Implementing Improved Analytical Methods To Support Vaccine Quality

By Maura C. Kibbey, Ph.D., U.S. Pharmacopeia (USP)

Advancements in analytical technology can improve testing paradigms in vaccine development, while also supporting existing vaccine manufacturing and quality control. A key impediment to implementing new methods for characterizing and releasing licensed vaccines, however, is submitting updates to regulators across multiple jurisdictions. This impediment and perceived regulatory risk can deter manufacturers from adopting new technologies to ensure quality in older products; nonetheless, these tools can be very powerful, bringing efficiencies and better data to help inform decisions and speed new vaccines and therapeutics to patients.

In less than one year since the emergence of the devastating global pandemic due to the coronavirus SARS-CoV-2, scientists have developed almost 200 candidate vaccines against this virus at an unprecedented speed.¹ This pace of vaccine development is supported by significant advances in multiple manufacturing platforms and analytical technologies. While these vaccines deliver SARS-CoV-2 antigens to patients in diverse ways, each one must be rigorously characterized and controlled in order to ensure their safety, efficacy, and quality prior to marketing. Some of the new COVID-19-targeted vaccines are spurring the use of novel analytical technologies to support rapid and thorough characterization, which may help pave the way for other vaccine manufacturers to implement these technologies as well. Now more than ever, it is important to develop and improve tests and analytical solutions that ensure the quality of vaccines and gain the public’s trust.

For over 200 years, USP’s mission has been focused on quality standards and tools that support the public’s trust and access to quality medicines. In addition to its standard setting role, USP has recently offered analytical support to companies that are developing new COVID-19-targeted therapeutics and vaccines. USP has also convened an interdisciplinary Vaccine Advisory Group to advise USP on the current pandemic and how USP can support the industry moving forward to ensure that vaccines are available to patients without delay. USP is partnering with stakeholders globally to support adoption of improved analytical solutions for testing and demonstrating the quality of vaccines and other biologics.

As part of this initiative, USP convened a two-day webinar series, Implementing Improved Analytical Methods to Support Vaccine Quality, in September 2020. The webinars were part of the 2020 World Vaccine Congress and included a panel of experts who brought diverse perspectives from both industry and regulatory authorities.

The summary below includes presentation highlights as well as key takeaways from the panel discussions.

International Standards Supporting Vaccine Testing

Earl Zablackis, Ph.D., director-principal scientist at Sanofi Pasteur and currently chair of the USP Biologics 3 Expert Committee for Complex Biologics & Vaccines, provided opening remarks on day one. To help frame the workshop, Zablackis reviewed the existing compendial chapters and international guidance documents that support vaccine testing. Currently, the USP–NF does not contain product-specific vaccine monographs but does include one test chapter for bacterial polysaccharide identity testing as well as several informational general chapters that provide guidance on development, testing, and manufacturing of several classes of vaccine products (summarized in Table 1). There are also additional test chapters that support vaccines and other therapeutics such as those found in USP chapters <85> Bacterial Endotoxins Test, <71> Sterility Tests, <791> pH, and others. Additional standards are needed to support the adoption of new analytical methods that provide benefits beyond traditional methods for testing vaccine quality. For example, reference standards are needed to support existing USP chapters as well as serve as controls for in-house tests developed by scientists. Additionally, reference standards, test chapters, and informational chapters are needed to support and advance the new vaccine platforms that are being introduced in response to COVID-19 (e.g., mRNA, virus-like particles, and others).

Table 1: USP Chapters That Support Vaccine Quality

USP–NF General Chapter	Status
<198> Nuclear Magnetic Resonance Spectroscopy Identity Testing of Bacterial Polysaccharides Used in Vaccine Manufacture	Official
<1234> Vaccines for Human Use—Polysaccharide and Glycoconjugate Vaccines	Official
<1235> Vaccines for Human Use—General Considerations	Official
<1238> Vaccines for Human Use—Bacterial Vaccines	Official
<1239> Vaccines for Human Use—Viral Vaccines	Will be official May 1, 2021, in USP-NF 2021, Issue 1
<1430.x> Analytical Methodologies Based on Scattering Phenomena	Official/PF*

PF, Pharmacopeial Forum; * <1430.3> and <1430.6> are currently available for public review and comment in PF.

