Tangential Flow Ultrafiltration Research Articles

Process Optimization of Scaled-Up Production and Biosafety Evaluation of the Dimethyl-Dioctadecyl-Ammonium Bromide/Poly(lactic acid) Nano-Vaccine.

Nano-adjuvant vaccines could induce immune responses and enhance immunogenicity. However, the application and manufacturing of nano-adjuvant is hampered by its challenging scale-up, poor reproducibility, and low security. Therefore, the present study aimed to optimize the preparation nanoparticles (NPs) using FDA-approved biopolymer materials poly(lactic acid) (PLA) and cationic lipid didodecyl-dimethyl-ammonium bromide (DDAB), develop the scale-up process, and evaluate the stability and biosafety of it. The optimum preparation conditions of DDAB/PLA NPs on a small scale were as follows: DDAB amount of 30 mg, aqueous phase volume of 90 mL, stirring rate at 550 rpm, and solidifying time of 12 h. Under the optimum conditions, the size of the NPs was about 170 nm. In scale-up preparation experiments, the vacuum rotary evaporation of 6 h and the Tangential flow ultrafiltration (TFU) method were the optimum conditions. The results suggested that DDAB/PLA NPs exhibited a uniform particle size distribution, with an average size of 150.3 ± 10.4 nm and a narrow polydispersity index (PDI) of 0.090 ± 0.13, coupled with a high antigen loading capacity of 85.4 ± 4.0%. In addition, the DDAB/PLA NPs can be stored stably for 30 days and do not have side effects caused by residual solvents. For biosafety, the acute toxicity experiments showed good tolerance of the vaccine formulation even at a high adjuvant dose. The local irritation experiment demonstrated the reversibility of muscular irritation, and the repeated toxicity experiment revealed no significant necrosis or severe lesions in mice injected with the high-dose vaccine formulation. Overall, the DDAB/PLA NPs exhibit potential for clinical translation as a safe candidate vaccine adjuvant.

Influence of suspended particles and dissolved organic matters on virus enrichment in reclaimed water by two-step tangential flow ultrafiltration: Phenomena and mechanisms

Enteric virus concentration in large-volume water samples is crucial for detection and essential for assessing water safety. Certain dissolution and suspension components can affect the enrichment process. In this study, tangential flow ultrafiltration (TFUF) was used as an enrichment method for recovering enteric virus in water samples. Interestingly, the bacteriophage MS2 recovery in reclaimed water and the reclaimed water without particles were higher than that in ultrapure water. The simulated reclaimed water experiments showed that humic acid (HA) (92.16% ± 4.32%) and tryptophan (Try) (81.50 ± 7.71%) enhanced MS2 recovery, while the presence of kaolin (Kaolin) inhibited MS2 recovery with an efficiency of 63.13% ± 11.17%. Furthermore, Atomic force microscopy (AFM) revealed that the MS2-HA cluster and the MS2-Try cluster had larger roughness values on the membrane surface, making it difficult to be eluted, whereas MS2-Kaolin cluster had compact surfaces making it difficult to be eluted. Additionally, the MS2-HA cluster is bound to the membrane by single hydrogen bond with SO, whereas both the MS2-Try cluster and the MS2-Kaolin cluster are bound to the membrane by two hydrogen bonds, making eluting MS2 challenging. These findings have potential implications for validating standardized methods for virus enrichment in water samples.

Production of Bioactive Porcine Lactoferrin through a Novel Glucose-Inducible Expression System in Pichia pastoris: Unveiling Antimicrobial and Anticancer Functionalities.

