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Characteristics of low molecular weight heparin production by an ultrafiltration membrane bioreactor using maltose binding protein fused heparinase I
Biochemical Engineering Journal 46 (2009) 193–198
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Characteristics of low molecular weight heparin production by an ultrafiltration membrane bioreactor using maltose binding protein fused heparinase I Fengchun Ye, Ying Kuang, Shuo Chen, Chong Zhang, Yin Chen, Xin-Hui Xing ∗ Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Haidian-district, Beijing 100084, China
a r t i c l e
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Article history: Received 4 March 2009 Received in revised form 21 April 2009 Accepted 9 May 2009
Keywords: Affinity Enzyme Fusion protein Heparinase Low molecular weight heparin Membrane Stirred-tank
a b s t r a c t 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 (A235 ) was used as an indicator for monitoring the enzymatic reaction and product quality. When A235 of the reaction mixture reached 45, the produced LMWH reached a weight-average molecular weight (Mw ) of approximately 5038 Da, a number-average molecular weight (Mn ) 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 A235 . These results indicated that the qualified LMWH could be produced by the UF membrane bioreactor using the fusion enzyme. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Heparin is a highly sulphated and polydispersed linear glycosaminoglycan of alternating 1 → 4 linked hexuronic acid and d-glycosamine residues, with a molecular weight (Mw ) of 3,000–37,000 daltons (Da) and an average Mw of 15,000 Da [1]. Although heparin has been used as an anticoagulant and antithrombotic agent for more than 70 years, its side effects such as hemorrhage and reduction of hematoblasts, have limited its long term use [2]. Low molecular weight heparin (LMWH), with a Mw less than 8000 Da and an average Mw of approximately 5000 Da [1], maintains anticoagulant activity while having fewer side effects. Also, LMWH has better pharmacokinetics on subcutaneous injection, a longer half-life, better bioavailability and higher safety, because of its lower molecular weight and reduced polydispersity [3]. Therefore, LMWH has widely replaced heparin in clinical use. LMWH can be produced by physical separation, or by controlled chemical/enzymatic cleavage of heparin [4]. The physical separation method isolates LMWH from unfractionated heparin (UFH) by addition of an organic solvent. As the proportion of LMWH in UFH is quite limited, this method is unsuitable for large-scale production of LMWH. Although chemical depolymerization using hydrogen peroxide or nitrous acid is a well-developed method and has been widely used in industry, it still have some disadvantages, includ-
∗ Corresponding author. Tel.: +86 10 62794771; fax: +86 10 62770304. E-mail address: [email protected] (X.-H. Xing). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.05.007
ing the environmental pollution, the difficulties in process control, and a decrease in the pharmaceutical activity of LMWH due to the reaction between strong oxidants and the sulphate groups in heparin molecules [5]. Compared to the chemical method, enzymatic depolymerization has increased in popularity because of its mild reaction conditions, high selectivity and lower environmental impact. Nielsen and Denmark (1992) have successfully produced LMWH by enzymatic depolymerization of heparin with heparinase I (EC 4.2.2.7), and have shown the feasibility of the enzymatic approach [6]. For example, tinzaparin, a low molecular weight heparin (LMWH) produced by heparinase digestion of heparin, showed no differences in the antithrombotic activity compared with heparin [7]. Although the enzymatic method has been proved to be feasible [6], bioprocess engineering work for the heparinase bioreactor is still deficient, due to the high cost of the enzyme. Until now, enzymatic depolymerization of heparin has used heparinase I (EC 4.2.2.7), which was first purified from Flavobacterium heparinum by Robert Langer in 1985 [8]. Nonetheless, the low productivity of heparinase I, and the co-production of heparinase II, heparinase III and other enzymes that degrade heparin and heparin-like polysaccharides [9], increases the difficulties and the high cost of the enzyme separation and purification. This has hindered the industrial use of the enzymatic method for LMWH production. An efficient expression system was constructed with the goal of producing soluble and active heparinase I in recombinant Escherichia coli in our previous study, and an enzymatic activity of 20,650 IU/L was achieved by fusion to maltose-binding protein
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(MBP) [10,11]. The functional fusion protein (MBP-heparinase I) was purified by a one-step affinity separation with an amylose resin, and the enzymatic characteristics of MBP-heparinase I is similar to the original heparinase I [12]. The aim of this study was to establish an efficient enzyme reactor and examine the operating conditions for production of LMWH from heparin by using MBP-heparinase I. The enzyme was simply prepared with low cost and a UF membrane enzyme reactor was designed for the depolymerization of heparin. The following parameters of LMWH production were characterized, including the weight-average molecular weight (Mw ), the number-average molecular weight (Mn ), the anti-factor Xa and IIa activity, and the product yield. The absorbance at 235 nm of the products was used as the feasible indicator to monitor the enzymatic depolymerization process of heparin. 2. Materials and methods
thrombin (T-4648) was from Sigma (USA). All other reagents were of commercial grade. 2.2. Methods 2.2.1. MBP-heparinase I enzyme activity assay The MBP-heparinase I activity assay was carried out according to the 232 nm UV method [10]. Typically, 500 L of 25 g L−1 heparin solution (25 g L−1 heparin, 40 mM NaCl, 3.5 mM CaCl2 and 17 mM Tris–HCl, pH 7.4) was mixed with 950 L reaction buffer (200 mM NaCl, 3.5 mM CaCl2 and 20 mM Tris–HCl, pH 7.4) and 50 L enzyme solution. The entire system was kept at 30 ◦ C for the 2 min reaction. The degree of heparin degradation was detected by UV absorbance at 232 nm and the enzymatic activity in international units (IU) was calculated from the increase in absorbance at 232 nm at the detection time, using a molar absorption coefficient of ε = 3800 M−1 cm−1 . One IU was defined as the amount of protein that formed 1 mol unsaturated uronic acid per minute at 30 ◦ C.
