Development and optimization of a mobile affinity selection chromatography method for analysis of therapeutic monoclonal antibody aggregates in the presence of human serum albumin.

Demonstration of biological drug product quality prior to first-in-human dosing is a regulatory requirement to ensure patient safety and product performance. Protein aggregates, including dimers and higher-order species, are classified as product-related impurities and must be adequately controlled. However, conventional Size-Exclusion High-Performance Liquid Chromatography (SE-HPLC) may not provide selective aggregate quantitation in samples prepared in diluent containing human serum albumin (HSA). To overcome this limitation, we adapted and optimized a mobile affinity selection chromatography (MASC) method that incorporates a fluorescent selector in the mobile phase with specific affinity for the Fc region of antibody molecules. Following SE-HPLC separation, fluorescence detection enables elective monitoring of Fc-containing species. This strategy effectively eliminates interference from HSA, which lacks an Fc region and therefore is not fluorescently labeled. The method demonstrated good accuracy and linearity for quantification of aggregates in complex HSA-containing matrices. It is suitable for routine application in pharmacy manual qualification and in-use stability studies, providing a robust and selective solution for aggregate analysis.