Protein purification is an increasingly important problem in the current era of bioengineering, medicine, and biological research. Modern biomanufacturing schemes attribute up to 80% of process costs to purification processes. Unfortunately, existing separation schemes rely on batch-process chromatography columns that are diffusion mass transport-limited and have already reached optimal maximum loading densities and throughputs for bioseparations. [1]

Our present work improves a previously-demonstrated bioinspired separations concept that utilizes chemically functionalized, mesoporous, gold electrodes decorating the surface of anodized alumina membranes with well-controlled pore sizes. This separations platform is inspired by cellular transmembrane ATP-binding cassette (ABC) transporters that enable selective transport of nutrients across the cell membrane. In ABC transporters, a nutrient first binds selectively to the transporter and induces conformational changes in the transporter that effectively blocks the transporter active site and closes the transporter. Subsequently, the transporter hydrolyzes ATP to induce nutrient release, eluting the nutrient into the cellular cytoplasm. Finally, the transporter reforms into its original conformation and is ready to repeat the cycle.

In our system, proteins are selectively separated across an alumina membrane in a similar two-step fashion that alternates between “open” pores and sterically “closed” pores. This system is enabled by three critical components: (1) the histidine tag-based affinity chromatography chemistry for selective binding and elution, (2) timed electrochemical pulses to trigger the two steps in the transport cycle, and (3) affinity chemistry-modified, gold nanoscale mesoporous electrodes on the alumina membrane with characteristic pore diameter on the same size scale as target protein size (typically 5-10nm).

In the first step, the binding step, an electrochemical pulse triggers electrophoretic transport of proteins to the surface of the membrane. At the membrane surface, proteins bind to the affinity chemistry and induce steric blocking of the mesopores. Captured target proteins simultaneously create a steric hindrance effect, blocking transmembrane transport of non-specifically bound proteins during the binding cycle. In the second step, the elution step, imidazole is electrophoretically pumped from the permeate side of the membrane towards the membrane surface with bound protein. Imidazole induces elution of the protein and thus the imidazole concentration is carefully controlled to keep the top surface of the membrane pores blocked while facilitating protein release at the bottom edge of the electrode inside the membrane pores.

This concept was successfully demonstrated and showed a separation factor for GFP:BSA of 16:1 (mass/mass). Additionally, the net transmembrane protein flux (320 ng cm^-2 hr^-1) yields a daily throughput for a 0.75 cm² area membrane as comparable to 1 mL of chromatography resin. However, the prior work was not optimized and was performed in a batch operation. [2]

This work further develops the concept into a scalable, continuous separation process optimized for increased throughput and improved separation factor. A laser-machined, thermally-fused thermoplastic acrylic flow cell enables continuous operation, pH control, and finer control of operational details. Process optimizations are realized by improvements in the membrane fabrication and preparation protocol, binding and release reaction kinetics modeling, and electrophoretic transport . The optimizations improved the separation factor >20 and increased flux from 320 ng cm^-2 hr^-1 to >1000 ng cm^-2 hr^-1.

[1] S. S. Farid, Journal of Chromatography B, 848, 8–18 (2007). [2] Z. Chen, T. Chen, X. Sun, and B. J. Hinds, Advanced Functional Materials, 24, 4317–4323 (2014).