title: N+1 Engineering of an Aspartate Isomerization Hotspot in the Complementarity-Determining Region of a Monoclonal Antibody.
abstract: 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.
authors: Lilly Research Laboratories, Biotechnology Discovery Research, Eli Lilly and Company, Indianapolis, Indiana 46285. Electronic address: patelcc@lilly.com.
authors: Chetan N
authors: CN
authors: Patel
authors: Lilly Research Laboratories, Biotechnology Discovery Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
authors: Scott P
authors: SP
authors: Bauer
authors: Lilly Biotechnology Center, Biotechnology Discovery Research, Eli Lilly and Company, San Diego, California 92121.
authors: Julian
authors: J
authors: Davies
authors: Lilly Research Laboratories, Biotechnology Discovery Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
authors: Jim D
authors: JD
authors: Durbin
authors: Lilly Research Laboratories, Biotechnology Discovery Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
authors: Tatiyana L
authors: TL
authors: Shiyanova
authors: Lilly Biotechnology Center, Biotechnology Discovery Research, Eli Lilly and Company, San Diego, California 92121.
authors: Kai
authors: K
authors: Zhang
authors: Lilly Research Laboratories, Biotechnology Discovery Research, Eli Lilly and Company, Indianapolis, Indiana 46285.
authors: Jason X
authors: JX
authors: Tang
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry* / Q000378:metabolism; D001224:Aspartic Acid / Q000737:chemistry* / Q000378:metabolism; D002616:Chemical Engineering / Q000379:methods*; D022801:Complementarity Determining Regions / Q000737:chemistry* / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007536:Isomerism
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D022801:Complementarity Determining Regions; D001224:Aspartic Acid
keywords: biotechnology; critical quality attribute; developability; engineering; isomerization; monoclonal antibody