Porcine Intestinal Heparin Research Articles

A Unique Enoxaparin Derived from Bovine Intestinal Heparin: A Single Purification Step of the Starting Material Assures a Bovine Enoxaparin Like the Standard from Porcine Origin.

Low-molecular-weight heparin represent a significant advancement in anticoagulant therapy with enoxaparin being a prominent example obtained exclusively through the fragmentation of porcine intestinal heparin. However, escalating demand and limited resources have raised concerns about enoxaparin supplementation. The current challenge involves exploring alternative heparin sources for large-scale enoxaparin production with bovine intestinal heparin emerging as a promising option. Our study demonstrates that enoxaparin derived from the available bovine heparin preparation differs significantly from the reference compound. Yet, the implementation of a straightforward purification step yields a preparation termed "high-anticoagulant bovine heparin". Fragmentation of this purified product through β-elimination produces enoxaparin akin to the standard from a porcine origin. To ensure physicochemical similarity, we employed various spectroscopic, enzymatic, and chromatographic tests to compare the new bovine-derived enoxaparin with the original porcine compound. Biological activity was confirmed through in vitro coagulation assays and assessments using an animal model of venous thrombosis. Our study affirms that the β-elimination reaction cleaves the bovine heparin chain without preferential breaks in regions with different sulfation patterns. Additionally, we scrutinized decasaccharides purified from enoxaparin preparations, providing a comprehensive demonstration of the similarity between products obtained from porcine and bovine heparin. In summary, our findings indicate that an enoxaparin equivalent to the original porcine-derived product can be derived from bovine heparin, given that the starting material undergoes a simple purification step.

Quantification of Bovine and Porcine Heparins Utilizing the Heparin Red Assay, Applications in the Study of Pharmacokinetics and Pharmacodynamics

Introduction Currently there is a major interest in developing bovine intestinal heparin for anticoagulant purposes. It is well known that bovine intestinal heparins exhibit a relatively lower potency than porcine intestinal heparins. The pharmacokinetic behavior of heparins has traditionally been based on antithrombin-dependent anti-Xa and anti-IIa activities. Heparin Red is a polycationic substance whose intrinsic fluorescence is quenched upon binding to heparin. As such, assays utilizing Heparin Red detect all heparin oligosaccharides in a sample: those that bind to AT and produce anti-Xa and anti-IIa activities, and those that do not. These non-AT binding heparin oligosaccharides can still impact coagulation through their interaction with heparin cofactor II, TFPI and platelets. Thus, the pharmacokinetic behavior of heparin (half-life, etc.) as determined using a Heparin Red assay may more closely reflect the clinical behavior of heparin (antithrombotic, bleeding) than pharmacodynamic measures where heparin levels are determined based only on AT-dependent activities (anti-Xa or anti-IIa). Methods Primates were administered PMH (Medefil, Glendale Heights, IL) or BMH (KinMaster, Passo Fundo, Brazil) at a dose of 100 anti-Xa U/kg IV. Blood samples were collected prior to and at 15, 30, 60 and 120 minutes post-heparin administration. Blood samples were centrifuged to produce platelet poor plasma which was aliquoted and stored frozen until analysis. Heparin levels were assessed using a chromogenic anti-Xa assay and a Heparin Red assay relative to product-specific calibration curves. Pharmacokinetic parameters were assessed using a non-compartmental model. Results Circulating functional drug levels based on anti-Xa activity were the same in PMH and BMH-treated primates. Peak levels of 1.45 ± 0.11 and 1.48 ± 0.08 U/ml were observed in PMH and BMH-treated primates, respectively. Using drug levels determined by anti-Xa assay, AUCs for bovine and porcine heparin treated animals were calculated to be 111.5 ± 11.0 and 108.8 ± 26.7 U*min*ml-1, respectively. By Heparin Red assay, peak circulating heparin levels were higher following BMH administration (10.7 ± 0.5 vs. 9.0 ± 0.2 mg/ml; t-test p<0.001) and the AUC for BMH-treated primates was approximately 22% larger than for PMH-treated primates (728.2 ± 35.3 vs. 594.9 ± 5.4 mg*min*ml-1; Mann-Whitney test p=0.029). Conclusions BMH, when administered at equivalent anti-Xa unit doses, produces comparable pharmacodynamic effects as PMH despite the presence of higher circulating GAG levels as measured by the Heparin Red assay. Measurement of BMH levels using the Heparin Red assay may be useful for studying its absolute pharmacokinetics and optimization of protamine sulfate dosage for its neutralization.