2.1. Materials Pharmaceutical grade heparin (Mn 22,370) was a gift from the Changshan Biotechnology Corporation (Hebei Province, China). MBP-heparinase I was produced and purified according to the procedure established by Chen et al. [10]. Recombinant E. coli TB1 expressing MBP-heparinase I was grown and induced in LB medium for 21 h, and the cells (18 mL) were then harvested by centrifugation, suspended in Tris–HCl buffer (200 mM NaCl, 3.5 mM CaCl2 and 20 mM Tris–HCl, pH 7.4) and disrupted by sonication. After the centrifugation to remove the cell debris, the supernatant containing the target enzyme was frozen and reserved for the use. Standard LMWH, heparin low-molecular-mass calibration BRP (Biological Reference Preparation) (H0190000, Mn 3700, for molecular weight detection) and heparin low-molecular-mass for assay BRP (H0185000, for detection of anti-factor Xa activity and anti-factor IIa activity) were purchased from EDQM (European Directorate for the Quality of Medicines). The UF filtration device was Pellicon XL Ultrafiltration Module Biomax 8 kDa (nominal molecular weight limit) with the filtration area of 0.005 m2 (Millipore Co., USA). The HPLC system (Shimadzu Co., Japan) consisted of a computer control, pump (LC-10ATvp), autoinjector (SIL-10ADvp), GPC column (TSK Gel G3000SW, 30 mm × 750 mm, Tosoh Co., Japan), and a RI (RID-10A) detector linked after a UV detector (SPD-M10Avp). This HPLC system was used to detect the molecular weight distribution of the reaction solution. Antithrombin III, chromophore substrate S-2765 (N-␣-benzyloxycarbonyl-d-arginyll-glycyl-l-arginine-p-nitroaniline-dihydrochloride), S-2238 (H-dphenylalanyl-l-pipecolyl-arginine-p-nitroaniline dihydrochloride) and bovine factor Xa were from Chromogenix (Sweden), while
2.2.2. Production of LMWH by MBP-heparinase I LMWH products were produced through the controlled degradation of original heparin by MBP-heparinase I in the UF membrane reactor (Fig. 1). The reaction was carried out in a 250 mL reactor with a thermostatic water-jacket. 1.5 mL enzyme solution at 7.5 IU mL−1 enzyme activity, was added to 100 mL reaction solution containing 50 g L−1 heparin, 200 mM NaCl, 3.5 mM CaCl2 , pH 7.4, adjusted with 1 M NaOH. The mixture was incubated at 30 ◦ C under magnetic stirring for different times, during which the absorbance of the solution at 235 nm (A235 ) was measured every 10 min. At various predetermined A235 values (varying from 20 to 90), 1 M HCl was added to the system to stop the enzyme reaction by adjusting the pH to approximately 2.0. The mixture was then incubated for approximately 5 min, after which it was filtered by the connected UF membrane using a peristaltic pump (0.03–0.05 L/min) and the filtrate was collected in a 250 mL storage container. The LMWH product was obtained as a precipitate after the addition of 2.5 times volume of ethanol to the filtrate, which was subsequently washed with acetone and dried under vacuum at 40 ◦ C overnight to obtain the LMWH powder. The LMWH yield was the mass fraction of the LMWH powder obtained from the original raw heparin (5 g in this study). 2.2.3. Measurements of molecular weight and polydispersity index of LMWH The Mw , Mn and polydispersity index (D) measurements were performed using a HPLC method with gel permeation chromatography (GPC) [13,14] according to the European Pharmacopoeia 5.0 [15]. The mobile phase was 28.4 g L−1 solution of anhydrous sodium
Fig. 1. The schematic diagram of the LMWH production by UF membrane enzyme reactor with the nominal molecular weight limit of 8000.
F. Ye et al. / Biochemical Engineering Journal 46 (2009) 193–198
sulphate adjusted to pH 5.0 using dilute sulphuric acid. Samples were 20 mg of the standard low-molecular-mass heparin for calibration CRS (Chemical Reference Sample) or the LMWH powder sample obtained from enzymatic degradation, dissolved in 2 mL of the mobile phase. The flow rate was 0.5 mL min−1 . UV detection was at 235 nm. Both the reference solution and test solution injections were 25 L. To accurately calculate molecular weight, the time lapse between the 2 detectors must be accurately measured, so the chromatograms can be aligned [15]. The retention times used in the calibration were from the RI detector. After calculating the total area from the UV235 and the RI curves by numerical integration over the target range, the ratio r was estimated, using the following equation: r=
RI
UV235
(1)
The factor f was calculated by using the following equation: f =
Mna r
(2)
where Mna = 3700 (the known number-average molecular mass of the standard low-molecular-mass heparin for calibration CRS). Provided that the UV235 and the RI responses were aligned, the molecular mass M of LMWH (Mi ) at any point was calculated from the following equation: Mi = f
(RI)i (UV235 )i
(3)
The weight-average molecular weight Mw is defined by Eq. (4): Mw =
(RI M ) i i RIi
(4)
The number-average molecular weight Mn is defined by Eq. (5): Mn =
RIi
(RIi /Mi )
(5)
where RIi = mass of substance eluting in the fraction i; Mi = molecular mass corresponding to fraction i. The polydispersity index (D) of the LMWH is defined by Mw /Mn . 2.2.4. Anti-factor Xa and IIa activity assays The detection of anti-factor Xa and anti-factor IIa activities was according to the European Pharmacopoeia 5.0 [15]. Briefly, four independent series of four dilutions each, of the LMWH sample and the heparin low-molecular-mass for BRP assay were prepared in Tris–HCl buffer (25 g L−1 BSA, 150 mM NaCl and 50 mM Tris–HCl, pH 7.4). For anti-factor Xa detection, the concentration range was within 0.025–0.200 IU mL−1 , and for anti-factor IIa detection, it was 0.015–0.075 IU mL−1 . The concentrations of the four heparin lowmolecular-mass for BRP assay dilutions were 0.027, 0.042, 0.065, 0.100 IU mL−1 for anti-factor Xa detection, while the values were 0.015, 0.023, 0.036, 0.055 IU mL−1 for anti-factor IIa detection. The dilutions chosen gave a linear response when the absorbance was plotted vs. log concentration. To each of the 16 samples and reference dilution series tubes, 50 L of antithrombin III solution and 50 L of the diluted sample or reference were added and mixed, avoiding bubbles [15]. Tubes were equilibrated in a 37 ◦ C water-bath for 1 min before adding 100 L of bovine factor Xa solution. After incubation for 1 min, 250 L of chromophore substrate containing 175 mM NaCl, 8 mM EDTA, 50 mM Tris–HCl, 0.5 mM S-2765 for anti-factor, Xa or S-2238 for anti-factor IIa detection was added. The reaction was stopped after 4 min by adding 375 L of acetic acid. Mixtures were transferred to semimicro cuvettes for 405 nm absorbance measurements in a visible light spectrophotometer. Blank amidolytic activity was determined