Lactoferrin (LF) stands as one of the extensively investigated iron-binding glycoproteins within milk, exhibiting diverse biological functionalities. The global demand for LF has experienced consistent growth. Biotechnological strategies aimed at enhancing LF productivity through microbial expression systems offer substantial cost-effective advantages and exhibit fewer constraints compared to traditional animal bioreactor technologies. This study devised a novel recombinant plasmid, wherein the AOX1 promoter was replaced with a glucose-inducible G1 promoter (PG1) to govern the expression of recombinant porcine LF (rpLF) in Pichia pastoris GS115. High-copy-number PG1-rpLF yeast clones were meticulously selected, and subsequent induction with 0.05 g/L glucose demonstrated robust secretion of rpLF. Scaling up production transpired in a 5 L fermenter, yielding an estimated rpLF productivity of approximately 2.8 g/L by the conclusion of glycerol-fed fermentation. A three-step purification process involving tangential-flow ultrafiltration yielded approximately 6.55 g of rpLF crude (approximately 85% purity). Notably, exceptional purity of rpLF was achieved through sequential heparin and size-exclusion column purification. Comparatively, the present glucose-inducible system outperformed our previous methanol-induced system, which yielded a level of 87 mg/L of extracellular rpLF secretion. Furthermore, yeast-produced rpLF demonstrated affinity for ferric ions (Fe3+) and exhibited growth inhibition against various pathogenic microbes (E. coli, S. aureus, and C. albicans) and human cancer cells (A549, MDA-MB-231, and Hep3B), similar to commercial bovine LF (bLF). Intriguingly, the hydrolysate of rpLF (rpLFH) manifested heightened antimicrobial and anticancer effects compared to its intact form. In conclusion, this study presents an efficient glucose-inducible yeast expression system for large-scale production and purification of active rpLF protein with the potential for veterinary or medical applications.

SARS-CoV-2 in freshwaters used for irrigation in the agro-urban environment of Milan (Northen Italy)

Abstract Background During the COVID-19 pandemic, water has played a key role in epidemiological monitor of SARS-CoV-2. Our study aimed to verify if flood spillways activity during heavy rain events could be a source of SARS-CoV-2 contamination of a rural canal and consequently of human infection. Methods A total of 12 water samples (2L) were collected in four sampling campaigns conducted in 2022 (April, September, November, and December) during heavy rainfall. An automated sampler located in an irrigation stream collected 250mL of water at intervals of 10’ for 80’ since the spillways activation (sample A), then every 20’ for 160’ (sample B), and finally every 30’ for 240’ (sample C). Each sample was pre-filtered and concentrated by tangential flow ultrafiltration according to an in-house protocol. The presence of SARS-CoV-2 and Pepper Mild Mottle Virus (PMMoV, internal control) RNA was investigated by real time RT-PCR. SARS-CoV-2 viability was tested on VERO E6 cell culture, verifying the cytopathic effects and the viral load by real time RT-PCR every 24 hours for 7 days. Results PMMoV RNA was detected in all specimens assessing RNA quality. Although SARS-CoV-2 RNA was detected in all samples, except in those of November and in April sample C, there was no evidence of cytopathic effect or active replication in cell culture. RNA concentrations were generally low (0.27-6.95 gc/µL) but slightly higher in samples A than in B and C. Conclusions Despite the limited number of samples collected during the experimental campaign and due to the severe drought that affected Northen Italy, our results suggest that spillways activation could be a source of SARS-CoV-2 contamination in freshwaters, but there was no evidence of risk to human health. The detection of small concentrations may be associated with the circulation, during the monitoring, of the Omicron variants, which rarely cause gastrointestinal symptoms. Key messages • Spillways activation could be a source of SARS-CoV-2 contamination. • Both environmental diffusion and infectivity of SARS-CoV-2 were investigated to better understand the risk of disease for humans.

Deep neural network for prediction and control of permeability decline in single pass tangential flow ultrafiltration in continuous processing of monoclonal antibodies

Single-pass tangential flow filtration (SPTFF) is a crucial technology enabling the continuous manufacturing of monoclonal antibodies (mAbs). By significantly increasing the membrane area utilized in the process, SPTFF allows the mAb process stream to be concentrated up to the desired final target in a single pass across the membrane surface without the need for recirculation. However, a key challenge in SPTFF is compensating for flux decline across the membrane due to concentration polarization and surface fouling phenomena. In continuous downstream processing, flux decline immediately impacts the continuous process flowrates. It reduces the concentration factor achievable in a single pass, thereby reducing the final concentration attained at the outlet of the SPTFF module. In this work, we develop a deep neural network model to predict the NWP in real-time without the need to conduct actual NWP measurements. The developed model incorporates process parameters such as pressure and feed concentrations through inline sensors and a spectroscopy-coupled data model (NIR-PLS model). The model determines the optimal timing for membrane cleaning steps when the normalized water permeability (NWP) falls below 60%. Using SCADA and PLC, a distributed control system was developed to integrate the monitoring sensors and control elements, such as the NIRS sensor for concentration monitoring, the DNN model for NWP prediction, weighing balances, pressure sensors, pumps, and valves. The model was tested in real-time, and the NWP was predicted within <5% error in three independent test cases, successfully enabling control of the SPTFF step in line with the Quality by Design paradigm.

