Biophysical characterization of Met-G-CSF: effects of different site-specific mono-pegylations on protein stability and aggregation.
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), intrinsic fluorescence spectroscopies and dynamic light scattering (DLS). Interestingly, the two pegylation strategies were found to drastically reduce the polydispersity of Met-G-CSF, when incubated under conditions favouring protein aggregation, as indicated by DLS measurements. Our in vitro results are in agreement with preclinical studies, underlining that preliminary biophysical analyses, performed in the early stages of the development of new biopharmaceutical variants, might offer a useful tool for the identification of protein variants with improved therapeutic values.
Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milan, Italy.
Antonino
A
Natalello
Diletta
D
Ami
Maddalena
M
Collini
Laura
L
D'Alfonso
Giuseppe
G
Chirico
Giancarlo
G
Tonon
Silvia
S
Scaramuzza
Rodolfo
R
Schrepfer
Silvia Maria
SM
Doglia
D001703:Biophysics / Q000379:methods*; D002942:Circular Dichroism; D016179:Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D006801:Humans; D008027:Light; D008715:Methionine / Q000737:chemistry*; D015233:Models, Statistical; D011487:Protein Conformation; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence / Q000379:methods; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D013696:Temperature
D011994:Recombinant Proteins; D016179:Granulocyte Colony-Stimulating Factor; D008715:Methionine