Production Of Low Molecular Weight Heparin Research Articles

Cloning, Expression, and Characterization of a Highly Stable Heparinase I from Bacteroides xylanisolvens.

Heparinase I (Hep I), which specifically degrades heparin to oligosaccharide or unsaturated disaccharide, has an important role in the production of low molecular weight heparin (LMWH). However, low productivity and stability of heparinase I hinders its applications. Here, a novel heparinase I (BxHep-I) was cloned from Bacteroides xylanisolvens and overexpressed in soluble form in Escherichia coli. The expression conditions of BxHep-I were optimized for an activity of 7144 U/L. BxHep-I had a specific activity of 57.6 U/mg at the optimal temperature and pH of 30 °C and pH 7.5, with the Km and Vmax of 0.79 mg/mL and 124.58 U/mg, respectively. BxHep-I catalytic activity could be enhanced by Ca2+ and Mg2+, while strongly inhibited by Zn2+ and Co2+. Purified BxHep-I displayed an outstanding thermostability with half-lives of 597 and 158 min at 30 and 37 °C, respectively, which are the highest half-lives ever reported for heparinases I. After storage at 4 °C for one week, BxHep-I retained 73% of its initial activity. Molecular docking revealed that the amino acids Asn25, Gln27, Arg88, Lys116, His156, Arg161, Gln228, Tyr356, Lys358, and Tyr362 form 13 hydrogen bonds with the substrate heparin disaccharides in the substrate binding domain and are mainly involved in the substrate binding of BxHep-I. These results suggest that the BxHep-I with high stability could be a candidate catalyst for the industrial production of LMWH.

Considerations for the Bioanalytical Part of Equivalence Studies of Biosimilar Nadroparin Calcium

According to current regulatory views, a comparative study of the pharmacodynamics (PD) of low molecular weight heparin (LMWH) products and confirmation of their equivalence require comparing three PD markers: the anti-Xa activity, the anti-IIa activity, and the tissue factor pathway inhibitor (TFPI) concentration. The aim of this study was to analyse the features specific to the bioanalytical part of an equivalence study of a nadroparin calcium biosimilar after single subcutaneous administration. Material and methods: the anti-Xa and anti-IIa activity values and TFPI content were determined in human plasma samples obtained after single subcutaneous administration of the test and the reference product in the same dose, using commercially available reagent kits and pre-validated assays. The authors calculated the main PD parameters (surrogate pharmacokinetic markers), namely the maximum activity or concentration (Amax or Cmax), time to maximum activity or concentration (Tmax), area under the activity–time (or concentration–time) curve (AUC ), and half-life period (T1/2), by means of model-independent statistical moment analysis and carried out further statistical testing of the parameters. Results: the anti-Xa activity and TFPI concentration results provided for the possibility of calculating and comparing the PD parameters (Amax or Cmax, AUC0-24, AUC0-∞, Tmax, T1/2) and estimating the confidence intervals that are necessary to confirm the bioequivalence of the studied products. The anti-IIa activity data had a characteristic pattern of slight fluctuations around one level, which prevented the calculation and comparison of PD parameters. Conclusion: the study identified specific features to consider when planning comparative PD studies of nadroparin calcium products. Firstly, it is feasible to divide samples into two test aliquots (one for anti-Xa and anti-IIa activity determination, the other for TFPI analysis) at the moment of collection in order to perform the analytical step correctly. Secondly, there is no need in full validation for the bioanalytical assays of the anti-Xa and anti-II activity and TFPI content in human plasma validated in the concentration ranges of 0.024–0.182 IU/mL, 0.0069–0.052 IU/mL and 1.56–100 ng/mL, respectively; a confirmation that the active ingredient does not interfere with the analytical procedure is adequate for the purpose. Finally, the data obtained may not allow for calculating PD parameters and comparing confidence intervals for all three markers. The listed considerations may be relevant for other LMWH products as well.

