Interaction mechanism of mono‐PEGylated proteins in electrostatic interaction chromatography

Abstract

The retention and binding mechanisms in electrostatic interaction-based chromatography (ion-exchange chromatography) of PEGylated proteins (covalent attachment of polyethylene glycol chains to protein) were investigated using our previously developed model. Lysozyme and bovine serum albumin were chosen as model proteins. The retention volume of PEGylated proteins shifted to lower elution volumes with increasing PEG molecular weight compared with the non-modified (native) protein retention volume. However, PEGylation did not affect the number of binding sites appreciably. The enzyme activity of PEGylated lysozyme measured with a standard insoluble substrate in suspension decreased considerably, whereas the activity with a soluble small-molecule substrate did not drop significantly. These findings indicate that when a protein is mono-PEGylated, the binding site is not affected and the elution volume reduces due to the steric hindrance between PEGylated protein and ion-exchange ligand.

Optimization of random PEGylation reactions by means of high throughput screening

“…Lysozyme PEGylated at position lysine 33 provided with 35% the highest residual activity, followed by PEG‐lys 1 with 24%. Studies by Abe et al () suggest a residual activity of about 30% for 5 kDa mono‐PEG lysozyme isoform mixtures, which is thus in the line with the results made in this study. As can be seen, the PEG conjugation site has an influence on the residual activity, which corresponds to Monkarsh et al () where different activities in the range of 6% and 40% of mono‐PEGylated interferon were found.…”

Monoliths for the purification of whey protein–dextran conjugates

“…A similar occurrence was observed with PEGylated conjugates where these species bound weakly to the cation exchanger and this weak binding was attributed to the charge-shielding effect of the neutral polymer, poly(ethylene glycol) (PEG) [26][27][28][29]. During the rise in salt concentration from 0.0 to 0.3 M for peak P2, multi-dextran conjugates would elute first, followed by di-dextran conjugates, and lastly mono-dextran conjugates [28]. This selective elution sequence would broaden peak P2.…”

Microcalorimetric study of the adsorption of PEGylated lysozyme on a strong cation exchange resin

“…This effect can be explained by the increased molecular size of the higher PEGylated forms which leads to an increased distance between the protein's charges and the counterions on the adsorber surface. A detailed description and further experimental investigation of this phenomenon is given by Abe et al [21].…”