Preparation of Low Molecular Weight Heparin from a Remodeled Bovine Intestinal Heparin.

Bovine intestinal heparins are structurally distinct from porcine intestinal heparins and exhibit lower specific anticoagulant activity (units/mg). The reduced content of N-sulfo, 3-O-sulfo glucosamine, the central and critical residue in heparin's antithrombin III binding site, is responsible for bovine intestinal heparin's reduced activity. Previous studies demonstrate that treatment of bovine intestinal heparin with 3-O-sulfotransferase in the presence of 3'-phosphoadenosine-5'-phosphosulfate afforded remodeled bovine heparin with an enhanced activity reaching the United States Pharmacopeia's requirements. Starting from this remodeled bovine intestinal heparin, we report the preparation of a bovine intestinal low molecular weight heparin having the same structural properties and anti-factor IIa and anti-factor Xa activities of Enoxaparin. Moreover, this bovine intestinal heparin-derived "Enoxaparin" showed comparable platelet factor-4 binding affinity, suggesting that it should exhibit similarly low levels of heparin induced thrombocytopeneia, HIT.

Processing bovine intestinal mucosa to active heparin removes spiked BSE agent

Heparin is an anticoagulant sourced from animal tissues. In the 1990s, bovine-sourced heparin was withdrawn from the U.S. market due to a theoretical concern that the bovine spongiform encephalopathy (BSE) agent might contaminate crude heparin and spread to humans as variant Creutzfeldt-Jakob disease. Only porcine intestinal heparin is now marketed in the U.S. FDA has encouraged the reintroduction of bovine heparin. We applied a scaled-down laboratory model process to produce heparin as an active pharmaceutical ingredient (API) starting from bovine intestinal mucosa. The process consisted of two phases. To model the first phase, we applied enzymatic proteolysis, anionic resin separation and methanol precipitation of crude heparin. Bovine intestinal mucosa was spiked with BSE or scrapie agents. We assayed BSE- or scrapie-associated prion protein (PrPTSE) using the Real-Time Quaking-Induced Conversion (RT-QuIC) assay at each step. The process reduced PrPTSE by 4 log10 and 6 log10 from BSE-spiked and scrapie-spiked mucosa, respectively. To model the entire process, we spiked mucosa with scrapie agent and produced heparin API, reducing PrPTSE by 6.7 log10. The purification processes removed large amounts of PrPTSE from the final products. Heparin purification together with careful sourcing of raw materials should allow safely reintroducing bovine heparin in the U.S.

Quantitative Analysis of Impurities in Unfractionated Heparin of Bovine Origin.

The USP heparin sodium monograph lists impurities with specifications developed for porcine derived products. Most of these impurities are of biological origin and are present in porcine intestinal mucosa, the tissue source used in the production of porcine heparin. One of the specified impurities, oversulfated chondroitin sulfate (OSCS), has been introduced in the monograph to detect intended adulteration of heparin products with this impurity. The evaluation of bovine intestinal heparin as an alternative source of pharmaceutical heparin included an evaluation of bovine heparin with the current USP heparin sodium monograph methods. This evaluation included a comparison of impurity quantities observed in multiple bovine intestinal heparin samples against the specifications found in the USP heparin sodium monograph. The impurities investigated in this study were protein, galactosamine, nucleotidic impurities, and OSCS. Bovine intestinal heparin met the requirements in the tests for protein, galactosamine, and nucleotidic impurities. A potential issue was observed with the strong anion exchange high performance liquid chromatography (SAX-HPLC) used to analyze for the presence of OSCS. While the OSCS was well-resolved from the bovine heparin peak, the resolution of dermatan sulfate from heparin did not consistently meet system suitability requirements in the current USP Heparin sodium monograph. The overall levels of impurities observed in bovine intestinal mucosal heparin were comparable to those observed in porcine intestinal mucosal heparin. Bovine intestinal mucosal heparin can be produced with acceptable impurity levels that align with these important quality attributes found in porcine heparin.

