去酰胺
化学
色谱法
离子色谱法
质谱法
快速蛋白质液相色谱法
天冬酰胺
单克隆抗体
等电聚焦
等电点
重组DNA
高效液相色谱法
氨基酸
生物化学
抗体
酶
基因
免疫学
生物
作者
Alyssa Neill,Christian Nowak,Rekha Patel,Gomathinayagam Ponniah,Nidia Gonzalez,Dino Miano,Hongcheng Liu
标识
DOI:10.1021/acs.analchem.5b01452
摘要
Recombinant monoclonal antibody charge heterogeneity has been commonly observed as multiple bands or peaks when analyzed by charge-based analytical methods such as isoelectric focusing electrophoresis and cation or anion exchange chromatography. Those charge variants have been separated by some of the above-mentioned methods and used for detailed characterization. The utility of a combination of OFFGEL fractionation and weak anion exchange chromatography to separate the charge variants of a recombinant monoclonal antibody was demonstrated in the current study. Charge variants were separated into various fractions of high purity and then analyzed thoroughly by liquid chromatography mass spectrometry. Analysis of intact molecular weights identified the presence of heavy chain leader sequence, C-terminal lysine, and C-terminal amidation. The identified modifications were further localized into different regions of the antibody from analysis of antibody fragments obtained from FabRICATOR digestion. Analysis of tryptic peptides from various fractions further confirmed the previously identified modifications in the basic variants. Asparagine deamidation and aspartate isomerization were identified in acidic fractions from analysis of tryptic peptides. Basic variants have been fully accounted for by the identified modifications. However, only a portion of the acidic variants can be explained by deamidation and isomerization, suggesting that additional modifications are yet to be identified or acidic variants are an ensemble of molecules with different structures.
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