Microarray platform affords improved product analysis in mammalian cell growth studies

High throughput (HT) platforms serve as a cost‐efficient and rapid screening method for evaluating the effect of cell‐culture conditions and screening of chemicals. We report the development of a HT cell‐based microarray platform to assess the effect of culture conditions on Chinese hamster ovary (CHO) cells. Specifically, growth, transgene expression and metabolism of a GS/methionine sulphoximine (MSX) CHO cell line, which produces a therapeutic monoclonal antibody, was examined using a microarray system in conjunction with a conventional shake flask platform in a non‐proprietary medium. The microarray system consists of 60‐nL spots of cells encapsulated in alginate and separated in groups via an 8‐well chamber system attached to the chip. Results show the non‐proprietary medium developed allows cell growth, production, and normal glycosylation of recombinant antibody and metabolism of the recombinant CHO cells in both the microarray and shake flask platforms. In addition, 10.3 mM glutamate addition to the defined base medium results in lactate metabolism shift in the recombinant GS/MSX CHO cells in the shake flask platform. Ultimately, the results demonstrate that the HT microarray platform has the potential to be utilized for evaluating the impact of media additives on cellular processes, such as cell growth, metabolism, and productivity.     

Elucidating the role of copper in CHO cell energy metabolism using 13C metabolic flux analysis

¹³C‐metabolic flux analysis was used to understand copper deficiency‐related restructuring of energy metabolism, which leads to excessive lactate production in recombinant protein‐producing CHO cells. Stationary‐phase labeling experiments with U‐¹³C glucose were conducted on CHO cells grown under high and limiting copper in 3 L fed‐batch bioreactors. The resultant labeling patterns of soluble metabolites were measured by GC‐MS and used to estimate metabolic fluxes in the central carbon metabolism pathways using OpenFlux. Fluxes were evaluated 300 times from stoichiometrically feasible random guess values and their confidence intervals calculated by Monte Carlo simulations. Results from metabolic flux analysis exhibited significant carbon redistribution throughout the metabolic network in cells under Cu deficiency. Specifically, glycolytic fluxes increased (25%–79% relative to glucose uptake) whereas fluxes through the TCA and pentose phosphate pathway (PPP) were lower (15%–23% and 74%, respectively) compared with the Cu‐containing condition. Furthermore, under Cu deficiency, 33% of the flux entering TCA via the pyruvate node was redirected to lactate and malate production. Based on these results, we hypothesize that Cu deficiency disrupts the electron transport chain causing ATP deficiency, redox imbalance, and oxidative stress, which in turn drive copper‐deficient CHO cells to produce energy via aerobic glycolysis, which is associated with excessive lactate production, rather than the more efficient route of oxidative phosphorylation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1179–1186, 2015     

Comparative metabolite analysis to understand lactate metabolism shift in Chinese hamster ovary cell culture process

A metabolic shift from lactate production (LP) to net lactate consumption (LC) phenotype was observed in certain Chinese hamster ovary (CHO) cell lines during the implementation of a new chemically defined medium (CDM) formulation for antibody production. In addition, this metabolic shift typically leads to process performance improvements in cell growth, productivity, process robustness, and scalability. In our previous studies, a correlation between a key media component, copper, and this lactate metabolism shift was observed. To further investigate this phenomenon, two complementary studies were conducted. In the first study, a single cell line was cultivated in two media that only differed in their copper concentrations, yet were known to generate an LP or LC phenotype with that cell line. In the second study, two different cell lines, which were known to possess inherently different lactate metabolic characteristics, were cultivated in the same medium with a high level of copper; one cell line produced lactate throughout the duration of the culture, and the other consumed lactate after an initial period of LP. Cell pellet and supernatant samples from both studies were collected at regular time intervals, and their metabolite profiles were investigated. The primary finding from the metabolic analysis was that the cells in LP conditions exhibited a less efficient energy metabolism, with glucose primarily being converted into pyruvate, sorbitol, lactate, and other glycolytic intermediates. This decrease in energy efficiency may be due to an inability of pyruvate and acetyl-CoA to progress into the TCA cycle. The lack of progression into the TCA cycle or overflow metabolism in the LP phenotype resulted in the inadequate supply of ATP for the cells. As a consequence, the glycolysis pathway remained the major source of ATP, which in turn, resulted in continuous LP throughout the culture. In addition, the accumulation of free fatty acids was observed; this was thought to be a result of phospholipid catabolism that was being used to supplement the energy produced through glycolysis in order to meet the needs of LP cells. A thorough review of the metabolic profiles indicated that the lactate metabolic shift could be related to the oxidative metabolic capacity of cells.     

Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes

The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO₂ concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO₂ in batch and fed‐batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO₂ values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO₂. The presented results are especially interesting for large‐scale and perfusion processes where increased pCO₂ concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO₂ accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO₂ might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.     

Combining mechanistic and data‐driven approaches to gain process knowledge on the control of the metabolic shift to lactate uptake in a fed‐batch CHO process

A growing body of knowledge is available on the cellular regulation of overflow metabolism in mammalian hosts of recombinant protein production. However, to develop strategies to control the regulation of overflow metabolism in cell culture processes, the effect of process parameters on metabolism has to be well understood. In this study, we investigated the effect of pH and temperature shift timing on lactate metabolism in a fed‐batch Chinese hamster ovary (CHO) process by using a Design of Experiments (DoE) approach. The metabolic switch to lactate consumption was controlled in a broad range by the proper timing of pH and temperature shifts. To extract process knowledge from the large experimental dataset, we proposed a novel methodological concept and demonstrated its usefulness with the analysis of lactate metabolism. Time‐resolved metabolic flux analysis and PLS‐R VIP were combined to assess the correlation of lactate metabolism and the activity of the major intracellular pathways. Whereas the switch to lactate uptake was mainly triggered by the decrease in the glycolytic flux, lactate uptake was correlated to TCA activity in the last days of the cultivation. These metabolic interactions were visualized on simple mechanistic plots to facilitate the interpretation of the results. Taken together, the combination of knowledge‐based mechanistic modeling and data‐driven multivariate analysis delivered valuable insights into the metabolic control of lactate production and has proven to be a powerful tool for the analysis of large metabolic datasets. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1657–1668, 2015     

