PEG-GCSF的毛细管电泳分离

Arch Pharm Res Vol 33, No 3, 491-495, 2010 DOI 10.1007/s12272-010-0320-4 491 Capillary Electrophoretic Separation of Poly(ethylene glycol)- Modified Granulocyte-Colony Stimulating Factor Kyung Soo Lee and Dong Hee Na College of Pharmacy, Kyungsung University, Busan 608-736, Korea (Received November 17, 2009/Revised December 23, 2009/Accepted January 18, 2010) We evaluated the utility of capillary electrophoretic methods for analyzing poly(ethylene gly- col) (PEG)-modified granulocyte-colony stimulating factor (G-CSF), a long-acting form of G- CSF for the treatment of cancer therapy-induced neutropenia. Low- and high-molecular- weight PEG-G-CSF conjugates prepared with aldehyde-activated PEG-5K and PEG-20K were separated by high-performance size-exclusion chromatography (HP-SEC), capillary zone elec- trophoresis (CZE), and sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE). HP- SEC showed low resolution for separating mono- and di-PEG-G-CSFs. SDS-CGE had higher resolution, but required a long analysis and had low peak efficiency. CZE could successfully separate both PEG-5K- and PEG-20K-conjugated G-CSFs with a running time of 20 min and high peak efficiency. In conclusion, CZE was better than SDS-CGE for separating PEG-G-CSF conjugates and will be useful for PEGylation studies, such as reaction monitoring for optimi- zation of the PEGylation reaction, and purity and stability tests of PEG-G-CSF. Key words: PEGylation, Granulocyte-colony stimulating factor, Capillary electrophoresis INTRODUCTION Granulocyte-colony stimulating factor (G-CSF) is a growth factor that regulates proliferation and differ- entiation of neutrophilic granulocytes. Recombinant human G-CSF is marketed by Amgen as Neupogen® (Filgrastim), and is widely used to treat neutropenia induced by myelosuppressive chemotherapy (Buchsel et al., 2002). However, the clinical use of Neupogen is limited due to a short circulation half-life (3.5~3.8 h) that requires a daily injection (Frampton et al., 1994). The covalent modification of protein with poly(ethy- lene glycol) (PEG), termed PEGylation, is useful for increasing the circulation half-life and decreasing the dosing frequency (Kang et al., 2009). Neulasta® (pegfi- lgrastim), also developed by Amgen, is PEGylated G- CSF produced by the attachment of a 20 kDa mono- methoxy PEG-aldehyde to the N-terminal amine of G- CSF (Piedmonte and Treuheit, 2008). This modifica- tion significantly increases circulation half-life and only needs to be dosed once per chemotherapy cycle, as opposed to repeated daily injection for G-CSF (Kinstler et al., 2002; Molineux, 2003). PEGylated proteins are among the most challenging products in pharmaceutical analysis, because the PEGylation process results in molecular heterogeneity in terms of the number and positions of attached PEG molecules (Park and Na, 2008; Park et al., 2009). As these heterogeneities may confer different biological properties, the development of analytical methods for PEGylated proteins is becoming more important (Fee and Van Alstine, 2006). Capillary electrophoresis (CE) shows high separation capacity for proteins and their conjugates (Na and Lee, 2007). Several investigators have used capillary zone electrophoresis (CZE) for the analysis of several PEGylated forms of proteins, such as superoxide dismutase, lysozyme, ribonuclease A, and human parathyroid hormone (1-34) (Bullock et al., 1996; Roberts and Harris, 1998; Li et al., 2001; Na and Lee, 2004). Recently, we reported the use of sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using a hydrophilic replaceable polymer network matrix for the separation of PEGylated inter- ferons (Na et al., 2004, 2008). Correspondence to: Dong Hee Na, College of Pharmacy, Kyung- sung University, Busan 608-736, Korea Tel: 82-51-663-4881, Fax: 82-51-663-4809 