Protocol for bevacizumab purification using Ac-PHQGQHIGVSK-agarose

Gabriela R Barredo,Silvana L Giudicessi,María C Martínez Ceron,Soledad L Saavedra,Santiago Rodríguez,Lucas Filgueira Risso,Rosa Erra-Balsells,Gustavo Mahler,Fernando Albericio,Osvaldo Cascone,Silvia A Camperi

doi:10.1016/j.mex.2019.12.010

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.

Highlights

The peptide Ac-PHQGQHIGVSK contained in the VEGF fragment binds bevacizumab with high affinity
- Method details e Bevacizumab is an anti-vascular endothelial growth factor that blocks the growth of tumor blood vessels
P peptides consisting of few amino acids represent ideal affinity chromatography ligands because they are much more physically and chemically stable than protein A and can be readily synthesized by standard chemistry in l bulk amounts at a lower cost

Summary

Subject Area

A short peptide fragment of the vascular endothelial growth factor as a novel ligand for bevacizumab purification. P peptides consisting of few amino acids represent ideal affinity chromatography ligands because they are much more physically and chemically stable than protein A and can be readily synthesized by standard chemistry in l bulk amounts at a lower cost. Gly-92 VEGF segment [1], a peptide ligand for bevacizumab purification by AC was designed [2]. Wash resin with DMF (2 × 1 min) and repeat coupling reaction with fresh reagents as indicated in r steps 5-9. Remove Fmoc group, wash the resin and couple the Fmoc protected amino acid as indicated in steps 3-9. To test the completion of the acetylation reaction, perform the chloranil test with a small amount or resin (1-3 l mg) [4].

Procedure

Eluate fraction