Formulation and Scale-Up of Fast-Dissolving Lumefantrine Nanoparticles for Oral Malaria Therapy

Lumefantrine (LMN) is one of the first-line drugs in the treatment of malaria due to its long circulation half-life, which results in enhanced effectiveness against drug-resistant strains of malaria. However, LMN's therapeutic efficacy is diminished due to its low bioavailability when dosed as a crystalline solid. The goal of this work was to produce low-cost, highly bioavailable, stable LMN powders for oral delivery that would be suitable for global health applications. We report the development of a LMN nanoparticle formulation and the translation of that formulation from laboratory to industrial scale. We applied Flash NanoPrecipitation (FNP) to develop nanoparticles with 90% LMN loading and sizes of 200-260 nm. The integrated process involves nanoparticle formation, concentration by tangential flow ultrafiltration, and then spray drying to obtain a dry powder. The final powders are readily redispersible and stable over accelerated aging conditions (50°C, 75% RH, open vial) for at least 4 weeks and give equivalent and fast drug release kinetics in both simulated fed and fasted state intestinal fluids, making them suitable for pediatric administration. The nanoparticle-based formulations increase the bioavailability of LMN 4.8-fold in vivo when compared to the control crystalline LMN. We describe the translation of the laboratory-scale process at Princeton University to the clinical manufacturing scale at WuXi AppTec.

Isolation and Identification of Anti-Inflammatory Peptide from Goose Blood Hydrolysate to Ameliorate LPS-Mediated Inflammation and Oxidative Stress in RAW264.7 Macrophages.

This study was designed to isolate an anti-inflammatory activity oligopeptide from goose blood (GBP) for ameliorating LPS-mediated inflammation response and oxidative stress in RAW264.7 macrophages. In this study, GBP was isolated by tangential flow ultrafiltration system (TFUS) combined with size exclusion chromatography (SEC), ion exchange chromatography (IEC), and reversed-phase liquid chromatography (RP-LC), and then identified by liquid chromatography mass spectrometry (LC-MS/MS). The experiment results indicated that the amino acid sequence of oligopeptide with the best anti-inflammatory activity was IIe-Val-Tyr-Pro-Trp-Thr-Gln-Arg (IVYPWTQR), which had a molecular weight of 1062.5720 Da, and was derived from haemoglobin subunit beta OS in goose blood. In addition, IVYPWTQR was confirmed to have satisfactory stability and maintained high anti-inflammatory activity in a simulated gastrointestinal digestion. The mechanism by which the IVYPWTQR protected against LPS-mediated inflammation response was attributed to downregulating the TLR4/NF-kB/iNOS pathway. Moreover, IVYPWTQR ameliorated oxidative stress damage in inflammatory state was attributed to activating antioxidant defence system, which was regulated by Keap-1/NRF2/HO-1 signalling pathway for decreasing the accumulation of reactive oxide species (ROS). In summary, these results indicated GBP could serve as a potential functional factor for prevention and improvement of inflammation mediated by LPS and provided an affordable dietary intervention strategy to prevent inflammation.

Isolation and Identification of Antioxidative Peptide from Goose Liver Hydrolysate to Ameliorate Alcohol-Mediated Oxidative Stress Damage in HHL-5 Hepatocytes.

