavirus vaccine development using NGS with appropriate bioinformatics

● the critical need to follow up positive results to confirm they are ‘true’ positives

● the use of nuclease treatments to enrich the selection of encapsidated nucleic acids, and

● the evolution of the Ph. Eur. monographs and eventual harmonization of requirements and guidance

[A link to the panel’s discussion of these issues is provided below.]

Reinforced during the panel discussion was the importance of engaging with regulators before deployment of new and complex analytical technology like NGS to align on expectations. Also critical, participants agreed, is keeping up with and engaging in the open dialogue between academia, industry and regulators in various fora and interest groups.

Several of the issues debated by the panel – including validation, standardization, bioinformatics expertise and need for follow up of positive results – are being considered by AVDTIG. These include ● sample selection, preparation, and processing ● reference materials and virus standards ● development and evaluation of a publicly available virus reference database, and ● bioinformatics and follow up strategies to confirm an NGS “hit.”

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A Decade of Regulator/Industry Collaboration on NGS

CBER’s Arifa Khan, presenting at the December 2019 CASSS CMC Strategy Forum Japan, outlined the increasing focus on advanced virus detection technologies over the last decade and shared FDA’s collaborative efforts in developing and promoting the use of NGS for biological product safety testing.

Khan shared highlights from recent key conferences and detailed her department’s work on development of model virus reference standards, modernization of a virus-specific database, and outreach activities to manufacturers.

She began by walking the audience through the safety concerns raised by the introduction of novel cell substrates for vaccines two decades ago, and how the need for advanced virus detection technologies including NGS to test for previously undetected viral contaminants became clear with the 2010 discovery of porcine circovirus (PCV) in rotavirus vaccine.

Khan then went on to consider the challenges around the use of NGS in virus detection, including validation and bioinformatics – especially the handling of large amounts of data and follow-up strategies to determine the biological relevance and significance of positive signals.

Applying NGS to adventitious agent testing has been a major focus of her lab team. The Office of Vaccines Research and Review (OVRR) has been active in agency genomic working groups and in external collaborations and organizing conferences.

Outcomes of Recent NGS Meetings Highlighted

Discussing the outcomes of the IABS workshops on NGS for adventitious virus detection in biologics held in Rockville, Maryland in 2017 and in Ghent, Belgium in November 2019, Khan stressed the clear need for “continued collaborative efforts and scientific exchange” in meeting the challenges of applying NGS in the regulatory arena.

She further highlighted a September 2019 workshop on the progress made and gaps remaining to be filled in “standards for NGS detection of viral adventitious agents in biologics and biomanufacturing,” organized by FDA and the National Institute of Standards and Technology and held at NIST’s Maryland headquarters.  

The many collaborative initiatives in which Khan and her group have been involved have included publications, developing the panel of reference standards (see Part I), and modernizing the reference virus database (RVDB) to allow the information to be more widely accessible.

She explained that the RVDB was designed “to have complete representation of diverse virus families” – facilitating the general use of NGS for broad virus detection and aiding in detection of emerging viruses. The database was moved to the University of Delaware in October 2019, she noted, and the goal is to make it more user-friendly, blast-searchable, and regularly updated.

Concluding with a roundup of current and future regulatory guidance and the experience of the OVRR with NGS, Khan stressed the importance of engaging early with her office through technical working group discussions “to reach a consensus prior to initiating lengthy, expensive studies.”

The power of NGS and its benefits for potential applications in biologics is recognized, she said, and “therefore, regardless of the challenges, there has been much progress made towards the standardization and applications of NGS.” [A link to Khan’s full presentation at the CASSS Japan forum is provided below.]

Evolution of European and International Guidance on NGS

The evolution of international guidance and the Ph. Eur. monographs regarding NGS was further discussed by Italy National Center for the Control and Evaluation of Medicines Senior Researcher Domenico Genovese at the CASSS Europe strategy forum in May.

Genovese focused on the requirements for biological manufacturers to demonstrate the capability of the process to remove or inactivate known contaminants. He pointed out that various EMA guidelines provide recommendations on viral inactivation validation and also set specific values for viral clearance levels.

He discussed the limitations of conventional assays to detect viral contaminants, noting – as did Khan – the “evolving regulatory expectations on viral safety” over the past decade and that NGS “is a sensitive and un-biased detection method for adventitious agents.”

He then outlined the evolution of the EP monographs. These were updated in 2018 to encourage supplementation and replacement of conventional testing by newer, sensitive molecular methods with broad detection capabilities, and are supported by WHO technical report guidance.

The new/updated chapters include: ● Chapter 5.2.14 – “Substitution of in vivo method(s) by in vitro method(s) for the quality control of vaccines” ● Chapter 5.2.3 –  “Cell Substrates for the production of vaccines for human use” and ● Chapter 2.6.16 – “Tests for extraneous agents in viral vaccines for human use.”

Acknowledging the challenges of using NGS to detect adventitious agents, Genovese agreed with other speakers at the CASSS session that coordinated work among specialists is important. As examples, he also cited the efforts of the Advanced Virus Detection Technologies Group (AVDTIG), which is gathering together government agencies, industry, service providers, technology developers and academics worldwide, and conferences like that held by IABS in November 2019.  

Genovese echoed many of the messages that Khan presented in the December CASSS strategy forum in Tokyo – referencing her office’s work on upstream sample processing and library preparation as well as the efforts by the UK National Institute for Biological Standards and Control (NIBSC) on development of candidate reference material for adventitious virus detection in vaccine and biologicals manufacturing.

Looking to the future, Genovese reiterated the need for further dialogue between researchers, developers, companies and regulators to overcome current hurdles in approving the implementation of NGS and to develop harmonized international guidance on validation, for the benefit of both industry and agency assessors.

During the panel discussion that followed his talk at the CASSS Europe session, Genovese commented that his agency has not yet reviewed applications with NGS, but is preparing to do so through this dialogue. [See Part II for the full panel discussion.]

[CLICK HERE for Genovese’s slides from his CASSS Europe forum presentation.]

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Stakeholder Engagement Begins on ICH Q5A Revision

Members of the Expert Working Group (EWG) set up for revising ICH’s Q5A guideline on viral safety evaluation are moving forward in their efforts to engage with stakeholders as much and as early as possible to ensure that the revised document is fit for purpose for the future.

Among the venues where that engagement has already taken place are the CASSS CMC Strategy Forum Japan, held in Tokyo in December 2019, and a workshop session at the CASSS WCBP conference in Washington D.C. at the end of January.

