Highly selective Protein A resin allows for mild sodium chloride-mediated elution of antibodies

Abstract

The manufacturability of therapeutic monoclonal antibodies is limited by the harsh conditions that antibodies are subjected to during the purification procedure, which in turn restricts the development of novel acid-sensitive antibodies. The gold standard for antibody purification, Protein A affinity chromatography, offers the selective capture of antibodies with great yields, but also poses a threat to the quality of the antibodies. Antibodies and Fc-fusion proteins risk forming aggregates as a consequence of the acidic elution from the Protein A ligands, compromising the potency and safety of the drug. Here, we present a novel, mild purification strategy based on a calcium-dependent ligand derived from Protein A, called ZCa. Antibodies captured on a high-capacity tetrameric ZCa resin in the presence of calcium can be eluted by removing the calcium ions through the addition of a chelator, and we describe the strive to find a sustainable alternative to the previously applied chelator EDTA. The naturally occurring chelator citrate is shown to seamlessly replace EDTA. Further buffer optimization reveals that the elution can be considerably improved by increasing the conductivity through the addition of 300 mM sodium chloride, leading to a very concentrated eluate. Remarkably, merely sodium chloride at a concentration of 50 mM is proven to be sufficient for calcium-dependent antibody release in a cost-efficient manner. Antibodies of subclasses IgG2 and IgG4 are eluted with sodium chloride at neutral pH and IgG1 at pH 6, due to varying affinities for the tetrameric ZCa, ranging between 90-780 nM. The mild elution of an IgG4 antibody eliminated the formation of aggregates, which constituted as much as 34% of all eluted antibody from MabSelect SuRe at pH 3. This novel purification strategy thus combines the valuable qualities of a Protein A resin, by providing high selectivity and a recovery of 88-99%, with an exceptionally mild elution step similar to ion-exchange chromatography, rendering considerably more functional antibody.