Measuring Molecular Size — Not as Easy as You Would Think

Zablackis also presented Sanofi’s strategy to improve measurements of vaccine conjugate size, from classical methods such as determining distribution coefficients in gel filtration to direct measures of molecular size. High-pressure size-exclusion chromatography in combination with ultraviolet, viscometry, refractive index, and light-scattering detectors (e.g., multiangle laser light scattering) are faster methods that require less material. Furthermore, these methods can measure multiple characteristics, including polydispersity, molecular weight, hydrodynamic radius, and radius of gyration. To obtain accurate measurements, however, these methods require specific refractive index increment (dn/dc) values. These methods are precise, stability indicating, and robust when used in combination with appropriate sizing standards.

Using NMR Assays for Characterization of Polysaccharide-based Vaccines

Francesco Berti, Ph.D., scientific director, Technical R&D, GSK, reviewed the use of nuclear magnetic resonance (NMR) assays for characterizing polysaccharide-based vaccines both early in development as well as part of the control strategy. Berti stated that NMR spectroscopy can be implemented to confirm structural identity and conformity of polysaccharide antigens, to quantitate decorative groups (e.g., O-acetyl), and to measure polysaccharide content. NMR can also be used to quantify process- and product-related impurities (e.g., ethanol and dimethyl sulfoxide). Based on the work described by Berti as well as other scientists in the USP Expert Panel for NMR Identity Testing of Glycoconjugate Vaccines led by Chris Jones, Ph.D., a USP–NF test chapter <198> was developed. This chapter includes tests, system suitability criteria, and supporting reference standards for NMR identity testing of bacterial polysaccharides used in vaccines. Future revisions of the chapter are in progress that will incorporate additional acceptance criteria for specific bacterial polysaccharides used in vaccine products.

Substituting In Vitro for In Vivo Vaccine Potency and Safety Assays

Dean Smith, Ph.D., senior scientific valuator for vaccines, Health Canada, reviewed a new strategy described in Ph. Eur. general chapter 5.2.14 to substitute in vitro methods for existing in vivo potency and safety assays, where traditional one-to-one assay replacement is not feasible or scientifically justified. The primary focus for implementing any proposed in vitro method(s) within a quality control (QC) strategy should be the scientific relevance of the in vitro assay(s) to control critical quality attributes (CQAs). The guidance recognizes that using appropriate in vitro methods, manufacturers can develop quality data to demonstrate that CQAs for a product will be well controlled over the vaccine’s shelf life in the absence of in vivo methods. The general chapter explicitly acknowledges the limitations of in vivo methods, such as their inherent variability, that make them less suitable for QC than appropriately designed in vitro alternatives. Multiple in vitro methods may be required to adequately characterize the CQAs measured by an existing in vivo method; however, appropriate in vitro assays would still be more precise, robust, and efficient in doing so relative to existing in vivo methods. With the implementation of Ph. Eur. 5.2.14, the scientific relevance of long-standing in vivo methods has been challenged and several have now been removed from the Ph. Eur. Additionally, “substitution” provides a new framework for the approval of alternate potency and safety assays under development for DPT vaccines by the VAC2VAC consortium.

Regulatory Perspective on NGS for Adventitious Virus Detection in Biologics

Arifa Khan, Ph.D., supervisory microbiologist, Center for Biologics Evaluation and Research, U.S. FDA, provided a regulatory perspective on using next-generation-sequencing (NGS) for detection of adventitious viruses in vaccines. There is a need for improved analytical methods for sensitive and broad virus detection, including known viruses, unknown, and unexpected viruses, and for investigations of reverse transcriptase activity for presence of retroviruses. NGS is a powerful, relatively new technology capable of detecting known and unknown viruses without prior sequence knowledge. The FDA has developed reference viruses, which are under consideration by the World Health Organization as International Reference Standards for adventitious virus detection by NGS,² and a reference viral database³ to enhance detection of known and novel viruses. Conventional assays for detecting adventitious viruses can be supplemented, substituted, or even replaced by NGS—with proper justification for suitability and fit for purpose.

