Biotechnology Letters (2005) 27: 213–217
Springer 2005
Novel polyethylene glycol derivative suitable for the preparation of mono-PEGylated protein Qiang Yun1, Ting Chen2, Guifeng Zhang1, Jingxiu Bi1, Guanghui Ma1 & Zhiguo Su1,* 1
National Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, P.O. Box 353, Beijing 100080, P.R. China 2 Civil & Environmental Engineering School, University of Science and Technology of Beijing *Author for correspondence (Fax: +86-10-62561813; E-mail:
[email protected]) Received 9 September 2004; Revisions requested 30 September 2004/16 November 2004; Revisions received 12 November 2004/14 December 2004; Accepted 16 December 2004
Key words: consensus interferon, granulocyte-colony stimulating factor, PEGylation, polyethylene glycol Abstract A novel methoxypolyethylene glycol (mPEG) derivative, containing a reactive group of 1-methyl pyridinium toluene-4-sulfonate, was synthesized and characterized. The mPEG derivative was successfully conjugated with two proteins: recombinant human granulocyte-colony stimulating factor (rhG-CSF) and consensus interferon (C-IFN). Homogeneous mono-PEGylated proteins were obtained which were identified by high performance size-exclusion chromatography and MALDI-TOF mass spectrometry. The biological activities of the mono-PEGylated rhG-CSF and the mono-PEGylated C-IFN were maintained at 90% and 88%, respectively.
Introduction PEGylation, the process of attaching polyethylene glycol (PEG) to protein and peptides as well as other molecules, improves their pharmacological properties, most often making them more clinically effective (Veronese & Harris 2002). The improved properties include better physical and thermal stability, longer in vivo circulating half-life, decreased clearance and enhanced potency (Abuchowski et al. 1977, Harris & Veronese 2003). In traditional PEGylation, however, due to the variation in number and location of lysine residues in proteins and the conjugating reaction being random, the stoichiometry of protein-PEG conjugation and the attachment sites are not easily and precisely controlled (Veronese 2001). The resulting heterogeneous products are unacceptable in therapeutic use.
Site-directed modification in regions far away from the bioactivity domain has been considered as a method to circumvent these limitations (Pettit & Gombotz 1998). Some coupling strategies have been developed to achieve site-directed PEGylation. The most popular site-directed method is the thiol-selective PEGylation of an introduced cysteine residue by using site-directed mutagenesis and either maleimide-based or haloacetic-based PEG derivatives (Benhar et al. 1994). Another frequent target for site-directed PEGylation is the polysaccharides of glycoproteins, which can be oxidized under mild conditions and then reacted with a variety of activated PEG derivatives (Uchio et al. 1999). Although these strategies are superior to traditional PEGylation procedures with respect to the specificity of the reaction, they do not always successfully modify the proteins, since the mutant
214 protein may have a different structure and function as compared to the native protein and a lot of proteins do not contain polysaccharide residues, which probably limit their general application. Alternatively, a variety of methods for monoPEGylation of protein have been developed and several mono-PEGylated proteins have been licensed by US Food and Drug Administration (FDA) (Kinstler et al. 2002). As an example, Pegasys (mono-PEGylated interferon a-2a), which was prepared by branched succinimidyl carbonate PEG, has an increased half-life from 9 to 77 h, a 100-fold decrease in renal clearance and an increase in antiviral activity ranging from 12- to 135-fold (Bailon et al. 2001). Mono-PEGylated growth hormonereleasing factor (GRF), which was prepared by PEG succinimidyl esters (PEG-OSu5000), has a prolonged half-life in plasma and a single injection produced a sustained pharmacodynamic response (Esposito et al. 2003). Therefore, mono-PEGylation is a promising strategy for developing clinical useful PEG-protein conjugates, where reproducibility and homogeneity are essential and minimal loss of bioactivity is highly desirable. In this paper, a convenient approach for preparing homogeneous mono-PEGylated protein is described. The selectivity is achieved by conducting the alkylation of protein with 2-fluoro-1methylpyridinium toluene-4-sulfonate activated methoxypolyethylene glycol (FMP-mPEG), a new PEG derivative, which was firstly synthesized in our laboratory. Recombinant human granulocyte-colony stimulating factor (rhG-CSF) and consensus interferon (C-IFN) were used as model proteins, respectively.
