Preferential Interactions and the Effect of Protein PEGylation

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.

Semisynthetic prion protein (PrP) variants carrying glycan mimics at position 181 and 197 do not form fibrils

“… 23 , 24 , 40 , 41 Even though there is literature evidence that PEGylated proteins retain their native-like folding and PEGylation provides an increase in conformational stability, 40 , 42 , 43 Natalello et al reported a decrease in conformational stability and slight increase in aggregation sensitivity of PEGylated G-CSF. 44 Furthermore, PEGylated hen egg white lysozyme, featured a minor loss of α-helical content compared to wt lysozyme, 45 making any rational generalized conclusions about the effect of PEGylation on peptide and protein secondary structure very challenging. Therefore, a case by case analysis is required for each PEGylated species as different effects of PEGylation on structure are observed in full length PrP (less helicity and more β-sheet) when compared to the C-terminal peptides (increased helicity) and non-PEGylated PrP.…”

PEGylation of Biopharmaceuticals: A Review of Chemistry and Nonclinical Safety Information of Approved Drugs

“…Comparative studies of PEGylated proteins versus the non-PEGylated counterpart generally conclude that the addition of PEG does not significantly change the protein structure when assessed by circular dichroism (CD), ultraviolet absorption, or nuclear magnetic resonance (NMR) spectroscopy. 18,[31][32][33][34][35][36] Reported changes in protein temperature stability have been both positive [37][38][39] and negative 40,41 depending on the coupling chemistry, the degree of PEGylation, the number of protein subunits, and formulation. 42 Only a few systematic studies have been reported.…”

“…51 In addition, the container closure and formulation may also affect conjugate stability. 41,52 Based on the literature cited previously, if one performs the PEGylation and purification of a PEGylated protein in accordance with known sensitivities to temperature, pH, and other physical parameters, the resultant conjugate should not display any significant difference in the folded structure compared with native protein as monitored by CD, NMR, and complementary techniques to measure protein structure. Changes in thermal stability or subunit dissociation (either positively or negatively) may be observed which reflect characteristics of the protein but are not necessarily related to PEG.…”

A novel approach enables imaged capillary isoelectric focusing analysis of PEGylated proteins

“…PEGylation is a process through which PEG chains are conjugated to proteins or other molecules . PEGylation has been used as a strategy to enhance pharmacokinetic properties of therapeutic proteins as well as reduce immunogenicity and toxicity , which are appealing attributes for therapeutically relevant proteins. PEG‐protein conjugations can be formed by chemical or enzymatic reactions , and semisynthetic methods such as expressed protein ligation .…”