Monoclonal Antibody Bevacizumab purification by Peptide Affinity Chromatography

G. R. BARREDO; S. L. GIUDICESSI; M. C. MARTÍNEZ CERON; SOLEDAD L. SAAVEDRA; G. MAHLER; F. ALBERICIO; O.CASCONE; S. A. CAMPERI

Dublin

Simposio; 35th European Peptide Symposium; 2018

Europen Peptide Society

Therapeutic Monoclonal Antibodies (mAbs) are widely used in many diseases? treatments such as cancers, rheumatoid arthritis, multiple sclerosis, between others. Bevacizumab (trade name: Avastin), inhibits vascular endothelial growth factor, hence the angiogenesis and it is applied in brain, breast, colorectal, lung and renal cancers. Its biotechnological production process involves host cell optimization, cultivation and mAb biosynthesis (upstream processing) and the mAb recuperation and purification from the culture broth (downstream processing). Due to its parenteral administration, its degree of purity must be extremely high. Nowadays, that is achieved with affinity chromatography (AC) with immobilized protein A, a high expensive ligand that increases the total cost of the process. In addition, due to the high affinity between mAb and protein A, harsh elution conditions are required, impairing the protein ligand and reducing the chromatographic matrix half life. On the other hand, short peptides are ideal ligands for AC due to their higher stability, easier synthesis and lower cost in comparison to protein A. In this work, short peptides ligands with affinity for Bevacizumab were selected from a short one-bead-one-peptide combinatorial library obtained by the divide-couple-recombine method using the HMBA-ChemMatrix resin gently donated by Matrix innovation Inc. and using the Fmoc strategy. Bevacizumab, gently donated by Dr, Gustavo Mahler from AGC Biologist (USA) was labeled with Texas-Red and after mixing it with the library, fluorescent beads were selected and the peptide immobilized in each bead was identified by MALDI-TOF MS/MS. Those peptides that appeared most frequently were synthesized in larger quantities on Rink Amide resin and immobilized by Fmoc strategy, separated from the solid support with TFA cocktail, and afterwards immobilized on NHS-activated agarose. The peptide affinity chromatographic matrices were evaluated with adsorption isotherms. Affinities in the order of 106 M were obtained. Those peptides that selectively adsorbed Bevacizumab without interacting with the CHO cell supernatants proteins were selected for future process scale-up. In conclusion, an efficient one-step-method based on AC with immobilized short peptide allows the purification of the mAb Bevacizumab not only in a single step, but also at low cost.