Monoclonal antibody purification by affinity chromatography with ligands derived from the screening of peptide combinatory libraries

SILVIA A. CAMPERI; NANCY B. IANNUCCI; GUILLERMO J. ALBANESI; MARCOS OGGERO EBERHARDT; MARINA ETCHEVERRIGARAY; ANGEL MESSEGUER; FERNANDO ALBERICIO; OSVALDO CASCONE

Basilea, Suiza

Congreso; 11th European Congress on Biotechnology; 2003

ObjectivesMonoclonal antibody (Mab) purification by fast, economical and easy-to-scale-up methods is highly required. Small peptides as affinity chromatography ligands are more stable that antibodies and more selective than dyes and metals. The aim of this study was to purify a Mab against Granulocyte Macrophage- Colony Stimulating Factor (GM-CSF) by affinity chromatography with a peptide ligand selected from the screening of a combinatorial synthetic library attached to agarose and to characterise its chromatographic behaviour.ResultsA ?one bead-one peptide? library composed of 132.000 tetrapeptides was developed by the method of coupling and mixing. Its screening allowed identification of peptide APAR as the best ligand for the anti GM-CSF Mab. The peptide was coupled to Sepharose 4B through a cystein residue to obtain the chromatographic matrix for the anti GM-CSF Mab purification from ascitis.A maximum capacity of 9.1mg Mab/ml matrix and a dissociation constant of 0.014 mg/ml were calculated from isotherms developed with ascitis dilutions. Mab concentration in the supernatants and eluates was determined by specific indirect ELISA. From the breaktrough curve, a dynamic capacity of 6.9 mg Mab/ml matrix was calculated. To check the usefulness of the technique, a column (1 mL) was loaded with 15 ml of ascitis diluted ¼ in adsorption buffer. This corresponds to 95% of the dynamic capacity for Mab under optimal conditions. After washing to baseline, Mab was eluted with 5M LiCl, eluent selected from the screening of 5 alternative eluents. SDS-PAGE of the eluate showed only one band of MW around 160000, thus evidencing that the matrix allows one-step purification to homogeneity of the Mab.ConclusionsLigands selected from the screening of peptide libraries allow a very efficient purification of Mab in a single step.