Investigation of monoclonal antibody fragmentation artifacts in non-reducing SDS-PAGE

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• 作者：Zhiqing C. Zhu ; Yingchen Chen ; Michael S. Ackerman ; Bei Wang ; Wei Wu ; Bo Li ; Linda Obenauer-Kutner ; Rulin Zhao ; Li Tao ; Peter M. Ihnat ; Jinping Liu ; Rajesh B. G ; hi ; Bo Qiu
• 关键词：mAb ; monoclonal antibody ; SDS-PAGE ; sodium dodecylsulfate-polyacrylamide gel electrophoresis ; IAM ; iodoacetamide ; NEM ; N-ethylmaleimide ; CE ; capillary gel electrophoresis.
• 刊名：Journal of Pharmaceutical and Biomedical Analysis
• 出版年：2013
• 出版时间：September, 2013
• 年：2013
• 卷：83
• 期：Complete
• 页码：89-95
• 全文大小：933 K

文摘

Fragmentation of monoclonal antibodies has been routinely observed in non-reducing SDS-PAGE, mainly due to disulfide-bond scrambling catalyzed by free sulfhydryl groups, resulting in a method induced artifact. To minimize this artifact, alkylating agents like iodoacetamide (IAM) and N-ethylmaleimide (NEM) were commonly included in SDS sample buffer to block free sulfhydryls. However, the selection of agents and the applied concentrations differ from study to study. In addition, there is no direct comparison of these agents thus far, resulting in difficulties in selecting the suitable agent. To address these questions, we have tested the activities of IAM and NEM in inhibiting the fragment-band artifact of IgG₄ monoclonal antibodies. Our data suggest that the inhibition activity of both agents is concentration dependent. Interestingly, 5 mM NEM can achieve the same inhibition effect as 40 mM IAM. In addition, NEM still retained strong activity after prolonged sample heating, whereas IAM lost most of its activity. Overall, NEM appears to have a better inhibition effect than IAM on all tested IgG₄ proteins, either with SDS-PAGE or CE-SDS methods. These observations demonstrate that NEM has stronger fragmentation inhibition activity than IAM, and thus is a more suitable alkylating agent for both SDS-PAGE and CE-SDS method to reduce this fragmentation artifact.