Driving the dialogue around revision of the ICH Q5A guideline on the “viral safety evaluation of biotechnology products derived from cell lines of human or animal origin” is a concept paper,  endorsed by the ICH Management Committee in November 2019, which spells out the rationale and objectives for the revision and the issues that EWG views as most important to be considered in the update.

The concept paper stresses that the current 1999 version, “while still useful, requires revision to allow for a consistent global understanding of viral safety within the biopharmaceutical landscape.”

The revisions, the paper explains, will be aimed at supporting the new product development, use of state-of-the-art technologies, and a more harmonized regulatory approach for newer classes of biotechnology products. Alternative validation approaches are expected to provide increased flexibility for viral safety assessment.

A little over a month after the concept paper was released, the issues of virus control in bioprocessing came into sharp relief with the emergence of the coronavirus global pandemic (see the first section of this story).

Biotech manufacturers are now wrestling with the challenges of making sure that the three pillars of virus control defined in Q5A are in place in the SARS-CoV-2 virus context: ● avoiding contamination via raw materials ● ensuring viral elimination techniques are appropriate and satisfactory, and ● preventing potential contamination via personnel.

The additional complexities of the Covid-19 situation mean that both manufacturers and regulators are having to develop contingency plans to deal with reduced staffing levels, increased need of specialised protective equipment that may be in short supply, and adapting control strategies as more information on the novel coronavirus becomes available.

ISPE Webinar and PDA Virus Forum Continue the Q5A Dialogue

At an ISPE webinar on June 11 moderated by ISPE’s new CEO Tom Hartman, GlaxoSmithKline (GSK) TSE and Virus Control Director Anne Stokes shed significant light on the operational controls needed to mitigate the SARS-CoV-2 challenge to the Q5A virus control framework. Stokes represents the Pharmaceutical Research and Manufacturers of America (PhRMA) on the ICH Q5A (R2) EWG.

Along with the control framework, she discussed the planning activities required to prepare for a novel virus challenge to manufacturing facilities and the ongoing virus risk management and mitigations needed.

The Q5A revision dialogue between EWG members and stakeholders is continuing at the virtual PDA Europe Virus Forum June 22-23, where Stokes is again presenting.

Regulators from the EWG participating in the sessions are Paul-Ehrlich-Institut (PEI) Viral Safety Section Head Johannes Blümel and FDA CBER Office of Vaccines Research and Review (OVRR) Senior Investigator Arifa Khan.

Also presenting is FDA CDER Office of Biotechnology Products (OBP) CMC Product Quality Researcher/Reviewer Scott Lute – a colleague of the EWG rapporteur, Joel Welch, who serves as Review Chief at OBP’s Division of Biotechnology Review and Research IV.

In addition to representatives from major biopharmaceutical firms, service providers experienced in virus testing and clearance techniques will also be presenting and engaging in discussions at the forum. Service providers are mentioned in the Q5A (R2) concept paper as key stakeholders in the revision process.

[Editor’s note: IPQ coverage of the ISPE webinar and the PDA virus forum will follow.]

Japan’s EWG Member Outlines Plans for ICH Q5A(R2)

At the mid-December 2019 CASSS Japan Forum, PMDA’s Office of Vaccines and Blood Products Deputy Review Director Akira Sakurai, who serves on the ICH Q5A(R2) EWG, presented on the recently released concept paper and shared information on the composition and plans of the working group.

He outlined the drivers for the revision, progress made at the November ICH meeting in Singapore, and the anticipated development timelines. An initial draft is targeted to be completed by November 2020, with sign-off by topic leaders and endorsement of a Step 2 document called for in June 2021, and adoption of the final guideline in November 2022.

These key milestones were outlined prior to the COVID-19 pandemic, although it was noted in the slide presentation by Sakurai that the formal ICH procedures allow for sign off, endorsement and adoption to be achieved either by face-to-face meetings or electronically. The high relevance of the COVID-19 experience does add to the EWG’s challenges and the importance of making the update as valuable as possible.

Sakurai stressed that a common message in the concept paper and all ICH communications is the need for early engagement with stakeholders via public conferences. [A link to the CASSS Japan agenda and Sakurai’s slide set is provided at the end of the story. The concept paper is also provided in full below.]

 

 

 

Sakurai led off a session looking at technologies related to viral safety and the ICH Q5A revision, co-chaired by PEI Monoclonal and Polyclonal Antibodies Section Head Steffen Gross and Kyowa Kirin’s Kazuhisa Uchida.

Other speakers included FDA’s Khan and Kobe University researcher Keisuke Yusa, who spoke on the collaborative efforts to develop and use next-generation sequencing (NGS) for viral safety control in biologics. [See Part III of this story for Khan’s full presentation at the CASSS Japan forum.]

Yusa demonstrated that a cell specific database can reduce false positive results and the versatility of NGS as a platform for viral safety control of a range of biologic products. Contributing to the research was Japan National Institute of Health Sciences Division of Cell-Based Therapeutic Products Head Yosi Sato, who serves on the ICH Q5A EWG alongside Sakurai.

An industry perspective on CHO cell product virus safety in the context of the Q5A revision was then given by Genentech Process Virology Associate Director Qi Chen. She highlighted the “tremendous amount of virus safety knowledge” accumulated over the 20 years that the current guideline has been in use, which provides strong support for the proposed revisions.

ICH and PIC/S Collaborating to Tighten Review/Inspection Alignment

Joining the panel discussion following the presentations, Health Canada Centre for Evaluation of Radiopharmaceuticals and Biotherapeutics Senior Regulatory Scientist Anthony Ridgway – who had served on the original ICH Q5A EWG – shared his experience on a question relating to platform approaches and modular clearance.

Ridgway commented that he thought modular approaches would stand a good chance of being allowed for in the updated document, noting that it often happens that the scope of a guideline broadens over the years as more information and experience becomes available.

He suggested that regulators are already accepting a modular approach in many ways – for instance, in not requesting validation studies provided by the filter suppliers to be repeated by each product manufacturer. “So, in principle, the concepts of accepting these things are there.” Panel participants agreed that appropriate justification would be key in successful regulatory applications.

Another valuable discussion was around the interplay with other ICH guidelines – in particular, ICH Q7 on active substance GMPs.

In the context of this discussion, Genentech Regulatory Policy and International Operations VP Wassim Nashabeh, who was chairing the forum and represents the Biotechnology Innovation Organization (BIO) on the ICH Assembly, pointed out that ICH was in the process of entering into a collaboration agreement with the Pharmaceutical Inspection Co-Operation Scheme (PIC/S).

He explained that the collaboration would be focused on the elements of the quality guidelines that may have an impact on inspection practices or GMP expectations.

