A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: Production and physicochemical characterization

作者：Scaramuzza Silvia^*; Tonon Giancarlo; Olianas Alessandra; Messana Irene; Schrepfer Rodolfo; Orsini Gaetano; Caliceti Paolo

来源：Journal of Controlled Release, 2012, 164(3): 355-363.

DOI：10.1016/j.jconrel.2012.06.026

• Filgrastim
• G-CSF
• PEG
• Transglutaminase PEGylation
• rh-Met-G-CSF-Q(135)-PEG20kDa
• BK0026

We describe the preparation and characterization of a new monoPEGylated derivate of a recombinant form of filgrastim (methionyl human granulocite colony stimulating factor, rh-Met-G-CSF), BK0026, prepared by enzymatic site-specific 20 kDa PEG conjugation to glutamine 135 residue by microbial transglutaminase catalyzed reaction. BK0026 was purified to a clinical grade by a single cation exchange chromatography step and characterized by using a panel of physicochemical analyses. NH2-terminal sequence and peptidemapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim. The circular dichroism and fluorescence spectroscopy showed the preservation of high order protein structure. The single conjugation site on glutamine 135 was identified by endoproteinase Glu-C peptide mapping combined with mass spectrometry analysis and NH2-terminal sequence of the PEGylated peptides. BK0026 purity as well as product- and process-related contaminants was determined by several analytical methods, which showed that BK0026 is stable for more than 2 years when stored at 4-8 degrees C. The advantages of enzymatic PEGylation of filgrastim are the absolute specificity of glutamine 135 conjugation combined with high PEGylation yields under very mild reaction conditions. The new site specific monoPEGylated filgrastim is a promising candidate for preclinical and clinical studies aimed at developing a long-lasting treatment of neutropenia in oncological patients under chemotherapy treatments.

• 出版日期2012-12-28