Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media.
The effect of PEGylation on cation exchange chromatography was studied with poly(ethylene glycol) of different chain lengths (5kDa, 10kDa and 30kDa) using lysozyme as a model system. A stable binding via reduction of a Schiff base was formed during random PEGylation on lysine residues with methoxy-PEG-aldehyde. A purification method for PEGylated proteins using cation exchange chromatography was developed, and different isoforms of mono-PEGylated lysozyme were isolated. TSKgel SP-5PW and Toyopearl GigaCap S-650M showed the best performance of all tested cation exchange resins, and the separation of PEGylated lysozyme could be also scaled up to semi-preparative level. Size-exclusion chromatography, SDS-PAGE and MALDI-TOF mass spectrometry were used for analysis. Separated mono-PEGylated lysozyme of different sizes was used to determine dynamic binding capacities (DBC) and selectivity of cation exchange chromatography resins. An optimization of binding conditions resulted in a more than 20-fold increase of DBC for Toyopearl GigaCap S-650M with 30kDa mono-PEGylated lysozyme.
Moosmann
Anna
A
Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. anna.moosmann@izi.uni-stuttgart.de
Christel
Jessica
J
Boettinger
Heiner
H
Mueller
Egbert
E
D002411:Cation Exchange Resins / Q000737:chemistry*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D009113:Muramidase / Q000737:chemistry* / Q000302:isolation & purification; D011092:Polyethylene Glycols / Q000737:chemistry* / Q000302:isolation & purification; D011485:Protein Binding; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002411:Cation Exchange Resins; D011092:Polyethylene Glycols; D009113:Muramidase