Poly(ethylene glycol) conjugation of G-CSF using cleavable oligo-lactic acid spacer

Abstract

A new class of mPEG derivatives having cleavable oligo lactic acid was synthesized by ring opening polymerization of L-lactide using a terminal hydroxyl end of PEG as an initiator. The synthesized mPEG derivatives had 0.8 and 3.6 lactic acid units based on the MALDI-TOF data. A terminal end group of mPEG-OLA was further activated with p-nitrophenyl chloroformate to produce mPEG-OLA-p-nitrophenyl carbonate (PC). This mPEG-OLA-PC can conjugate to primary amine groups in proteins or drugs. In mPEG-OLA-fluorescein model system, the OLA spacer was cleaved over 40% by 12 h and most fluorescein moieties were detached from mPEG by 84 h. G-CSF was pegylated with this activated mPEG-OLA-PC. Pegylated G-CSF was consisted of native, mono-, di-, and tri-pegylated species. When incubating in pH 4 buffer at 4℃ for 2 weeks, mPEG-OLA-G-CSF liberated mPEG by cleaving OLA spacer, resulting in the reduced fraction of multi-pegylated G-CSF with concomitantly increasing the fraction of native G-CSF. In vitro NFS60 cell proliferation assay revealed that mPEG-OLA-G-CSF retained similar activity to native G-CSF.