LC-MS-based metabolic characterization of high monoclonal antibody-producing Chinese hamster ovary cells.
The selection of suitable mammalian cell lines with high specific productivities is a crucial aspect of large-scale recombinant protein production. This study utilizes a metabolomics approach to elucidate the key characteristics of Chinese hamster ovary (CHO) cells with high monoclonal antibody productivities (q(mAb)). Liquid chromatography-mass spectrometry (LC-MS)-based intracellular metabolite profiles of eight single cell clones with high and low q(mAb) were obtained at the mid-exponential phase during shake flask batch cultures. Orthogonal projection to latent structures discriminant analysis (OPLS-DA) subsequently revealed key differences between the high and low q(mAb) clones, as indicated by the variable importance for projection (VIP) scores. The mass peaks were further examined for their potential association with q(mAb) across all clones using Pearson's correlation analysis. Lastly, the identities of metabolites with high VIP and correlation scores were confirmed by comparison with standards through LC-MS-MS. A total of seven metabolites were identified-NADH, FAD, reduced and oxidized glutathione, and three activated sugar precursors. These metabolites are involved in key cellular pathways of citric acid cycle, oxidative phosphorylation, glutathione metabolism, and protein glycosylation. To our knowledge, this is the first study to identify metabolites that are associated closely with q(mAb). The results suggest that the high producers had elevated levels of specific metabolites to better regulate their redox status. This is likely to facilitate the generation of energy and activated sugar precursors to meet the demands of producing more glycosylated recombinant monoclonal antibodies.
Bioprocessing Technology Institute, Agency for Science, Technology and Research (A*STAR), 20 Biopolis Way, #06-01 Centros, Singapore 138668, Singapore.
William Pooi Kat
WP
Chong
Shu Hui
SH
Thng
Ai Ping
AP
Hiu
Dong-Yup
DY
Lee
Eric Chun Yong
EC
Chan
Ying Swan
YS
Ho
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D019149:Bioreactors; D016466:CHO Cells / Q000166:cytology / Q000378:metabolism*; D002853:Chromatography, Liquid; D006224:Cricetinae; D003412:Cricetulus; D005978:Glutathione / Q000378:metabolism; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways; D055442:Metabolome; D055432:Metabolomics / Q000379:methods*; D009711:Nucleotides / Q000378:metabolism; D025341:Principal Component Analysis
D000911:Antibodies, Monoclonal; D009711:Nucleotides; D005978:Glutathione