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PEGylation of Hirudin and Analysis of Its Antithrombin Activity in vitro
Chin. J . Chem. Eng., 15(4) 586-590
(2007)
PEGylation of Hirudin and Analysis of Its AntithrombinActivity in v h o QIN Haina(%&?!$)a, XIU Zhilong(@ & , ZHANG Daijia( %&@)a, (647k % a, LI Xiaohui($ d @?)a and HAN Guozhu( B&)b
BAO Yongming
Department of Bioscience and Biotechnology, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, China Department of Pharmacology, Dalian Medical University, Dalian 116027, China
a
Abstract Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical application. The PEGylation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEGylated hirudin were investigated. When the PEGylation reaction was conducted under 4°C after 10h, in the borate buffer at pH 8.5, with the molar ratio 250 : 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro. Compared with unmodified hirudin, 26% of anti-thrombin activity was retained. Keywords PEGylated protein, hirudin, analysis, anti-thrombin activity
1 INTRODUCTION Hirudin has become the most specific tight-binding thrombin inhibitor since it was isolated by Markwardt in the late 195Os[l]. It is a polypeptide containing 65 amino acids and produces its anticoagulant effect by blocking the thrombin anion binding site responsible for thrombin-fibrinogen interaction as well as inhibiting platelet receptors and protease activity. However, the rapid clearance of hirudin from the circulation decreases its bioavailability and limits its clinical use[2-41. Hirudin can be covalently conjugated with polyethylene glycol (PEG) to prolong its half-life time and increase its clinical use[5 -71. When PEG is properly linked to the polypeptide, the biodistribution and solubility of polypeptide drugs would be modified by reducing their renal clearance, preventing the approach of antibodies or antigen to cells, and reducing the degradation by proteolytic enzymes[8,9]. Various PEGylated proteins have been commercially available or are waiting at various stages of clinical trials[lD-l4]. According to the investigation of Esslinger et al., r-Hir containing two lysine residues (three in native hirudin) was used to minimize heterogeneity. Monodisperse product containing two moles of mPEG5000 per mole of r-Hir was the predominant product (PEG-hirudin). The half-life time of PEGylated hirudin prolongs from 0.5h to 24h. Furthermore, it has little or no immunogenicity. PEGylated hirudin has finished the trial of pharmacodynamics and safety investigation in healthy volunteers[151. During PEGylation of protein, the analysis of PEGylated proteins is one of major problems. Traditional analysis methods, such as 2,4,6-trinitrobenzenesulfonic acid spectrophotometry (TNBS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [ 16,171 cannot achieve accurate results because the chemical and physical properties of protein have changed after chemical modification. In this article, hirudin was covalently conjugated with PEG. The abovementioned two analytical methReceived 2006-08-3 1, accepted 2007-01-29.
ods were employed and improved in the analysis of PEGylated hirudin. The optimized conditions for PEGylation of hirudin were investigated on basis of improved TNBS and SDS-PAGE. Finally, the bioactivity of modified hirudin was also measured in vitro.
2 EXPERIMENTAL 2.1 Materials Methoxyl-polyethylene glycol (mPEG)-5000, carboxyl diimidazole (CDI), and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were purchased from Sigma. Sephadex G-50, Superdex 75, and Coomassie Brilliant Blue R250 were purchased from Aldrich and Fluka, respectively. Barium chloride from Tianhe Factory, Tianjin, iodine from Shenyang 1st Reagent Factory, China, and acetonitrile from Tedia Company of USA were also purchased. Hirudin, a recombinant polypeptide with 65 amino acid residues, was kindly donated by High-Tech. Biomedicine Ltd. Co. of Dalian, China. The standard proteins included ribonuclease (M,13700), carbonic anhydrase (Mr29000), ovalbumin (M,45000), bovine albumin (M,66000), and phosphorylase B (M, 97400). Standard polyethylene glycol was from several sources and of the best available grade. They were prepared as stock solution and lop1 were loaded directly on a SDS-PAGE. Thrombin was purchased from Hacon Oceanic Biomedicine Ltd. Co., Zhejiang, China. 2.2 Equipment High-performance liquid chromatography (HPLC) was equipped with JASCO pu- 1580 intelligent HPLC pump, JASCO uv- 1575 intelligent UVNis detector, and DG- 1580-54 online degasser. JASCO UVNIS Spectrophotometer V-560 was used to measure the optical density (OD) value of the reaction between hirudin and TNBS. SDS-PAGE experiments were carried out by a DYY-2C system produced by Beijing Liuyi Factory of China. The system was programmed to apply at a constant current of 45mA. Ultrafiltration
* To whom correspondence should be addressed. E-mail: zhlxiu9dlut.edu.cn
PEGylation of Hirudin and Analysis of Its AntithrombinActivity in vih.0
membrane (cut-off molecular weight 3000) was purchased from Millipore. Purification was performed by AKTA Purifier 100 (Amersham Biosciences, Sweden); anti-thrombin test was performed on C2000-4 Four-channel coagulation time measurement.
