Novel Strategy to Enhance Human Mesenchymal Stromal Cell Immunosuppression: Harnessing Interferon-Gamma Presentation in Metal-Organic Frameworks Embedded on Heparin/Collagen Multilayers.

The immunomodulatory potential of human mesenchymal stromal cells (hMSCs) can be boosted when exposed to interferon-gamma (IFN-γ). While pretreating hMSCs with IFN-γ is a common practice to enhance their immunomodulatory effects, the challenge lies in maintaining a continuous IFN-γ presence within cellular environments. Therefore, in this research, we investigate the sustainable presence of IFN-γ in the cell culture medium by immobilizing it in water-stable metal-organic frameworks (MOFs) [PCN-333(Fe)]. The immobilized IFN-γ in MOFs was coated on top of multilayers composed of combinations of heparin (HEP) and collagen (COL) that were used as a bioactive surface. Multilayers were created by using a layer-by-layer assembly technique, with the final layer alternating between collagen (COL) and heparin (HEP). We evaluated the viability, differentiation, and immunomodulatory activity of hMSCs cultured on (HEP/COL) coated with immobilized IFN-γ in MOFs after 3 and 6 days of culture. Cell viability, compared to tissue culture plastic, was not affected by immobilized IFN-γ in MOFs when they were coated on (HEP/COL) multilayers. We also verified that the osteogenic and adipogenic differentiation of the hMSCs remained unchanged. The immunomodulatory activity of hMSCs was evaluated by examining the expression of indoleamine 2,3-dioxygenase (IDO) and 11 essential immunomodulatory markers. After 6 days of culture, IDO expression and the expression of 11 immunomodulatory markers were higher in (HEP/COL) coated with immobilized IFN-γ in MOFs. Overall, (HEP/COL) multilayers coated with immobilized IFN-γ in MOFs provide a sustained presentation of cytokines to potentiate the hMSC immunomodulatory activity.

The effects of flux on the clearance of minute virus of mice during constant flux virus filtration.

Constant flux virus filtration experiments were conducted to evaluate minute virus of mice retention behavior of four commercial virus filters for continuous bioprocessing applications. Fluxes chosen were guided by the Peclet number and the processing logistics as well as based on the filter characteristics. At the low flux condition of 5 LM-2H-1 (LMH) when diffusive force dominates, a significant breakthrough was observed for all the filtrate fractions for the filtration of a low fouling monoclonal antibody for three of the four filters. When both diffusive and convective forces are equally important at 40 LMH, virus breakthrough in buffer chase was observed only in one of the four filters investigated. When convective force dominates at 60 LMH or above, a high degree of virus clearance was observed for all three parvovirus filters investigated. Our work shed light on virus clearance during constant flux virus filtration for future continuous biomanufacturing.

A Nanofiltration Decision Tool for Potable Reuse: A New Rejection Model for Recalcitrant CECs.

Effluent samples from three water resource recovery facilities (WRRFs) were individually characterized for presence and concentration of 94 different chemicals of emerging concern (CECs), using analytical methods with reporting limits in the low-parts-per-trillion. Following CEC analysis, each sample was subjected to dead-end, pressurized membrane filtration with either a nanofiltration (NF) or reverse osmosis (RO) membrane. The majority of the measured CECs were rejected by both membranes by 1-log removal (90%) or greater. However, nine of the 94 CECs had average rejection rates by the NF membrane less than 90%. A multilevel, multivariable model was developed to predict the probable rejection coefficients of CECs with the studied NF membrane. The resulting Quantitative Molecular Properties Model (QMPM) predicted the NF rejection of CECs based on size, ionic charge, and hydrophobicity. The model parameters that successfully predicted NF rejection in bench testing were log (Kow/Kaw) and the polar surface area of the CEC molecule.

Characterization of a Thermoresponsive Chitosan Derivative as a Potential Draw Solute for Forward Osmosis.

