Physicochemical methods were developed for quality and consistency evaluation of in-process material and complex final human and veterinary vaccines.
Mass spectrometry (MS) assays for Leptospira and Diphtheria, tetanus, and acellular pertussis (DTaP) vaccines confirmed suitability of MS for in-process or final product testing.
Two MS methods for relative and absolute quantification of proteins (LC-MSE and LC-MRM) were developed and tested.
LC-MS methodology developed for DTaP vaccines was performed with non-booster and booster lots. Antigens in non-booster DTaP vaccines were reliably quantitated and LC-MS was shown to be applicable for DTaP vaccines of different manufacturers. Batch-to-batch consistency was demonstrated in booster vaccines, but absolute quantification was not.
Comparison of different analytical approaches (LC-MS and ELISA, multiplex immunoassay) to monitor batch-to-batch consistency of DTaP non-booster vaccine was completed. All methods performed well with less variability than current animal assays.
Software-supported circular dichroism and fluorescence spectroscopy provided rapid assessment of structural conformation and stability of monovalent and trivalent adjuvanted veterinary tetanus vaccines. Vaccines adjuvanted with Alhydrogel or Carbopol showed good spectra but saponin seemed to interfere with spectroscopy. Limits of detection for each physicochemical technique were still determined. Assessing different vaccine batches supplied via VAC2VAC enabled demonstration of abilities and limitations in antigen quality analysis based on higher order structures.
Enzymatic assays simulating antigen degradation by immune cells were successfully set up for some antigens used in DTaP vaccine. For some, the approach successfully identified sub-standard material, enabling further development for in-process controls and detection of issues with antigens before blending with other components.
Immunochemical methods were developed for different vaccines to measure consistency of antigen content and quality, potentially indicating potency, thus negating the need for animal potency tests. ELISAs were developed for diphtheria and tetanus vaccine, Clostridium chauvoei vaccine and Tick-Borne Encephalitis Virus (TBEV) vaccine and were shown to be specific, precise and reproducible. The immunoassays identified changes in antigen content and antigen quality in the vaccine (for example after heat treatment), suggesting they may indicate stability. A Luminex assay for DTaP vaccine antigens yielded similar results, with the aim to multiplex the assay to reduce total product evaluation time. Following proof of concept for the immunoassay approach within VAC2VAC, these assays will be further developed and validated by manufacturers.
Cell based assays were developed and optimized to allow in vitro monitoring of parameters linked to the safety and biological function of vaccines.
The monocyte-activation test (MAT) using human peripheral blood mononuclear cells (h-PBMC) was transferred to industry, validated, approved by the competent regulatory authorities, and implemented in industry, replacing the Rabbit Pyrogen Test (RPT) for TBEV vaccine.
A proposal to revise Ph. Eur. monograph 1375 on TBEV by replacing the RPT with the MAT was submitted, acknowledged and included in the more extensive revision of all monographs to replace the RPT. Also, novel biomarkers for TBEV vaccine potency testing were identified, including a link between type I interferon expression upon stimulation of human PBMC with inactivated TBEV and Ig production by B cells (
https://doi.org/10.1371/journal.ppat.1009505(si apre in una nuova finestra)). Further, development of a novel assay based on identified biomarker, TBEV-induced CXCL-10 secretion, yielded promising results.
Assays to assess immune-potentiating properties of Infectious Bovine Rhinotracheitis (IBR) vaccine, inactivated poultry vaccines (IBV), and Leptospira vaccines were also developed (
https://doi.org/10.3389/fimmu.2022.823058(si apre in una nuova finestra))
Feline Leukaemia Virus (FeLV) vaccine component Quil-A was found to specifically induce NF-κB-signalling in a dose-dependent and reproducible manner in THP-1 cells.
It was demonstrated that the B-cell assay based on h-PBMC is compatible with the use of final vaccine product (tetanus). However, the B cell assay did not show specific reactivity toward pertussis toxin (doi: 10.1016/j.jim.2021.113081 doi: 10.1038/s41541-021-00344-1)
Assays evaluating T cell activation induced by veterinary IBV and Leptospira vaccines in chickens and dogs, respectively, were optimized.
An in vitro assay based on THP-1 cells to detect active β-toxin was modified to detect active β-toxin in the presence of inactivated C. perfringens C antigen. Also, THP-1-based assay demonstrated proof-of-concept for absence of toxicity testing of toxoided C. perfringens C preparations.
Multiparametric assays and bioinformatics were developed and optimized for vaccine strain characterization and vaccine quality evaluation.
Characterization and validation of Clostridium tetani seed strains using DNA, RNA and protein analysis was completed (
https://doi.org/10.3390/toxins14010031(si apre in una nuova finestra) https://doi.org/10.1016/j.talanta.2021.122883(si apre in una nuova finestra))
For the development of platform technology to study vaccine/adjuvant interactions with APC, transcriptomics and/or proteomics studies were performed for DTaP, Leptospira, TBEV, and IBV vaccines (
https://doi.org/10.3390/vaccines9060664(si apre in una nuova finestra) https://doi.org/10.1371/journal.ppat.1009505
https://doi.org/10.14573/altex.2010081(si apre in una nuova finestra) https://doi: 10.1038/s41598-021-89810-3)
A template was created to guide method development and reporting, with parameters and characteristics of development and validation based on analytical target profiles. A summary was published in 2018 on designing multi-centre validation studies (
https://doi.org/10.1016/j.biologicals.2018.01.003(si apre in una nuova finestra)) Discussions were regularly held internally and with external experts on shifting toward a consistency approach-based control strategy. A document to be published open-access was finalized and presented during the VAC2VAC annual meeting 2021, and the Regulatory Stakeholders webinar 2021.
Awareness of VAC2VAC is quickly growing due to a coordinated approach with global organisations, the Bangkok meeting (10.1016/j.biologicals.2020.07.010) and participation at congresses and workshops. Stakeholders’ webinar meetings led to wide outreach to stakeholders and regulatory authorities (NC3Rs, WHO, EDQM, USDA, FDA, OIE, ICH/VICH and national regulatory authorities from 22 countries). A significant outcome was the acceptance of the need for global harmonization of vitro batch control in a consistency approach.