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at the beginning and end of the procedure, using Tris–HCl buffer (25 g L−1 BSA, 150 mM NaCl and 50 mM Tris–HCl, pH 7.4). Regression of the absorbance against log concentrations of the sample or reference was used to calculate the potency, using standard statistical methods for parallel-line assays. 3. Results 3.1. Characterization of heparin enzymatic depolymerization by MBP-heparinase I MBP-heparinase I degrades heparin through an elimination mechanism on the oxygen–aglycone bond adjacent to the uronic acid moiety at the position C5, and results in a product with a C4–C5 double bond that has strong absorption at around 235 nm [16]. The value of A235 thus reflects the degree of the cleavage reaction of heparin. In order to understand the behavior of the heparin cleavage process by MBP-heparinase I, the A235 was measured over time until it ceased to change. The A235 increased linearly at first and slowly flattened as shown in Fig. 2. Further analysis found that the time course profile of A235 followed approximate first-order reaction kinetics, suggesting that the depolymerization rate of heparin is proportional to the substrate concentration at a given enzyme concentration (Fig. 2). The apparent first-order reaction kinetics of the A235 time course profile could be explained by a depolymerization process that formed an enzyme-substrate binding intermediate, that then decomposed into enzyme and oligosaccharides. The A235 reflected the degree of enzymatic reaction. Detailed information on the distribution of oligosaccharides during the reaction was obtained by GPC (Fig. 3). The main chromatographic peaks formed later and later and split into several smaller, higher peaks as the A235 increased over time (Fig. 3A), indicating the heparin molecular weight became smaller. The late efflux had the greatest total A235 , divided into two or three single peaks, for example S6 –S9 in Fig. 3B. This result indicated that the heparin had been depolymerized into smaller oligosaccharides with a strong absorption at 235 nm. In fact, the GPC data for the reaction mixtures obtained at different reaction time (Fig. 3) gave some information about the reaction sequence of the enzymatic depolymerization of heparin. Previous research indicated that the enzyme may cleave exolytically at either the reducing or the non-reducing end of heparin, or may cut each linkage that meets the specificity with equal probability (random endolytic cleavage) [17]. Subsequent to the initial (exolytic or endolytic) cleavage of a substrate molecule, the
Fig. 2. A235 change during enzymatic depolymerization of heparin by MBPheparinase I at 30 ◦ C.
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3.2. Average molecular weight change and LMWH products from the heparin mixture during the reaction Mw and the logarithm of Mn of the reaction mixture samples showed a linear relationship (linear correlation coefficient R = 0.9779, Fig. 4A). A relationship (as shown below) was found between Mn and increase in absorption at 235 nm (A235 ) (Fig. 4B), consistent with the report published previously [6]. 1 1 A235 = + Mn Mnu Cε Mnu = the number average molecular weight of the heparin substrate; C = the substrate concentration (g L−1 ); ε = the molar absorption coefficient (M−1 cm−1 ). The average molecular weight (including Mw and Mn ) of the LMWH produced in different batches was determined (Table 1), which was different from the average molecular weight of the reaction mixture samples mentioned above. The LMWH products were purified from the filtrate of the reaction mixture which contained not only the filtrated LMWH products, but also larger fragments of heparin. With the increase of A235 from 20 to 90, the average molecular weight of the LMWH product decreased and the polydispersity index (D) became lower (Table 1). According to the European Pharmacopoeia 5.0 [15], the batches whose A235 was between 30 and 50 met the standards as the anticoagulant (Table 1). Moreover, there existed a linear relationship either between the Mw and Mn of the LMWH products (Fig. 4C), or between the Mn of the reaction mixture samples and the Mw of the LMWH products (Fig. 4D). All the linear relationships in Fig. 4 were fitted from the exact molecular weight values measured by HPLC. 3.3. Bioactivities of the LMWH products Fig. 3. GPC analysis of the heparin enzymatic depolymerization mixture in different A235 (S0 refers to the raw heparin, S1 –S9 refer to the heparin enzymatic depolymerization mixture samples obtained at different A235 as follows: 18.8, 46.5, 63.2, 86.5, 100.9, 136.5, 168.9, 227.0, 255.3). (A) Analysis results by RI detector; (B) analysis results by UV detector.
enzyme may proceed to cleave the same heparin molecule several times before releasing it, or it may cleave non-processively by releasing the substrate immediately after cleavage and binding to a new heparin molecule [18]. Fig. 3 supported the hypothesis that cleavage is mostly by the random endolytic process. Further research on the enzyme reaction enzyme remains to be elucidated.
After precipitation with ethanol, washing with acetone and vacuum drying, the LMWH products were obtained. Oligosaccharide distribution of the LMWH products was found to be similar to that of the mixture samples (data not shown). The average molecular weight index, anti-Xa and anti-IIa activities, activity ratio, and the yield of purified LMWH products were examined (Table 1). Both the anti-Xa and anti-IIa activity decreased with the increase of A235 . According to the European Pharmacopoeia 5.0 [15], the potency of LMWH should not be less than 70 IU of anti-factor Xa activity per milligram, calculated with reference to the dried substance. The ratio of anti-factor Xa activity to anti-factor IIa activity should not be less than 1.5. The results in Table 1 suggested that the batches whose final A235 was below 50 met the activity requirement of European Pharmacopoeia 5.0 [15], indicating that the reaction time
Table 1 Characteristics of different batches of LMWH products (n = 2). A235
20.6 30.8 40.7 46.7 48.4 51.5 64.2 75.2 88.9
Average molecule weight Mw
Mn
7578 6883 6282 5038 4916 4641 3979 3938 3314
4982 4674 4550 3498 3379 3230 3063 3163 2828
Polydispersity indexa (D)
Anti-Xa (IU/mg)
Anti-IIa (IU/mg)
Activity ratiob
Yield (%)
1.52 1.47 1.38 1.44 1.45 1.44 1.30 1.25 1.17
412.18 ± 15.71 385.29 ± 11.36 262.62 ± 11.42 154.95 ± 5.80 133.86 ± 4.17 116.69 ± 1.27 65.56 ± 0.53 55.21 ± 0.55 44.99 ± 0.45
356.08 ± 24.93 184.22 ± 19.77 109.26 ± 28.12 70.01 ± 2.21 57.72 ± 1.14 28.56 ± 0.75 29.48 ± 1.75 13.30 ± 0.27 12.33 ± 0.27
1.2 2.1 2.4 2.2 2.3 4.1 2.2 4.2 3.6
36.12 35.53 57.58 61.24 62.34 66.21 87.71 86.79 94.41
Note: According to the European Pharmacopoeia 5.0 [15], the potency of LMWH should not be less than 70 IU of anti-factor Xa activity per milligram, while the ratio of anti-factor Xa activity to anti-factor IIa activity should not be less than 1.5. a Polydispersity index is the ratio of weight-average molecular weight to number-average molecular weight. b Activity ratio is the ratio of anti-Xa activity to anti-IIa activity.