E-mail: dhna2@ks.ac.kr 492 K. S. Lee and D. H. Na Here, we used both CZE and SDS-CGE methods for the analysis of PEGylated G-CSF produced by a reac- tion with aldehyde-activated PEG. PEGylated G-CSFs were prepared with low (5 kDa)- and high (20 kDa)- molecular-weight-PEGs and the two methods were compared based on analysis time, peak efficiency, and resolution. MATERIALS AND METHODS Materials and reagents Recombinant human granulocyte-colony stimulating factor (G-CSF) (MW 18,800) was a gift from Dong-A Pharmaceutical Co. Ltd. Monomethoxy PEG-butyral- dehydes (mPEG-ALD, MW 5 or 20 kDa) were pur- chased from Nektar Therapeutics. A micro BCA pro- tein assay kit was obtained from Pierce. Capillary electrophoresis buffer (0.1 M phosphate buffer, pH 2.5) and sodium cyanoborohydride were obtained from Sigma. All other chemicals were of analytical grade and were used as obtained commercially. PEGylation of G-CSF The PEGylation of G-CSF was performed with mPEG-ALD (MW 5 or 20 kDa) in the presence of sodium cyanoborohydride (NaCNBH3) at pH 5, as described previously (Kinstler et al., 2002; Na et al., 2006). Briefly, PEG solutions were prepared by adding 4.4 mg of mPEG-ALD-5K (MW 5 kDa) or 17.6 mg of mPEG-ALD-20K (MW 20,000) to 1 mL of 0.1 M sodium acetate buffer (pH 5.0) containing 40 mM NaCNBH3. The PEG solutions were added to 1 mL of G-CSF solution (3.3 mg/mL in 0.1 M sodium acetate buffer, pH 5.0) at a molar ratio of G-CSF:mPEG-ALD=1:5, and the reaction was continued for 18 h at 4oC. The mono- PEGylated G-CSF (mono-PEG-G-CSF) was isolated by size-exclusion chromatography on a Superose 12 10/300 GL column (Amersham Biosciences) with 10 mM sodium phosphate buffer saline (PBS) (pH 7.4) as the mobile phase. The flow rate was 0.4 mL/min and the UV absorbance was monitored at 215 nm. The purified mono-PEG-G-CSF was concentrated with Amicon centricon 10 (molecular weight cut-off 10 kDa, Millipore) and the protein concentration was deter- mined by the micro BCA assay (Smith et al., 1985). High-performance size-exclusion chromato- graphy (HP-SEC) Analytical HP-SEC was performed using a Dionex HPLC system (Dionex Co.) consisted of a quaternary gradient pump with an on-line vacuum degasser (Model P680A), an automated sample injector (Model ASI-100), thermostatic column compartment (Model TCC-100), and 4-channel multi UV-Vis detector (Model 170U). Separations were performed on a Shodex Protein KW 802.5 column (8.0 mm i.d. ×300 mm, silica particle 5 µm, Showa Denko) using 10 mM sodium phosphate buffer (pH 7.4) as a mobile phase. Sample injection volume was 20 µL and the flow rate was 1 mL/min. UV absorbance was monitored at 215 nm. Capillary zone electrophoresis (CZE) CE experiments were performed on a P/ACETM MDQ capillary electrophoresis system (Beckman Coulter Inc.). CZE was performed with a BioCap bare silica capillary, 75 µm i.d., 32 cm total length, and 22 cm to the detector (Bio-Rad). Separation of each sample was performed in 100 mM phosphate buffer (pH 2.5) as the electrolyte for 20 min, and the UV absorbance was measured at 214 nm. The capillary was rinsed with 0.1 M NaOH, deionized water and 100 mM phosphate buffer (pH 2.5) for 120, 120, and 180 sec, respectively, prior to each injection. Samples were loaded by applying a nitrogen pressure of 0.5 psi for 5 sec, and the voltage across the ends of the capillary was set at 10 kV. The temperature of the capillary and samples was maintained at 20oC by a liquid cooling system. SDS-capillary gel electrophoresis (SDS-CGE) SDS-CGE was performed with an eCAP SDS 14-200 Kit using a coated capillary with 100 µm i.d., 30 cm total length, and 20 cm to the detection window (Beckman Coulter). Prior to each sample injection, the capillary was rinsed with 1 M HCl and gel buffer for 60 and 180 sec, respectively. Two additional purges for 0 sec with a