Low culture temperature is known to enhance the specific productivity of Chinese hamster ovary (CHO) cells expressing erythropoietin (EPO) (LGE10-9-27). Genomic and proteomic approaches were taken to better understand the intracellular responses of these CHO cells resulting from use of low culture temperature (33 degrees C). For transcriptome analysis, commercially available rat and mouse cDNA microarrays were used. The data obtained from the rat and mouse cDNA chips were only somewhat informative in understanding the gene expression profile of CHO cells because of their different sequence homologies with CHO transcriptomes. Overall, transcriptome analysis revealed that low culture temperature could lead to changes in gene expression in various cellular processes such as metabolism, transport, and signaling pathways. Proteome analysis was carried out using 2-D PAGE. Based on spot intensity, 60 high intensity protein spots, from a total of more than 800, were chosen for MS analysis. Forty of the 60 protein spots, which represent 26 different kinds of proteins, were identified by MALDI-TOF-MS and validated by MS/MS. Compared to the reference temperature (37 degrees C), the expression levels of seven proteins (PDI, vimentin, NDK B, ERp57, RIKEN cDNA, phosphoglycerate kinase, and heat shock cognate 71 kDa protein) were increased over twofold at 33 degrees C and those of two proteins (HSP90-beta and EF2) were decreased over twofold at 33 degrees C. Taken together, the results demonstrate the potential of combined analysis of transcriptome and proteome analyses as a tool for the systematic comprehension of cellular mechanisms in CHO cells.

Heparin is the most widely used pharmaceutical to control blood coagulation in modern medicine. A health crisis that took place in 2008 led to a demand for production of heparin from non-animal sources. Chinese hamster ovary (CHO) cells, commonly used mammalian host cells for production of foreign pharmaceutical proteins in the biopharmaceutical industry, are capable of producing heparan sulfate (HS), a related polysaccharide naturally. Since heparin and HS share the same biosynthetic pathway, we hypothesized that heparin could be produced in CHO cells by metabolic engineering. Based on the expression of endogenous enzymes in the HS / heparin pathways of CHO-S cells, human N-deacetylase/N-sulfotransferase (NDST2) and mouse heparan sulfate 3-O-sulfotransferase 1 (Hs3st1) genes were transfected sequentially into CHO host cells growing in suspension culture. Transfectants were screened using quantitative RT-PCR and Western blotting. Out of 120 clones expressing NDST2 and Hs3st1, 2 clones, Dual-3 and Dual-29, were selected for further analysis. An antithrombin III (ATIII) binding assay using flow cytometry, designed to recognize a key sugar structure characteristic of heparin, indicated that Hs3st1 transfection was capable of increasing ATIII binding. An anti-factor Xa assay, which affords a measure of anticoagulant activity, showed a significant increase in activity in the dual-expressing cell lines. Disaccharide analysis of the engineered HS showed a substantial increase in N-sulfo groups, but did not show a pattern consistent with pharmacological heparin, suggesting that further balancing the expression of transgenes with the expression levels of endogenous enzymes involved in HS / heparin biosynthesis might be necessary.

A high resolution method for the separation and analysis of disaccharides prepared from heparin and heparan sulfate (HS) using heparin lyases is described. Ultraperformance liquid chromatography in a reverse-phase, ion-pairing mode efficiently separates eight heparin/HS disaccharides. The disaccharides can then be detected and quantified using electrospray ionization mass spectrometry. This method is particularly useful in the analysis of small amounts of biological samples, including cells, tissues and biological fluids, as it provides high sensitivity without being subject to interference from proteins, peptides and other sample impurities.