The minutes from the ICH November 2019 meeting in Singapore indicate that its Management Committee received positive feedback on a proposal for more routine engagement with PIC/S and that there would be a pilot phase for this collaboration.

 “As part of this proposal and in line with ICH processes, PIC/S would be involved in ICH guideline work during the public consultation following Step 2b,” the ICH Assembly said in its minutes. “Additionally, as an ICH Observer, PIC/S could also request to be part of Plenary Working Parties (PWPs) which would allow an involvement prior to step 1.”

Q5A(R2) EWG Engagement Continues at CASSS WCBP Workshop

The discussion on revising Q5A continued at a workshop during the CASSS WCBP meeting in late January. The workshop was led by the two EWG members from FDA, CBER’s Khan and CDER OBP Product Quality Team Leader Cecilia Tami, and by AbbVie Director of Cellular and Molecular Biology Science Lianchun Fan, who represents PhRMA on the EWG along with GSK’s Stokes.

Khan began by providing a brief background on the Q5A revision and how the ICH Q5A-E biotechnology product series relates to the other ICH quality guidelines.

She then outlined the five key issues highlighted in the concept paper that the EWG members were particularly looking for feedback on, involving areas where advances have been made over the 20 years since the existing guideline was written.

To set up the discussion on each of the issues, the workshop leaders invited participants to share their related experiences, concerns they may have related to the updates, and suggestions for gaps that warranted attention.

Khan stressed that it was important for the EWG to get the feedback requested, because different manufacturers use the guideline for different types of products, including vaccines. “In the early days,” she pointed out, it was mostly used by monoclonal antibody manufacturers.

Which New Classes of Biotechnology Products Does the Guideline Apply to?

The discussion at the CASSS WCBP workshop began with a focus on the considerations that come into play for the new classes of biotechnology products.

Khan clarified for “those who only work on monoclonal antibodies” that the guideline will also apply to some of the other biologics, including vaccines and gene therapy products. However, she stressed that Q5A “is specifically for those that can undergo clearance of virus vector and adventitious agents.” 

“Currently there are baculovirus-expressed VLPs [virus-like particles], subunit proteins, and viral-vectored products that are already using the principles laid out in the Q5A,” she explained. “So we felt that if we were updating the document it is timely to be current and forward thinking to include this category of products.”

Responding to a question about whether there had been consideration of opening up the guidance to cell therapies – which cannot undergo clearance but still have to follow many of the other principles in Q5A – Khan commented that the EWG felt that those aspects were covered by other existing documents. 

CDER’s Tami explained that the guideline was for products derived from cell lines as indicated in the title. While it “took a lot of discussion,” she said, “ultimately the decision was not to include cell therapies.”

Khan asked if people were actually using the Q5A guideline for cell therapies. Biologics Consulting’s Barngrover replied that in her experience they were, and that while the clearance may not be applicable, “certainly some of the new viral assays may very well be relevant.”

Khan clarified that the new assays were being introduced to catch up with some of the other documents, noting that the WHO and EP have already introduced the newer assay methods and that the FDA cell substrate document is being updated concurrently. “So, we are introducing it here to be prospective in terms of where technologies are going.”

The CBER official stressed that the EWG will preserve the guideline’s current format. The aim would be to give some relevant examples. However, she highlighted the challenge of achieving the right balance, “because then people tend to think this is the only way. So, we do want to keep it more in terms of philosophy” allowing people to apply the guidance “according to what they feel best fits for what they are doing.”

Tami agreed, commenting that “the idea is that it doesn’t need updating in two years when things evolve so rapidly. So it is really going to deal with concepts, principles, and maybe examples, but I don’t think it is going to be very specific.”

Khan emphasized that “this has been a great document. It has and is still serving its purpose very well. We want to continue in the same spirit, so it will be good at least for another 20 years or more.”

How Are New Virus Assays and Alternative Analytical Methods Justified?

The second topic for discussion was the methodology advances since the publication of the guideline – specifically the nucleic acid-based assays polymerase chain reaction (PCR) and NGS, and their use in characterization, detection, and viral clearance validation.   

“You may be surprised to see PCR there” Kahn remarked, “when we have maybe been using it for almost as long as the document has been there. Well, PCR is not in the document,” so it is being updated to formally include it. “NGS is an evolving, emerging technology and there has been a great deal of progress made on it, so now we do feel comfortable putting it in the document.”

She mentioned again that in CBER’s office of vaccines “we are also updating our document. It has already been introduced in the European documents, the EP – they are already talking about using NGS to replace some of the in vivo testing. So it is moving rapidly. We are not saying you must use it. It will be introduced for you to decide how best it can be applied in the appropriate manner.”

One of the participants asked for clarification around the methods and whether they could be recommended for testing of unprocessed bulk.

“Clearly,” he said, PCR can be used for viral clearance studies instead of an infectivity method. “With NGS you certainly could do that – I don’t know if we all do it – but my question really is if this guidance is considering supporting the use of alternate methods for virus detection in unprocessed bulk or the equivalent in some of these new modalities? Today it is restricted to some kind of infectivity assay, which takes a long time and is pretty challenging. So is that within the scope of this, because I think it would be hugely valuable if it were?”

Khan replied: “Your question is being addressed in various areas and data is being generated in terms of how NGS can be used to complement, supplement or replace the current assays. What we are expecting is that as we are working on the document there will be more information generated before we finalise it, and we will be able to update it with the current information at that time.”

Kahn was asked about the timeline for the additional supportive data versus the guideline’s completion. She replied that the data is expected “to be there by the end of next year or sooner. There will be some data at the end of this year. There are studies ongoing to evaluate NGS with the other assays.”

She pointed to the extensive research and interest group efforts on NGS and in vitro/in vivo correlations, and noted that the Office of Vaccines had already received submissions in which NGS is being used to supplement and even replace some assays. “So we are looking at it on a case-by-case basis even right now, and all of that knowledge…will build towards information that will go into the document.” [See Part III.]

Fan added that PhRMA was collecting feedback from across the industry. “This is definitely one of the points under pretty intense discussion. We are aware that both PCR and NGS have their pros and cons – they could be too sensitive, and how do you deal with false positives? So there will be lots of discussion, and hopefully some of the data will help us to make some decisions.”

The Revised Guideline Will Need to Continue to Be Flexible

Asked whether the new guideline would help sponsors with defining some of the criteria for them to do the replacement, Khan responded that it was too early to discuss the details of what the revision will contain. “At the moment,” she said, “these are the topics we want to include, and we will have to see how much detail can go in. We don’t want to be too specific.”

“We still want to keep it flexible so that people can use it for different purposes, but the data package will need to support that [the method] is fit for purpose. We can indicate what should be in the package, but not to be specific in terms of the exact criteria for the methodology, because it is evolving.”

