title: Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media.
abstract: 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.
authors: Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. anna.moosmann@izi.uni-stuttgart.de
authors: Anna
authors: A
authors: Moosmann
authors: Jessica
authors: J
authors: Christel
authors: Heiner
authors: H
authors: Boettinger
authors: Egbert
authors: E
authors: Mueller
mesh_terms: 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
chemical_list: D002411:Cation Exchange Resins; D011092:Polyethylene Glycols; D009113:Muramidase