The aim of this study was to isolate and identify antioxidative peptide from goose liver hydrolysate (GLHP) for ameliorating oxidative stress damage by alcohol in HHL-5 hepatocytes. In this research, the target antioxidative peptides in GLHP were separated, purified, and identified via a tangential flow ultrafiltration system combined with size exclusion chromatography (SEC), ion exchange chromatography (IEC), reversed-phase liquid chromatography (RP-LC), and LC-MS/MS. The results suggested that the amino acid sequence of the target antioxidative peptide for ameliorating alcohol-mediated oxidative stress damage in HHL-5 hepatocytes was Leu-Pro-Leu-Pro-Phe-Pro (LPLPFP), which had a molecular weight of 683.41 Da, and was derived from NADH-ubiquinone oxidoreductase chain 1 in goose liver. In addition, LPLPFP was confirmed to have a satisfactory stability and maintained high hepatic protective activity in a simulated gastrointestinal digestion. Moreover, the mechanism of LPLPFP prevented against oxidative stress damage in HHL-5 hepatocytes was attributed to inhibiting the production of reactive oxide species (ROS) by upregulating genes expression in the Ahr-NQO1 signal pathway. In conclusion, these results indicated that dietary GLHP supplementation could ameliorate alcohol-mediated oxidative stress damage and provide an affordable dietary intervention strategy to prevent alcohol-mediated hepatocyte damage.

Feasibility of membrane ultrafiltration as a single-step clarification and fractionation of microalgal protein hydrolysates.

Protein hydrolysates are one of the most valuable products that can be obtained from lipid-extracted microalgae (LEA). The advantages of protein hydrolysates over other protein products encompass enhanced solubility, digestibility, and potential bioactivity. The development of an economically feasible process to produce protein hydrolysates depends on maximizing the recovery of hydrolyzed native protein from the lipid-extracted algal biomass and subsequent fractionation of hydrolyzed protein slurry. Previously, we reported a method for fractionation of enzymatically generated protein hydrolysates by acidic precipitation of algal cell debris and unhydrolyzed protein, precipitate wash, centrifugation, and depth filtration. The present study evaluates tangential flow ultrafiltration as a single-step alternative to centrifugation, precipitate wash, and depth filtration. The results demonstrate that the tangential flow ultrafiltration process has a potential that deserves further investigation. First, the membrane diafiltration process uses a single and easily scalable unit operation (tangential flow filtration) to separate and “wash out” hydrolyzed protein from the algal residue. Second, the protein recovery yield achieved with the tangential flow process was >70% compared to 64% previously achieved by centrifugation and depth filtration methods. Finally, protein hydrolysates obtained by membrane ultrafiltration exhibited slightly better heat and pH stability.

A Methodological Approach to Water Concentration to Investigate the Presence of SARS-CoV-2 RNA in Surface Freshwaters.

During the COVID-19 public health emergency, an increasing number of studies reported the occurrence of SARS-CoV-2 in wastewaters worldwide, but little is known about the presence of the virus in surface freshwaters. The aim of the current study was to develop and validate an appropriate and scalable methodological approach for the concentration and detection of SARS-CoV-2 from surface freshwater samples, collected within the Milan rural network subjected to flood spillways activity. Overall, both surface water and distilled water samples spiked with inactivated SARS-CoV-2 were used to validate the concentration method for pathogens determination. Two pre-filtration systems, filter paper and Sartolab® P20 (Sartorius, Germany) and two concentration methods, two-phase (PEG-dextran method) separation and tangential flow ultrafiltration (UF), were compared. The effects of pre-filtration and concentration on viral nucleic acid recovery were assessed through real time RT-PCR targeting SARS-CoV-2 and the internal viral control PMMoV (Pepper Mild Mottle Virus). Our results showed that UF is more sensitive than the PEG-dextran method in viral acid nucleic recovery from surface water samples. Better results were obtained pre-filtering samples with Sartolab® P20 and extracting the nucleic acids with undiluted silica, rather than diluted as required by the standard protocol. The proposed method will be used for the monitoring of surface waters in the Milan area.

Process Analytical Technology (PAT) Implementation for Membrane Operations in Continuous Manufacturing of mAbs: Model-Based Control of Single-Pass Tangential Flow Ultrafiltration.

