Affinity Chromatography based on a Combinatorial Strategy for rErythropoietin Purification

MARTÍNEZ CERON MARÍA C.; MARANI MARIELA M.; TAULÉS MARTA; ETCHEVERRYGARAY MARINA; ALBERICIO FERNANDO; CASCONE OSVALDO; CAMPERI SILVIA A.

ACS Combinatorial Science

American Chemical Society

Año: 2011 vol. 13 p. 251 - 258

2156-8952

Abstract Small peptides containing fewer than ten amino acids are promising ligand candidates to build affinity chromatographic systems for industrial protein purification. The application of combinatorial peptide synthesis strategies greatly facilitates the discovery of suitable ligands for any given protein of interest. Here we sought to identify peptide ligands with affinity for recombinant human erythropoietin (rhEPO), which is used for the treatment of anemia. A combinatorial library containing the octapeptides X-X-X-Phe-X-X-Ala-Gly, where X=Ala, Asp, Glu, Phe, His, Leu, Asn, Pro, Ser or Thr, was synthesized on HMBA-ChemMatrix resin by the divide-couple-recombine method. For the library screening, rhEPO was coupled to either Texas Red or biotin. Fluorescent beads or beads showing a positive reaction with streptavidin-peroxidase were isolated. After cleavage, peptides were sequenced by MALDI-TOF MS. Fifty-seven beads showed a positive reaction. Peptides showing more consensuses were synthesized and their affinity to rhEPO was assessed using a plasma resonance biosensor. Dissociation constant values in the range of 1-18 µM were obtained. The best two peptides were immobilized on Sepharose and the resultant chromatographic matrices showed affinity for rhEPO with dissociation constant values between 1.8 and 2.7 µM. CHO cell culture supernatant was spiked with rhEPO and the artificial mixture was loaded on Peptide-Sepharose columns. The rhEPO was recovered in the elution fraction with a yield of 90 % and a purity of 95% and 97% for P1-Sepharose and P2-Sepharose respectively.