A thermoresponsive chitosan derivative was synthesized by reacting chitosan (CS) with butyl glycidyl ether (BGE) to break the inter- and intramolecular hydrogen bonds of the polymer. An aqueous solution of the thermoresponsive CS derivative exhibits a lower critical solution temperature (LCST) than CS, and it undergoes a phase transition separation when the temperature changes. Successful incorporation of BGE into the CS was confirmed by FTIR and XPS analyses. Varying the BGE content and the concentration of the aqueous solution produced different LCST ranges, as shown by transmittance vs temperature curves. The particle size was observed by scanning electron microscopy, which revealed that the particles were smaller and well dispersed at 15 °C, whereas the particles became larger and tended to aggregate at 60 °C. A similar trend was observed with the mean particle size measured using dynamic light scattering. Positron annihilation lifetime spectroscopy data also revealed the reversibility of the particle properties as a function of temperature. Microstructure analysis showed that the particles had larger free-volume sizes at 15 °C than at 60 °C. The particles were also found to be nontoxic with 92% cell survival. A simple forward osmosis (FO) test for dye dehydration revealed the potential use of the thermoresponsive chitosan derivative as a draw solute with a flux of 8.6 L/m2 h and rejection of 99.8%.

Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography.

Membrane surface engineering for protein separations: experiments and simulations.

A bisphosphonate derived ligand was successfully synthesized and grafted from the surface of regenerated cellulose membrane using atom transfer radical polymerization (ATRP) for protein separations. This ligand has a remarkable affinity for arginine (Arg) residues on protein surface. Hydrophilic residues N-(2-hydroxypropyl) methacrylamide (HPMA) was copolymerized to enhance the flexibility of the copolymer ligand and further improve specific protein adsorption. The polymerization of bisphosphonate derivatives was successful for the first time using ATRP. Static and dynamic binding capacities were determined for binding and elution of Arg rich lysozyme. The interaction mechanism between the copolymer ligand and lysozyme was elucidated using classical molecular dynamics (MD) simulations.

Proteomic analysis of host cell protein fouling during bioreactor harvesting.

Chinese hamster ovary (CHO) cells are among the most common cell lines used for therapeutic protein production. Membrane fouling during bioreactor harvesting is a major limitation for the downstream purification of therapeutic proteins. Host cell proteins (HCP) are the most challenging impurities during downstream purification processes. The present work focuses on identification of HCP foulants during CHO bioreactor harvesting using reverse asymmetrical commercial membrane BioOptimal™ MF-SL. In order to investigate foulants and fouling behavior during cell clarification, for the first time a novel backwash process was developed to effectively elute almost all the HCP and DNA from the fouled membrane filter. The isoelectric points (pIs) and molecular weights (MWs) of major HCP in the bioreactor harvest and fouled on the membrane were successfully characterized using two-dimensional gel electrophoresis (2D SDS-PAGE). In addition, a total of 8 HCP were identified using matrix-assisted laser desorption/ionization-mass spectroscopy (MALDI-MS). The majority of these HCP are enzymes or associated with exosomes, both of which can form submicron-sized particles which could lead to the plugging of the filters.

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.

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.

Use of scanning electron microscopy and energy dispersive X-ray spectroscopy to identify key fouling species during alternating tangential filtration.

Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, membrane fouling can cause product sieving losses that limit the performance of these systems. This study used scanning electron microscopy and energy dispersive X-ray spectroscopy to identify the nature and location of foulants on 0.2 µm polyethersulfone hollow fiber membranes after use in industrial Chinese hamster ovary cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet/exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.

Process- and Product-Related Foulants in Virus Filtration.

Regulatory authorities place stringent guidelines on the removal of contaminants during the manufacture of biopharmaceutical products. Monoclonal antibodies, Fc-fusion proteins, and other mammalian cell-derived biotherapeutics are heterogeneous molecules that are validated based on the production process and not on molecular homogeneity. Validation of clearance of potential contamination by viruses is a major challenge during the downstream purification of these therapeutics. Virus filtration is a single-use, size-based separation process in which the contaminating virus particles are retained while the therapeutic molecules pass through the membrane pores. Virus filtration is routinely used as part of the overall virus clearance strategy. Compromised performance of virus filters due to membrane fouling, low throughput and reduced viral clearance, is of considerable industrial significance and is frequently a major challenge. This review shows how components generated during cell culture, contaminants, and product variants can affect virus filtration of mammalian cell-derived biologics. Cell culture-derived foulants include host cell proteins, proteases, and endotoxins. We also provide mitigation measures for each potential foulant.

Retention and Fouling during Nanoparticle Filtration: Implications for Membrane Purification of Biotherapeutics.

