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1
المؤلفون: Silva, Luís C. V., Sousa, Maria I., Correia, Andreia, Freire, Mara G., Silva, Francisca A. e, Pedro, Augusto Q.
مصطلحات موضوعية: mRNA vaccines, Process integration, In vitro transcription, Thermoreversible aqueous biphasic systems, Ionic liquids
الوصف: COVID-19 reinforced the potential of messenger RNA (mRNA) vaccines as effective platforms to control disease outbreaks, given their rapid development, safety and efficacy. Nevertheless, mRNA manufacturing can be optimized by combining production with initial clarification steps. This work aims to integrate mRNA production and initial clarification steps, using thermoreversible ionic liquid-based aqueous biphasic systems (IL-ABS), ultimately simplifying mRNA vaccine production. Quality control methods (dot-bot, electrophoresis, among others) were designed to evaluate mRNA production by in vitro transcription, accessing its purity and integrity. Polymer-based ABS composed of dextran from Leuconostoc spp. with an average molecular weight of 450.000-650.000 g/mol (Dex 500) and polyethylene glycol (PEG), 3350 g/mol, with and without ionic liquid as adjuvants (Tetrabutylphosphonium bromide; Tetrabutylammonium bromide; 1-butyl-3-methylimidazolium chloride; Tetrabutylphosphonium chloride) were studied. Polymer-ABS thermoreversability is achieved by lowering the monophasic system temperature from 37 to 25 degrees, enabling phase separation. Preliminary extraction experiments with Dex-PEG ABS revealed mRNA can be recovered with high integrity in the Dex-rich phase as well as common contaminants such as double-stranded mRNA and T7 RNA Polymerase. However, the addition of ILs as adjuvants to the ABS changed the mRNA partition profile, being mainly found in the phase enriched in PEG and IL. Promising systems for mRNA purification have been identified. Using the most promising IL-ABS previously identified, current work focusses of designing the integrated mRNA production-clarification process. In conclusion, thermoreversible IL-ABS can be used as production-clarification platforms to diminish costs, ecological footprint and mRNA production time, while maintaining mRNA integrity and yield.
وصف الملف: application/pdf
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2دورية أكاديمية
المؤلفون: Marguerita E. Rosa, Maria S.M. Mendes, Diana C.V. Belchior, João A.P. Coutinho, Francisca A. e Silva, Mara G. Freire
المصدر: Advances in Sample Preparation, Vol 10, Iss , Pp 100116- (2024)
مصطلحات موضوعية: CYFRA 21–1, Human serum pretreatment, Aqueous biphasic systems, Three-phase partitioning system, Polyethylene glycol, Chemistry, QD1-999
الوصف: Analysing biomarkers in human serum could be used as an effective and less invasive approach for the diagnosis of lung cancer; however, biomarker detection reliability is highly limited due to matrix effects. Herein, aqueous biphasic systems (ABS) are studied as platforms for human serum pretreatment, allowing the simultaneous depletion of high abundant proteins and biomarker extraction. By using ABS varying the polyethylene glycol (PEG) molecular weight between 400 and 6000 g·mol−1 and adopting phosphate buffer as the other phase-forming component, the depletion of the high abundance serum proteins immunoglobulin G (IgG) and human serum albumin (HSA) is induced at the ABS interphase, through precipitation, forming a three-phase partitioning system (ABS-TPP). Maximum depletion efficiencies of 99 % for IgG and 70 % for HSA were achieved in one step using PEG 1500-based ABS-TPP. On the other hand, lung cancer biomarkers, such as CYFRA 21–1, are extracted to the PEG-rich phase of the same ABS-TPP with recovery yields of 91 %. This work shows that a proper selection of the PEG molecular weight in the ABS composition leads to the efficient depletion of high-abundance proteins and extraction of cancer biomarkers from human serum, in a single step, confirming the potential of ABS for sample pretreatment to improve biomarker analysis.
