Flow-through polishing of Protein A eluates: Removal of high and low molecular weight impurities by...

The sustained growth of monoclonal antibody (mAb)-based therapies and the goal of reducing production costs to below $10 per gram of mAb by the end of the 2020 decade pose an urgent need for novel purification technologies that boost process intensification and cost reduction. While upstream advances now routinely deliver high harvest titers and convective Protein A adsorbents provide superior binding capacity, the efficient removal of high and low molecular weight (HMW and LMW) product-related impurities remains a key challenge in downstream bioprocessing. These impurities, often associated with host cell proteins and nucleic acids, compromise product purity and safety. Addressing the need for rapid and disposable bioprocess solutions, our team introduced a single-use adsorbent - SEMM-silica resin - that integrates size-exclusion and mixed-mode mechanisms for antibody polishing in flow-through mode. A panel of industrial mAbs, including IgG₁ and IgG₄ with pIs ranging between 6.1 and 9.2, was purified by Protein A chromatography and subjected to controlled enzymatic digestion to generate model feedstocks with diverse impurity profiles. The SEMM-silica resin achieved robust removal of LMW species, capturing up to 67 % of F(ab)₂, 44 % of Fab/Fc, and 44 % of polypeptide fragments, as well as up to 36 % of aggregates. This resulted in polished pools with monomeric mAb purities up to 99 %, and average LMW and HMW levels below 2 % and 1 %, respectively. Notably, the synergism of size-exclusion and mixed-mode mechanisms enabled product yields above 90 % at a residence time of 6 min, translating into productivities of up to 50 g/L-h.