Strategies and Examples for Vaccine Development and Lifecycle Management

Sabrina Restrepo, Ph.D., director, Global Vaccines Technical Operations, Merck, reviewed a draft technical report that is being developed by the Vaccines Interest Group of the Parenteral Drug Association. The report will provide strategies and examples for both vaccine development and life cycle management. Restrepo emphasized that ideally the CQAs should be defined in discovery and drive process development and analytical testing strategies. She also reviewed considerations for a hypothetical situation: rapidly responding to an emerging threat. By leveraging existing process and product knowledge, manufacturers can expedite development timelines while making improvements to formulation (e.g., moving away from frozen storage requirements) post-licensure if necessary.

Using Overall Control Strategy to Develop and Advance New Vaccine Platforms

Marta Germano, Ph.D., scientific director and group head of product characterization, Janssen Vaccines, presented on the overall control strategy as a tool to develop and advance new vaccine platforms. Advanced analytical technologies are instrumental for gaining sufficient product and process knowledge for a well-controlled process and for developing an effective control strategy; however, due to their increased sensitivity and occasional difficulty in accurately measuring the diverse sample matrices encountered during in-process testing, their use must be carefully considered. For example, if a new minor product variant is detected when a sensitive technology is added, determining its effect, if at all, on product quality can be challenging. Nonetheless, the knowledge gained from these technologies can decrease the residual risk of the overall control strategy. Germano also reviewed the development of the AdVac/PER.C6 vaccine platform, which is used in the company’s licensed Ebola vaccines as well as other vaccines in development, including one for SARS-CoV-2. She shared the analytical testing strategy, of which most were common tests based on the platform, with only a few tests that are specific to production of the viral antigen of interest (e.g., sequencing for genome identity and others). This approach streamlines development, manufacturing, and testing of new vaccines tremendously.

Potency Testing for Vaccines: Challenges and Opportunities

Jean-Francois Dierick, Ph.D., global subject matter expert, analytical method validation, and Xianzhi Zhou, Ph.D., bioassay manager, GSK, presented challenges and opportunities—from development to commercial life cycle—for vaccine potency testing. Dierick used examples of products in development and commercial stages as case studies to describe the replacement of in vivo assays with in vitro assays. For licensed products, some in vivo and in vitro potency tests were run in parallel during both clinical and stability testing and then these data were used to justify the advantageous replacement of the in vivo assay at licensure. These methods are also complemented with a robust antigen characterization package that supports the approach. In another example, an in vitro assay was introduced shortly after the clinical testing and run in parallel for a time with the in vivo potency test before justifying removal of the in vivo test. In this case, clinical samples were still available to go back and test with the in vitro assay as well. Moving forward, in most recent product development strategies, in vitro potency assays are developed and used solely for clinical and stability data after analysis of preclinical in vivo studies to justify the strategy and critical reagents selected for the in vitro potency assay.  Dierick emphasized that collaboration between manufacturers and regulatory authorities was a key enabler for replacement of in vivo assays. Zhou presented a specific example of replacing an in vivo potency assay with a robust 24-hour assay using a recombinant virus that encodes firefly luciferase. This approach allows rapid assessment of neutralization activity in a robust, precise manner without the biosafety concerns of the live target virus.

Implementing Novel Analytical Technologies for Viral Vector-based Vaccines

Mark van Ooij, Ph.D., scientific director, PER.C6 technical lead and head of platform innovation and implementation, Janssen, presented an industry perspective on implementing novel analytical technologies for viral vector-based vaccines. Van Ooij reviewed technological advances in analytical equipment, considerations for method selection, and regulatory aspects related to novel analytical technologies. Particle aggregation and protein analyses were reviewed to demonstrate how to select appropriate analytical technologies. He also shared that despite some of the hurdles implementing new technologies, some, such as those using detection by mass spectrometry (MS), can be used to measure several quality attributes at one time (“multi-attribute methods”) and could replace several tests so the effort can pay off over the long term.

It Takes A Village To Advance New Initiatives

During panel sessions at the close of each day, Dirk Redlich, Ph.D., vice president, global head technical development and clinical trial material manufacturing vaccines, Infectious Diseases and Vaccines Therapeutic Area, at Janssen, led discussions with the speakers that emphasized a village of industry, academia, regulatory partners, CRO/CMOs, instrument vendors, and standard setting organizations like USP are all important partners to advance new initiatives. In this spirit and based on audience questions, the following points were highlighted.

