King Cory, Patel Rekha, Ponniah Gomathinayagam, Nowak Christine, Neill Alyssa, Gu Zhenyu, Liu Hongcheng
Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven CT 06510, United States.
Product Characterization, Alexion Pharmaceuticals, 100 College Street, New Haven CT 06510, United States.
J Chromatogr B Analyt Technol Biomed Life Sci. 2018 May 15;1085:96-103. doi: 10.1016/j.jchromb.2018.03.049. Epub 2018 Apr 3.
In-depth characterization of the commonly observed variants is critical to the successful development of recombinant monoclonal antibody therapeutics. Multiple peaks of a recombinant monoclonal antibody were observed when analyzed by hydrophobic interaction chromatography and imaged capillary isoelectric focusing. The potential modification causing the heterogeneity was localized to F(ab')2 region by analyzing the antibody after IdeS digestion using hydrophobic interaction chromatography. LC-MS analysis identified asparagine deamidation as the root cause of the observed multiple variants. While the isoelectric focusing method is expected to separate deamidated species, the similar profile observed in hydrophobic interaction chromatography indicates that the single site deamidation caused differences in hydrophobicity. Forced degradation demonstrated that the susceptible asparagine residue is highly exposed, which is expected as it is located in the light chain complementarity determining region. Deamidation of this single site decreased the mAb binding affinity to its specific antigen.
深入表征常见变体对于重组单克隆抗体治疗药物的成功开发至关重要。当通过疏水相互作用色谱法和成像毛细管等电聚焦分析时，观察到重组单克隆抗体有多个峰。通过使用疏水相互作用色谱法分析IdeS消化后的抗体，将导致异质性的潜在修饰定位到F(ab')2区域。液相色谱-质谱分析确定天冬酰胺脱酰胺化是观察到的多个变体的根本原因。虽然等电聚焦方法有望分离脱酰胺化物种，但在疏水相互作用色谱中观察到的相似图谱表明，单位点脱酰胺化导致了疏水性差异。强制降解表明，易感天冬酰胺残基高度暴露，鉴于其位于轻链互补决定区，这是预期的。该单位点的脱酰胺化降低了单克隆抗体与其特异性抗原的结合亲和力。
