The purification of blood-derived biotherapeutics, including clotting factors and protein hormones, is an emerging area in current bioseparations. Our group has recently discovered cyclic peptide ligands targeting human erythropoietin (EPO) by screening a rationally designed library of cyclic depsipeptides. The library design was based upon the study of the affinity interaction between EPO and the EPO receptor, while the screening was performed following a radiological method previously validated by our group. The screening resulted in identification of ligands displaying a predominantly hydrophobic binding character, similar to those observed in the EPO/EPOr complex. To elucidate binding mechanisms and develop sequences enabling EPO elution under mild conditions, several studies were performed, including single amino acid mutagenesis studies and computational binding studies using the docking software HADDOCK. Affinity adsorbents were generated by direct synthesis of selected leads onto chromatographic resins. The resulting adsorbents were characterized via bind-and-elute studies in both non-competitive and competitive conditions. Further binding studies indicated that, in agreement with the hydrophobicity of their sequences, the binding strength of the selected ligand increases with increasing the ionic strength of the binding buffer, and that, unlike EPO-binding lectins, retain their binding in presence of sugars. Binding and elution conditions were optimized to achieve an elution of EPO higher than 90%. Peptide-based affinity adsorbents were also subjected to multiple rounds of EPO binding, elution, and sanitization with 0.1 M NaOH, and found to be stable at these conditions. Finally, leading affinity adsorbents were tested for purification of recombinant EPO from artificial mixtures of proteins and CHO cell culture supernatant. Our results demonstrate that the selected cyclic peptides show some promise as affinity ligands and upon further development, may be useful for purification and/or detection of EPO.

Cyclic peptide ligands for the purification of erythropoietin by affinity chromatography

251st National Meeting (2016)

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