title: Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.
abstract: Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans was found to be limited by UDP-Gal biosynthesis, which was observed to be both cell line and cultivation condition-dependent. Extracellular glucose and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality.
authors.affiliation: Network Engineering of Eukaryotic Cell Factories, Department of Systems Biology, Technical University of Denmark, Building 223, 2800 Kgs, Lyngby, Denmark; Symphogen A/S, Pederstrupvej 93, 2750, Ballerup, Denmark.
authors.forename: Yuzhou
authors.initials: Y
authors.lastname: Fan
authors.forename: Ioscani
authors.initials: I
authors.lastname: Jimenez Del Val
authors.forename: Christian
authors.initials: C
authors.lastname: Müller
authors.forename: Jette
authors.initials: J
authors.lastname: Wagtberg Sen
authors.forename: Søren Kofoed
authors.initials: SK
authors.lastname: Rasmussen
authors.forename: Cleo
authors.initials: C
authors.lastname: Kontoravdi
authors.forename: Dietmar
authors.initials: D
authors.lastname: Weilguny
authors.forename: Mikael Rørdam
authors.initials: MR
authors.lastname: Andersen
mesh_terms: D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D061249:Batch Cell Culture Techniques / Q000379:methods*; D019149:Bioreactors; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005947:Glucose / Q000378:metabolism*; D006031:Glycosylation; D007074:Immunoglobulin G / Q000737:chemistry* / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D011134:Polysaccharides / Q000378:metabolism
chemical_list: D000596:Amino Acids; D007074:Immunoglobulin G; D011134:Polysaccharides; D005947:Glucose
keywords: Chinese hamster ovary cells; IgG; amino acids; fed-batch; glucose; glycosylation; metabolism; upstream process optimization