F. Ye et al. / Biochemical Engineering Journal 46 (2009) 193–198
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Fig. 4. Linear relationship between (A) the logarithm of Mn and Mw of the reaction mixture at different times; (B) the Mn of reaction mixture samples and increasing A235 at different times; (C) the average molecular weight (including Mw and Mn ) of LMWH products; (D) the Mn of the reaction mixture samples and Mw of LMWH products.
was a key factor in the LMWH production by MBP-heparinase I. The yield of LMWH was improved as A235 increased, which could be explained by the increase in smaller oligosaccharides. Polyacrylamide gel electrophoresis analysis demonstrated that the LMWH products consisted of 4–22 repeat disaccharide units (determined from the standard polysaccharide samples with the known repeat disaccharide units number prepared by Prof. Guangli Yu of China Ocean University of Qing Dao), while the raw heparin has 45 units (data not shown). 4. Discussion The enzymatic method to prepare LMWH has been proved to be feasible [6], but the systematic study on the bioprocess and the operating conditions for the production of LMWH are still deficient due to the high cost of the conventional heparinase I. The present study has developed an efficient bioprocess to produce LMWH by MBP-heparinase I which can be produced easily by our over-expression system [10]. The fusion of MBP to the heparinase I is of great significance to the industrial application of heparinase I in LMWH preparation. MBP can dramatically enhance the solubility of the fusion protein and reach high enzymatic activity when MBP-heparinase I is expressed in recombinant E. coli [10]. MBP-heparinase I is easily purified [10] and could be immobilized directly by the affinity separation using maltose-coating particles. This will allow the reuse of the enzyme to be possible in a bioreactor, which can reduce LMWH production costs. The low productivity and high cost of the commercial heparinase I purified from F. heparinum have limited
the application of this enzyme in LMWH production [9]. MBPheparinase I used in the present study could be a useful form for the bioprocess integration of enzyme separation/purification and biocatalysis. The average molecular weight of LMWH could be predicted by using an empirically determined relationship between A235 and the average molecular weight of either the reaction samples or the LMWH products. With the value of A235 , the Mn and Mw of the LMWH products can be determined. However, the HPLC method is still needed to obtain the exact molecular weight values of the LMWH product for the standard curve. Furthermore, the ability to obtain LMWH products with different molecular weights at different reaction time suggested a method to direct the production of specific classes of LMWH. Also, the value of A235 is an important indicator to evaluate the activity of LMWH: the anti-factor Xa and anti-factor IIa activity. Heparin pentasaccharide, which binds antithrombin III (AT III) and changes its conformation, forms the complex that inhibits factor Xa activity [19]. For anti-factor IIa function, the pentasaccharide and an adjacent tridecasaccharide are necessary for factor IIa inhibition [20]. The longer the heparin is digested, the higher the value of A235 becomes and the lower the percentage of remaining pentasaccharide and tridecasaccharide remains, leading to a reduction in LMWH’s anti-factor Xa, and especially anti-factor IIa activity. Therefore, A235 is a key parameter for controlling the production of LMWH and the quality of the products by the enzymatic depolymerization, including the molecular weight, the anti-factor Xa activity, and the anti-factor IIa activity. Using A235 as a sim-
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ple indicator, it will be easy to determine the reaction time as well as the reaction conditions for the production of the qualified LMWH. The yield of LMWH is an essential index to evaluate the efficiency of the bioprocess. Firstly, controlling the reaction time at any given enzyme activity is important. Longer reaction time produced smaller oligosaccharides (more filtrates), which increased the product yield. However, anti-factor Xa and anti-factor IIa activity decreased inversely with the reaction progress. Thus, optimization of the enzyme reaction will be required to maximize the LMWH yield while maintaining the high activity. Another factor is the nominal molecular weight limit of the UF membrane. The appropriate nominal molecular weight limit should be optimized by taking the yield and the Mw restrictions based on European Pharmacopoeia 5.0 [15] (the LMWH product must meet the requirement that at least 60% of the total product (w/w) has a molecular weight less than 8000 Da). Finally, the yield could also be influenced by the operating conditions. For example, the LMWH produced by the enzymatic degradation of heparin can be continuously separated from the mixture by the UF filtration, which could increase the efficiency of residual heparin depolymerization by the enzyme. Further optimization for the enzyme bioreactor of MBP- MBP-heparinase I is needed. 5. Conclusions Production of the qualified LMWH by UF membrane bioreactor using MBP-heparinase I was studied, and the reaction process was evaluated by absorbance at 235 nm. When the A235 of the reaction solution reached 30–50, the produced LMWH could well meet the standards of the European Pharmacopoeia 5.0 [15]. The present enzyme bioreactor has the following advantages: the low cost of the fusion enzyme of MBP-heparinase I, the easy control of the product quality by the detection of A235 and the controllability of the UF membrane bioreactor. Acknowledgements This research was supported by the Grant of NSF Key Projects of China (Grant Nos. 20336010 and 20836004). Grateful acknowledgement is given to Prof. Guangli Yu of China Ocean University of Qing Dao, China for his kind help in the PAGE analysis of LMWH and heparin.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Characteristics of low molecular weight heparin production by an ultrafiltration membrane bioreactor using maltose binding protein fused heparinase I Fengchun Ye, Ying Kuang, Shuo Chen, Chong Zhang, Yin Chen, Xin-Hui Xing ∗ Institute of Biochemical Engineering, Department of Chemical Engineering, Tsinghua University, Haidian-district, Beijing 100084, China