A retrospective review on importance and various preparation of low molecular weight heparin for cardio vascular diseases

Low-molecular-weight heparin (LMWH) are most importantly used in the clinical practice for cardio vascular diseases from 1990. The LMWH was a sulfated polysaccharide obtained from animal sources and some in natural especially from marine sources. The actual anticoagulant has the molecular weight about 25000 Daltons which have bleeding as side effect in the cardiovascular diseases. The LMWH are prepared by enzymatic or chemical hydrolysis the length of the heparin chain is reduced and also in same manner the molecular weight also reduced below 10000 Daltons. So, it has main advantage to reduce the bleeding in the cardiovascular diseases. The LMWH eliminated through the renal and it was not given to the patients with renal dysfunction. The LMWH of some product are still in clinical trials in order to reduce its side effects. The commercial LMWH preparation has concentrated in animal sources by killing them and lungs, intestine etc are used to prepare heparin. Here the alternate sources are discussed in order from killing the animal. This review summarizes the importance, difference between commercial heparin and LMWH, mechanism, preparation, LMWH products, clinical trials and LMWH from marine sources, Enzymatic degradation in shrimp species, Preparation of LMW heparin by chemically modified Fractions, Chromatography separation of LMW Heparin are discussed in this review.

Analysis of heparinase derived LMWH products using a MHC 2D LC system linked to Q-TOF MS

Low molecular weight heparins (LMWHs), depolymerized from unfractionated heparin (UFH), are widely used as anticoagulant drugs in clinic. The variable degradation methods result the different types of LMWHs, such as enoxaparin prepared by alkaline degradation following benzylation and nadroparin degraded by nitrous acid and subsequent reduction. They have different anticoagulant activities, molecular weight and special oligosaccharide sequences. Oligosaccharide analysis of the heparinase-catalyzed digestion products of heparin and LMWHs is an important way to explore the fine structural composition. In this work, a MHC-2D-LC-MS system using SAX followed by SEC and tandem to MS was applied to analyze the heparinase-products of LMWHs. 15 components of enoxaparin and 20 components of nadroparin were separated and unambiguously characterized with mass spectrum, including eight common disaccharides, and the special structural domains resistant to enzyme digestion which have the 3-O sulfated residue and/or characteristic terminal residues and the linkage region tetrasaccharides.

Analysis of Heparinase Derived LMWH Products Using a MHC 2D LC System Linked to Q-TOF MS

Low molecular weight heparins (LMWHs), depolymerized from unfractionated heparin (UFH), are widely used as anticoagulant drugs in clinic. The variable degradation methods result the different types of LMWHs, such as enoxaparin prepared by alkaline degradation following benzylation and nadroparin degraded by nitrous acid and subsequent reduction. They have different anticoagulant activities, molecular weight and special oligosaccharide sequences. Oligosaccharide analysis of the heparinase-catalyzed digestion products of heparin and LMWHs is an important way to explore the fine structural composition. In this work, a MHC-2D-LC-MS system using SAX followed by SEC and tandem to MS was applied to analyze the heparinase-products of LMWHs. 15 components of enoxaparin and 20 components of nadroparin were separated and unambiguously characterized with mass spectrum, including eight common disaccharides, and the special structural domains resistant to enzyme digestion which have the 3-O sulfated residue and/or characteristic terminal residues and the linkage region tetrasaccharides.

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.

Characterization of Low-Molecular-Weight Heparins by Strong Anion-Exchange Chromatography.

Currently, detailed structural characterization of low-molecular-weight heparin (LMWH) products is an analytical subject of great interest. In this work, we carried out a comprehensive structural analysis of LMWHs and applied a modified pharmacopeial method, as well as methods developed by other researchers, to the analysis of novel biosimilar LMWH products; and, for the first time, compared the qualitative and quantitative composition of commercially available drugs (enoxaparin, nadroparin, and dalteparin). For this purpose, we used strong anion-exchange (SAX) chromatography with spectrophotometric detection because this method is more helpful, easier, and faster than other separation techniques for the detailed disaccharide analysis of new LMWH drugs. In addition, we subjected the obtained results to statistical analysis (factor analysis, t-test, and Newman-Keuls post hoc test).

Comprehensive Identification and Quantitation of Basic Building Blocks for Low-Molecular Weight Heparin.

