Affilin™ Molecules: Novel Ligands for Bioseparation

With the success of biotherapeutics the need for new ligands for affinity purification is growing. A novel approach to generate customized ligands is the use of alternative binding proteins. Here, we describe the generation, selection, and the use of specific Affilin™ molecules as small and robust ligands for recombinant protein purification. The Affilin™ molecules were isolated from a complex phage display library based on the randomization of eight surface exposed amino acids of the human eye lens protein γ-B-crystallin. Characterization of Affilin™ candidates by surface plasmon resonance (SPR) revealed dissociation constants in the nanomolar range. The E9 Affilin™ variant which was characterized in more detail has nanomolar affinity to the pro-form of human nerve growth factor (proNGF) and was used for matrix coupling to test proNGF recovery. The use of E9 Affilin™ as a ligand in affinity chromatography has demonstrated efficient recovery of its target protein, proNGF, from complex mixtures, such as spiked CHO supernatant or E. coli crude extract. Further, it has been shown that the E9 Affilin™ ligand can withstand denaturing conditions standard for cleaning processes in affinity chromatography. These findings suggest the application of Affilin™ molecules as potent and versatile ligands for affinity chromatography in the field of bioseparation.