Protocol for bevacizumab purification using Ac-PHQGQHIGVSK-agarose

Abstract

Bevacizumab is a monoclonal antibody, produced in CHO cells, used for the treatment of many human cancers. It is an anti-vascular endothelial growth factor (antsi-VEGF) that blocks the growth of tumor blood vessels. Nowadays its purification is achieved by affinity chromatography (AC) using protein A which is a very expensive ligand. On the other hand, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment binds bevacizumab with high affinity. This short peptide ligand has higher stability and lower cost than protein A and it can be prepared very easily by solid phase peptide synthesis. The present protocol describes the synthesis of Ac-PHQGQHIGVSK-agarose and its use for affinity chromatography purification of bevacizumab from a clarified CHO cell culture. • Ac-PHQGQHIGVSK-agarose capacity and selectivity are equivalent to those of protein A matrices. • The peptide ligand shows a greater stability and lower cost. The lack of Trp, Met or Cys in the peptide ligand prevents its oxidation and extends the useful life of the chromatographic matrix. • Mild conditions used during chromatography preserved the integrity of bevacizumab.

Keywords

• Peptide
• Ligand
• Downstream processing
• Biopharmaceuticals
• Monoclonal antibodies
• Affinity chromatography