A Metabolomics Approach to Increasing Chinese Hamster Ovary (CHO) Cell Productivity

Abstract

Much progress has been made in improving the viable cell density of bioreactor cultures in monoclonal antibody production from Chinese hamster ovary (CHO) cells; however, specific productivity (qP) has not been increased to the same degree. In this work, we analyzed a library of 24 antibody-expressing CHO cell clones to identify metabolites that positively associate with qP and could be used for clone selection or medium supplementation. An initial library of 12 clones, each producing one of two antibodies, was analyzed using untargeted LC-MS experiments. Metabolic model-based annotation followed by correlation analysis detected 73 metabolites that significantly correlated with growth, qP, or both. Of these, metabolites in the alanine, aspartate, and glutamate metabolism pathway, and the TCA cycle showed the strongest association with qP. To evaluate whether these metabolites could be used as indicators to identify clones with potential for high productivity, we performed targeted LC-MS experiments on a second library of 12 clones expressing a third antibody. These experiments found that aspartate and cystine were positively correlated with qP, confirming the results from untargeted analysis. To investigate whether qP correlated metabolites reflected endogenous metabolic activity beneficial for productivity, several of these metabolites were tested as medium additives during cell culture. Medium supplementation with citrate improved qP by up to 490% and more than doubled the titer. Together, these studies demonstrate the potential for using metabolomics to discover novel metabolite additives that yield higher volumetric productivity in biologics production processes.

Temporal Effects of Galactose and Manganese Supplementation on Monoclonal Antibody N-Linked Glycosylation in Fed-Batch and Perfusion Bioreactor Operation

“…When comparing the extracellular amino acid profiles between culture conditions (Supplemental Figure S4), the amino acids all followed a similar trend, however, the +Gal/Mn culture had a higher level of aspartate in the culture which is correlated with higher cell productivity but not with cell growth. 45 These results are consistent with the increased mAb productivity, as explained in the next section.…”

Understanding the effect of temperature downshift onCHOcell growth, antibody titer and product quality by intracellular metabolite profiling and in vivo monitoring of redox state

“…This is consistent with previous findings that aspartate consumption has been observed to be positively associated with antibody productivity. 21 The consumption of cystine in temperature downshift cultures was significantly lower than in the control, which indicated there was more pronounced metabolic stress in the presence of high temperature. Furthermore, sufficient extracellular cysteine concentration under temperature downshift culture might allow to cope with high productivity associated stress.…”

Optimizing VLP production in gene therapy: Opportunities and challenges for in silico modeling

“…The use of cell line specific GSMMs for studying bioprocesses has been increasing over the last years. However, accurate dry weight measurements are often ignored in these models [44,45] , despite solid evidence that time-resolved extracellular metabolome quantification [26][27][28] combined with dry weight determination of biomass to calculate exchange fluxes [46][47][48][49] improves cell line specific models even further. Most often, the measured exometabolome is limited to metabolites with well-established quantification protocols, like amino acids, glucose, lactate, and ammonia.…”

“…Established concepts for biomarker identification for diagnosis can be adapted for bioprocesses based on multi‐omics data. [ 88 ] For example, comprehensive quantitative metabolomics analyses, as performed for CHO [ 45 ] and HEK‐293 cells, [ 44 ] could form starting points to more efficient and sustainable bioprocesses.…”