Scott M. Husson

Anion exchange membrane adsorbers for flow-through polishing steps: Part II. Virus, host cell protein, DNA clearance, and antibody recovery.

Anion exchange membrane adsorbers are used for contaminant removal in flow‐through polishing steps in the manufacture of biopharmaceuticals. This contribution describes the clearance of minute virus of mice, DNA, and host cell proteins by three commercially available anion‐exchange membranes: Sartobind Q, Mustang Q, and ChromaSorb. The Sartobind Q and Mustang Q products contain quaternary amine ligands; whereas, ChromaSorb contains primary amine based ligands. Performance was evaluated over a range of solution conditions: 0–200 mM NaCl, pH 6.0–9.0, and flow rates of 4–20 membrane volumes/min in the presence and absence of up to 50 mM phosphate and acetate. In addition contaminant clearance was determined in the presence and absence of 5 g/L monoclonal antibody. The quaternary amine based ligands depend mainly on Coulombic interactions for removal of negatively charged contaminants. Consequently, performance of Sartobind Q and Mustang Q was compromised at high ionic strength. Primary amine based ligands in ChromaSorb enable high capacities at high ionic strength due to the presence of secondary, hydrogen bonding interactions. However, the presence of hydrogen phosphate ions leads to reduced capacity. Monoclonal antibody recovery using primary amine based anion‐exchange ligands may be lower if significant binding occurs due to secondary interactions. The removal of a specific contaminant is affected by the level of removal of the other contaminants. The results of this study may be used to help guide selection of commercially available membrane absorbers for flow‐through polishing steps. Biotechnol. Bioeng. 2013; 110: 500–510.

Membrane chromatography: Protein purification from E. coli lysate using newly designed and commercial anion-exchange stationary phases

This contribution describes the purification of anthrax protective antigen (PA) protein from Escherichia coli lysate using bind-and-elute chromatography with newly designed weak anion-exchange membranes. Protein separation performance of the new AEX membrane adsorber was compared with the commercial Sartobind D membrane adsorber and HiTrap DEAE FF resin column under preparative scale conditions. Dynamic protein binding capacities of all three stationary phases were determined using breakthrough curve analysis. The AEX membrane showed higher binding capacities than the Sartobind D membrane at equivalent volumetric throughput and higher capacities than the HiTrap DEAE FF resin column at 15 times higher volumetric throughput. Anion-exchange chromatography was performed using all three stationary phases to purify PA protein. Quantitative SDS-PAGE analysis of effluent fractions showed that the purity of PA protein was higher for membrane adsorbers than the HiTrap DEAE FF resin column and was the same for the new AEX membrane and Sartobind D membrane adsorbers. The effects of E. coli lysate load volume and volumetric flow rate on PA protein separation resolution using the membrane adsorbers were minor, and the peak elution profile remained un-changed even under conditions where >75% of the total protein dynamic binding capacity of the membranes had been utilized. PA protein peak resolution was higher using pH-gradient elution than with ionic strength gradient elution. Overall, the results clearly demonstrate that membrane chromatography is a high-capacity, high-throughput, high-resolution separation technique, and that resolution in membrane chromatography can be higher than resin column chromatography under preparative conditions and at much higher volumetric throughput.

Anion exchange membrane adsorbers for flow‐through polishing steps: Part I. clearance of minute virus of mice

Membrane adsorbers may be a viable alternative to the packed‐bed chromatography for clearance of virus, host cell proteins, DNA, and other trace impurities. However, incorporation of membrane adsorbers into manufacturing processes has been slow due to the significant cost associated with obtaining regulatory approval for changes to a manufacturing process. This study has investigated clearance of minute virus of mice (MVM), an 18–22 nm parvovirus recognized by the FDA as a model viral impurity. Virus clearance was obtained using three commercially available anion exchange membrane adsorbers: Sartobind Q®, Mustang Q®, and ChromaSorb®. Unlike earlier studies that have focused on a single or few operating conditions, the aim here was to determine the level of virus clearance under a range of operating conditions that could be encountered in industry. The effects of varying pH, NaCl concentration, flow rate, and other competing anionic species present in the feed were determined. The removal capacity of the Sartobind Q and Mustang Q products, which contain quaternary ammonium based ligands, is sensitive to feed conductivity and pH. At conductivities above about 20 mS/cm, a significant decrease in capacity is observed. The capacity of the ChromaSorb product, which contains primary amine based ligands, is much less affected by ionic strength. However the capacity for binding MVM is significantly reduced in the presence of phosphate ions. These differences may be explained in terms of secondary hydrogen bonding interactions that could occur with primary amine based ligands. Biotechnol. Bioeng. 2013; 110: 491–499.

