Maintaining the quality of vaccines through the use of standards: Current challenges and future opportunities.

An international hybrid meeting held 21-22 June 2023 in Ottawa, Canada brought together regulators, scientists, and industry experts to discuss a set of principles and best practices in the development and implementation of standards. Although the use of international standards (ISs) and international units (IUs) has been an essential part of ensuring human and animal vaccine quality in the past decades, the types and uses of standards have expanded with technological advances in manufacture and testing of vaccines. The needs of stakeholders are evolving in response to the ever-increasing complexity, diversity, and number of vaccine products as well as increasing efforts to replace animal-based potency tests with in vitro assays that measure relevant quality attributes. As such, there must be a concomitant evolution in the design and implementation of both international and in-house standards. Concomitantly, greater harmonization of regulatory expectations must be achieved through collaboration with standard-setting organizations, national control laboratories and manufacturers. Stakeholders provided perspectives on challenges and several recommendations emerged as essential to advancing agreed upon objectives.

Collaborative study for the calibration of a replacement International Standard for Diphtheria Antitoxin Equine.

The International Standard for Diphtheria Antitoxin Equine is essential for the standardisation of assays used to determine the potency of therapeutic diphtheria antitoxin products produced from equine serum. This paper describes the production and characterization of the 2nd International Standard for Diphtheria Antitoxin Equine and its calibration in International Units. Calibration was performed by toxin neutralization test in vivo and in vitro (Vero cell assay), and potency was expressed relative to the 1st International Standard to ensure continuity of the International Unit. The candidate standard (NIBSC product code 18/180) was assigned a unitage of 57 IU/ampoule based on results from 14 laboratories in 9 different countries and was established by the World Health Organisation Expert Committee on Biological Standardization in 2021.

Collaborative study for the calibration of a replacement International Standard for tetanus toxoid for use in flocculation test.

Here we report the results of a study to establish a replacement WHO International Standard (IS) for tetanus toxoid for use in flocculation test. The standard was calibrated in flocculation units (Lf) against the 2nd IS using the Ramon flocculation method. At its 70th meeting in October 2019, WHO ECBS established the material (coded 16/302) as the 3rd WHO IS, with an assigned value of 970 Lf/ampoule from the results of seventeen laboratories across ten different countries. The study also provided an opportunity to assess the use of alternative methods for measuring Lf. Participants were asked to use an in-house Enzyme Linked Immunosorbent Assay (ELISA) developed at NIBSC, or other suitable in-house methods, to determine ELISA-specific Lf values (Lf-eq units are specific only for pre-calibration of antitoxin in the flocculation test) of 16/302 to compare to those of the flocculation test. Nine laboratories participated by performing the NIBSC ELISA, one laboratory performed flocculation by laser light-scattering following an in-house protocol, and three laboratories performed ELISA following in-house protocols. The results intimate that these alternative methods could be useful for monitoring consistency of production at different stages of vaccine manufacturing.

Integrating 3Rs approaches in WHO guidelines for the batch release testing of biologicals: Responses from a survey of vaccines and biological therapeutics manufacturers.

The UK National Centre for the Replacement, Refinement, and Reduction of Animals in Research (NC3Rs) has been tasked by the World Health Organization (WHO) to review the extent to which animal-based testing methods are described in their manuals, guidelines and recommendations for vaccines and biotherapeutics. The aim is to identify and recommend where updates to these documents can lead to an increased and more harmonised adoption of 3Rs principles (i.e. Replacement, Reduction and Refinement of animal tests) in the quality control and batch release testing requirements for vaccines and biotherapeutics. Developing recommendations that are widely applicable by both the manufacturers and national regulatory authorities for vaccines and biologicals globally requires a detailed understanding of how different organisations view the opportunities and barriers to better integration of the 3Rs. To facilitate this, we developed and distributed a survey aimed at vaccine and biotherapeutics manufacturers in July 2021. In this paper, we present the key findings from this survey and how these will help inform the recommendations for wider integration of 3Rs approaches by WHO in their guidance documents applicable to the quality control and batch testing of vaccines and biotherapeutics.

3Rs implementation in veterinary vaccine batch-release testing: Current state-of-the-art and future opportunities. A webinar and workshop report.

