Rapid affinity-based purification of multi-specific antibodies using Kappa Select and Protein L

Multispecific antibodies (msAbs) are becoming more prevalent as formats of choice for therapeutic antibody development due to their ability to modulate multiple biological targets. However, msAbs present unique protein production challenges due to product-related impurities, which are difficult to remove without loss of the protein of interest. Here, we report a versatile approach to remove product-related impurities by altering the binding affinity of light chains to Kappa Select (KS) or Protein L (Pro-L) resins. Introduction of amino acid mutations in the constant light chain domain or Framework 1 of the light chain abolished binding to KS and Pro-L resins, respectively, while antigen binding affinity remained intact. These purification-enabling mutations (PEMs) did not affect the thermal stability or purity of the proteins tested. In conjunction with PEMs, we demonstrate the design and application of an entirely affinity-based purification scheme employing Protein A (Pro-A), followed by KS and Pro-L affinity resins, to remove light chain mispaired species in Y-shaped bispecific antibodies and crossover dual variable domain (CODV) tri-specific antibodies. In principle, this purification scheme could be applied to any IgG-like msAb since it is compatible with Fc knobs-into-holes mutations and Fab arm charge-pair mutations. Moreover, it should be adaptable across a range of production scales and medium to high-throughput purification workflows within early-stage research.