2.3 Procedure 2.3.1 Preparation of PEGylated hirudin mPEG was activated with CDI according to the method described in Ref.[18]. A quantity of 56mg recombinant hirudin and 2g activated mPEG were added into 20ml O.lmol.L-' borate buffer (pH 8.5). The reaction was stopped after 10 hours and the mixture was filtered by the membrane with average pore diameter of 0.22pm and was then separated by Sephadex G-50 into two peaks to remove the impurity. The first peak was collected and then condensed by an ultra-filtration membrane with a cut-off molecular weight of 3000. The condensed section was then separated by Superdex 75. 2.3.2 TNBS spectrophotometry According to the method described in Ref.[ 191, the concentration of hirudin in the solution can be determined by the standard curve of hirudin concentration versus OD424 value and the modification extent is calculated according to the following equation: modification extent (%) = ( C , - Ci)/C2X 100%
(1) CI is the concentration of hirudin corresponding to the OD424 value of PEGylated hirudin. PEGylated hirudin was reacted with TNBS according to Ref.[19], and then corresponding to the OD424, the concentration was obtained from the standard curve; C2 represents the concentration of hirudin corresponding to the OD424 value of unmodified hirudin. Activated mPEG was added into water overnight to make it inactive. The inactivated mPEG was then added into the mixture of lmg-ml-' hirudin, 0.1% TNBS solution and 0.lmol.L-' borate buffer (pH 8.5). The mixture was kept in water bath at 40°C for 2h, and measured at 424nm every 5min. The measurable results were compared with the case without inactivated mPEG. 2.3.3 SDS-PAGE The typical Coomassie Brilliant Blue staining for SDS-PAGE cannot detect the PEGylated hirudin at a low protein concentration due to the absence of lysine residue; while the staining of PEG chains with barium iodide complex will be more sensitive and specific, according to the previous report[20]. After electrophoresis, the gel was put into the 5% glutaraldehyde solution for 2Omin at room temperature, and then into 0.lmol.L - perchloric acid solutions for 15min. Thereafter, the gel was stained with 5% barium chloride solution and 0.1mo1.L - iodine solutions for 15min. The staining bands would appear after a few minutes. At last, the gel was washed with water for another 15min. 2.3.4 Optimization of modification condition The PEGylation of proteins is usually achieved by covalently attaching mPEG to free amino groups of protein via (especially &-amino group of lysine resi-
'
'
587
due). Recombinant hirudin and activated mPEG were added to 20ml 0.lmol.L-' borate buffer to react at different pH, temperature, molar ratio and the reaction was quenched in scheduled time. The mixture was filtered by the 0 . 2 2 microfiltration ~ membrane, and then was analyzed with TNBS spectrophotometry and SDS-PAGE, respectively. The optimized conditions were determined by comparing the modification extents. 2.3.5 Assay of anticoagulant activity The product was purified by Sephadex G50 and Superdex 75. The PEGylated hirudin was collected and freeze-dried to evaluate the bioactivity. The sodium citrate (3.8%) was added to the rabbit plasma with a molar ratio of 1 9, and then thrombin solution (5NIHunit.d-') was also addedi 0, 10, 20, 40, 80, 160p1 mPEG-hirudin (100pg.ml- ) were added to the above plasma, respectively, and the thrombin time ('IT) was measured. When TT of hirudin was equal to the TT of PEGylated hirudin, the retained anti-thrombin activity could be got by the following equation: ratio of reserved antithrombin ability of modified hirudin (%) = C, / C , ~ 1 0 0 % (2) where C1 is the concentration of PEGylated hirudin, and CZis the concentration of hirudin.
3 RESULTS 3.1 TNBS spectrophotometry Hirudin reacted with TNBS for IOOmin, and the mixture was scanned from the wavelength of 300nm to 500nm. From Fig.1, the wavelength of maximum absorbance could be determined as 424nm (peak 1).