Low-molecular weight heparins (LMWHs) are widely used anticoagulant drugs. They inherit the heterogeneous backbone sequences of the parent heparin, while the chemical depolymerization process modifies the nonreducing end (NRE) and reducing end (RE) of their sugar chains. Some side reactions may also occur and increase the structural complexity of LMWHs. It is important to precisely characterize the structures of LMWHs, especially their chemical modifications, to ensure drug quality and safety. Compositional analysis provides a powerful approach to reveal the building blocks that make up the LMWHs, which are the mutual consequence of the heparin starting materials and the manufacturing process. Here, we introduce a comprehensive analytical method to recover the most basic building blocks of LMWHs. A strategy of combining both enzymatic digestion and oxidative degradation of LMWH was used to make the NRE, RE, and backbone structures differentiable from one another. Satisfactory separation, identification, and quantitation were achieved by coupling hydrophilic interaction chromatography with a triple quadrupole mass spectrometer operating under the multiple reaction monitoring mode. After enzymatic digestion, over 30 species were detected, with both natural and chemically modified heparin basic building blocks. Two novel structures, including a trisaccharide containing two glucosamine residues and a tetrasaccharide containing a 3-O-sulfated uronic acid residue, were discovered. Reduced and oxidatively degraded samples were analyzed to provide the complementary information on both termini of LMWHs. The reproducibility of this method was evaluated, and enoxaparin injections were analyzed to demonstrate the application of this method for evaluating the sameness of LMWH products.

Heparins in cancer-associated venous thrombosis.

A close causal relationship between cancer and venous thrombosis gives rise to questions about the effect of treatment modalities, in particular of the administered drugs, in patients with cancer-related venous thrombosis. An increased risk of chemotherapy-associated venous thromboembolism (VTE) has been well documented, while the effect of heparins used in VTE treatment on the disease course and prognosis in cancer patients has not been fully elucidated. This paper discusses the outcomes of the studies conducted so far investigating the role of heparins, in particular, low-molecular-weight heparins (LMWHs), in the prevention of thrombosis in cancer patients. It also focuses on such aspects of the treatment for cancer-associated VTE as treatment duration and drugs used. The paper summarizes the often discrepant results of long-term therapies with various LMWH products, emphasising that in this specific case the class effect is rather unlikely. It also presents the possible effects of heparins administered as part of cancer treatment, and points to the effects of LMWHs on cancer that are not related to an antithrombotic effect. On the 100th anniversary of heparin discovery, it can be said that heparin is irreversibly connected with thrombosis in the course of cancer.

Exploring bacterial heparinase II activities with defined substrates.

Bacterial heparinases that cleave heparan sulfate (HS) and heparin are widely used to generate low-molecular-weight heparins (LMWHs) and to structurally and functionally characterise heparin and HS biomolecules. We provide novel insights into the substrate specificity of heparinase II from two different bacteria: Pedobacter heparinus (formerly Flavobacterium heparinum) and Bacteroides eggerthii. The activity towards various well-defined HS oligosaccharides was investigated by (1) H NMR spectroscopy; this revealed distinct specificities for the two heparinases. Heparinase II from P. heparinus appears to be more active and displays a broader substrate specificity than B. eggerthii heparinase II. Furthermore, HS di- and tetrasaccharides inhibited B. eggerthii heparinase II activity. A better understanding of heparinase substrate specificity will contribute to the production of homogenous LMWHs, provide better characterisation of heparin and HS and assist therapeutic applications.

Venous thromboembolism in cancer patients.