Regulatory authorities require veterinary batch-release testing to confirm vaccine potency and safety, but these tests have traditionally relied on large numbers of laboratory animals. Advances in vaccine research and development offer increasing opportunities to replace in vivo testing, and some stakeholders have made significant progress in incorporating 3Rs elements in quality control strategies. A three-part event series entitled "3Rs Implementation in Veterinary Vaccine Batch-Release Testing: Current state-of-the-art and future opportunities" was jointly organized by the Animal-Free Safety Assessment Collaboration, HealthforAnimals, and the International Alliance of Biological Standardization. Two webinars and a workshop aimed to outline the state-of-the-art non-animal approaches for veterinary batch-release testing. The events included information on the state of the deletion of obsolete safety testing and the current initiatives implemented by European, North American, and Asian-Pacific stakeholders on 3Rs implementation and regulatory acceptance. The events contributed to a better understanding of the barriers to 3Rs implementation. Participants highlighted the need for open communication, continued collaboration between stakeholders, and international harmonization of regulatory requirements to help accelerate acceptance. Despite the challenges, the countries represented at this three-part event have shared their commitments to advancing the acceptance of alternative methods.

Characterisation of diphtheria monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Immunoassays are used for routine potency assessment of several vaccines, in some cases having been specifically developed as alternatives to in vivo potency tests. These methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against diphtheria with a view to select antibodies that can be used for development of an in vitro potency immunoassay for diphtheria vaccines. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined by a cell-based toxin neutralisation test and diphtheria toxin-domain recognition was determined by Western blotting. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Characterisation of tetanus monoclonal antibodies as a first step towards the development of an in vitro vaccine potency immunoassay.

Batch release testing for human and veterinary tetanus vaccines still relies heavily on methods that involve animals, particularly for potency testing. The quantity and quality of tetanus antigen present in these products is of utmost importance for product safety and clinical effect. Immunochemical methods that measure consistency of antigen content and quality, potentially as an indicator of potency, could be a better choice and negate the need for an in vivo potency test. These immunochemical methods require at least one well characterised monoclonal antibody (mAb) that is specific for the target antigen. In this paper we report the results of the comprehensive characterisation of a panel of mAbs against tetanus with a view to select antibodies that can be used for development of an in vitro potency immunoassay. We have assessed binding of the antibodies to native antigen (toxin), detoxified antigen (toxoid), adsorbed antigen and heat-altered antigen. Antibody function was determined using an in-house cell-based neutralisation assay to support prior in vivo potency data that was available for some, but not all, of the antibodies. In addition, antibody affinity was measured, and epitope competition analysis was performed to identify pairs of antibodies that could be deployed in a sandwich immunoassay format. Not all characterisation tests provided evidence of "superiority" of one mAb over another, but together the results from all characterisation studies allowed for selection of an antibody pair to be taken forward to assay development.

Animal testing for vaccines. Implementing replacement, reduction and refinement: challenges and priorities.

Transition to in vitro alternative methods from in vivo in vaccine release testing and characterization, the implementation of the consistency approach, and a drive towards international harmonization of regulatory requirements are most pressing needs in the field of vaccines. It is critical for global vaccine community to work together to secure effective progress towards animal welfare and to ensure that vaccines of ever higher quality can reach the populations in need in the shortest possible timeframe. Advancements in the field, case studies, and experiences from Low and Middle Income Countries (LMIC) were the topics discussed by an international gathering of experts during a recent conference titled "Animal Testing for Vaccines - Implementing Replacement, Reduction and Refinement: Challenges and Priorities". This conference was organized by the International Alliance for Biological Standardization (IABS), and held in Bangkok, Thailand on December 3 and 4 2019. Participants comprised stakeholders from many parts of the world, including vaccine developers, manufacturers and regulators from Asia, Europe, North America, Australia and New Zealand. In interactive workshops and vibrant panel discussions, the attendees worked together to identify the remaining barriers to validation, acceptance and implementation of alternative methods, and how harmonization could be promoted, especially for LMICs.

Evaluation of a capture antigen ELISA for the characterisation of tetanus vaccines for veterinary use.

We previously developed an ELISA assay for detection of tetanus toxoid antigen in tetanus vaccines for human use. Tetanus vaccines for veterinary use are qualitatively different to those used in humans, often containing a larger number and variety of non-tetanus antigens in the multi-valent products, and adjuvants that are not found in human vaccines. We assessed performance of the capture ELISA with a range of veterinary tetanus vaccines as a first step towards development of an immunoassay as a potential in vivo potency substitute. Nine tetanus vaccines were tested and all produced a good dose response in the ELISA. The shape of the dose response curve for the whole vaccine compared to a matched non-adjuvanted tetanus toxoid antigen was more comparable for vaccines containing a non-aluminium adjuvant than products containing aluminium adjuvants. Elution of the antigen from aluminium adjuvant did not improve the comparability of the dose response curve but did increase the total amount of tetanus antigen available for detection. The ELISA was highly specific for tetanus with no signal obtained for a large number of non-tetanus antigens. These results suggest that a capture ELISA assay can be applied to a control strategy for veterinary tetanus vaccines.

