Throughout the last two decades, bacterial-derived proteins have been employed as ligands for the purification of biotherapeutics by affinity chromatography. The structural complexity of these ligands, finely tuned by nature, guarantees high binding affinity and specificity. On the other hand, the same complexity also causes low biochemical stability and dramatic loss of binding upon exposure to harsh pH, solvents, and enzymes. To overcome these issues and develop ligands that combine the affinity and specificity of protein ligands with high biochemical stability, we have introduced and developed novel poly-cyclic peptide ligands for protein targets. Three different poly-cyclic structures are proposed, which are produced by introducing multiple intramolecular tethers and display a complex 3D structure. Virtual combinatorial libraries of these compounds were computationally generated and screened against two important therapeutic proteins, the granulocyte-colony stimulating factor (G-CSF) and the bone morphogenic protein BMP-7. A number of ligands with high predicted affinity (K<sub>D</sub> &lt; 1nM) were selected for each protein target. The candidate sequences were synthesized and coupled on chromatographic resins and tested for protein purification from cell culture fluids. Several sequences showed excellent performance, both in measured affinity and purification efficiency. In particular, the average K<sub>D</sub> approximated the target 1nM, and the purity of recovered G-CSF and BMP-7 was above 98%. Notably, a BMP-targeting ligand was found able to inhibit the BMP binding by its natural receptor, when displayed on yeast cells. Two ligands, one for each target, were also exposed to alkaline (0.5M NaOH) and organic (100% methanol) solvents to test their chemical stability, and, as expected, both retained their binding capacity. This work thus provides a validated strategy for the easy and high throughput identification of synthetic ligands with antibody-like affinity and specificity for the purification of protein therapeutics.

Best of both worlds: High affinity and structural stability in 3D-structured synthetic ligands for protein purification by affinity chromatography

251st National Meeting (2016)

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.