a r t i c l e
i n f o
Article history: Received 4 March 2009 Received in revised form 21 April 2009 Accepted 9 May 2009
Keywords: Affinity Enzyme Fusion protein Heparinase Low molecular weight heparin Membrane Stirred-tank
a b s t r a c t 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 (A235 ) was used as an indicator for monitoring the enzymatic reaction and product quality. When A235 of the reaction mixture reached 45, the produced LMWH reached a weight-average molecular weight (Mw ) of approximately 5038 Da, a number-average molecular weight (Mn ) 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 A235 . These results indicated that the qualified LMWH could be produced by the UF membrane bioreactor using the fusion enzyme. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Heparin is a highly sulphated and polydispersed linear glycosaminoglycan of alternating 1 → 4 linked hexuronic acid and d-glycosamine residues, with a molecular weight (Mw ) of 3,000–37,000 daltons (Da) and an average Mw of 15,000 Da [1]. Although heparin has been used as an anticoagulant and antithrombotic agent for more than 70 years, its side effects such as hemorrhage and reduction of hematoblasts, have limited its long term use [2]. Low molecular weight heparin (LMWH), with a Mw less than 8000 Da and an average Mw of approximately 5000 Da [1], maintains anticoagulant activity while having fewer side effects. Also, LMWH has better pharmacokinetics on subcutaneous injection, a longer half-life, better bioavailability and higher safety, because of its lower molecular weight and reduced polydispersity [3]. Therefore, LMWH has widely replaced heparin in clinical use. LMWH can be produced by physical separation, or by controlled chemical/enzymatic cleavage of heparin [4]. The physical separation method isolates LMWH from unfractionated heparin (UFH) by addition of an organic solvent. As the proportion of LMWH in UFH is quite limited, this method is unsuitable for large-scale production of LMWH. Although chemical depolymerization using hydrogen peroxide or nitrous acid is a well-developed method and has been widely used in industry, it still have some disadvantages, includ-
∗ Corresponding author. Tel.: +86 10 62794771; fax: +86 10 62770304. E-mail address: [email protected] (X.-H. Xing). 1369-703X/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2009.05.007
ing the environmental pollution, the difficulties in process control, and a decrease in the pharmaceutical activity of LMWH due to the reaction between strong oxidants and the sulphate groups in heparin molecules [5]. Compared to the chemical method, enzymatic depolymerization has increased in popularity because of its mild reaction conditions, high selectivity and lower environmental impact. Nielsen and Denmark (1992) have successfully produced LMWH by enzymatic depolymerization of heparin with heparinase I (EC 4.2.2.7), and have shown the feasibility of the enzymatic approach [6]. For example, tinzaparin, a low molecular weight heparin (LMWH) produced by heparinase digestion of heparin, showed no differences in the antithrombotic activity compared with heparin [7]. Although the enzymatic method has been proved to be feasible [6], bioprocess engineering work for the heparinase bioreactor is still deficient, due to the high cost of the enzyme. Until now, enzymatic depolymerization of heparin has used heparinase I (EC 4.2.2.7), which was first purified from Flavobacterium heparinum by Robert Langer in 1985 [8]. Nonetheless, the low productivity of heparinase I, and the co-production of heparinase II, heparinase III and other enzymes that degrade heparin and heparin-like polysaccharides [9], increases the difficulties and the high cost of the enzyme separation and purification. This has hindered the industrial use of the enzymatic method for LMWH production. An efficient expression system was constructed with the goal of producing soluble and active heparinase I in recombinant Escherichia coli in our previous study, and an enzymatic activity of 20,650 IU/L was achieved by fusion to maltose-binding protein
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(MBP) [10,11]. The functional fusion protein (MBP-heparinase I) was purified by a one-step affinity separation with an amylose resin, and the enzymatic characteristics of MBP-heparinase I is similar to the original heparinase I [12]. The aim of this study was to establish an efficient enzyme reactor and examine the operating conditions for production of LMWH from heparin by using MBP-heparinase I. The enzyme was simply prepared with low cost and a UF membrane enzyme reactor was designed for the depolymerization of heparin. The following parameters of LMWH production were characterized, including the weight-average molecular weight (Mw ), the number-average molecular weight (Mn ), the anti-factor Xa and IIa activity, and the product yield. The absorbance at 235 nm of the products was used as the feasible indicator to monitor the enzymatic depolymerization process of heparin. 2. Materials and methods
thrombin (T-4648) was from Sigma (USA). All other reagents were of commercial grade. 2.2. Methods 2.2.1. MBP-heparinase I enzyme activity assay The MBP-heparinase I activity assay was carried out according to the 232 nm UV method [10]. Typically, 500 L of 25 g L−1 heparin solution (25 g L−1 heparin, 40 mM NaCl, 3.5 mM CaCl2 and 17 mM Tris–HCl, pH 7.4) was mixed with 950 L reaction buffer (200 mM NaCl, 3.5 mM CaCl2 and 20 mM Tris–HCl, pH 7.4) and 50 L enzyme solution. The entire system was kept at 30 ◦ C for the 2 min reaction. The degree of heparin degradation was detected by UV absorbance at 232 nm and the enzymatic activity in international units (IU) was calculated from the increase in absorbance at 232 nm at the detection time, using a molar absorption coefficient of ε = 3800 M−1 cm−1 . One IU was defined as the amount of protein that formed 1 mol unsaturated uronic acid per minute at 30 ◦ C.