وصف الملف: electronic resource
العلاقة: http://www.sciencedirect.com/science/article/pii/S2772582024000159Test; https://doaj.org/toc/2772-5820Test
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3
المؤلفون: Sousa, Maria I., Silva, Luís C. V., Correia, Andreia, Freire, Mara G., Silva, Francisca A. e, Pedro, Augusto Q.
مصطلحات موضوعية: mRNA nanomedicines, Ionic liquids, Thermoreversible aqueous biphasic systems, In vitro transcription
الوصف: It is undeniable the potential of messenger RNA (mRNA) vaccines as effective tools to manage infectious disease outbreaks. However, mRNA nanomedicines production is complex and expensive, requiring improved technologies to produce stable and widely accessible products, whilst meeting required production times and fulfilling market demands. Ionic liquids (ILs) are composed of organic cations and organic or inorganic anions. Given their remarkable structural diversity, ILs can be properly designed to improve RNA stability. Moreover, as phase-forming agents of aqueous biphasic systems (ABS), ILs contribute to develop highly selective purification processes. This work aims to integrate production-clarification steps of mRNA vaccines, using thermoreversible IL-based ABS, simplifying subsequent purification steps. Several quality control methods were designed to evaluate integrity and purity of mRNA manufactured resorting to in vitro transcription. ABS formed by dextran from Leuconostoc spp. with an average molecular weight of 450.000-650.000 g/mol (Dex 500) and polyethylene glycol (PEG) 3350 g/mol, containing ILs as adjuvants were characterized regarding their thermoreversible nature. Preliminary mRNA partition experiments using IL-based ABS indicate that mRNA is preferentially partitioned toward the IL-PEG-Rich phase. Promising systems for mRNA purification have been identified. Ongoing work focuses on selecting the most promising IL-based ABS for initial mRNA clarification. In the future, mRNA production-clarification will be integrated into the best performing system to overcome current challenges in mRNA nanomedicine manufacturing, such as sustainability and production speed, whilst improving mRNA stability and yield.
وصف الملف: application/pdf
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4
المؤلفون: Pereira, Ana F., Pedro, Augusto Q., Castro, Leonor S., Quental, Maria J., Tavares, Ana P. M., Branco, Luís C., Coutinho, João A. P., Sousa, Fani, Freire, Mara G.
مصطلحات موضوعية: Biopharmaceuticals, Aqueous biphasic systems, Amino acid ionic liquid, Purification, Stability
الوصف: The emergence of RNA as a promising biopharmaceutical paved the way for the development of innovative medicines with broad therapeutic and prophylactic efficiencies in infectious diseases and cancer. Notwithstanding their relevance on this field, the labile nature and intrinsic low stability of RNA coupled with the laborious and costly methods of extraction and purification of this biopolymer constitute the main challenge for its widespread application1. To surpass the described bottlenecks, more competitive and sustainable strategies for purifying RNA are of crucial relevance, where amino-acid-based ILs (AA-ILs) may play a prominent role on this field. In fact, the high affinity between amino-acids and RNA as well as the favourable nucleic acids-stabilization properties exhibited by AA-ILs justifies the investigation of AA-ILs as RNA purification platforms2. From the exposed, this work aims to develop alternative cost-effective and sustainable purification-preservation platforms for RNA, taking advantage of the tunable character of ILs, with the ultimate goal of purifying RNA from a complex recombinant lysate. AA-ILs comprising cholinium, L-arginine, L-lysine and L-histidine as cations combined with chloride, DL-aspartate, L-tyrosine or L-phenylalanine were synthesized, characterized, and their ability to form two phases with distinct salts and polymers investigated. All AA-ILs in study formed ABS with polypropylene glycol with a molecular weight of 400 g.mol-1 (PPG 400) and were further investigated as potential extraction and preservation platforms for RNA. It was demonstrated that RNA was majorly extracted to the IL-rich phase while simultaneously preserving its integrity and stability. Ongoing work is focusing on the application of the most promising IL-based ABS for the separation of RNA from complex recombinant lysates. Overall, the approach herein developed represents a promising strategy to surpass the critical demand of RNA with high integrity, purity and biological activity, envisaging its potential use as biotherapeutics.
