High throughput purification of monoclonal recombinant antibodies using a Protein-A coated membrane plate system.

The therapeutic use of monoclonal antibodies (mAbs) ranges from cancer treatment to immune-mediated conditions, covering infectious and cardiovascular disorders, among others. The development of improved methods for therapeutic antibody discovery has accelerated the identification of numerous mAbs: a discovery campaign can be deeply mined, resulting in hundreds, even thousands, of potential antibody leads for a given target of interest. High throughput mAb expression and purification methods are required for the rapid validation of those leads. In this work, we describe the implementation of a Protein-A coated membrane plate system, the Purexa™ AHT membrane plate, for robust preparative purification of hundreds of recombinant mAbs, without the need for automation. The high efficiency (>80%) recovery generated sufficient mAb for downstream screening analyses such as ELISA and surface plasmon resonance (SPR). This new system allows the functional validation of hundreds of lead antibodies from discovery campaigns in a timely manner regardless of operational size.

Stability control of high-speed magnetic levitation turbomolecular pumps with shock-excited disturbance.

The disturbance suppression of magnetic levitation turbomolecular pumps is a critical problem in industrial applications. This work addresses the stability control of high-speed magnetic levitation turbomolecular pumps with shock-excited disturbance. A disturbance suppression method based on improved linear extended state observer is proposed to attenuate the impact of external low-frequency disturbing force on a magnetic levitation turbomolecular pump. Firstly, a linear extended state observer of an active magnetic bearing is obtained by analyzing the rotor dynamics model. Then, the detailed descriptions of external disturbance suppression method using linear extended state observers and adaptive notch filters are discussed for the system. The boundary condition of the parameters of the improved linear extended state observer is determined. The root loci of the closed-loop system with improved linear extended state observers is also investigated. Finally, simulation and experimental results on a magnetic levitation turbomolecular pump show the applicability of the proposed method. The results show that the proposed method can attenuate the rotor vibration displacement caused by impact by 46.9%.

High-capacity multimodal anion-exchange membranes for polishing of therapeutic proteins.

This contribution reports on a study using Purexa™-MQ multimodal anion-exchange (AEX) membranes for protein polishing at elevated solution conductivities. Dynamic binding capacities (DBC10 ) of bovine serum albumin (BSA), human immunoglobulins, and salmon sperm DNA (ss-DNA) are reported for various salt types, salt concentrations, flowrates, and pH. Using 1 mg/ml BSA, DBC10 values for Purexa™-MQ were >90 mg/ml at conductivities up to 15 mS/cm. The membranes maintained a high, salt-tolerant BSA DBC10 of 89.8 ± 2.7 (SD) over the course of 100 bind-elute cycles. Polishing studies with acidic and basic monoclonal antibodies at >2 kg/L loads showed that Purexa™-MQ had higher clearance of host cell proteins and aggregate species at high conductivity (13 mS/cm) and in the presence of phosphate than other commercial AEX media. Purexa™-MQ also had a high ss-DNA DBC10 of 50 mg/ml at conductivities up to 15 mS/cm, markedly outperforming other commercial products. In addition to the effectiveness of Purexa™-MQ for protein polishing at elevated solution conductivities, its unusually high binding capacity for ss-DNA indicates potential applications for plasmid DNA purification.

Low-Energy Membrane Process for Concentration of Stick Water.

This communication describes the application of forward osmosis (FO) to concentrate stick water, a nutrient-rich water byproduct of meat rendering operations. The objectives of the study were to carry out a set of batch FO runs in concentration mode to determine the maximum achievable stick water concentration and to perform a preliminary cost analysis for operating a FO/reverse osmosis membrane separation process for comparison to an evaporative concentration process. The study examined the roles of feed and draw solution stir rates, temperature, feed concentration, and draw solution ionic strength on flux using commercial cellulose triacetate membranes. Results show that FO could concentrate the stick water up to 45 wt %; however, concentrations above about 30 wt % would be difficult to process through conventional membrane configurations. Preliminary operating cost estimations show that the energy cost of the FO process is about 5.3% of the energy costs for a single-effect thermal evaporation process; and, assuming a 2-year membrane lifetime, the total operating cost using FO membranes was estimated to be about 23.1% of the operating cost using such a thermal evaporation process.

Antibody purification from CHO cell supernatant using new multimodal membranes.

This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG1 following a size-exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two-step MMM chromatography process achieved high selectivity for capturing hIgG1 from the CHO cell culture supernatant, though the desalting step resulted in product dilution. Product purity and host cell protein (HCP) level in the elution pool were analyzed and compared to results from a commercial Protein A column. The product purity was >98% and HCP levels were <20 ppm for both purification methods. In addition, hIgG1 could be eluted from the MMM chromatography column at neutral pH, which is important for limiting the formation of aggregates; although slow elution dilutes the product. Overall, this paper shows that MMMs are highly effective for capture step purification of proteins and should be considered when Protein A cannot be used, e.g., for pH sensitive mAbs or proteins lacking an Fc binding domain. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:658-665, 2017.

Polishing Step Purification of High-Strength Wastewaters by Nanofiltration and Reverse Osmosis.

This article reports findings on the use of nanofiltration (NF) and reverse osmosis (RO) for secondary treatment of high-strength rendering facility wastewaters following an ultrafiltration step. These wastewaters present significant challenges to classical treatment technologies. Constant-pressure, direct-flow membrane filtration experiments were done to screen for flux and effluent water permeate quality of ten commercial NF and RO membranes. All membranes tested were effective in reducing total dissolved salts (TDS) and chemical oxygen demand (COD); however, only two membranes (Koch MPF-34 and Toray 70UB) gave sufficiently stable flux values to warrant longer term cross-flow filtration studies. Cross-flow flux measurements, scanning electron microscopy (SEM), X-ray dispersive spectroscopy (EDS), and attenuated total reflectance-Fourier-transform infrared spectroscopy (ATR-FTIR) indicated that both membranes were eventually fouled by organic and inorganic foulants; however, the Toray 70UB RO membrane yielded a capacity of 1600 L/m² prior to cleaning. A preliminary economic analysis compared the estimated costs of energy and consumables for a dual-stage UF/RO membrane process and dissolved air floatation (DAF) and found membrane process costs could be less than about 40% of the current DAF process.