Biophysical characterization and applications of PEGylated proteins
Reza Hosseinzadeh*
Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran
Objective: To investigate the characteristics of PEGylated proteins and potential applications
of these proteins. Introduction: PEGylation (approach that a activated PEG binds to the free
amino group situated in the amino acid side chains of the proteins particularly lysine) is a
strategy that has been used to improve the biophysical and biochemical properties of proteins and
also is a clinically proven strategy for increasing the therapeutic efficacy of protein-based
medicines. PEGylation of pharmaceutical proteins is an established method for prolonging the
circulatory half-life of proteins, reducing self-aggregation, increasing water solubility and
decreasing antibody recognition, and it has been used successfully in several marketed proteins.
Methods: Model protein bovine serum albumin(BSA), was considered for our study. The stable
linkage between the proteins and PEG was made by using intermediate heterocyclic compounds
such as cyanuric chloride, succinimide and any other intermediates which allow a reaction
between PEG and proteins in mild conditions of pH and temperature. PEGylated proteins were
characterized using various spectroscopic and thermal methods such as fluorescence, CD, UVVis and DLS techniques. DSC and ITC also were used for protein stability and interactions
studies. Results: The secondary and tertiary structure of BSA measured by CD and was
independent of PEGylation. In contrast, the thermal stability of BSA was affected by
PEGylation. The unfolding temperature and the temperature of aggregation were both
independent of the molecular weight of the PEG chain. Discussion: PEGylation can increase the
apparent unfolding temperature compared to the unmodified protein. The formation of
aggregates will contribute exothermally to the total heat signal. As the heat measured by DSC is
the total contribution from both unfolding and aggregation, an extensive formation of aggregates
may shift the location of the peak in the thermogram towards a lower temperature. The
hypothesis that Glyco-PEGylation prevents or postpones the aggregation of protein is supported
by the results of the CD heating scans. Conclusion: By PEGylation, BSA appears to be
unaffected structurally, slightly destabilized thermally, stabilized kinetically and has an altered
functionality (binding profile). These biophysical characteristics are all independent of the
molecular weight of the attached polymer chain.
Keywords: Protein stability; PEGylation; Thermal unfolding; Spectroscopy; DSC
References:
[1] B. Plesner, C. J. Fee, P. Westh, A. D. Nielsen, “Effects of PEG size on structure, function and
stability of PEGylated BSA”, European J. Pharm.and Biopharm., 79 (2011) 399–405.
[2] D. S. Freitas, J. Abrahao-Neto, “Biochemical and biophysical characterization of lysozyme
modified by PEGylation”, Int. J. of Pharmaceutics, 392 (2010) 111–117.
* Corresponding author: Reza Hosseinzadeh
E-mail: chem.reza@ibb.ut.ac.ir