A single nutrient feed supports both chemically defined NS0 and CHO fed‐batch processes: Improved productivity and lactate metabolism

Abstract

A chemically defined nutrient feed (CDF) coupled with basal medium preloading was developed to replace a hydrolysate-containing feed (HCF) for a fed-batch NS0 process. The CDF not only enabled a completely chemically defined process but also increased recombinant monoclonal antibody titer by 115%. Subsequent tests of CDF in a CHO process indicated that it could also replace the hydrolysate-containing nutrient feed in this expression system as well as providing an 80% increase in product titer. In both CDF NS0 and CHO processes, the peak lactate concentrations were lower and, more interestingly, lactate metabolism shifted markedly from net production to net consumption when cells transitioned from exponential to stationary growth phase. Subsequent investigations of the lactate metabolic shift in the CHO CDF process were carried out to identify the cause(s) of the metabolic shift. These investigations revealed several metabolic features of the CHO cell line that we studied. First, glucose consumption and lactate consumption are strictly complementary to each other. The combined cell specific glucose and lactate consumption rate was a constant across exponential and stationary growth phases. Second, Lactate dehydrogenase (LDH) activity fluctuated during the fed-batch process. LDH activity was at the lowest when lactate concentration started to decrease. Third, a steep cross plasma membrane glucose gradient exists. Intracellular glucose concentration was more than two orders of magnitude lower than that in the medium. Fourth, a large quantity of citrate was diverted out of mitochondria to the medium, suggesting a partially truncated tricarboxylic acid (TCA) cycle in CHO cells. Finally, other intermediates in or linked to the glycolytic pathway and the TCA cycle, which include alanine, citrate, isocitrate, and succinate, demonstrated a metabolic shift similar to that of lactate. Interestingly, all these metabolites are either in or linked to the pathway downstream of pyruvate, but upstream of fumarate in glucose metabolism. Although the specific mechanisms for the metabolic shift of lactate and other metabolites remain to be elucidated, the increased understanding of the metabolism of CHO cultures could lead to future improvements in medium and process development.

The availability of glucose to CHO cells affects the intracellular lipid-linked oligosaccharide distribution, site occupancy and the N-glycosylation profile of a monoclonal antibody

“…Reducing lactate production has shown to increase peak cell density, extend process duration, and promote protein productivity (Ma et al, 2009). …”

Software Techniques for Enabling High-Throughput Analysis of Metabolomic Datasets

“…The applications are not only in demonstrating simple metabolic comparisons between treated and control groups but in studies involving biomarker discovery, drug development/MOA/recovery, bio-processing, agricultural applications, consumer products, diagnostics, and so on (Sreekumar, Poisson et al 2009;Berger, Kramer et al 2007;Barnes, Teles et al 2009;Boudonck, Mitchell et al 2009;Ma, Ellet et al 2009;Ohta, Masutomi et al 2009;Watson, Roulston et al 2009;Oliver, Guo et al). The ability to run metabolomic studies in highthroughput has been a challenge thus far, not so much because of the complexity or size of the data, but because of the difficulty in generating reproducible data having low process variation that can be quantified, is devoid of artifactual components, and provides high confidence in the identification of metabolites.…”

Scale-down of CHO cell fed-batch cultures

“…Moreover, the variation of the specific glucose consumption rate does not always correlate with the specific amino acid consumption rate, in other words, they do not shift up or down at the same time and to the same extent. Thus, as was done in other studies 15,49 , it is better to add glucose through a separate feed from the amino acids. Part of the variation in glucose consumption rate may be caused by uncontrolled glucose levels.…”

“…A feed is generally more concentrated than a basal medium to maximize culture volumetric productivity and product titer. Studies have shown that the composition of culture media and feeds can influence cell growth, protein productivity 44 , gene expression 45 , product quality 10,46,47 , and metabolism of lactate and ammonia 6,45,48,49 .…”