Effect of divalent polyethylene glycol units, conjugated on human granulocyte colony-stimulating factor, on biological activities in vitro and in vivo.

New divalent (two-chain type) polyethylene glycol (PEG) conjugates of a derivative of human recombinant granulocyte colony-stimulating factor (rhG-CSF), ND28, were synthesized by a novel conjugation method using triazine ring and amino butyric acid, and separated into mono-, di- and tri-PEG2(two chains)-ND28 with high purity of more than 90%, to examine the effect of the number of PEG units on their biological properties. Three species of PEG2-ND28 conjugates showed reduced but clear in vitro bioactivity and receptor binding inhibitory activity, and an inverse correlation between the number of PEG units and the activity was seen. On the other hand, the in vivo granulopoietic effect of tri-PEG2-ND28 in mice was observed to be most potent and long-lasting for 6 days after only one administration, and was followed by di-PEG2-ND28 and mono-PEG2-ND28. The plasma concentration of tri-PEG2-ND28 was maintained at a high level for 3 days after administration, while that of PEG-unbound ND28 disappeared within 30 h. There was a positive correlation between the number of PEG units and both the granulopoietic effect and the plasma half-life. These results suggest that the number of PEG units attached to the rhG-CSF can increase their stability during circulation in the plasma of mice, in turn resulting in a long-lasting granulopoietic effect in vivo.