title: Monolith disk chromatography separates PEGylated protein positional isoforms within minutes at low pressure.
abstract: 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.
authors.affiliation: Bio-Process Engineering Laboratory, School of Engineering and Graduate School of Medicine, Yamaguchi University, Ube, Japan.
authors.forename: Yu
authors.initials: Y
authors.lastname: Isakari
authors.affiliation: Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia.|Center of Excellence COBIK, Ajdovščina, Slovenia.
authors.forename: Ales
authors.initials: A
authors.lastname: Podgornik
authors.affiliation: Bio-Process Engineering Laboratory, School of Engineering and Graduate School of Medicine, Yamaguchi University, Ube, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Yoshimoto
authors.affiliation: Bio-Process Engineering Laboratory, School of Engineering and Graduate School of Medicine, Yamaguchi University, Ube, Japan. shuichi@yamaguchi-u.ac.jp.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Yamamoto
mesh_terms: D001274:Atmospheric Pressure; D002852:Chromatography, Ion Exchange / Q000379:methods*; D011092:Polyethylene Glycols / Q000737:chemistry*; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*
chemical_list: D020033:Protein Isoforms; D011506:Proteins; D011092:Polyethylene Glycols
keywords: Gradient elution; Ion-exchange chromatography; Monolith; PEGylation; Process analytical technology (PAT)