Biologics Consulting’s Barngrover noted that the current guidance is silent on how much qualification or validation of the virus assay is needed and asked if there was any intention to change that. Khan replied that “again, those are questions that we are taking notes on and will discuss in detail with the EWG.”

She reiterated that “the current document has held strong for so long because it was not very prescriptive. It was flexible, so it could be used across product categories.” How it is revised will depend on the different product types, she said. “I think for the newer products we might keep it broader, and maybe for the more experienced products we can learn and leverage experience and provide details for that.”

In response to a query from Tami about what sort of validation details would be helpful, Barngrover shared that in her experience, some sponsors query whether any validation was needed for assays used for qualifying cells. When Khan referred to the clear validation requirements laid out in ICH Q2, Barngrover pointed out that the Q2 guidance was specifically for assays used for drug substance and product release.

Tami agreed the point was well taken. Further support came from another workshop participant remarking that “some guidance documents talk about using scientifically sound methods, or sometimes validated methods.” There can be “uncertainty about which way to go,” he said. 

Encouraging workshop delegates to provide input, a participant urged: “If you have a strong opinion on what it needs to say, this is the chance to really highlight it for the group, because there will never be a time more open than now to try to hear about what perspectives should be captured.”

Decision Tree Suggested for Evaluating PCR/NGS Findings

Daiichi Sankyo CMC Biologics Regulatory Affairs Director Roman Drews, a former CBER official,  suggested that a decision tree would be helpful for verifying the results of PCR and NGS used in the GMP/QC context.

Khan and Tami agreed that yes, that would be useful and could be provided as an appendix. Khan referred to the work of the Advanced Virus Detection Technologies Interest Group (AVDTIG), mentioning that this was one of the topics being followed up by them. [See Part III.]

Khan also highlighted that with any nucleic acid-based assay, before embarking on the technology there should be a follow-up strategy in place. Tami added that it was important to have a decision tree “to confirm any positive before you discard a lot.”

A participant requested that the discussion around NGS should be described as specifically as possible in the guideline out of concern that everyone would start to do NGS because it is mentioned in the guideline without understanding why, “and this should be avoided.”

Khan agreed that it was important to be thoughtful about using NGS and whether it is the best approach. “It will be your decision, but we can lay out the principles for potential applications.” Tami again emphasised the concern that examples in a guideline can sometimes lead to being regarded as the best way to go “when it is not necessarily so.”

Khan explained that the EWG had been thinking of reducing examples, but acknowledged that it may be important to have some, “like the decision tree or different situations in which these new assays might be applied.”

How Can Prior Knowledge be Leveraged in Viral Clearance?

Another area of inquiry identified by the EWG in the concept paper regards the use of prior knowledge and learnings from similar processes and products in validating the viral clearance for a new product.

Chen, who introduced the topic, noted that Genentech has been using modular viral validation for clinical products for many years, and the approach has successfully sped up clinical development. Industry has been accumulating “a lot of knowledge” in this area, she said – raising the potential for Q5A to recognize its viability also for commercial products.

Biologics Consulting’s Barngrover commented that she could see where knowledge leveraging might work for viral validation, but questioned how it would work in other areas, for instance chromatography. A concern would be how similar the chromatography would have to be – for example, in terms of protein load and flow rates.

CDER’s Tami commented that there are some steps that are very clearly understood, including the parameters that need to be controlled. “Maybe for those, modular clearance works. But with other examples it is more challenging.”

A discussion followed from Tami’s request for thoughts from the audience on what was needed. “Can you use experience from others in industry, from literature, confirmation runs? What would be the most appropriate to claim modular clearance?”

A session attendee suggested the question is really a scientific one. “It doesn’t necessarily relate to how many marketed products you have, but it is more about how many experiments you have performed or have access to the data from – not how much your company did, rather how big the database is, including data from both marketed and clinical products.”

CMOs, he pointed out, may also be able to provide the data they have accumulated. “Maybe it wasn’t your data, but as long as you have access to all the data – beyond just a log reduction of virus (LRV) value – I think you are able to judge what it means.”

Khan stressed the importance of publishing as much data as possible so that the information can be leveraged – noting that this is something she encourages and is seeing happen more often.

Chen commented that the principles should be the same for both clinical and commercial products – involving the need to define your conditions. The approach should apply to well-characterised steps, such as low pH, inactivation by detergent, and retrovirus removal by small virus retentive filters, she maintained. 

“You need to define the conditions that are most robust so you can reliably get the same log reduction every time – becoming product independent. When you actually have that knowledge, you can apply this approach. There has already been a lot of data shared by the industry and there are white papers published for all of these well-characterized steps.”

Another audience member commented that in-house data for modular clearance could be supported by literature data and white papers as a knowledge base for risk analyses for quality-by-design approaches to process development. “Firm A will not know exactly how Firm B will run their columns or their unit operations, but it certainly contributes to the knowledge base that feeds into risk analyses.”

ICH Can’t Be Overly Prescriptive, Health Canada’s Ridgway Comments

Health Canada’s Ridgway weighed in with his experience on multiple ICH EWG’s, including that on the lifecycle management guideline Q12 – commenting that the details of what would constitute sufficient information are beyond the scope of an ICH guideline.

What is needed, he said, is to “achieve harmonization on whether that type of evidence will be accepted and considered by regulators. But it is not for the guideline to try and define exactly what is needed to satisfy the regulators. The information is going to change over time and new information will come in after the guideline is finished.”

“So I think there are dangers in setting defined parameters about what information would be acceptable or not. The important thing is to determine whether the regulators involved in drafting the guidance document are willing to consider this and leave it at that.”

Another participant commented on the international acceptance of certain robust, effective steps. He noted that there are certain conditions that have been shown to be very robust and hold up against a wide variety of manufacturing processes, and that some of these steps have been accepted by health authorities internationally.

“It would be nice for the guidance to mention these steps again – we have had this in other documents – of what is a robust, effective step. Maybe even some of those international references could be included in the guidance, to help people look at and be familiar with them, and how they can apply them to mitigate certain studies.”

How Should Virus Clearance Validation Guidance be Updated?

The next issue receiving attention was aspects of viral clearance validation that have emerged or evolved over the lifetime of the current guideline.

Genentech’s Chen provided some examples to stimulate discussion, such as whether the mention of the six-log safety margin for retroviral clearance in the current version should be reconsidered.

Echoing concerns expressed earlier about the risks of including examples that later become expectations, an audience member commented that he believed there was a misunderstanding because “people take it as a target.” 