One major challenge in the development of nanoparticle-based therapeutics, including viral vectors for the delivery of gene therapies, is the development of cost-effective purification technologies. The objective of this study was to examine fouling and retention behaviors during the filtration of model nanoparticles through membranes of different pore sizes and the effect of solution conditions. Data were obtained with 30 nm fluorescently labeled polystyrene latex nanoparticles using both cellulosic and polyethersulfone membranes at a constant filtrate flux, and both pressure and nanoparticle transmission were evaluated as a function of cumulative filtrate volume. The addition of NaCl caused a delay in nanoparticle transmission and an increase in fouling. Nanoparticle transmission was also a function of particle hydrophobicity. These results provide important insights into the factors controlling transmission and fouling during nanoparticle filtration as well as a framework for the development of membrane processes for the purification of nanoparticle-based therapeutics.

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.

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.

Magnetically activated micromixers for separation membranes.

Presented here is a radically novel approach to reduce concentration polarization and, potentially, also fouling by colloids present in aqueous feeds: magnetically responsive micromixing membranes. Hydrophilic polymer chains, poly(2-hydroxyethyl methacrylate) (PHEMA), were grafted via controlled surface-initiated atom transfer radical polymerization (SI-ATRP) on the surface of polyamide composite nanofiltration (NF) membranes and then end-capped with superparamagnetic iron oxide magnetite (Fe(3)O(4)) nanoparticles. The results of all functionalization steps, that is, bromide ATRP initiator immobilization, SI-ATRP, conversion of PHEMA end groups from bromide to amine, and carboxyl-functional Fe(3)O(4) nanoparticle immobilization via peptide coupling, have been confirmed by X-ray photoelectron spectroscopy (XPS) and field emission scanning electron microscopy (FESEM). These nanoparticles experience a magnetic force as well as a torque under an oscillating external magnetic field. It has been shown, using particle image velocimetry (PIV), that the resulting movement of the polymer brushes at certain magnetic field frequencies induces mixing directly above the membrane surface. Furthermore, it was demonstrated that with such membranes the NF performance could significantly be improved (increase of flux and salt rejection) by an oscillating magnetic field, which can be explained by a reduced concentration polarization in the boundary layer. However, the proof-of-concept presented here for the active alteration of macroscopic flow via surface-anchored micromixers based on polymer-nanoparticle conjugates has much broader implications.

Modeling flux in tangential flow filtration using a reverse asymmetric membrane for Chinese hamster ovary cell clarification.

Tangential flow filtration is advantageous for bioreactor clarification as the permeate stream could be introduced directly to the subsequent product capture step. However, membrane fouling coupled with high product rejection has limited its use. Here, the performance of a reverse asymmetric hollow fiber membrane where the more open pore structure faces the feed stream and the barrier layer faces the permeate stream has been investigated. The open surface contains pores up to 40 µm in diameter while the tighter barrier layer has an average pore size of 0.4 µm. Filtration of Chinese hamster ovary cell feed streams has been investigated under conditions that could be expected in fed batch operations. The performance of the reverse asymmetric membrane is compared to that of symmetric hollow fiber membranes with nominal pore sizes of 0.2 and 0.65 µm. Laser scanning confocal microscopy was used to observe the locations of particle entrapment. The throughput of the reverse asymmetric membrane is significantly greater than the symmetric membranes. The membrane stabilizes an internal high permeability cake that acts like a depth filter. This stabilized cake can remove particulate matter that would foul the barrier layer if it faced the feed stream. An empirical model has been developed to describe the variation of flux and transmembrane pressure drop during filtration using reverse asymmetric membranes. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor clarification.

Remote Performance Modulation of Ultrafiltration Membranes by Magnetically and Thermally Responsive Polymer Chains.

