Cross-flow Filtration Research Articles

Enrichment, Characterization, and Proteomic Profiling of Small Extracellular Vesicles Derived from Human Limbal Mesenchymal Stromal Cells and Melanocytes.

Limbal epithelial progenitor cells (LEPC) rely on their niche environment for proper functionality and self-renewal. While extracellular vesicles (EV), specifically small EVs (sEV), have been proposed to support LEPC homeostasis, data on sEV derived from limbal niche cells like limbal mesenchymal stromal cells (LMSC) remain limited, and there are no studies on sEVs from limbal melanocytes (LM). In this study, we isolated sEV from conditioned media of LMSC and LM using a combination of tangential flow filtration and size exclusion chromatography and characterized them by nanoparticle tracking analysis, transmission electron microscopy, Western blot, multiplex bead arrays, and quantitative mass spectrometry. The internalization of sEV by LEPC was studied using flow cytometry and confocal microscopy. The isolated sEVs exhibited typical EV characteristics, including cell-specific markers such as CD90 for LMSC-sEV and Melan-A for LM-sEV. Bioinformatics analysis of the proteomic data suggested a significant role of sEVs in extracellular matrix deposition, with LMSC-derived sEV containing proteins involved in collagen remodeling and cell matrix adhesion, whereas LM-sEV proteins were implicated in other cellular bioprocesses such as cellular pigmentation and development. Moreover, fluorescently labeled LMSC-sEV and LM-sEV were taken up by LEPC and localized to their perinuclear compartment. These findings provide valuable insights into the complex role of sEV from niche cells in regulating the human limbal stem cell niche.

Compact SiOC ceramic composite nanofiltration membranes by slow dip coating for water purification

In this work, novel silicon oxycarbide (SiOC) ceramic composite nanofiltration membranes with high performance were successfully synthesized at low sintering temperatures by gradual dip-coating approach for the first time. The slow dip-coating process facilitated improved wetting and even spreading of the precursor solution across the substrate. This controlled method ensured the formation of uniform and continuous SiOC ceramic membranes without the introduction of substantial defects or irregularities. Consequently, the systematic approach minimized the occurrence of pinholes, cracks and other structural imperfections in the SiOC ceramic membranes. The synthesis of the SiOC separation layer involved the sintering of polymeric polysilicon as precursor, which caused a lower decomposition temperature in contrast to the conventional method employing silicon carbide powder for sintering. The specific feature enabled a sharp decrease in the sintering temperature which was required for the formation of the separation layer. The prepared SiOC ceramic composite nanofiltration membranes exhibited good permeability with high rejection of dye molecules (>95%) and maintained stable water flux for at least 24 h, showing good long-term stability during cross-flow filtration. This progress provides new approach for the fabrication of compact ceramic nanofiltration membranes with low sintering temperatures, high separation efficiency and enhanced stability.

305 Extracellular Vesicular mRNA and miRNA Biomarkers in Patients With Glioblastoma

INTRODUCTION: Glioblastoma (GBM) is a highly lethal primary brain cancer that is resistant to conventional immunotherapies. However, currently, there is a lack of reliable biomarkers for clinical practice. Extracellular vesicles (EVs) present in biofluids are a potential biomarker for GBM diagnosis. METHODS: EVs from cell culture medium, healthy donor and GBM patient serum were purified using tangential flow filtration (TFF). Morphology, size, and numbers of EVs were characterized by TEM and qNano, respectively. EVs from healthy donor and GBM patient serum were compared against other cancer EV samples using bulk mRNA and miRNA sequencing analysis. Selected mRNA and miRNA name are not mentioned due to patents being drafted. mRNA and miRNA expression in EVs were quantified by a fluorescent nucleic acid detection method using a biochip platform at a single-EV level. Fluorescence images were recorded using high-resolution total internal reflection fluorescence (TIRF) microscopy. RESULTS: From bulk mRNA and miRNA sequencing and bioinformatics analysis, three miRNA and two mRNA were selected as potential candidate biomarkers for GBM. Subsequently, EVs from the serum of GBM patients (n = 39) were evaluated through our biochip platform. mRNA and miRNA expression of these biomarkers were positive in single EVs, for which we detected colocalization of protein/RNAs or multiple RNAs in subpopulations of EVs. Our platform was benchmarked with a standard PCR method showing 100X more sensitivity. Moreover, levels of expression of the different RNAs were compared to a cohort of serum from healthy donors (n=20), and showing significant differences between the two groups. CONCLUSIONS: Our non-invasive biochip assay outperformed qRT-PCR for EV RNA detection. Selected mRNAs and miRNAs in subpopulations of EVs could be used as potential GBM biomarkers.

