Abstract
BackgroundPEGylation 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.Methodology/Principal FindingsThe 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.Conclusion/SignificanceThe 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.
Highlights
Next-generation protein drugs are proteins with altered amino acid sequence or altered glycosylation patterns, or proteins that are covalently modified with chemical moieties such as polyethylene glycol (PEG)
The far-UV circular dichroism (CD) spectra show that LyzPEG (Fig 1A) has a lower (85% at 205 nm) signal at the same molar concentration as Lyz indicating a change in the secondary structure compared to the nonPEGylated, native protein [44]
Addition of 2.0 M guanidine hydrochloride (GdnHCl) has a marginal effect on Lyz (Fig 1B) and a stronger effect on LyzPEG (Fig 1C)
Summary
Next-generation protein drugs are proteins with altered amino acid sequence or altered glycosylation patterns, or proteins that are covalently modified with chemical moieties such as polyethylene glycol (PEG) These modifications are generally aimed at improving the pharmacokinetics of the protein, most commonly an increase in circulation half-life. PEGylation of proteins has led to significantly improved possibilities for drug administration; for example, in treatment of chronic hepatitis C a 7-fold increase in circulation half-life is observed upon PEGylation of the native protein drug [1]. This allows once-weekly injections with improved clinical outcome compared to the thriceweekly injections of the unmodified drug, despite the fact that receptor binding is reduced by more than a factor 10 for the PEGylated product [2]. We investigate the role of PEGylation in protein stabilization by formulation excipients that preferentially interact with the protein
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