وصف الملف: application/pdf
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5دورية أكاديمية
المصدر: International Journal of Molecular Sciences, Vol 25, Iss 11, p 5766 (2024)
مصطلحات موضوعية: lysozyme, BSA, aqueous biphasic systems, biocompatible fluorinated ionic liquids, selective partition, Biology (General), QH301-705.5, Chemistry, QD1-999
الوصف: This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [N1112(OH)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [N1112(OH)][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs’ biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and FILs aggregates. Two of the FIL-based ABSs (30 %wt [C2C1Im][C4F9SO3] + 2 %wt K3PO4 and 30 %wt [C2C1Im][C4F9SO3] + 25 %wt sucrose) allowed the simultaneous purification of Lys and BSA in a single ABS extraction step with high yield (extraction efficiency up to 100%) for both proteins. The purity of both recovered proteins was validated by SDS-PAGE analysis. Even with a high-charge density salt, the FIL-based ABSs developed in this work seem more amenable to be tuned. Lys and BSA were purified through selective partition to opposite phases in a single FIL-based ABS extraction step. FIL-based ABSs are proposed as an improved extraction step for proteins, based on their biocompatibility, customizable properties, and selectivity.
وصف الملف: electronic resource
العلاقة: https://www.mdpi.com/1422-0067/25/11/5766Test; https://doaj.org/toc/1661-6596Test; https://doaj.org/toc/1422-0067Test
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6دورية أكاديمية
المساهمون: LAQV@REQUIMTE, DQ - Departamento de Química
مصطلحات موضوعية: aqueous biphasic systems, biocompatible fluorinated ionic liquids, BSA, lysozyme, selective partition, Catalysis, Molecular Biology, Spectroscopy, Computer Science Applications, Physical and Theoretical Chemistry, Organic Chemistry, Inorganic Chemistry
الوصف: Funding Information: This research was funded by FCT/MCTES (Portugal) through the project PTDC/EQU-EQU/2223/2021 (DOI 10.54499/PTDC/EQU-EQU/2223/2021). Funding Information: This research was supported by FCT/MCTES (Portugal), through grant SFRH/BD/139677/2018 (S.F.C.), and the Individual Call to Scientific Employment Stimulus 2020.00835.CEECIND (J.M.M.A.) and 2021.01432.CEECIND (A.B.P.). This work also received support from FCT/MCTES (LA/P/0008/2020 DOI https://doi.org/10.54499/LA/P/0008/2020Test , UIDP/50006/2020 DOI https://doi.org/10.54499/UIDP/50006/2020Test and UIDB/50006/2020 DOI https://doi.org/10.54499/UIDB/50006/2020Test ), through national funds. Publisher Copyright: © 2024 by the authors. ; This work unfolds functionalized ABSs composed of FILs ([C2C1Im][C4F9SO3] and [(Formula presented.)][C4F9SO3]), mere fluoro-containing ILs ([C2C1Im][CF3SO3] and [C4C1Im][CF3SO3]), known globular protein stabilizers (sucrose and [(Formula presented.)][C4F9SO3]), low-molecular-weight carbohydrate (glucose), and even high-charge density salt (K3PO4). The ternary phase diagrams were determined, stressing that FILs highly increased the ability for ABS formation. The functionalized ABSs (FILs vs. mere fluoro-containing ILs) were used to extract lysozyme (Lys). The ABSs’ biphasic regions were screened in terms of protein biocompatibility, analyzing the impact of ABS phase-forming components in Lys by UV-VIS spectrophotometry, CD spectroscopy, fluorescence spectroscopy, DSC, and enzyme assay. Lys partition behavior was characterized in terms of extraction efficiency (% EE). The structure, stability, and function of Lys were maintained or improved throughout the extraction step, as evaluated by CD spectroscopy, DSC, enzyme assay, and SDS-PAGE. Overall, FIL-based ABSs are more versatile and amenable to being tuned by the adequate choice of the phase-forming components and selecting the enriched phase. Binding studies between Lys and ABS phase-forming components were attained by MST, demonstrating the strong interaction between Lys and ...
