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.

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.

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.

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.

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.

The Influence of Moisture Content and Temperature on the Long-Term Storage Stability of Freeze-Dried High Concentration Immunoglobulin G (IgG).

High protein concentration products for targeted therapeutic use are often freeze-dried to enhance stability. The long-term storage stability of freeze-dried (FD) plasma-derived Immunoglobulin G (IgG) from moderate to high concentrations (10-200 mg/mL) was assessed. Monomer content, binding activity and reconstitution times were evaluated over a 12-month period under accelerated and real-term storage conditions. In the first case study it was shown that FD IgG from 10 to 200 mg/mL had minimal monomer/activity losses at up to ambient temperature after 12 months of storage. However, at 45 °C the sucrose-to-protein ratio played a significant impact on IgG stability above 50 mg/mL. All IgG concentrations witnessed moisture ingress over a 12-month period. The impact of moisture ingress from environmental exposure (between 0.1% and 5% w/w moisture) for IgG 50 mg/mL was assessed, being generated by exposing low moisture batches to an atmospheric environment for fixed time periods. Results showed that at -20 °C and 20 °C there was no significant difference in terms of monomer or antigen-binding activity losses over 6 months. However, at 45 °C, there were losses in monomer content, seemingly worse for higher moisture content samples although model binding activity indicated no losses. Finally, the difference between a low moisture product (0.1-1% w/w) and a moderately high moisture (3% w/w) product generated by alternative freeze-drying cycles, both stoppered under low oxygen headspace conditions, was evaluated. Results showed that at -20 °C and 20 °C there was no difference in terms of binding activity or monomer content. However, at 45 °C, the low moisture samples had greater monomer and binding activity losses than samples from the highest moisture cycle batch, indicating that over-drying can be an issue.

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.