Long-acting Recombinant Human Granulocyte Colony Stimulating Factor (rhG-CSF) with a Trimer-Structured Polyethylene Glycol

Abstract

ABSTRACT − Mono PEGylated rhG-CSF (PEG-G-CSF) prepared by utilizing unique PEG was purified and characterizedby cation-exchange chromatography. A unique, trimer-structured PEG was chosen for PEGylation of rhG-CSF among var-ious PEG moieties. The in-vitro bioactivity, stability, and pharmacokinetics of mono-PEG-G-CSF were examined and com-pared to those of native rhG-CSF. Mono PEG-G-CSF exhibited reduced in-vitro bioactivity to native rhG-CSF but showedan excellent in-vivo bioactivity and stability. Furthermore, it showed markedly reduced clearance in rats, thereby increasingthe biological half-life by about 4.5-fold compared to that of native rhG-CSF. The results suggest that this unique, trimer-structured 23 kDa PEG can provide advantages to improve the bioactivity of therapeutic proteins in clinical use.Key words − G-CSF, PEG, Structure, Characteristics Granulocyte colony-stimulating factor (G-CSF) is a growthfactor that serves as a major regulator of the proliferation anddifferentiation of neutrophilic granulocytes. Recombinanthuman G-CSF (rhG-CSF) produced in E. coli is a 175-residueprotein that folds into a 4 helical bundle, typical of a cytokine(Hill et al., 1993; Manavalan et al., 1992; Souza et al., 1986).It has been generally known that the proteins such as rhG-CSF have short life in the body due to their rapid clearance(Bronchud et al., 1988). The short half-life of a recombinantprotein such as rhG-CSF can be increased by covalent mod-ification with the polyethylene glycol (PEG), in a proceduretermed ‘PEGylation’ (Molineux, 2002; Yowell and Blackwell,2002). Covalent modification of therapeutic proteins with PEGcan result in increased serum half life due to decreased renalclearance (Crawford, 2002). PEGylation is known to improvethe physicochemical properties of protein stability, increasedsolubility and decreased immunogenicity compared to theirparent molecule by increasing protein molecular size andshielding the metabolic sites (Rajan et al., 2006; Lee et al.,2005; Yang et al., 2004; Tsuji et al., 1985). Due to these advan-tages, PEGylated protein therapeutics can enhance therapeuticefficacy and reduce undesirable effects.The increasing size of the attached PEG, however, mayresult in loss of bioactivity (Bowen et al., 1999). Therefore, thesuccessful development of a PEGylated therapeutic proteinrequires an optimized balance between enhanced pharmaco-kinetics and reduced bioactivity by the judicious selection ofPEG size. In addition, PEG structure also plays a crucial rolein bioactivity and pharmacokinetic behavior reported that lin-ear PEG was distributed with a larger distribution volume,whereas branched PEG was distributed with a smaller dis-tribution volume (Caliceti, 2004). These reports showed theadvantages of branched PEG in the therapeutic use of PEGy-lated proteins by minimizing the loss of in-vitro bioactivity andmaximizing the blood residual time. Therefore, the properPEGylation, the selection of size and shape for PEG is one ofthe most important points. In this study, a unique, trimer-structured, 23 kDa PEG wasconjugated to rhG-CSF by forming an amide bond to improvethe pharmacokinetic properties and minimize the loss of nativeprotein bioactivity. in-vitro bioactivity, in-vivo stability, andpharmacokinetics of 23 kDa mono PEG-G-CSF prepared withthis PEG molecule were assessed and compared to those of