Biopharmaceutical manufacturing has been using ultrafiltration (UF) and diafiltration (DF) for buffer exchange, desalting, and formulation of biologics. The legacy UF/DF is commonly a two-step batch process that is challenging to integrate into end-to-end continuous biomanufacturing. Here, we introduce asymmetric dialysis, a novel one-step continuous process that combines UF and DF. It works by utilizing asymmetric flow between the inlet and outlet of the retentate and complementary flow of the dialysate solution, achieving product concentration, buffer exchange, and salt removal using a commercially available hollow fiber device. Asymmetric dialysis can achieve product concentrations of 105 g/L (3.8x), 200 g/L (10x), 64 g/L (9.4x) starting from feed concentrations of 30 g/L, 20 g/L, and 7 g/L, respectively, with modest pressures of 6-7 psi. The interplay between feed and exchange buffer flow rates was exploited to make the process sustainable by reducing buffer consumption by 75% (25 L/kg mAb) compared to conventional batch UF/DF (100 L/kg, mAb). We successfully processed 7 kg of mAb at 20 g/L feed using 5-day asymmetric dialysis with a daily productivity of 0.8 kg/m 2/day to product concentration of 200 g/L. These results demonstrate the potential of asymmetric dialysis a simple, sustainable, and low-cost bioprocessing technology continuous bioprocessing.

In the manufacture of therapeutic monoclonal antibodies (mAbs), the clarified cell culture fluid is typically loaded onto an initial protein A affinity capture column. Imperfect mass transfer and loading to maximum capacity can risk antibody breakthrough and loss of valuable product, but conservative underloading wastes expensive protein A resin. In addition, the effects of column fouling and ligand degradation require the frequent optimization of IgG loading to avoid wastage. Therefore, continuous real-time monitoring of IgG flowthrough is of great interest. We previously developed a fluorescence-based monitoring technology that allows mix-and-read mAb detection in cell culture fluid. Here we report the use of reporters immobilized on CNBr-activated Sepharose 4B resin for continuous detection of IgG in column breakthrough. The column effluent is continuously contacted with immobilized fluorescein-labeled Fc-binding ligands to produce an immediately detectable change in fluorescence intensity. The technology allows rapid and reliable monitoring of IgG in a flowing stream of clarified cell culture fluid emerging from a Protein A column, without prior sample preparation. We observed a significant change in fluorescence intensity at 0.5 g/L human IgG, sufficient to detect a 5% breakthrough of a 10 g/L load, within 2 minutes at a flow rate of 0.5 mL/min.