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 NH 4 + 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 3 intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality.

Altering the central carbon metabolism of HEK293 cells: Impact on recombinant glycoprotein quality

“…For example, in fedbatch culture of the human cell line rF2N78 producing a human IgG, Sin Seok Seo and colleagues (2014) observed that the increase of non-glycosylated antibody at the end of a culture without glucose feeding was not due to a deficiency in the expression of glycosylation enzymes but rather to insufficient precursor for glycosylation (Seo et al, 2014). Indeed, low glucose concentration leads to reduced intracellular concentration of UDP-GlcNAc and UDP-GalNAc (Fan et al, 2015;Villacrés et al, 2015). In our study, glucose depleted virtually at the same time of the culture and yet we observed a delayed production of IFNα2b of lower quality between the two types of cells thus suggesting an effect linked to the difference in the nutrient metabolism.…”

“…However, such approach can adversely impact protein quality, since it reduces the availability of nucleotide sugars, the key precursors for N-and O-glycosylations (Chee Furng Wong et al, 2005;Liu et al, 2014;Seo et al, 2014;Villacrés et al, 2015). On the other hand, the use of nutrient-rich media and concentrated feed solutions also present drawbacks as they may be associated with the generation of high amounts of metabolic by-products that can be detrimental to both productivity and protein quality (Andersen and Goochee, 1995;Fan et al, 2015). These observations highlight the important role of the central carbon metabolism on protein glycosylation.…”

Elucidating the effects of arginine and lysine on a monoclonal antibody C-terminal lysine variation in CHO cell cultures

“…Amino acids have been found to be associated with product quality, including glycosylation patterns (Aghamohseni et al 2014;Chen and Harcum 2005;Crowell et al 2007;Fan et al 2015), sequence variants (Yu et al 2009), aggregates, and fragments (Jing et al 2012;Yang and Butler 2000). However, the effect of amino acid on mAb charge variants has not been investigated.…”

A protein chimera strategy supports production of a model “difficult‐to‐express” recombinant target

“…Alternatively, high mannose/hybrids forms may be released early as a result of cell lysis. The detection of high mannose/hybrid glycoforms has been reported for other secreted recombinant proteins in mammalian cells 55, 56, 57, 58.…”