العلاقة: PURE: 93818133; PURE UUID: c82a43f2-cfe5-47b6-89e6-eefc6b9adcf3; Scopus: 85195832763; WOS: 001245413100001; PubMed: 38891953; PubMedCentral: PMC11172314; ORCID: /0000-0002-8648-7539/work/162433946; http://hdl.handle.net/10362/169111Test; https://doi.org/10.3390/ijms25115766Test
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7دورية أكاديمية
المؤلفون: Dimitrijević, Aleksandra, Marić, Slađana, Jocić, Ana, Tekić, Danijela, Mušović, Jasmina, Amaral, Joana S.
مصطلحات موضوعية: Aqueous biphasic systems, Extraction, Food waste, Gallic acid, Grape by-products, Ionic liquids, Polyphenols, Quercetin, Resveratrol
الوصف: Polyphenols are natural compounds with enhanced antioxidant properties. They are present in relatively high concentrations in fruit/vegetable by-products. Therefore, there is a need for the development of efficient and cost-effective methods for the separation and purification of these valuable compounds. Traditional extraction with organic solvents needs to be switched to novel methods that are more efficient, with reduced extraction times and low consumption of organic solvents. Aiming at developing sustainable processes for the separation and purification of phenolic compounds, we used three model compounds, namely resveratrol, quercetin, and gallic acid, to investigate ionic liquid-based aqueous biphasic systems (IL-ABSs) formed by cholinium-based IL in combination with polypropylene glycol with a molecular mass of 400 g/mol (PPG400). The ABS composition in the two-phase region was selected according to a previously determined phase diagram. Extraction studies indicated the preferential partition of resveratrol and quercetin toward the hydrophobic PPG-rich phase that is mainly dominated by its hydrophobic nature and the strong salting-out effect of ILs. On the other hand, due to its considerably hydrophilic nature, gallic acid preferentially migrates toward the IL phase. The achieved results from grape stem extract demonstrated high extraction efficiencies of cholinium dihydrogen phosphate (~99% for resveratrol for the PPG phase and 78% for gallic acid for the IL phase), with considerable selectivity, demonstrating promising outcomes for potential applications. ; This research received support from the Ministry of Science, Technological Development and Innovation of the Republic of Serbia under Contract number 451-03-66/2024-03/200017. Additionally, it was supported by a Short-Term Scientific Mission (STSM) Grant (Reference: E-COST-GRANT-CA20133-975d1689) from the COST Action CA20133 (Fullrec4us—Cross Border Transfer and Development of Sustainable Resource Recovery Strategies Towards Zero Waste). J.S. Amaral ...
