High Level Of Purity Research Articles

Investigating Sustainable Materials for AEM Electrolysers: Strategies to Improve the Cost and Environmental Impact

In recent years, the EU policy identified the hydrogen as one of the main energy vectors to support the power production from renewable sources. Coherently, electrolysis is suitable to convert energy in hydrogen with no carbon emission and high purity level. Among the electrolysis technologies, the anion exchange membrane (AEM) seems to be promising for the performance and the development potential at relatively high cost. In the present work, AEM electrolysers, and their technological bottlenecks, have been investigated, in comparison with other electrolysers’ technology such as alkaline water electrolysis and proton exchange membranes. Major efforts and improvements are investigated about innovative materials design and the corresponding novel approach as main focus of the present review. In particular, this work evaluated new materials design studies, to enhance membrane resistance due to working cycles at temperatures close to 80 °C in alkaline environment, avoiding the employment of toxic and expensive compounds, such as fluorinated polymers. Different strategies have been explored, as tailored membranes could be designed as, for example, the inclusion of inorganic nanoparticles or the employment of not-fluorinated copolymers could improve membranes resistance and limit their environmental impact and cost. The comparison among materials’ membrane is actually limited by differences in the environmental conditions in which tests have been conducted, thereafter, this work aims to derive reliable information useful to improve the AEM cell efficiency among long-term working periods.

Ultrafast and Controlled Capturing, Loading, and Release of Extracellular Vesicles by a Portable Microstructured Electrochemical Fluidic Device.

Extracellular vesicles (EVs) are secreted by all living cells and are found in body fluids. They exert numerous physiological and pathological functions and serve as cargo shuttles. Due to their safety and inherent bioactivity, they have emerged as versatile therapeutic agents, biomarkers, and potential drug carriers. Despite the growing interest in EVs, current progress in this field is, in part, limited by relatively inefficient isolation techniques. Conventional methods are indeed slow, laborious, require specialized laboratory equipment, and may result in low yield and purity. This work describes an electrochemically controlled "all-in-one" device enabling capturing, loading, and releasing of EVs. The device is composed of a fluidic channel confined within antibody-coated microstructured electrodes. It rapidly isolates EVs with a high level of purity from various biofluids. As a proof of principle, the device is applied to isolate EVs from skin wounds of healthy and diabetic mice. Strikingly, it is found that EVs from healing wounds of diabetic mice are enriched in mitochondrial proteins compared to those of healthy mice. Additionally, the device improves the loading protocol of EVs with polyplexes, and may therefore find applications in nucleic acid delivery. Overall, the electrochemical device can greatly facilitate the development of EVs-based technologies.

Research on the production of 99mTc and 99Mo using multi-layer targets at APEP

One of the most effective methods for producing radioisotopes aimed at Nuclear Medicine applications, such as 99mTc and 99Mo, is to use a compact proton accelerator located near hospitals. Target utilization efficiency is a critical factor in the production of these isotopes, through the 100Mo(p, 2n)99mTc and 100Mo(p, pn)99Mo nuclear reaction routes. To maximize the utilization efficiency, a multi-layer target structure was developed aimed at achieving a high radionuclidic purity (RNP) level. The yield and RNP of different target structures were calculated and compared by using FLUKA code, and benchmark experiments were conducted using a proton beam at the China Spallation Neutron Source (CSNS) Associated Proton Experimental Platform (APEP). Results showed that the multi-layer target structure has the potential to significantly reduce production costs compared to a single-layer target. The multi-layer target structure is being adopted for the production of both 99mTc and 99Mo at APEP, with an expected 99mTc yield of up to 60 MBq enough for preclinical studies after 100Mo target being irradiated for about 2 h at a beam intensity of 62.5 μA.

