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.

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.

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.

Factors affecting robustness of anion exchange chromatography: Selective retention of minute virus of mice using membrane media.

Mobile and stationary phase factors were investigated in order to identify conditions for effective capture of minute virus of mice (MVM), a potential adventitious contaminant in biomanufacturing, using anion exchange membrane chromatography (AEX). The initial study was conducted for Membrane A for a range of feed conditions using bovine serum albumin (BSA) as a model protein mimicking acidic host-cell proteins (HCPs) competitive for virus binding. The effects of pH (6-8), salt concentration (0-150 mM NaCl) and level of BSA (0-10 g/L) were systematically investigated. It was found that higher BSA concentration has the most negative impact on MVM binding followed by the increased conductivity of the feed solution. The effect of pH on MVM binding is also detected but has a less impact compared to other two factors in the range of feed conditions investigated. In addition to Membrane A, three other AEX membranes (Membrane B, C and D) were investigated for MVM binding at a selected feed condition. Based on properties of the membranes investigated, it was found that ligand charge density has the most significant impact on MVM binding performance of AEX membranes from stationary phase perspective.

Unusual case of post-operative suicide left ventricle in a patient with dynamic LVOT obstruction.

Suicide left ventricle (SLV) is a well-documented complication after surgical or transcatheter aortic valve replacement. We present an unusual case of a patient who developed left ventricular outflow tract (LVOT) obstruction with a native aortic valve, resulting in SLV after routine non-cardiac surgery. A 45-year-old male presented to the emergency room with abdominal pain and was diagnosed with acute cholecystitis. The patient had a known medical history of severe left ventricular hypertrophy. The patient underwent an uncomplicated laparoscopic cholecystectomy. Post-operatively, he went into shock during weaning from anesthesia. He was started on norepinephrine followed by epinephrine and vasopressin, without much improvement. Increasing doses of vasopressors failed to improve the patient's hemodynamics. A presumptive diagnosis of SLV was made. This was secondary to hemodynamic collapse due to vasoplegia from anesthesia, worsening LVOT obstruction and subsequent right ventricular failure. Despite being in shock, the patient was taken off pressors and started on esmolol infusion to increase diastolic filling and epoprostenol to decrease the right ventricle strain by pulmonary vasodilation. The patient responded promptly to these measures. A repeat echocardiogram showed a significant improvement in right and left ventricular function. Learning objective: Suicide left ventricle (SLV) is commonly seen in patients post aortic valve replacement. It presents as shock which does not respond to pressors and instead is treated by beta-blockers. Our patient developed SLV pathophysiology despite having native aortic valve. He developed shock which did not improve with pressors but responded to esmolol. This emphasizes the importance of fluid management in patients with severe left ventricular outflow tract obstruction. It also gives a different perspective to managing shock in such patients who are not responding to pressors.

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.

Recent developments in porous ceramic membranes for wastewater treatment and desalination: A review.

The development of membrane technology has proved vital in providing a sustainable and affordable supply of clean water to address the ever-increasing demand. Though liquid separation applications have been still dominated by polymeric membranes, porous ceramic membranes have gained a commercial foothold in microfiltration (MF) and ultrafiltration (UF) applications due to their hydrophilic nature, lower fouling, ease of cleaning, reliable performance, robust performance with harsh feeds, relative insensitivity to temperature and pH, and stable long-term flux. The enrichment of research and development on porous ceramic membranes extends its focus into advanced membrane separation technologies. The latest emerging nanofiltration (NF) and membrane distillation (MD) applications have witnessed special interests in constructing porous membrane with hydrophilic/functional/hydrophobic properties. However, NF and MD are relatively new, and many shortcomings must be addressed to compete with their polymeric counterparts. For the last three years (2018-2020), state-of-the-art literature on porous ceramic membranes has been collected and critically reviewed. This review highlights the efficiency (permeability, selectivity, and antifouling) of hydrophilic porous ceramic membranes in a wide variety of wastewater treatment applications and hydrophobic porous ceramic membranes in membrane distillation-based desalination applications. A significant focus on pores characteristics, pore sieving phenomenon, nano functionalization, and synergic effect on fouling, the hydrophilic porous ceramic membrane has been discussed. In another part of this review, the role of surface hydrophobicity, water contact angle, liquid entry pressure (LEP), thermal properties, surface micro-roughness, etc., has been discussed for different types of hydrophobic porous ceramic membranes -(a) metal-based, (b) silica-based, (c) other ceramics. Also, this review highlights the potential benefits, drawbacks, and limitations of the porous membrane in applications. Moreover, the prospects are emphasized to overcome the challenges in the field.

