Analyzing Clonal Variation of Monoclonal Antibody-Producing CHO Cell Lines Using an In Silico Metabolomic Platform

Abstract

Monoclonal antibody producing Chinese hamster ovary (CHO) cells have been shown to undergo metabolic changes when engineered to produce high titers of recombinant proteins. In this work, we have studied the distinct metabolism of CHO cell clones harboring an efficient inducible expression system, based on the cumate gene switch, and displaying different expression levels, high and low productivities, compared to that of the parental cells from which they were derived. A kinetic model for CHO cell metabolism was further developed to include metabolic regulation. Model calibration was performed using intracellular and extracellular metabolite profiles obtained from shake flask batch cultures. Model simulations of intracellular fluxes and ratios known as biomarkers revealed significant changes correlated with clonal variation but not to the recombinant protein expression level. Metabolic flux distribution mostly differs in the reactions involving pyruvate metabolism, with an increased net flux of pyruvate into the tricarboxylic acid (TCA) cycle in the high-producer clone, either being induced or non-induced with cumate. More specifically, CHO cell metabolism in this clone was characterized by an efficient utilization of glucose and a high pyruvate dehydrogenase flux. Moreover, the high-producer clone shows a high rate of anaplerosis from pyruvate to oxaloacetate, through pyruvate carboxylase and from glutamate to α-ketoglutarate, through glutamate dehydrogenase, and a reduced rate of cataplerosis from malate to pyruvate, through malic enzyme. Indeed, the increase of flux through pyruvate carboxylase was not driven by an increased anabolic demand. It is in fact linked to an increase of the TCA cycle global flux, which allows better regulation of higher redox and more efficient metabolic states. To the best of our knowledge, this is the first time a dynamic in silico platform is proposed to analyze and compare the metabolomic behavior of different CHO clones.

A quantitative metabolomics study of high sodium response in Clostridium acetobutylicum ATCC 824 acetone-butanol-ethanol (ABE) fermentation

“…All analytical protocols for metabolites quantification were described in Ghorbaniaghdam’s article51. A Perkin-Elmer Clarus 480 GC with a FID detector (PerkinElmer, Quebec, Canada) equipped with the Elite-WAX ETR (30 m, 0.32 mm I.D.)…”

Mechanisms driving the lactate switch in Chinese hamster ovary cells

“…In these instances, often another major carbon source, glutamine, is instead depleted (Ghorbaniaghdam, Chen, Henry, & Jolicoeur, 2014;Nolan & Lee, 2011;Wahrheit et al, 2014). In these instances, often another major carbon source, glutamine, is instead depleted (Ghorbaniaghdam, Chen, Henry, & Jolicoeur, 2014;Nolan & Lee, 2011;Wahrheit et al, 2014).…”

Multiplexed Digital mRNA Expression Analysis Profiles System‐Wide Changes in mRNA Abundance and Responsiveness of UPR‐Specific Gene Expression Changes During Batch Culture of Recombinant Chinese Hamster Ovary Cells

“…Unlike standard qRT‐PCR, the Nanostring nCounter allows the quantitative measurement of expression and relative abundance of up to 800 transcripts simultaneously without staged conversion of mRNA to cDNA or the amplification of the resulting cDNA by PCR . Cell‐to‐cell variation have been shown for a variety of gross CHO cell characteristics including cell growth, protein glycosylation, response to culture environment, metabolism, and UPR activation (timing and extent) . In this context, omics studies of clonal derivatives have found that clonal variation may be more important than recombinant protein expression to the differences observed in metabolomics or transcriptomics studies .…”