200
300 400 wavelength, nm
500
Figure 1 Spectrum of the product of reaction between hirudin and TNBS (lml O.l%TNBS and lml lrngm- hirudin reacted in 0 . l m o l L ' borate buffer with pH 8.5 under 40°C for 100min)
The analytical results might be influenced by the presence of inactivated mPEG in the mixture because it disturbed the nucleophilic reaction between protein and TNBS. For this reason, it should be removed[21]. However, this would undoubtedly bring trouble for analysis, especially for the analysis of polypeptide with a lower molecular weight. Fortunately, the experiments showed that the analytical results were reproducible and credible only if the reaction was stopped within an appropriate time, as shown in Fig.2. The error was caused by the impurity in activated Chin. J. Ch. E. 15(4) 586 (2007)
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i"lfl
Chin. J. Ch. E. (Vol. 15, No.4)
5
9
010
s:' L
9
-
0
-20 0 20 40 60 801001201401 time, min
Figure 2 Effect of inactivated mPEG on the reaction (Both reactions were carried out in 0.lmol.L-l borate buffer with pH 8.5 under 40'C for 2h) 1 r n g . d ' Hir; add inactivated mPEG in Hir
mPEG, for example, CDI and its hydrolyzed products, which could be reduced by taking the hydrolyzed activated mPEG as reference sample. From Fig.2, it can be seen that the OD424 value with mPEG increased only 0.87% when the reaction completed 98.2% in 100min. The deviation can be ignored if the reaction is stopped at 100min. In this experiment, it was found that within loomin, even without removing the impurity, the analytical results were accurate. So the removing procedure of TNBS spectrophotometry could be omitted.
3.2 Optimization of modification conditions The activity of i?-amino group is affected by pH value. The modification extent is related with temperature, reaction time, pH value of the buffer, and the molar ratio of reactants. Therefore, it is necessary to investigate the above reaction conditions. 3.2.1 Effect of p H value on the reaction Adjusted pH of O.lmol.L-' borate buffer to 7.0, 7.5, 8.0, 8.5, 9.0, 21mg recombinant hirudin and 450mg activated mPEG were put into the buffers, respectively, and reacted under 4'C. Finally, the modification extent was determined by TNBS spectrophotometry. From Fig.3 and Table 1, it could be seen that the modification extent was the highest when pH value was 8.5. Therefore, pH 8.5 is preferred for the reaction.
Table 1 Modification ratio of PEGylated hirudin measured by TNBS spectrophotometery
DH
OD*?*
7.5
0.17
16.3
8.0
0.15
33.6
Modification ratio. %
45.7 8.5 0.14 33.6 9.0 0.15 Note: Molar ratio of hirudin to mPEG was 1 :250, in 2011-11 O.lmol.L-' borate buffer with different pH value under 4'C.
3.2.2 Effect of the molar ratio of reactants on reaction Hirudin and activated mPEG reacted with different molar ratio of 1 : 150, 1 : 200, 1 : 250, 1 : 300 (recombinyt hirudin: activated mPEG) in 20ml 0.lmol.L- borate buffer with pH 8.5 under 4"C, and the modification extent was measured. Fig.4 showed that when the molar ratios were 1 :250 and 1 300, the modification extent was highest. Molar ratio of 1 250 is preferable considering the cost. PEG20000
PEG I0000
PEGS000
1
2
3
s
4
Figure 4 Effect of molar ratio of hirudin to mPEG on the modification extent (0.lmol.L-' borate buffer of pH 8.5,4'C, reacted for 10h) Lane 1: PEG standards; Lane 2: 1 100; Lane3: 1 :200; Lane 4: 1 :250; Lane5 1 :300
3.2.3 Efect of temperature on the reaction Figure 5 and Table 2 showed that the lower temperature was better for the reaction, possibly because the lower temperature might slow down the hydrolysis of activated mPEG, and more activated mPEG will react with hirudin. Furthermore, the bioactivity of hirudin will be protected under lower temperature. PEG20000
PEG20000
+biPEG 10000
PEG I0000
e mono-
PEGS000
PEG5000 1
2
3
4
5
Figure 3 PEGylated hirudin produced under different pH values (0.lmol.L-l borate buffer, 4'C, reacted for 10h, molar ratio of hirudin to mPEG was 1 :250) Lane 1: PEG standards; Lane 2: pH=7.5; Lane 3: pH=8.0; Lane 4: pH=8.5; Lane 5: pH=9.0 August, 2007
I
2
3
Figure 5 Effect of temperature on modiflation extent (O.lmol.L-' borate buffer of pH 8.5, reacted for 10h, molar ratio of hirudin to mPEG was 1 :250) Lane 1: PEG standards; Lane 2: 20'C; Lane 3: 4°C
PEGylation of Hirudin and Analysis of Its AntithrombinActivity in vifro Table 2 Modification ratio of PEGylated hirudin measured by TNBS spectrophotometery T, “C 20 4
OD424 Modification ratio, % 0.24 15.4 41.4 0.2 1 Note: Molar ratio of hirudin to mPEG was 1 :250 in 20ml O.lmol.L-’ pH8.5 borate buffer under 4°C and 20°C.
3.2.4
Effect of reaction time on modijkation extent From Fig.6, it could be seen that when hirudin reacted with activated mPEG for 10h, the modification extent was the greatest. When reacted for 16h, the modification extent decreased. As a result, 10h is best for our reaction.
PEG20000
PEG 10000
PEG5000
3
2
I
Table 3 Comparison of anti-thrombinactivity between unmodified and PEGylated hirudin Hirudin Ratio of pro- Concentralonged p, % tion,lgm-l 179 0.3 33 0.15 17 0.08 0 TT means thrombin time.
