[4S06a] 17. Downstream processing - Part 2

2:45 PM - 3:10 PM JST(5:45 AM - 6:10 AM UTC)

[4DSP-07] Rabbit scFv-based affinity chromatography for purification of biopharmatheuticals

○YOICHI KUMADA¹, YODAI YAMAMOTO¹, KENSHIRO YABUUCHI¹, JUN-ICHI HORIUCHI¹ (1. Kyoto Institute of Technology (Japan))

Keywords:

affinity chromatography,rabbit single-chain variable fragment,affinity ligand development,site-directed immobilization,CDR-grafting technology

Affinity chromatography (AC) has played an important role in the downstream processing of biopharmaceuticals since it enables isolating the target molecules efficiently with significantly high selectivity from the complex culture broth. Recombinant protein A has been utilized as an affinity ligand for the purification of humanized monoclonal antibodies as well as Fc fusion proteins, while the effective and versatile affinity ligands for the other target substances including non-Fc proteins, bio-nanoparticles including Exosomes and viral vectors, have been aggressively developed. Here, we propose and demonstrate a novel rabbit single-chain variable fragment (scFv) -based affinity ligand for purification of biologics at the capture step. Rabbit antibodies potentially possessing extremely high specificity and affinity have been exclusively utilized in the fields of clinical diagnostics, allergy tests and biochemical research. Target-specific scFvs in which variable domains of VH and VL are genetically connected via the flexible linker could be isolated, identified, and characterized by the original screening systems based on the phage display system. Furthermore, rabbit scFvs with different target specificity could be produced on a large scale by DO-stat fed-batch culture of recombinant E. coli cells, while molecular evolution as well as CDR-grafting were possible due to their simple framework structure. Consequently, we designed and successfully developed the original framework structure of rabbit scFv-based affinity ligand. The lysine residues in the ligand, which were the potential coupling sites to the chromatography resin, were substituted with other amino acids without loss of antigen-binding activity, and multiple lysine residues were additionally introduced as the coupling sites at the C-terminal position. Consequently, both the binding capacity and the ligand utilization of the affinity ligand became significantly improved. The affinity ligand could maintain high acidic (pH 1.5) and alkaline (pH 12) stabilities. Furthermore, target-binding selectivity could be altered by the originally developed CDR-grafting technology without diminishing their target-binding affinity levels. Thus, the rabbit scFv-based affinity ligand developed in the present study is highly available for a variety of biologics at the capture step in downstream processing.