Analytical and preparative separation of PEGylated lysozyme for the characterization of chromatography media |
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| Authors: | Anna Moosmann Jessica Christel Heiner Boettinger Egbert Mueller |
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| Institution: | 1. Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany;2. Institute for Interfacial Engineering, University of Stuttgart, Nobelstraße 12, 70569 Stuttgart, Germany;3. Tosoh Bioscience GmbH, Zettachring 6, 70567 Stuttgart, Germany |
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| Abstract: | 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. |
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| Keywords: | Lysozyme PEGylation Cation exchange chromatography Analytical separation Preparative separation Dynamic binding capacity Selectivity PEG |
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