Facile fabrication of polymeric-inorganic hybrid coated membrane with super-hydrophilic and underwater super-oleophobic properties for highly efficient crude oil-in-water emulsion separation

The oily wastewater is a huge wastewater stream that can potentially serve as an enormous source of clean water for water reuse applications such as groundwater recharge or irrigation. Given the huge potential of cleaning oily wastewater, we have developed an amino-functionalized nickel oxide nanoparticles coated inorganic-polymeric hybrid membrane with superhydrophilic and underwater superoleophobic properties for excellent crude oil-in-water (O/W) emulsion separation potential using a cross-flow filtration system. The use of ceramic alumina (Al2O3) support offers increased chemical, thermal, and mechanical stability while nickel oxide (NiO) decoration equips the membrane with special surface wettability to avoid membrane fouling. Moreover, the amino-functionalized NiO-coated polyamide network (F-NiO/PA) offers an active layer dense enough to separate the oil from water when the surfactant-stabilized crude oil-in-water emulsion was used as feed during separation experiments. Hence fabricated F-NiO/PA@Alumina membrane was studied by using different membrane characterization techniques to explore the features and understand the structure of F-NiO/PA@Alumina membrane. The F-NiO/PA@Alumina membrane possessed a superhydrophilic (water contact angle; θW, A = 0°) and underwater superoleophobic (oil contact angle; θO, W = 161°) surface wettability which is an ideal feature required for O/W emulsion separation. The F-NiO/PA@Alumina membrane demonstrated a high O/W emulsion separation efficiency of >99 % even with crude oil concentrations as high as 200 ppm using a cross-flow filtration system. The stability test of the separation performance of the F-NiO/PA@Alumina membrane also revealed a separation performance of >99 % for the duration of the test using a cross-flow filtration system.

Engineered extracellular vesicles carrying let-7a-5p for alleviating inflammation in acute lung injury

BackgroundAcute lung injury (ALI) is a life-threatening respiratory condition characterized by severe inflammation and lung tissue damage, frequently causing rapid respiratory failure and long-term complications. The microRNA let-7a-5p is involved in the progression of lung injury, inflammation, and fibrosis by regulating immune cell activation and cytokine production. This study aims to use an innovative cellular electroporation platform to generate extracellular vesicles (EVs) carring let-7a-5p (EV-let-7a-5p) derived from transfected Wharton’s jelly-mesenchymal stem cells (WJ-MSCs) as a potential gene therapy for ALI.MethodsA cellular nanoporation (CNP) method was used to induce the production and release of EV-let-7a-5p from WJ-MSCs transfected with the relevant plasmid DNA. EV-let-7a-5p in the conditioned medium were isolated using a tangential flow filtration (TFF) system. EV characterization followed the minimal consensus guidelines outlined by the International Society for Extracellular Vesicles. We conducted a thorough set of therapeutic assessments, including the antifibrotic effects using a transforming growth factor beta (TGF-β)-induced cell model, the modulation effects on macrophage polarization, and the influence of EV-let-7a-5p in a rat model of hyperoxia-induced ALI.ResultsThe CNP platform significantly increased EV secretion from transfected WJ-MSCs, and the encapsulated let-7a-5p in engineered EVs was markedly higher than that in untreated WJ-MSCs. These EV-let-7a-5p did not influence cell proliferation and effectively mitigated the TGF-β-induced fibrotic phenotype by downregulating SMAD2/3 phosphorylation in LL29 cells. Furthermore, EV-let-7a-5p regulated M2-like macrophage activation in an inflammatory microenvironment and significantly induced interleukin (IL)-10 secretion, demonstrating their modulatory effect on inflammation. Administering EVs from untreated WJ-MSCs slightly improved lung function and increased let-7a-5p expression in plasma in the hyperoxia-induced ALI rat model. In comparison, EV-let-7a-5p significantly reduced macrophage infiltration and collagen deposition while increasing IL-10 expression, causing a substantial improvement in lung function.ConclusionThis study reveals that the use of the CNP platform to stimulate and transfect WJ-MSCs could generate an abundance of let-7a-5p-enriched EVs, which underscores the therapeutic potential in countering inflammatory responses, fibrotic activation, and hyperoxia-induced lung injury. These results provide potential avenues for developing innovative therapeutic approaches for more effective interventions in ALI.

