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

被引：29

作者：

Moosmann, Anna ^{[1
]}

Christel, Jessica ^{[2
]}

Boettinger, Heiner ^{[1
]}

Mueller, Egbert ^{[3
]}

机构：

[1] Univ Stuttgart, Inst Cell Biol & Immunol, D-70569 Stuttgart, Germany

[2] Univ Stuttgart, Inst Interfacial Engn, D-70569 Stuttgart, Germany

[3] Tosoh Biosci GmbH, D-70567 Stuttgart, Germany

来源：

JOURNAL OF CHROMATOGRAPHY A | 2010年 / 1217卷 / 02期

关键词：

Lysozyme; PEGylation; Cation exchange chromatography; Analytical separation; Preparative separation; Dynamic binding capacity; Selectivity; PEG; PORE-SIZE DISTRIBUTIONS; POLYETHYLENE-GLYCOL; ION-EXCHANGE; COVALENT ATTACHMENT; PROTEINS; PURIFICATION; PEPTIDE;

D O I：

10.1016/j.chroma.2009.11.031

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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. (C) 2009 Elsevier B.V. All rights reserved.

引用

页码：209 / 215

页数：7

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