ACKNOWLEDGEMENT The authors wish to thank Ms.Hou Beibei for her revising this research paper.
REFERENCES 2
3 4
5
6
1
Ratio of pro- Concentration, longed TT, % 1g.ml-l 52 3 26 1.5 15 0.75
100min; As far as SDS-PAGE is concerned, the PEG chains are staiped with 5% barium chloride solution and O.lmo1-L- iodine solutions, which is much more specific and sensitive than Coomassie Brilliant Blue staining for protein. When hirudin reacted with activated mPEG for 10h, the modification extent was the greatest because the PEGylated proteins begin to precipitate after 10h. Moreover, the bioactivity of modified hirudin was measured. Although only 26% anti-thrombin activity is retained when compared with unmodified hirudin, it represents only the bioactivity in vitro; in vivo, when the mPEG is hydrolyzed from the active site, its bioactivity may recover.
1
3.3 Bioactivity of the PEGylated hirudin The product was first separated by Sephadex G50 and further purified by Superdex 75. As shown in Fig.7, peak 2 was the target product. It was collected and freeze-dried to evaluate the bioactivity. The anti-thrombin activity of unmodified and PEGylated hirudin was shown in Table 3.
I
PEGylated hirudin
4
Figure 6 PEGylated hirudin reacted for different times (O.1mol.L-l borate buffer of pH 8.5,4”C, molar ratio of hirudin to mPEG was 1 : 250) Lane 1: PEG Standards; Lane 2: reacted for 4h; Lane 3: reacted for 10h; Lane 4: reacted for 16h
40
589
I
I
60 80 100 retention volume, ml
Figure 7 SEC separation of PEGylated reaction mixture by Superdex 75 (0.lmol.L-l pH 8.5 borate buffer for 10h under 4’C, the molar ratio of hirudin to mPEG was 1 250) 1-himdin; 2-ITPEG-hirudin; 3-hirudin
4 DISCUSSION TNBS will achieve accurate results without the removal of inactivated mPEG only when the reaction between PEGylated hirudin and TNBS is stopped after
7
8
9
10
Markwart, F., “The development of hirudin as an antithrombotic drug”, Thmmb. Res., 74, 1-23( 1994). Rydel, T.J., Ravichandran, K.G, Tulinsky, A., Bode, W., Huber, R., Roitsch, C., Fenton, J.W., “The structure of a complex of recombinant hirudin and human a-thrombin”, Science, 249,277-280( 1990). Rydel, T.J., Tulinsky, A., Bode, W., Huber, R., “Refined structure of the hirudin-thrombin complex”, J . Mol. Biol., 221,583-601(1991). Markwardt, F., “Past, present and future of hirudin”, Huemost. 21(Suppl. I), 11-26( 199 1). Ulbricht, K., Bucha, E., Poschel, KA., Stein, G, Wolf, G, Nowak, G, ‘The use of PEG-Hirudin in chronic hemodialysis monitored by the Ecarin Clotting Time: Influence on clotting of the extracorporeal system and hemostatic parameters”, Clin. Nephrol., 65(3), 180-190(2006). Alexander, B., Burnand, K.G, Lattimer, C.L., Humphries, J., Gaffney, P.J., Eastham, D., Smith, A., “The effect of anticoagulation with subcutaneously delivered polyethylene glycol conjugated hirudin and recombinant tissue plasminogen activator on recurrent stenosis in the rabbit double-balloon injury model”, Thromb. Res., 113(2), 155-16 l(2004). Poschel, K.A., Bucha, E., Esslinger, H.U., Ulbricht, K., Nortersheuser, P., Stein, G, Nowak, G, “Anticoagulant efficacy of PEG-Hirudin in patients on maintenance hemodialysis”, Kidney Int., 65(2), 666-674(2004). Roberts, M.J., Bentley, M.D., Harris, J.M., “Chemistry for peptide and protein PEGylation”, Adv. Drug. Deliv. Rev., 54,459-476(2002). Veronese, F., “Peptide and protein PEGylation: A review of problems and solutions”, Biomafer.,22,405-417(2001). Monkarsh, S.P., Ma, Y.,Aglione, A., Bailon, P., Ciolek, D., DeBarbieri, B., Graves, M.C., Hollfelder, K., Michel, H., Palleroni, A., Porter, J.E., Russoman, E., Roy, S., Pan,
Chin. J. Ch. E. 15(4) 586 (2007)
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Y.E., “Positional isomers of monopegylated interferon a-2a: Isolation, characterization, and biological activity”, Anal. Biochem., 247,434-40( 1997). 11 Uchio, T.,Baudys, M., Liu, F., Song, S.C., Kim, S.W., “Site-specific insulin conjugates with enhanced stability and extended action profile”, Adv. Drug. Deliv. Rev., 35, 289-306( 1999). 12 Kinstler, O.B., Gabriel, N.E., Farrar, C.E., DePrince, R.B., “N-terminally chemically modified protein composition methods”, US Pat., 5985265 (1999). 13 Goffin, V., Bernichtein, S., Caniere, 0.. Bennett, W.F., Kopchick, J.J., Kelly, P.A., “The human growth hormone antagonist B2036 does not interact with the prolactin receptor”, Endocrinol., 140.3853-3856( 1999). 14 Giardino, R., Capelli, S., Fini, M., Giavaresi, G, Orienti, L., Veronese, EM., Caliceti, P., Rocca, M., “Biopolymeric modification of superoxide dismutase (mPEG-SOD) to prevent muscular ischemia-reperfusion damage”, Inr. J . Arfif. Organs., 18, 167-172(1995). 15 Esslinger, H.U., Haas, S., Mauer, R., Lassmann, A., Dubbers, K., Muler-Pelzer, H., “Pharmacolodynamic and safety results of PEG-himdin in healthy volunteers”, Thromb. Haemost., 5,911-919(1997). 16 Li, W.J., Lin, B.C., Su, Z.G, “Analytical methods for
