(237e) On-Column (solid-phase) Pegylation and Separation of Pegylated Proteins

PEGylation is known to be an effective method for making a smart protein drug. It prolongs the residence time in the body and makes proteins more stable and soluble. However, producing a PEGylated protein is not an easy process since the reaction yield is not very high and consequently, the reaction mixture contains non-reacted protein and PEG as well as PEGylated proteins. In addition, due to over PEGylation reaction unfavorable, mutli-PEGylated proteins are produced. Separation of a target mono-PEGylated protein from other PEGylated protein forms and native protein is also a difficult process.

Solid phase or on-column PEGylation is claimed to be a promising method for improving the reaction selectivity. We carried out random PEGylation of lysozyme on various cation-exchange chromatography columns. After the PEGylation reaction, linear gradient elution experiments were performed to separate PEGylated protein forms.

Production of multi-PEGylated forms was reduced significantly. However, the yield was relatively low. In order to understand the reason for the low yield, various different chromatography media as well as mobile phase conditions were tested. Porous beads of different particle size (SP-Sepharose FF and HP), hydroxyapatite, Phosphate-Cellufine, Sulfate-Cellufine, SP-Toyopearl650M, Resource S and SO3-monolithic disk were used. Large beads (Sepharose FF) resulted in much lower yield values most likely due to the mass transfer resistance. However, convention-aided chromatography media, monolithic disk did not improve the yield significantly. By choosing a suitable salt concentration of the mobile phase, lysozyme was rather weakly adsorbed onto the disk entirely before PEGylation. This condition increased the yield.