mixture of sample buffer (0.12 M Tris/ HCl/1% SDS) and deionized water (1:1) was perform- ed to wash any residual running buffer from the outlet surface of the capillary prior to sample injection. The samples for CE analysis were prepared by mixing 100 mL of the sample with 100 mL of sample buffer. Samples were heated at 100oC in a water bath for 10 min and then cooled on ice for 3 min, followed by centrifugation. Samples were injected by applying a nitrogen pressure of 0.5 psi for 30 sec, and the voltage across the ends of the capillary was set at 8-15 kV. UV absorbance was monitored at 214 nm for 20-40 min. The capillary temperature was maintained at 20oC by the liquid cooling system. RESULTS AND DISCUSSION G-CSF has five primary amines in the N-terminus and four lysine residues (Lys17, Lys24, Lys35, and Lys41) that are potential PEGylation sites. Neulasta (pegfilgrastim) is PEGylated G-CSF produced by the Capillary Electrophoresis of PEG-G-CSF 493 attachment of mPEG-ALD-20K to the N-terminal amine of G-CSF. In this study, PEGylation of G-CSF was performed by a similar method with mPEG-ALD- 5K or 20K (Fig. 1). Fig. 2 shows the HP-SEC chromatograms of native G-CSF and PEGylation reaction mixtures between G- CSF and mPEG-ALD-5K or 20K at a molar ratio of G- CSF:PEG=1:5 for 18 h at 4oC. G-CSF was converted to the mono-PEG conjugate and di-PEG conjugate by the reaction with mPEG-ALD. Native G-CSF was detect- ed at a retention time of approximately 8.9 min, and the mono- and di-PEG-5K-G-CSFs were detected at 7.6 and 7.0 min, respectively (Fig. 2B). The mono- and di-PEG-20K-G-CSFs were detected at 6.4 and 6.0 min, respectively (Fig. 2C). The production yields of mono- PEG-G-CSF were 87% with mPEG-ALD-5K and 80.4% with mPEG-ALD-20K. Capillary zone electrophoresis (CZE) was performed with an uncoated silica capillary using 100 mM phos- phate buffer (pH 2.5) as the electrolyte. The standard G-CSF was detected as a single peak at a migration time of 4.3 min (Fig. 3). The calibration curve of G- CSF was obtained from 18 to 1130 µg/mL, with a correlation coefficient greater than 0.998. Fig. 4 shows the CE electropherograms of the PEGylation reaction mixture of PEG-5K-G-CSF and the isolated mono- PEG-5K-G-CSF. As demonstrated previously, the separation in CZE using acidic buffer is dominated by the size of PEGylated protein because the acidic pH produces complete protein protonation and minimizes the charge difference (Na and Lee, 2004). The PEG-G- CSF showed lower electrophoretic mobility because of the increased hydrodynamic radius. The mono- and di-PEG-5K-G-CSFs were detected at migration times of 7.5 and 9.6 min, respectively, with a resolution of 1.62 (Fig. 4A). The migration times of PEG-20K-G- CSF conjugates were significantly prolonged (Fig. 5), with mono- and di-PEG-20K-G-CSFs detected at migration times of 10.3 and 16.4 min, respectively. The isolated mono-PEG-20K-G-CSF had satisfactory purity (Fig. 5B), with mono-PEG-5K- and mono-PEG- 20K-G-CSFs concentrations of 45.5 and 139.9 µg/mL, respectively. SDS-capillary gel electrophoresis (SDS-CGE) can separate PEGylated interferons, with advantages of speed, minimal sample consumption, and higher Fig. 1. Reaction of primary amines of G-CSF with mPEG- ALD in the presence of sodium cyanoborohydride (NaCNBH3) Fig. 3. CZE electropherogram of G-CSF Fig. 2. HP-SEC chromatograms of native G-CSF (A), re- action mixture between G-CSF and mPEG-ALD-5K (B), and reaction mixture between G-CSF and mPEG-ALD-20K (C). Peak 1: unmodified G-CSF, 2: mono-PEG-5K-G-CSF, 3: di- PEG-5K-G-CSF, 4: mono-PEG-20K-G-CSF, and 5: di-PEG- 20K-G-CSF. 494 K. S. Lee and D. H. Na resolution than conventional SDS-PAGE (Na et al., 2004, 2008). Fig. 6 shows the SDS-CGE electropherog- rams of the PEGylation reaction mixture between G- CSF and mPEG-ALD-5K obtained by different