Bevacizumab Purification by Peptide Affinity Chromatography

GABRIELA R. BARREDO; SILVANA L. GIUDICESSI; MARA C. MARTINEZ-CERON; SOLEDAD L. SAAVEDRA; GUSTAVO MAHLER; FERNANDO ALBERICIO; OSVALDO CASCONE; , SILVIA A. CAMPERI

Proceedings of the 35th European Peptide Symposium

European Peptide Society

Lugar: Dublin; Año: 2018; p. 130 - 132

IntroductionTherapeutic Monoclonal Antibodies (mAbs) are widely used in the treatment of many diseases. Bevacizumab (trade name: Avastin) inhibits the vascular endothelial growth factor and hence the angiogenesis process. It is used in brain, breast, colorectal, lung and renal cancer treatments (1). Due to its parenteral administration, its degree of purity must be extremely high. Nowadays, that is achieved by affinity chromatography (AC) with immobilized protein A, a highly expensive ligand that increases the cost of the purification process. 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 (2). In this work, a short peptide ligand with affinity for Bevacizumab wasselected from a peptide library.MethodsA one-bead-one-peptide combinatorial library was developed by the divide-couple-recombine method, using the HMBA-ChemMatrix resin as solid support and the Fmoc strategy (3). Bevacizumab (AGC Biologist, USA) was labeled with Texas-Red. After mixing it with the library, fluorescent beads were selected, and the peptidesidentified by MALDI-TOF MS/MS. Those that appeared most frequently were synthesized in larger quantities on Rink Amide resin and separated from the solid support with TFA cleavage cocktail. Chromatographies werecarried out after immobilizing the peptides on PierceTM NHS-activated dry agarose resin. One of the peptides was capable to adsorb IgG from CHO cell supernatants better than the others, while all the contaminantspassed through without interacting with the chromatographic matrix. Adsorption isotherms were performed at room temperature to characterize the generated affinity resin.Results and DiscussionChromatographies with the peptide ligand were performed using pure IgG, CHO supernatant, and CHO supernatant spiked with IgG (Fig. 1). Chromatographicsamples were loaded on SDS-PAGE (Fig. 2). The peptide AC showed a good selectivity for IgG and no interaction with CHO supernatant proteins was verified.