title: 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.
abstract: 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.
authors.affiliation: College of Life Sciences and Biotechnology, Korea University, Anam-dong, Seoungbuk-gu, Seoul 136-701, Republic of Korea.
authors.forename: Jin Pub
authors.initials: JP
authors.lastname: Son
authors.forename: Seoung-Wook
authors.initials: SW
authors.lastname: Jun
authors.forename: Yun-Kyu
authors.initials: YK
authors.lastname: Choi
authors.forename: Hyoung Seo
authors.initials: HS
authors.lastname: Park
authors.forename: Mi Kyoung
authors.initials: MK
authors.lastname: Son
authors.forename: Mee Yong
authors.initials: MY
authors.lastname: Lee
authors.forename: Soo Hyoung
authors.initials: SH
authors.lastname: Kang
authors.forename: Jung Seok
authors.initials: JS
authors.lastname: Kang
authors.forename: Young In
authors.initials: YI
authors.lastname: Park
mesh_terms: D000595:Amino Acid Sequence; D001681:Biological Assay; D020134:Catalytic Domain; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007536:Isomerism; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D055503:Protein Multimerization; D020539:Sequence Analysis, Protein; D013388:Succinimides / Q000737:chemistry*
chemical_list: D013388:Succinimides; C486981:pegylated granulocyte colony-stimulating factor, human; D016179:Granulocyte Colony-Stimulating Factor; D011092:Polyethylene Glycols; C001426:N-hydroxysuccinimide