Venous thromboembolism (VTE) is a common complication in cancer patients. This review summarizes some of the most current knowledge of the epidemiology, risk factors, risk models, prophylaxis, and treatment of VTE in cancer patients. A literature search was conducted using PubMed; the search terms were venous thromboembolism, anticoagulation, and cancer. The bibliographies of pertinent studies and review articles were reviewed for additional references. Venous thromboembolism is the second leading cause of death in patients with cancer. Cancer patients with VTE have poorer outcomes compared with noncancer patients with VTE. Many risk factors have been identified for VTE in patients with cancer that are patient-related, cancer-related, or treatment-related. Several biomarkers have been identified as potentially predictive of VTE risk. Risk assessment models such as the Khorana Risk Score stratify cancer patients with low, intermediate, and high risk of developing VTE based on baseline clinical and laboratory variables. Currently, enoxaparin is the preferred anticoagulant for initial VTE treatment in cancer patients. Low molecular weight heparin (LMWH) is recommended for both initial and long-term management of cancer-related VTE. Because the optimal duration of anticoagulation in cancer patients with VTE is unknown, the decision to extend anticoagulation requires weighing the risk of recurrent thrombosis against the risk of major bleeding. Patients with recurrent VTE can be bridged with LMWH, transitioned to full-dose LMWH or treated with LMWH dose escalation. While there is insufficient data to determine whether anticoagulation should be held in the setting of thrombocytopenia, full-dose anticoagulation is typically considered unsafe when platelets are < 50 000/μL. Inferior vena cava filters are currently recommended only for patients with acute VTE and contraindications to anticoagulation. Although management of catheter-associated thrombosis has not been well studied in cancer patients, it is recommended that cancer patients with catheter-associated thrombosis be treated with therapeutic anticoagulation for ≥ 3 months. Venous thromboembolism prophylaxis with UFH, LMWH, or fondaparinux is recommended in all hospitalized nonsurgical cancer patients and cancer patients undergoing major cancer surgery. Primary thromboprophylaxis is only currently recommended in high-risk ambulatory cancer patients such as multiple myeloma patients receiving thalidomide- or lenalidomide- based therapy. Cancer-associated thrombosis is a common problem. As we begin to better understand the risk factors and biomarkers for cancer-associated VTE, we can further refine and develop risk-assessment models to determine those patients who would most likely benefit from anticoagulation. While LMWH products are generally preferred in cancer-related VTE, more research will continue to evolve our understanding of treatment and thrombopprophylaxis in cancer-associated VTE.

Venous Thromboembolism in Cancer Patients

Objective: Venous thromboembolism (VTE) is a common complication in cancer patients. This review summarizes some of the most current knowledge of the epidemiology, risk factors, risk models, prophylaxis, and treatment of VTE in cancer patients. Methods: A literature search was conducted using Pub Med; the search terms were venous thromboembolism, anticoagulation, and cancer. The bibliographies of pertinent studies and review articles were reviewed for additional references. Results: Venous thromboembolism is the second leading cause of death in patients with cancer. Cancer patients with VTE have poorer outcomes compared with noncancer patients with VTE. Many risk factors have been identified for VTE in patients with cancer that are patient-related, cancer-related, or treatment-related. Several biomarkers have been identified as potentially predictive of VTE risk. Risk assessment models such as the Khorana Risk Score stratify cancer patients with low, intermediate, and high risk of developing VTE based on baseline clinical and laboratory variables. Currently, enoxaparin is the preferred anticoagulant for initial VTE treatment in cancer patients. Low molecular weight heparin (LMWH) is recommended for both initial and long-term management of cancer-related VTE. Because the optimal duration of anticoagulation in cancer patients with VTE is unknown, the decision to extend anticoagulation requires weighing the risk of recurrent thrombosis against the risk of major bleeding. Patients with recurrent VTE can be bridged with LMWH, transitioned to full-dose LMWH or treated with LMWH dose escalation. While there is insufficient data to determine whether anticoagulation should be held in the setting of thrombocytopenia, full-dose anticoagulation is typically considered unsafe when platelets are < 50 000/μL. Inferior vena cava filters are currently recommended only for patients with acute VTE and contraindications to anticoagulation. Although management of catheter-associated thrombosis has not been well studied in cancer patients, it is recommended that cancer patients with catheter-associated thrombosis be treated with therapeutic anticoagulation for ≥ 3 months. Venous thromboembolism prophylaxis with UFH, LMWH, or fondaparinux is recommended in all hospitalized nonsurgical cancer patients and cancer patients undergoing major cancer surgery. Primary thromboprophylaxis is only currently recommended in high-risk ambulatory cancer patients such as multiple myeloma patients receiving thalidomide-or lenalidomide- based therapy. Conclusion: Cancer-associated thrombosis is a common problem. As we begin to better understand the risk factors and biomarkers for cancer-associated VTE, we can further refine and develop risk-assessment models to determine those patients who would most likely benefit from anticoagulation. While LMWH products are generally preferred in cancer-related VTE, more research will continue to evolve our understanding of treatment and thrombopprophylaxis in cancer-associated VTE.

