N+1 Engineering of an Aspartate Isomerization Hotspot in the Complementarity-Determining Region of a Monoclonal Antibody

• Published in: Journal of pharmaceutical sciences 2016-02, Vol.105 (2), p.512-518
• Main Authors: Patel, Chetan N., Bauer, Scott P., Davies, Julian, Durbin, Jim D., Shiyanova, Tatiyana L., Zhang, Kai, Tang, Jason X.
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal, Humanized - chemistry; Antibodies, Monoclonal, Humanized - metabolism; Aspartic Acid - chemistry; Aspartic Acid - metabolism; biotechnology; Chemical Engineering - methods; Complementarity Determining Regions - chemistry; Complementarity Determining Regions - metabolism; critical quality attribute; developability; engineering; HEK293 Cells; Humans; Isomerism; isomerization; monoclonal antibody
• ISSN: 0022-3549; 1520-6017
• DOI: 10.1016/S0022-3549(15)00185-9

Aspartate (Asp) isomerization is a common degradation pathway and a potential critical quality attribute that needs to be well characterized during the optimization and development of therapeutic antibodies. A putative Asp–serine (Ser) isomerization motif was identified in the complementarity-determining region of a humanized monoclonal antibody and shown to be a developability risk using accelerated stability analyses. To address this issue, we explored different antibody engineering strategies. Direct engineering of the Asp residue resulted in a greater than 5× loss of antigen-binding affinity and bioactivity, indicating a critical role for this residue. In contrast, rational engineering of the Ser residue at the n+1 position had a negligible impact on antigen binding affinity and bioactivity compared with the parent molecule. Furthermore, the n+1 engineering strategy effectively eliminated Asp isomerization as determined by accelerated stability analysis. This outcome affirms that the rate of Asp isomerization is strongly dependent on the identity of the n+1 residue. This report highlights a systematic antibody engineering strategy for mitigating an Asp isomerization developability risk during lead optimization.