Control of single pass tangential flow ultrafiltration (SPTFF) is crucial for continuous manufacturing of monoclonal antibodies (mAbs). Integrating SPTFF technology into continuous manufacturing trains requires successful resolution of several challenges that arise due to the complexity of mass transfer interactions across multi-membrane configurations, the significant effect of feed material attributes and process variability on flux, and the need for advanced scheduling. In this paper, we propose a real-time, automated monitoring and control strategy for SPTFF in continuous processing of mAbs. The approach leverages a previously developed model for predicting the VCF across an SPTFF module based on the gel polarization model of protein ultrafiltration. A distributed control system (DCS) architecture was created for integrating the monitoring sensors and control elements, including NIRS sensors for concentration monitoring, as well as weighing balances, pressure sensors, pumps, and valves. Two different SPTFF control strategies were developed, firstly for final formulation of the drug product into the drug substance (ultrafiltration and diafiltration), and secondly for in-line concentration between two chromatography steps. Case studies were designed with 15 runs to test the strategy with a range of deviations induced in the feed and process conditions. The retentate concentration was controlled to within 10% of the target value in all runs. The combination of real-time sensor data and model-based control effectively enabled automated and tightly controlled operation of the SPTFF step and is a key enabler of quality by design in continuous mAb manufacturing.

Efficient Small Extracellular Vesicles (EV) Isolation Method and Evaluation of EV-Associated DNA Role in Cell–Cell Communication in Cancer

Simple SummarySmall extracellular vesicles (sEVs) released by all cell types function as a mediator in intercellular communication that can promote cell division and survival to remodel the tumor microenvironment to develop tumor invasion and metastasis. Even though dsDNA baggage is associated with all small EV populations, the functional role of EV-DNA in cancer remains poorly understood. This is due to a lack of methods allowing the efficient separation of small EVs (sEVs) from other non-sEV components. The main aim of our study was to develop an efficient sEV isolation method along with EV-associated DNA (EV-DNA) monitoring tool to evaluate the role of EV-DNA as a mediator of cell–cell communication in cancer. Our detailed small EV-DNA characterization confirmed that isolated sEVs using the TSU method (Tangential flow filtration + Size exclusion chromatography + Ultrafiltration) are free from contaminants such as cell-free and apoptotic bodies DNA, making TSU ideal for performing EV-DNA functional studies. Next, we revealed the exact EV-DNA distribution in the recipient cells using 3D image analysis and the association of EV-DNA with key cellular proteins, which may have an essential role in cancer. In the leukemia model, EV-DNA isolated from leukemia cell lines associated with mesenchymal stromal cells (MSCs), a crucial factor in the bone marrow (BM) microenvironment.Small extracellular vesicles (sEVs) play essential roles in intercellular signaling both in normal and pathophysiological conditions. Comprehensive studies of dsDNA associated with sEVs are hampered by a lack of methods, allowing efficient separation of sEVs from free-circulating DNA and apoptotic bodies. In this work, using controlled culture conditions, we enriched the reproducible separation of sEVs from free-circulated components by combining tangential flow filtration, size-exclusion chromatography, and ultrafiltration (TSU). EV-enriched fractions (F2 and F3) obtained using TSU also contained more dsDNA derived from the host genome and mitochondria, predominantly localized inside the vesicles. Three-dimensional reconstruction of high-resolution imaging showed that the recipient cell membrane barrier restricts a portion of EV-DNA. Simultaneously, the remaining EV-DNA overcomes it and enters the cytoplasm and nucleus. In the cytoplasm, EV-DNA associates with dsDNA-inflammatory sensors (cGAS/STING) and endosomal proteins (Rab5/Rab7). Relevant to cancer, we found that EV-DNA isolated from leukemia cell lines communicates with mesenchymal stromal cells (MSCs), a critical component in the BM microenvironment. Furthermore, we illustrated the arrangement of sEVs and EV-DNA at a single vesicle level using super-resolution microscopy. Altogether, employing TSU isolation, we demonstrated EV-DNA distribution and a tool to evaluate the exact EV-DNA role of cell–cell communication in cancer.