Ultrafiltration membranes, that respond to an external magnetic field and local temperature have been developed. Surface-initiated activator-generated electron transfer (AGET) atom transfer radical polymerization (ATRP) has been used to graft poly(N-isopropylacrylamide) (PNIPAm) from the surface of 300 kDa regenerated cellulose membranes. The polymerization initiator was selectively attached to the entire membrane surface, only the outer membrane surface or only the inner pore surface. A superparamagnetic nanoparticle was attached to the end of the polymer chain. The DI water flux as well as the flux and rejection of bovine serum albumin were investigated in the absence and presence of a 20 and 1000 Hz oscillating magnetic field. In an oscillating magnetic field, the tethered superparamagnetic nanoparticles can cause movement of the PNIPAm chains or induce heating. A 20 Hz magnetic field maximizes movement of the chains. A 1000 Hz magnetic field leads to greater induced heating. PNIPAm displays a lower critical solution temperature at 32 °C. Heating leads to collapse of the PNIPAm chains above their Lower Critical Solution Temperature (LCST). This work highlights the versatility of selectively grafting polymer chains containing a superparamagnetic nanoparticle from specific membrane locations. Depending on the frequency of the oscillating external magnetic field, membrane properties may be tuned.

Removal of Emerging Contaminants from Wastewater Streams Using Membrane Bioreactors: A Review.

Water is a very valuable natural resource. As the demand for water increases the presence of emerging contaminants in wastewater has become a growing concern. This is particularly true when one considers direct reuse of wastewater. Obtaining sufficient removal of emerging contaminants will require determining the level of removal for the various unit operations in the wastewater treatment process. Membrane bioreactors are attractive as they combine an activated sludge process with a membrane separation step. They are frequently used in a wastewater treatment process and can operate at higher solid loadings than conventional activated sludge processes. Determining the level of removal of emerging contaminants in the membrane bioreactor step is, therefore, of great interest. Removal of emerging contaminants could be by adsorption onto the biomass or membrane surface, biotransformation, size exclusion by the membrane, or volatilization. Given the fact that most emerging contaminants are low molecule weight non-volatile compounds, the latter two methods of removal are usually unimportant. However, biotransformation and adsorption onto the biomass are important mechanisms of removal. It will be important to determine if the microorganisms present at given treatment facility are able to remove ECs present in the wastewater.

Application of Zwitterions in Forward Osmosis: A Short Review.

Forward osmosis (FO) is an important desalination method to produce potable water. It was also used to treat different wastewater streams, including industrial as well as municipal wastewater. Though FO is environmentally benign, energy intensive, and highly efficient; it still suffers from four types of fouling namely: organic fouling, inorganic scaling, biofouling and colloidal fouling or a combination of these types of fouling. Membrane fouling may require simple shear force and physical cleaning for sufficient recovery of membrane performance. Severe fouling may need chemical cleaning, especially when a slimy biofilm or severe microbial colony is formed. Modification of FO membrane through introducing zwitterionic moieties on the membrane surface has been proven to enhance antifouling property. In addition, it could also significantly improve the separation efficiency and longevity of the membrane. Zwitterion moieties can also incorporate in draw solution as electrolytes in FO process. It could be in a form of a monomer or a polymer. Hence, this review comprehensively discussed several methods of inclusion of zwitterionic moieties in FO membrane. These methods include atom transfer radical polymerization (ATRP); second interfacial polymerization (SIP); coating and in situ formation. Furthermore, an attempt was made to understand the mechanism of improvement in FO performance by zwitterionic moieties. Finally, the future prospective of the application of zwitterions in FO has been discussed.

Electrospun Weak Anion-exchange Fibrous Membranes for Protein Purification.

Membrane based ion-exchange (IEX) and hydrophobic interaction chromatography (HIC) for protein purification is often used to remove impurities and aggregates operated under the flow-through mode. IEX and HIC are also limited by capacity and recovery when operated under bind-and-elute mode for the fractionation of proteins. Electrospun nanofibrous membrane is characterized by its high surface area to volume ratio and high permeability. Here tertiary amine ligands are grafted onto the electrospun polysulfone (PSf) and polyacrylonitrile (PAN) membrane substrates using UV-initiated polymerization. Static and dynamic binding capacities for model protein bovine serum albumin (BSA) were determined under appropriate bind and elute buffer conditions. Static and dynamic binding capacities in the order of ~100 mg/mL were obtained for the functionalized electrospun PAN membranes whereas these values reached ~200 mg/mL for the functionalized electrospun PSf membranes. Protein recovery of over 96% was obtained for PAN-based membranes. However, it is only 56% for PSf-based membranes. Our work indicates that surface modification of electrospun membranes by grafting polymeric ligands can enhance protein adsorption due to increased surface area-to-volume ratio.