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PEGylated protein drugs”, Analysis, 29, 228-23 l(2001). (in Chinese) Mcgoff, P., Baziotis, A.C., Maskiewicz, R., “Analysis of polyethylene glycol modified superoxidedismutase by chromatographic, electrophoretic, light scattering, chemical and enzymatic methods”, Chem. Pharm. Bull., 36,3079-3091(1988). Biscoff, R., “Polyethylene glycol-himdin conjugates, process for preparing them and their use for the treatment of thromboses”, U.S. Pat., 5362858( 1994). Snyder, S.L., Sobocinski, P.Z., “An improved 2,4,6-trinitrobenzenesulfonicacid method for determination of amines”, Anal. Biochem., 64,284-288(1975). Manfred, M.K., “Detection and molecular weight determination of polyethylene glycol-modified hirudin by staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis”, Anal. Biochem., 200,244-248( 1992). Bullock, J., Chowdhury, S., Severdia, A., Sweeney, J., Johnston, D., Pachla, L., “Comparison of results of various methods used to determine the extent of modification of methoxy polyethylene glycol 5000-modified bovine cupri-zinc superoxide dismutase”, Anal. Biochem., 254, 254-262( 1997).
(2007)
PEGylation of Hirudin and Analysis of Its AntithrombinActivity in v h o QIN Haina(%&?!$)a, XIU Zhilong(@ & , ZHANG Daijia( %&@)a, (647k % a, LI Xiaohui($ d @?)a and HAN Guozhu( B&)b
BAO Yongming
Department of Bioscience and Biotechnology, School of Environmental and Biological Science and Technology, Dalian University of Technology, Dalian 116024, China Department of Pharmacology, Dalian Medical University, Dalian 116027, China
a
Abstract Hirudin is the most anticoagulant drug found in nature, but its short serum half-life significantly inhibits its clinical application. The PEGylation of hirudin, the most promising anticoagulant drug, was performed in this paper. The optimal reaction conditions for PEGylated hirudin were investigated. When the PEGylation reaction was conducted under 4°C after 10h, in the borate buffer at pH 8.5, with the molar ratio 250 : 1 of PEG to hirudin, a higher modification extent was achieved. Finally, the bioactivity of PEGylated hirudin was measured in vitro. Compared with unmodified hirudin, 26% of anti-thrombin activity was retained. Keywords PEGylated protein, hirudin, analysis, anti-thrombin activity
1 INTRODUCTION Hirudin has become the most specific tight-binding thrombin inhibitor since it was isolated by Markwardt in the late 195Os[l]. It is a polypeptide containing 65 amino acids and produces its anticoagulant effect by blocking the thrombin anion binding site responsible for thrombin-fibrinogen interaction as well as inhibiting platelet receptors and protease activity. However, the rapid clearance of hirudin from the circulation decreases its bioavailability and limits its clinical use[2-41. Hirudin can be covalently conjugated with polyethylene glycol (PEG) to prolong its half-life time and increase its clinical use[5 -71. When PEG is properly linked to the polypeptide, the biodistribution and solubility of polypeptide drugs would be modified by reducing their renal clearance, preventing the approach of antibodies or antigen to cells, and reducing the degradation by proteolytic enzymes[8,9]. Various PEGylated proteins have been commercially available or are waiting at various stages of clinical trials[lD-l4]. According to the investigation of Esslinger et al., r-Hir containing two lysine residues (three in native hirudin) was used to minimize heterogeneity. Monodisperse product containing two moles of mPEG5000 per mole of r-Hir was the predominant product (PEG-hirudin). The half-life time of PEGylated hirudin prolongs from 0.5h to 24h. Furthermore, it has little or no immunogenicity. PEGylated hirudin has finished the trial of pharmacodynamics and safety investigation in healthy volunteers[151. During PEGylation of protein, the analysis of PEGylated proteins is one of major problems. Traditional analysis methods, such as 2,4,6-trinitrobenzenesulfonic acid spectrophotometry (TNBS) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) [ 16,171 cannot achieve accurate results because the chemical and physical properties of protein have changed after chemical modification. In this article, hirudin was covalently conjugated with PEG. The abovementioned two analytical methReceived 2006-08-3 1, accepted 2007-01-29.