Chen agreed that although the six-log safety factor is included in the appendix, for some reason it has become a standard, “so people always talk about less than one particle in a million doses.”

“But that then translates to about 15 to 20 logs of clearance you have to validate for your purification process. And for well-characterized cell lines such as CHO cells, we know that these particles are not infectious. Over the 30-40 years of history, these particles have never been shown to be infectious. So,” she questioned, “is that clearance target reasonable?”

A session participant added that the list of evolving aspects of virus clearance validation provided in the concept paper were “excellent choices – exactly the ones that require an update.”

He remarked that the evaluation of resin at the lifetime was particularly relevant, noting that the studies that need to be performed currently could sometimes lead to a delay in the submission of a BLA if there were any technical issues.

He stressed that there is a lot of data available showing that “there is no big additional value coming out of these studies, as the resin at the end of lifetime always, in my experience, has the same clearance capacity as at the beginning of the lifetime.”

BMS Downstream Process Development Associate Director Angela Lewandowski joined the discussion, adding, “I am actually pretty excited about these potential updates – the first one in particular…. There is a lot of data collectively within the industry showing that end of lifetime resin has no impact on viral clearance LRVs.”

Referring to a recent multi-company evaluation done through the BioPhorum Operations Group (BPOG) of cycled resin in viral clearance studies, Lewandowski agreed that these kinds of studies involve “a huge amount of effort, material, and FTEs, when we have never seen a safety impact.”   [A link to the BPOG study report published in the PDA journal is provided below.]

Daiichi Sankyo’s Drews suggested that the guidance could include defined criteria in cases where lifetime studies for virus clearance could be avoided. Another audience member offered that the guidance could indicate “that if certain criteria are met, then these studies can be mitigated,” and that regulators are not expecting to see them.

He added: “In looking at viral clearance and what levels of reduction are needed, it also depends on what the load is. So maybe having some description of how you determine the load and the process to decide how much more you need to clear in the studies would be helpful.”

Noting that the specific changes being discussed by the participants related to CHO cell production, Khan suggested that the EWG “may have to indicate the basis for why the changes are being made, so the principles are not applied to the new types of product being considered.”

Parexel consultant Kurt Brorson, a former CBER official, pointed out that CHO cell-derived products use similar columns and are an “enormous” class, which includes both antibodies and recombinant therapeutic proteins.

Khan agreed and suggested that the format of the guideline could be modified “to distinguish the product categories where there is more experience versus the newer, emerging products where we have less experience. We will have to figure out how to present that so it is clear and not confusing.”

Drews offered that the risk of the class of products would also be an important factor to consider. The panel agreed, acknowledging that even though the guidance was specifically directed at cell-derived products, the principles on qualification of the columns were also being applied to plasma-derived products.

Biologics Consulting’s Barngrover suggested that there might be value in combining the wisdom and expertise of the EWG to include some of the reasons for failure of viral clearance studies – for instance, if the manufacturer didn’t take into account aggregation of the virus stock that was being used or the need to use a high enough titer. These are things that people should be aware of and “that might be helpful guidance to add.”

What About Continuous Manufacturing?

The last of the five areas put out for discussion at the CASSS WCBP session was how the guideline should be updated regarding assessing the risks of and validating viral clearance for continuous processes.

Bayer Regulatory Affairs Senior Director Bob Kozak began the discussion by suggesting that “it might just be helpful in general to outline some of the expectations for residence time. What are some of the parameters that need to be shown and developed to support continuous manufacturing clearance?”

Khan explained that the EWG felt the issue of continuous manufacturing (CM) needed to be considered so that the guideline keeps pace with the field and is forward thinking. “Even if we don’t have the answers, maybe we can think in terms of what questions we need to address and the expectations.”

Chen agreed, noting that Genentech’s exploration of elements of CM has led to questions about viral clearance validation when the process steps are connected. She suggested that the linkage to ICH’s Q13 guideline on CM “will also be very helpful.”

Khan commented that the Q5A EWG was in conversation with the Q13 EWG to make sure Q5A “is not duplicating but complementary.”

Another participant at the session added that, “for this area, I think the equipment vendors are going to be very important in terms of providing examples of how to validate a continuous chromatography system, for example. They are starting to publish on this area, so the scientific literature will be useful to follow. It will be an area likely needing a lot of flexibility.”

Health Canada’s Ridgway suggested that “there will not be a huge body of real data available with regard to viral clearance during continuous manufacturing by the time this guidance document is coming to completion.” As such, it “could allude to possible considerations that regulatory agencies might be willing to consider, but it might be very difficult and a little too early for the guidance document to try to make some firm conclusions on how this can be applied to continuous manufacturing. I think you will need to be cautious.”

International Harmonization Needed on Viral Clearance

After considering the different areas for inquiry called out in the concept paper, the audience was asked if there were any other concerns that they would like the EWG to consider.

Daiichi Sankyo’s Drews underscored the regulatory differences in different regions of the world regarding viral clearance and the dedicated steps involved.  He asked if the guidance may be able to help clarify specific requirements. 

Khan responded that Q5A is “an international document that needs consensus and agreement by all of the different regions. So the one thing that is important in addition to all the discussions we are having is that we are made aware of the different publications from the different regions, and we want to make sure we are consistent with the other guidelines that are published or being published.”

The differing requirements do make the guideline process challenging. “But, again, based on the experience and the knowledge, perhaps the others may now come to the same conclusions. So, I think that is an important point that we can discuss with the working group.”

Khan concluded the session by noting that the EWG had begun working on the specifics of the revision, and was holding regular teleconferences at which the points raised can be more broadly discussed. The face-to-face ICH meeting planned for Vancouver in May 2020 took place virtually.

Links:

●  CASSS CMC Strategy Forum Japan 2019 Agenda

●  Sakurai’s Slides on ICH Q5A(R2) from the Japan Forum

●  PDA Journal Article (Sept/Oct 2019) on BPOG Viral Clearance Study 

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Effort to Reduce Animal Testing for Vaccines Includes Global Health Fund Support for NGS

The collaborative work that FDA, industry and academia have been doing on next-generation sequencing (NGS) for adventitious virus detection and control in vaccines will get an important helping hand from the Global Health Fund (GHF) – set up through the National Institute for Innovation in Manufacturing Biopharmaceuticals (NIIMBL) with the support of the Bill and Melinda Gates Foundation (BMGF).

The NGS project was one of 14 that NIIMBL announced in May 2020 had been chosen to receive funding from among those submitted in response to its mid-2019 Project Call 3.1.

The Global Health Fund initiative will focus on advancing collaboration on: ● comparing NGS with currently used in vivo animal assays and in vitro cell culture and PCR assays to determine the applications of NGS as alternative assays, and ● developing a well-qualified reference virus database (RVDB II) that will facilitate specific detection of viruses for accurate interpretation of NGS data for adventitious virus detection.

