This article is published in Journal of Molecular Recognition as a special issue on Affinity 2009, edited by Gideon Fleminger, Tel-Aviv University, Tel-Aviv, Israel and George Ehrlich, Hoffmann-La Roche, Nutley, NJ, USA.
Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography†
Version of Record online: 28 OCT 2010
Copyright © 2010 John Wiley & Sons, Ltd.
Journal of Molecular Recognition
Special Issue: Affinity 2009 – The 18th biennial meeting of the International Society for Molecular Recognition
Volume 23, Issue 6, pages 569–576, November/December 2010
How to Cite
Azevedo, A. M., Gomes, A. G., Borlido, L., Santos, I. F. S., Prazeres, D. M. F. and Aires-Barros, M. R. (2010), Capture of human monoclonal antibodies from a clarified cell culture supernatant by phenyl boronate chromatography. J. Mol. Recognit., 23: 569–576. doi: 10.1002/jmr.1068
- Issue online: 28 OCT 2010
- Version of Record online: 28 OCT 2010
- Manuscript Accepted: 11 JUN 2010
- Manuscript Revised: 28 APR 2010
- Manuscript Received: 28 NOV 2009
- monoclonal antibodies;
- phenyl boronate;
- affinity chromatography;
- downstream processing;
In this work, we investigated the feasibility of using phenyl boronate (PB) chromatography for the direct capture of monoclonal antibodies from a CHO cell supernatant. Preliminary results, using pure protein solutions have shown that PB media can bind to human antibodies, not only at strong alkaline conditions but also at acidic pH values. In fact, antibodies have been found to bind in the pH range 5.5–8.5. On the other hand, insulin and human serum albumin did not bind at alkaline pH but at lower pH, which reflects the importance of non-specific interactions with the matrix. Different binding and eluting buffers were evaluated for the capture of immunoglobulin G (IgG) from a CHO cell supernatant and the most promising results were obtained using 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid at pH 8.5 as binding buffer and 1.5 M Tris–HCl as eluting buffer. Using a step elution, all IgG was recovered in the elution pool with a maximum purification factor of 56. A gradient elution allowed a further increase of the final purity, yet achieving a slightly lower yield. IgG recovery was around 85% and the purification factor was 76. The highest purity was obtained when the pH of the cell supernatant feed was previously adjusted to 8.5. Starting from an initial protein purity of 1.1% and high-performance liquid chromatography (HPLC) purity of 2.2%, after PB adsorption, a final protein purity of 85% and a HPLC purity of 88% was achieved. Copyright © 2010 John Wiley & Sons, Ltd.