Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies: Development and optimization of a washing step

dos Santos, Raquel; Rosa, Sara A.S.L.; Aires-Barros, M Raquel; Tover, Andres; Azevedo, Ana M.

Phenylboronic acid as a multi-modal ligand for the capture of monoclonal antibodies: Development and optimization of a washing step

2014

dos Santos, Raquel | Rosa, Sara A.S.L. | Aires-Barros, M Raquel | Tover, Andres | Azevedo, Ana M.

In this work, phenylboronic acid (PBA) was thoroughly investigated as a synthetic ligand for the purification of an immunoglobulin G (IgG) from a clarified cell supernatant from Chinese Hamster Ovary (CHO) cell cultures. In particular, the study was focused on the development of a washing step and in the optimization of the elution step using a serum containing supernatant. From the different conditions tested, best recoveries – 99% – and purifications – protein purity of 81% and a purification factor of 16 out of a maximum of 20 – were achieved using 100mM d-sorbitol in 10mM Tris–HCl as washing buffer and 0.5M d-sorbitol with 150mM NaCl in 10mM Tris–HCl as elution buffer. The purification outcome was also compared with protein A chromatography that revealed a recovery of 99%, 87% protein purity and 29 out of a maximum of 33 purification factor. Following the main purification, purified IgG was characterized in terms of isoelectric point, size and activity. In the end, a proof of concept was performed using two different mAbs from serum-free CHO cell cultures.

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