Ultrasonic-assisted preparation of a low molecular weight heparin (LMWH) with anticoagulant activity

Low molecular weight heparin (LMWH) is currently used as an anticoagulant agent and constitutes an alternative to unfractionated heparin, which is the cause of serious adverse drug reaction such as heparin-induced thrombocytopenia (HIT). Commercially available LMWH is produced by enzymatic depolymerization that is costly or by chemical methods that are generally carried out under conditions that could imply side reactions that reduce final product efficiency and yields. In this work, we present the use of a physicochemical method for the production of LMWH. This method consists in the use of hydrogen peroxide-catalyzed radical hydrolysis assisted by ultrasonic waves. LMWH that are produced using this physicochemical method have an average molecular weight and anticoagulant properties (Anti-Xa and Anti-IIa) that are comparable to some of commercial LMWH that are currently used. Ultrasonic-assisted radical depolymerization of heparin leads to products with a remarkably low polydispersity index. Moreover, in comparison to other LMWH such as those produced by enzymatic β-elimination, this physicochemical depolymerization of heparin induces fewer oligosaccharides with less than five monosaccharide units. This contributes to the better preservation of the ATIII pentasaccharide binding sequence, which results in a high Anti-Xa/Anti-IIa ratio (1.86). However, LMWH obtained using this physicochemical method have a lower degree of sulfation than other LMWH, which seems to be the cause of a lower Anti-Xa and Anti-IIa activity (143.62±5.42 and 77.07±4.4, respectively).

Reversed-phase ion-pair ultra-high-performance-liquid chromatography–mass spectrometry for fingerprinting low-molecular-weight heparins

Heparin is a complex mixture of sulfated linear carbohydrate polymers. It is widely used as an antithrombotic drug, though it has been shown to have a myriad of additional biological activities. Heparin is often partially depolymerized in order to decrease the average molecular weight, as it has been shown that low molecular weight heparins (LMWH) possess more desirable pharmacokinetic and pharmacodynamic properties than unfractionated heparin (UFH). Due to the prevalence of LMWHs in the market and the emerging availability of generic LMWH products, it is important that analytical methods be developed to ensure the drug quality. This work explores the use of tributylamine (TrBA), dibutylamine (DBA), and pentylamine (PTA) as ion-pairing reagents in conjunction with acetonitrile and methanol modified mobile phases for reversed-phase ion-pairing ultraperformance liquid chromatography coupled to mass spectrometry (RPIP-UPLC–MS) for fingerprint analysis of LMWH preparations. RPIP-UPLC–MS fingerprints are presented and compared for tinzaparinand enoxaparin.

Magnetic nanoparticles for the affinity adsorption of maltose binding protein (MBP) fusion enzymes

By the combination of fusion protein technology and nanoparticle surface modification technology, a maltose-modified magnetic support has been successfully developed for the integration of separation/purification, and immobilization of maltose binding protein–Heparinase I fusion enzyme (MBP–HepA) for enzymatic reaction and recycle. HepA is an important enzyme to degrade heparin and heparan sulfate to produce low molecular weight heparin (LMWH). This magnetic support may play an important role for process simplification and cost reduction on the industrial production of LMWH. Further, this magnetic support also can be used for the immobilization of other MBP fusion proteins, which implies that this magnetic support may have wide applications in biotechnology and enzyme engineering.

Low Molecular Weight Heparins Differ Substantially: Impact on Developing Biosimilar Drugs

Generic drugs are an important component for meaningful health-care reform currently being debated in the United States. Aside from defining the period of drug exclusivity, however, there is a critical need to ensure that generics of biologic medicines (biosimilars) are safe and effective. For low molecular weight heparins (LMWHs), the standard of care for management of venous thromboembolism, their complex structure and polypharmacological actions make producing a generic LMWH more challenging than a generic small molecule medicine. Because biosimilar LMWHs will be used interchangeably with their branded product, inherent variability between products could lead to important differences in potency, safety, or effectiveness, including unanticipated immune responses. Awareness of the specific problems associated with biosimilar LMWH development led to new recommendations from several expert bodies. This article discusses the implications of these differences for the production of biosimilar LMWHs and provides recommendations to address the limitations in the pending U.S. Congress legislation, a well-intentioned undertaking but one that must preserve the health and welfare of citizens who require these critical care medications.

