Selective purification of antibody-based protein scaffolds with a novel variable heavy chain 3 affinity resin

• MabSelect VH3 resin removes IgG Fc binding while improving human VH3 binding. • MabSelect VH3 works effectively across a range of mAb and mAb-like scaffolds. • MabSelect VH3 has similar or better product quality attributes compared to PrismA. • MabSelect VH3 shows targeted impurity removal in asymmetric antibody purification. • MabSelect VH3 can bind human VH3 sequences shown to disrupt ProA binding. The evolution of biotherapeutics to effectively treat diseases has resulted in complex molecule scaffolds compared to traditional monoclonal antibodies (mAbs). While Protein A (ProA) affinity chromatography continues to be the predominant choice for affinity-based purification of traditional mAbs, it has also been deployed for primary capture of Fc-fusions, Fabs, single variable domain on a variable heavy chain (VHHs), and heteromAbs. When binding these different molecular scaffolds, ProA offers a high-affinity interaction with the Fc-domain, or in the case of antibody-like fragments such as Fabs and VHHs, a weaker affinity interaction with certain human VH3 sequences. As a result of the dual binding mechanism of ProA, the removal of product-related impurities, such as homodimer and half-body, can be challenging when deployed in the primary purification step of a heteromAb process. To address some of the key challenges faced in the purification of these novel scaffolds, we evaluated the unique purification capabilities of the MabSelect™ VH3 affinity resin compared to MabSelect PrismA™ for the capture of VHHs, Fab-VHH fusions, mAbs, and an asymmetric antibody. Our studies showed that the purification of mAb-based scaffolds with the new MabSelect VH3 resin has similar binding capacity with comparable product quality, decreased host cell protein (HCP) content of the eluted product pool, and increased binding affinity for human VH3 sequence compared to MabSelect PrismA. Furthermore, we highlighted the advantages of utilizing this novel selective affinity resin to remove molecule-specific impurities to purify an asymmetric antibody.