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 mols. as small and robust ligands for recombinant protein purificationThe Affilin mols. 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 chromatog. has demonstrated efficient recovery of its target protein, proNGF, from complex mixtures, such as spiked CHO supernatant or E. coli crude extractFurther, it has been shown that the E9 Affilin ligand can withstand denaturing conditions standard for cleaning processes in affinity chromatog.These findings suggest the application of Affilin mols. as potent and versatile ligands for affinity chromatog. in the field of biosepn.