title: Efficient antibody production in the methylotrophic yeast Ogataea minuta by overexpression of chaperones.
abstract: A production system for a therapeutic monoclonal antibody was developed using the methylotrophic yeast Ogataea minuta IFO10746. The genetically engineered O. minuta secreted a detectable amount of anti-TRAIL receptor antibody into the culture supernatant, and the secreted antibody was purified by multiple column chromatography steps. In the purification process, both fully and partially assembled antibodies were detected and isolated. The fully assembled antibody from O. minuta showed almost the same biological activity as that derived from mammalian cells despite the distinct glycosylation profile, whereas the partially assembled antibody showed no cytotoxic activity. To increase the production of active antibody in O. minuta, we overexpressed selected chaperone proteins (included protein disulfide isomerase (OmPDI1), thiol oxidase (OmERO1), and immunoglobulin heavy chain binding protein (OmKAR2)) known to assist in the proper folding (in the endoplasmic reticulum) of proteins destined for secretion. Each of these chaperones enhanced antibody secretion, and together these three factors yielded 16-fold higher antibody accumulation while increasing the ratio of the fully assembled antibody compared to that from the parental strain. Supplementation of a rhodanine-3-acetic acid derivative (R3AD_1c), an inhibitor of O-mannosylation, further increased the secretion of the correctly assembled antibody. These results indicated that the co-overexpression of chaperones is an effective way to produce the correctly assembled antibody in O. minuta.
authors.affiliation: Daiichi Sankyo Co., Ltd., Biologics Technology Research Laboratories, 2716-1 Kurakake, Akaiwa, Chiyoda, Gunma 370-0503, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Daiichi Sankyo Co., Ltd., Biologics Technology Research Laboratories, 2716-1 Kurakake, Akaiwa, Chiyoda, Gunma 370-0503, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Baba
authors.affiliation: Daiichi Sankyo Chemical Pharma Co., Ltd., 389-4 Ohtsurugi, Shimokawa, Izumi, Iwaki, Fukushima 971-8183, Japan.
authors.forename: Minako
authors.initials: M
authors.lastname: Ono
authors.affiliation: Daiichi Sankyo Co., Ltd., Biologics Technology Research Laboratories, 2716-1 Kurakake, Akaiwa, Chiyoda, Gunma 370-0503, Japan.
authors.forename: Koichi
authors.initials: K
authors.lastname: Nonaka
authors.affiliation: Daiichi Sankyo Co., Ltd., Biologics Technology Research Laboratories, 2716-1 Kurakake, Akaiwa, Chiyoda, Gunma 370-0503, Japan.
authors.forename: Kimihisa
authors.initials: K
authors.lastname: Ichikawa
authors.affiliation: Daiichi Sankyo Co., Ltd., Biologics Technology Research Laboratories, 2716-1 Kurakake, Akaiwa, Chiyoda, Gunma 370-0503, Japan.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Yabuta
authors.affiliation: Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan.
authors.forename: Rie
authors.initials: R
authors.lastname: Ito
authors.affiliation: Biotechnology Research Institute for Drug Discovery, Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan. Electronic address: y-chiba@aist.go.jp.
authors.forename: Yasunori
authors.initials: Y
authors.lastname: Chiba
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D018832:Molecular Chaperones / Q000235:genetics* / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D004718:Saccharomycetales / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D018832:Molecular Chaperones
keywords: Antibody production; Chaperones; Correct protein assembly; Ogataea minuta; Protein folding