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重组抗体互补决定区(CDR)位点化学修饰的评估:关键质量属性的敏感性与功能性

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies: Susceptibility vs. functionality of critical quality attributes.

作者信息

Haberger Markus, Bomans Katrin, Diepold Katharina, Hook Michaela, Gassner Jana, Schlothauer Tilman, Zwick Adrian, Spick Christian, Kepert Jochen Felix, Hienz Brigitte, Wiedmann Michael, Beck Hermann, Metzger Philipp, Mølhøj Michael, Knoblich Constanze, Grauschopf Ulla, Reusch Dietmar, Bulau Patrick

机构信息

Pharma Technical Development Penzberg; Roche Diagnostics GmbH; Penzberg, Germany.

Pharma Research and Early Development; Roche Diagnostics GmbH; Penzberg, Germany.

出版信息

MAbs. 2014 Mar-Apr;6(2):327-39. doi: 10.4161/mabs.27876. Epub 2014 Jan 17.

Abstract

Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.

摘要

天冬酰胺脱酰胺、天冬氨酸异构化、蛋氨酸氧化和赖氨酸糖基化等修饰是重组抗体典型的降解方式。为了鉴定和功能评估互补决定区(CDR)和保守区化学修饰产生的抗体关键质量属性(CQA),采用了一种特定应激条件的方法,包括升高温度、pH值、使用氧化剂以及通过葡萄糖孵育进行强制糖基化。将特定应激条件与离子交换色谱、蛋白酶解肽图谱分析、定量液相色谱质谱联用以及表面等离子体共振分析进行功能评估相结合,足以鉴定和功能评估重组IgG1的CDR中的化学修饰位点。LC-Met-4、LC-Asn-30/31、LC-Asn-92、HC-Met-100c和HC Lys-33被鉴定为潜在的CQA。然而,如果仅天然抗体的一条轻链或重链受到影响,所评估的降解产物均不会导致功能完全丧失。

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