Biophysical Characterization of Met-G-CSF: Effects of Different Site-Specific Mono-Pegylations on Protein Stability and Aggregation

Dafydd Jones,Antonino Natalello,Diletta Ami,Giancarlo Tonon,Maddalena Collini,Rodolfo Schrepfer,Laura D’Alfonso,Giuseppe Chirico,Silvia Scaramuzza,Silvia Maria Doglia

doi:10.1371/journal.pone.0042511

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), 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.

Highlights

The advent of recombinant DNA technologies allowed the production of a large number of proteins with potential applications as therapeutic drugs
We report a biophysical characterization of the non-pegylated and of two different site-specific pegylated forms of recombinant human methionyl-granulocyte colony stimulating factor (G-CSF), known as Filgrastim, a protein of 18,799 Dalton belonging to the family of hematopoietic cytokines
The selectivity of enzymatic pegylation is demonstrated by the fact that Met-G-CSF-Gln135-polyethylene glycol (PEG), obtained with more than 90% yields, is mono-pegylated only on the glutamine residue 135 over the seventeen glutamine residues of Met-G-CSF

Summary

Introduction

The advent of recombinant DNA technologies allowed the production of a large number of proteins with potential applications as therapeutic drugs. Proteins can be degraded in vivo by cellular proteases and/or rapidly excreted by kidneys, leading to a short circulating half-life that reduces their therapeutic efficacy [1]. To overcome these problems several strategies have been explored, as the use of protectants to increase the protein storage stability, the sitespecific mutations to reduce the aggregation propensity and enzymatic proteolysis, and the incorporation of the protein within delivery vehicles. PEG is recognized as a nontoxic and non-immunogenic polymer, approved by regulatory authorities for modification of biopharmaceuticals It should be, noted that the final effects of pegylation will depend on the polymer characteristics (size and shape), the conjugation chemistry and the pegylation site, which, for instance, should be distant from the binding site of the protein with its target

Methods

Results

Discussion

Conclusion