Heavy Metal Remediation by Dry Mycelium Membranes: Approaches to Sustainable Lead Remediation in Water.

Lead contamination poses significant and lasting health risks, particularly in children. This study explores the efficacy of dried mycelium membranes, distinct from live fungal biomass, for the remediation of lead (Pb(II)) in water. Dried mycelium offers unique advantages, including environmental resilience, ease of handling, biodegradability, and mechanical reliability. The study explores Pb(II) removal mechanisms through sorption and mineralization by dried mycelium hyphae in aqueous solutions. The sorption isotherm studies reveal a high Pb(II) removal efficiency, exceeding 95% for concentrations below 1000 ppm and ∼63% above 1500 ppm, primarily driven by electrostatic interactions. The measured infrared peak shifts and the pseudo-second-order kinetics for sorption suggests a correlation between sorption capacity and the density of interacting functional groups. The study also explores novel surface functionalization of the mycelium network with phosphate to enhance Pb(II) removal, which enables remediation efficiencies >95% for concentrations above 1500 ppm. Scanning electron microscopy images show a pH-dependent formation of Pb-based crystals uniformly deposited throughout the entire mycelium network. Continuous cross-flow filtration tests employing a dried mycelium membrane demonstrate its efficacy as a microporous membrane for Pb(II) removal, reaching remediation efficiency of 85-90% at the highest Pb(II) concentrations. These findings suggest that dried mycelium membranes can be a viable alternative to synthetic membranes in heavy metal remediation, with potential environmental and water treatment applications.

Isolation and Characterization of Extracellular Vesicles Through Orthogonal Approaches for the Development of Intraocular EV Therapy.

Isolating extracellular vesicles (EVs) with high yield, replicable purity, and characterization remains a bottleneck in the development of EV therapeutics. To address these challenges, the current study aims to establish the necessary framework for preclinical and clinical studies in the development of stem cell-derived intraocular EV therapeutics. Small EVs (sEVs) were separated from the conditioned cell culture medium (CCM) of the human embryogenic stem cell-derived fully polarized retinal pigment epithelium (hESC-RPE-sEV) by a commercially available microfluidic tangential flow filtration (TFF) device ExoDisc (ED) or differential ultracentrifugation (dUC). The scaling and concentration capabilities and purity of recovered sEVs were assessed. Size, number, and surface markers of sEVs were determined by orthogonal approaches using multiple devices. ED yielded higher numbers of sEVs, ranging from three to eight times higher depending on the measurement device, compared to dUC using the same 5 mL of CCM input. Within the same setting, the purity of ED-recovered hESC-RPE-sEVs was higher than that for dUC-recovered sEVs. ED yielded a higher concentration of particles, which is strongly correlated with the input volume, up to 10 mL (r = 0.98, P = 0.016). Meanwhile, comprehensive characterization profiles of EV surface markers between ED- and dUC-recovered hESC-RPE-sEVs were compatible. Our study supports TFF as a valuable strategy for separating sEVs for the development of intraocular EV therapeutics. However, there is a growing need for diverse devices to optimize TFF for use in EV preparation. Using orthogonal approaches in EV characterization remains ideal for reliably characterizing heterogeneous EV.

