Preferential Interactions and the Effect of Protein PEGylation

<div><p>Background</p><p>PEGylation is a strategy used by the pharmaceutical industry to prolong systemic circulation of protein drugs, whereas formulation excipients are used for stabilization of proteins during storage. Here we investigate the role of PEGylation in protein stabilization by formulation excipients that preferentially interact with the protein.</p><p>Methodology/Principal Findings</p><p>The model protein hen egg white lysozyme was doubly PEGylated on two lysines with 5 kDa linear PEGs (mPEG-succinimidyl valerate, MW 5000) and studied in the absence and presence of preferentially excluded sucrose and preferentially bound guanine hydrochloride. Structural characterization by far- and near-UV circular dichroism spectroscopy was supplemented by investigation of protein thermal stability with the use of differential scanning calorimetry, far and near-UV circular dichroism and fluorescence spectroscopy. It was found that PEGylated lysozyme was stabilized by the preferentially excluded excipient and destabilized by the preferentially bound excipient in a similar manner as lysozyme. However, compared to lysozyme in all cases the melting transition was lower by up to a few degrees and the calorimetric melting enthalpy was decreased to half the value for PEGylated lysozyme. The ratio between calorimetric and van’t Hoff enthalpy suggests that our PEGylated lysozyme is a dimer.</p><p>Conclusion/Significance</p><p>The PEGylated model protein displayed similar stability responses to the addition of preferentially active excipients. This suggests that formulation principles using preferentially interacting excipients are similar for PEGylated and non-PEGylated proteins.</p></div

Far- and near- UV CD spectra measured at 20°C, pH 7.4 in HEPES buffer.

<p>Excipients are 1.0 M sucrose and 2.0 M GdnHCl. (A-C) Far-UV CD spectra of (A) Lyz and LyzPEG without excipients, (B) Lyz with excipients and (C) LyzPEG with excipients; (D-F) near-UV CD spectra of (D) Lyz and LyzPEG without excipients, (E) Lyz with excipients and (F) LyzPEG with excipients.</p

Peak maximum of fluorescence spectra as function of temperature and excipient.

<p>A) Lyz B) LyzPEG.</p

Ribbon structure of hen egg white lysozyme (pdb entry 1E8L).

<p>The PEGylated lysines are indicated in yellow and the fluorescence active tryptophans are indicated in green. The sequence of lysozyme is given below the structure with the same color indications for the mentioned lysines and tryptophans.</p