title: Temperature reduction in cultures of hGM-CSF-expressing CHO cells: effect on productivity and product quality.
abstract: We have demonstrated that temperature reduction from 37 to 33 degrees C in the culture of a CHO cell line producing recombinant human granulocyte macrophage colony stimulating factor (CHO-K1-hGM-CSF) leads to a reduced growth rate, increased cell viability, improved cellular productivity, and decreased cell metabolism. In the present study, CHO-K1-hGM-CSF cells were cultured in a biphasic mode: first, a 37 degrees C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 degrees C. The maximum cell density was not affected after temperature reduction while cell viability remained above 80% for a further 3.7 days in the culture kept at the lower temperature, when compared to the control culture maintained at 37 degrees C. Furthermore, the total rhGM-CSF production increased 6 times in the culture shifted to 33 degrees C. Because the quality and hence the in vivo efficacy of a recombinant protein might be affected by numerous factors, we have analyzed the N- and O-glycosylation of the protein produced under both cell culture conditions using high-pH anion-exchange chromatography and complementary mass spectrometry techniques. The product quality data obtained from the purified protein preparations indicated that decreasing temperature had no significant effect on the rhGM-CSF glycosylation profiles, including the degree of terminal sialylation. Moreover, both preparations exhibited the same specific in vitro biological activity. These results revealed that the employed strategy had a positive effect on the cell specific productivity of CHO-K1-hGM-CSF cells without affecting product quality, representing a novel procedure for the rhGM-CSF production process.
authors.affiliation: Laboratorio de Cultivos Celulares, Facultad de Bioquímica y Ciencias Biológicas, Universidad Nacional del Litoral, Ciudad Universitaria--C.C. 242, S3000ZAA Santa Fe, Argentina.
authors.forename: Mariela
authors.initials: M
authors.lastname: Bollati-Fogolín
authors.forename: Guillermina
authors.initials: G
authors.lastname: Forno
authors.forename: Manfred
authors.initials: M
authors.lastname: Nimtz
authors.forename: Harald S
authors.initials: HS
authors.lastname: Conradt
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; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D006224:Cricetinae; D046508:Culture Techniques / Q000379:methods; D006031:Glycosylation; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000096:biosynthesis* / Q000302:isolation & purification; D006801:Humans; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D013696:Temperature; D013997:Time Factors
chemical_list: D011994:Recombinant Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D007770:L-Lactate Dehydrogenase