Performance of tumour microenvironment deconvolution methods in breast cancer using single-cell simulated bulk mixtures

Cells within the tumour microenvironment (TME) can impact tumour development and influence treatment response. Computational approaches have been developed to deconvolve the TME from bulk RNA-seq. Using scRNA-seq profiling from breast tumours we simulate thousands of bulk mixtures, representing tumour purities and cell lineages, to compare the performance of nine TME deconvolution methods (BayesPrism, Scaden, CIBERSORTx, MuSiC, DWLS, hspe, CPM, Bisque, and EPIC). Some methods are more robust in deconvolving mixtures with high tumour purity levels. Most methods tend to mis-predict normal epithelial for cancer epithelial as tumour purity increases, a finding that is validated in two independent datasets. The breast cancer molecular subtype influences this mis-prediction. BayesPrism and DWLS have the lowest combined numbers of false positives and false negatives, and have the best performance when deconvolving granular immune lineages. Our findings highlight the need for more single-cell characterisation of rarer cell types, and suggest that tumour cell compositions should be considered when deconvolving the TME.

CPVL suppresses metastasis of nasopharyngeal carcinoma through inhibiting epithelial-mesenchymal transition.

Distant metastasis is the main obstacle to treating nasopharyngeal carcinoma (NPC). Tumor distance metastasis is a complex process involving the jointly participation of multiple oncogenes, tumor suppressor genes, and metastasis-associated genes. Enough accurate prognostic genes for evaluating metastasis risk are lacking. We aimed to identify more precise biomarkers for NPC metastasis. We performed weighted gene co-expression network analysis, differentially expressed gene analysis, univariate and multivariate stepwise Cox regression, and Kaplan-Meier (K-M) survival analyses, on data obtained from RNA sequencing of 10 NPC samples and the public database, to identify key genes correlated with NPC metastasis. Wound healing assays, transwell assays, and immunohistochemistry were conducted to validate our bioinformatic conclusions. Western blotting was performed to evaluate and quantify the effect of identified EMT genes on epithelial-mesenchymal transition (EMT) of NPC. Combined our own RNA sequencing data and public data, we determined carboxypeptidase vitellogenic-like protein (CPVL) as a tumor suppressor for NPC. Pathway enrichment analyses indicated that genes associated with CPVL are involved in EMT. NPC with low CPVL expression had high tumor purity and low levels of immune cells. Experimental results showed that CPVL protein predominantly expressed in cytoplasmic and membranous and it exhibited higher expression levels in NPC tissues without distant metastasis than those with distant metastasis. CPVL inhibits the migration and invasive capability of NPC cells. Overexpression of CPVL upregulates E-cadherin and ZO-1, whereas it downregulates vimentin, suggesting that CPVL suppresses tumor metastasis by inhibiting EMT. CPVL inhibits migration and invasion of NPC cells and is associated with tumor metastasis suppression through upregulating epithelial marker and inhibiting mesenchymal marker expression and could be a prognostic biomarker for metastasis risk evaluation in NPC.

A practical approach to enhancing energy efficiency in dual-stage membrane processes for various industrial gas separations: Membrane combination optimization

Membrane-based separation technology offers notable advantages in terms of its compact size and energy efficiency. However, single-stage membrane processes encounter limitations in achieving high levels of purity for target gases, particularly when dealing with low feed concentrations, due to the constrained separation performance of polymeric membranes. Herein, we report on the optimization of dual-stage membrane processes for diverse gas separation applications, including CO2/N2, CO2/CH4, H2/N2, and H2/CH4 separation. The optimization process involves the utilization of various membrane combinations, which are carefully selected using a user-friendly apparent selectivity model. In particular, our evaluation includes an assessment of the separation performance of the proposed membrane combinations for different molar fractions in the feed while maintaining a fixed target purity. Additionally, we determine their practical feasibility by estimating other process parameters such as the required membrane area, energy demand, and material cost. Through process analysis, it is observed that a combination of more selective membranes in the first stage and more permeable membranes in the second stage can yield economic benefits for various gas separation applications. The present study provides a practical approach to enhancing the process efficiency of a dual-stage membrane process for various gas separation applications.

