Leveraging HRMS to Characterize Critical Reagents for Biologics Drug Development

Ligand binding and immunoaffinity purification-based LC-MS (liquid chromatography – mass spectrometry) assays are vital in pre-clinical and clinical drug development, relying on conjugated critical reagents for accurate and reproducible results. Understanding the incorporation ratio (IR) of the conjugation is crucial for confirming lot-to-lot consistency and its impact on assay performance. Traditional spectrophotometric methods like dye-based (HABA) kits, used to determine IR of biotinylated reagents have several disadvantages, including failure to assess the amount of unconjugated reagent and lack of accuracy, resulting in misleading information. In contrast, HRMS (high-resolution mass spectrometry) based intact mass analysis method has been adapted for IR determination of critical reagents. It requires smaller sample amounts, tolerates diverse buffers, enables real-time monitoring, and assesses unconjugated reagents. This presentation will elucidate the advantages of IR analysis using HRMS over dye-based kits (with case studies from clinical sample analysis) and recommend alternate dye-based options when HRMS is not feasible.