| 19963219 |
Moosmann A, Christel J, Boettinger H, Mueller E: Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media. J Chromatogr A. 2010 Jan 8;1217(2):209-15. Epub 2009 Nov 17.
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. |
11(0,0,1,6) |