Anion exchange HPLC monitoring of mRNA in vitro transcription reactions to support mRNA manufacturing process development.

mRNA technology has recently demonstrated the ability to significantly change the timeline for developing and delivering a new vaccine from years to months. The potential of mRNA technology for rapid vaccine development has recently been highlighted by the successful development and approval of two mRNA vaccines for COVID-19. Importantly, this RNA-based approach holds promise for treatments beyond vaccines and infectious diseases, e.g., treatments for cancer, metabolic disorders, cardiovascular conditions, and autoimmune diseases. There is currently significant demand for the development of improved manufacturing processes for the production of mRNA therapeutics in an effort to increase their yield and quality. The development of suitable analytical methods for the analysis of mRNA therapeutics is critical to underpin manufacturing development and the characterisation of the drug product and drug substance. In this study we have developed a high-throughput, high-performance liquid chromatography (HPLC) workflow for the rapid analysis of mRNA generated using in vitro transcription (IVT). We have optimised anion exchange (AEX) HPLC for the analysis of mRNA directly from IVT. Chromatography was performed in under 6min enabling separation of all of the key components in the IVT, including nucleoside triphosphates (NTPs), Cap analogue, plasmid DNA and mRNA product. Moreover, baseline separation of the NTPs was achieved, which facilitates accurate quantification of each NTP such that their consumption may be determined during IVT reactions. Furthermore, the HPLC method was used to rapidly assess the purification of the mRNA product, including removal of NTPs/Cap analogue and other contaminants after downstream purification, including solid phase extraction (SPE), oligo deoxythymidine (oligo-dT) affinity chromatography and tangential flow filtration (TFF). Using the developed method excellent precision was obtained with calibration curves for an external mRNA standard and NTPs giving correlation coefficients of 0.98 and 1.0 respectively. Intra- and inter-day studies on retention time stability of NTPs, showed a relative standard deviation ≤ 0.3% and ≤1.5% respectively. The mRNA retention time variability was ≤0.13%. This method was then utilised to monitor the progress of an IVT reaction for the production of Covid spike protein (C-Spike) mRNA to measure the increasing yield of mRNA alongside the consumption of NTPs during the reaction.

Metal-organic framework-intercalated graphene oxide nanofiltration membranes for enhanced treatment of wastewater effluents

Graphene oxide membranes (GO) hold immense potential in the field of water purification. However, when applied directly to real wastewater effluents, pure GO membranes suffer from drawbacks such as fouling sensitivity and limited stability. To address these challenges and unlock the full potential of GO membranes, novel nanocomposite membranes have been developed by the intercalation of GO with nanoparticles of ZIF-8 (a type of zeolitic imidazolate framework). The prepared GO/ZIF-8 (GZ) nanocomposite membranes have exhibited enhanced hydrophilicity and exceptional water purification capabilities. Specifically, the GZ membranes have demonstrated a permeance enhancement of over two-fold when compared to the pristine GO reference membrane. This enhancement is coupled with anti-fouling performance and competitive rejection rates for both salts and organic pollutants. GZ membranes have been effectively employed for the purification by cross-flow filtration of 3 industrial wastewater effluents. They have shown improved separation performance compared to the pristine GO reference membrane, and high stability under cross-flow conditions. The origin of the high performances of the GZ membrane has been clarified using structural and morphological analyses. This work highlights the significant progress made in the field of water treatment using graphene-based membranes.

Characterization of the Outer Membrane Vesicles of Pseudomonas aeruginosa Exhibiting Growth Inhibition against Acinetobacter baumannii.

