Bevacizumab Purification by Peptide Affinity Chromatography

GABRIELA R. BARREDO; SILVANA L. GIUDICESSI; MARÍA C. MARTÍNEZ CERON; SOLEDAD L. SAAVEDRA; GUSTAVO MAHLER; FERNANDO ALBERICIO; OSVALDO CASCONE; SILVIA A. CAMPERI

Dublin

Simposio; 35th European Peptide Symposium; 2018

European Peptide Society

Therapeutic Monoclonal Antibodies (mAbs) are widely used in many diseases? treatments such as cancer, rheumatoid arthritis, multiple sclerosis,among others. Bevacizumab (trade name: Avastin) inhibits the vascular endothelial growth factor and hence the angiogenesis process and itis applied to treat brain, breast, colorectal, lung and renal cancers. Itsbiotechnological production process involves host cell optimization, cultivation and mAb biosynthesis (upstream processing) and finally, the mAbrecovery and purification from the culture broth (downstream processing).Due to its parenteral administration, its degree of purity must be extremelyhigh. Nowadays, that is achieved with affinity chromatography (AC) withimmobilized protein A, a highly expensive ligand that increases the costof the process. In addition, due to the high affinity between antibodiesand protein A, harsh elution conditions are required, impairing the proteinligand and reducing the chromatographic matrix half life. On the otherhand, 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 peptide ligands with affinity for Bevacizumab were selected from ashort one-bead-one-peptide combinatorial library obtained by the dividecouple-recombine method using the HMBA-ChemMatrix resin and usingthe Fmoc strategy. Bevacizumab (AGC Biologist (USA) was labeled withTexas-Red, and after mixing it with the library, fluorescent beads were selected and the peptide immobilized in each bead was identified by MALDITOF MS/MS. Those peptides that appeared most frequently were synthesized in larger quantities on Rink Amide resin by Fmoc strategy, separatedfrom the solid support with TFA cocktail, and afterwards immobilized onNHS-activated agarose. The affinity chromatographic matrices were evaluated by measuring the adsorption isotherms and affinities between 105-106M were obtained. Those peptides that selectively adsorbed Bevacizumabwithout interacting with the CHO cell supernatants proteins were selectedfor future process scale-up. In conclusion, an efficient one-step-methodbased on AC with an immobilized short peptide allows the purification ofthe mAb Bevacizumab not only in a single step, but also at low cost.