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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

Authors
Son, Jin PubJun, Seoung-WookChoi, Yun-KyuPark, Hyoung SeoSon, Mi KyoungLee, Mee YongKang, Soo HyoungKang, Jung SeokPark, Young In
Issue Date
15-4월-2012
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Keywords
Recombinant human granulocyte colony-stimulating factor (rhG-CSF); Trimer-structured mPEG-NHS; PEGylation; Individual positional isomers; In vitro biological activity
Citation
ANALYTICAL BIOCHEMISTRY, v.423, no.2, pp.286 - 293
Indexed
SCIE
SCOPUS
Journal Title
ANALYTICAL BIOCHEMISTRY
Volume
423
Number
2
Start Page
286
End Page
293
URI
https://scholar.korea.ac.kr/handle/2021.sw.korea/108729
DOI
10.1016/j.ab.2011.12.014
ISSN
0003-2697
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 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. (C) 2012 Elsevier Inc. All rights reserved.
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