Analysis of processing systems involving reaction and distillation : the synthesis of ethyl acetate

The integration of reaction and separation into a single process unit, i. e., reactive destillation, may offer several advantages over conventional systems that use a reactor followed by a distillation column. In this paper we explore the operational characteristics of reactive distillation and highlight some of this potential benefits, using the production of ethyl acetate as an illustrative example. With this aim, the two types of system are compared employing different reactor types and a number of performance indicators, such as yield, conversion, purity, specific energy consumption and residence time. A sensitivity analysis is carried out on some variables and parameters, in order to explore and define the distillation columns operating conditions. As expected, results point to a clear advantage of reactive distillation allowing for the azeotrope to be surpassed and for the overcoming of chemical equilibrium, favouring an increase in conversion and product purity, along with reduced operating costs

Progress in the Development of Aqueous Two-Phase Systems Comprising Ionic Liquids for the Downstream Processing of Protein- Based Biopharmaceuticals

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LP-based heuristic procedure for the optimal design of water using networks with multi-contaminants

This paper proposes a new strategy for the optimal design of water-using networks in industrial systems featuring possibly more than a single water source and multiple contaminants. The model formulation is supported on a superstructure that exploits reuse opportunities and gives rise to a non-convex nonlinear which often leads to local optimal solutions. To overcome this, the new approach generates multiple initialization points, one for each possible sequence of operations, where a particular starting point is obtained by the sequential solution of a small set of related linear programs. The best solution of the several non-linear problems that are solved is then assumed to be the global optimal solution. The results obtained for a set of case studies have shown that the best initialization point is often the global optimal solution and that the procedure as a whole is efficient in escaping local optima

Ionic-liquid-based approaches to improve biopharmaceuticals downstream processing and formulation

The emergence of biopharmaceuticals, including proteins, nucleic acids, peptides, and vaccines, revolutionized the medical field, contributing to significant advances in the prophylaxis and treatment of chronic and life-threatening diseases. However, biopharmaceuticals manufacturing involves a set of complex upstream and downstream processes, which considerably impact their cost. In particular, despite the efforts made in the last decades to improve the existing technologies, downstream processing still accounts for more than 80% of the total biopharmaceutical production cost. On the other hand, the formulation of biological products must ensure they maintain their therapeutic performance and long-term stability, while preserving their physical and chemical structure. Ionicliquid (IL)-based approaches arose as a promise alternative, showing the potential to be used in downstream processing to provide increased purity and recovery yield, as well as excipients for the development of stable biopharmaceutical formulations. This manuscript reviews the most important progress achieved in both fields. The work developed is critically discussed and complemented with a SWOT analysis.publishe

Improving the downstream processing of interferon alfa-2b using alternative purification platforms based on ionic liquids

Improvements on human life expectancy and the lack of effective therapies has led to an increment of chronic diseases, being the application of biopharmaceuticals an efficient strategy to mitigate this scenario. Among the current available biopharmaceuticals, the role of interferon α-2b (IFNα-2b) should be highlighted, as it has been marketed over 30 years with a considerable impact on the global therapeutic proteins market (Castro et al, Vaccines, 2021). IFN manufacturing requires the use of the recombinant DNA technology, involving two main stages, the upstream and downstream stages. The first includes recombinant protein production in a suitable host microorganism, such as Escherichia coli (Castro et al, Sep. Purif. Technol., 2020), while the second comprises protein recovery, isolation, purification and polishing. Due to the high demands of the pharmaceutical industry for products with high purity and biological activity, the downstream stage is responsible for the majority of the production costs of biopharmaceuticals (50–90%), often including time-consuming and multi-step processes. Therefore, there is an immediate need to develop more efficient, cost-effective, and sustainable protein purification methodologies. In this work, two ionic-liquid-(IL)-based strategies were investigated for the purification of IFNα-2b recombinantly produced from E. coli fermentation broth, namely as adjuvants in aqueous biphasic systems or as chromatographic ligands immobilized in solid materials. Overall, the obtained results demonstrate that by tailoring IL’s chemical structures, improved protein purification processes are obtained and that the secondary structure of proteins is preserved.publishe

Sustainable ionic-liquid-based strategies for the downstream processing of interferon α-2b from Echerichia coli