We investigated the Pseudomonas aeruginosa (PA) outer membrane vesicles (OMVs) and their effect on Acinetobacter baumannii (AB) growth in vitro. The inhibitory effects of PA on AB were assessed using a cross-streak assay. The OMVs were extracted through high-speed centrifugation, tangential flow filtration, and ultracentrifugation and characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transmission electron microscopy (TEM), and nanoparticle tracking assays (NTAs). Proteomic analysis was conducted to compare the OMVs of different PA strains. PA022 exhibited more pronounced inhibition of AB growth compared with PA ATCC 27853. TEM confirmed the presence of OMVs in both PA022 and PA ATCC 27853, revealing phospholipid bilayer structures. The NTA revealed similar sizes and concentrations. Proteomic analysis identified 623 and 538 proteins in PA022 and PA ATCC 27853 OMVs, respectively, with significant proportions of the outer membrane and extracellular proteins, respectively. Importantly, PA022 OMVs contained six known virulence factors and motility-associated proteins. This study revealed the unique characteristics of PA OMVs and their inhibitory effects on AB growth, shedding light on their role in bacterial interactions. Proteomic analysis provides valuable insights into potential pathogenic functions and therapeutic applications against bacterial infections.

Residence time distribution of continuous capture of recombinant antibody by precipitation and two stage tangential flow filtration

AbstractBACKGROUNDThe determination of the residence time distribution of an entire process or process step in integrated continuous biomanufacturing is required for process understanding, traceability determination and to efficiently handle process disturbances. When recombinant proteins such as monoclonal antibodies are captured by continuous precipitation and two‐stage filtration, the question arises whether simple salt is suitable as a tracer or whether a labeled protein is required.RESULTSWe have investigated the use of various inert tracers, sodium nitrate (NaNO3) due to its absorbance at UV 280 nm, and fluorescently labeled or unlabeled antibodies to determine the RTD of the product at steady‐state and in the start‐up and shut‐down phases. All tracers are suitable for measuring the RTD of the precipitated antibody for individual unit operations. For interconnected unit operations, if the product is concentrated, the RTD can be determined with labeled or unlabeled antibodies, because it is not possible to concentrate salts in a microfiltration unit. The duration of the start‐up and shut‐down phases depends on the concentration factor applied in two‐stage filtration, and the number of reactor volumes required to reach steady state corresponds to the concentration factor. Around 1.6 reactor volumes are required for the shutdown phase for 99% wash‐out.CONCLUSIONThe integrated process has a plug flow characteristic, which is advantageous as the process can react quickly when process deviations occur. © 2024 The Authors. Journal of Chemical Technology and Biotechnology published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry (SCI).

Accelerated Development of AAV Purification Process Using a High-Throughput and Automated Crossflow System

Adeno-associated viruses (AAV) are currently predominant viral transfer tools for gene therapy, and efforts are being made to design faster and more efficient methods and technologies for their manufacturing. The early selection of high-performing filters is essential for developing an ultrafiltration and diafiltration (UF/DF) process, especially when feed material is scarce, and timelines are short. However, few methods and technologies exist to enable process optimization with multiple variations in a single run. In this study, we explored the potential of Ambr® Crossflow for high-throughput, automated screening of different membrane materials, pore sizes and different process conditions for the UF/DF step of AAV8. The best overall performance was obtained with a 100 kDa PES flat sheet cassette. The UF/DF process was further transferred to a larger scale to the Sartoflow® Smart Tangential Flow Filtration (TFF) system using a 100 kDa PES Sartocon® Slice 200 cassette and compared to a 100 kDa PES hollow fiber. Virus recovery, permeate flux and total protein removal values of the flat sheet cassette were similar to those achieved in small-scale devices, and higher than those of the hollow fiber, thus demonstrating similar performance at a larger process scale. The high-throughput, automated method described herein allowed to screen multiple materials and process parameters of a UF/DF process in a time- and resource-efficient way, making it a useful tool to accelerate early-stage downstream process development of AAV.

Quantification of Pluronic F127 used on PLGA nanoparticle preparation and comparing purification techniques by centrifugation, tangential flow filtration, and ultrafiltration

