Ion exchange chromatography of antibody fragments
Article first published online: 9 NOV 2006
Copyright © 2006 Wiley Periodicals, Inc.
Biotechnology and Bioengineering
Volume 96, Issue 3, pages 515–524, 15 February 2007
How to Cite
Ljunglöf, A., Lacki, K. M., Mueller, J., Harinarayan, C., van Reis, R., Fahrner, R. and Van Alstine, J. M. (2007), Ion exchange chromatography of antibody fragments. Biotechnol. Bioeng., 96: 515–524. doi: 10.1002/bit.21124
- Issue published online: 27 DEC 2006
- Article first published online: 9 NOV 2006
- Manuscript Accepted: 30 JUN 2006
- Manuscript Received: 31 MAR 2006
- antibody fragment;
- ion exchange chromatography;
- confocal microscopy
Effects of pH and conductivity on the ion exchange chromatographic purification of an antigen-binding antibody fragment (Fab) of pI 8.0 were investigated. Normal sulfopropyl (SP) group modified agarose particles (SP Sepharose™ Fast Flow) and dextran modified particles (SP Sepharose XL) were studied. Chromatographic measurements including adsorption isotherms and dynamic breakthrough binding capacities, were complemented with laser scanning confocal microscopy. As expected static equilibrium and dynamic binding capacities were generally reduced by increasing mobile phase conductivity (1–25 mS/cm). However at pH 4 on SP Sepharose XL, Fab dynamic binding capacity increased from 130 to 160 (mg/mL media) as mobile phase conductivity changed from 1 to 5 mS/cm. Decreasing protein net charge by increasing pH from 4 to 5 at 1.3 mS/cm caused dynamic binding capacity to increase from 130 to 180 mg/mL. Confocal scanning laser microscopy studies indicate such increases were due to faster intra-particle mass transport and hence greater utilization of the media's available binding capacity. Such results are in agreement with recent studies related to ion exchange of whole antibody molecules under similar conditions. Biotechnol. Bioeng. 2007;96: 515–524. © 2006 Wiley Periodicals, Inc.