The Use of Myelinating Cultures as a Screen of Glycomolecules for CNS Repair.

In vitro cell-based assays have been fundamental in modern drug discovery and have led to the identification of novel therapeutics. We have developed complex mixed central nervous system (CNS) cultures, which recapitulate the normal process of myelination over time and allow the study of several parameters associated with CNS damage, both during development and after injury or disease. In particular, they have been used as a reliable screen to identify drug candidates that may promote (re)myelination and/or neurite outgrowth. Previously, using these cultures, we demonstrated that a panel of low sulphated heparin mimetics, with structures similar to heparan sulphates (HSs), can reduce astrogliosis, and promote myelination and neurite outgrowth. HSs reside in either the extracellular matrix or on the surface of cells and are thought to modulate cell signaling by both sequestering ligands, and acting as co-factors in the formation of ligand-receptor complexes. In this study, we have used these cultures as a screen to address the repair potential of numerous other commercially available sulphated glycomolecules, namely heparosans, ulvans, and fucoidans. These compounds are all known to have certain characteristics that mimic cellular glycosaminoglycans, similar to heparin mimetics. We show that the N-sulphated heparosans promoted myelination. However, O-sulphated heparosans did not affect myelination but promoted neurite outgrowth, indicating the importance of structure in HS function. Moreover, neither highly sulphated ulvans nor fucoidans had any effect on remyelination but CX-01, a low sulphated porcine intestinal heparin, promoted remyelination in vitro. These data illustrate the use of myelinating cultures as a screen and demonstrate the potential of heparin mimetics as CNS therapeutics.

Chemometric analysis of porcine, bovine and ovine heparins

Heparin is a polysaccharide anticoagulant drug isolated from animal tissues. There have been concerns on the safety and security of the heparin supply chain since 2007-8 when a contamination crisis led to its disruption. The current study applies a suite of modern analytical techniques to porcine, bovine and ovine intestinal mucosal heparins. These techniques include structural analysis by nuclear magnetic resonance spectrometry, disaccharide compositional analysis, bottom-up analysis of tetrasaccharides corresponding to heparin’s antithrombin III binding site. Chemometric analysis was then applied to understand how these structural differences to predict the animal/tissue source of heparin and to help detect blending of heparins from various sources.

Preparation of low molecular weight heparins from bovine and ovine heparins using nitrous acid degradation

Low molecular weight heparins (LMWHs) are important anticoagulant drugs. Nitrous acid degradation is a major approach to produce LMWHs, such as dalteparin. Due to the foreseeable shortage of porcine intestinal mucosa heparin and other potential risks, expansion of other animal tissues for heparin preparation is necessary. Heparins from different tissues differ in structure and bioactivity potency, and these variations may be carried over to the LMWH products. Sophisticated analytical techniques have been applied to compare various versions of dalteparins produced from porcine intestinal, bovine lung and ovine intestinal heparins to elucidate the effects of different animal tissues starting materials and processing conditions on the properties of final dalteparin products. With adjusted depolymerization conditions, versions of dalteparins that qualify under the European Pharmacopeia (EP) specifications were manufactured using non-porcine heparins. Dissimilarities among the three interspecies animal tissue heparin-derived dalteparins regarding fine structures are also disclosed, and their origins are discussed.

