Monolith disk chromatography separates PEGylated protein positional isoforms within minutes at low pressure

• Published in: Biotechnology journal 2016-01, Vol.11 (1), p.100-106
• Main Authors: Isakari, Yu, Podgornik, Ales, Yoshimoto, Noriko, Yamamoto, Shuichi
• Format: Article
• Language: English
• Subjects: Atmospheric Pressure; Chromatography, Ion Exchange - methods; Gradient elution; Ion-exchange chromatography; Monolith; PEGylation; Polyethylene Glycols - chemistry; Process analytical technology (PAT); Protein Isoforms - chemistry; Protein Isoforms - isolation & purification; Proteins - chemistry; Proteins - isolation & purification
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.201500294

Although PEGylation makes proteins drugs more effective, the PEGylation reaction must be controlled carefully in order to obtain a desired PEGylated protein form since various different PEGylated forms may be produced during the reaction. For monitoring the PEGylation reaction, a method with monolith disk ion exchange chromatography, which can separate positional isomers as well as PEGmers, has been developed as a process analytical tool (PAT). The method was optimized for separation of randomly PEGylated protein (lysozyme) isoforms 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 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 ca. 4 min while the pressure drop was below 1 MPa. As the method was successfully applied to monitoring mono and di‐PEGylated positional isoforms in the reaction mixture of random PEGylation of lysozyme, it is expected to be an efficient PAT tool. Various different PEGylated protein forms must be monitored in order to obtain the desired PEGylated protein during PEGylation reaction. A high‐speed, high‐resolution method using monolith disk ion exchange chromatography, which can separate positional isomers as well as PEGmers, has been developed as a process analytical tool (PAT). Under the optimal conditions, the analysis can be completed within ca. 4 min while the pressure drop DP is below 1 MPa.