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

• Direct capture of monoclonal antibodies from a serum culture medium by PBA chromatography.
• Final purity was improved by designing a washing step to remove weakly bound impurities.
• The biological activity was enhanced by optimising the elution buffer composition.
• PBA chromatography generated yields comparable to protein A chromatography.

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 100 mM d-sorbitol in 10 mM Tris–HCl as washing buffer and 0.5 M d-sorbitol with 150 mM NaCl in 10 mM 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.