(M1130-01-07) Characterization of Critical Reagents Used in Immunogenicity Analysis: Case Studies from NAb Assays

Purpose: Neutralizing antibody (NAb) assays play a crucial role in biologics drug development if immunogenicity is observed in patients. Ligand binding assays that use critical reagents such as biotin and ruthenium tag are often employed in identifying NAbs, thus placing higher emphasis on thoroughly characterizing such reagents. Here we present 2 case studies, where the NAb assay performance changed significantly after switching to a new lot of biotinylated capture reagent. Our investigation used aSEC (analytical size exclusion chromatography) and HRMS (high resolution mass spectrometry) to characterize these critical reagents. The results revealed insufficient conjugation and improper characterization to be the reason for change in assay performance. Possible strategies to prevent such failures are presented.

Methods: Critical reagent stability was assessed using aSEC, which was performed on a standalone UPLC equipped with a PDA detector. Biotin incorporation ratio (IR) of the critical reagent was determined by HRMS based intact mass analysis method after subjecting the reagents to enzymatic deglycosylation and separation using a reversed phase HPLC column.

Results: The case studies revealed insufficient biotinylation of the capture reagent, to be the reason behind unsatisfactory assay performance. In case study 1, the 2^nd lot was severely under biotinylated compared to the 1^st lot (IR 4.99 vs. IR 0.64 for lots 1 and 2, respectively) and contained significant amount of unconjugated reagent, which competes with the biotinylated reagent. In case study 2, although there was no unconjugated reagent, the overall biotinylation efficiency of the 2^nd lot was less than 50%, when compared to the 1^st lot (IR 9.22 vs. IR 4.12 for lots 1 and 2, respectively). In both cases, a 3^rd lot was generated by carefully monitoring the conjugation process and ensuring that IR matches the 1^st lot, resulting in satisfactory assay performance. Our analysis revealed that lot to lot inconsistency of biotinylation arose most likely due to interference from buffer components present in the unconjugated reagent. Thus, adequate buffer exchange before conjugation is critical for consistent coupling. Applying HRMS based real time IR determination during conjugation could help ensure reagent reproducibility. Unlike spectrophotometric methods, HRMS based method is less prone to interference from reaction components and require minimal amount of reagents for analysis.

Conclusion: The investigations presented here emphasize the importance of carefully characterizing the critical reagents during their generation, using appropriate techniques and quality control measures. This will lead to reagent consistency and better assay outcomes.