Emerging research in polymeric nanoparticles (PNP) in the field of drug delivery systems focuses on the preferable use of biocompatible materials like poly(lactic-co-glycolic) acid (PLGA). However, the substantial use of surfactant in achieving colloidal stability requires its subsequent elimination to avoid side effects. This study aimed to establish suitable conditions for detecting and quantifying Pluronic® F127 (F127), a non-ionic surfactant widely used for this specific purpose, through a precise Size Exclusion Chromatography (SEC) method. Then, comparing the F127 removal efficacy via three purification techniques: Centrifugation (Cf), Ultrafiltration (UF), and tangential flow filtration (TFF), determining the minor influence of surfactant removal on the colloidal and morphological properties of surfactant-free PLGA suspensions. Thus, to determine the most suitable strategy for purifying PNP. The initial results of the SEC method revealed two distinct molar mass distributions of F127, associated with a blend of triblock alongside a minor fraction of homopolymer or diblock. The method exhibited a high linear correlation (R2 0.99991) for surfactant concentrations ranging from 0.3 to 14.7 mg/mL. While assessing the three purification techniques, UF demonstrated proficiency in concentrating nanoparticle suspensions, albeit tending to retain a significant portion of the surfactant. In contrast, Cf displayed a remarkable capability in removing more than 90 % of the incorporated F127. However, Cf recovered only 60 % of the initial nanoparticles, predominantly eliminating smaller diameter particles. Conversely, TFF demonstrated high efficacy in surfactant removal, requiring an extended washing process and leading to minimal nanoparticle loss while leaving the colloidal variables unaffected.

Anti-Listerial Activity of Bacteriocin-like Inhibitory Substance Produced by Enterococcus lactis LBM BT2 Using Alternative Medium with Sugarcane Molasses.

Listeria monocytogenes is a foodborne pathogen that contaminates food-processing environments and persists within biofilms on equipment, thus reaching final products by cross-contamination. With the growing demand for clean-label products, the search for natural antimicrobials as biopreservants, such as bacteriocins, has shown promising potential. In this context, this study aimed to evaluate the anti-listerial action of bacteriocins produced by Enterococcus lactis LBM BT2 in an alternative medium containing sugarcane molasses (SCM). Molecular analyses were carried out to characterize the strain, including the presence of bacteriocin-related genes. In the kinetic study on SCM medium E. lactis, LBM BT2 showed biomass and bacteriocin productions similar to those observed on a sucrose-based medium (control), highlighting the potential of the sugarcane molasses as a low-cost substrate. Stability tests revealed that the molecule remained active in wide ranges of pH (4-10) and temperature (60-100 °C). Furthermore, the proteolytic treatment reduced the biomolecule's antimicrobial activity, highlighting its proteinaceous nature. After primary purification by salting out and tangential flow filtration, the bacteriocin-like inhibitory substance (BLIS) showed bacteriostatic activity on suspended L. monocytogenes cells and against biofilm formation at a concentration of 0.625 mg/mL. These results demonstrate the potential of the produced BLIS as a biopreservative in the food industry.

Desalting Plasma Protein Solutions by Membrane Capacitive Deionization.

Plasma protein therapies are used by millions of people across the globe to treat a litany of diseases and serious medical conditions. One challenge in the manufacture of plasma protein therapies is the removal of salt ions (e.g., sodium, phosphate, and chloride) from the protein solution. The conventional approach to remove salt ions is the use of diafiltration membranes (e.g., tangential flow filtration) and ion-exchange chromatography. However, the ion-exchange resins within the chromatographic column as well as filtration membranes are subject to fouling by the plasma protein. In this work, we investigate the membrane capacitive deionization (MCDI) as an alternative separation platform for removing ions from plasma protein solutions with negligible protein loss. MCDI has been previously deployed for brackish water desalination, nutrient recovery, mineral recovery, and removal of pollutants from water. However, this is the first time this technique has been applied for removing 28% of ions (sodium, chloride, and phosphate) from human serum albumin solutions with less than 3% protein loss from the process stream. Furthermore, the MCDI experiments utilized highly conductive poly(phenylene alkylene)-based ion exchange membranes (IEMs). These IEMs combined with ionomer-coated nylon meshes in the spacer channel ameliorate Ohmic resistances in MCDI improving the energy efficiency. Overall, we envision MCDI as an effective separation platform in biopharmaceutical manufacturing for deionizing plasma protein solutions and other pharmaceutical formulations without a loss of active pharmaceutical ingredients.

Regression Metamodel-Based Digital Twin for an Industrial Dynamic Crossflow Filtration Process.

