Structural identification and biological activity of positional isomers of long-acting and mono-PEGylated recombinant human granulocyte colony-stimulating factor with trimeric-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group

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• 作者：Jin Pub Son^a ; ^{jinpubs@donga.co.kr} ; Seoung-Wook Jun^b ; Yun-Kyu Choi^b ; Hyoung Seo Park^b ; Mi Kyoung Son^b ; Mee Yong Lee^b ; Soo Hyoung Kang^b ; Jung Seok Kang^b ; Young In Park^a ; ^c ; ^{yipark@korea.ac.kr}
• 关键词：Recombinant human granulocyte colony-stimulating factor (rhG-CSF) ; Trimer-structured mPEG-NHS ; PEGylation ; Individual positional isomers ; In vitro biological activity
• 刊名：Analytical Biochemistry
• 出版年：2012
• 期刊代码：93_00032697
• 类别：imm
• 出版时间：15 April, 2012
• 卷：423
• 期：2
• 页码：286-293
• 文件大小：1099 K

摘要

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 Lys³⁵, Met^N-terminal, and Lys¹⁷ of the rhG-CSF molecule with a 23-kDa trimer-structured methoxy polyethylene glycol N-hydroxysuccinimidyl functional group (mPEG-NHS). All individual positional isomers (Lys³⁵-PEGylated rhG-CSF, Met^N-terminal-PEGylated rhG-CSF, and Lys¹⁷-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 Lys³⁵, Met^N-terminal, and Lys¹⁷. This characterization of the individual positional isomers would be critical to provide a basis for establishing consistency in the manufacturing process.