Parent heparin and daughter LMW heparin correlation analysis using LC-MS and NMR

Heparin is a structurally complex, polysaccharide anticoagulant derived from livestock, primarily porcine intestinal tissues. Low molecular weight (LMW) heparins are derived through the controlled partial depolymerization of heparin. Increased manufacturing and regulatory concerns have provided the motivation for the development of more sophisticated analytical methods for determining both their structure and pedigree. A strategy, for the comprehensive comparison of parent heparins and their LMW heparin daughters, is described that relies on the analysis of monosaccharide composition, disaccharide composition, and oligosaccharide composition. Liquid chromatography-mass spectrometry is rapid, robust, and amenable to automated processing and interpretation of both top-down and bottom-up analyses. Nuclear magnetic resonance spectroscopy provides complementary top-down information on the chirality of the uronic acid residues and glucosamine substitution. Principal component analysis (PCA) was applied to the normalized abundance of oligosaccharides, calculated in the bottom-up analysis, to show parent and daughter correlation in oligosaccharide composition. Using these approaches, six pairs of parent heparins and their daughter generic enoxaparins from two different manufacturers were comprehensively analyzed. Enoxaparin is the most widely used LMW heparin and is prepared through controlled chemical β-eliminative cleavage of porcine intestinal heparin. Lovenox®, the innovator version of enoxaparin marketed in the US, was analyzed as a reference for the daughter LMW heparins. The results, show similarities between LMW heparins from two different manufacturers with Lovenox®, excellent lot-to-lot consistency of products from each manufacturer, and detects a correlation between each parent heparin and daughter LMW heparin.

Analytical comparison of a US generic enoxaparin with the originator product: The focus on comparative assessment of antithrombin-binding components

Enoxaparin sodium, a low-molecular-weight heparin (LMWH) prepared from porcine intestinal heparin, is widely used for the prevention and treatment of venous thromboembolism. The antithrombotic activity of heparin is mediated mainly through its activation of antithrombin (AT) and subsequent inhibition of coagulation factors. Heparin is a complex heteropolymer and the sulfation pattern of its alternating uronic acid and glucosamine sugar units is a major factor influencing its biological activity. The manufacturing process itself is associated with the introduction of exogenous microheterogeneities that may further affect its biological efficacy. This is important since enoxaparin is prepared by depolymerizing the heparin with the aim of optimizing its biological activity and safety. Changes during its manufacture could thus affect its biological activity and safety. The current study was performed to assess potential differences between the originator enoxaparin and a new generic enoxaparin commercialized by Teva. Heparinase digestion, AT affinity chromatography, gel permeation chromatography, anion exchange chromatography, and nuclear magnetic resonance methodologies were used. The results indicated differences in oligosaccharides related to the cleavage selectivity around the heparin AT-binding sequences of the Teva Enoxaparin Sodium Injection, USP and the originator Sanofi enoxaparin. These differences influence the strength of the AT-binding affinity of the individual oligosaccharides, their ability to activate AT and, therefore, the inhibitory potency on the proteases of the coagulation cascade. This study, together with other published analytical reports, describes specific compositional differences between generics and originator LWMHs. However, it is yet to be established whether such variations might have any clinical relevance.

Low anticoagulant heparin oligosaccharides as inhibitors of BACE-1, the Alzheimer’s β-secretase

Heparin (HP) is a promising agent for anti-Alzheimer’s disease (AD), but its anticoagulant activity limits its applications. So a low anticoagulant heparin (LAH) with anti-AD effect is needed. A novel LAH and heparan sulfate (HS) were purified from crude porcine intestinal heparin. Their structures were characterized by nuclear magnetic resonance and liquid chromatography-mass spectrometry. LAH had a relatively high degree of sulfation, but lower than that of HP. 3-O-Sulfated-containing glucosamine residues further confirmed the low anticoagulant activity of LAH. Sixteen oligosaccharides of LAH and HS were prepared and assigned. Evaluation of anti-BACE-1 activities suggested that their potencies were positively correlated with degree of sulfation and polymerization of oligosaccharides. Besides, LAH-derived hexa- to dodecasaccharides was promised to be administrated in vitro as BACE-1 inhibitors. This study presented ideal BACE-1 inhibitors, LAH-derived oligosaccharides, with virtually no anticoagulant activities, which were promised to be excellent leads for treatment of AD.