Modification of Nanofiltration Membranes by Surface-Initiated Atom Transfer Radical Polymerization for Produced Water Filtration

Abstract Poly(N-isopropylacrylamide) and poly(N-isopropylacrylamide-block-ethylene glycol methacrylate) were grafted from commercial polyamide thin-film composite nanofiltration membranes using surface-initiated atom transfer radical polymerization. The results from Fourier-transform infrared spectroscopy confirmed the successful grafting of both polymers from the membrane surfaces. Contact angle measurements were done to illustrate the temperature responsive wettability of the modified membrane surfaces. Modified membranes were used in flux measurements of wastewater produced during coal bed methane gas exploration. The degree of fouling of the original and the modified membranes was examined by pure water flux recovery measurements after produced water filtration and a temperature-controlled water rinse.

The role of polymer nanolayer architecture on the separation performance of anion‐exchange membrane adsorbers: I. Protein separations

This contribution describes the preparation of strong anion‐exchange membranes with higher protein binding capacities than the best commercial resins. Quaternary amine (Q‐type) anion‐exchange membranes were prepared by grafting polyelectrolyte nanolayers from the surfaces of macroporous membrane supports. A focus of this study was to better understand the role of polymer nanolayer architecture on protein binding. Membranes were prepared with different polymer chain graft densities using a newly developed surface‐initiated polymerization protocol designed to provide uniform and variable chain spacing. Bovine serum albumin and immunoglobulin G were used to measure binding capacities of proteins with different size. Dynamic binding capacities of IgG were measured to evaluate the impact of polymer chain density on the accessibility of large size protein to binding sites within the polyelectrolyte nanolayer under flow conditions. The dynamic binding capacity of IgG increased nearly linearly with increasing polymer chain density, which suggests that the spacing between polymer chains is sufficient for IgG to access binding sites all along the grafted polymer chains. Furthermore, the high dynamic binding capacity of IgG (>130 mg/mL) was independent of linear flow velocity, which suggests that the mass transfer of IgG molecules to the binding sites occurs primarily via convection. Overall, this research provides clear evidence that the dynamic binding capacities of large biologics can be higher for well‐designed macroporous membrane adsorbers than commercial membrane or resin ion‐exchange products. Specifically, using controlled polymerization leads to anion‐exchange membrane adsorbers with high binding capacities that are independent of flow rate, enabling high throughput. Results of this work should help to accelerate the broader implementation of membrane adsorbers in bioprocess purification steps. Biotechnol. Bioeng.

Synthesis and characterization of molecularly imprinted polymers for chromatographic separations

Abstract: The primary objective of this investigation was to discover the optimum solution-phase synthesis conditions for preparing molecularly imprinted polymers (MIPs) against Boc-L-Trp using a binary monomer system. Methacrylic acid (MAA) and 2-vinylpyridine (2-Vpy) were used as the two functional monomers with ethylene glycol dimethacrylate (EGDMA) as the cross-linker. Polymers prepared from two monomers showed improved binding strength and recognition capabilities compared to polymers that were prepared using a single monomer. The synergistic effect on binding was most pronounced for polymers prepared from a starting MAA : 2-Vpy molar ratio of about 1 :5. A correlation was developed based on molecular interaction scales to relate the template-MIP binding strength to M1P composition. This correlation provides insights on the most important interaction types for the system studied; it also provides an explanation for the improved binding seen with a mixed monomer system. Polymerization temperature and cross-linking density were also investigated for their effects on the MIP recognition properties.

The role of polymer nanolayer architecture on the separation performance of anion-exchange membrane adsorbers: part II. DNA and virus separations.