العلاقة: info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F00690%2F2020/PT; info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F00690%2F2020/PT; info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/LA%2FP%2F0007%2F2020/PT; Dimitrijević, Aleksandra; Marić, Slađana; Jocić, Ana; Tekić, Danijela; Mušović, Jasmina; Amaral, Joana S. (2024). Green Extraction Strategy Using Bio-Based Aqueous Biphasic Systems for Polyphenol Valorization from Grape By-Product. Foods. ISSN 2304-8158. 13:6, p. 1-21; http://hdl.handle.net/10198/29976Test
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8دورية أكاديمية
المصدر: Separation and Purification Technology , 334 , Article 125468. (2024)
مصطلحات موضوعية: Liquid-liquid, Plug flow, Extraction, Ionic liquid, Aqueous-biphasic-systems, Biomolecules, Two-phase flows, Process intensification, Small channels
الوصف: A double process intensification of the conventional solvent extraction was accomplished by employing two strategies. Firstly, by utilizing continuous small channels and secondly by replacing organic solvents with novel ionic liquid based aqueous biphasic systems (IL-ABS). The flow extraction of the amino acid L-tryptophan was investigated in small channels with internal diameters of 0.5 and 0.8 mm and three different IL-ABS. The partition coefficients for two different L-tryptophan concentrations (0.325 and 0.5 wt%) were measured for IL-ABS compositions 18, 20 and 22 wt% K3PO4 and C4mimCl. Increasing the weight composition of the conventional salt and the ionic liquid increased the partition of the amino acid for the top ionic liquid rich phase. The continuous extractions were carried out in the plug flow regime. Correlations were proposed for the plug length and the film thickness as functions of the capillary number. It was found that the extraction efficiency with the 20 wt% ABS reached 95 % in under 15 s in the 0.5 mm channel and in about 40 s in the 0.8 mm channel. When comparing the IL-ABS, the 22 wt% reached 96 % extraction efficiency in 14 s compared to the 70 s of the 20 wt% in the 0.8 mm channel. The overall mass transfer coefficients varied between 0.05 and 0.2 s−1 for all cases studied, which were significantly higher than those in conventional solvent extraction systems.
وصف الملف: text
العلاقة: https://discovery.ucl.ac.uk/id/eprint/10184474/1/1-s2.0-S1383586623023766-main.pdfTest; https://discovery.ucl.ac.uk/id/eprint/10184474Test/
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9دورية أكاديمية
المؤلفون: Almeida, Mafalda R., Rufino, Ana F. C. S., Belchior, Diana C. V., Carvalho, Pedro J., Freire, Mara G.
مصطلحات موضوعية: Aqueous biphasic systems, Phase diagrams, NRTL, Cholinium carboxylate, Ionic liquids, Alkaloids
الوصف: In the past years large efforts have been placed in the development of novel separation techniques with improved resolution, simplicity, speed and easy to scale-up. Among these, ionic-liquid-based (IL-based) aqueous biphasic systems (ABS) have been broadly proposed for the separation of high-value compounds, allowing improved extraction performance and purification. More recently, significant efforts have been placed on the synthesis and use of novel ILs with both an acceptable environmental footprint and enhanced biocompatibility. In this sense, this work aims to characterize ABS composed of cholinium carboxylate ILs ([Ch][CnO2], with n = 2 to 7), K3PO4 and water. The respective ternary phase diagrams, including binodal curves, tie-lines and tie-line lengths, were determined at (298 ± 1) K and at atmospheric pressure. The ability to form ABS (or of the IL to be salted-out) increases with the increase of the alkyl chain length of the IL anion, up to [Ch][C5O2]; nevertheless, for longer anion alkyl chain lengths ([Ch][C6O2] and [Ch][C7O2]) the ILs self-aggregation leads to a decrease of the ILs ability to form ABS. The liquid−liquid equilibrium data experimentally determined were modeled using the local composition activity coefficient model NRTL (Non-Random Two Liquid). Finally, the partition behavior of three alkaloids (nicotine, caffeine and theobromine), used here as hydrophobicity probes, was evaluated. In all studied systems, alkaloids preferentially migrate to the IL-rich phase, with partition coefficients (K) ranging between 2.23 and complete extraction in a single-step. Furthermore, the set of ILs investigated allowed identifying an odd-even effect in the alkaloids partitioning derived from the IL anion alkyl chain length. These results support the salting-out effect exerted by K3PO4 and favorable dispersive interactions established between the IL-rich phase-forming components and the alkaloids. ; published
العلاقة: info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F50011%2F2020/PT; info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F50011%2F2020/PT; LA/P/0006/2020; info:eu-repo/grantAgreement/FCT/9471 - RIDTI/PTDC%2FBII-BBF%2F30840%2F2017/PT; info:eu-repo/grantAgreement/FCT/POR_CENTRO/SFRH%2FBD%2F138997%2F2018/PT; http://hdl.handle.net/10773/34457Test; 113458
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10دورية أكاديمية
المؤلفون: Carvalho, Sara F., Pereiro, Ana B., Araújo, João M. M.