Dynamic crossflow filtration (DCF) is the state-of-the-art technology for solid-liquid separation from viscous and sensitive feed streams in the food and biopharma industry. Up to now, the potential of industrial processes is often not fully exploited, because fixed recipes are usually applied to run the processes. In order to take the varying properties of biological feed materials into account, we aim to develop a digital twin of an industrial brownfield DCF plant, allowing to optimize setpoint decisions in almost real time. The core of the digital twin is a mechanistic-empirical process model combining fundamental filtration laws with process expert knowledge. The effect of variation in the selected process and model parameters on plant productivity has been assessed using a model-based design-of-experiments approach, and a regression metamodel has been trained with the data. A cyclic program that bidirectionally communicates with the DCF asset serves as frame of the digital twin. It monitors the process dynamics membrane torque and transmembrane pressure and feeds back the optimum permeate flow rate setpoint to the physical asset in almost real-time during process runs. We considered a total of 24 industrial production batches from the filtration of grape juice from the years 2022 and 2023 in the study. After implementation of the digital twin on site, the campaign mean productivity increased by 15% over the course of the year 2023. The presented digital twin framework is a simple example how an industrial established process can be controlled by a hybrid model-based algorithm. With a digital process dynamics model at hand, the presented metamodel optimization approach can be easily transferred to other (bio)chemical processes.

Decoupling synthesis and crystallization of covalent organic frameworks (COFs) for more crystalline layer targeting high efficiency dye separation

Covalent organic frameworks (COFs) are crystalline porous materials composed of organic building blocks linked by covalent bonds, which have attracted great attention for membrane–based separation applications. However, fabrication of COF membranes with high quality and performance remains a challenge, especially for the β–ketoenamine linked COFs. In the conventional IP methods, the synthesis process (the Schiff–base reaction of the monomers and the tautomerism of the oligomers) and the crystallization process (the self–assembly of the oligomers) of the COFs materials occur simultaneously on the surface of the support membrane, which can result in low crystallinity, high defect density, and poor separation performance of the COF layer. In this study, a novel strategy for fabricating large-area, defect–free COFs composite membranes by interfacial polymerization (IP) was presented to overcome this problem. By incorporating both the amine and aldehyde monomers in one organic phase, and dissolving organic acid catalyst in another phase (the water), the synthesis process mostly take place in the bulk of the organic phase, while the crystallization occurs on the surface of the support membrane under the influence of the organic acid catalyst. This strategy effectively decoupled the synthesis process with the crystallization, providing a stable growth region for crystalline layer, ultimately leading to the formation of a thin, defect–free and highly ordered COF layer on the support membrane. The strategy was demonstrate by synthesis of triformylphloroglucinol phenylenediamine (TpPa)–Ⅱ/Poly(m–phenylene isophthalaminde) (PMIA) composite membrane. We characterize the structure and morphology of the TpPa–Ⅱ/PMIA composite membrane by various analytical techniques to verify our hypothesis, and evaluate its separation performance using pure water permeance and dyes retention rate as indicators. The results show that the TpPa–Ⅱ/PMIA composite membrane has an 80 nm thick top-layer with regular COF structure and orderly pores. It exhibits an excellent dye separation performance, with a (pure water permeance) PWP of 105.6 L·m−2·h−1·bar−1 and CR rejection of 99.2 %, which surpasses most of the TpPa–type COFs composite membranes reported in literature, as well as the reference (TpPa–I/PMIA membrane) in this work. Moreover, the TpPa–Ⅱ/PMIA composite membrane maintains both its performance and structural stability during a 240–hour cross–flow filtration of the dye solution, demonstrating its excellent potential in the dye wastewater treatment.

Zeolite Imidazolate Frameworks-8@SiO2-ZrO2 Crystal-Amorphous Hybrid Core-Shell Structure as a Building Block for Water Purification Membranes.

