Effect of lysozyme solid-phase PEGylation on reaction kinetics and isoform distribution

• A high-throughput method for solid-phase PEGylation was developed.
• Different reaction kinetics (solid vs. liquid-phase PEGylation) were determined.
• Different isoform distributions (solid vs. liquid-phase PEGylation) were determined.
• By using solid-phase PEGylation mainly two isoforms (lys 97 + 116) were produced.
• By varying the amount of bound protein the isoform distribution was manipulated.

The combination of PEG–protein conjugation and chromatographic separation is generally known as solid-phase or on-column PEGylation and can provide advantages compared to commonly applied batch PEGylation. Even though the concept was already applied by several authors, changes in the isoform distribution compared to liquid-phase PEGylation due to sterically hindered PEGylation sites could not be confirmed. In this manuscript, a method for solid-phase PEGylation experiments in a 96-well plate format, using an automated liquid handling station is described. Applying size exclusion chromatography (SEC) and highly sensitive isoform analytics for mono-PEGylated lysozyme, we were able to investigate the differences in reaction kinetics and isoform distribution between adsorber-based PEGylation and modifications in free solution. Accordingly, solid-phase PEGylation with SP Sepharose FF and XL generally showed a reduced PEGylation reaction. In contrast to the predominant N-terminal PEGylation of lysozyme in liquid phase, a main modification of lys 97 and lys 116 was found for solid-phase experiments, which could be explained by binding orientations on corresponding adsorbent materials. Further experiments with varying amounts of bound protein additionally showed an influence on the isoform distribution of mono-PEGylated lysozyme.

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