Abstract
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.
Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