المساهمون: LAQV@REQUIMTE, DQ - Departamento de Química
مصطلحات موضوعية: aqueous biphasic systems, bioactive proteins, bioprivileged fluorinated ionic liquids, BSA, human IFN-α2b, purification, serum albumin, Catalysis, Molecular Biology, Spectroscopy, Computer Science Applications, Physical and Theoretical Chemistry, Organic Chemistry, Inorganic Chemistry, SDG 3 - Good Health and Well-being
الوصف: Publisher Copyright: © 2024 by the authors ; Interferon alpha-2b (IFN- (Formula presented.) 2b) is an essential cytokine widely used in the treatment of chronic hepatitis C and hairy cell leukemia, and serum albumin is the most abundant plasma protein with numerous physiological functions. Effective single-step aqueous biphasic system (ABS) extraction for the simultaneous purification of IFN- (Formula presented.) 2b and BSA (serum albumin protein) was developed in this work. Effects of the ionic liquid (IL)-based ABS functionalization, fluorinated ILs (FILs; [C (Formula presented.) C (Formula presented.) Im][C (Formula presented.) F (Formula presented.) SO (Formula presented.)] and [N (Formula presented.)][C (Formula presented.) F (Formula presented.) SO (Formula presented.)]) vs. mere fluoro-containing IL ([C (Formula presented.) C (Formula presented.) Im][CF (Formula presented.) SO (Formula presented.)]), in combination with sucrose or [N (Formula presented.)][H (Formula presented.) PO (Formula presented.)] (well-known globular protein stabilizers), or high-charge-density salt K (Formula presented.) PO (Formula presented.) were investigated. The effects of phase pH, phase water content (%wt), phase composition (%wt), and phase volume ratio were investigated. The phase pH was found to have a significant effect on IFN- (Formula presented.) 2b and BSA partition. Experimental results show that simultaneous single-step purification was achieved with a high yield (extraction efficiency up to 100%) for both proteins and a purification factor of IFN- (Formula presented.) 2b high in the enriched IFN- (Formula presented.) 2b phase (up to 23.22) and low in the BSA-enriched phase (down to 0.00). SDS-PAGE analysis confirmed the purity of both recovered proteins. The stability and structure of IFN- (Formula presented.) 2b and BSA were preserved or even improved (FIL-rich phase) during the purification step, as evaluated by CD spectroscopy and DSC. Binding studies of IFN- (Formula presented.) 2b and BSA with the ABS ...
العلاقة: info:eu-repo/grantAgreement/FCT/OE/SFRH%2FBD%2F139677%2F2018/PT; info:eu-repo/grantAgreement/FCT/CEEC IND 3ed/2020.00835.CEECIND%2FCP1586%2FCT0014/PT; info:eu-repo/grantAgreement/FCT/CEEC IND4ed/2021.01432.CEECIND%2FCP1657%2FCT0044/PT; info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/LA%2FP%2F0008%2F2020/PT; info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDP%2F50006%2F2020/PT; info:eu-repo/grantAgreement/FCT/6817 - DCRRNI ID/UIDB%2F50006%2F2020/PT; PURE: 86390388; PURE UUID: 3625c775-47d4-4d39-9e5b-94a14b47ab30; Scopus: 85187723358; WOS: 001183284500001; PubMed: 38473998; PubMedCentral: PMC10931833; ORCID: /0000-0002-8648-7539/work/157689447; http://hdl.handle.net/10362/166248Test; https://doi.org/10.3390/ijms25052751Test
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