Purpose

A major difficulty in monoclonal antibody (mAb) therapeutic development is product aggregation. In this study, intermolecular isopeptide bonds in mAb aggregates were characterized for the first time. We aim to propose a mechanism of covalent aggregation in a model antibody using stressed studies at raised temperatures to aid in the understanding of mAb aggregation pathways.

Methods

Aggregate fractions were generated using raised temperature and were purified using size-exclusion chromatography (SEC). The fractions were tryptically digested and characterized using liquid chromatography hyphenated to tandem mass-spectrometry (LC–MS/MS).

Results

An increased amount of clipping between aspartic acid and proline in a solvent accessible loop in the constant heavy 2 (CH2) domain of the mAb was observed under these conditions. Detailed peptide mapping revealed 14 isopeptide bonds between aspartic acid at that cleavage site and lysine residues on adjacent antibodies. Two additional isopeptide bonds were identified between the mAb HC N-terminal glutamic acid or a separate aspartic acid to lysine residues on adjacent antibodies.

Conclusions

Inter-protein isopeptide bonds between the side chains of acidic amino acids (aspartate and glutamate) and lysine were characterized for the first time in mAb aggregates. A chemical mechanism was presented whereby spontaneous isopeptide bond formation could be facilitated via either the aspartic acid side chain or C-terminus.

中文翻译：

---

检测单克隆抗体聚集体中的异肽键

目的

单克隆抗体 (mAb) 治疗开发的一个主要困难是产品聚集。在这项研究中，首次对 mAb 聚集体中的分子间异肽键进行了表征。我们的目标是提出一种模型抗体中的共价聚集机制，使用升高温度下的压力研究来帮助理解 mAb 聚集途径。

方法

使用升高的温度产生聚集级分，并使用尺寸排阻色谱 (SEC) 进行纯化。使用与串联质谱联用的液相色谱法 (LC-MS/MS) 对级分进行胰蛋白酶消化和表征。

结果

在这些条件下，观察到 mAb 恒定重质 2 (CH2) 结构域中溶剂可接近环中天冬氨酸和脯氨酸之间的剪切量增加。详细的肽图分析揭示了该切割位点的天冬氨酸与相邻抗体上的赖氨酸残基之间的 14 个异肽键。在 mAb HC N-末端谷氨酸或单独的天冬氨酸与相邻抗体上的赖氨酸残基之间鉴定了两个额外的异肽键。

结论

首次在 mAb 聚集体中表征了酸性氨基酸（天冬氨酸和谷氨酸）和赖氨酸侧链之间的蛋白质间异肽键。提出了一种化学机制，由此可以通过天冬氨酸侧链或 C 末端促进自发异肽键的形成。