Low glucose concentrations within typical industrial operating conditions have minimal effect on the transcriptome of recombinant CHO cells

Abstract

Typically, mammalian cell culture medium contains high glucose concentrations that are analogous to diabetic levels in humans, suggesting that mammalian cells are cultivated in excessive glucose. Using RNA-Seq, this study characterized the Chinese hamster ovary (CHO) cell transcriptome under two glucose concentrations to assess the genetic effects associated with metabolic pathways, in addition to other global responses. The initial extracellular glucose concentrations used represented high (30 mM) and low (10 mM) glucose conditions, where at the time the transcriptomes were compared, the glucose concentrations were approximately 24 and 4.4 mM for the mid-exponential cultures, where 4.4 mM represents a common target concentration in the biopharmaceutical industry for controlled fed-batch cultures. A recombinant CHO cell line producing a monoclonal antibody was used, such that the impact on glycosylation genes could be evaluated. Relatively few genes were identified as being significantly different (FDR ≤ 0.01) between the high and low glucose conditions, for example, only 575 genes, and only 40 of these genes had 2-fold or greater differences. Gene expression differences for glycolysis, TCA cycle, and glycosylation-related reactions were minimal and unlikely to have biological significance. This transcriptome study indicates that low glucose concentrations in the culture medium are unlikely to cause any biologically significant or detrimental changes to CHO cells at the transcriptome level. Furthermore, it is well-known that maintaining low glucose concentrations in fed-batch cultures can reduce lactate production, which in turn improves process outcomes. Taken together, the transcriptome data supports the continued development of low glucose-based processes to control lactate. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:771-785, 2017.

RNA‐Seq Highlights High Clonal Variation in Monoclonal Antibody Producing CHO Cells

“…Numerous CHO transcriptomics studies have been performed over the past decade comparing changes in culture conditions including hyperosmolarity, temperature shift, glucose concentration, lactate concentration, and sodium butyrate treatment . Differences in growth phases, growth rates, productivity, and protein glycosylation have also been studied.…”

High glucose and low specific cell growth but not mild hypothermia improve specific r-protein productivity in chemostat culture of CHO cells

“…Many strategies aiming to enhance recombinant protein production focus on maximizing specific protein productivity while maintaining high viable cell density in culture for long periods. In this context, the operational conditions (e.g., temperature or medium composition) play a significant role in culture performance and proper handling of the cultures may indeed enable considerable increases in r-protein production [ 5 – 12 ].…”

“…Nowadays, most industrially relevant culture media for mammalian cells contain a glucose concentration from 25 to 35 mM [ 29 , 32 ]. Therefore, 30 mM is the average glucose concentration for standard mammalian cell culture media, and concentrations below 20 mM are considered low [ 12 , 28 ], while concentrations above 40 mM are considered high [ 11 , 32 ], as compared with typical culture media. In cultures under very glucose-limited conditions (below 2 mM), cells have a drastically reduced intracellular concentration of ATP, amino acids and TCA cycle metabolites [ 31 , 33 ].…”

Elucidating the Impact of CHO Cell Culture Media on Tryptophan Oxidation of a Monoclonal Antibody Through Gene Expression Analyses

“…Gowtham et al also reported their transcriptome analysis of recombinant CHO cells grown under two glucose concentrations to assess the genetic effects associated with metabolic pathways and global responses. They indicated that the transcriptome data support the continued development of low glucose‐based processes to control lactate . Another transcriptome analysis using both microarray and RNA‐Seq was reported by Yuk et al to explore the MOA of high copper concentrations’ impact on lactate metabolism .…”

“…The advent of Next Generation Sequencing (NGS) technologies and the availability of CHO reference genomes have enabled the systematic analysis of CHO biology and its capacity for recombinant protein production . Transcriptome analyses by microarray and RNA‐Seq have become commonly applied to unveil physiological insights into global gene expression related to cell culture performance . As an example, Shridhar et al recently reported using a high‐performance microarray platform to examine the transcriptome of CHO cells grown in the presence and absence of serum.…”