Octapeptides ligands with affinity for recombinant erythropoietin derived from the screening of combinatorial libraries.

M. C. MARTÍNEZ-CERON; M. TAULÉS; M. M. MARANI; M. ETCHEVERRIGARAY; F. ALBERICIO; O. CASCONE; S. A. CAMPERI

Peptides. Copenhagen 2010: Tales of peptides.

European Peptide Society

Lugar: Copenhagen; Año: 2010; p. 194 - 195

Recombinant erythropoietin (rhEPO) is used for therapeutics of anemia associated with chronic renal failure and for AZT-induced anemia of AIDS. Monoclonal antibody (mAb) affinity chromatography and dye affinity chromatography are alternative techniques nowadays in use for its purification. MAb are expensive, thus increasing the cost of the final product. Affinity chromatography with Cibacron Blue as the ligand is widely used, but the selectivity is not high. The use of short peptides as affinity chromatography ligands would result in a more economic and selective purification process. Divide-couple-recombine (DCR) method allows obtaining a library with all possible combinations of the amino acids in the form of "one bead-one peptide". Peptide ligands can be selected from the library screening. We have developed a rapid and non-expensive strategy for the identification of peptides contained on positive beads, by using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS), and 4-hydroxymethylbenzoic acid (HMBA). In this work the method designed was applied for identification of peptide ligands for rhEPO purification. In previous works we have obtained low-affinity tetrapeptide ligands for rhEPO. Taking into account those results, a combinatorial library containing the octapeptides XXXFXXAG where X=A, D, E, F, H, L, N, P, S or T was synthesised on HMBA-ChemMatrix resin by the DAR method using the Fmoc chemistry. Side-chain deprotection was carried out with TFA. For the library screening the rhEPO was coupled with either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. Fifty beads showed positive reactions. After peptides cleavage with NH4OH, they were sequenced by MALDI-TOF-MS. and those sequences showing more consensus were synthesised and their affinity to rhEPO evaluated using a plasma resonance biosensor. Kd values between 1-10 uM. Peptides with the highest affinity were immobilised on agarose. All peptide-agarose matrices showed affinity for rhEPO. Also, the affinity of the peptide-agarose matrices for bovine seroalbumin (BSA) - usually present in the culture supernatants - was assessed. Those peptides with the highest selectivity between rhEPO and BSA were selected for future development of a rhEPO purification chromatographic matrix.