Analysis of 3-O-sulfo group-containing heparin tetrasaccharides in heparin by liquid chromatography–mass spectrometry

Complete heparin digestion with heparin lyase 2 affords a mixture of disaccharides and resistant tetrasaccharides with 3-O-sulfo group-containing glucosamine residues at their reducing ends. Quantitative online liquid chromatography–mass spectrometric analysis of these resistant tetrasaccharides is described in this article. The disaccharide and tetrasaccharide compositions of seven porcine intestinal heparins and five low-molecular-weight heparins were analyzed by this method. These resistant tetrasaccharides account for from 5.3 to 7.3wt% of heparin and from 6.2 to 8.3wt% of low-molecular-weight heparin. Because these tetrasaccharides are derived from heparin’s antithrombin III-binding sites, we examined whether this method could be applied to estimate the anticoagulant activity of heparin. The content of 3-O-sulfo group-containing tetrasaccharides in a heparin correlated positively (r=0.8294) to heparin’s anticoagulant activity.

Heparin-derived supersulfated disaccharide inhibits allergic airway responses in sheep

The tetrasaccharide sequence of heparin oligosaccharides is the minimum chain length possessing anti-allergic activity, as the disaccharide fraction is inactive. Since sulfation pattern can modify the biological actions of heparin, we hypothesized that “supersulfation” of the inactive heparin disaccharide could confer anti-allergic activity to this molecule. To test this, we produced a supersulfated heparin disaccharide (Hep-SSD) and evaluated its anti-allergic activity in sheep with documented antigen-induced early and late airway responses (EAR and LAR) and airway hyperresponsiveness (AHR). Porcine intestinal heparin was depolymerized with nitrous acid, the disaccharide fraction separated by size exclusion chromatography, and then treated with pyridine-sulfur trioxide complex to yield Hep-SSD. Its chemical structure [IdoU2′,3′,4′S (1→4) AMan1,3,6S] was confirmed by HPLC, Mass Spectrometry and NMR analysis. Inhaled doses of 5 mg, 10 mg and 20 mg Hep-SSD produced inhibition of EAR (8%, 35% and 35%), LAR (50%, 80%, and 77%) and AHR (67%, 100% and 75%), respectively. A single oral dose of 2 mg/kg Hep-SSD given 90 min before challenge significantly inhibited EAR, LAR and AHR, but 1 mg/kg was ineffective. Multi dose oral treatment with Hep-SSD had a cumulative effect, as a once daily dose of 2 mg/kg for 3 days (last dose, 16 h before antigen) inhibited EAR, LAR and AHR by 30%, 75% and 74%, respectively. Finally, the oral activity of Hep-SSD could be enhanced 4 fold by formulating it with Carbopol®934P, in an enteric coated capsule. These data demonstrate that “supersulfation” can confer biological activity to the inactive heparin disaccharide. Both inhaled and oral Hep-SSD demonstrate significant anti-allergic activity and, therefore, may have therapeutic potential.

Bovine and porcine heparins: different drugs with similar effects on human haemodialysis

BackgroundHeparins from porcine and bovine intestinal mucosa differ in their structure and also in their effects on coagulation, thrombosis and bleeding. However, they are used as undistinguishable drugs.MethodsWe compared bovine and porcine intestinal heparin administered to patients undergoing a particular protocol of haemodialysis. We compared plasma concentrations of these two drugs and also evaluated how they affect patients and the dialyzer used.ResultsCompared with porcine heparin, bovine heparin achieved only 76% of the maximum plasma concentration as IU mL-1. This observation is consistent with the activities observed in the respective pharmaceutical preparations. When the plasma concentrations were expressed on weight basis, bovine heparin achieved a maximum concentration 1.5 fold higher than porcine heparin. The reduced anticoagulant activity and higher concentration, on weight basis, achieved in the plasma of patients under dialysis using bovine instead of porcine heparin did not affect significantly the patients or the dialyzer used. The heparin dose is still in a range, which confers security and safety to the patients.DiscussionDespite no apparent difference between bovine and porcine intestinal heparins in the haemodialysis practice, these two types of heparins should be used as distinct drugs due to their differences in structure and biological effects.ConclusionsThe reduced anticoagulant activity achieved in the plasma of patients under dialysis using bovine instead of porcine heparin did not affect significantly the patients or the dialyzer.

Challenges for Heparin Production: Artificial Synthesis or Alternative Natural Sources?

