title: Responses of CHO cell lines to increased pCO2 at normal (37 °C) and reduced (33 °C) culture temperatures.
abstract: The correlation between dissolved carbon dioxide (pCO2) and cell growth, cell metabolism, productivity and product quality has often been reported. However, since pCO2 values in bioprocesses always vary concurrently with other bioprocess variables, it is very difficult to distinguish only the effect of pCO2. The aim of our work was to investigate further the specific effect of pCO2 and cell response on a proteome level. Proteome responses of three different CHO-Der3 cell lines in the exponential growth phase at normal (37 °C) and reduced (33 °C) culture temperatures, with normal (10%) and increased (20%) pCO2, were studied by comparative proteomic analysis (2D-DIGE). Cell viability and cell density, and the concentration of glucose, glutamine and lactate monitored over 72-h cultures showed that elevated pCO2 did not affect cell viability or productivity at either culture temperature, while metabolic activity was reduced. The specific metabolic profile also indicated altered glucose metabolism toward a less efficient anaerobic metabolism. Two-way ANOVA of proteomic data discriminated many more pCO2-specific changes in protein abundance (p<0.01) at 33 °C than at 37 °C and PCA analysis was able to distinguish clusters distinguishing cell lines and culture conditions at low temperature and elevated pCO2, indicating substantial proteome changes under these culture conditions. Cell sensitivity to increased pCO2 at the lower temperature was further confirmed by a significantly increased abundance of twelve proteins involved in anti- oxidative mechanisms and increased abundance of six proteins involved in glycolysis, including L-lactate dehydrogenase. Proteomic results support the metabolic data and the proposed pCO2 invoked metabolic switch toward anaerobic pathways. Anti- oxidative mechanisms, together with the anaerobic metabolism, allow the cells to detoxify while maintaining sufficient energy levels to preserve their vitality and functionality. This study provides further insight into the proteome responses of CHO cell lines to increased pCO2 at the two culture temperatures.
authors.affiliation: Lek Pharmaceuticals d.d., 1000 Ljubljana, Slovenia. Electronic address: darja.obrstar@sandoz.com.
authors.forename: Obrstar
authors.initials: O
authors.lastname: Darja
authors.affiliation: Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia. Electronic address: stanislav.mandelc@sandoz.com.
authors.forename: Mandelc
authors.initials: M
authors.lastname: Stanislav
authors.affiliation: Lek Pharmaceuticals d.d., 1000 Ljubljana, Slovenia. Electronic address: sasa.stojkovic@sandoz.com.
authors.forename: Stojković
authors.initials: S
authors.lastname: Saša
authors.affiliation: Lek Pharmaceuticals d.d., 1000 Ljubljana, Slovenia. Electronic address: andrej.francky@sandoz.com.
authors.forename: Francky
authors.initials: F
authors.lastname: Andrej
authors.affiliation: Lek Pharmaceuticals d.d., 1000 Ljubljana, Slovenia. Electronic address: lea.bojic@sandoz.com.
authors.forename: Bojić
authors.initials: B
authors.lastname: Lea
authors.affiliation: Biotechnical Faculty, University of Ljubljana, 1000 Ljubljana, Slovenia. Electronic address: branka.javornik@bf.uni-lj.si.
authors.forename: Javornik
authors.initials: J
authors.lastname: Branka
mesh_terms: D000818:Animals; D016466:CHO Cells; D002245:Carbon Dioxide / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003412:Cricetulus; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D020543:Proteome / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*; D013696:Temperature
chemical_list: D020543:Proteome; D005973:Glutamine; D002245:Carbon Dioxide; D019344:Lactic Acid; D005947:Glucose
keywords: 2D-DIGE; Anaerobic metabolism; CHO cells; Oxidative stress; Temperature; pCO(2)