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

Figures & data

Table 1. Psychological and regulatory hurdles for replacement/substitution of in vivo methods reported by Romberg et al. [4] and counterarguments as discussed in the current paper.

Hurdle	Counterargument
‘Comfort’ with the current in vivo test, which seems to work well (as vaccine failures are rare).	There is sufficient evidence that in vivo assay in fact suffer from poor precision and high variability making them unsuitable for use in a routine control strategy where the main objective is to demonstrate batch-to-batch consistency [6,7] (and references therein). The fact that vaccine failures are rare is more because of the consistent manufacturing process and its control that ensure product quality.
Public health impacts-concerns over consequences that would be associated with release of sub-potent medicinal products. Current conventional products were authorized using the established in vivo test.	Quality control with modern analytical (non-animal) tests is likely to be superior to existing animal tests for the purpose of monitoring manufacturing consistency and identifying substandard batches and is the accepted strategy for most newly introduced vaccine.
Conventional wisdom: validation would require that a new assay is directly compared against the existing animal test, which is costly and for veterinary vaccines this includes trials in target species.	The regulatory approach has changed in recent years (as mentioned in European Pharmacopoeia monographs and EMA Guidelines [8,9]. New clinical trials are no longer required when in vitro tests shall replace in vivo tests for QC purpose and the inherent variability of many in vivo assays means that direct comparison with an in vitro assay to demonstrate a correlation is not scientifically justified.
Regulators want a single format for all manufacturers. Current industrial and regulatory dogma calls for a single assay to determine product potency.	This approach requires more and more reevaluation. The variety of products, especially so-called modern products, does not allow a single format testing of all products. This is also recognized in the European Pharmacopoeia text on substitution of in vivo methods [8].
Unilateral acceptance by one regulatory body will not incentivize the industry and acceptance of a new approach must be global. Manufacturers are reluctant to invest in an alternative test without assurance of regulatory acceptance.	There is strong effort on a better communication and harmonization of decisions involving all competent authorities and organizations (e.g. ICH, EDQM, ICMRA, and WHO)*.

Note: *(ICH= International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use, EDQM= European Directorate for the Quality of Medicines, ICMRA= International Coalition of Medicines Regulatory Authorities, WHO = World Health Organization).

Table 2. Technical hurdles encountered and expected as reported by Van den Biggelaar et al. [5] and counterarguments or potential solutions as discussed in the current paper.

Hurdle	Counterargument/Potential Solution
It is difficult to mimic vaccine-induced immune responses using in vitro test methods	Animal in vivo testing is also a model for the immune response in humans. For sufficiently characterized vaccines, in vitro testing of CQA related to potency/safety are state of art for more recently developed and authorized vaccines
Knowledge about critical quality attributes of vaccines and critical process parameters of vaccination is often limited	Only true for legacy vaccines, but reverse characterization will allow to obtain this knowledge [6,7]. Efforts on e.g. diphtheria and tetanus have started in the last decades, and publications demonstrate good progress [11–14]
Measuring vaccine properties of adjuvanted vaccines is complex	Testing can be performed at stages prior to adjuvant addition, there is the option of desorption and not all assays show interference of e.g. aluminum adjuvant
There is a need to create subpotent formulations for method validation due to a lack of appropriate non- compliant batches to test (and validate) in vitro methods	This is not always needed, and there are accepted strategies to obtain these when needed e.g. as indication in the European Pharmacopoeia text on substitution of in vivo methods [8]

Romberg J, Lang S, Balks E, et al. Potency testing of veterinary vaccines: the way from in vivo to in vitro. Biologicals. 2012;40:100–106.

 PubMed Web of Science ®Google Scholar

Stalpers CAL, Retmana IA, Pennings JLA, et al. Variability of in vivo potency tests of Diphtheria, Tetanus and acellular Pertussis (DTaP) vaccines. Vaccine. 2021;39(18):2506–2516. DOI:10.1016/j.vaccine.2021.03.078.

 PubMed Web of Science ®Google Scholar

Stalpers CAL, Retmana IA, Pennings JLA, et al. Corrigendum to “Variability of in vivo potency tests of Diphtheria, Tetanus and acellular Pertussis (DTaP) vaccines” [Vaccine, 39(18)(2021), 2506-2516]. Vaccine. 2022;40(23):3272–3273. DOI:10.1016/j.vaccine.2022.04.047

 PubMed Web of Science ®Google Scholar

EDQM. General chapter 5.2.14 Substitution of in vivo methods by in vitro methods for the quality control of vaccines. In: European Pharmacopoeia 11th. Strasbourg France: European Department for the Quality of Medicines; 2023. p. 716–718.

 Google Scholar

European Medicines Agency (2016). EMA/CHMP/CVMP/JEG-3Rs/450091/2012. Guideline on the principles of regulatory acceptance of 3rs (replacement, reduction, refinement) testing approaches. https://www.ema.europa.eu/en/regulatory-acceptance-3r-replacement-reduction-refinement-testing-approaches#current-effective-version-section

 Google Scholar