The commentary emphasizes the immediate necessity to find alternative sources for the production of pharmaceutical heparin to meet the ton-amount world's demand of the glycosaminoglycan. The recent development of a synthetic approach brings considerable new technical and scientifically relevant knowledge to the field, with a strong potential for application in the future. However, the artificial approach does not offer a rapid alternative for the current world crisis affecting the production of heparin, which has to respond to an increasing worldwide demand. It is important to call attention for the availability of marine organisms that are rich sources of heparin analogs with significant anticoagulant activity, low bleeding effect that have been cultivated in very large amounts for years in different parts of the world. Additionally, alternative sources of mammalian heparin, such as bovine intestine and lung have been continually used in countries from South America, Africa and Asia, since the outbreak of the BSE without any report of prion contamination in humans. Recently, it has been shown that bovine and porcine intestinal heparins are composed by different proportions of a mixture of the same fractions that can be simply separated by anion exchange chromatography. In other words, high quality porcine heparin can be obtained from bovine tissue. We believe that alternative animal sources of heparin are currently a more realistic solution than artificial heparin to respond to the increasing demand for heparin.

Heparin from bovine intestinal mucosa: Glycans with multiple sulfation patterns and anticoagulant effects

Pharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide →4-α-IdoA2S-1→4-α-GlcNS6S-1→. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N -sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3- and 6-trisulfated α-glucosamine (lower proportions) and α-GlcNS-1→4-α-GlcA and α-IdoA2S-1→4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the α-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical heparin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro . Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.

Heparin from bovine intestinal mucosa: Glycans with multiple sulfation patterns and anticoagulant effects

SummaryPharmaceutical grade heparins from porcine intestine and bovine lung consist mainly of repeating tri-sulfated units, of the disaccharide →4-α-IdoA2S-1 →4-α-GlcNS6S-1 →. Heparin preparations from bovine intestine, in contrast, are more heterogeneous. Nuclear magnetic resonance (NMR) and disaccharide analysis after heparinase digestions show that heparin from bovine intestine contains α-glucosamine with significant substitutive variations: 64% are 6-O-sulfated and N-sulfated, as in porcine intestinal heparin while 36% are 6-desulfated. Desulfated α-iduronic acid units are contained in slightly lower proportions in bovine than in porcine heparin. NMR data also indicate N-, 3-and 6-trisulfated a-glucosamine (lower proportions) and α-GlcNS-1→4-α-GlcA and α-IdoA2S-1→4-α-GlcNAc (higher amounts) in bovine than in porcine heparin. Porcine and bovine heparins can be fractionated by anion exchange chromatography into three fractions containing different substitutions on the a-glucosamine units. Each individual fraction shows close disaccharide composition and anticoagulant activity, regardless of their origin (bovine or porcine intestine). However, these two heparins differ markedly in the proportions of the three fractions. Interestingly, fractions with the typical he-parin disaccharides of porcine intestine are present in bovine intestinal heparin. These fractions contain high in vitro anticoagulant activity, reduced antithrombotic effect and high bleeding tendency. These observations indicate that the prediction of haemostatic effects of heparin preparations cannot rely exclusively on structural analysis and anticoagulant assays in vitro. Minor structural components may account for variations on in vivo effects. In conclusion, we suggest that pharmaceutical grade bovine intestinal heparin, even after purification procedures, is not an equivalent drug to porcine intestinal heparin.

