biophysical characterization of met-g-csf effects of different site-specific mono-pegylations on protein stability and aggregation生物物理表征met-g-csf不同特定站点mono-pegylations对蛋白质稳定性的影响和聚合.pdf

Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation 1,2 1,2 3 3 3 Antonino Natalello , Diletta Ami *, Maddalena Collini , Laura D’Alfonso , Giuseppe Chirico , 4 5 5 1,2 Giancarlo Tonon , Silvia Scaramuzza , Rodolfo Schrepfer , Silvia Maria Doglia 1 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy, 2 Consorzio Nazionale Interuniversitario per le Scienze Fisiche della Materia (CNISM), UdR Milano-Bicocca, Milan, Italy, 3 Department of Physics, University of Milano-Bicocca, Milan, Italy, 4 IRCSS Multimedica, Milan, Italy, 5 Bio-Ker srl c/o Sardinia Scientific and Technological Park, Pula, Italy Abstract The limited stability of proteins in vitro and in vivo reduces their conversion into effective biopharmaceuticals. To overcome this problem several strategies can be exploited, as the conjugation of the protein of interest with polyethylene glycol, in most cases, improves its stability and pharmacokinetics. In this work, we report a biophysical characterization of the non- pegylated and of two different site-specific mono-pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (Met-G-CSF), a protein used in chemotherapy and bone marrow transplantation. In particular, we found that the two mono-pegylations of Met-G-CSF at the N-terminal methionine and at glutamine 135 increase the protein thermal stability, reduce the aggregation propensity, preventing also protein precipitation, as revealed by circular dichroism (CD), Fourier transform infrared (FTIR),