The results from the project, NIIMBL stresses, will “enhance confidence in use of NGS for adventitious virus testing of biologics and aid regulatory agencies in decision-making and policy development regarding use of NGS for replacing the in vivo animal assays and for replacing or supplementing the in vitro assays that are currently used for virus testing. These changes will reduce animal use and facilitate rapid and broad detection of adventitious viruses to enhance vaccine safety and accelerate new product development.”

The project will be led by FDA, which, under the direction of CBER Office of Vaccines Research and Review (OVRR) Senior Investigator Arifa Khan, has been actively involved in advancing NGS. The many collaborative initiatives in which Khan and her team have been involved over the past decade have included publications, organizing symposiums, developing a panel of reference standards, and modernizing the RVDB to allow the information to be more widely accessible (see Part III).

Reviewing CBER’s  engagement with NGS at the CASSS CMC strategy forum in Japan last December, she explained that the RVDB was designed “to have complete representation of diverse virus families” – facilitating the general use of NGS for broad virus detection and aiding in detection of emerging viruses.

The database was moved to the University of Delaware (UD) in October 2019, with the goal of making it more user-friendly, blast-searchable, and regularly updated. The University of Delaware serves as the host of NIIMBL and has recently opened a new headquarters facility to provide a nexus for bringing industry/government/academic project teams together to pursue their collaborative work (see IPQ May 15, 2020).

Along with FDA and UD, participating in the Global Health Fund NGS project will be MilliporeSigma and GlaxoSmithKline.

A second GHF project approved in May will target the multi-laboratory validation of the serological test for in vitro batch testing of the potency of whole-cell pertussis containing vaccines. The test is intended to reduce the variability inherent in the intracerebral mice tests, the numbers of animals used, and the level of their distress.

The project will be handled by the Developing Countries Vaccine Manufacturers Network. DCVMN is an international alliance of manufacturers formed in 2000 with a mission “to provide a consistent and sustainable supply of quality vaccines at an affordable price to developing countries.” The network now includes over 40 vaccine manufacturers in 14 countries and territories. The members produce and supply over 40 different types of vaccines in several presentations and use a variety of technology platforms totaling around 200 products, over 70 of which are prequalified by the World Health Organization (WHO).

GHF Projects Flow from 2019 Workshop on Replacing Animal Testing  

With an initial grant of $1.5 million to NIIMBL from the Gates Foundation, the Global Health Fund was established in January 2019. Its mission is to support “leading scientists and subject matter experts from the NIIMBL community” in developing and implementing “manufacturing technology solutions that increase speed to market and lower costs for biopharmaceuticals to improve healthcare outcomes for all patients, including those in underserved markets and developing nations.”

The GHF represents an extension of the BMGF’s focus on vaccines as part of its overall efforts to develop new tools and strategies and high impact health products and services that can reduce the burden of infectious diseases in low and middle income countries (LMICs).

With the support of NIIMBL and the National Institute of Standards and Technology (NIST), which oversees NIIMBL, a Global Health Fund workshop was held in April 2019 focused on reducing and replacing animal-based testing for vaccines.

The workshop was designed to bring biomanufacturing, academic, non-profit, and government stakeholders together to discuss the issues and help in formulating viable proposals for the forthcoming NIIMBL 3.1 project call.

The focus of the workshop reflected a survey the Gates Foundation had conducted through the Developing Countries Vaccine Manufacturers Network (DCVMN). The survey found a very high level of interest among the respondents in the development of alternative in vitro assays to reduce the amount of animal testing required for lot release in the effort to assure a sustainable supply of affordable vaccines.

[Editor’s Note: A link to the informative proceedings prepared by the workshop coordinating committee, which includes the agenda and a synopsis of each of the presentations and the four breakout topic discussions, is provided below. Links to the presentation slides are included, where available.]  

Extreme Diligence Needed for Both Vaccine and Animal Production

Highlighting the survey in his opening remarks at the workshop, BMGF Global Health Vaccine Development Senior CMC Advisor Ray Prasad – whose career has included senior management roles in vaccine manufacturing – gave an eye-opening description of the variety of pain points, scientific limitations, and stumbling blocks that exist in the current animal testing approaches and expectations.

Issues of concern include: ● the variability across labs, animals, operators, environments, and animal food ● the precision of testing ● the sourcing and maintaining of a sufficient number of animals ● the differing requirements from different regulatory bodies, and ● the expensive and time-consuming nature of the studies and inconsistencies in the results.

Prasad drew from his decade of experience working in India as head of operations for vaccine manufacturer Biological E. (BE). In trying to deliver 100 million doses of a diphtheria, tetanus, pertussis, Hib, and hepatitis vaccine every year, “one of the biggest nightmares I had was animals,” he stressed.

While there was a lot of innovation from the companies to reduce the number of injections through the pentavalent vaccine, the manufacturing and releasing of this vaccine “was extremely complicated, particularly with animals and animal tests.”

Among the “nightmares” is making sure that there are a sufficient number of animals available with the right health, age, and weight needed to manage the actual release. And the Indian government labs have to test and release the vaccines as well, also requiring the animals.

BE had to work “extremely diligently” to produce both the vaccines and the animals for testing them. The animals also had to be supplied to the government labs to avoid the variability involved in different vendor sourcing. “So it was a massive undertaking to be able to manufacture and release close to 100 million doses every year.”

Prasad noted that 25 million babies are born each year in India and the pentavalent vaccine is given three times – at six weeks, ten weeks, and 14 weeks – which equates to 75 million doses, with an extra 25 million doses needed for wastage.

A significant percentage of this wastage, he explained, has to do with animal tests that are judged invalid. “It is not a failure, it is not a pass, but it is invalid” because of the behavior or variability of the results. “This kills us. When you talk about 20% of the entire supply is affected by invalid tests, that is a huge cost.”

When vaccines are being supplied at $1 to $2 per dose, “in many cases the cost of the animals is higher than the cost of the vaccine itself. So it has been a terrible ordeal for all the manufacturers in that part of the world.”

Looking at this situation, the Gates Foundation felt that “we had to do something. With all the collective knowledge and understanding and the technology and the development, there has to be a better way for us to do this.”

Animal Testing Involves a Lot of Prayer

The survey among the DCVMN members confirmed that the animal testing “is what keeps them up at night. Because the manufacturer can control the process and control the product. But you never know once you put it in the animal. You just pray.”