NMR methods to monitor the enzymatic depolymerization of heparin

Heparin and the related glycosaminoglycan, heparan sulfate, are polydisperse linear polysaccharides that mediate numerous biological processes due to their interaction with proteins. Because of the structural complexity and heterogeneity of heparin and heparan sulfate, digestion to produce smaller oligosaccharides is commonly performed prior to separation and analysis. Current techniques used to monitor the extent of heparin depolymerization include UV absorption to follow product formation and size exclusion or strong anion exchange chromatography to monitor the size distribution of the components in the digest solution. In this study, we used 1H nuclear magnetic resonance (NMR) survey spectra and NMR diffusion experiments in conjunction with UV absorption measurements to monitor heparin depolymerization using the enzyme heparinase I. Diffusion NMR does not require the physical separation of the components in the reaction mixture and instead can be used to monitor the reaction solution directly in the NMR tube. Using diffusion NMR, the enzymatic reaction can be stopped at the desired time point, maximizing the abundance of larger oligosaccharides for protein-binding studies or completion of the reaction if the goal of the study is exhaustive digestion for characterization of the disaccharide composition. In this study, porcine intestinal mucosa heparin was depolymerized using the enzyme heparinase I. The unsaturated bond formed by enzymatic cleavage serves as a UV chromophore that can be used to monitor the progress of the depolymerization and for the detection and quantification of oligosaccharides in subsequent separations. The double bond also introduces a unique multiplet with peaks at 5.973, 5.981, 5.990, and 5.998 ppm in the 1H-NMR spectrum downfield of the anomeric region. This multiplet is produced by the proton of the C-4 double bond of the non-reducing end uronic acid at the cleavage site. Changes in this resonance were used to monitor the progression of the enzymatic digestion and compared to the profile obtained from UV absorbance measurements. In addition, in situ NMR diffusion measurements were explored for their ability to profile the different-sized components generated over the course of the digestion. FigureDOSY spectra of intact (blue) and digested (red) heparin illustrating the differences in their diffusion coefficients.

Heparinase 1 selectivity for the 3,6-di-O-sulfo-2-deoxy-2-sulfamido-α-D-glucopyranose (1,4) 2-O-sulfo-α-L-idopyranosyluronic acid (GlcNS3S6S-IdoA2S) linkages

Porcine intestinal mucosa heparin was partially depolymerized by recombinant heparinase 1 (heparin lyase 1, originating from Flavobacterium heparinum and expressed in Escherichia coli) and then fractionated, leading to the isolation of 22 homogeneous oligosaccharides with sizes ranging from disaccharide to hexadecasaccharide. The purity of these oligosaccharides was determined by gel electrophoresis, strong anion exchange and reversed-phase ion-pairing high-performance liquid chromatography. The molecular mass of oligosaccharides was determined using electrospray ionization-mass spectrometry and their structures were elucidated using one- and two-dimensional nuclear magnetic resonance spectroscopy at 600MHz. Five of the characterized oligosaccharides represent new compounds. The most prominent oligosaccharide comprises the common repeating unit of heparin, ΔUA2S-[-GlcNS6S-IdoA2S-](n)-GlcNS6S, where ΔUA is 4-deoxy-α-l-threo-hex-4-eno-pyranosyluronic acid, GlcN is 2-deoxy-2-amino-d-glucopyranose, IdoA is l-idopyranosyluronic acid, S is sulfate and n=0-7. A second prominent heparin oligosaccharide motif corresponds to ΔUA2S-[GlcNS6S-IdoA2S](n)-GlcNS6S-IdoA-GlcNAc6S-GlcA-GlcNS3S6S (where n=0-5 and GlcA is d-glucopyranosyluronic acid), a fragment of the antithrombin III binding site in heparin. The prominence of this second set of oligosaccharides and the absence of intact antithrombin III binding sites suggest that the -GlcNS3S6S-IdoA2S- linkage is particularly susceptible to heparinase 1.

Analysis of E. coli K5 capsular polysaccharide heparosan

Heparosan is the key precursor for the preparation of bioengineered heparin, a potential replacement for porcine intestinal heparin, an important anticoagulant drug. The molecular weight (MW) distribution of heparosan produced by the fermentation of E. coli K5 was investigated. Large-slab isocratic and mini-slab gradient polyacrylamide gel electrophoresis (PAGE) were used to analyze the MW and polydispersity of heparosan. A preparative method that allowed fractionation by continuous-elution PAGE was used to obtain heparosan MW standards. The MWs of the heparosan standards were determined by electrospray ionization Fourier-transform mass spectrometry (ESI-FT-MS). A ladder of the standards was then used to determine the MW properties of polydisperse heparosan samples. Unbleached and bleached heparosan produced by fermentation of E. coli K5 had similar number-averaged MWs (M(N)), weight-averaged MWs (M(W)), and MW ranges of 3,000 to 150,000Da.