Metal-organic frameworks (MOFs) are emerging as promising materials for water purification membranes, owing to their uniform microporous structures and chemical functionalities. Here, we report a simple procedure for depositing MOF-based nanofiltration membranes on commercial TiO2 ceramic tubular supports, completely avoiding the use of dispersants or binders. Zeolite imidazolate frameworks-8 (ZIF-8) nanocrystals were synthesized in methanol at room temperature and subsequently coated with an amorphous SiO2-ZrO2 gel to generate a dispersion of ZIF-8@SiO2-ZrO2 core-shell nanoparticles. The amorphous SiO2-ZrO2 gel served as a binding agent for the ZIF-8 nanocrystals, thus forming a defect-free continuous membrane layer. After repeating the coating twice, the active layer had a thickness of 0.96 μm, presenting a rejection rate >90% for the total organic carbon in an aquaculture effluent and in a wastewater treatment plant, while reducing the concentration of trimethoprim, here used as a target pollutant. Moreover, the oxide gel provided the MOF-based active layer with good adhesion to the support and enhanced its hydrophilicity, resulting in a membrane with excellent mechanical stability and resistance to fouling during the crossflow filtration of the real wastewater samples. These results implied the high potential of the MOF-based nanocomposite membrane for effective treatment of actual wastewater streams.

Contributions of Chinese hamster ovary cell derived extracellular vesicles and other cellular materials to hollow fiber filter fouling during perfusion manufacturing of monoclonal antibodies.

Hollow fiber filter fouling is a common issue plaguing perfusion production process for biologics therapeutics, but the nature of filter foulant has been elusive. Here we studied cell culture materials especially Chinese hamster ovary (CHO) cell-derived extracellular vesicles in perfusion process to determine their role in filter fouling. We found that the decrease of CHO-derived small extracellular vesicles (sEVs) with 50-200 nm in diameter in perfusion permeates always preceded the increase in transmembrane pressure (TMP) and subsequent decrease in product sieving, suggesting that sEVs might have been retained inside filters and contributed to filter fouling. Using scanning electron microscopy and helium ion microscopy, we found sEV-like structures in pores and on foulant patches of hollow fiber tangential flow filtration filter (HF-TFF) membranes. We also observed that the Day 28 TMP of perfusion culture correlated positively with the percentage of foulant patch areas. In addition, energy dispersive X-ray spectroscopy-based elemental mapping microscopy and spectroscopy analysis suggests that foulant patches had enriched cellular materials but not antifoam. Fluorescent staining results further indicate that these cellular materials could be DNA, proteins, and even adherent CHO cells. Lastly, in a small-scale HF-TFF model, addition of CHO-specific sEVs in CHO culture simulated filter fouling behaviors in a concentration-dependent manner. Based on these results, we proposed a mechanism of HF-TFF fouling, in which filter pore constriction by CHO sEVs is followed by cake formation of cellular materials on filter membrane.

Selective hydrophobic interaction chromatography for high purity of supercoiled DNA plasmids.

High purity of plasmid DNA (pDNA), particularly in supercoiled isoform (SC), is used for various biopharmaceutical applications, such as a transfecting agent for production of gene therapy viral vectors, for pDNA vaccines, or as a precursor for linearized form that serves as a template for mRNA synthesis. In clinical manufacturing, pDNA is commonly extracted from Escherichia coli cells with alkaline lysis followed by anion exchange chromatography or tangential flow filtration as a capture step for pDNA. Both methods remove a high degree of host cell contaminants but are unable to generically discriminate between SC and open-circular (OC) pDNA isoforms, as well as other DNA impurities, such as genomic DNA (gDNA). Hydrophobic interaction chromatography (HIC) is commonly used as polishing purification for pDNA. We developed HIC-based polishing purification methodology that is highly selective for enrichment of SC pDNA. It is generic with respect to plasmid size, scalable, and GMP compatible. The technique uses ammonium sulfate, a kosmotropic salt, at a concentration selective for SC pDNA binding to a butyl monolith column, while OC pDNA and gDNA are removed in flow-through. The approach is validated on multiple adeno-associated virus- and mRNA-encoding plasmids ranging from 3 to 12 kbp. We show good scalability to at least 300 mg of >95% SC pDNA, thus paving the way to increase the quality of genomic medicines that utilize pDNA as a key raw material.