ods were employed and improved in the analysis of PEGylated hirudin. The optimized conditions for PEGylation of hirudin were investigated on basis of improved TNBS and SDS-PAGE. Finally, the bioactivity of modified hirudin was also measured in vitro.
2 EXPERIMENTAL 2.1 Materials Methoxyl-polyethylene glycol (mPEG)-5000, carboxyl diimidazole (CDI), and 2,4,6-trinitrobenzenesulfonic acid (TNBS) were purchased from Sigma. Sephadex G-50, Superdex 75, and Coomassie Brilliant Blue R250 were purchased from Aldrich and Fluka, respectively. Barium chloride from Tianhe Factory, Tianjin, iodine from Shenyang 1st Reagent Factory, China, and acetonitrile from Tedia Company of USA were also purchased. Hirudin, a recombinant polypeptide with 65 amino acid residues, was kindly donated by High-Tech. Biomedicine Ltd. Co. of Dalian, China. The standard proteins included ribonuclease (M,13700), carbonic anhydrase (Mr29000), ovalbumin (M,45000), bovine albumin (M,66000), and phosphorylase B (M, 97400). Standard polyethylene glycol was from several sources and of the best available grade. They were prepared as stock solution and lop1 were loaded directly on a SDS-PAGE. Thrombin was purchased from Hacon Oceanic Biomedicine Ltd. Co., Zhejiang, China. 2.2 Equipment High-performance liquid chromatography (HPLC) was equipped with JASCO pu- 1580 intelligent HPLC pump, JASCO uv- 1575 intelligent UVNis detector, and DG- 1580-54 online degasser. JASCO UVNIS Spectrophotometer V-560 was used to measure the optical density (OD) value of the reaction between hirudin and TNBS. SDS-PAGE experiments were carried out by a DYY-2C system produced by Beijing Liuyi Factory of China. The system was programmed to apply at a constant current of 45mA. Ultrafiltration
* To whom correspondence should be addressed. E-mail: zhlxiu9dlut.edu.cn
PEGylation of Hirudin and Analysis of Its AntithrombinActivity in vih.0
membrane (cut-off molecular weight 3000) was purchased from Millipore. Purification was performed by AKTA Purifier 100 (Amersham Biosciences, Sweden); anti-thrombin test was performed on C2000-4 Four-channel coagulation time measurement.
2.3 Procedure 2.3.1 Preparation of PEGylated hirudin mPEG was activated with CDI according to the method described in Ref.[18]. A quantity of 56mg recombinant hirudin and 2g activated mPEG were added into 20ml O.lmol.L-' borate buffer (pH 8.5). The reaction was stopped after 10 hours and the mixture was filtered by the membrane with average pore diameter of 0.22pm and was then separated by Sephadex G-50 into two peaks to remove the impurity. The first peak was collected and then condensed by an ultra-filtration membrane with a cut-off molecular weight of 3000. The condensed section was then separated by Superdex 75. 2.3.2 TNBS spectrophotometry According to the method described in Ref.[ 191, the concentration of hirudin in the solution can be determined by the standard curve of hirudin concentration versus OD424 value and the modification extent is calculated according to the following equation: modification extent (%) = ( C , - Ci)/C2X 100%
(1) CI is the concentration of hirudin corresponding to the OD424 value of PEGylated hirudin. PEGylated hirudin was reacted with TNBS according to Ref.[19], and then corresponding to the OD424, the concentration was obtained from the standard curve; C2 represents the concentration of hirudin corresponding to the OD424 value of unmodified hirudin. Activated mPEG was added into water overnight to make it inactive. The inactivated mPEG was then added into the mixture of lmg-ml-' hirudin, 0.1% TNBS solution and 0.lmol.L-' borate buffer (pH 8.5). The mixture was kept in water bath at 40°C for 2h, and measured at 424nm every 5min. The measurable results were compared with the case without inactivated mPEG. 2.3.3 SDS-PAGE The typical Coomassie Brilliant Blue staining for SDS-PAGE cannot detect the PEGylated hirudin at a low protein concentration due to the absence of lysine residue; while the staining of PEG chains with barium iodide complex will be more sensitive and specific, according to the previous report[20]. After electrophoresis, the gel was put into the 5% glutaraldehyde solution for 2Omin at room temperature, and then into 0.lmol.L - perchloric acid solutions for 15min. Thereafter, the gel was stained with 5% barium chloride solution and 0.1mo1.L - iodine solutions for 15min. The staining bands would appear after a few minutes. At last, the gel was washed with water for another 15min. 2.3.4 Optimization of modification condition The PEGylation of proteins is usually achieved by covalently attaching mPEG to free amino groups of protein via (especially &-amino group of lysine resi-