Hierarchical Structure of SiO<sub>2</sub> by <i>In Situ</i> Modification Combining Quartz Sand and Methyltrimethoxysilane

Hierarchical Structure of SiO<sub>2</sub> by <i>In Situ</i> Modification Combining Quartz Sand and Methyltrimethoxysilane

Synthesis of nano-silica as a promising route of recycling phosphate waste rocks and its incorporation in mortars

This article is devoted to the synthesis of nano-silica from phosphate waste rocks as a promising route of recycling these silica-rich phosphate by-products. Furthermore, their reactivity potential will be investigated while incorporating them as nano-additive and as a partial replacement to cement in the conventionally used recipes of mortars. It has been shown that the synthesized nano-silica has a higher level of purity and a large specific surface area, which is about 8 times higher than the initial raw material. Various formulations of mortars were tested after adding different percentages of nano-silica to the ordinarily used recipe, at a constant water-to-cement ratio, and their microstructure, mineralogical composition, and mechanical properties were investigated. It was shown that compressive strength and flexural strength increased, and the fluidity of the mortars decreased when the ratio of nano-silica increased from 1% to 5%.

Cosmological Constraints from the BOSS DR12 Void Size Function

We present the first cosmological constraints derived from the analysis of the void size function. This work relies on the final Baryon Oscillation Spectroscopic Survey (BOSS) Data Release 12 (DR12) data set, a large spectroscopic galaxy catalog, ideal for the identification of cosmic voids. We extract a sample of voids from the distribution of galaxies, and we apply a cleaning procedure aimed at reaching high levels of purity and completeness. We model the void size function by means of an extension of the popular volume-conserving model, based on two additional nuisance parameters. Relying on mock catalogs specifically designed to reproduce the BOSS DR12 galaxy sample, we calibrate the extended size function model parameters and validate the methodology. We then apply a Bayesian analysis to constrain the Lambda cold dark matter (ΛCDM) model and one of its simplest extensions, featuring a constant dark energy equation of state parameter, w. Following a conservative approach, we put constraints on the total matter density parameter and the amplitude of density fluctuations, finding Ωm = 0.29 ± 0.06 and . Testing the alternative scenario, we derive w = −1.1 ± 0.2, in agreement with the ΛCDM model. These results are independent and complementary to those derived from standard cosmological probes, opening up new ways to identify the origin of potential tensions in the current cosmological paradigm.

Lipid membrane modulated control of magnetic nanoparticles within bacterial systems

Bacterial magnetosomes synthesized by the magnetotactic bacterium Magnetospirillum magneticum are suitable for biomedical and biotechnological applications because of their high level of chemical purity of mineral with well-defined morphological features and a biocompatible lipid bilayer coating. However, utilizations of native magnetosomes are not sufficient for maximum effectiveness in many applications as the appropriate particle size differs. In this study, a method to control magnetosome particle size is developed for integration into targeted technological applications. The size and morphology of magnetosome crystals are highly regulated by the complex interactions of magnetosome synthesis-related genes; however, these interactions have not been fully elucidated. In contrast, previous studies have shown a positive correlation between vesicle and crystal sizes. Therefore, control of the magnetosome vesicle size is tuned by modifying the membrane lipid composition. Exogenous phospholipid synthesis pathways have been genetically introduced into M.magneticum. The experimental results show that these phospholipids altered the properties of the magnetosome membrane vesicles, which yielded larger magnetite crystal sizes. The genetic engineering approach presented in this study is shown to be useful for controlling magnetite crystal size without involving complex interactions of magnetosome synthesis-related genes.

Prognostic value of amino acid metabolism-related gene expression in invasive breast carcinoma.

