title: Impact of autoclave sterilization on the activity and structure of formulated heparin.
abstract: The stability of a formulated heparin was examined during its sterilization by autoclaving. A new method to follow loss in heparin binding to the serine protease inhibitor, antithrombin III, and the serine protease, thrombin, was developed using a surface plasmon resonance competitive binding assay. This loss in binding affinity correlated well with loss in antifactor IIa (thrombin) activity as well as antifactor Xa activity as measured using conventional amidolytic assays. Autoclaving also resulted in a modest breakdown of the heparin backbone as confirmed by a slight reduction in number-averaged and weight-averaged molecular weight and an increase in polydispersity. Although no clear changes were observed by nuclear magnetic resonance spectroscopy, disaccharide composition analysis using high-performance liquid chromatography-electrospray ionization-mass spectrometry suggested that loss of selected sulfo groups had taken place. It is this sulfo group loss that probably accounts for a decrease in the binding of autoclaved heparin to antithrombin III and thrombin as well as the observed decrease in its amidolytic activity.
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Julie M
authors.initials: JM
authors.lastname: Beaudet
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Amanda
authors.initials: A
authors.lastname: Weyers
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Kemal
authors.initials: K
authors.lastname: Solakyildirim
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180.
authors.forename: Bo
authors.initials: B
authors.lastname: Yang
authors.affiliation: The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, New York 12208.
authors.forename: Majde
authors.initials: M
authors.lastname: Takieddin
authors.affiliation: The Pharmaceutical Research Institute, Albany College of Pharmacy and Health Sciences, Albany, New York 12208.
authors.forename: Shaker
authors.initials: S
authors.lastname: Mousa
authors.affiliation: Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180. Electronic address: zhangf2@rpi.edu.
authors.forename: Fuming
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Chemistry and Chemical Biology, Rensselaer Polytechnic Institute, Troy, New York 12180; Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, New York 12180; Department of Biomedical Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York 12180; Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180. Electronic address: linhar@rpi.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Linhardt
mesh_terms: D000925:Anticoagulants / Q000737:chemistry* / Q000494:pharmacology; D000990:Antithrombin III / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D015180:Electrophoresis, Gel, Two-Dimensional; D015951:Factor Xa / Q000737:chemistry; D006493:Heparin / Q000737:chemistry* / Q000494:pharmacology; D006358:Hot Temperature; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D013242:Sterilization / Q000379:methods*; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000737:chemistry
chemical_list: D000925:Anticoagulants; D000990:Antithrombin III; D006493:Heparin; D013917:Thrombin; D015951:Factor Xa