(117d) Monitoring of Pegylation Reaction By Monolith Chromatography

PEGylation has been used for making a smart protein drug as it prolongs the residence time in the body and makes proteins more stable and soluble. However, PEGylation reaction is so complicated that undesired PEGylated protein isoforms are also produced unless the process is carefully controlled.  It is therefore needed to develop a very fast and high-resolution analytical method for monitoring  PEGylation reaction.

Separation of randomly PEGylated protein (lysozyme) isoforms by monolith disk ion exchange chromatography (IEC) with a CIM SO3 strong cation-exchange monolith disk was optimized based on the number of resolved peaks, peak resolution, analysis time and pressure drop. In order to increase the retention of mono- and di-PEGylated protein isomers the mobile phase was decreased to pH 4.5, where a large number of mono- and di-PEGylated isomers were resolved within a few minutes. Based on the linear gradient elution (LGE) optimization model, the following values were determined; gradient slope =0.016 M/mL, disk thickness = 3 mm (single disk) and flow rate = 10 mL/min. Under these optimal conditions, the analysis was completed within 4 minutes while the pressure drop was below 1 MPa. We compared the performance of this method with pH gradient elution IEC. The number of peaks retained and resolved in the method developed (salt LGE) was much larger that in pH gradient elution. This method was successfully applied to monitoring mono and di-PEGylated positional isoforms in the reaction mixture of random PEGylation of lysozyme.