Materials and methods Chemicals and reagents Methoxypolyethylene glycol 5000 (mPEG5000) was from Union Carbide (Danbury, CT). 2-Fluoro1-methylpyridinium toluene-4-sulfonate (FMP) was from Aldrich. rhG-CSF and C-IFN were obtained from genetically engineered Escherichia coli as described previously (Souza et al. 1986, Blatt et al. 1996). All other organic solvents were of analytical grade. Ultra-pure water was prepared by a Rios ultra-pure water system (Millipore).
Synthesis and characterization of FMP-mPEG Five g (1 mmol) mPEG5000 were dissolved in 120 ml toluene and dried, by removing the water/ toluene azeotrope at 140 C, and then cooled to room temperature. About 0.56 g (2 mmol) FMP was added to the toluene solution with stirring. The reaction pH was maintained at 10 by adding 0.28 ml (2 mmol) triethylamine (TEA) within 10 min. After standing overnight at room temperature, the reaction mixture was concentrated under vacuum to about 10 ml, filtered, and then added dropwise into 100 ml cold diethyl ether (0 C) with stirring. The resulting precipitate was collected by filtration and then crystallized twice from ethyl acetate. The activity degree of mPEG was 98% calculated from the absorbance at 297 nm in alkaline. IR (KBr): 3325 (s, tC–H, aryl), 2928 (s, tC–H, CH3), 2850 (s, tC–H, CH2CH2O), 1626 (s, tC=C, pyridinium), 1574 (s, tC=C, toluene); 1H NMR (400 MHz in CDCl3 (ppm)): d2.33 (s, 3H, CH3-toluene), d3.38 (s, 3H, CH3O ), d3.64 (br s, nH, CH2CH2O), d4.23 (s, 3H, CH3-pyridinium), d7.12 (d, 2H, toluene), d7.47 (t, 2H, toluene), d7.76 (dd, 2H, pyridinium), d8.37 (t, 1H, pyridinium), d8.87 (d, 1H, pyridinium).
Covalent attachment of polyethylene glycol to proteins RhG-CSF and C-IFN were dialyzed overnight against 100 mM borate buffer (pH 7.6). FMPmPEG was added with stirring to 10 ml of the corresponding protein solution (2 mg ml)1) and the final molar ratio of FMP-mPEG to protein was about 2:1. The pH was maintained at 7.6 with 1 M NaOH. The reaction was carried out at 4 C for 1 h, and then 2 g glycine was added to the solution to quench the reaction. Excess free mPEG was removed by dialysis against 20 mM phosphate saline buffer (pH 7.3), using Amicon ultrafiltration membrane XM-10 (Millipore). Analysis of the PEGylated proteins A Superdex 75 (HR 10/30) size-exclusion column (GE Healthcare Biosciences) connected to an A¨KTA Explorer100 liquid chromatography system (GE Healthcare Biosciences) was used to analyze the heterogeneity of the PEGylated rhG-CSF and C-IFN.
215 Identification of the molecular weight of the PEGylated proteins The molecular weights of the PEGylated proteins were identified by matrix assisted laser desorption/ ionization-time of flight mass spectrometry (MALDI-TOF MS). In vitro bioactivity assay
Scheme 1. The synthetic route of 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP) activated methoxypolyethylene glycol (mPEG) and its attachment to protein through amino groups. In the presence of a slight excess of triethylamine and in dry polar organic solvents such as acetonitrile or tetrahydrofuran, FMP rapidly reacts with the hydroxyl group of mPEG at room temperature to form 2-alkoxyl-1-methylpyridinium salts, which react readily with the terminal amino group of a protein under mild alkaline aqueous conditions (pH 7–9).
The PEGylated rhG-CSF and C-IFN were purified by ion-exchange chromatography as described elsewhere (Seely & Richey 2001). The in vitro activity of the PEGylated rhG-CSF was determined by cell proliferation assay with the G-CSF dependent cell line, murine myeloid leukemia cells (NFS-60) (Naoki et al. 1989). The antiviral assay of PEGylated C-IFN for cytopathic effect was carried out in 96-well microtitre plate seeded with human foreskin fibroblast cells infected with encephalomyocarditis (EMC) virus as previously described (Forti et al. 1986).
