| Although most pharmaceutical heparin used today is obtained from porcine intestine, heparin has historically been prepared from bovine lung and ovine intestine. There is some regulatory concern about establishing the species origin of heparin. This concern began with the outbreak of mad cow disease in the1990s and was exacerbated during the heparin shortage in the2000s and the heparin contamination crisis of2007-2008. Three heparins from porcine, ovine, and bovine were characterized through state-of-the-art carbohydrate analysis methods with a view profiling their physicochemical properties. Differences in molecular weight, monosaccharide and disaccharide composition, oligosaccharide sequence, and antithrombin Ⅲ-binding affinity were observed. These data provide some insight into the variability of heparins obtained from these three species and suggest some analytical approaches that may be useful in confirming the species origin of a heparin active pharmaceutical ingredient.The development of a bioengineered heparin from a non-animal source is in response to a health crisis that took place in early2008. This crisis involved the introduction of an over sulfated chondroitin sulfate into heparin produced from hogs in China leading to the death of nearly100Americans. The bioengineered heparin synthesized in this dissertation was undertaken at100-mg scale using soluble enzymes and cofactor recycling to prepare sufficient material for in vivo evaluation. This bioengineered heparin, obtained in～3%yield by ion exchange fractionation, had a Trisulfated disaccharide composition and an antithrombin-binding site tetrasaccharide very close to USP heparin. The in vitro anticoagulant activity of this bioengineered heparin was in compliance with the USP. In conclusion, the completion of the this bioengineered heparin demonstrates that successful synthesis of a BRP heparin that is generically equivalent to heparin is possible. The standard process for preparing the low molecular weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield due to the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-Ⅱ, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-Ⅱ. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin’s core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by1D and2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity.The thermal instability of the anticoagulant heparin is associated, in part, with the solvolytic loss of N-sulfo groups. This study describes a new method to assess the content of unsubstituted amino groups present in bioengineered heparin and the increased content of unsubstituted amino groups present in thermally-stressed and autoclave-sterilized heparin formulations. N-acetylation of heparin samples with acetic anhydride-d6is followed by exhaustive heparinase treatment, and disaccharide analysis by hydrophilic interaction chromatography mass spectrometry. The introduction of stable isotopic label provides a sensitive probe for the detection and localization of the lost N-sulfo groups potentially providing valuable insights into degradation mechanism and the reasons for anticoagulant potency loss. |
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