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

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

Peptides. Copenhaguen 2010: Tales of peptides. Proceedings of the thirty-first European Peptide Symposium.

European Peptide Society

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

Erythropoietin (Epo), glycoprotein hormone produced  in mammalian kidney and  liver, is the main inducer and regulator of red cell proliferation and differentiation in bone marrow. Recombinant erythropoietin (rhEpo) is used for therapeutics of anemia associated with chronic renal disease, AZT-induced anemia of AIDS and for the treatment of cancer patients on chemotherapy and surgical patients to avoid  the need for a  red blood cell transfusion [1]. Affinity Chromatography (AC) is ideally suited for  the  purification of therapeutic proteins as  it is the most effective method for the direct isolation and purification of biomolecules from complex mixtures. Its  good  selectivity minimizes contamination and yields samples of high purity in a single step [2]. Successful separation by AC requires the availability of a ligand with satisfactory affinity and selectivity. Small peptides consisting of a few amino acids represent promising affinity ligand candidates for industrial separations. Peptide ligands are much more physically and chemically stable than antibody ligands and  are  very resistant to  proteolytic cleavage. They can be readily synthesized in bulk amounts at a lower cost under good manufacturing practices (GMP) by standard chemistry. Also, peptides can be easily modified by chemical methods to facilitate product elution under mild conditions. Furthermore,  peptides allow site-directed immobilization and high ligand density and the matrices are more robust during elution and regeneration than protein-based affinity matrices such as monoclonal antibodies. Moreover, in the  case of leakage into the  product,  peptides  have  low toxicity and generate low immune responses compared with proteins, dyes and transition metal ion ligands [3]. When leakage does occur, small peptide molecules can be easily removed from a macromolecular product. The combinatorial synthesis of peptide libraries allows the production of millions of peptides, thus greatly facilitating the discovery of suitable ligands for a given protein of interest. The preparation of combinatorial libraries by the divide–couple–recombine (DCR) or mix and split solid-phase method assures a theoretically even representation of the library members and a one-bead-one-compound distribution [4,5]. In previous  studies, we developed a rapid and inexpensive strategy for the identification of peptides contained on positive beads, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and 4-hydroxymethylbenzoic acid (HMBA) immobilized in ChemMatrix resin [6-8]. The aim  of this study was to apply  that strategy  for the identification of peptide ligands with affinity for rhEpo and  to  attach these peptides to agarose  in order  to purify rhEPO by AC.