Results and discussion Synthesis and characterization of new mPEG derivative The synthetic route of 2-fluoro-1-methylpyridinium toluene-4-sulfonate activated methoxypolyethylene glycol (FMP-mPEG) was shown in Scheme 1. The synthesized FMP-mPEG was characterized by IR and 1H-NMR, and the data demonstrated that the designed structure was obtained by this experimental procedure. Heterogeneity characterization of PEGylated proteins
Fig. 1. Analysis of the conjugates by high performance sizeexclusion chromatography. Superdex 75 (HR 10/30) column was equilibrated by 20 mM phosphate/saline buffer, and 40 ll samples were loaded. The flow rate was 1 ml min)1 and the elution profile was monitored at 280 nm. (a) rhG-CSF; (b) PEGylated rhG-CSF; (c) C-IFN; (d) PEGylated C-INF.
Size-exclusion chromatography was used to analyze the PEGylated rhG-CSF and C-IFN. The native proteins, rhG-CSF and C-IFN, were used as controls (shown in Figures 1a, c), while the elution profiles of the reaction mixture of FMPmPEG and rhG-CSF or C-IFN were shown in Figures 1b, d, where the first peaks corresponded to PEGylated rhG-CSF and PEGylated C-IFN, respectively. The retarded mobility of PEGylated proteins (resulting in higher molecular weight estimates) on SDS-PAGE gels and high perfor-
216
Fig. 2. MALDI-TOF MS of the PEGylated proteins. The assay was performed on Voyager–Elite mass spectrometer (Perseptive Biosystem) with delayed extraction. The samples were irradiated by nitrogen laser at 337 nm and 4-hydroxy-a-cyano-cinnamic acid was used as matrix (5 mg ml)1 in 70% acetonitrile/water solution). (a) The reaction mixture of intact recombinant human granulocyte colony stimulating factor (rhG-CSF) and PEGylated rhG-CSF; (b) the reaction mixture of intact C-INF and PEGylated C-IFN.
mance size-exclusion chromatography has been previously discussed elsewhere (Snider et al. 1992). Therefore, the accurate molecular weight of PEGylated rhG-CSF (23.6 kDa) and PEGylated C-IFN (24.6 kDa) were confirmed by MALDITOF MS (Figures 2a, b) and compared to native rhG-CSF (18.4 kDa) or C-IFN (19.4 kDa). The
two homogenous mono-PEGylated proteins were, thus, successfully prepared by FMP-mPEG. In vitro biological activity assay The in vitro cell proliferation activity of the purified mono-PEGylated rhG-CSF and the antiviral
217 Table 1. The comparison of in vitro bioactivities of mono-PEGylated proteins and responding native proteins. Intact rhG-CSFa c
)1
Specific bioactivity (ml ) Conserved bioactivityd (%)
1.33 · 10 100
7
Mono-PEGylated rhG-CSF 7
1.19 · 10 90
Intact C-IFNb 9
4.21 · 10 100
Mono-PEGylated C-IFN 3.71 · 109 88
a
rhG-CSF: recombinant human granulocyte-colony stimulating factor. C-IFN: consensus interferon. c The in vitro cell proliferation activities of native rhG-CSF and purified mono-PEGylated rhG-CSF were examined by methylthiazoletetrazolium (MTT) assay using murine myeloid leukemia cells (NFS-60). The in vitro antiviral activities of native CIFN and purified mono-PEGylated C-IFN were examined by using human foreskin fibroblast cells (FS-71) infected with encephalomyocarditis (EMC) virus. d The native rhG-CSF and C-IFN were taken as controls, respectively. The conserved bioactivities of mono-PEGylated rhG-CSF and mono-PEGylated C-IFN were evaluated by comparing the specific bioactivity of purified PEGylated proteins and responding native proteins. b
activity of purified mono-PEGylated C-IFN were assayed. The native rhG-CSF and C-INF were taken as controls, and the results are shown in Table 1. About 90% of cell proliferation activities and 88% of antiviral activities were maintained after mono-PEGylation. Although the new synthesized FMP-mPEG was demonstrated to be a promising mPEG derivative for preparing homogeneous monoPEGylated proteins with high level of bioactivity in present studies, the further investigations, such as the physical and thermal stability, susceptibility to enzymatic degradation and in vivo circulating half-life of PEGylated proteins should be performed to develop successful therapeutic biodrugs. Acknowledgements The authors are thankful for the financial support from the National Nature Science Foundation of China (Contract Nos. 20125616 and 20136020).
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