Heparin is the most widely used anticoagulant drug in modern medicine. It is a highly sulfated polysaccharide (glycosaminoglycan) found covalently attached to a core protein stored in intracellular granules of mast cells that are found in large numbers in the intestines and lungs of many animals. The heparan sulfate glycosaminoglycan is a less sulfated version of heparin that is attached to core proteins of proteoglycans, ubiquitously found on the external cell membrane of all animal tissues. In response to a health crisis that took place in early 2008, we propose to develop a metabolically engineered heparin from a non-animal source. This health crisis involved the adulteration of heparin produced from hogs in China with an oversulfated chondroitin sulfate, leading to the death of nearly 100 Americans. The proposed 3-year project is a translational and multi-disciplinary research effort aimed at producing heparin in metabolically engineered Chinese hamster ovary (CHO) cells. By engineering the CHO cell glycosylation pathway, which normally affords the related polysaccharide heparan sulfate, a metabolically engineered heparin will be prepared with a structure identical to the pharmaceutical heparin prepared from animals. Chemical and in vitro bioequivalence studies will provide the necessary pre-clinical data required to carry metabolically engineered heparin forward as a generic heparin. CHO cells are widely used in the biotechnology/biopharmaceutical industry for the production of recombinant therapeutic proteins. They are accepted by the FDA as a host for the production of human therapeutics, and protocols exist for removal of host cell proteins, nucleic acids, and viral contaminants. Despite the widespread use of CHO cells for production of recombinant proteins, there are no reports of CHO cells used to produce therapeutic carbohydrates. Hence, the intellectual merit of the proposed work is to demonstrate that the biosynthetic pathway for heparan sulfate can be modified to produce heparin, and that this heparin can be secreted by the CHO cells on a heparin sulfated core protein into the culture medium for collection. To achieve this goal, we will perform the following four tasks: 1. Engineer the CHO cell pathway of heparan sulfate biosynthesis to afford the more highly sulfated glycosaminoglycan, heparin 2. Engineer the CHO cell so that it makes this highly sulfated glycosaminoglycan attached to a core protein normally carrying heparan sulfate and export this proteoglycan to the external cell surface and then shed it into the medium 3. Recover and confirm chemical and in vitro bioequivalence of CHO cell metabolically engineered heparin with USP heparin 4. Scale-up the production and recovery of CHO cell metabolically engineered heparin while maintaining chemical and bioequivalence.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM090127-01
Application #
7780684
Study Section
Special Emphasis Panel (ZGM1-PPBC-X (ME))
Program Officer
Jones, Warren
Project Start
2009-08-17
Project End
2010-03-31
Budget Start
2009-08-17
Budget End
2010-03-31
Support Year
1
Fiscal Year
2009
Total Cost
$167,070
Indirect Cost
Name
Rensselaer Polytechnic Institute
Department
Type
Organized Research Units
DUNS #
002430742
City
Troy
State
NY
Country
United States
Zip Code
12180
Lin, Lei; Liu, Xinyue; Zhang, Fuming et al. (2017) Analysis of heparin oligosaccharides by capillary electrophoresis-negative-ion electrospray ionization mass spectrometry. Anal Bioanal Chem 409:411-420
Oduah, Eziafa I; Linhardt, Robert J; Sharfstein, Susan T (2016) Heparin: Past, Present, and Future. Pharmaceuticals (Basel) 9:
Sun, Xiaojun; Lin, Lei; Liu, Xinyue et al. (2016) Capillary Electrophoresis-Mass Spectrometry for the Analysis of Heparin Oligosaccharides and Low Molecular Weight Heparin. Anal Chem 88:1937-43
Wang, Xiaohua; Liu, Xinyue; Li, Lingyun et al. (2016) GlycCompSoft: Software for Automated Comparison of Low Molecular Weight Heparins Using Top-Down LC/MS Data. PLoS One 11:e0167727
Li, Guoyun; Li, Lingyun; Tian, Fang et al. (2015) Glycosaminoglycanomics of cultured cells using a rapid and sensitive LC-MS/MS approach. ACS Chem Biol 10:1303-10
Baik, Jong Youn; Dahodwala, Hussain; Oduah, Eziafa et al. (2015) Optimization of bioprocess conditions improves production of a CHO cell-derived, bioengineered heparin. Biotechnol J 10:1067-81
Sun, Xiaojun; Li, Lingyun; Overdier, Katherine H et al. (2015) Analysis of Total Human Urinary Glycosaminoglycan Disaccharides by Liquid Chromatography-Tandem Mass Spectrometry. Anal Chem 87:6220-7
Datta, Payel; Yang, Bo; Linhardt, Robert J et al. (2015) Modulation of heparan sulfate biosynthesis by sodium butyrate in recombinant CHO cells. Cytotechnology 67:223-35
Li, Guoyun; Li, Lingyun; Xue, Changhu et al. (2015) Profiling pneumococcal type 3-derived oligosaccharides by high resolution liquid chromatography-tandem mass spectrometry. J Chromatogr A 1397:43-51
Gasimli, Leyla; Glass, Charles A; Datta, Payel et al. (2014) Bioengineering murine mastocytoma cells to produce anticoagulant heparin. Glycobiology 24:272-80

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