Evaluation of reduction efficiencies of pepper mild mottle virus and human enteric viruses in full-scale drinking water treatment plants employing coagulation-sedimentation–rapid sand filtration or coagulation–microfiltration

Here, we evaluated the reduction efficiencies of indigenous pepper mild mottle virus (PMMoV, a potential surrogate for human enteric viruses to assess virus removal by coagulation-sedimentation–rapid sand filtration [CS–RSF] and coagulation–microfiltration [C–MF]) and representative human enteric viruses in four full-scale drinking water treatment plants that use CS–RSF (Plants A and B) or C–MF (Plants C and D). First, we developed a virus concentration method by using an electropositive filter and a tangential-flow ultrafiltration membrane to effectively concentrate and recover PMMoV from large volumes of water: the recovery rates of PMMoV were 100% when 100-L samples of PMMoV-spiked dechlorinated tap water were concentrated to 20 mL; even when spiked water volume was 2000 L, recovery rates of >30% were maintained. The concentrations of indigenous PMMoV in raw and treated water samples determined by using this method were always above the quantification limit of the real-time polymerase chain reaction assay. We therefore were able to determine its reduction ratios: 0.9–2.7-log10 in full-scale CS–RSF and 0.7–2.9-log10 in full-scale C–MF. The PMMoV reduction ratios in C–MF at Plant C (1.0 ± 0.3-log10) were lower than those in CS–RSF at Plants A (1.7 ± 0.5-log10) and B (1.4 ± 0.7-log10), despite the higher ability of MF for particle separation in comparison with RSF owing to the small pore size in MF. Lab-scale virus-spiking C–MF experiments that mimicked full-scale C–MF revealed that a low dosage of coagulant (polyaluminum chloride [PACl]) applied in C–MF, which is determined mainly from the viewpoint of preventing membrane fouling, probably led to the low reduction ratios of PMMoV in C–MF. This implies that high virus reduction ratios (>4-log10) achieved in previous lab-scale virus-spiking C–MF studies are not necessarily achieved in full-scale C–MF. The PMMoV reduction ratios in C–MF at Plant D (2.2 ± 0.6-log10) were higher than those at Plant C, despite similar coagulant dosages. In lab-scale C–MF, the PMMoV reduction ratios increased from 1-log10 (with PACl [basicity 1.5], as at Plant C) to 2–4-log10 (with high-basicity PACl [basicity 2.1], as at Plant D), suggesting that the use of high-basicity PACl probably resulted in higher reduction ratios of PMMoV at Plant D than at Plant C. Finally, we compared the reduction ratios of indigenous PMMoV and representative human enteric viruses in full-scale CS–RSF and C–MF. At Plant D, the concentrations of human norovirus genogroup II (HuNoV GII) in raw water were sometimes above the quantification limit; however, whether its reduction ratios in C–MF were higher than those of PMMoV could not be judged since reduction ratios were >1.4-log10 for HuNoV GII and 2.3–2.9-log10 for PMMoV. At Plant B, the concentrations of enteroviruses (EVs) and HuNoV GII in raw water were above the quantification limit on one occasion, and the reduction ratios of EVs (>1.2-log10) and HuNoV GII (>1.5-log10) in CS–RSF were higher than that of PMMoV (0.9-log10). This finding supports the usefulness of PMMoV as a potential surrogate for human enteric viruses to assess virus removal by CS–RSF.

Modelling and optimization of single-pass tangential flow ultrafiltration for continuous manufacturing of monoclonal antibodies

• Single pass ultrafiltration is an emerging technology in continuous bioprocessing. • Single pass modules are configured with multiple membranes in series and/or parallel. • Approaches for module configuration are currently not well-defined for the end-user. • A single-membrane model is used as a building block for modelling complex configurations. • An in-silico optimization approach is developed, facilitating membrane area savings. Formulation of biotherapeutics using single pass tangential-flow filtration (SPTFF) is a critical step in continuous manufacturing processes for many drugs including monoclonal antibodies (mAbs). The concentration of the mAb in the final formulation is a critical quality attribute which affects safety and efficacy and is determined by the concentration factor achieved by the process material stream in a single pass through an SPTFF module. Modern SPTFF modules consist of multiple smaller membranes connected in different series and parallel configurations to increase the overall membrane area and residence time of the process material in order to achieve high concentration factors in a single pass. The present work leverages the gel polarization model of protein ultrafiltration to develop a model for the permeate flux vs. time profile of a single membrane inside an SPTFF module based on three key resistances, namely the boundary layer resistance, resistance of the deposited protein layer over time, and intrinsic membrane resistance. Correlations are developed to predict the key mechanistic parameters across the typical operating range of flow rate, bulk concentration and transmembrane pressure in downstream mAb processing steps. The single membrane model is then used as a building block to model complex SPTFF configurations with diverse tree structures, and to facilitate in-silico design of customized SPTFF configurations based on operating targets.

