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Optimization of PEGylation reaction time and molar ratio of rhG-CSF toward increasing bioactive potency of monoPEGylated protein
- Source :
- International Journal of Biological Macromolecules, International Journal of Biological Macromolecules, Elsevier, 2018, 109, pp.888--895. ⟨10.1016/j.ijbiomac.2017.11.070⟩
- Publication Year :
- 2018
- Publisher :
- HAL CCSD, 2018.
-
Abstract
- International audience; PEGylation is one of the strategies used for enhancing in vivo residence time of recombinant human Granulocyte Colony-Stimulating Factor (rhG-CSF) and therefore reducing in dose frequency to better fit with patient comfort treatment. In this study, three methoxy polyethylene glycol propionaldehydes (mPEG- ALD) of 10, 20 and 30kDa MW were utilized to produce biologically active monoPEGylated rhG-CSF with enhanced molecular weight. PEGylation reactions were carried out at room temperature and pH 5.0 in the presence of cyanoborohydride and two mPEG-ALD: protein molar ratios (3:1 and 5:1). The reactions were monitored with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion chromatography (SEC-HPLC). The results showed that a 2h reaction with 5:1 mPEG-ALD: protein molar ratio was sufficient to direct the reaction toward optimal yields of monoPEGylated protein (86%). Subsequently, isolation of the monoPEGylated forms was successfully achieved. The purified products were compared with respect to their purity (≥95%), identity and isoelectric focusing parameter characteristics. Biological potencies were measured by cell proliferation assay and showed 20.80-42.73% retention of bioactivities. This study highlights the possible improvement of rhG-CSF efficiency by PEGylation. Further studies will investigate in vitro and in vivo immunogenicity and toxicity of monoPEGylated conjugates.
- Subjects :
- 0106 biological sciences
0301 basic medicine
Characterization
Size-exclusion chromatography
Polyethylene glycol
01 natural sciences
Biochemistry
Cell Line
Polyethylene Glycols
03 medical and health sciences
chemistry.chemical_compound
Mice
Structure-Activity Relationship
Structural Biology
In vivo
010608 biotechnology
Granulocyte Colony-Stimulating Factor
Animals
Humans
Sodium dodecyl sulfate
Molecular Biology
Polyacrylamide gel electrophoresis
Chromatography, High Pressure Liquid
Cell Proliferation
Chromatography
Isoelectric focusing
Biological activity
PEGylation
General Medicine
Purity
Chromatography, Ion Exchange
Recombinant Proteins
Molecular Weight
030104 developmental biology
chemistry
[SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Conjugate
rhG-CSF
Subjects
Details
- Language :
- English
- ISSN :
- 01418130
- Database :
- OpenAIRE
- Journal :
- International Journal of Biological Macromolecules, International Journal of Biological Macromolecules, Elsevier, 2018, 109, pp.888--895. ⟨10.1016/j.ijbiomac.2017.11.070⟩
- Accession number :
- edsair.doi.dedup.....eca4c99b790b75f3044997d6e0263274