An increasing number of studies indicated that metabolic reprogramming of amino acid metabolism may either promote or inhibit tumor progression. The purpose of this study was to investigate the ability of a gene risk signature associated with amino acid metabolism to predict the prognosis and immune characteristics of invasive breast carcinoma. LASSO Cox regression analysis was performed to construct and validate the prognostic risk signature based on the expression of 9 amino acid metabolism-related genes. The predictive value of the signature, immune characteristics, and chemotherapeutic drugs was also predicted. Finally, 9 significant genes were examined in MDA-MB-231 and MCF-7 cells, and the predicted chemotherapeutic drugs were also verified. The prognosis of the low-risk group was better than that of the high-risk group. The areas under the curve (AUCs) at 1, 2, and 3 years were 0.852, 0.790, and 0.736, respectively. In addition, the GSEA results for KEGG and GO revealed that samples with a high-risk score exhibited a variety of highly malignant manifestations. The high-risk group was characterized by an increased number of M2 macrophages, a high level of tumor purity, low levels of APC co-stimulation, cytolytic activity, HLA, para-inflammation, and type I IFN response. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) confirmed that MDA-MB-231 and MCF-7 cells express 9 amino acid metabolism-related genes differently. In addition, cell experiments were conducted to examine the effect of cephaeline-induced on cell viability, migration ability, and protein expression of the PI3K/AKT signaling pathway and HIF-1α. We established a risk signature based on 9 amino acid metabolism-related genes for invasive breast carcinoma. Further analyses revealed that this risk signature is superior to other clinical indexes in survival prediction and that the subgroups identified by the risk signature exhibit distinct immune characteristics. Cephaeline was determined to be a superior option for patients in high-risk groups.

Partial Characterization and Immunomodulatory Effects of Exopolysaccharides from Streptococcus thermophilus SBC8781 during Soy Milk and Cow Milk Fermentation.

The immunomodulatory properties of exopolysaccharides (EPSs) produced by Streptococcus thermophilus have not been explored in depth. In addition, there are no comparative studies of the functional properties of EPSs produced by streptococci in different food matrices. In this work, EPSs from S. thermophilus SBC8781 were isolated after soy milk (EPS-s) or cow milk (EPS-m) fermentation, identified, and characterized in their abilities to modulate immunity in porcine intestinal epithelial cells. Fresh soy milk and cow milk were inoculated with S. thermophilus SBC8781 (7 log CFU/mL) and incubated at 37 °C for 24 h. The extraction of EPSs was performed by the ethanol precipitation method. Analytical techniques, including NMR, UV-vis spectroscopy, and chromatography, identified and characterized both biopolymer samples as polysaccharides with high purity levels and similar Mw. EPS-s and EPS-m had heteropolysaccharide structures formed by galactose, glucose, rhamnose, ribose, and mannose, although with different monomer proportions. On the other hand, EPS-s had higher quantities of acidic polymer than EPS-m. The biopolymer production of the SBC8781 strain from the vegetable culture broth was 200-240 mg/L, which was higher than that produced in milk, which reached concentrations of 50-70 mg/L. For immunomodulatory assays, intestinal epithelial cells were stimulated with 100 µg/mL of EPS-s or EPS-m for 48 h and then stimulated with the Toll-like receptor 3 agonist poly(I:C). EPS-s significantly reduced the expression of IL-6, IFN-β, IL-8, and MCP-1 and increased the negative regulator A20 in intestinal epithelial cells. Similarly, EPS-m induced a significant reduction of IL-6 and IL-8 expressions, but its effect was less remarkable than that caused by EPS-s. Results indicate that the structure and the immunomodulatory activity of EPSs produced by the SBC8781 strain vary according to the fermentation substrate. Soy milk fermented with S. thermophilus SBC8781 could be a new immunomodulatory functional food, which should be further evaluated in preclinical trials.

Chondroitin Sulfate Supplements for Osteoarthritis: A Critical Review.

