title: Efficient folding/assembly in Chinese hamster ovary cells is critical for high quality (low aggregate content) of secreted trastuzumab as well as for high production: stepwise multivariate regression analyses.
abstract: When developing cell culture processes for therapeutic antibodies, the low content of aggregated proteins is the most critical because administering aggregated antibody molecules might result in adverse effects such as immunogenicity. To characterize cells with high productivity and quality, we determined factors that are closely related to antibody titer, which is a productivity indicator, and the area percentage of high molecular weight species in cultivated media, which is equivalent to aggregate content and is used as a quality indicator. We examined the factors influencing antibody titer and aggregate content using various data from 28 cell lines throughout their culture periods from growth to death phases. Our study using correlation analysis revealed that statistically significant correlations between factors and indicators changes with sampling points, hence we thought that various factors would influence each indicator simultaneously. To understand the relationship between these factors and titer/aggregates contents, we performed stepwise multiple linear regression analyses and deduced a multiple linear model for each indicator. The titer was found to positively associate with specific growth rate and specific production rate and negatively with intracellular heavy chain content. The aggregate content was found to positively associate with protein disulfide isomerase mRNA level and negatively with light chain secreted into culture media, specific production rate, intracellular light chain content, and specific growth rate. Our observations suggest that correct and efficient assembling and/or folding of an antibody molecule in an endoplasmic reticulum are important for high titer and low aggregates contents.
authors.affiliation: Bio-process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan; Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu-shi, Gunma 376-8515, Japan. Electronic address: yoichi.ishii@kyowa-kirin.co.jp.
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Ishii
authors.affiliation: Bio-process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan.
authors.forename: Junko
authors.initials: J
authors.lastname: Murakami
authors.affiliation: Bio-process Research and Development Laboratories, Kyowa Hakko Kirin Co., Ltd., 100-1 Hagiwara-machi, Takasaki-shi, Gunma 370-0013, Japan.
authors.forename: Kazue
authors.initials: K
authors.lastname: Sasaki
authors.affiliation: Kyowa Hakko Kirin Co., Ltd., 1-6-1 Ohte-machi, Chiyoda-ku, Tokyo 100-8185, Japan.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Tsukahara
authors.affiliation: Graduate School of Engineering, Gunma University, 1-5-1 Tenjin-cho, Kiryu-shi, Gunma 376-8515, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Wakamatsu
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000096:biosynthesis* / Q000235:genetics; D000970:Antineoplastic Agents / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics; D016014:Linear Models; D008970:Molecular Weight; D015999:Multivariate Analysis; D066329:Protein Aggregates; D019704:Protein Disulfide-Isomerases / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D055503:Protein Multimerization; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000068878:Trastuzumab
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D066329:Protein Aggregates; D012333:RNA, Messenger; D019704:Protein Disulfide-Isomerases; D000068878:Trastuzumab
keywords: Antibody aggregate; Antibody productivity; Antibody quality; Cell line; Chinese hamster ovary cell; Endoplasmic reticulum stress; Heavy chain binding protein; Protein disulfide isomerase; Stepwise multiple linear regression; Therapeutic antibody