RESUMO
Heparins have been invaluable therapeutic anticoagulant polysaccharides for over a century, whether used as unfractionated heparin or as low molecular weight heparin (LMWH) derivatives. However, heparin production by extraction from animal tissues presents multiple challenges, including the risk of adulteration, contamination, prion and viral impurities, limited supply, insecure supply chain, and significant batch-to-batch variability. The use of animal-derived heparin also raises ethical and religious concerns, as well as carries the risk of transmitting zoonotic diseases. Chemoenzymatic synthesis of animal-free heparin products would offer several advantages, including reliable and scalable production processes, improved purity and consistency, and the ability to produce heparin polysaccharides with molecular weight, structural, and functional properties equivalent to those of the United States Pharmacopeia (USP) heparin, currently only sourced from porcine intestinal mucosa. We report a scalable process for the production of bioengineered heparin that is biologically and compositionally similar to USP heparin. This process relies on enzymes from the heparin biosynthetic pathway, immobilized on an inert support and requires a tailored N-sulfoheparosan with N-sulfo levels similar to those of porcine heparins. We also report the conversion of our bioengineered heparin into a LMWH that is biologically and compositionally similar to USP enoxaparin. Ultimately, we demonstrate major advances to a process to provide a potential clinical and sustainable alternative to porcine-derived heparin products.
Assuntos
Heparina de Baixo Peso Molecular , Heparina , Animais , Suínos , Heparina/metabolismo , Heparina de Baixo Peso Molecular/química , Anticoagulantes/química , Peso Molecular , Contaminação de MedicamentosRESUMO
Keratan sulfate (KS) was isolated from chicken egg white in amounts corresponding to â¼0.06 wt% (dry weight). This KS had a weight-average molecular weight of â¼36-41 kDa with a polydispersity of â¼1.3. The primary repeating unit present in chicken egg white KS was â4) ß-N-acetyl-6-O-sulfo-d-glucosamine (1 â 3) ß-d-galactose (1â with some 6-O-sulfo galactose residues present. This KS was somewhat resistant to depolymerization using keratanase 1 but could be depolymerized efficiently through the use of reactive oxygen species generated using copper (II) and hydrogen peroxide. Of particular interest was the presence of substantial amounts of 2,8- and 2,9-linked N-acetylneuraminic acid residues in the form of oligosialic acid terminating the non-reducing ends of the KS chains. Most of the KS appears to be N-linked to a protein core as evidenced by its sensitivity to PNGase F.
Assuntos
Clara de Ovo/química , Sulfato de Queratano/química , Proteoglicanas/química , Animais , Galinhas , Galactose/química , Glicosídeo Hidrolases/química , Sulfato de Queratano/isolamento & purificação , Peso Molecular , Ácido N-Acetilneuramínico/química , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/química , Ácidos Siálicos/químicaRESUMO
The separation and quantification of glycosaminoglycan (GAG) chains with different levels of sulfation from cells and media, and prepared through chemoenzymatic synthesis or metabolic engineering, pose a major challenge in glycomics analysis. A method for microscale separation and quantification of heparin, heparan sulfate, and heparosan from cells is reported. This separation relies on a mini strong anion exchange spin column eluted stepwise with various concentrations of sodium chloride. Disaccharide analysis by LC-MS was used to monitor the chemical structure of the various GAG chains that were recovered.
Assuntos
Dissacarídeos/análise , Heparina/análise , Heparitina Sulfato/análise , Animais , Células CHO , Técnicas de Química Analítica , Cricetinae , Dissacarídeos/química , Heparina/química , Heparitina Sulfato/químicaRESUMO
Anticoagulant heparin has been shown to possess important biological functions that vary according to its fine structure. Variability within heparin's structure occurs owing to its biosynthesis and animal tissue-based recovery and adds another dimension to its complex polymeric structure. The structural variations in chain length and sulfation patterns mediate its interaction with many heparin-binding proteins, thereby eliciting complex biological responses. The advent of novel chemical and enzymatic approaches for polysaccharide synthesis coupled with high throughput combinatorial approaches for drug discovery have facilitated an increased effort to understand heparin's structure-activity relationships. An improved understanding would offer potential for new therapeutic development through the engineering of polysaccharides. Such a bioengineering approach requires the amalgamation of several different disciplines, including carbohydrate synthesis, applied enzymology, metabolic engineering, and process biochemistry.
Assuntos
Biotecnologia/métodos , Heparina/metabolismo , Engenharia Metabólica , Animais , Heparina/química , Relação Estrutura-AtividadeRESUMO
Heparin is a widely used clinical anticoagulant that is prepared from pig intestine. A contamination of heparin in 2008 has led to a reexamination of animal-derived pharmaceuticals. A bioengineered heparin prepared by bacterial fermentation and chemical and enzymatic processing is currently under development. This study examines the challenges of reducing or removing endotoxins associated with this process that are necessary to proceed with preclinical in vivo evaluation of bioengineered heparin. The current process is assessed for endotoxin levels, and strategies are examined for endotoxin removal from polysaccharides and enzymes involved in this process.