Over the years, chondroitin sulfate (CS) has been used as a slow-acting drug for the treatment of osteoarthritis, for the reduction of pain and improvement of function, and for its disease-modifying properties by limiting cartilage volume loss and joint space narrowing progression. However, there have been inconsistencies in published trials regarding clinical efficacy, with reports of a lack of significant effects compared to placebo. The therapeutic effects of chondroitin sulfate may depend on many variables, such as the source of origin, purity, and contamination with by-products. Another source of confusion may be related to the fact that CS is commonly combined with glucosamine, which makes it challenging to isolate the specific contribution of chondroitin to the therapeutic outcome. This is aggravated by the fact that CS supplements, used in many countries, are not regulated, and labels wrongly claim high levels of purity. Many of these inferior CS products may have been used in clinical trials, which may have had limited but significant results. This has led to recent recommendations to opt for higher-purity pharmacologic-grade CS for the treatment of OA. This article aims to provide an up-to-date view of the current literature regarding the biological effects and efficacy of CS and discusses the quality of available chondroitin sulfate supplements and the current direction in CS investigation. This review concludes that pharmacologic-grade CS supplements may have clinically significant benefits when properly standardized; however, high-quality evidence from properly designed clinical trials is still needed to draw definitive conclusions about clinical efficacy in osteoarthritis.

Towards net zero emissions, recovered silicon from recycling PV waste panels for silicon carbide crystal production

The high rate of PV adaptation around the world requires a strategy for recovery of the materials from PV waste panels and a circular market development. In particular, the silicon recovered from the PV cells can be used in different applications. A valuable acquisition is to refine the recovered silicon at metallurgical grade to a high level of purity namely electronic grade silicon which in conjunction with graphite can be the raw material for silicon carbide crystal production. Based on a comprehensive and critical review of the latest research and development of relevant technologies, a manufacturing process is proposed to increase the usage of renewable resources, reduce the energy consumption and CO2e emissions. The recovered silicon from recycling PV waste can reduce the CO2e emissions to a third of what is involved in the current carbothermic reduction of quartz in an induction furnace. Based upon the thorough evaluation of available evidence, a continuous coupled process is envisaged where the upgraded metallurgical grade silicon from recycling PV waste undergoes further vacuum and gas refining to the electronic grade silicon. Then the purified liquid silicon enters a vertical Bridgeman furnace for silicon carbide crystal production through a bottom seeded solution growth process. Considering the silicon melting point at 1410 °C, omitting a solidification/ meting step after vacuum/gas refining leads to energy and material saving. The continuous method can reduce the CO2e emissions up to 75% compared with the current silicon carbide production through Acheson process.

Efficiency of Biological Typing Methods in Maize Hybrid Genetic Purity Estimation.

A high level of genetic purity in crop varieties must be achieved and maintained for agronomic performance, encouraging investment and innovation in plant breeding and ensuring that the improvements in productivity and quality imparted by breeders are delivered to the consumer. Since the success of hybrid seed production is dependent upon the genetic purity of the parental lines, in this study, the experimental F1exp maize hybrid and its parental inbreeds were used as a model system to examine the discriminative power of morphological, biochemical and SSR markers for seed purity assay. The highest number of off-type plants was estimated by morphological markers. According to the comparison of prolamins and albumins banding patterns of parental and derived F1exp seeds, genetic impurities could not be detected. Molecular analysis detected two types of genetic profile irregularity. Beside its use for verifying varieties of maize, report on umc1545 primer pair ability to detect non-specific bands (i.e., off-types), in both the maternal component and F1exp, which is the first report on this issue yet, strongly supports the recommendation of this SSR marker use for more accurate and time-efficient maize hybrids and parental lines genetic pyrity testing.

Effectively removing the homodimer in bispecific antibodies by weak partitioning mode of anion exchange chromatography

Small amounts of by-products are nevertheless created during the recombinant production of IgG-like bispecific antibodies due to imbalanced chain expression and improper chain pairing, despite the employment of molecular strategy techniques to promote accurate pairing. Among them, homodimers represent the species that are more difficult to remove due to their physical and chemical properties being similar to the target antibody. Homodimer by-products are always produced even though various technologies can significantly increase the expression of heterodimers, so a robust purification process to recover high-purity heterodimers is required. Most of the chromatography methods commonly adopt the bind-and-elute mode or two-step to separate homodimers, which has numerous drawbacks such as prolonged process times and limited dynamic binding capacity. Flow-through mode of anion exchange is a frequently-used polishing step for antibodies, but it is typically regarded as being more effective for host-cell protein or host-cell DNA removal rather than other product-related impurities such as homodimers and aggregates. This paper demonstrated that single-step anion exchange chromatography allows high capacity and effective clearance of the homodimer byproduct to be simultaneously achieved, suggesting that weak partitioning was a better polishing strategy for achieving a high level of heterodimer purity. And robust operation range of anion exchange chromatography steps for homodimer removal was also developed by leveraging the design of experiments.

