Cell Culture and Gene Transcription Effects of Copper Sulfate on Chinese Hamster Ovary Cells Yueming Qian, Sarwat F. Khattak, Zizhuo Xing Biologics Process and Product Development, Technical Operations, Bristol-Myers Squibb Company, Syracuse, NY 13057 Aiqing He and Paul S. Kayne Dept. of Functional Genomics, Bristol-Myers Squibb Company, Pennington, NJ 08534 Nan-Xin Qian, Shih-Hsie Pan and Zheng Jian Li Biologics Process and Product Development, Technical Operations, Bristol-Myers Squibb Company, Syracuse, NY 13057 DOI 10.1002/btpr.630 Published online May 18, 2011 in Wiley Online Library (wileyonlinelibrary.com). This study reports the effects of varying concentrations of copper sulfate on the metabolic and gene transcriptional profile of a recombinant Chinese hamster ovary (CHO) cell line producing an immunoglobulin G (IgG)-fusion protein (B0). Addition of 50 lM copper sulfate significantly decreased lactate accumulation in the cultures while increasing viable cell den- sity and protein titer. These changes could be seen from day 6 and became increasingly evi- dent with culture duration. Reducing the copper sulfate concentration to 5 lM retained all the above beneficial effects, but with the added benefit of reduced levels of the aggregated form of the B0 protein. To profile the cellular changes due to copper sulfate addition at the transcriptional level, AffymetrixV R CHO microarrays were used to identify differentially expressed genes related to reduced cellular stresses and facilitated cell cycling. Based on the microarray results, down-regulation of the transferrin receptor and lactate dehydrogen- ase, and up-regulation of a cytochrome P450 family-2 polypeptide were then confirmed by Western blotting. These results showed that copper played a critical role in cell metabolism and productivity on recombinant CHO cells and highlighted the usefulness of microarray data for better understanding biological responses on medium modification. V V C 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 1190–1194, 2011 Keywords: Chinese hamster ovary cell, microarray, lactate, cell growth, cell cycle Introduction Historically, copper sulfate has not been evaluated as a supplement to cell culture media, because it was contained in serum, and thus additional copper was not needed. The concentration of copper is 0.7 lg mL 1 in bovine serum and 1.2 lg mL 1 in human plasma. 1–3 In cell culture media with 10% serum, the copper concentration can range from 50 to 120 ng mL 1 (0.8–2.3 lM) which is in the acceptable physi- ological range. In serum-free media, copper can be added as ceruloplasmin and/or copper-loaded albumin. Ceruloplasmin is expensive and in general, is not a recommended method for supplementing copper, as cell culture process develop- ment is moving away from using large proteins as media supplements. On the other hand, adding large amounts of copper salts directly to media can also be undesirable, as amino acids may chelate copper and facilitate potentially toxic free-radical reactions. There have been several reports on the specific effects of copper in cell culture applications. Chaderjian et al. 4 reported that increasing copper sulfate at concentration of 5, 50, or 100 lM in the basal media reduced protein aggregation by lowering free thiol levels from 3- to 10-fold. Supplementing cell culture media with 50–800 nM copper sulfate increased protein production of recombinant factor VIII in baby ham- ster kidney cells and Chinese hamster ovary (CHO) cells. 5 The current study of copper sulfate stemmed from recent CHO cell culture medium optimization efforts that showed additional copper in culture medium to result in reduced lac- tate accumulation, increased viable cell density, and improved recombinant protein titer. This study was subsequently extended to include an investigation at the gene transcrip- tional level and identified differentially expressed genes related to cellular stress reduction and cell cycle stimulation that in turn may contribute to the increased cell growth. Materials and Methods Cell line and culture condition A proprietary CHO cell line was used to produce recombi- nant immunoglobulin G (IgG)-fusion protein B0. The cells were cultured in a chemically defined medium that contained Additional Supporting Information may be found in the online ver- sion of this article. Yueming Qian, Sarwat F. Khattak, and Zizhuo Xing contributed equally to this work. Correspondence concerning this article should be addressed to Z. J. Li at zhengjian.li@bms.com. 1190 V V C 2011 American Institute of Chemical Engineers