Over the last decades, society has been facing an increment of chronic diseases due to the higher human life expectancy and the lack of efficient treatments for several pathologies. In this regard, biopharmaceuticals have become one of the most effective clinical treatments for a broad range of diseases, including cancer, metabolic and neurodegenerative disorders [1]. Among biopharmaceuticals, the role of interferons, particularly interferon α-2b (IFNα-2b), should be underlined, as they have been marketed for over 30 years with a considerable impact on the global therapeutic proteins market [2]. Usually based on the recombinant DNA technology, the manufacturing process of biopharmaceuticals encompasses two main stages: the upstream and downstream stages. Typically, the upstream phase includes recombinant protein production processes in a suitable host microorganism, such as Escherichia coli [3], while the general downstream processing of biopharmaceuticals comprises four stages - recovery, isolation, purification and polishing -, which are responsible for the majority of the production costs of biopharmaceuticals (50–90%) [3]. The downstream processing is a time-consuming and multi-step process, for which the development of cost-effective purification processes is mandatory to decrease their costs and environmental impact. In this context, two ionic-liquid-(IL)-based strategies were investigated in this work for the purification of IFNα-2b recombinantly produced from E. coli fermentation broth. ILs have been used as adjuvants in aqueous two-phase systems (ATPS) and applied in supported materials as alternative ligands. The obtained results demonstrate that ILs have a tailoring ability and contribute to the development of more effective and sustainable downstream processes of biopharmaceuticals.publishe

Purification of interferon alpha 2b-based biopharmaceuticals using ionic liquid-based technologies

Protein biopharmaceuticals, among which interferon alpha-2b (IFNα-2b) that can be used in the treatment of chronic hepatitis C, have become an indispensable product of current medicine. Aiming at finding new cost-effective, efficient and sustainable technologies for recombinant IFNα-2b purification, ionic liquids were investigated as adjuvants in polymer-polymer aqueous two-phase systems (ATPS) or as chromatographic ligands covalently attached in silica (Supported ionic liquids, SILs). The application of ionic liquids as adjuvants in ATPS composed of polyethylene glycol (PEG 600 g/mol) and polypropylene glycol (PPG 400 g/mol) enhanced the purity of IFNα-2b recovered in the PEG-rich phase. On the other hand, SILs exhibited promising results toward IFNα-2b purification both in conditions favouring ionic or hydrophobic interactions, reinforcing the multimodal character of these novel stationary phases. Also, the secondary structure of IFNα-2b is preserved with both purification processes, as appraised by circular dichroism and western-blot studies. Overall, our results demonstrate the high potential exhibited by ionic liquids toward the preparative purification of the recombinant IFNα-2b biopharmaceuticals.publishe

Development of an integrated messenger RNA manufacturing process using thermoreversible aqueous biphasic systems

The promising potential of messenger RNA (mRNA) vaccines as effective approaches to contain the dissemination of infectious diseases was fully disclosed during the combat to the COVID-19 pandemic. Over conventional vaccines, mRNA-based vaccines exhibit improved safety and efficacy profiles, and the possibility of repeatedly administration [1]. However, the manufacturing of mRNA vaccines is complex, costly and requires multi-step purification strategies to produce high quality products. If properly designed, ionic liquids (ILs) can act as RNA stabilizing agents [2] and enhance the selectivity of purification processes when used to form aqueous biphasic systems (ABS) [3]. Aiming to improve mRNA manufacturing, this work proposes the use of thermoreversible ABS based on ILs to integrate the production and clarification steps, further simplifying subsequent purification steps. Up to date, we have achieved the production of mRNA by in vitro transcription and its purification using conventional methods and gathered insights on mRNA stability and integrity in several structurally distinct ILs. According to these previous results, current attention is being placed on the identification of the best thermoreversible IL-based ABS to integrate production and clarification steps. Overall, the proposed integrated production-clarification platform is expected to tackle current challenges of mRNA manufacturing, especially by improving the cost-efficiency and technological simplicity of existing manufacturing processes and enhancing the stability and yield of the final product.publishe

Advances in Membrane-Bound Catechol-O-Methyltransferase Stability Achieved Using a New Ionic Liquid-Based Storage Formulation