While there are other common biomanufacturing needs related to staffing, analytics, automation, the supply chain, and training that present opportunities for the Gates Foundation and NIIMBL to work together to address,  Prasad said, “the one that wins the prize is reducing the animal testing. It directly impacts our global health initiative,” and was picked as the first target to work on accordingly.

Among the challenges is getting the buy-in across regulators for alternative methods. That involves having a reliable, standardized, and cost-effective in vitro assay that does not compromise quality, and clarity and agreement around the comparative data required.

Prasad explored in more detail the key issues with the current animal testing regimens.

Citing the operator-dependence problem, he explained that “one of the tests for pertussis is actually injecting into the head of the animal to try to precisely hit the target to get the right amount of dose. And this could change. When you are talking about a slight difference in the way it is tested, it can have a major impact on the outcome of the test results.”

Another problem is the sourcing and maintaining of the animals. Pharma’s expertise is “in making the vaccines, not breeding the animals,” Prasad explained. Reliance is then placed on others whose main focus is breeding for outside the vaccine needs, which means that “many times” the process does not work as well as expected. “A lot of controls are necessary to ensure that that is sufficient, and the right health, the right quantities, the right environment is maintained.”

The differing requirements from regulatory bodies is also a significant issue.

While there have been some examples of backstepping among Chinese regulators in asking for animal testing, “India has been a little more progressive in looking at openly applying some of these in vitro methods to replace in vivo models.”

Where there has been some success in regulatory advancement and convergence is in the hepatitis B and Hib components of the pentavalent vaccine.

The hepatitis B vaccine is a recombinant protein, “almost like a well-characterized biologic,” Prasad explained. However, while the tools are available, “it still took a long time to do the collaborative study with the WHO to get the in vitro method approved as the release step.” As a conjugate vaccine, Hib is “more chemistry than biology” and “significant parts of its analytics can be done with HPLC and things of that nature,” allowing for more consensus.

A Huge Volume of Animals Are Used in DTP Potency Testing

However, Prasad noted, “there still remains the issue of the legacy vaccines – diphtheria, tetanus, and pertussis – for which we do not have good answers at this point.”

Exacerbating the problems in the animal potency tests is that the results take two months to become available. This creates “a lot of shelf life issues,” and if the test has to be repeated, “that takes away almost half of your shelf life with many of these vaccines.”

As such, the BMGF’s focus is on diptheria, tetanus and pertussis (DTP) in trying to implement the 3R principles of “refine, reduce, and replace” for animal testing.

Also in focus as part of the foundation’s efforts to eradicate polio, Prasad noted, is the release testing for the polio vaccine in which large volumes of animals are also required.  Many countries are still requiring a rat immunogenicity test, which has significant variability.

In turn, the objective of the GHF workshop, he stressed, is “to review the status of what is currently being used, what is currently available,” and to look for opportunities to promote alternative methods that meet the 3R objectives.

Prasad then reviewed the huge volume of animals involved in the DTP potency analyses.

Involved in tetanus and diptheria vaccines are lethal intradermal and paralytics challenge tests and serological tests in mice and guinea pigs. In the case of hepatitis B, about 180 animals per batch are still being used, which for 100 million doses “is a ton of animals to deal with,” Prasad stressed.

He went on to point out the wide variability in the allowed safety testing parameters for these vaccines, which is in the 50-200% range. “You can drive a truck through that. Are we really establishing the quality of the vaccine with this testing or not? But that is the standard we have.”

The overall challenge is to maintain a sustainable supply of these vaccines at the highest quality possible, and keep up with the technology and innovation, the Gates official concluded.

He praised the ongoing global collaborative efforts that organizations attending the workshop like FDA, WHO, the International Alliance for Biological Standardization (IABS), and the Humane Society International are engaged in to further the understanding and find ways to progress and harmonize the testing requirements.

During his presentation, Prasad highlighted the close partnership the Gates Foundation has with WHO in supporting global health and the availability of quality medicines around the world. He noted that Gates support for WHO was only exceeded by the US, which currently provides about 15% of the overall WHO funding.

Following up on comments made beginning in April, US President Donald Trump announced at the end of May his decision that the US will drop out of the WHO and cease the funding due to his dissatisfaction with WHO’s early handling of the pandemic and alleged favoring of China in its response. However, health policy leaders on Capitol Hill have expressed strong disagreement with the decision and are asserting that the President does not have the right to make the decision without Congressional approval.

Are There True Positives in Animal Studies?   

During the Q&A that followed his remarks, Prasad had a chance to explain that the survey that was done spotlighting the animal testing problem was among 14 of the vaccine manufacturers in the DCVMN that were the biggest suppliers of the legacy vaccines and had WHO prequalification status.

Noting that the survey did not include testing labs, Prasad said that he would be interested in getting further feedback from the workshop and its breakout sessions on the efforts that have been working in moving away from in vivo testing. “Collectively we will look for the right approaches. The foundation is extremely interested in this area. We support those efforts so that it helps our partners to reduce their costs.”

Another workshop participant asked the provocative question of whether in all the testing that Prasad has done he had ever seen a true positive in an animal study.

“Not really,” he responded. The problem is if one animal dies on the table, “you do not know what to do with it, so you call it invalid.”

While there are ways to repeat the test with an additional number of animals following a protocol, “many times what happens is, by the time you resolve it, you have already lost the batch anyway. It is actually in many cases cheaper to throw away the batch than to do the repeat testing – do the investigation necessary to satisfy the regulators – even for an invalid test. So like I said, we were budgeting for about 20% wastage overall for vaccines.”

Industry consultant Tim Schofield, who would later present on the scientific/statistical issues around bridging the gap between in vivo and in vitro assays, commented that among the reasons for replacing the animal tests is to have in vitro methods that would be more sensitive to finding the true positives.

In his talk, Schofield stressed that the inherent variability of the in vivo assays does make the bridging to in vitro methods difficult. He went on to propose a “consistency approach” using a holistic understanding of the process and market experience to establish in vitro limits.

Insights Provided on WHO’s Priorities and Collaborative Efforts

Presenting virtually at the GHF workshop after Prasad was WHO Norms and Standards Biologicals Group Lead Ivana Knezevic.

Her presentation encompassed insights on: ● WHO’s organization and priorities, including its efforts to assist agencies in regulatory system strengthening ● how WHO helps with norms and standards setting through guidance documents, measurement standards, collaboration with partners, and implementation efforts ● current practice and regulatory divergence for animal testing ● developments in rabies vaccine testing and the discontinuance of the innocuity test, and ● the opportunities and challenges of global regulatory convergence around reducing animal testing. [A link to the full remarks by Knezevic at the workshop.]

