Abstract
We evaluated the utility of capillary electrophoretic methods for analyzing poly(ethylene glycol) (PEG)-modified granulocyte-colony stimulating factor (G-CSF), a long-acting form of GCSF for the treatment of cancer therapy-induced neutropenia. Low- and high-molecularweight 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 electrophoresis (CZE), and sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE). HPSEC 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 optimization of the PEGylation reaction, and purity and stability tests of PEG-G-CSF.
Similar content being viewed by others
References
Buchsel, P. C., Forgey, A., Grape, F. B., and Hamann, S. S., Granulocyte macrophage colony-stimulating factor: current practice and novel approaches. Clin. J. Oncol. Nurs., 6, 198–205 (2002).
Bullock, J., Chowdhury, S., and Johnston, D., Characterization of poly(ethylene glycol)-modified superoxide dismutase: comparison of capillary electrophoresis and matrixassisted 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 conjugates. 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. Anticancer Drugs, 14, 259–264 (2003).
Na, D. H. and Lee, K. C., Capillary electrophoretic characterization of PEGylated human parathyroid hormone with matrix-assisted laser desorption/ionization time-off-light 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 Biotechnology. 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 polyethylene 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 recombinant 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 positional 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).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Lee, K.S., Na, D.H. Capillary electrophoretic separation of poly(ethylene glycol)-modified granulocyte-colony stimulating factor. Arch. Pharm. Res. 33, 491–495 (2010). https://doi.org/10.1007/s12272-010-0320-4
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12272-010-0320-4