'
'
587
due). Recombinant hirudin and activated mPEG were added to 20ml 0.lmol.L-' borate buffer to react at different pH, temperature, molar ratio and the reaction was quenched in scheduled time. The mixture was filtered by the 0 . 2 2 microfiltration ~ membrane, and then was analyzed with TNBS spectrophotometry and SDS-PAGE, respectively. The optimized conditions were determined by comparing the modification extents. 2.3.5 Assay of anticoagulant activity The product was purified by Sephadex G50 and Superdex 75. The PEGylated hirudin was collected and freeze-dried to evaluate the bioactivity. The sodium citrate (3.8%) was added to the rabbit plasma with a molar ratio of 1 9, and then thrombin solution (5NIHunit.d-') was also addedi 0, 10, 20, 40, 80, 160p1 mPEG-hirudin (100pg.ml- ) were added to the above plasma, respectively, and the thrombin time ('IT) was measured. When TT of hirudin was equal to the TT of PEGylated hirudin, the retained anti-thrombin activity could be got by the following equation: ratio of reserved antithrombin ability of modified hirudin (%) = C, / C , ~ 1 0 0 % (2) where C1 is the concentration of PEGylated hirudin, and CZis the concentration of hirudin.
3 RESULTS 3.1 TNBS spectrophotometry Hirudin reacted with TNBS for IOOmin, and the mixture was scanned from the wavelength of 300nm to 500nm. From Fig.1, the wavelength of maximum absorbance could be determined as 424nm (peak 1).
200
300 400 wavelength, nm
500
Figure 1 Spectrum of the product of reaction between hirudin and TNBS (lml O.l%TNBS and lml lrngm- hirudin reacted in 0 . l m o l L ' borate buffer with pH 8.5 under 40°C for 100min)
The analytical results might be influenced by the presence of inactivated mPEG in the mixture because it disturbed the nucleophilic reaction between protein and TNBS. For this reason, it should be removed[21]. However, this would undoubtedly bring trouble for analysis, especially for the analysis of polypeptide with a lower molecular weight. Fortunately, the experiments showed that the analytical results were reproducible and credible only if the reaction was stopped within an appropriate time, as shown in Fig.2. The error was caused by the impurity in activated Chin. J. Ch. E. 15(4) 586 (2007)
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010
s:' L
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-20 0 20 40 60 801001201401 time, min
Figure 2 Effect of inactivated mPEG on the reaction (Both reactions were carried out in 0.lmol.L-l borate buffer with pH 8.5 under 40'C for 2h) 1 r n g . d ' Hir; add inactivated mPEG in Hir
mPEG, for example, CDI and its hydrolyzed products, which could be reduced by taking the hydrolyzed activated mPEG as reference sample. From Fig.2, it can be seen that the OD424 value with mPEG increased only 0.87% when the reaction completed 98.2% in 100min. The deviation can be ignored if the reaction is stopped at 100min. In this experiment, it was found that within loomin, even without removing the impurity, the analytical results were accurate. So the removing procedure of TNBS spectrophotometry could be omitted.
3.2 Optimization of modification conditions The activity of i?-amino group is affected by pH value. The modification extent is related with temperature, reaction time, pH value of the buffer, and the molar ratio of reactants. Therefore, it is necessary to investigate the above reaction conditions. 3.2.1 Effect of p H value on the reaction Adjusted pH of O.lmol.L-' borate buffer to 7.0, 7.5, 8.0, 8.5, 9.0, 21mg recombinant hirudin and 450mg activated mPEG were put into the buffers, respectively, and reacted under 4'C. Finally, the modification extent was determined by TNBS spectrophotometry. From Fig.3 and Table 1, it could be seen that the modification extent was the highest when pH value was 8.5. Therefore, pH 8.5 is preferred for the reaction.
Table 1 Modification ratio of PEGylated hirudin measured by TNBS spectrophotometery
DH
OD*?*
7.5
0.17
16.3
8.0
0.15
33.6
Modification ratio. %
45.7 8.5 0.14 33.6 9.0 0.15 Note: Molar ratio of hirudin to mPEG was 1 :250, in 2011-11 O.lmol.L-' borate buffer with different pH value under 4'C.
