This Small Business Technology Transfer (STTR) Phase I project proposes to combine a novel metabolically engineered Chinese hamster ovary (CHO) cell line with a unique, high performance, continuous cell-culture bioreactor to demonstrate the commercial viability of producing a bioengineered heparin from CHO cells. This CHO cell line overexpresses critical enzymes in the heparin biosynthesis pathway, resulting in increased levels of a product that is the pharmacological equivalent to heparin. These cells will be grown in the company's proprietary wicking matrix bioreactor that can potentially enable high density, continuously producing cultures in a small footprint, and thus have a multifold reduction in manufacturing cost of goods. The goal is to demonstrate that this engineered CHO cell line can be grown for an extended duration in this bioreactor while maintaining high productivity and product quality. This is expected to be accomplished by optimizing various physical and chemical parameters, developing a unique monitoring system, and continuously assessing metabolic, productivity, and quality attributes associated with heparin production.
The broader/commercial impacts of the proposed research, if successful, will be to advance the development of a novel bioreactor system, potentially with a multifold reduction in manufacturing cost of making therapeutics, and the development of a bioengineered heparin, a substantially safer version of a widely used therapeutic. Heparin is the most widely used anticoagulant drug in modern medicine (~$7B) with >100 tons of heparin being produced annually. However, current production methods rely on purification from animal tissues with known and documented deadly incidences of contamination and disease. Achievement of these objectives will have a substantial impact on human health by facilitating the introduction of novel pharmaceuticals in a more cost-effective manner.
Sepragen Inc. and the Colleges of Nanoscale Science and Engineering (CNSE) at SUNY Polytechnic Institute are collaborating on the development of a novel cell line/reactor system capable of producing a bioengineered heparin that is structurally and pharmaceutically equivalent to current heparin purified from animal tissues. This project seeks to leverage Sepragen’s novel Express® bioreactor and the expertise in metabolic engineering of Chinese hamster ovary (CHO) cell lines of Professor Susan Sharfstein at CNSE and her long-time collaborator, Professor Robert Linhardt at Rensselaer Polytechnic Institute (RPI). The Express® bioreactor is a high performance, cell-culture bioreactor in which the cells are grown in a honey-comb mesh, continually bathed by a thin film of gently flowing culture media, experiencing essentially no shear-stress, while simultaneously exposed to optimized gas exchange. A CHO cell line in which the naturally produced heparan sulfate (HS) had been altered by metabolic engineering to produce a glycosaminoglycan (GAG) that more closely resembles anticoagulant heparin. If successful, this bioengineered heparin will ultimately be used as a substitute for animal-derived heparin, reducing the risks of adventitious agents and adulteration. A critical aspect of the success of creating a bioengineered heparin for commercial use is development of process conditions that will yield an adequate amount of product at a competitive cost. We hypothesized that the Express® bioreactor could be used to increase the productivity of the cell lines with no loss of product quality. Our original proposal had the following objectives and milestones. Project Milestones: Grow CHO cell line Dual-29 (described below) in Sepragen’s reactors and establish that comparable or better productivity is achieved compared with shake flask and fed batch bioreactors. Demonstrate that the product quality remains unchanged upon culture in Sepragen bioreactors Demonstrate that the cell line (and product) remain stable during extended culture Summary of Outcomes We have successfully shown proof of principle that a CHO cell line producing a bioengineered heparin can be cultured for an extended period, maintaining productivity and product quality. We have repeatedly, successfully cultured Dual-29 cells in the Sepragen Express® bioreactor for over one month and demonstrated that the product has anticoagulant activity and that the rate of production (as measured by anticoagulant activity) is nearly identical to that of stirred tank bioreactor cultures. Disaccharide analysis shows that the GAG compositions are similar to that seen in fed-batch, stirred-tank bioreactors and that there is no significant change in composition throughout the run. While we did not successfully complete a two-month run as intended, cultures were terminated due to engineering issues (gas line clogging, contamination) rather than a loss of cell viability, suggesting that a two-month culture would be feasible. We have made engineering modifications to address the issues that have arisen during the bioreactor runs. We have also developed more rapid assays to address product concentration and activity so that they can be measured on a daily basis and used for process control and optimization. We explored extended culture without medium removal (comparable to standard fed-batch cultures), which had never been previously employed in the Sepragen Express® bioreactor. We demonstrated that this was a viable option, permitting a more concentrated product than can be obtained with complete medium changes.