The surface‐initiated polymerization protocol developed in part I was used to prepare strong anion‐exchange membranes with variable polymer chain graft densities and degrees of polymerization for DNA and virus particle separations. A focus of part II was to evaluate the role of polymer nanolayer architecture on DNA and virus binding. Salmon sperm‐DNA (SS‐DNA) was used as model nucleic acid to measure the dynamic‐binding capacities at 10% breakthrough. The dynamic‐binding capacity increases linearly with increasing poly ([2‐(methacryloyloxy)ethyl]trimethylammonium chloride) chain density up to the highest chain density used in this study. The new membranes yielded threefold higher SS‐DNA‐binding capacity (30 mg/mL) than a leading commercial membrane with the same functional group chemistry. Elution of bound DNA yielded a sharp peak, and resulted in a 13‐fold increase relative to the feed concentration. This concentration effect further demonstrates the highly favorable transport properties of the newly designed Q‐type membranes. However, unlike findings in part I on protein binding, SS‐DNA binding was not fully reversible. Minute virus of mice (MVM) was used as model virus to evaluate the virus clearance performance of newly designed Q‐type membranes. Log reduction of virus (LRV) of MVM increased with increasing polymer chain density. Membranes exhibited >4.5 LRV for the given MVM impurity load and may be capable of higher LRV values, as the MVM concentration in the flow‐through fraction of these samples was below the limit of detection of the assay. Biotechnol. Bioeng. 2011;108: 2654–2660.

Preparation of polymer-coated, scintillating ion-exchange resins for monitoring of 99Tc in groundwater.

The present study was oriented to prepare new scintillating anion-exchange resins for measurement of (99)TcO(4)(-) in natural waters. The organic fluor 2-(1-naphthyl)-5-phenyloxazole was diffused into (chloromethyl)polystyrene resin. Thereafter, a thin layer of poly[[2-(methacryloyloxy)ethyl]trimethylammonium chloride] was grafted from the resin surface by surface-initiated atom transfer radical polymerization as an attempt to overcome potential problems related to the leaching of fluor molecules during usage. The residual chloromethyl groups of the polymer-coated resin were aminated by reaction with two different tertiary amines, triethylamine (TEA) and methyldioctylamine (MDOA). Off- and on-line quantification of (99)Tc was achieved with high detection efficiencies of 60.72 ± 1.93% and 72.83 ± 0.81% for resin with TEA and MDOA functional groups, respectively. The detection limit was determined to be less than the maximum contaminant level (33 Bq L(-1)) established under the Safe Drinking Water Act. The two functionalized resins were demonstrated to be selective for pertechnetate from synthetic groundwater containing up to 1000 ppm Cl(-), SO(4)(2-), and HCO(3)(-) and up to 1200 ppb Cr(2)O(7)(2-) in an acidic medium.

Development of polymerizable 2-(1-naphthyl)-5-phenyloxazole scintillators for ionizing radiation detection

The synthesis, chemical characterization and optical properties of 2-(1-naphthyl)-4-vinyl-5-phenyloxazole (vNPO) and 2-(1-naphthyl)-4-allyl-5-phenyloxazole (allylNPO) monomers are reported. Starting with the organic fluor 2-(1-naphthyl)-5-phenyloxazole (αNPO), the vNPO and allylNPO monomers were synthesized using Stille coupling followed by purification. The final products were obtained with yields of ∼95% and ∼55% for vNPO and allylNPO. The absorption/emission spectra of αNPO, vNPO and allylNPO revealed that vNPO has the largest red-shifted in emission with an average wavelength of ∼420 nm, which is an advantage for increasing photomultiplier tube sensitivity without the need to add a wavelength shifter. Stable scintillating resin beads were prepared through copolymerization of the newly synthesized fluor monomers with styrene or 4-methylstyrene and divinylbenzene in the presence of toluene porogen. The resin beads were chemically stable and retained the ability to scintillate efficiently after energy deposition of beta particles from 99Tc. This result indicates efficient energy transfer occurs from the base polymer to the covalently attached fluors with subsequent fluorescence in the presence of ionizing radiation.

GAS ANTISOLVENT-INDUCED REGENERATION OF LACTIC ACID-LADEN EXTRACTANTS

Extraction equilibria are presented for reversible chemical complexation of lactic acid by the liquid tertiary amine extractant, trin-octylamine, in mixed diluents composed of 1-octanol and the gas antisolvent, propane. Experimental data were collected that show that the effect of increasing propane partial pressure during extraction is to decrease the acid loading of tri-n-octylamine. This result is discussed. A simple complexation model was employed to predict and to fit the experimental results. The model uses the Law of Mass Action to describe reaction equilibria and Wohls expansion to estimate the solubility of propane in the organic-phase mixture. Finally, estimated energy requirements are presented for a gas antisolvent-induced regeneration process for acid-laden basic extractants; results are compared with those for a conventional diluentswing regeneration process.