Modern science for better quality control of medicinal products "Towards global harmonization of 3Rs in biologicals": The report of an EPAA workshop.

This article summarizes the outcome of an international workshop organized by the European Partnership for Alternative Approaches to Animal Testing (EPAA) on Modern science for better quality control of medicinal products: Towards global harmonization of 3Rs in biologicals. As regards the safety testing of biologicals, the workshop participants agreed to actively encourage the deletion of abnormal toxicity tests and target animal batch safety tests from all relevant legal requirements and guidance documents (country-specific guidelines, pharmacopoeia monographs, WHO recommendations). To facilitate the global regulatory acceptance of non-animal methods for the potency testing of, e.g., human diphtheria and tetanus vaccines and veterinary swine erysipelas vaccines, international convergence on the scientific principles of the use of appropriately validated in vitro assays for replacing in vivo methods was identified as an overarching goal. The establishment of scientific requirements for new assays was recognized as a further means to unify regulatory approaches in different jurisdictions. It was recommended to include key regulators and manufacturers early in the corresponding discussions. Manufacturers and responsible expert groups, e.g. at the European Directorate for the Quality of Medicines and Health Care of the Council of Europe or the European Medicines Agency, were invited to consider leadership for international collaboration.

Collaborative study for the calibration of a replacement International Standard for diphtheria toxoid for use in flocculation test.

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for diphtheria toxoid for use in flocculation test and its calibration in Lf units. Calibration was performed using Ramon flocculation method, standardized using the 2nd IS. The candidate standard was assigned a unitage of 1870 Lf/ampoule based on results from 25 laboratories in 15 different countries and was established as the 3rd IS for diphtheria toxoid for use in flocculation test by the WHO Expert Committee on Biological Standardization (ECBS) in October 2015. The study also assessed the use of alternative methods for measuring Lf. Participants were asked to determine the Lf value of the candidate standard using an Enzyme Linked Immunosorbent Assay (ELISA) established at NIBSC, or other suitable in-house method. 10 laboratories performed ELISA according to the NIBSC protocol, 1 laboratory performed flocculation using laser-light scattering according to an in-house protocol, and 1 laboratory performed another in-house ELISA. Results suggest these methods may provide suitable alternatives to the Ramon flocculation test, subject to validation, and that the new standard could act as a suitable reference preparation in these methods.

Development of a monoclonal antibody sandwich ELISA for the determination of antigen content and quality in diphtheria vaccines.

At present, quality control of diphtheria vaccines by both manufacturers and national control laboratories relies heavily on in vivo assays to confirm potency. As part of the VAC2VAC project we have developed a monoclonal antibody (mAb) enzyme-linked immunosorbent assay (ELISA) to measure the relative amount and quality of diphtheria toxoid (DTxd) in diphtheria-tetanus based vaccines and believe this test has the potential to play a key role in a control strategy no longer including an in vivo potency test. The mAb ELISA is highly specific, has good dilutional linearity, and is suitable for detecting DTxd in a range of different human vaccine products. We demonstrate the ability of the assay to discriminate between batches of different content and quality using vaccine batches that were prepared to contain differing amounts of DTxd or were altered by exposure to heat or oxidative stress. We also demonstrate successful transfer of the method to other laboratories and show that different diphtheria antigen materials may be able to serve as a reference antigen for local standardization of the method. The assay is ideally suited for incorporation into a consistency approach for routine diphtheria vaccine quality control testing and may be suitable to serve as the stability indicating test in replacement of the current in vivo potency test.

Diphtheria vaccines help to protect against diphtheria infection. Currently, animal tests are used to ensure the potency of such vaccines. Since these tests were first introduced, there have been improvements in non-animal technologies that can be used to ensure consistent production of potent vaccine batches. To demonstrate that a new batch of diphtheria vaccine is consistent with a previous batch of known potency, the quality and amount of the component that stimulates the immune response upon vaccination must be assessed in comparison. We have developed an assay that can measure the quality of a range of different diphtheria vaccine product types. The assay is very specific and reliable, and different laboratories obtained comparable results, showing that the assay is suited for routine use. Once validated by manufacturers and recognized by regulators, this assay will greatly reduce the number of animals needed for batch release of diphtheria vaccines.