Effect of copper variation in yeast hydrolysate on C‐terminal lysine levels of a monoclonal antibody

The ability to control charge heterogeneity in monoclonal antibodies is important to demonstrate product quality comparability and consistency. This article addresses the control of C‐terminal lysine processing through copper supplementation to yeast hydrolysate powder, a raw material used in the cell culture process. Large‐scale production of a murine cell line exhibited variation in the C‐terminal lysine levels of the monoclonal antibody. Analysis of process data showed that this variation correlated well with shifts in cell lactate metabolism and pH levels of the production culture. Small‐scale studies demonstrated sensitivity of the cells to copper, where a single low dose of copper to the culture impacted cell lactate metabolism and C‐terminal lysine processing. Subsequent analytical tests indicated that the yeast hydrolysate powder, added to the basal media and nutrient feed in the process, contained varying levels of trace copper across lots. The measured copper concentrations in yeast hydrolysate lots correlated well with the variation in lactate and pH trends and C‐terminal lysine levels of the batches in manufacturing. Small‐scale studies further demonstrated that copper supplementation to yeast hydrolysate lots with low concentrations of copper can shift the metabolic performance and C‐terminal lysine levels of these cultures to match the control, high copper cultures. Hence, a strategy of monitoring, and if necessary supplementing, copper in yeast‐hydrolysate powders resulted in the ability to control and ensure product quality consistency. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:463–468, 2017     

Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed‐batch CHO cell culture

Cell metabolism can vary considerably over the course of a typical fed‐batch antibody production process. However, the intracellular pathway alterations associated with various phases of growth and antibody production have yet to be fully elucidated using industrially relevant production hosts. Therefore, we performed ¹³C labeling experiments and metabolic flux analysis (MFA) to characterize CHO cell metabolism during four separate phases of a fed‐batch culture designed to closely represent industrial process conditions. First, we found that peak specific growth rate was associated with high lactate production and minimal TCA cycling. Conversely, we found that lactate metabolism switched from net production to net consumption as the culture transitioned from peak growth to peak antibody production. During the peak antibody production phase, energy was primarily generated through oxidative phosphorylation, which was also associated with elevated oxidative pentose phosphate pathway (oxPPP) activity. Interestingly, as TCA cycling and antibody production reached their peaks, specific growth rate continued to diminish as the culture entered stationary phase. However, TCA cycling and oxPPP activity remained high even as viable cell density began to decline. Overall, we found that a highly oxidative state of metabolism corresponded with peak antibody production, whereas peak cell growth was characterized by a highly glycolytic metabolic state. Biotechnol. Bioeng. 2013; 110: 2013–2024. © 2013 Wiley Periodicals, Inc.     

Hyperosmotic stimulus study discloses benefits in ATP supply and reveals miRNA/mRNA targets to improve recombinant protein production of CHO cells

Biopharmaceuticals are predominantly produced by Chinese hamster ovary (CHO) cells cultivated in fed‐batch mode. Hyperosmotic culture conditions (≥ 350 mOsmol kg^∑1) resulting from feeding of nutrients may enhance specific product formation rates (q_p). As an improved ATP supply was anticipated to enhance q_p this study focused on the identification of suitable miRNA/mRNA targets to increase ATP levels. Therefor next generation sequencing and a compartment specific metabolomics approach were applied to analyze the response of an antibody (mAB) producing CHO cell line upon osmotic shift (280 → 430 mOsmol kg^‐1). Hyperosmotic culture conditions caused a ～2.6‐fold increase of specific ATP formation rates together with a ～1.7‐fold rise in cytosolic and mitochondrial ATP‐pools, thus showing increased ATP supply. mRNA expression analysis identified several genes encoding glycosylated proteins with strictly tissue related function. In addition, hyperosmotic culture conditions induced an upregulation of miR‐132‐3p, miR‐132‐5p, miR‐182, miR‐183, miR‐194, miR‐215‐3p, miR‐215‐5p which have all been related to cell cycle arrest/proliferation in cancer studies. In relation to a previous independent CHO study miR‐183 may be the most promising target to enhance q_p by stable overexpression. Furthermore, deletion of genes with presumably dispensable function in suspension growing CHO cells may enhance mAB formation by increased ATP levels.     

On‐line glucose and lactate monitoring in rat striatum: effect of malonate and correlation with histological damage

Microdialysis of glucose, lactate and glycerol was performed to monitor brain insults and to predict brain injury in a rat model using the mitochondrial toxin malonate (5–100 mm ). Striatal dialysates were analyzed off‐line using a CMA 600 microdialysis analyzer or on‐line using flow‐injection analysis and biosensors for glucose and lactate. Histological damage was evaluated using stereological principles. Lactate (baseline ca. 1 mm ) was dose‐dependently increased, reaching a maximum of five‐ to six‐fold increase, whereas glucose (baseline 1–2 mm ) was decreased (>50%) by malonate >20 mm . These changes were reversible upon perfusion with normal Ringer's. Transient increases in glycerol (four‐ to eight‐fold) were only observed in some rats, and were not dose‐dependent. Histological damage was related to the perfused malonate concentration, but was not significantly correlated with lactate or glycerol changes.