Magnetic affinity nanoparticles for bevacizumab adsorption

GR BARREDO VACCHELLI; SL GIUDICESSI; F ALBERICIO; O CASCONE; SA CAMPERI

Simposio; 36th European and 12th International Peptide Symposium (EPS 2022).; 2022

European peptide society

The IgG monoclonal antibody bevacizumab, produced in CHO cells, is used for treating cancer due to its capability of targeting the endothelial growth factor A (VEGF-A) and inhibiting angiogenesis. Sold as Avastin, it is on the World Health Organization´s List of Essential Medicines as one of the safest and most effective medicines in the health system. Nowadays, its purification is achieved by protein A affinity chromatography (AC). However, protein A is a very expensive ligand and harsh elution conditions are required to recover bevacizumab from the AC column, which can damage the mAb and the protein A. We have reported that a short peptide contained in VEGF binds bevacizumab with high affinity and selectivity while immobilized on agarose can be used for bevacizumab purification at lower cost and avoiding harsh elution conditions. However, conventional chromatography requires a previous clarification step to avoid clogging the column. On the other hand, the use of magnetic supports is a promising alternative to conventional methods in downstream processing as target materials can be extracted directly from crude cell lysates in a single step by magnetic nanoadsorbents. In this work, bevacizumab purification with magnetic nanoparticles (MNPs) is assessed.The peptide Ac-PHQGQHIGVSK was synthesized by Fmoc solid phase synthesis on Rink Amide and characterized by MALDS MS. Lys was incorporated at the C-termini to facilitate its subsequent immobilization on Pierce NHS-activated magnetic beads. MNPs with the peptide immobilized were added to nanotubes with a solution of purified bevacizumab or CHO cell filtrate. The MNPs were separated using a magnetic rack and adsorbed protein was eluted. Samples were evaluated by SDS-PAGE, which demonstrated that the MNPs can adsorbe bevacizumab selectively.Peptide-MNPs can be useful for bevacizumab purification from culture media without prior clarification and for easy sampling for quality control during the production process.