Interaction between humic substances and arsenic species simulating environmental conditions

The importance of evaluating how natural organic matter influences the mobility of arsenic species in an ecosystem is an environmental concern. This work aimed to evaluate the interaction between humic substances (HS) and four arsenic species of high toxicity [As(III), As(V), MMA(V), and DMA(V)] (HS-As) under the influence of HS concentration and pH. Next, the complexing capacity (CC) of HS by As(III) was determined in function of pH, ionic strength and co-existing ions. The free arsenic (Asfree) was determined after a tangential flow ultrafiltration procedure by hydride generation atomic fluorescence spectrometry. The better HS-As interactions for As(III) and As(V) at pH 10.5 reached 52% and 61%, respectively. The increase in pH and ionic strength, as well as co-existing ions increased the CC, which reached 1.57 mg of As(III) g−1 HS. We proposed a HS-As interaction model based on the inner and outer binding sites of HS from these results. The inner sites were occupied through hydrogen bonds, Pearson acid-base, hydrophobic, and van der Waals interactions for trivalent arsenic species, while the interactions through the outer sites for pentavalent arsenic species were mostly by hydrogen bonds and electrostatic forces. According to ecotoxicological studies against Artemia salina, the presence of HS decreased the toxicity of As(III) and As(V) as the lethal concentration increased from 5.81 to 8.82 mg L−1 and from 8.82 to 13.37 mg L−1, respectively. From the results through the proposed model, it was possible to successfully understand the interaction dynamic between soil HS and As(III), As(V), MMA(V) and DMA(V) under simulated environmental conditions.

Establishment and clinical application of a detection method for the infectious hypodermal and hematopoietic necrosis virus in water

To better understand the distribution of viruses in water, we established a method of concentrating the infectious hypodermal and hematopoietic necrosis virus (IHHNV) in aquaculture. In our study, tangential flow ultrafiltration was established to concentrate the virus from water samples. The results showed that the best filtration columns for the concentration of IHHNV were microfiltration columns (CFP-2-E-4MA) and ultrafiltration columns (UFP-50-C-4MA). The results showed that the average concentration recovery of IHHNV was 67.11%. The method was then used to detect viruses in aquaculture water samples, water samples carried with imported parent shrimp and the dissolved water of imported frozen shrimp. The results showed that the sensitivity of virus detection in water is 46copies / μL, and our methods can be an important tool for aquatic biological security in shrimp ponds and the risk assessment of water carried with imported live animals.

A method for characterizing dissolved DNA and its application to the North Pacific Subtropical Gyre

AbstractDissolved DNA (D‐DNA) is a ubiquitous component of dissolved organic matter in aquatic systems. It is operationally defined as the DNA that passes a membrane filter and thus includes pools of truly dissolved “free” DNA (F‐DNA), virion encapsidated DNA, DNA within membrane vesicles, and possibly other bound forms, each with different sources and lability. We investigated whether filtration (< 0.1 μm), concentration by tangential flow ultrafiltration (> 30 kDa), and fractionation in an equilibrium buoyant density gradient could be used to discriminate the mass contributions of the different pools of filterable DNA in seawater. Spike‐in experiments with a known range of DNA standards (75–20,000 bp) indicated that this method results in high recoveries of F‐DNA (68–86%) with minimal degradation. Application of the fractionation method to seawater samples collected from the oligotrophic North Pacific Ocean followed by analysis of fractions (epifluorescence and electron microscopy, DNase digestion) suggested that the low‐density fractions (1.30–1.35 g mL−1) were dominated by vesicle‐like particles, mid‐density fractions (1.45–1.55 g mL−1) by virus‐like particles, and high‐density fractions (1.60–1.70 g mL−1) by F‐DNA. The estimated concentration of DNA that is either F‐DNA, in viruses, or in vesicles was 0.13, 0.14, and 0.08 μg L−1, respectively in the euphotic zone and 0.09, 0.04, and 0.03 μg L−1, respectively in the mesopelagic zone. The approach described should be useful for more detailed investigations of the abundance, dynamics, and sources of DNA in the distinct pools that comprise filterable DNA in aquatic environments.

