Metabolic analysis of antibody producing CHO cells in fed-batch production

• Published in: Biotechnology and bioengineering Vol. 110; no. 6; pp. 1735 - 1747
• Main Authors: Dean, Jason, Reddy, Pranhitha
• Format: Journal Article
• Language: English
• Published: Hoboken Wiley Subscription Services, Inc., A Wiley Company 01.06.2013; Wiley Subscription Services, Inc
• Subjects: Amino Acids - metabolism; Animals; Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - biosynthesis; Antibodies, Monoclonal - chemistry; Cell Culture Techniques - methods; Cells; Chinese hamster ovary cells; CHO Cells; Cricetinae; Cricetulus; fed-batch mAb production; Genotype & phenotype; Glucose - metabolism; Industrial production; Isotope Labeling; Isotopes; Lactic Acid - metabolism; metabolic flux analysis; Metabolism; Metabolites; Metabolome; Phosphoenolpyruvate Carboxykinase (ATP) - metabolism; Proteins; Pyruvic Acid - metabolism; Recombinant Proteins - analysis; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Ribose - metabolism; Rodents; stable isotope tracers; Stable isotopes
• DOI: 10.1002/bit.24826
• ISSN: 0006-3592; 1097-0290

Chinese hamster ovary (CHO) cells are commonly used for industrial production of recombinant proteins in fed batch or alternative production systems. Cells progress through multiple metabolic stages during fed‐batch antibody (mAb) production, including an exponential growth phase accompanied by lactate production, a low growth, or stationary phase when specific mAb production increases, and a decline when cell viability declines. Although media composition and cell lineage have been shown to impact growth and productivity, little is known about the metabolic changes at a molecular level. Better understanding of cellular metabolism will aid in identifying targets for genetic and metabolic engineering to optimize bioprocess and cell engineering. We studied a high expressing recombinant CHO cell line, designated high performer (HP), in fed‐batch productions using stable isotope tracers and biochemical methods to determine changes in central metabolism that accompany growth and mAb production. We also compared and contrasted results from HP to a high lactate producing cell line that exhibits poor growth and productivity, designated low performer (LP), to determine intrinsic metabolic profiles linked to their respective phenotypes. Our results reveal alternative metabolic and regulatory pathways for lactate and TCA metabolite production to those reported in the literature. The distribution of key media components into glycolysis, TCA cycle, lactate production, and biosynthetic pathways was shown to shift dramatically between exponential growth and stationary (production) phases. We determined that glutamine is both utilized more efficiently than glucose for anaplerotic replenishment and contributes more significantly to lactate production during the exponential phase. Cells shifted to glucose utilization in the TCA cycle as growth rate decreased. The magnitude of this metabolic switch is important for attaining high viable cell mass and antibody titers. We also found that phosphoenolpyruvate carboxykinase (PEPCK1) and pyruvate kinase (PK) are subject to differential regulation during exponential and stationary phases. The concomitant shifts in enzyme expression and metabolite utilization profiles shed light on the regulatory links between cell metabolism, media metabolites, and cell growth. Biotechnol. Bioeng. 2013; 110: 1735–1747. © 2013 Wiley Periodicals, Inc. In this study Dean and Reddy investigated two mAb producing CHO cell lines with contrasting metabolic and productivity phenotypes using stable isotope metabolic tracers and biochemical methods. The authors found that the low growth mAb production phase of fed batch productions was accompanied by increased glucose incorporation into TCA cycle metabolites, and this shift was more prominent in the CHO cell line with elevated mAb expression. In addition, alternative sources of lactate from those typically reported were observed throughout a fed batch process.