2.1. Materials Pharmaceutical grade heparin (Mn 22,370) was a gift from the Changshan Biotechnology Corporation (Hebei Province, China). MBP-heparinase I was produced and purified according to the procedure established by Chen et al. [10]. Recombinant E. coli TB1 expressing MBP-heparinase I was grown and induced in LB medium for 21 h, and the cells (18 mL) were then harvested by centrifugation, suspended in Tris–HCl buffer (200 mM NaCl, 3.5 mM CaCl2 and 20 mM Tris–HCl, pH 7.4) and disrupted by sonication. After the centrifugation to remove the cell debris, the supernatant containing the target enzyme was frozen and reserved for the use. Standard LMWH, heparin low-molecular-mass calibration BRP (Biological Reference Preparation) (H0190000, Mn 3700, for molecular weight detection) and heparin low-molecular-mass for assay BRP (H0185000, for detection of anti-factor Xa activity and anti-factor IIa activity) were purchased from EDQM (European Directorate for the Quality of Medicines). The UF filtration device was Pellicon XL Ultrafiltration Module Biomax 8 kDa (nominal molecular weight limit) with the filtration area of 0.005 m2 (Millipore Co., USA). The HPLC system (Shimadzu Co., Japan) consisted of a computer control, pump (LC-10ATvp), autoinjector (SIL-10ADvp), GPC column (TSK Gel G3000SW, 30 mm × 750 mm, Tosoh Co., Japan), and a RI (RID-10A) detector linked after a UV detector (SPD-M10Avp). This HPLC system was used to detect the molecular weight distribution of the reaction solution. Antithrombin III, chromophore substrate S-2765 (N-␣-benzyloxycarbonyl-d-arginyll-glycyl-l-arginine-p-nitroaniline-dihydrochloride), S-2238 (H-dphenylalanyl-l-pipecolyl-arginine-p-nitroaniline dihydrochloride) and bovine factor Xa were from Chromogenix (Sweden), while
2.2.2. Production of LMWH by MBP-heparinase I LMWH products were produced through the controlled degradation of original heparin by MBP-heparinase I in the UF membrane reactor (Fig. 1). The reaction was carried out in a 250 mL reactor with a thermostatic water-jacket. 1.5 mL enzyme solution at 7.5 IU mL−1 enzyme activity, was added to 100 mL reaction solution containing 50 g L−1 heparin, 200 mM NaCl, 3.5 mM CaCl2 , pH 7.4, adjusted with 1 M NaOH. The mixture was incubated at 30 ◦ C under magnetic stirring for different times, during which the absorbance of the solution at 235 nm (A235 ) was measured every 10 min. At various predetermined A235 values (varying from 20 to 90), 1 M HCl was added to the system to stop the enzyme reaction by adjusting the pH to approximately 2.0. The mixture was then incubated for approximately 5 min, after which it was filtered by the connected UF membrane using a peristaltic pump (0.03–0.05 L/min) and the filtrate was collected in a 250 mL storage container. The LMWH product was obtained as a precipitate after the addition of 2.5 times volume of ethanol to the filtrate, which was subsequently washed with acetone and dried under vacuum at 40 ◦ C overnight to obtain the LMWH powder. The LMWH yield was the mass fraction of the LMWH powder obtained from the original raw heparin (5 g in this study). 2.2.3. Measurements of molecular weight and polydispersity index of LMWH The Mw , Mn and polydispersity index (D) measurements were performed using a HPLC method with gel permeation chromatography (GPC) [13,14] according to the European Pharmacopoeia 5.0 [15]. The mobile phase was 28.4 g L−1 solution of anhydrous sodium
Fig. 1. The schematic diagram of the LMWH production by UF membrane enzyme reactor with the nominal molecular weight limit of 8000.
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sulphate adjusted to pH 5.0 using dilute sulphuric acid. Samples were 20 mg of the standard low-molecular-mass heparin for calibration CRS (Chemical Reference Sample) or the LMWH powder sample obtained from enzymatic degradation, dissolved in 2 mL of the mobile phase. The flow rate was 0.5 mL min−1 . UV detection was at 235 nm. Both the reference solution and test solution injections were 25 L. To accurately calculate molecular weight, the time lapse between the 2 detectors must be accurately measured, so the chromatograms can be aligned [15]. The retention times used in the calibration were from the RI detector. After calculating the total area from the UV235 and the RI curves by numerical integration over the target range, the ratio r was estimated, using the following equation: r=
RI
UV235
(1)
The factor f was calculated by using the following equation: f =
Mna r
(2)
where Mna = 3700 (the known number-average molecular mass of the standard low-molecular-mass heparin for calibration CRS). Provided that the UV235 and the RI responses were aligned, the molecular mass M of LMWH (Mi ) at any point was calculated from the following equation: Mi = f
(RI)i (UV235 )i
(3)
The weight-average molecular weight Mw is defined by Eq. (4): Mw =
(RI M ) i i RIi
(4)
The number-average molecular weight Mn is defined by Eq. (5): Mn =
RIi
(RIi /Mi )
(5)
where RIi = mass of substance eluting in the fraction i; Mi = molecular mass corresponding to fraction i. The polydispersity index (D) of the LMWH is defined by Mw /Mn . 2.2.4. Anti-factor Xa and IIa activity assays The detection of anti-factor Xa and anti-factor IIa activities was according to the European Pharmacopoeia 5.0 [15]. Briefly, four independent series of four dilutions each, of the LMWH sample and the heparin low-molecular-mass for BRP assay were prepared in Tris–HCl buffer (25 g L−1 BSA, 150 mM NaCl and 50 mM Tris–HCl, pH 7.4). For anti-factor Xa detection, the concentration range was within 0.025–0.200 IU mL−1 , and for anti-factor IIa detection, it was 0.015–0.075 IU mL−1 . The concentrations of the four heparin lowmolecular-mass for BRP assay dilutions were 0.027, 0.042, 0.065, 0.100 IU mL−1 for anti-factor Xa detection, while the values were 0.015, 0.023, 0.036, 0.055 IU mL−1 for anti-factor IIa detection. The dilutions chosen gave a linear response when the absorbance was plotted vs. log concentration. To each of the 16 samples and reference dilution series tubes, 50 L of antithrombin III solution and 50 L of the diluted sample or reference were added and mixed, avoiding bubbles [15]. Tubes were equilibrated in a 37 ◦ C water-bath for 1 min before adding 100 L of bovine factor Xa solution. After incubation for 1 min, 250 L of chromophore substrate containing 175 mM NaCl, 8 mM EDTA, 50 mM Tris–HCl, 0.5 mM S-2765 for anti-factor, Xa or S-2238 for anti-factor IIa detection was added. The reaction was stopped after 4 min by adding 375 L of acetic acid. Mixtures were transferred to semimicro cuvettes for 405 nm absorbance measurements in a visible light spectrophotometer. Blank amidolytic activity was determined
195
