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.
Highlights
Monoclonal antibodies are among the largest segment of today’s therapeutic proteins market, with a 21% annual increase rate in launching into clinical trial [1]
Proteomic and genomic studies on various NS0 [11,12,13] and murine cell lines [14], and of their recombinant derivative clones, allowed to clearly demonstrate that clones differing in their Monoclonal antibodies (mAbs) productivities differ in the abundance of proteins involved in cellular functions such as energetic metabolism, mAb folding/ assembly, and cytoskeletal organization
Metabolic flux analysis (MFA) techniques and mathematical models, different metabolic patterns in Chinese hamster ovary (CHO) cell clones were observed such as a higher metabolic efficiency as a result of lower by-products production
Summary
Monoclonal antibodies (mAbs) are among the largest segment of today’s therapeutic proteins market, with a 21% annual increase rate in launching into clinical trial [1]. Metabolic flux analysis (MFA) techniques and mathematical models, different metabolic patterns in CHO cell clones were observed such as a higher metabolic efficiency as a result of lower by-products production Taken together, these works have significantly improved our knowledge on CHO cell behavior, as well as our conviction on the need for developing tools allowing a more indepth capacity to describe cell metabolic behavior. The model, which is based on cell energetic and redox states [36], was implemented with metabolic regulation aspects and applied as an in silico platform to the characterization of clonal variation comparing a parental CHO cell line to its high- and low-producer derived clones. We present a descriptive model as well as evaluating its predictive capacity
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