Moving New Analytical Technologies into QC Labs

NMR and MS have recently gained acceptance in QC environments as the instruments have become more robust, the software compliant with requirements such as those in 21 CFR part 11 and others, and the newer versions amenable to a QC environment. In addition to identity testing of polysaccharides in vaccines, NMR has also become important in a GMP environment for convenient and sensitive control of raw materials. The technology is faster and produces higher-quality results than traditional methods. Many technological advances also start in academic laboratories (e.g., the use of capillary electrophoresis to test adenovirus-based vaccines) and then are further developed and validated in pharmaceutical laboratories, so it is important to bring together all parties to address these problems.

Leveraging Existing Knowledge to Quickly Respond to COVID-19

Over the past 10 years, industry and regulators have been gaining experience and sharing knowledge about new analytical technologies, including NGS. Just like the current COVID-19 acceleration of supporting technologies, discovery of a porcine circovirus in a rotavirus vaccine about 10 years ago by using NGS spurred recognition of its power, and many discussions since then brought NGS to where it is today. While these technologies are still evolving, specific applications can be realized. For example, NGS was sufficiently developed to be deployed as a rapid assay for adventitious virus detection in COVID-19 vaccines. 

The pandemic also created an environment that advanced new potency assay development within existing regulatory frameworks. To support COVID-19 vaccines, many sponsors have shifted from in vivo to in vitro potency assays. Manufacturers were encouraged to continue this trend of pivoting away from highly variable in vivo potency assays in the QC environment.

Interagency Collaboration Can Expedite the Replacement of Conventional QC Assays

To replace a conventional QC method with a new technology (e.g., MS), manufacturers have traditionally been expected to run both methods side by side for a while, which is a resource-intensive and time-consuming activity. However, informal interagency collaboration and information sharing can help expedite this process, reducing the parallel track of analogous testing across multiple jurisdictions. Manufacturers were encouraged to let regulators share data among themselves when licensing applications are submitted in multiple jurisdictions to help speed up approvals and harmonize requirements.

Another driver for advancing new technologies and harmonizing specifications is collaboration across regulatory agencies. While agencies may not be in the position to conduct joint reviews, sponsors should consider simultaneous licensing applications in multiple jurisdictions and allow each regulatory body to exchange positions and share information with each other. The International Coalition of Medicines Regulatory Authorities (ICMRA) has had success sharing approaches to COVID-19 vaccine regulation, and such collaboration would be valuable if extended to other areas following the pandemic.

Next Steps: Advancing Quality Tests, Standards, And Knowledge

The panel members encouraged leveraging the knowledge of all stakeholders through both open forums and confidential discussions to advance new analytical technologies. USP is committed to supporting stakeholders by efforts to not only increase public trust in vaccines but also to facilitate the adoption of new methods that demonstrate quality and expedite vaccine development. Vaccine development and manufacturing experts are encouraged to engage with USP on developing new and revising existing standards that support advanced analytical technologies to further the quality and efficient development of vaccines. As new approaches evolve, USP will support the spread and implementation of these new technologies and approaches by additional educational offerings that help ensure quality from the very beginning of development to global distribution of licensed products.

References:

1. https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines
2. https://www.who.int/publications/m/item/WHOBS2020-2394
3. https://rvdb.dbi.udel.edu/

About The Author:

Maura Kibbey, Ph.D., is senior scientific fellow for education and training in USP’s Global Biologics department. She leads development of courses, workshops, and forums to engage USP’s biologics stakeholders. This role builds on her previous responsibilities directing USP scientists developing compendial standards. Before joining USP, Kibbey worked for several biotechnology and diagnostic companies in the Washington, D.C., area in scientific, management, marketing, and business development roles, as well as performing cancer research at the National Institutes of Health. She has published over 40 peer-reviewed articles and has been an invited speaker or workshop organizer for numerous scientific conferences. You can reach her at her at mck@usp.org.

The US Pharmacopeia (USP) is an independent, nonprofit, scientific organization that collaborates with the world's top experts in health and science to develop quality standards for medicines, dietary supplements, and food ingredients. Through our standards, advocacy and education, USP helps increase the availability of quality medicines, supplements and food for billions of people worldwide.

Conflict of interest: Francesco Berti, Jean-François Dierick, and Xianzhi Zhou are employees of the GSK group of companies.