Calibration and commutability assessment of the 1st International Standard for Diphtheria Antitoxin Human.

The 1st International Standard for Diphtheria Antitoxin Human (coded 10/262) was established by the World Health Organization Expert Committee on Biological Standardization in 2012. This paper describes the production, characterization and calibration of the new standard which is intended for use in the standardization of assays used to measure diphtheria antibody responses in human serum. The new standard was calibrated in terms of the International Standard for Diphtheria Antitoxin Equine in an international collaborative study. A total of 8 participants from 8 different countries performed in vivo and/or in vitro toxin neutralization tests and returned data that was used to assign units to the proposed new standard. The new standard has a diphtheria antitoxin potency of 2 IU/ampoule and is predicted to be stable. A follow up study was performed to assess commutability of the new standard. The follow up study was an existing external quality assessment, modified to include the new standard. Results obtained suggest that the new standard is commutable, showing comparable behaviour to native human serum samples in the majority of the assays compared, and is therefore suitable for use as a reference preparation in assays used to measure the level of anti-diphtheria antibodies in human serum.

Rational arguments for regulatory acceptance of consistency testing: benefits of non-animal testing over in vivo release testing of vaccines.

INTRODUCTION: There are rational arguments to replace existing in vivo potency and safety assays for batch release testing of vaccines with more advanced non-animal techniques to measure critical quality attributes. However, the introduction of in vitro alternatives to replace in vivo release assays of authorized vaccines is challenging. AREAS COVERED: This report describes the hurdles encountered in substituting in vivo assays and ways to overcome these and provides arguments why more advanced in vitro alternatives are superior, not only as a tool to monitor the quality of vaccines but also from a practical, economical, and ethical point of view. The rational arguments provided for regulatory acceptance can support a strategy to replace/substitute any in vivo batch release test if an appropriate non-animal testing strategy is available. EXPERT OPINION: For several vaccines, in vivo release assays have been replaced leading to an optimized control strategy. For other vaccines, new assays are being developed that can expect to be introduced within 5-10 years. From a scientific, logistical, and animal welfare perspective, it would be beneficial to substitute all existing in vivo batch release assays for vaccines. Given the challenges related to development, validation, and acceptance of new methods, and considering the relatively low prices of some legacy vaccines, this cannot be done without government incentives and supportive regulatory authorities from all regions.

Variability of in vivo potency assays of whole-cell pertussis, inactivated polio, and meningococcal B vaccines.

For the batch release of vaccines, potency release assays are required. Non-animal in vitro tests have numerous advantages and are preferred; however, several vaccines are still released using in vivo assays. Their major drawback is the inherent variability with its practical implications. We quantified the variability of in vivo potency release assays for whole-cell pertussis, inactivated polio and meningococcal B (MenB) vaccines which showed large CV (Coefficient of Variation) ranging from 34% to 125%. As inherent variability might potentially be attributed to the highly variable immune system between individual animals, we evaluated the antibody titres to four MenB antigens in 344 individual outbred mice. These varied strongly, with more than 100-fold differences in antibody titres in responsive mice. Furthermore, within individual mice there was generally no correlation between the strengths of the responses to the four antigens. A mouse with a very low or no response to one antigen in many cases exhibited a strong response to another antigen. The large differences between individual animals is likely a considerable contributor to the inherent variability of in vivo potency assays. Our data again support the notion that it is preferred to move away from in vivo potency assays for monitoring batch to batch consistency as part of vaccine batch release testing.

Development of a multiplex-based immunoassay for the characterization of diphtheria, tetanus and acellular pertussis antigens in human combined DTaP vaccines.

Routine batch quality testing before vaccine release, notably for potency evaluation, still relies on animal use for several animal and human vaccines. In this context, the VAC2VAC project is a public-private consortium of 22 partners funded by EU whose the main objective is to reduce the number of animal used for batch testing by developing immunoassays that could be implemented for routine potency assessment of vaccines. This paper focused on the development of a Luminex-based multiplex assay to monitor the consistency of antigen quantity and quality throughout the production process of DTaP vaccines from two human vaccine manufacturers. Indepth characterized monoclonal antibody pairs were used for development and optimization of the Luminex assay with non-adsorbed and adsorbed antigens and with complete vaccine formulations from both manufacturers. The multiplex assay demonstrated good specificity, reproducibility and absence of cross-reactivity. Analysis of over and underdosed formulations, heat and H2O2-degraded products as well as batch to batch consistency of vaccines from both manufacturers brought the proof of concept for a future application of the multiplex immunoassay as a useful tool in the frame of DTaP vaccine quality control.