3.2.2 Effect of the molar ratio of reactants on reaction Hirudin and activated mPEG reacted with different molar ratio of 1 : 150, 1 : 200, 1 : 250, 1 : 300 (recombinyt hirudin: activated mPEG) in 20ml 0.lmol.L- borate buffer with pH 8.5 under 4"C, and the modification extent was measured. Fig.4 showed that when the molar ratios were 1 :250 and 1 300, the modification extent was highest. Molar ratio of 1 250 is preferable considering the cost. PEG20000
PEG I0000
PEGS000
1
2
3
s
4
Figure 4 Effect of molar ratio of hirudin to mPEG on the modification extent (0.lmol.L-' borate buffer of pH 8.5,4'C, reacted for 10h) Lane 1: PEG standards; Lane 2: 1 100; Lane3: 1 :200; Lane 4: 1 :250; Lane5 1 :300
3.2.3 Efect of temperature on the reaction Figure 5 and Table 2 showed that the lower temperature was better for the reaction, possibly because the lower temperature might slow down the hydrolysis of activated mPEG, and more activated mPEG will react with hirudin. Furthermore, the bioactivity of hirudin will be protected under lower temperature. PEG20000
PEG20000
+biPEG 10000
PEG I0000
e mono-
PEGS000
PEG5000 1
2
3
4
5
Figure 3 PEGylated hirudin produced under different pH values (0.lmol.L-l borate buffer, 4'C, reacted for 10h, molar ratio of hirudin to mPEG was 1 :250) Lane 1: PEG standards; Lane 2: pH=7.5; Lane 3: pH=8.0; Lane 4: pH=8.5; Lane 5: pH=9.0 August, 2007
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Figure 5 Effect of temperature on modiflation extent (O.lmol.L-' borate buffer of pH 8.5, reacted for 10h, molar ratio of hirudin to mPEG was 1 :250) Lane 1: PEG standards; Lane 2: 20'C; Lane 3: 4°C
PEGylation of Hirudin and Analysis of Its AntithrombinActivity in vifro Table 2 Modification ratio of PEGylated hirudin measured by TNBS spectrophotometery T, “C 20 4
OD424 Modification ratio, % 0.24 15.4 41.4 0.2 1 Note: Molar ratio of hirudin to mPEG was 1 :250 in 20ml O.lmol.L-’ pH8.5 borate buffer under 4°C and 20°C.
3.2.4
Effect of reaction time on modijkation extent From Fig.6, it could be seen that when hirudin reacted with activated mPEG for 10h, the modification extent was the greatest. When reacted for 16h, the modification extent decreased. As a result, 10h is best for our reaction.
PEG20000
PEG 10000
PEG5000
3
2
I
Table 3 Comparison of anti-thrombinactivity between unmodified and PEGylated hirudin Hirudin Ratio of pro- Concentralonged p, % tion,lgm-l 179 0.3 33 0.15 17 0.08 0 TT means thrombin time.
ACKNOWLEDGEMENT The authors wish to thank Ms.Hou Beibei for her revising this research paper.
REFERENCES 2
3 4
5
6
1
Ratio of pro- Concentration, longed TT, % 1g.ml-l 52 3 26 1.5 15 0.75
100min; As far as SDS-PAGE is concerned, the PEG chains are staiped with 5% barium chloride solution and O.lmo1-L- iodine solutions, which is much more specific and sensitive than Coomassie Brilliant Blue staining for protein. When hirudin reacted with activated mPEG for 10h, the modification extent was the greatest because the PEGylated proteins begin to precipitate after 10h. Moreover, the bioactivity of modified hirudin was measured. Although only 26% anti-thrombin activity is retained when compared with unmodified hirudin, it represents only the bioactivity in vitro; in vivo, when the mPEG is hydrolyzed from the active site, its bioactivity may recover.
1
3.3 Bioactivity of the PEGylated hirudin The product was first separated by Sephadex G50 and further purified by Superdex 75. As shown in Fig.7, peak 2 was the target product. It was collected and freeze-dried to evaluate the bioactivity. The anti-thrombin activity of unmodified and PEGylated hirudin was shown in Table 3.
I
PEGylated hirudin
4
Figure 6 PEGylated hirudin reacted for different times (O.1mol.L-l borate buffer of pH 8.5,4”C, molar ratio of hirudin to mPEG was 1 : 250) Lane 1: PEG Standards; Lane 2: reacted for 4h; Lane 3: reacted for 10h; Lane 4: reacted for 16h
40
589
I
I
60 80 100 retention volume, ml
Figure 7 SEC separation of PEGylated reaction mixture by Superdex 75 (0.lmol.L-l pH 8.5 borate buffer for 10h under 4’C, the molar ratio of hirudin to mPEG was 1 250) 1-himdin; 2-ITPEG-hirudin; 3-hirudin
4 DISCUSSION TNBS will achieve accurate results without the removal of inactivated mPEG only when the reaction between PEGylated hirudin and TNBS is stopped after
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