Mass spectra of the peptides corresponding to the epitopes, positive and negative controls of ForteBio binding studies, binding rate comparison of mAb 2−25 binding to DTx and to DTx/HBEGF complex, and Western blot analysis of the mAbs binding to full-length and partially hydrolyzed DTx (PDF)
Background: Epitope mapping is an increasingly important aspect of biotherapeutic and vaccine development. Recent advances in therapeutic antibody design and production have enabled candidate mAbs to be identified at a rapidly increasing rate, resulting in a significant bottleneck in the characterization of "structural" epitopes, that are challenging to determine using existing high throughput epitope mapping tools. Here, a Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) epitope screening workflow was introduced that is well suited for accelerated characterization of epitopes with a common antigen.
Main methods and major results:The method is demonstrated on set of six candidate mAbs targeting Pertactin (PRN). Using this approach, five of the six epitopes were unambiguously determined using two HDX mixing timepoints in 24 h total run time, which is equivalent to the instrument time required to map a single epitope using the conventional workflow.
Conclusion:An accelerated HDX-MS epitope screening workflow was developed. The "screening" workflow successfully characterized five (out of six attempted) novel epitopes on the PRN antigen; information that can be used to support vaccine antigenicity assays.
We have developed an accurate, precise and stability-indicating flow cytometry (FC) based assay to directly measure antigenicity of H4 protein (also known as HyVac4) in a vaccine formulation of H4-IC31, without desorbing the H4 protein from the IC31 adjuvant. This method involves immuno-staining of H4-IC31 complex with anti-H4 monoclonal antibodies (mAbs) followed by FC analysis. The assay is not only able to consistently measure H4 antigenicity levels in H4-IC31 stored under normal condition at 2-8°C, but also able to detect changes in H4 antigenicity after H4-IC31 undergoes heat stress or freeze-thawing. In addition, the FC method is able to characterize particle morphology while measuring antigenicity. The biological relevance of the changes in H4 antigenicity detected by the FC assay was supported by an in vitro cell based functional assay using human PBMCs to measure IFN-gamma (IFN-γ) secretion upon re-stimulation with H4-IC31. Our results show that the FC based antigenicity assay can efficiently monitor the biological and physicochemical properties of H4-IC31 and is an indicator for adjuvanted vaccine product stability.
In the vaccine industry, multiple physicochemical, immunological, in vitro and in vivo analytical methods are applied throughout the manufacturing process to characterize and monitor the quality of vaccines. Presented here is the Single Epitope Antigenicity Test (SEAT), an innovative, quantitative epitope profiling method which provides an extended immunochemical analysis for diphtheria toxoid (DTxd) to be used for consistency testing during manufacturing process changes. The method uses BioLayer Interferometry (BLI) and a panel of monoclonal antibodies (mAbs) to independently assess nine individual antigenic sites of DTxd. The panel includes mAbs which are functional, bind distinct sites on DTxd and are able to distinguish intact DTxd from that which has been exposed to heat treatment. The SEAT method was qualified for precision, accuracy, and linearity, and was used to define a preliminary comparability range for DTxd made using the current manufacturing process. DTxd lots manufactured using alternate processes were assessed in the context of this range to determine the impact on DTxd antigenicity. Epitope profiling by SEAT provides quantitative information on the integrity of multiple important antigenic regions of DTxd, and therefore represents a valuable tool in a comprehensive analytical test package which can be used to support manufacturing process changes for vaccines.
There is increasing interest to replace animal-based potency assays used routinely to test vaccines, since they are highly variable, are costly, and present ethical concerns. The development of relevant in vitro assays is part of the solution. Using pertactin (PRN) antigen as an example in DTaP-IPV (diphtheria, tetanus, acellular pertussis, and inactivated poliovirus) vaccines, a PRN antigenicity ELISA was developed using two monoclonal antibodies with a high affinity to unique PRN epitopes, relevance to human immune responses, and evidence of functionality. The ELISA measured consistent PRN antigenicity between the vaccine lots and was validated to demonstrate its accuracy, precision, linearity, and specificity. Notably, the PRN antigenicity ELISA was more sensitive than the mouse-based potency test and could more effectively differentiate between degraded and intact vaccine lots compared to the in vivo test. From these studies, the PRN antigenicity ELISA is proposed as an in vitro replacement for the in vivo potency test for PRN in DTaP-IPV-based formulations. Important considerations in this study included comprehensive antibody characterization, testing of multiple vaccine lots, method validation, and comparison to animal-based potency. Together, these factors form part of an overall strategy that ensures reliable and relevant in vitro assays are developed to replace animal tests.
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