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.

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.

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.

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.

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.

Methods for the Assembly and Characterization of Polyelectrolyte Multilayers as Microenvironments to Modulate Human Mesenchymal Stromal Cell Response.

Thin films are of interest in materials design because they allow for the modification of surface properties of materials while the bulk properties of the material are largely unaffected. In this work, we outline methods for the assembly of thin films using a technique known as layer-by-layer (LbL). Furthermore, their interactions with human mesenchymal stromal cells (hMSCs) are discussed. hMSCs are a subject of growing interest because of their potential to treat or cure diseases, given their immunosuppressive properties, multipotent differentiation capabilities, and tissue regeneration capabilities. Numerous improvements and modifications have been suggested for the harvesting, treatment, and culture of hMSCs prior to their administration in human subjects. Here, we discuss methods to assess the interactions of hMSCs with thin LbL-assembled films of heparin and collagen. Three different methods are discussed. The first details the preparation of heparin/collagen multilayers on different surfaces and the seeding of cells on these multilayers. The second method details the characterization of multilayers, including techniques to assess the thickness, roughness, and surface charge of the multilayers, as well as in situ deposition of multilayers. The third method details the analysis of cell interactions with the multilayers, including techniques to assess proliferation, viability, real-time monitoring of hMSC behavior, analysis of hMSC-adhesive proteins on the multilayers, immunomodulatory factor expression of hMSCs, and cytokine expression on heparin/collagen multilayers. We propose that the methods described in this work will assist in the design and characterization of LbL-assembled thin films and the analysis of hMSCs cultured on these thin films.

A "Graft to" Electrospun Zwitterionic Bilayer Membrane for the Separation of Hydraulic Fracturing-Produced Water via Membrane Distillation.

Simultaneous fouling and pore wetting of the membrane during membrane distillation (MD) is a major concern. In this work, an electrospun bilayer membrane for enhancing fouling and wetting resistance has been developed for treating hydraulic fracture-produced water (PW) by MD. These PWs can contain over 200,000 ppm total dissolved solids, organic compounds and surfactants. The membrane consists of an omniphobic surface that faces the permeate stream and a hydrophilic surface that faces the feed stream. The omniphobic surface was decorated by growing nanoparticles, followed by silanization to lower the surface energy. An epoxied zwitterionic polymer was grafted onto the membrane surface that faces the feed stream to form a tight antifouling hydration layer. The membrane was challenged with an aqueous NaCl solution containing sodium dodecyl sulfate (SDS), an ampholyte and crude oil. In the presence of SDS and crude oil, the membrane was stable and displayed salt rejection (>99.9%). Further, the decrease was much less than the base polyvinylidene difluoride (PVDF) electrospun membrane. The membranes were also challenged with actual PW. Our results highlight the importance of tuning the properties of the membrane surface that faces the feed and permeate streams in order to maximize membrane stability, flux and salt rejection.

Cu(I/II) Metal-Organic Frameworks Incorporated Nanofiltration Membranes for Organic Solvent Separation.

Copper-based metal-organic frameworks (MOFs) with different oxidation states and near-uniform particle sizes have been successfully synthesized. Mixed-matrix polyimide membranes incorporating 0.1-7 wt% of Cu(II) benzene-1,2,5-tricarboxylic acid (Cu(II)BTC), Cu(I/II)BTC and Cu(I) 1,2-ethanedisulfonic acid (EDS) (Cu(I)EDS) MOFs were fabricated via non-solvent-induced phase inversion process. These membranes are found to be solvent resistant and mechanically stable. Liquid phase nanofiltration experiments were performed to separate toluene from n-heptane at room temperature. These membranes demonstrate preferential adsorption and permeation of the aromatic toluene over aliphatic n-heptane. The amount of MOF particles incorporated, the oxidation state of the Cu ion and membrane, and barrier layer thickness have a significant impact on the separation factor. Toluene/heptane separation factor at 1.47, 1.67 and 1.79 can be obtained for membranes incorporating 7 wt% Cu(II)BTC, Cu(I/II)BTC and Cu(I)EDS respectively at room temperature.

High-Performance Polyacrylic Acid-Grafted PVDF Nanofiltration Membrane with Good Antifouling Property for the Textile Industry.