Knezevic explained how WHO is responsible for providing leadership for the UN and its 194 member states on global health matters. In addition to setting and implementing norms and standards, this includes shaping the health research agenda, articulating evidence-based policy options, providing technical support to countries, and monitoring and assessing health trends.

Vaccine lot release, she stressed, represents “a huge undertaking in terms of animal testing [and] has been identified as potentially the most important [area] if we want to make an impact and really help countries change the current practice.”

Also the issue of post-approval changes, she stressed, “is now becoming more and more important” in the wake of the guideline WHO released for post-approval changes of vaccines, which has high relevance to the issue of animal testing.

Moving From Regulatory Divergence to Convergence

In her concluding remarks on the opportunities and challenges around regulatory convergence, she pointed out that WHO sees “quite a lot of space and opportunity to improve the situation and move from divergence to convergence by using the definitions that WHO provides, international standards, and also more and more educational and training tools for improving expertise – because the expertise of regulators in countries is really critical.”

Having the relevant expertise allows regulators to be more flexible in applying the principles in WHO guidance documents. “They can think in the context of case-by-case scenarios and make relevant decisions for each case of vaccine evaluation.”

International and regional organizations also present the opportunity to take the vaccine testing issues forward, Knezevic stressed.

Those with which WHO has been working with include the Pan American Network for Drug Regulatory Harmonization (PANDRH), the African Vaccine Regulatory Forum (AVAREF), the Association of Southeast Asian Nations (ASEAN), the Asia-Pacific Economic Cooperation (APEC) Harmonization Center, and the International Pharmaceutical Regulators Programme (IPRP). On the industry association side, these include the DCVMN and the International Federation of Pharmaceutical Manufacturers and Associations (IFPMA). Pharmacopeias, she noted, “are also very good collaborators in these areas of work,” as also is IABS.

She pointed out that WHO has wanted for several years to review and compare the current approaches in UN member states as a way forward in the animal testing arena, but the project has been postponed due to a lack of resources. Needed, she said, is the establishment of a WHO working group that could initiate systematic work with regulators, manufacturers, academia, and other relevant expert groups on the topic.

WHO sees the way forward as including further development and implementation of alternatives to in vivo potency assays – such as serological assays, ELISA, and biochemical methods – and methodological advances – such as the use of synthetic materials instead of animal based substances, and NGS for adventitious agent control.

Of importance is the strengthening of collaborations to promote science-based regulation and optimal testing. Also beneficial would be expanding the concept of mutual regulator reliance, which the International Conference of Drug Regulatory Authorities (ICDRA) has recognized as a very important mechanism for reducing redundancy and duplication at all levels – “not just in the context of animal testing, but really in all areas of regulatory function.”

FDA Has Made Substantial Efforts to Support In Vitro Assay Development

Following Knezevic’s was a presentation by BioReliance Principal Scientist Kathy Remington on replacing animal-based safety tests.

Remington explored the degenerate PCR approach that FDA has recommended for immune end-point detection as an alternative to animal-based testing. She highlighted the advantages the approach offers, including accelerated detection, increased specificity and sensitivity, as well as the potential to discover new variants of viruses.

CBER Division of Viral Products Deputy Director Robin Levis then reviewed the “smorgasbord” of collaborative activities with which FDA has been engaged to advance in vitro assay development for vaccines.

She discussed: ● the removal of the general safety test (GST) ● moving from monkey to rat-based mumps virus vaccine safety tests ● conventional adventitious agent testing versus NGS, and the collaborative work the Advanced Virus Detection Technologies Interest Group (AVDTIG) has been doing to advance NGS ● the changes that have been adopted in acellular pertussis vaccine safety testing, and ● the ongoing efforts to replace the animal-based immunogenicity assays for rabies vaccine potency vaccine testing with in vitro ELISA-based assays. [A link to Levis’ complete remarks at the GHF workshop is provided below.]

PATH Center for Vaccine Innovation and Access Senior Scientist Bob Sitrin followed Knezevic to the podium to discuss the replacement of the animal-based potency test for lot release of Merck’s HPV vaccine Gardasil as a case study.

The development of mononclonal antibody-based assays for evaluating vaccine potency was then discussed by Wadsworth Center’s Nicholas Mantis, using the vaccine RiVax against ricin toxin-A as an example.

Consultant Schofield then followed with his review of the issues in bridging in vivo and in vitro assays.

The final presentation at the workshop was given by former CBER and NIH vaccine expert Rebecca Sheets, now with the consulting firm Grimalkin Partners. Sheets explored the problems with the current in vivo adventitious agent testing, and the need for moving to cell-based production and reviewing the whole rationale for why toxicology studies are accepted as the appropriate scientific approach.

Modern techniques such as genomics, transcriptomics, sequencing and micro-array exist, she pointed out, that – while presenting validation and data analysis challenges – are capable of improved sensitivity and detecting a greater variety of adventitious agents and need to reach acceptance by regulatory agencies around the world.

Four breakout sessions then followed at the workshop to further explore the issues around: ● the advances made by biopharmaceutical manufacturers for vaccine release ● the in vitro adventitious release testing for vaccines ● regulatory considerations for non-animal-based release tests, and ● vaccine potency assays.

NIIMBL CARES Act Grant Further Supports Testing Efforts

At the end of May 2020 – shortly after the 14 new 3.1 project grants were announced, including the two supported by the GHF – NIIMBL announced that it had been awarded about $9 million from NIST to fund high-impact projects that will support the US response to the COVID-19 pandemic. The funding is being made available through the Coronavirus Aid, Relief, and Economic Security (CARES) Act passed by Congress in late March.

The NIIMBL release explains that along with enhancing coronavirus diagnostic, protection, and facility and worker contamination control,  the projects will focus on “the development of flexible manufacturing capabilities for biologic therapies and rapid release testing to position the nation for faster medical product scale-up in response to pandemics.”

With its focus on biopharmaceutical manufacturing innovation, the release states, “NIIMBL is uniquely positioned to assist with the pandemic response. Its network of more than 150 members nationwide — including 10 leading global pharmaceutical manufacturers, small businesses, and academic and federal scientists — can leverage flexible, agile and cost-effective advanced manufacturing technology.”

NIIMBL Director Kelvin Lee commented that “never in our lifetime have we experienced a global crisis of this magnitude. It presents a massive challenge…that I deeply believe we are positioned to help meet.”

NIIMBL’s governing committee includes federal agency representatives from FDA, the National Cancer Institute, the Department of Defense, the National Science Foundation, and NIST. NIIMBL and NIST are also working with the National Institutes of Health (NIH) and Biomedical Advanced Research and Development Authority (BARDA) “to help ensure maximum impact from this investment,” the release explains.