voltages across the capillary. As SDS-CGE separates proteins by size, peaks 2 and 3 were assigned to mono- and di- PEG-5K-G-CSF, respectively. As capillary voltage in- creases from 8 to 12 kV, the migration times of every peaks became faster, but the resolutions were not significantly changed. The resolutions of peaks 1-2 and 2-3 at 8 kV were 1.86 and 1.25, respectively, whereas those of peaks 1-2 and 2-3 at 12 kV were 1.87 and 1.30, respectively. Although the recommended voltage is 8 kV, 12 kV showed faster run time with no loss in resolution. The SDS-CGE separation of PEG- 20K-G-CSF conjugates required more time time (Fig. 7): at 12 kV, mono-PEG-20K-G-CSF was detected at 25.0 min and di-PEG-20K-G-CSF was not detected until 40 min. At 15 kV, mono- and di-PEG-20K-G-CSFs were detected at 19.5 and 33.7 min, respectively. Higher voltage caused excessive Joule heat in the capillary. In conclusion, CE methods showed better separation capacity than HP-SEC for the separation of PEG-G- CSFs. The CZE method could separate successfully Fig. 4. CZE electropherograms of reaction mixtures between G-CSF and mPEG-ALD-5K (A), and the isolated mono- PEG-5K-G-CSF (B). Peak 1: unmodified G-CSF, 2: mono- PEG-5K-G-CSF, and 3: di-PEG-5K-G-CSF. Fig. 5. CZE electropherograms of reaction mixtures between G-CSF and mPEG-ALD-20K (A), and the isolated mono- PEG-20K-G-CSF (B). Peak 1: unmodified G-CSF, 2: mono- PEG-20K-G-CSF, and 3: di-PEG-20K-G-CSF. Fig. 6. SDS-CGE electropherograms of reaction mixtures between G-CSF and mPEG-ALD-5K obtained with running voltages of 8 kV (A), 10 kV (B), and 12 kV (C). Peak 1: un- modified G-CSF, 2: mono-PEG-5K-G-CSF, and 3: di-PEG- 5K-G-CSF. Fig. 7. SDS-CGE electropherograms of reaction mixtures between G-CSF and mPEG-ALD-20K obtained with runn- ing voltages of 12 kV (A) and 15 kV (B). Peak 1: unmodified G-CSF, 2: mono-PEG-20K-G-CSF, and 3: di-PEG-20K-G-CSF. Capillary Electrophoresis of PEG-G-CSF 495 both PEG-5K- and PEG-20K-conjugated G-CSFs with a running time of 20 min. The SDS-CGE method was useful for the separation of low-molecular-weight PEG-5K-conjugated G-CSFs, but not for high-mole- cular-weight PEG-20K-conjugated G-CSF because of long migration times and low peak efficiency. The sensitivity of the CZE method was superior to the SDS-CGE method. The CZE method will be useful for studies of G-CSF PEGylation, such as reaction moni- toring for optimization of PEGylation reaction, purity tests, and stability tests. ACKNOWLEDGEMENTS This work was financially supported by the Ministry of Knowledge Economy (MKE) and Korea Institute for Advancement in Technology (KIAT) through the Work- force Development Program in Strategic Technology. REFERENCES Buchsel, P. C., Forgey, A., Grape, F. B., and Hamann, S. S., Granulocyte macrophage colony-stimulating factor: cur- rent practice and novel approaches. Clin. J. Oncol. Nurs., 6, 198-205 (2002). Bullock, J., Chowdhury, S., and Johnston, D., Characteri- zation of poly(ethylene glycol)-modified superoxide dis- mutase: comparison of capillary electrophoresis and matrix- assisted laser desorption/ionization mass spectrometry. Anal. Chem., 68, 3258-3264 (1996). Fee, C. J. and Van Alstine, J. M., PEG-proteins: reaction engineering and separation issues. Chem. Eng. Sci., 61, 924-939 (2006). Frampton, J. E., Lee, C. R., and Faulds, D., Filgrastim. A review of its pharmacological properties and therapeutic efficacy in neutropenia. Drugs, 48, 731-760 (1994). Kang, J. S., Deluca, P. P., and Lee, K. C., Emerging PEGylated drugs. Expert Opin. Emerg. Drugs, 14, 363- 380 (2009). Kinstler, O. B., Brems, D. N., Lauren, S. L., Paige, A. G., Hamburger, J. B., and Treuheit, M. J., Characterization and stability of N-terminally PEGylated rhG-CSF. Pharm. Res., 13, 996-1002 (1996). Kinstler, O., Molineux, G., Treuheit, M., Ladd, D., and Gegg, C., Mono-N-terminal