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.

Modulation of heparan sulfate biosynthesis by sodium butyrate in recombinant CHO cells

“…AT and FGF-2 were labeled with BODIPY R6G (SE, Invitrogen) as described previously (Baik et al 2012).…”

“…The cells were fixed with freshly prepared 4 % paraformaldehyde and analyzed by flow cytometry. A minimum of 10,000 cells per sample were analyzed on a BD LSRII flow cytometer (BD, San Jose, CA, USA) as described previously (Baik et al 2012).…”

“…CHO cells produce HS and express NDSTs, HS2sts, GLCE, and HS6sts enzymes involved in HS/HP biosynthesis (Zhang et al 2006;Xu et al 2011;Kobayashi et al 1996Kobayashi et al , 1997Habuchi et al 1995Habuchi et al , 1998. However, NDST2 and HS3st1, which are critical for the synthesis of anticoagulant HP, are absent in CHO cells (Xu et al 2011;Zhang et al 2006;Baik et al 2012). …”

“…A clone with a high level of NSDT2 expression, as determined by RT-PCR and Western blotting, was transfected with HS3st1 (Baik et al 2012). From this study, *40 stable CHO-S dual-transfected clones were isolated.…”

“…This analysis showed an increase in the amount of GAGs produced when compared with the wild-type CHO-S cells, particularly for the Dual-29 clone. While the sulfation pattern was altered and an increase in anticoagulant activity was observed, the HS/HP produced still had a much lower anticoagulant activity and a sulfation pattern distinctly different from pharmaceutical heparin (Baik et al 2012), suggesting incomplete activity of the transgenes and the importance of balancing exogenous and endogenous genes in metabolic engineering of CHO cells for the production of anticoagulant HP.…”