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Yongin si, South Korea

Son J.P.,Korea University | Jun S.-W.,Dong A Pharmaceutical | Choi Y.-K.,Dong A Pharmaceutical | Park H.S.,Dong A Pharmaceutical | And 5 more authors.
Analytical Biochemistry | Year: 2012

The individual positional isomers from the mono-PEGylated recombinant human granulocyte colony-stimulating factor (rhG-CSF) were successfully isolated with additional strong cation exchange chromatography using Source 15S. The three isolated individual positional isomers were found to be homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical size exclusion high-performance liquid chromatography (SE-HPLC), and analytical cation exchange HPLC (CIE-HPLC) and were also characterized with respect to site of PEGylation by enzymatic digestion with endoproteinase Lys-C and N-terminal sequencing. In addition, in vitro biological activity was determined by cell proliferation assay. It was determined that the three isolated individual positional isomers were PEGylated at Lys 35, Met N-terminal, and Lys 17 of the rhG-CSF molecule with a 23-kDa trimer-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group (mPEG-NHS). All individual positional isomers (Lys 35-PEGylated rhG-CSF, Met N-terminal-PEGylated rhG-CSF, and Lys 17-PEGylated rhG-CSF) retained in vitro biological activity and were found to be 18.5%, 37.6%, and 7.1%, respectively, compared with the rhG-CSF molecule. The significantly different in vitro biological activities observed in the individual positional isomers could be presumably due to interference of receptor binding or active sites on the rhG-CSF molecule. In conclusion, the individual positional isomers isolated from the mono-PEGylated rhG-CSF were well characterized with respect to the site of PEGylation involving Lys 35, Met N-terminal, and Lys 17. This characterization of the individual positional isomers would be critical to provide a basis for establishing consistency in the manufacturing process. © 2012 Elsevier Inc. All rights reserved.

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