Implementing PAT for single-pass tangential flow ultrafiltration for continuous manufacturing of monoclonal antibodies

Control of single pass tangential flow ultrafiltration is crucial for continuous manufacturing of monoclonal antibodies. We propose a strategy to control the output concentration of a continuous ultrafiltration step regardless of variations in the volume or concentration of the feed material. Scheduling algorithms for the filtration runs and cleaning cycles are developed, accounting for the expected cycle times of different unit operations in the continuous train. The complete design space for the membrane module is characterized experimentally. An empirical model of the design space is developed to predict the logarithmic relationship between the feed flowrate and the maximum concentration factors achievable in a single pass for feed concentrations in the range of 1–50 g/L and feed flowrates in the range of 10–210 mL/min. The control strategy leverages in-line concentration, flowrate and pressure sensors, including near infrared spectroscopy (NIRS) flow cells to measure the concentration of mAb in the feed and retentate streams in the range 0.5–200 g/L. The control elements are the permeate pump and a variable control valve on the retentate line, which are used to obtain the desired concentration factor across the module in a single pass. Two case studies are designed to test the control system under conditions of high feed flow rate and low feed concentration, and low feed flow rate and high feed concentration, respectively. Successful control is demonstrated in both cases. The proposed system is the first complete approach that integrates process understanding, advanced monitoring sensors, and control strategies to consistently achieve concentration targets over long continuous campaigns for production of monoclonal antibodies.

Isolation, purification, and structure identification of antioxidant peptides from embryonated eggs

Antioxidant peptides are increasingly attracting researchers in medicine and foods. In the present study, egg white hydrolysates of embryonated eggs hatched on the sixth day (EWHD) were segmented consecutively by ultrafiltration membranes with small tangential flow ultrafiltration system. Four segments with more than 30, 30 to 10kDa, 10 to 5kDa, and less than 5kDa of molecular weight cut-off (MWCO) values were separated and were labeled as MWCOI, MWCOII, MWCOIII, and MWCOIV, respectively. The antioxidant activities of segments were investigated by performing DPPH•, •OH radical scavenging, ultra oxygen anion (O2-•), total antioxidant capacity, and reducing power experiments. The results indicated that MWCOI has the strongest scavenging activities on DPPH• radical. However, MWCOIV has the strongest scavenging activities on •OH, O2-•, total antioxidant capacity, and reducing power, which revealed that MWCOIV has strong antioxidant activity. MWCOIV was further separated into 14 fractions via semipreparative reverse-phased high-performance liquid chromatography (RP-HPLC), and their antioxidant activity was evaluated by different antioxidant assays in vitro. The fractions 10 and 7 had strong antioxidant activities. The purities of these 2 fractions were determined by analytical RP-HPLC. Moreover, both fractions 10 and 7 displayed high purity levels, and they were identified by quadrupole time-of-flight tandem mass spectrometry. Only fraction 10, with a molecular weight of 204Da, can be identified to be Ser-Val. EWHD can be considered as a promising source of natural food antioxidants for the development of functional food.

Membrane selection and optimisation of tangential flow ultrafiltration of Cyclopia genistoides extract for benzophenone and xanthone enrichment

Ultrafiltration of Cyclopia genistoides extract was optimised to increase its benzophenone and xanthone content as quantified using HPLC-DAD. Regenerated cellulose (RC) and polyethersulphone membranes with molecular weight cut-offs of 10 and 30 kDa were evaluated in terms of compound enrichment, permeate flux and permeate yield, using dead-end ultrafiltration. Compound enrichment was subsequently optimised using the 10 kDa RC membrane and tangential flow ultrafiltration (TFU). The effect of extract composition on compound enrichment, due to natural variation in the source material, was assessed using extracts from different batches of plant material (n = 11). Transmembrane pressure and feed flow rate affected (p < 0.05) process efficiency (mean permeate flux, compound enrichment and membrane fouling). TFU achieved ≥20% enrichment of the target compounds, proving its suitability for preparation of a nutraceutical extract of C. genistoides.