In the textile industry, a high-efficiency dye removal and low-retention of salt is demanded for recycling wastewater. In this study, polyvinylidene fluoride (PVDF) ultrafiltration membrane was transformed to a negatively charged loose nanofiltration (NF) membrane through UV-grafting of acrylic acid. At the optimal exposure of PVDF membrane in UV light for 5 min, the membrane had a high dye recovery above 99% (Congo red and Eriochrome® Black T) and a low sodium chloride (NaCl) rejection of less than 15% along with pure water flux of 26 L∙m-2∙h-1∙bar-1. Its antifouling and oleophobicity surface properties were verified using fluorescent- bovine serum albumin (BSA) and underwater mineral oil contact angle, respectively. According to the fluorescent microscopic images, the modified membrane had ten times lower adhesion of protein on the surface than the unmodified membrane. The underwater oil contact angle was raised from 110° to 155°. Moreover, the salt rejection followed this sequence: Na2SO4 > MgSO4 > NaCl > MgCl2, which agreed with the typical negatively charged NF membrane. In addition, the physicochemical characterization of membranes was further investigated to understand and link to the membrane performance, such as surface functional group, surface elements analysis, surface roughness/morphology, and surface hydrophilicity.

Tailoring and Remotely Switching Performance of Ultrafiltration Membranes by Magnetically Responsive Polymer Chains.

Magnetically responsive ultrafiltration membranes were prepared by grafting poly(2-hydroxyethyl methacrylate) chains from the outer surface of 100-kDa regenerated cellulose ultrafiltration membranes. Surface-initiated atom transfer radical polymerization was used to graft the polymer chains. Grafting from the internal pore surface was suppressed by using glycerol as a pore-filling solvent during initiator immobilization at varied densities. Glycerol suppresses the initiator attachment to the pore surface. Polymerization times of up to four hours were investigated. Superparamagnetic nanoparticles were covalently attached to the chain end. Membrane performance was determined using bovine serum albumin and dextran as model solutes. Increasing the grafted polymer chain density and length led to a decrease in the permeate flux and an increase in the apparent rejection coefficient. In an oscillating magnetic field, movement of the grafted polymer chains led to a decrease in the permeate flux, as well as an increase in the apparent rejection coefficient of the model solutes.

Zwitterionic Polymer Brush Grafted on Polyvinylidene Difluoride Membrane Promoting Enhanced Ultrafiltration Performance with Augmented Antifouling Property.

Superhydrophilic zwitterions on the membrane surface have been widely exploited to improve antifouling properties. However, the problematic formation of a <20 nm zwitterionic layer on the hydrophilic surface remains a challenge in wastewater treatment. In this work, we focused on the energy consumption and time control of polymerization and improved the strong hydrophilicity of the modified polyvinylidene difluoride (PVDF) membrane. The sulfobetaine methacrylate (SBMA) monomer was treated with UV-light through polymerization on the PVDF membrane at a variable time interval of 30 to 300 s to grow a poly-SBMA (PSBMA) chain and improve the membrane hydrophilicity. We examined the physiochemical properties of as-prepared PVDF and PVDF-PSBMAx using numeric analytical tools. Then, the zwitterionic polymer with controlled performance was grafted onto the SBMA through UV-light treatment to improve its antifouling properties. The PVDF-PSBMA120s modified membrane exhibited a greater flux rate and indicated bovine serum albumin (BSA) rejection performance. PVDF-PSBMA120s and unmodified PVDF membranes were examined for their antifouling performance using up to three cycles dynamic test using BSA as foulant. The PVDF-modified PSBMA polymer improved the antifouling properties in this experiment. Overall, the resulting membrane demonstrated an enhancement in the hydrophilicity and permeability of the membrane and simultaneously augmented its antifouling properties.

Atrazine Removal from Municipal Wastewater Using a Membrane Bioreactor.

As the demand for potable water increases, direct potable reuse of wastewater is an attractive alternative method to produce potable water. However, implementation of such a process will require the removal of emerging contaminants which could accumulate in the drinking water supply. Here, the removal of atrazine, a commonly used herbicide, has been investigated. Using real and synthetic wastewater, as well as sludge from two wastewater treatment facilities in the United States in Norman, Oklahoma and Fayetteville, Arkansas, atrazine removal has been investigated. Our results indicate that about 20% of the atrazine is removed by adsorption onto the particulate matter present. Significant biodegradation of atrazine was only observed under aerobic conditions for sludge from Norman, Oklahoma. Next-generation sequencing of the activated sludge revealed the abundance of Noncardiac with known atrazine degradation pathways in the Norman aerobic sludge, which is believed to be responsible for atrazine biodegradation in our study. The detection of these bacteria could also be used to determine the likelihood of biodegradation of atrazine for a given wastewater treatment facility.