Assuntos
Bioengenharia/métodos , Contaminação de Medicamentos/prevenção & controle , Endotoxinas/análise , Endotoxinas/isolamento & purificação , Heparina/genética , Heparina/metabolismo , Cromatografia , Endotoxinas/química , Etanolaminas/química , Heparina/biossíntese , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Polimixina B/químicaRESUMO
Heparin, a sulfated polysaccharide, has been used as a clinical anticoagulant for over 90 years. Newer anticoagulants, introduced for certain specialized applications, have not significantly displaced heparin and newer heparin-based anticoagulants in most medical procedures. This chapter, while reviewing anticoagulation and these newer anticoagulants, focuses on heparin-based anticoagulants, including unfractionated heparin, low molecular weight heparins and ultra-low molecular weight heparins. Heparin's structures and its biological and therapeutic roles are discussed. Particular emphasis is placed on heparin's therapeutic application and its adverse effects. The future prospects are excellent for new heparins and new heparin-based therapeutics with improved properties.
Assuntos
Anticoagulantes/farmacologia , Heparina/farmacologia , Anticoagulantes/química , Anticoagulantes/classificação , Coagulação Sanguínea/efeitos dos fármacos , Coagulação Sanguínea/fisiologia , Coagulação Intravascular Disseminada/sangue , Coagulação Intravascular Disseminada/tratamento farmacológico , Circulação Extracorpórea/efeitos adversos , Heparina/química , Heparina/classificação , Humanos , Tromboembolia Venosa/sangue , Tromboembolia Venosa/tratamento farmacológicoRESUMO
Heparin is a widely used clinical anticoagulant. It is also a linear glycosaminoglycan with an average mass between 10 and 20 kDa and is primarily made up of trisulfated disaccharides comprised of 1,4-linked iduronic acid and glucosamine residues containing some glucuronic acid residues. Heparin is biosynthesized in the Golgi of mast cells commonly found in the liver, intestines, and lungs. Pharmaceutical heparin currently used in the United States is primarily extracted from porcine intestines. Other sources of heparin including bovine intestine and bovine lung are being examined as potential substitutes for porcine intestinal heparin. These additional sources are intended to serve to diversify the heparin supply, making this lifesaving drug more secure. The current study examines bovine heparins prepared from both intestines and lung and compares these to porcine intestinal heparin. The structural properties of these heparins are examined using nuclear magnetic resonance, gel permeation chromatography, and disaccharide analysis of heparinase-catalyzed depolymerized heparin. The in vitro functional activities of these heparins have also been determined. The goal of this study is to establish the structural and functional similarities and potential differences between bovine and porcine heparins. Porcine and bovine heparins have structural and compositional similarities and differences.
Assuntos
Heparina/análise , Animais , Anticoagulantes , Testes de Coagulação Sanguínea , Bovinos , Heparina/química , Heparina/normas , Intestinos/química , Pulmão/química , Estrutura Molecular , Peso Molecular , SuínosRESUMO
Contamination in heparin batches during early 2008 has resulted in a significant effort to develop a safer bioengineered heparin using bacterial capsular polysaccharide heparosan and recombinant enzymes derived from the heparin/heparan sulfate biosynthetic pathway. This requires controlled chemical N-deacetylation/N-sulfonation of heparosan followed by epimerization of most of its glucuronic acid residues to iduronic acid and O-sulfation of the C2 position of iduronic acid and the C3 and C6 positions of the glucosamine residues. A combinatorial study of multi-enzyme, one-pot, in vitro biocatalytic synthesis, carried out in tandem with sensitive analytical techniques, reveals controlled structural changes leading to heparin products similar to animal-derived heparin active pharmaceutical ingredients. Liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy analysis confirms an abundance of heparin's characteristic trisulfated disaccharide, as well as 3-O-sulfo containing residues critical for heparin binding to antithrombin III and its anticoagulant activity. The bioengineered heparins prepared using this simplified one-pot chemoenzymatic synthesis also show in vitro anticoagulant activity.
Assuntos
Anticoagulantes/farmacologia , Heparina/biossíntese , Sulfotransferases/metabolismo , Animais , Anticoagulantes/química , Bioengenharia/métodos , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , Fator Xa/química , Fator Xa/metabolismo , Heparina/química , Heparina/farmacologia , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Protrombina/antagonistas & inibidores , Protrombina/metabolismoRESUMO
The standard process for preparing the low-molecular-weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield because of 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 by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity.
Assuntos
Heparina/química , Dissacarídeos/química , Heparina de Baixo Peso Molecular/química , Peso Molecular , Tinzaparina , Ultrafiltração/métodosRESUMO
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 the 1990s and was exacerbated during the heparin shortage in the 2000s and the heparin contamination crisis of 2007-2008. 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.