title: Impact of temperature reduction and expression of yeast pyruvate carboxylase on hGM-CSF-producing CHO cells.
abstract: Recently, we demonstrated that a recombinant yeast pyruvate carboxylase expressed in the cytoplasm of BHK-21 cells was shown to partially reconstitute the missing link between glycolysis and TCA, increasing the flux of glucose into the TCA and achieving higher yields of recombinant erythropoietin. In the present study, a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor was used to evaluate the impact of PYC2 expression and reduced culture temperature. Temperature reduction from 37 to 33 degrees C revealed a reduced growth rate, a prolonged stationary phase and a 2.1-fold increase of the cell specific rhGM-CSF production rate for CHO-K1-hGM-CSF cells. The PYC2-expressing cell clones showed a decreased cell growth and a lower maximum cell concentration compared to the control expressing rhGM-CSF but no PYC2. However, only 65% lactate were produced in PYC2-expressing cells and the product yield was 200% higher compared to the control. The results obtained for CHO cells compared to BHK cells reported previously, indicated that the PYC2 expression dominantly reduced the lactate formation and increased the yield of the recombinant protein to be produced. Finally, the growth and productivity of PYC2-expressing CHO-K1-hGM-CSF cells under both temperature conditions were investigated. The average cell specific rhGM-CSF production increased by 3.2-fold under reduced temperature conditions. The results revealed that the expression of PYC2 and a reduced culture temperature have an additive effect on the cell specific productivity of CHO-K1-hGM-CSF cells.
authors.affiliation: Cell Culture Technology, German Research Centre for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany.
authors.forename: Mariela Bollati
authors.initials: MB
authors.lastname: Fogolín
authors.forename: Roland
authors.initials: R
authors.lastname: Wagner
authors.forename: Marina
authors.initials: M
authors.lastname: Etcheverrigaray
authors.forename: Ricardo
authors.initials: R
authors.lastname: Kratje
mesh_terms: D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus; D002470:Cell Survival; D003080:Cold Temperature; D006224:Cricetinae; D003412:Cricetulus; D005947:Glucose / Q000378:metabolism; D005973:Glutamine / Q000378:metabolism; D006801:Humans; D019344:Lactic Acid / Q000378:metabolism; D016173:Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D011766:Pyruvate Carboxylase / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013696:Temperature; D015003:Yeasts / Q000201:enzymology
chemical_list: D011994:Recombinant Proteins; D005973:Glutamine; D019344:Lactic Acid; D016173:Macrophage Colony-Stimulating Factor; D011766:Pyruvate Carboxylase; D005947:Glucose