Differences of Present Recommendations and Guidelines for Generic Low-Molecular-Weight Heparins: Is There Room for Harmonization

Based on the results of large clinical trials, several low-molecular-weight heparins (LMWHs) have been approved for prophylaxis and the treatment of venous and arterial thromboembolism. As a result of expiration or pending expiration of patent protection of the originator LMWHs, the first generic LMWH enoxaparin has been approved by the Food and Drug Administration for clinical use in all medical indications. The European Medicines Agency has set up guidelines for the production of generic LMWHs. The International Society of Thrombosis, the North American Thrombosis Forum and other scientific organizations raised concerns regarding the safety of generic LMWHs due to economic reasons. These organizations have published statements for the production of generic LMWHs to ensure the quality of the products and the safety for patients. Ideally, the differences between the actual recommendations and guidelines for the production of generic version of LMWHs should be harmonized.

Overview on guidelines and recommendations for generic low-molecular-weight heparins

The first generic low-molecular-weight heparin (LMWH) was approved by the Food and Drug Administration (FDA) end of July 2010 for clinical use in all medical indications where the branded product enoxaparin has reached approval. Many LMWHs have been approved for prophylaxis and the treatment of venous and arterial thromboembolism based on the results of large clinical trials. Generic LMWHs are now marketed in various countries. The European Medicines Agency (EMA) has set up guidelines for the production of generic LMWHs. The International Society of Thrombosis, the North American Thrombosis Forum and other scientific organisations raised concerns regarding the safety of generic LMWHs due to the possibility of a reduced quality of the anticoagulants to ensure a lower price compared to the branded LMWHs. They have published statements for the production of generic LMWHs to ensure the quality of the products and the safety for patients. The present review describes the differences between the actual guidelines and recommendations for the production of generic version of LMWHs.

Protamine neutralisation of low molecular weight heparins and their oligosaccharide components

Protamine sulphate is an effective inhibitor of heparin and is used clinically to neutralise both low molecular weight heparins (LMWH) and unfractionated heparin (UFH). However, protamine sulphate does not fully counter the anti-Xa effect of LMWH, even in excess (>40μg to 1IU/ml). To investigate the molecular basis for this observation, the residual potencies in the presence and absence of plasma as well as the molecular weight profiles of commercial LMWH neutralised with increasing amounts of protamine were measured. Materials over 5000Da are preferentially neutralised by protamine. To further investigate this molecular weight dependence, monodisperse oligosaccharides were prepared from three commercial LMWHs. The specific anti-Xa activity for the fractions increased with molecular weight, and was found to vary between the three preparations for oligosaccharides of the same molecular weight. Our results indicate that protamine sulphate neutralisation is largely dependent on molecular weight, leading to the implication that LMWHs containing a larger proportion of small oligosaccharides will not be as effectively neutralised. Protamine sulphate neutralisation of any given LMWH is also affected by the specific anticoagulant activities of its low molecular weight components, which varies between LMWH products, presumably with the method of manufacture.

Characteristics of low molecular weight heparin production by an ultrafiltration membrane bioreactor using maltose binding protein fused heparinase I

Low molecular weight heparin (LMWH) produced by the degradation of heparin has been widely used for clinical use, because it retains heparin's anticoagulant activity while has fewer side effects. In this study, a bioprocess for efficient production of LMWH was established by an ultrafiltration (UF) membrane bioreactor using maltose-binding protein (MBP)-heparinase I. The absorption of the reaction mixture at 235 nm ( A 235) was used as an indicator for monitoring the enzymatic reaction and product quality. When A 235 of the reaction mixture reached 45, the produced LMWH reached a weight-average molecular weight ( M w) of approximately 5038 Da, a number-average molecular weight ( M n) of 3498 Da, the anti-factor Xa activity of 154.95 ± 5.80 IU/mg, and the ratio of anti-factor Xa activity to anti-factor IIa activity of 2.2, which all meet the standard of European Pharmacopoeia 5.0. The average molecular weight of LMWH showed a good linear relationship with the increment in A 235. These results indicated that the qualified LMWH could be produced by the UF membrane bioreactor using the fusion enzyme.