Cell engineering and modulation of metabolic pathways in mammalian cells for the production of glycoprotein and proteoglycan based pharmaceuticals.

Abstract

The third aim of the study involved evaluating modulation of heparan sulfate and heparin biosynthesis in recombinant CHO cells. We successfully demonstrated that sodium butyrate could be used as an additional tool besides cell engineering strategy towards production of heparin in bioengineered CHO cells.

Mammalian cell lines such as Chinese hamster ovarian cells (CHO) have been ex-tensively used for industrial production of biologics. Most of the biopharmaceutical biologics produced in CHO cells have been glycoproteins. The critical goal of industrial production of biotherapeutic proteins from CHO cells is to produce copious amounts of properly glycosylated biotherapeutic proteins from robust cells in a cost-effective manner.

Media development and optimization has been one of the strategies to optimize growth of CHO cells and/or the transgene expression in CHO cell engineering bioprocesses. The first specific aim of the study was to development of a non-proprietary medium for the growth of a recombinant glycoprotein producing GS/MSX CHO cell line. The non-proprietary medium supplemented with hypoxanthine and thymidine solution helped cell growth of the GS/MSX CHO cell line, such that, the viable cell density did not start dropping by third day and continued to thrive till a week. To conclude, we demonstrated here that a serum-free non-proprietary media that was initially developed for the growth of DHFR/MTX CHO cells could be modified for the growth of a recombinant GS/MSX CHO cells. However, every cell line need customized optimization and therefore this base non-proprietary media has the potential to be further developed, altered and/or optimized for the growth of other cell lines. Further we demonstrate that the optimized media has potential application towards evaluating effect of media components on cell growth, metabolism and productivity in shake flasks and 3D high throughput platforms.

The second aim of the study was to evaluate metabolic engineering strategies towards restructuring glycome of CHO and mastocytoma cells for the production of proteoglycan-based products, such as, heparin. Heparin (HP) shares biosynthetic path-way with heparan sulfate (HS) and involves action of HS/HP biosynthesis initiation and polymerizing enzymes and a series of sulfotransferases. Two sulfotransferases, N-deacetylase/N-sulfotransferase (NDST2) and 3-O-sulfotransferase (HS3st1) are critical for anticoagulant HP biosynthesis. Overexpression of exogenous human HS3st1 in a mastocytoma cell line (MST10H) results in production of heparin-like glycosaminoglycan and the exogenous enzyme HS3st1 is properly localized in Golgi. The recombinant mastocytoma cells were utilized for developing fractionation and tetrasaccharide analysis of heparan/heparin sulfate from mammalian cells and these methods were utilized to analyze structural glycomics in bioengineered CHO cells. Metabolic engineering of CHO cells resulted in CHOSdual clones (Dual-3, Dual-10, Dual-20, Dual-22 and Dual-29) that demonstrate highly N-sulfated HS/HP. High expression of HS3st1 broadly distributes the HS3st1, while in low-expressing clone HS3st1 localizes in the Golgi. Further, in MST10H and Dual-29, there is an increase in total glycosaminoglycan production, most of which is due to increase in HS/HP glycosaminoglycans.

In conclusion the study suggests that metabolic engineering strategies may impact the structural glycome profile of CHO and mastocytoma cells, including glycosaminoglycan production. Moreover, we demonstrate that over-expression of Golgi-targeted HS3st1 in CHO cells results in formation of one of the 3-O-sulfated residues, as evidenced by tetrasaccharide analysis. This is a novel observation as CHO-S cells do not produce NDST2 and we report here that HS3st1 activity is dependent on substrate availability.