Funding: The authors acknowledge the support from FEDER funds through the POCI—COMPETE 2020—Operational Program Competitiveness and Internationalization in Axis I—Strengthening research, technological development, and innovation (Project POCI-01-0145-FEDER-007491) and National Funds (Project UID/Multi/00709/2013) LA/P/0006/2020, financed by national funds through the Portuguese Foundation for Science and Technology/MCTES (PIDDAC). This work was also supported by the Applied Molecular Biosciences Unit UCIBIO (UIDB/04378/2020 and UIDP/04378/2020) and the Associate Laboratory Institute for Health and Bioeconomy—i4HB (project LA/P/0140/2020) which are financed by National Funds from FCT/MCTES. Researchers also acknowledge funding by FEDER through COMPETE 2020—Programa Operacional Compet-itividade e Internacionalização (POCI), Augusto Q. Pedro research contract CEEC-IND/02599/2020 under the Scientific Stimulus Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.Membrane-bound catechol-O-methyltransferase (MBCOMT), present in the brain and involved in the main pathway of the catechol neurotransmitter deactivation, is linked to several types of human dementia, which are relevant pharmacological targets for new potent and nontoxic inhibitors that have been developed, particularly for Parkinson’s disease treatment. However, the inexistence of an MBCOMT 3D-structure presents a blockage in new drugs’ design and clinical studies due to its instability. The enzyme has a clear tendency to lose its biological activity in a short period of time. To avoid the enzyme sequestering into a non-native state during the downstream processing, a multi-component buffer plays a major role, with the addition of additives such as cysteine, glycerol, and trehalose showing promising results towards minimizing hMBCOMT damage and enhancing its stability. In addition, ionic liquids, due to their virtually unlimited choices for cation/anion paring, are potential protein stabilizers for the process and storage buffers. Screening experiments were designed to evaluate the effect of distinct cation/anion ILs interaction in hMBCOMT enzymatic activity. The ionic liquids: choline glutamate [Ch][Glu], choline dihydrogen phosphate ([Ch][DHP]), choline chloride ([Ch]Cl), 1-dodecyl-3-methylimidazolium chloride ([C12mim]Cl), and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl) were supplemented to hMBCOMT lysates in a concentration from 5 to 500 mM. A major potential stabilizing effect was obtained using [Ch][DHP] (10 and 50 mM). From the DoE 146% of hMBCOMT activity recovery was obtained with [Ch][DHP] optimal conditions (7.5 mM) at −80◦ C during 32.4 h. These results are of crucial importance for further drug development once the enzyme can be stabilized for longer periods of time.publishersversionpublishe

Bioprocessing of recombinant proteins using alternative purification platforms

The advent of biopharmaceuticals in modern medicine brought enormous benefits to diverse human diseases and improved the well-being of many people worldwide. Since the FDA approval of humulin (recombinant insulin) in 19821, remarkable advances in the treatment of chronic diseases have been achieved with biopharmaceutical-based therapies2. This sector represents 40% of ca. 6000 products currently in clinical development2, and is dominated by therapeutic proteins with over 200 protein drugs in the market1. Along the years, many improvements in the biopharmaceuticals upstream stage resulted in high titers of the desired product, and shifted the bioprocess bottleneck to the downstream processing, which is currently dominated by chromatography, accounting with more than 70% of total downstream costs3. Aiming at finding new cost-effective, efficient and sustainable technologies for proteins purification, novel polymer-polymer aqueous biphasic systems (ABS) with ionic liquids (ILs) as adjuvants are investigated as alternative purification platforms for the downstream of interferon alfa 2b (IFNα2b) from Escherichia coli BL21 cultures. Initial experiments showed that the production of IFNα2b is higher using the SOB culture medium and the western-blot analysis revealed that it is present in the inclusion body fraction. This fraction was washed, solubilized using a specific buffer and, finally dialyzed. After analyzing the stability of the target protein in several phase-forming components, the ternary phase diagrams of ABS composed by polyethylene glycol (PEG), polypropylene glycol (PPG) with ILs as adjuvants were determined at 25 °C, as well as the corresponding tie-lines, tie-line lengths and critical points. Chloride-based ILs combined with cholinium, imidazolium, pyrrolidinium, piperidinium, tetralkylammonium and tetralkylphosphonium cations were investigated. In summary, this study reports effective IFNα2b purification platforms from E. coli based on polymer-polymer-ABS, being highlighted the beneficial role of ILs in the downstream processing of proteins, either as adjuvants in ABS or by exploring new ILs features in protein stabilization.publishe