Characterization of protease inhibitor conjugated CAP256-VRC26.25 impurities

Authors

• XC Cai
• NA Schneck
• VB Ivleva
• W Zhao
• D Blackstock
• FJ Arnold
• JW Cooper
• QP Lei

Abstract

During cell culture development, heavy chain clipping of CAP256-VRC26.25 (CAP256), a highly potent HIV-1 broadly neutralizing antibody, was detected. The addition of a protease inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), to the cell culture media has shown to effectively reduce the clipping rate. However, AEBSF can potentially bind to CAP256 and generate an undesirable product (i.e., CAP256-AEBSF species), which may impact final product quality. Therefore, LC-MS analysis was applied at the subunit and peptide levels to assess the impact of the introduction of AEBSF to the developmental CAP256 product. Protein subunit analysis confirmed that (1) the negative control CAP256 was free from AEBSF modification; (2) the positive control (CAP256+AEBSF forced conjugated) contained CAP256-AEBSF conjugation species; and (3) no detectable CAP256-AEBSF conjugation was observed in the cell culture developmental CAP256 sample. For further characterization, peptide mapping was applied to pinpoint the exact sites of AEBSF conjugation. For the developmental CAP256 product with AEBSF fed into the cell culture media, conjugation was 3% @Y177 site. Overall, the results for the positive control sample successfully demonstrated that conjugation under forced condition can occur between CAP256 and AEBSF; meanwhile, for the developmental sample going through the AEBSF feeding-strategy in cell culture, levels of AEBSF conjugated to CAP256 were negligible. Furthermore, no potency change was detected for the developmental CAP256 sample compared to CAP256 standard. In summary, we conclude that feeding AEBSF into the cell culture media under controlled conditions can be a potential strategy to mitigate clipping during CAP256 production.

Scientific Focus Area: Research Support Services

This page was last updated on Friday, March 26, 2021