Metabolic engineering of Chinese hamster ovary cells: towards a bioengineered heparin

Abstract

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.

Fig. 1

The structures of the major (A) and variable (B) repeating disaccharides comprising heparin, where X = SO₃⁻ or H and Y = SO₃⁻ or COCH₃.

Fig. 2

Gene and protein expression of NDST2 and Hs3st1 among the selected dual-expressing clones. Based on their levels of gene and protein expression, out of 40 NDST2 and Hs3st1 dual-expressing cell lines, Dual-3 and Dual-29 (asterisks) were selected for further characterization.

Fig. 3

Flow cytometric analysis of dual NDST2 and Hs3st1 expressing cell lines. (A) Histogram of binding activity between fluorescently tagged ATIII and dual NDST2 and Hs3st1 expressing cell lines. (B) Normalized binding activity of ATIII and FGF-2. Error bars represent 95% confidence intervals.

Fig. 4

Factor Xa assay of dual NDST2 and Hs3st1 expressing cell lines. Pharmaceutical heparin was used as a positive control. Error bars represent 95% confidence intervals.

Fig. 5

Disaccharide analysis of HS / heparin from cell pellets by RPIP-UPLC-MS. (a) Extracted ion chromatography (EIC) of heparin disaccharide standards; (b), (c) and (d) EIC of HS / heparin disaccharides of the GAG samples from CHO-S, Dual-3 and Dual-29 cell pellets, respectively. Quantification analysis of heparin/HS disaccharides was performed using calibration curves (e).

Fig. 6

Disaccharide analysis of HS / heparin from culture media by RPIP-UPLC-MS. (a) EIC of heparin disaccharide standards; (b), (c) and (d) EIC of HS / heparin disaccharides of the GAG samples from culture media of CHO-S, Dual-3 and Dual-29 cell lines, respectively. Quantification analysis of heparin/HS disaccharides was performed using calibration curves (e).