Characterization of Bispecific Antibody Production in Cell Cultures by Unique Mixed Mode Size Exclusion ChromatographyClick to copy article linkArticle link copied!
- Haitao JiangHaitao JiangMerck Analytical R&D, Merck & Co., Inc., Kenilworth, New Jersey 07033, United StatesMore by Haitao Jiang
- Wei XuWei XuMerck Analytical R&D, Merck & Co., Inc., Kenilworth, New Jersey 07033, United StatesMore by Wei Xu
- Ren LiuRen LiuMerck Process R&D, MRL, Merck & Co., Inc., Kenilworth, New Jersey 07033, United StatesMore by Ren Liu
- Balrina GuptaBalrina GuptaMerck Process R&D, MRL, Merck & Co., Inc., Kenilworth, New Jersey 07033, United StatesMore by Balrina Gupta
- Bruce KilgoreBruce KilgoreMerck Analytical R&D, Merck & Co., Inc., Kenilworth, New Jersey 07033, United StatesMore by Bruce Kilgore
- Zhimei DuZhimei DuMerck Process R&D, MRL, Merck & Co., Inc., Kenilworth, New Jersey 07033, United StatesMore by Zhimei Du
- Xiaoyu Yang*Xiaoyu Yang*Email: [email protected]. Tel: 908-740-6568.Merck Analytical R&D, Merck & Co., Inc., Kenilworth, New Jersey 07033, United StatesMore by Xiaoyu Yang
Abstract
Bispecific antibodies have received wide attention as promising immunotherapeutic agents because of their high specificity and the ability to target immune cells to tumors. However, analysis of bispecific antibodies is challenging because multiple forms of antibodies are potentially generated during production in cell culture. Most analyses of bispecific antibodies rely on liquid chromatography with mass spectrometry (LC-MS), which could miss detection or becomes less quantitative if those forms are not physically separated. Here, we report a novel and sensitive mixed mode size exclusion chromatography (MM SEC) coupled with multiangle light scattering (MALS) to analyze different forms of bispecific IgG molecules under native conditions. The method displayed great ability to separate various antibody forms with peak resolutions unmatched by other methods we tested, isolating desired bispecific molecules, parental homodimers, half molecules, and antibodies with mispaired light and heavy chains. Each peak was analyzed by online MALS and then identified and confirmed by intact and reduced LC-MS of isolated forms. MM SEC in this study performs by a novel mechanism through the interactions of resin with protein surface hydrophobic clusters distributed across CDRs of light chains. This novel MM SEC allows quantitative detection of even low abundance forms and provides a new tool for screening expression profiles of cell culture clones, monitoring purification, and evaluating drug substance purity.
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