Purifying Bevacizumab via Affinity Precipitation Using Branched Peptide

The therapeutic monoclonal antibody bevacizumab is typically purified using protein A affinity chromatography, a highly effective but costly method. Affinity-based precipitation for antibody purification is a lower-cost approach. In this work, a precipitation protocol was developed for bevacizumab purification using a branched peptide (Ac-PHQGQHIG-Ahx3)2-K-Ahx3-PHQGQHIG-NH2, which contains the epitope PHQGQHIG that is responsible for interacting with bevacizumab. The peptide was synthesised by a microwave-assisted solid-phase peptide method, employing LiCl as an additive to prevent aggregation and ensure high purity and yield. Three molecules of 6-aminohexanoic acid were introduced between each epitope branch as spacer arms to promote the formation of cyclic complexes. Bevacizumab purification from cell-free culture broth was achieved through a fractional precipitation process. First, a negative precipitation step using (NH4)2SO4 1.18 M was performed to remove contaminants. Afterwards, 5 moles of peptide per mol of bevacizumab was added to the supernatant, together with additional (NH4)2SO4, to reach a final concentration of 1.20 M. Under these conditions, bevacizumab was recovered in the precipitate with 98% purity and a yield of 73%. In addition to being recyclable, the peptide’s relatively low production cost could enable the development of a single-use purification process, which would be particularly advantageous for biopharmaceutical manufacturing.