An integrated geochemical spatial and temporal survey of thermal springs to characterize the geothermal resource of a volcano (Piton des Neiges, Réunion Island)

Geothermal exploration can be challenging in the absence of obvious surface indicators of a high enthalpy geothermal resource such as geysers, fumaroles or thermal springs with temperatures near 100 °C. This is the case at the Piton des Neiges volcano (Réunion Island), which displays evidence of hydrothermal activity mostly as thermal springs with temperatures not exceeding 50 °C, affected by mixing and reequilibration, and emerging in a complex geological framework, resulting from the polygenic history of this dormant volcano. Yet, these thermal springs keep a partial record of the reservoir geochemistry. Here, we propose an exploration method to set this thermal signature apart from the contribution of other processes and trace back the equilibrium conditions, enabling us to locate thermal fluid circulations, and evaluate their temperature and availability. We report its application at Piton des Neiges volcano. We led an extensive geochemical survey which nearly doubled the number of thermal springs reported on the volcano and dramatically extended the existing geochemical database in space and time. In tandem, we also monitored δ18O and δD values in rainwaters and thermal springs over two years to compare their temporal variations.Our surveys show that the repartition of thermal springs is controlled by only one of the volcanic rift zones (the N30°E axis), which is thus identified as the heat source of hydrothermal circulations. This N30°E rift zone is also a preferential pathway for the degassing of mantellic CO2. In addition, the composition of thermal waters is influenced by syenite intrusions along the rim of the caldera, acting as drains for the uprise of hydrothermal waters. O, H and Sr isotopic signatures of springs show that the geothermal resource of Piton des Neiges is in fact made of two independent and unconnected hydrothermal systems on the northern and southern circular depressions of the volcano (the cirques of Salazie and Cilaos, respectively). Their recharge is meteoric, multi-year, and mostly achieved by cyclonic events. Thermal waters experience reequilibration during their ascent and various degrees of CO2 input or degassing. Last equilibrium temperatures of water/rock interaction, estimated by multicomponent geothermometry, reach up to 125 ± 6 °C in the cirque of Salazie, and up to 141 ± 15 °C in the cirque of Cilaos, with possibility for hotter waters before reequilibration. Taken together, our results suggest that the most promising target for future exploration is located in the cirque of Cilaos, along the caldera rim, and in proximity of syenite intrusions.With a thorough prospecting of thermal springs, a comprehensive geochemical study and a geochemical monitoring of the different water reservoirs, we improved our understanding of the geothermal resource and identified future exploration targets at Piton des Neiges volcano.A separation scheme for strontium and light rare earth elements and its application to the isotopic analysis of strontium and neodymium in silicate rocks are presented. This method benefits from the selectivity and high capacity of two newly introduced extraction Chromatographic materials, referred to as Sr.Spec and TRU.Spec, respectively. These afford a straightforward separation of Sr and Sm + Nd with high yield, good purity and satisfactory blank levels, on very small (0.25 ml) columns using small volumes of solutions of a single mineral acid, HNO3. The validity of the method is illustrated by the measurement of 87Sr/ 86Sr and 143Nd/144Nd ratios in twelve international standard reference materials.

Valuable pigments from microalgae: phycobiliproteins, primary carotenoids, and fucoxanthin.

