Increased CHO cell fed-batch monoclonal antibody production using the autophagy inhibitor 3-MA or gradually increasing osmolality |
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| Institution: | 1. Michael Smith Laboratories, University of British Columbia, 2185 East Mall, Vancouver, BC, Canada V6T 1Z4;2. Department of Chemical and Biological Engineering, UBC, 2360 East Mall, Vancouver, BC, Canada V6T 1Z3;3. Department of Microbiology, University of Manitoba, 79 Freedman Crescent, Fort Garry Campus, Winnipeg, MB, Canada R3T 2N2;1. Project Planning and Coordination Department, Project and Lifecycle Management Unit, Chugai Pharmaceutical Co., Ltd., 1-1 Nihonbashi-Muromachi 2-Chome, Chuo-ku, Tokyo 103-8324, Japan;2. API Process Development Department, Pharmaceutical Technology Division, Chugai Pharmaceutical Co., Ltd., 5-1 Ukima 5-Chome, Kita-ku, Tokyo 115-8543, Japan;3. Life Science and Bioengineering, Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1 Tennodai 1-Chome, Tsukuba, Ibaraki 305-8572, Japan;1. Biomedical Engineering and Biotechnology, University of Massachusetts Lowell, Lowell, MA 01850, USA;2. Division of Biotechnology Review and Research II, Office of Biotechnology Products, OPQ, CDER, FDA, Silver Spring, MD, USA;3. Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive, Singapore 117585, Singapore;1. Département de Biochimie et médecine moléculaire, Université de Montréal, Montréal, Québec H3C 3J7, Canada;2. Life Sciences, NRC Human Health Therapeutics Portfolio, Building Montreal-Royalmount, National Research Council Canada, Montréal, Québec H4P 2R2, Canada;3. Département de Génie Chimique, École Polytechnique de Montréal, C.P. 6079, Succ. Centre-ville, Montréal, Québec H3C 3A7, Canada;1. Advanced Drug Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan;2. Graduate School of Bioresources and Bioenvironmental Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan |
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| Abstract: | Modulating autophagy provides a new method to increase CHO cell protein production. A fed-batch protocol using the autophagy inhibitor 3-methyl adenine (3-MA), developed for a tissue-plasminogen activator (t-PA) expressing DHFR based CHO cell line, was successfully adapted to a monoclonal antibody (MAb) expressing CHOK1-SV based CHO cell line. By optimizing the timing and dose of 3-MA treatment, the cell-specific productivity was increased 4-fold, resulting in 2-fold increased total MAb production. The positive effect of the 3-MA treatment appeared to be reduced when the amino acid feed concentration was increased 5-fold. Further investigation revealed that by slowly increasing osmolality up to ∼450 mOsm/kg, both the cell-specific productivity and the total MAb almost doubled. This effect was replicated with a DUXB-based CHO cell line expressing a human–llama chimeric antibody. The positive effect of gradually increasing osmolality was then combined with the positive effects of the 3-MA treatment, however their combined effect were not additive. Thus, either increased osmolality or 3-MA treatment were equally effective in increasing MAb-CHO cell fed-batch production on the cell lines tested. Analysis of protein glycosylation showed that both of these fed-batch modifications did not substantially influence the overall glycan profiles of the MAb product. |
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| Keywords: | Autophagy 3-MA Osmolality Glycosylation Fed-Batch Recombinant protein production |
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