In vitro antigen ELISA for quality control of tetanus vaccines.

Consistency of production is recognised as an important aspect of vaccine manufacture and suitably validated in vitro assays are required for quality control testing of these products. For the manufacture and batch release of tetanus vaccines, antigen content and integrity, and degree of adsorption of antigen to the adjuvant are critical parameters that should be monitored for consistency. Here we describe the development and use of an Enzyme Linked Immunosorbent Assay (ELISA) to quantify tetanus antigen in combined vaccine products and to measure the degree of adsorption of antigen to adjuvant. Whilst the antigen assay cannot be assumed to predict potency for different products, it can be used as part of a panel of in vitro methods to provide a more informative product profile and to monitor trends in production. The antigen assay is particularly valuable for providing quantitative information on every final lot when modifications of in vivo potency tests, such as single dilution assays, are used.

Collaborative study for the calibration of a replacement International Standard for Tetanus Toxoid Adsorbed.

We present the results of a collaborative study for the establishment of a replacement International Standard (IS) for Tetanus Toxoid Adsorbed. Two candidate preparations were included in the study, one of which was established as the 4th IS for Tetanus Toxoid Adsorbed at the WHO Expert Committee on Biological Standardization meeting in October 2010. This preparation was found to have a unitage of 490 IU/ampoule, based on calibration in guinea pig challenge assays. Results from mouse challenge assays suggest that the relative performance of two candidate preparations may differ significantly between guinea pigs and mice. The authors note that the number of laboratories that performed guinea pig challenge assays, which are used to calibrate and assign IU, is much lower than in previous collaborative studies and this may have implications for calibration of replacement standards in the future. The issue of assigning separate units to the IS for guinea pig and mouse assays is discussed. The study also assessed performance of the replacement standard in serological assays which are used as alternative procedures to challenge assays for tetanus potency testing. Results suggest that the replacement standard is suitable for use as the reference vaccine in serological assays.

A novel single-domain antibody multimer that potently neutralizes tetanus neurotoxin.

Tetanus antitoxin, produced in animals, has been used for the prevention and treatment of tetanus for more than 100 years. The availability of antitoxins, ethical issues around production, and risks involved in the use of animal derived serum products are a concern. We therefore developed a llama derived single-domain antibody (VHH) multimer to potentially replace the conventional veterinary product. In total, 28 different tetanus neurotoxin (TeNT) binding VHHs were isolated, 14 of which were expressed in yeast for further characterization. Four VHH monomers (T2, T6, T15 and T16) binding TeNT with high affinity (KD < 1 nM), covering different antigenic domains as revealed by epitope binning, and including 3 monomers (T6, T15 and T16) that inhibited TeNT binding to neuron gangliosides, were chosen as building blocks to generate 11 VHH multimers. These multimers contained either 1 or 2 different TeNT binding VHHs fused to 1 VHH binding to either albumin (A12) or immunoglobulin (G13) to extend serum half-life in animals. Multimers consisting of 2 TeNT binding VHHs showed more than a 10-fold increase in affinity (KD of 4-23 pM) when compared to multimers containing only one TeNT binding VHH. The T6 and T16 VHHs showed synergistic in vivo TeNT neutralization and, when incorporated into a single VHH trimer (T6T16A12), they showed a very high TeNT neutralizing capacity (1,510 IU/mg).

Investigating Alternative Container Formats for Lyophilization of Biological Materials Using Diphtheria Antitoxin Monoclonal Antibody as a Model Molecule.

When preparing biological reference materials, the stability of the lyophilized product is critical for long-term storage, particularly in order to meet WHO International Standards, which are not assigned expiry dates but are expected to be in use for several decades. Glass ampoules are typically used by the National Institute for Biological Standards and Control (NIBSC) for the lyophilization of biological materials. More recently, a clear need has arisen for the filling of smaller volumes, for which ampoules may not be optimal. We investigated the use of plastic microtubes as an alternative container for small volume fills. In this study, a recombinant diphtheria antitoxin monoclonal antibody (DATMAB) was used as a model molecule to investigate the suitability of plastic microtubes for filling small volumes. The stability and quality of the dried material was assessed after an accelerated degradation study using a toxin neutralization test and size exclusion HPLC. While microtubes have shown some promise in the past for use in the lyophilization of some biological materials, issues with stability may arise when more labile materials are freeze-dried. We demonstrate here that the microtube format is unsuitable for ensuring the stability of this monoclonal antibody.