Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media

• Published in: Journal of Chromatography A Vol. 1217; no. 2; pp. 209 - 215
• Main Authors: Moosmann, Anna, Christel, Jessica, Boettinger, Heiner, Mueller, Egbert
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 08.01.2010; Amsterdam; New York: Elsevier
• Subjects: Analytical separation; Cation exchange chromatography; Cation Exchange Resins - chemistry; Chromatography, Gel; Chromatography, Ion Exchange - methods; Dynamic binding capacity; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Lysozyme; Muramidase - chemistry; Muramidase - isolation & purification; PEG; PEGylation; Polyethylene Glycols - chemistry; Polyethylene Glycols - isolation & purification; Preparative separation; Protein Binding; Selectivity; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; PEGylation; PEG; Cation exchange chromatography; Preparative separation; Selectivity; Lysozyme; Dynamic binding capacity; Analytical separation
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2009.11.031

The effect of PEGylation on cation exchange chromatography was studied with poly(ethylene glycol) of different chain lengths (5 kDa, 10 kDa and 30 kDa) 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 30 kDa mono-PEGylated lysozyme.