Quantification of residual AEBSF-related impurities by reversed-phase liquid chromatography

During research of a broadly neutralizing antibody (bNAb) for HIV-1 infection, site-specific clipping was observed during cell culture incubation. Protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), was supplemented to the cell culture feeding to mitigate clipping as one of the control strategies. It led to the need and development of a new assay to monitor the free AEBSF-related impurities during the purification process. In this work, a reversed-phase liquid chromatography (RPLC-UV) method was developed to measure the total concentration of AEBSF and its major degradant product, 4-(aminoethyl) benzenesulfonic acid (AEBS-OH). This quantitative approach involved hydrolysis pre-treatment to drive all AEBSF to AEBS-OH, a filtration step to remove large molecules, followed by RPLC-UV analysis. The method was qualified and shown to be capable of measuring AEBS-OH down to 0.5 microM with good accuracy and precision, which was then applied for process clearance studies. The results demonstrated that a Protein A purification step in conjunction with a mock ultrafiltration/diafiltration (UF/DF) step could remove AEBSF-related impurities below the detection level. Overall, this study is the first to provide a unique approach for monitoring the clearance of free AEBSF and its related degradant, AEBS-OH, in support of the bNAb research.