Investigation of the free heavy chain homodimers of a monoclonal antibody

• Published in: Biotechnology progress Vol. 34; no. 3; pp. 738 - 745
• Main Authors: Chung, Hyo (Helen), Buck, Lynette, Daris, Kristi, Welborn, Brent, Luo, Quanzhou, Wypych, Jette
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01-05-2018
• Subjects: aggregation; Amplification; Animals; Antibodies, Monoclonal - chemistry; Antibodies, Monoclonal - immunology; Capillary electrophoresis; Cation exchange; Cation exchanging; Cell culture; cell line development; Cells, Cultured; Chains; CHO Cells; Cricetulus; Culture media; Dimers; Electrophoresis; Endoplasmic reticulum; free HC homodimer; Immunoglobulin Heavy Chains - chemistry; Immunoglobulin Heavy Chains - immunology; Immunoglobulin Light Chains - chemistry; Immunoglobulin Light Chains - immunology; Immunoglobulins; Molecular weight; Monoclonal antibodies; monoclonal antibody; Organic chemistry; Polypeptides; re‐transfection; Sodium; Sodium dodecyl sulfate; Sodium lauryl sulfate; Surface charge; cell line development; aggregation; free HC homodimer; monoclonal antibody; re-transfection
• ISSN: 8756-7938; 1520-6033
• DOI: 10.1002/btpr.2611

Monoclonal antibodies (mAbs) are composed of two heavy chain (HC) and two light chain (LC) polypeptides. The proper folding and assembly of HC and LC is critical for antibody production. Current dogma indicates that the free HCs are retained in the endoplasmic reticulum (ER) unless assembled with LCs into antibodies, while the LCs on the other hand can be secreted as free monomer or dimer molecules. In this study, high levels of extracellular HC homodimers (7%‐45%) were observed in the cell culture media during cell line development for mAb1. Excellent correlation (R2 > 0.9) between the level of free HC homodimers and the percentage of high molecular weight species indicates that the free HC homodimers might be causative of unwanted aggregation. Due to the different surface charge of HC homodimer and fully assembled antibodies, the unwanted extracellular HC homodimers were successfully removed by downstream processing, through a cation exchange chromatography step. Reduced capillary electrophoresis‐sodium dodecyl sulfate (rCE‐SDS) analysis of the cell culture media from different MTX‐amplified pools indicated that insufficient expression of LC is one potential root cause for the high level of free HC homodimers. The level of free HC homodimers decreased significantly (3%‐25%) after retransfecting the MTX amplified pools with additional LC gene. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:738–745, 2018