at the beginning and end of the procedure, using Tris–HCl buffer (25 g L−1 BSA, 150 mM NaCl and 50 mM Tris–HCl, pH 7.4). Regression of the absorbance against log concentrations of the sample or reference was used to calculate the potency, using standard statistical methods for parallel-line assays. 3. Results 3.1. Characterization of heparin enzymatic depolymerization by MBP-heparinase I MBP-heparinase I degrades heparin through an elimination mechanism on the oxygen–aglycone bond adjacent to the uronic acid moiety at the position C5, and results in a product with a C4–C5 double bond that has strong absorption at around 235 nm [16]. The value of A235 thus reflects the degree of the cleavage reaction of heparin. In order to understand the behavior of the heparin cleavage process by MBP-heparinase I, the A235 was measured over time until it ceased to change. The A235 increased linearly at first and slowly flattened as shown in Fig. 2. Further analysis found that the time course profile of A235 followed approximate first-order reaction kinetics, suggesting that the depolymerization rate of heparin is proportional to the substrate concentration at a given enzyme concentration (Fig. 2). The apparent first-order reaction kinetics of the A235 time course profile could be explained by a depolymerization process that formed an enzyme-substrate binding intermediate, that then decomposed into enzyme and oligosaccharides. The A235 reflected the degree of enzymatic reaction. Detailed information on the distribution of oligosaccharides during the reaction was obtained by GPC (Fig. 3). The main chromatographic peaks formed later and later and split into several smaller, higher peaks as the A235 increased over time (Fig. 3A), indicating the heparin molecular weight became smaller. The late efflux had the greatest total A235 , divided into two or three single peaks, for example S6 –S9 in Fig. 3B. This result indicated that the heparin had been depolymerized into smaller oligosaccharides with a strong absorption at 235 nm. In fact, the GPC data for the reaction mixtures obtained at different reaction time (Fig. 3) gave some information about the reaction sequence of the enzymatic depolymerization of heparin. Previous research indicated that the enzyme may cleave exolytically at either the reducing or the non-reducing end of heparin, or may cut each linkage that meets the specificity with equal probability (random endolytic cleavage) [17]. Subsequent to the initial (exolytic or endolytic) cleavage of a substrate molecule, the
Fig. 2. A235 change during enzymatic depolymerization of heparin by MBPheparinase I at 30 ◦ C.
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3.2. Average molecular weight change and LMWH products from the heparin mixture during the reaction Mw and the logarithm of Mn of the reaction mixture samples showed a linear relationship (linear correlation coefficient R = 0.9779, Fig. 4A). A relationship (as shown below) was found between Mn and increase in absorption at 235 nm (A235 ) (Fig. 4B), consistent with the report published previously [6]. 1 1 A235 = + Mn Mnu Cε Mnu = the number average molecular weight of the heparin substrate; C = the substrate concentration (g L−1 ); ε = the molar absorption coefficient (M−1 cm−1 ). The average molecular weight (including Mw and Mn ) of the LMWH produced in different batches was determined (Table 1), which was different from the average molecular weight of the reaction mixture samples mentioned above. The LMWH products were purified from the filtrate of the reaction mixture which contained not only the filtrated LMWH products, but also larger fragments of heparin. With the increase of A235 from 20 to 90, the average molecular weight of the LMWH product decreased and the polydispersity index (D) became lower (Table 1). According to the European Pharmacopoeia 5.0 [15], the batches whose A235 was between 30 and 50 met the standards as the anticoagulant (Table 1). Moreover, there existed a linear relationship either between the Mw and Mn of the LMWH products (Fig. 4C), or between the Mn of the reaction mixture samples and the Mw of the LMWH products (Fig. 4D). All the linear relationships in Fig. 4 were fitted from the exact molecular weight values measured by HPLC. 3.3. Bioactivities of the LMWH products Fig. 3. GPC analysis of the heparin enzymatic depolymerization mixture in different A235 (S0 refers to the raw heparin, S1 –S9 refer to the heparin enzymatic depolymerization mixture samples obtained at different A235 as follows: 18.8, 46.5, 63.2, 86.5, 100.9, 136.5, 168.9, 227.0, 255.3). (A) Analysis results by RI detector; (B) analysis results by UV detector.
enzyme may proceed to cleave the same heparin molecule several times before releasing it, or it may cleave non-processively by releasing the substrate immediately after cleavage and binding to a new heparin molecule [18]. Fig. 3 supported the hypothesis that cleavage is mostly by the random endolytic process. Further research on the enzyme reaction enzyme remains to be elucidated.
After precipitation with ethanol, washing with acetone and vacuum drying, the LMWH products were obtained. Oligosaccharide distribution of the LMWH products was found to be similar to that of the mixture samples (data not shown). The average molecular weight index, anti-Xa and anti-IIa activities, activity ratio, and the yield of purified LMWH products were examined (Table 1). Both the anti-Xa and anti-IIa activity decreased with the increase of A235 . According to the European Pharmacopoeia 5.0 [15], the potency of LMWH should not be less than 70 IU of anti-factor Xa activity per milligram, calculated with reference to the dried substance. The ratio of anti-factor Xa activity to anti-factor IIa activity should not be less than 1.5. The results in Table 1 suggested that the batches whose final A235 was below 50 met the activity requirement of European Pharmacopoeia 5.0 [15], indicating that the reaction time
Table 1 Characteristics of different batches of LMWH products (n = 2). A235
20.6 30.8 40.7 46.7 48.4 51.5 64.2 75.2 88.9
Average molecule weight Mw
Mn
7578 6883 6282 5038 4916 4641 3979 3938 3314
4982 4674 4550 3498 3379 3230 3063 3163 2828
Polydispersity indexa (D)
Anti-Xa (IU/mg)
Anti-IIa (IU/mg)
Activity ratiob
Yield (%)
1.52 1.47 1.38 1.44 1.45 1.44 1.30 1.25 1.17
412.18 ± 15.71 385.29 ± 11.36 262.62 ± 11.42 154.95 ± 5.80 133.86 ± 4.17 116.69 ± 1.27 65.56 ± 0.53 55.21 ± 0.55 44.99 ± 0.45
356.08 ± 24.93 184.22 ± 19.77 109.26 ± 28.12 70.01 ± 2.21 57.72 ± 1.14 28.56 ± 0.75 29.48 ± 1.75 13.30 ± 0.27 12.33 ± 0.27
1.2 2.1 2.4 2.2 2.3 4.1 2.2 4.2 3.6
36.12 35.53 57.58 61.24 62.34 66.21 87.71 86.79 94.41
Note: According to the European Pharmacopoeia 5.0 [15], the potency of LMWH should not be less than 70 IU of anti-factor Xa activity per milligram, while the ratio of anti-factor Xa activity to anti-factor IIa activity should not be less than 1.5. a Polydispersity index is the ratio of weight-average molecular weight to number-average molecular weight. b Activity ratio is the ratio of anti-Xa activity to anti-IIa activity.