Phycobiliproteins, carotenoids and fucoxanthin are photosynthetic pigments extracted from microalgae and cyanobacteria with great potential biotechnological applications, as healthy food colorants and cosmetics. Phycocyanin possesses a brilliant blue color, with fluorescent properties making it useful as a reagent for immunological essays. The most important source of phycocyanin is the cyanobacterium Arthrospira platensis, however, recently, the Rhodophyta Galdieria sulphuraria has also been identified as such. The main obstacle to the commercialization of phycocyanin is represented by its chemical instability, strongly reducing its shelf-life. Moreover, the high level of purity needed for pharmaceutical applications requires several steps which increase both the production time and cost. Microalgae (Chlorella, Dunaliella, Nannochloropsis, Scenedesmus) produce several light harvesting carotenoids, and are able to manage with oxidative stress, due to their free radical scavenging properties, which makes them suitable for use as source of natural antioxidants. Many studies focused on the selection of the most promising strains producing valuable carotenoids and on their extraction and purification. Among carotenoids produced by marine microalgae, fucoxanthin is the most abundant, representing more than 10% of total carotenoids. Despite the abundance and diversity of fucoxanthin producing microalgae only a few species have been studied for commercial production, the most relevant being Phaeodactylum tricornutum. Due to its antioxidant activity, fucoxanthin can bring various potential benefits to the prevention and treatment of lifestyle-related diseases. In this review, we update the main results achieved in the production, extraction, purification, and commercialization of these important pigments, motivating the cultivation of microalgae as a source of natural pigments.

A closed, autologous bioprocess optimized for TCR-T cell therapies.

Autologous cell therapy has proven to be an effective treatment for hematological malignancies. Cell therapies for solid tumors are on the horizon, however the high cost and complexity of manufacturing these therapies remain a challenge. Routinely used open steps to transfer cells and reagents through unit operations further burden the workflow reducing efficiency and increasing the chance for human error. Here we describe a fully closed, autologous bioprocess generating engineered TCR-T cells. This bioprocess yielded 5-12 × 10e9 TCR-expressing T cells, transduced at low multiplicity of infections, within 7-10 days, and cells exhibited an enriched memory T-cell phenotype and enhanced metabolic fitness. It was demonstrated that activating, transducing, and expanding leukapheresed cells in a bioreactor without any T-cell or peripheral blood mononuclear cell enrichment steps had a high level of T-cell purity (~97%). Several critical process parameters of the bioreactor, including culturing at a high cell density (7e6 cells/mL), adjusting rocking agitations during phases of scale-up, lowering glycolysis through the addition of 2-deoxy- d-glucose, and modulating interleukin-2 levels, were investigated on their roles in regulating transduction efficiency, cell growth, and T-cell fitness such as T-cell memory phenotype and resistance to activation-induced cell death. The bioprocess described herein supports scale-out feasibility by enabling the processing of multiple patients' batches in parallel within a Grade C cleanroom.

A flow-through strategy using supported ionic liquids for L-asparaginase purification

L-asparaginase (ASNase) is an amidohydrolase enzyme widely distributed in nature, e.g., microorganisms, plants and tissues of several animals. Nevertheless, microorganisms are the preferential source of ASNase since they usually grow in simple substrates and culture conditions. However, a high level of enzyme purity is required by the pharmaceutical industry, in which the applied downstream process may account for up to 80% of the total ASNase production costs. Silica-based supported ionic liquid-like phase (SSILLP) materials are here proposed as alternative immobilization/capture or processing supports for the ASNase purification. SSILLP materials with different alkyl chain lengths at the cation source and Cl− as the counterion were investigated to purify ASNase by the flow-through like mode. Silica functionalized with dimethylbutylpropylammonium chloride ([Si][N3114]Cl) was selected as the most promising material since it displayed the highest purification factor (1.65) and specific activity of ASNase (0.026 U mg−1) achieved. The ASNase purification operating conditions were then optimized through Response Surface Methodology, using pH (range in which enzyme is active) and solid/liquid ratio (S/L ratio) as factors, achieving a maximum purification factor of 3.36. Semi-continuous purification of ASNase was finally performed under the optimized purification conditions (pH 3 and S/L ratio of 15), enabling a purification factor of 5.15. This corresponds to a 3.12- and a 1.53-fold increase in the purification factor obtained compared with the initial screening and batch assays under optimized purification conditions. These findings demonstrate that SSILLP materials can act as simple semi-continuous ASNase purification supports with potential in flow-through downstream processing.