poly(ethylene glycol)-protein con- jugates. Adv. Drug Deliv. Rev., 54, 477-485 (2002). Li, W., Zhong, Y., Lin, B., and Su, Z., Characterization of polyethylene glycol-modified proteins by semi-aqueous capillary electrophoresis. J. Chromatogr. A., 905, 299-307 (2001). Molineux G., Pegfilgrastim: using pegylation technology to improve neutropenia support in cancer patients. Anti- cancer Drugs, 14, 259-264 (2003). Na, D. H. and Lee, K. C., Capillary electrophoretic charac- terization of PEGylated human parathyroid hormone with matrix-assisted laser desorption/ionization time-of- flight mass spectrometry. Anal. Biochem., 331, 322-328 (2004). Na, D. H. and Lee, K. C., Capillary separation techniques, In Gad, S. C. (Ed.). Handbook of Pharmaceutical Biotechno- logy. John Wiley & Sons, Inc., New Jersey, pp. 469-510, (2007). Na, D. H., Park, E. J., Youn, Y. S., Moon, B. W., Jo, Y. W., Lee, S. H., Kim, W. B., Sohn Y., and Lee, K. C., Sodium dodecyl sulfate-capillary gel electrophoresis of polyethy- lene glycolylated interferon alpha. Electrophoresis, 25, 476-479 (2004). Na, D. H., Park, E. J., Jo, Y. W., and Lee, K. C., Capillary electrophoretic separation of high-molecular-weight poly (ethylene glycol)-modified proteins. Anal. Biochem., 373, 207-212 (2008). Na, D. H., Youn, Y. S., Lee, I. B., Park, E. J., Park, C. J., and Lee, K. C., Effect of molecular size of PEGylated recom- binant human epidermal growth factor on the biological activity and stability in rat wound tissue. Pharm. Dev. Technol., 11, 513-519 (2006). Park, E. J., Lee, K. C., and Na, D. H., Separation of posi- tional isomers of mono-poly(ethylene glycol)-modified octreotides by reversed-phase high-performance liquid chromatography. J. Chromatogr. A., 1216, 7793-7797 (2009). Park, E. J. and Na, D. H., Optimization of octreotide PEGylation by monitoring with fast reversed-phase high- performance liquid chromatography. Anal. Biochem., 380, 140-142 (2008). Piedmonte, D. M. and Treuheit, M. J., Formulation of Neulasta (pegfilgrastim). Adv. Drug Deliv. Rev., 60, 50-58 (2008). Roberts, M. J. and Harris, J. M., Attachment of degradable poly(ethylene glycol) to proteins has the potential to increase therapeutic efficacy. J. Pharm. Sci., 87, 1440- 1445 (1998). Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J., and Klenk, D. C., Measurement of protein using bicinchoninic acid. Anal. Biochem., 150, 76- 85 (1985). << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /None /Binding /Left /CalGrayProfile (Gray Gamma 2.2) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Error /CompatibilityLevel 1.3 /CompressObjects /Off /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages false /CreateJDFFile false /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends true /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams true /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveEPSInfo true /PreserveHalftoneInfo false /PreserveOPIComments false /PreserveOverprintSettings true /StartPage 1 /SubsetFonts false /TransferFunctionInfo /Apply /UCRandBGInfo /Remove /UsePrologue false /ColorSettingsFile (Color Management Off) /AlwaysEmbed [ true ] /NeverEmbed [ true ] /AntiAliasColorImages false /DownsampleColorImages true /ColorImageDownsampleType /Bicubic /ColorImageResolution 200 /ColorImageDepth 8 /ColorImageDownsampleThreshold 1.00000 /EncodeColorImages true /ColorImageFilter /FlateEncode /AutoFilterColorImages false /ColorImageAutoFilterStrategy /JPEG /ColorACSImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /ColorImageDict << /QFactor 0.15 /HSamples [1 1 1 1] /VSamples [1 1 1 1] >> /JPEG2000ColorACSImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /JPEG2000ColorImageDict << /TileWidth 256 /TileHeight 256 /Quality 30 >> /AntiAliasGrayImages false /DownsampleGrayImages true /GrayImageDownsampleType /Bicubic /G