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Fig. 4. Linear relationship between (A) the logarithm of Mn and Mw of the reaction mixture at different times; (B) the Mn of reaction mixture samples and increasing A235 at different times; (C) the average molecular weight (including Mw and Mn ) of LMWH products; (D) the Mn of the reaction mixture samples and Mw of LMWH products.
was a key factor in the LMWH production by MBP-heparinase I. The yield of LMWH was improved as A235 increased, which could be explained by the increase in smaller oligosaccharides. Polyacrylamide gel electrophoresis analysis demonstrated that the LMWH products consisted of 4–22 repeat disaccharide units (determined from the standard polysaccharide samples with the known repeat disaccharide units number prepared by Prof. Guangli Yu of China Ocean University of Qing Dao), while the raw heparin has 45 units (data not shown). 4. Discussion The enzymatic method to prepare LMWH has been proved to be feasible [6], but the systematic study on the bioprocess and the operating conditions for the production of LMWH are still deficient due to the high cost of the conventional heparinase I. The present study has developed an efficient bioprocess to produce LMWH by MBP-heparinase I which can be produced easily by our over-expression system [10]. The fusion of MBP to the heparinase I is of great significance to the industrial application of heparinase I in LMWH preparation. MBP can dramatically enhance the solubility of the fusion protein and reach high enzymatic activity when MBP-heparinase I is expressed in recombinant E. coli [10]. MBP-heparinase I is easily purified [10] and could be immobilized directly by the affinity separation using maltose-coating particles. This will allow the reuse of the enzyme to be possible in a bioreactor, which can reduce LMWH production costs. The low productivity and high cost of the commercial heparinase I purified from F. heparinum have limited
the application of this enzyme in LMWH production [9]. MBPheparinase I used in the present study could be a useful form for the bioprocess integration of enzyme separation/purification and biocatalysis. The average molecular weight of LMWH could be predicted by using an empirically determined relationship between A235 and the average molecular weight of either the reaction samples or the LMWH products. With the value of A235 , the Mn and Mw of the LMWH products can be determined. However, the HPLC method is still needed to obtain the exact molecular weight values of the LMWH product for the standard curve. Furthermore, the ability to obtain LMWH products with different molecular weights at different reaction time suggested a method to direct the production of specific classes of LMWH. Also, the value of A235 is an important indicator to evaluate the activity of LMWH: the anti-factor Xa and anti-factor IIa activity. Heparin pentasaccharide, which binds antithrombin III (AT III) and changes its conformation, forms the complex that inhibits factor Xa activity [19]. For anti-factor IIa function, the pentasaccharide and an adjacent tridecasaccharide are necessary for factor IIa inhibition [20]. The longer the heparin is digested, the higher the value of A235 becomes and the lower the percentage of remaining pentasaccharide and tridecasaccharide remains, leading to a reduction in LMWH’s anti-factor Xa, and especially anti-factor IIa activity. Therefore, A235 is a key parameter for controlling the production of LMWH and the quality of the products by the enzymatic depolymerization, including the molecular weight, the anti-factor Xa activity, and the anti-factor IIa activity. Using A235 as a sim-
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ple indicator, it will be easy to determine the reaction time as well as the reaction conditions for the production of the qualified LMWH. The yield of LMWH is an essential index to evaluate the efficiency of the bioprocess. Firstly, controlling the reaction time at any given enzyme activity is important. Longer reaction time produced smaller oligosaccharides (more filtrates), which increased the product yield. However, anti-factor Xa and anti-factor IIa activity decreased inversely with the reaction progress. Thus, optimization of the enzyme reaction will be required to maximize the LMWH yield while maintaining the high activity. Another factor is the nominal molecular weight limit of the UF membrane. The appropriate nominal molecular weight limit should be optimized by taking the yield and the Mw restrictions based on European Pharmacopoeia 5.0 [15] (the LMWH product must meet the requirement that at least 60% of the total product (w/w) has a molecular weight less than 8000 Da). Finally, the yield could also be influenced by the operating conditions. For example, the LMWH produced by the enzymatic degradation of heparin can be continuously separated from the mixture by the UF filtration, which could increase the efficiency of residual heparin depolymerization by the enzyme. Further optimization for the enzyme bioreactor of MBP- MBP-heparinase I is needed. 5. Conclusions Production of the qualified LMWH by UF membrane bioreactor using MBP-heparinase I was studied, and the reaction process was evaluated by absorbance at 235 nm. When the A235 of the reaction solution reached 30–50, the produced LMWH could well meet the standards of the European Pharmacopoeia 5.0 [15]. The present enzyme bioreactor has the following advantages: the low cost of the fusion enzyme of MBP-heparinase I, the easy control of the product quality by the detection of A235 and the controllability of the UF membrane bioreactor. Acknowledgements This research was supported by the Grant of NSF Key Projects of China (Grant Nos. 20336010 and 20836004). Grateful acknowledgement is given to Prof. Guangli Yu of China Ocean University of Qing Dao, China for his kind help in the PAGE analysis of LMWH and heparin.
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