title: A purification process for heparin and precursor polysaccharides using the pH responsive behavior of chitosan.
abstract: The contamination crisis of 2008 has brought to light several risks associated with use of animal tissue derived heparin. Because the total chemical synthesis of heparin is not feasible, a bioengineered approach has been proposed, relying on recombinant enzymes derived from the heparin/HS biosynthetic pathway and Escherichia coli K5 capsular polysaccharide. Intensive process engineering efforts are required to achieve a cost-competitive process for bioengineered heparin compared to commercially available porcine heparins. Towards this goal, we have used 96-well plate based screening for development of a chitosan-based purification process for heparin and precursor polysaccharides. The unique pH responsive behavior of chitosan enables simplified capture of target heparin or related polysaccharides, under low pH and complex solution conditions, followed by elution under mildly basic conditions. The use of mild, basic recovery conditions are compatible with the chemical N-deacetylation/N-sulfonation step used in the bioengineered heparin process. Selective precipitation of glycosaminoglycans (GAGs) leads to significant removal of process related impurities such as proteins, DNA and endotoxins. Use of highly sensitive liquid chromatography-mass spectrometry and nuclear magnetic resonance analytical techniques reveal a minimum impact of chitosan-based purification on heparin product composition.
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Ujjwal
authors.initials: U
authors.lastname: Bhaskar
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Anne M
authors.initials: AM
authors.lastname: Hickey
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Guoyun
authors.initials: G
authors.lastname: Li
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Ruchir V
authors.initials: RV
authors.lastname: Mundra
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Fuming
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Dept. of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Li
authors.initials: L
authors.lastname: Fu
authors.affiliation: Dept. of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Chao
authors.initials: C
authors.lastname: Cai
authors.affiliation: Dept. of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Zhimin
authors.initials: Z
authors.lastname: Ou
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY.|Dept. of Biology, Rensselaer Polytechnic Institute, Troy, NY.|Dept. of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY.|Dept. of Materials Science and Engineering, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Jonathan S
authors.initials: JS
authors.lastname: Dordick
authors.affiliation: Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY.|Dept. of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, NY.|Dept. of Biology, Rensselaer Polytechnic Institute, Troy, NY.|Dept of Biomedical Engineering, Rensselaer Polytechnic Institute, Troy, NY.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D016667:Bacterial Capsules / Q000737:chemistry; D057005:Bioengineering; D048271:Chitosan / Q000737:chemistry*; D002853:Chromatography, Liquid; D004187:Disaccharides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D005285:Fermentation; D006025:Glycosaminoglycans / Q000737:chemistry*; D006493:Heparin / Q000096:biosynthesis / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008279:Magnetic Resonance Imaging; D013058:Mass Spectrometry; D008970:Molecular Weight
chemical_list: D004187:Disaccharides; D006025:Glycosaminoglycans; C560363:heparosan; C050673:capsular polysaccharide K5; D006493:Heparin; D048271:Chitosan
keywords: United States Pharmacopeia (USP); bioengineered heparin; liquid chromatography-mass spectrometry (LC-MS); nuclear magnetic resonance (NMR); polyelectrolyte based purification