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

• Published in: Journal of pharmaceutical and biomedical analysis Vol. 83; pp. 89 - 95
• Main Authors: Zhu, Zhiqing C., Chen, Yingchen, Ackerman, Michael S., Wang, Bei, Wu, Wei, Li, Bo, Obenauer-Kutner, Linda, Zhao, Rulin, Tao, Li, Ihnat, Peter M., Liu, Jinping, Gandhi, Rajesh B., Qiu, Bo
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 01.09.2013
• Subjects: Alkylation agents; Antibodies, Monoclonal - chemistry; Electrophoresis, Polyacrylamide Gel - methods; Ethylmaleimide - chemistry; Fragment-band artifact; heat; IgG4; Immunoglobulin G - chemistry; Iodoacetamide - chemistry; monoclonal antibodies; Monoclonal antibody; polyacrylamide gel electrophoresis; proteins; Sulfhydryl Compounds - chemistry; sulfhydryl groups; SDS-PAGE; CE; Fragment-band artifact; IAM; NEM; IgG4; Monoclonal antibody; Alkylation agents; mAb
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2013.04.030

•First study that systematically compared IAM/NEM in SDS-PAGE artifact inhibition.•The artifact inhibition activity of both agents is concentration dependent.•5mM NEM achieves the same inhibition effect as 40mM IAM under same conditions.•NEM retained activity after prolonged sample heating, whereas IAM did not.•NEM has a better inhibition effect than IAM on all tested IgG4 proteins. 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, 5mM NEM can achieve the same inhibition effect as 40mM 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.