Ponniah Gomathinayagam, Kita Adriana, Nowak Christine, Neill Alyssa, Kori Yekaterina, Rajendran Saravanamoorthy, Liu Hongcheng
Product Characterization and ‡Biochemical Process Development, Alexion Pharmaceuticals Inc. , 352 Knotter Drive, Cheshire, Connecticut, 06410, United States.
Anal Chem. 2015 Sep 1;87(17):9084-92. doi: 10.1021/acs.analchem.5b02385. Epub 2015 Aug 11.
Charge variants, especially acidic charge variants, of recombinant monoclonal antibodies have been challenging to fully characterize despite the fact that several posttranslational modifications have already been identified. The acidic species of a recombinant monoclonal antibody were collected using weak cation exchange (WCX)-10 chromatography and characterized by LC-MS at multiple levels. In this study, methionine oxidation and asparagine deamidation are the only two modifications identified in the acidic species. Incubation of the collected main chromatographic peak with hydrogen peroxide generated acidic species, which confirmed that acidic species were enriched in oxidized antibody. Differences observed between the original acidic species and the oxidization-induced acidic species indicate that different mechanisms are involved in the formation of acidic species. Additionally, acidic species were generated by thermal stress of the collected main peak from the original sample. Thermal stress of the collected main peak in pH 9 buffer or ammonium bicarbonate generated chromatograms that are highly similar to those from the analysis of the original molecule. LC-MS analysis identified oxidation of the same methionine residue and deamidation of the same asparagine in the corresponding acidic fractions generated by thermal stress; however, relatively lower levels of methionine oxidation and higher levels of asparagine deamdiation were observed. The results support the use of stressed conditions to generate low abundance species for detailed characterization of recombinant monoclonal antibody charge variants, but with caution.
尽管已经鉴定出几种翻译后修饰，但重组单克隆抗体的电荷变体，尤其是酸性电荷变体，仍难以完全表征。使用弱阳离子交换（WCX）-10色谱法收集重组单克隆抗体的酸性组分，并通过液相色谱-质谱联用（LC-MS）在多个水平上进行表征。在本研究中，甲硫氨酸氧化和天冬酰胺脱酰胺是在酸性组分中鉴定出的仅有的两种修饰。将收集到的主要色谱峰与过氧化氢孵育产生了酸性组分，这证实了酸性组分富含氧化抗体。原始酸性组分与氧化诱导的酸性组分之间观察到的差异表明，酸性组分的形成涉及不同的机制。此外，通过对原始样品收集到的主峰进行热应激产生了酸性组分。在pH 9缓冲液或碳酸氢铵中对收集到的主峰进行热应激产生的色谱图与对原始分子分析得到的色谱图高度相似。LC-MS分析在热应激产生的相应酸性馏分中鉴定出相同甲硫氨酸残基的氧化和相同天冬酰胺的脱酰胺；然而，观察到甲硫氨酸氧化水平相对较低，天冬酰胺脱酰胺水平较高。结果支持使用应激条件来产生低丰度物种，以详细表征重组单克隆抗体的电荷变体，但要谨慎使用。
