CCE IX: Articles Temperature Reduction in Cultures of hGM-CSF-expressing CHO Cells: Effect on Productivity and Product Quality Mariela Bollati-Fogolı ´n, †,⊥,| Guillermina Forno, †,| Manfred Nimtz, ‡ Harald S. Conradt, § Marina Etcheverrigaray, † and Ricardo Kratje* ,† Laboratorio de Cultivos Celulares, Facultad de Bioquı ´mica y Ciencias Biolo ´gicas, Universidad Nacional del Litoral, Ciudad Universitaria - C.C. 242, S3000ZAA Santa Fe, Argentina, and Structural Biology and Protein Glycosylation, GBF German Research Center for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany We have demonstrated that temperature reduction from 37 to 33 °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 °C growth phase for achieving a high cell number, followed by a production phase where the culture temperature was shifted to 33 °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 °C. Furthermore, the total rhGM-CSF production increased 6 times in the culture shifted to 33 °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. Introduction Human granulocyte macrophage colony stimulating factor (hGM-CSF) is an early acting factor essential for the regulation and differentiation of hematopoietic pro- genitor cells as well as for stimulating functional activa- tion of mature cell populations (1). Natural and mam- malian-cell-derived hGM-CSF is a 127 amino acid protein, and it contains both N- and O-glycans. It is highly heterogeneous as a result of differences in site occupancy at the two N-glycosylation sites (2). This lymphokine is of clinical interest according to its potential to treat myeloid leukemia and its ability to stimulate the granu- locyte and macrophage production in patients suffering immunodeficiency or being suppressed by disease or radiation and chemotherapy (3). With the aim of designing an efficient process for glycoprotein production by a CHO cell culture, several groups have investigated the influence of temperature on cell growth and recombinant glycoprotein production. Recombinant CHO cells (rCHO) are normally cultivated at 37 °C. Although lowering the culture temperature in rCHO cells decreases the specific growth rate (μ), several beneficial effects have been reported, including improved cell viability and decreased protease activity (4-9). Moreover, lowering the culture temperature may also increase specific productivity (q), but its effect on q is variable among different rCHO cell lines. The problem of a slow growth rate associated with a lower culture temperature can be solved by using a biphasic process. Effectively, cells can be initially culti- vated at 37 °C for an adequate period of time in order to obtain a reasonably high cell density, and then the culture temperature can be reduced with the aim of decreasing cellular metabolism while achieving high * To whom correspondence should be addressed. Tel./Fax: +54- (0)342-4564397. E-mail: rkratje@fbcb.unl.edu.ar. † Universidad Nacional del Litoral. ‡ Structural Biology, GBF. § Protein Glycosylation, GBF. | These two authors contributed equally to this work. ⊥ Present address: Experimental Immunology, German Re- search Center for Biotechnology (GBF), Mascheroder Weg 1, D-38124 Braunschweig, Germany. 17 Biotechnol. Prog. 2005, 21, 17-21 10.1021/bp049825t CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers Published on Web 12/30/2004