期刊
JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
卷 149, 期 -, 页码 541-546出版社
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jpba.2017.11.046
关键词
Disulfide bonds; Free sulfhydryls; IdeS; IgdE; Mass spectrometry; Monoclonal antibody
资金
- LFB Biotechnologies
Despite significant analytical improvements during this last decade, characterizing the whole integrity of monoclonal antibodies during their bioproduction remains a challenge. In this study, we report a new analytical approach to evaluate the overall heterogeneity/integrity of mAbs by LC-MS after combined proteolysis at their lower- and upper-hinge sites using the immunoglobulin-degrading enzymes IdeS and IgdE respectively. The whole sample preparation did not use any harsh conditions such as low pH, high temperature or reductive conditions and enables the splitting of mAbs structure into three fragments, namely the hinge dimer, Fab and Fc/2. Using the NIST mAb reference material, this method was demonstrated to be particularly suited for the analysis of mAbs disulfide bridges. The three fragments as well as their corresponding free sulfhydryl forms were well separated by chromatography and identified online by mass spectrometry. The method was then successfully applied to several mAbs of variable hydrophobicities. (C) 2017 LFB BIOTECHNOLOGIES. Published by Elsevier B.V.
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