Electrospun nanofiber membranes surface functionalized with 3D nanolayers through ATRP provide adsorption capacities over 50-times higher than current commercial membrane adsorption systems and over 12-times higher than packed bed resins; additionally, the adsorption kinetics remain 10-times faster than packed bed resins and have over 15-times higher permeance.

No abstract

Fabrication of membrane adsorbers with elevated binding capacity and high throughput is highly desired for simplifying and improving purification efficiencies of bioproducts (biotherapeutics, vaccines, etc.) in the biotechnological and biopharmaceutical industries. Here we demonstrate the preparation of a novel class of self-supported, cellulose-graft-polypropionic acid (CL-g-PPA) cation-exchange nanofiber membrane adsorbers under mild reaction conditions for the purification of positively charged therapeutic proteins. In our fabrication method, acrylonitrile was first polymerized and surface grafted onto cellulose nanofibers using cerium ammonium nitrate as a redox initiator to form cellulose-g-polyacrylonitrile (CL-g-PAN). CL-g-PAN was then submitted to a hydrolyzation reaction to form CL-g-PPA cationic membrane adsorbers. Morphology and structural characterization illustrated the formation of CL-g-PPA membranes with uniform coating of polyacid nanolayers along the individual nanofibers without disturbing the nanofiber structure. Benefiting from these numerous cationic polyacid binding sites and inherent large surface area and open porous structure, CL-g-PPA nanofiber membrane adsorbers showed a lysozyme static adsorption capacity of 1664 mg/g of nanofibers. These membranes showed a lysozyme dynamic binding capacity of 508 mg/g of nanofibers at 10% breakthrough (equivalent to 206 g/L capacity), with a residence time of less than 6 s. Moreover, CL-g-PPA self-supported nanofibers displayed excellent structural stability and reversibility after several cycles of protein binding studies. This dynamic binding capacity of the CL-g-PPA nanofiber membranes was 3.2 times higher than that of macroporous cellulose membranes and 8.5 times higher than that of the Sartobind S commercial membrane adsorber. Considering the simple fabrication method employed, excellent protein adsorption capacity, remarkable structural stability, and reusability, CL-g-PPA nanofiber membranes provided a versatile platform for the chromatographic separations of biomolecules (e.g., proteins, nucleic acids, and viral vaccines) as well as water purification and similar ion-exchange applications.

Development of bioseparation media that can perform separations based on both ion exchange and size exclusion provide a transformative technology to purify therapeutic proteins with fewer operations in downstream processing. Thus, in this study, we have developed cellulose-graf tpolyethyleneamidoamine (CL-g-PEAA) anion-exchange nanofiber membrane adsorbers with a narrow effective pore size distribution using redox polymerization. Structural characterization illustrated the functionalization of each nanofiber of the CL-g-PEAA membrane adsorbers with three-dimensional nanolayers of amidoamine functionality. The ion-exchange separation efficiency of the CL-g-PEAA anion-exchange membranes, evaluated using bovine serum albumin (BSA), exhibited an excellent static adsorption of 239 mg/g of nanofibers. The BSA dynamic binding capacity in flow through mode at 10% breakthrough of these membranes was 69 mg/g (corresponding to a volume capacity of 31 g/L) with an approximate residence time of 8 s. Meanwhile, CL-g-PEAA membranes have shown 80−100% rejection for model bead particles of various sizes (200, 100, and 40 nm), which illustrated the potential in removing viruses, microbial agents, and cellular debris from the harvested cell culture fluid (HCCF) during downstream processing. Overall performance of the CL-g-PEAA membranes illustrated that undesired impurities (viruses, host cell proteins, and DNA) in HCCF could be removed in a single step by an anion-exchange membranes with ion-exchange and size-exclusion-based separation mechanisms.

In recent years, the market for therapeutic monoclonal antibodies (mAb) has grown exponentially, and with this there has been a desire to reduce the costs associated with production and purification of these high-value biological products. A typical mAb purification process involves three adsorption/chromatography steps [protein A, ion exchange (IEX), and hydrophobic interaction (HIC)], along with ultrafiltration, nanofiltration, and microfiltration. With the development of membrane adsorption/chromatography as a viable alternative to traditional pack bed systems, the opportunity exists to complete the entire downstream purification process using only membrane operations. In this study, the process simulation tool SuperPro Designer was used to evaluate the application of recently developed ultra-high capacity electrospun nanofibrous adsorption membranes as a replacement for conventional chromatographic media in the downstream mAb production process. The simulation showed that nanofibrous adsorption membranes in place of the three packed bed chromatography steps reduced the required volume of protein A, IEX, and HIC adsorptive medium by 25, 80, and 80%, respectively. In addition, the membrane-only process reduced the downstream processing time by 50%, decreased the number of labor hours associated with the purification steps by 40%, generated 40% less aqueous waste, and reduced the overall downstream process operating expenses per unit product by 23%. There were also significant savings in facility construction costs and the price of fixed equipment required for separations. With these savings not only is the membrane-only process economically competitive with the traditional packed bed operations, but it offers the possibility of moving toward more disposable process.