Supported ionic liquids used as chromatographic matrices in bioseparation: an overview

Liquid chromatography plays a central role in biomanufacturing, and, apart from its use as a preparative purification strategy, either in biopharmaceuticals or in fine chemicals industries, it is also very useful as an analytical tool for monitoring, assessing, and characterizing diverse samples. The present review gives an overview of the progress of the chromatographic supports that have been used in the purification of high-value products (e.g., small molecules, organic compounds, proteins, and nucleic acids). Despite the diversity of currently available chromatographic matrices, the interest in innovative biomolecules emphasizes the need for novel, robust, and more efficient supports and ligands with improved selectivity. Accordingly, ionic liquids (ILs) have been investigated as novel ligands in chromatographic matrices. Given herein is an extensive review regarding the different immobilization strategies of ILs in several types of supports, namely in silica, Sepharose, and polymers. In addition to depicting their synthesis, the main application examples of these supports are also presented. The multiple interactions promoted by ILs are critically discussed concerning the improved selectivity towards target molecules. Overall, the versatility of supported ILs is here considered a critical point to their exploitation as alternatives to the more conventional liquid chromatographic matrices used in bioseparation processes.publishe

Solid catalysts obtained from wastes for FAME production using mixtures of refined palm oil and waste cooking oils

More than 95% of biodiesel production feedstocks come from edible oils, however it may cause some problems such as the competition of land use between food production and biodiesel production. The waste cooking oils (WCO) are an alternative feedstock for biodiesel production; its usage reduces significantly the cost of biodiesel production and has environmental benefits, e.g., a waste recovery instead of its elimination. This work aims to produce a low-cost efficient solid catalyst for fatty acid methyl esters (FAME) production using mixtures of refined palm oil (RPO) and WCO. Four low cost catalysts were prepared (biomass fly ashes, natural dolomite rock, chicken eggshells and polyethylene terephthalate - PET), characterized (by SEM, EDX, XRD, BET, FT-IR and Hammett indicators) and tested regarding their performance in FAME production. The maximum yield of FAME achieved was around 96%wt. for biomass fly ashes catalyst at 60 °C, 9:1 (mol/mol) of methanol to oil mixture, 10%wt. catalyst to oil mixture, over 180 min in batch reactor. The results point out for promising bifunctional catalysts able to achieve also conversion of free fatty acids up to 100% using mixtures of RPO and WCO.publishe

Multimodal ionic liquid-based chromatographic supports for an effective RNA purification

Nucleic acids have been considered interesting molecules to be used as biopharmaceuticals for the treatment of various diseases, in gene therapy strategies. In particular, RNA arises as the most promising approach because it does not require access to the nucleus of cells to exert its function; however, it is quite challenging due to its labile nature. To increase the possibility of translating RNA-based technology to clinical protocols, the biomanufacturing of RNAs has been intensively exploited in the last few years. However, the standard RNA purif ication processes remain time-consuming and present limitations regarding recovery yield and purity. This work describes the functionalization of chromatographic silica-based supports with four ionic liquids (ILs) composed of functional moieties that can promote distinct interactions with nucleic acids. After an initial screening to evaluate the binding and elution behavior of nucleic acids in the IL-based supports, SSi[C 3 C 3NH2 Im]Cl has shown to be the most promising for further purification assays. This support was studied for the RNA purification from different samples (clarified or more complex) and has shown to be highly effective, for all the conditions studied. Generally, it is here presented a new method for RNA isolation in a single step, using an IL-based chromatographic support, able to eliminate the usage of hazardous compounds often included in standard RNA extraction protocols.publishe

Optimization of FAME production from blends of waste cooking oil and refined palm oil using biomass fly ash as a catalyst

One of the problems associated with biomass combustion is the amount of fly ashes generated and its subsequent management. The search for ways of valorizing these ashes has been a challenge for the academic and industrial community. On the other hand, used cooking oils are wastes which management is quite difficult, by they have a very important energetic potential. The goal of this work was to optimize the Fatty Acid Methyl Esters (FAME) process, recovering two residual materials (waste cooking oils (WCO), and biomass fly flash (BFA)). The optimization of the process was achieved using the response surface methodology and a Box-Benhken experimental design applied to mixtures of WCO and refined palm oil (RPO), using BFA as catalyst. The influence on FAME yield of four variables (catalyst loading, methanol/oil molar ratio, RPO/WCO ratio and reaction temperature) was studied. The higher FAME yield achieved was 73.8% for the following operating conditions: 13.57 wt% of catalyst loading, 6.7 of methanol/oil molar ratio, 28.04 wt% of RPO in the oil mixture with WCO and 55 °C for the reaction temperature. The reusability of the BFA catalyst in the process was also studied through three successive usage cycles finding no loss of catalytic activity.publishe

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

Sustainable lysis of Bacillus subtilis biomass to recover the biopharmaceutical L-asparaginase

The first-line biopharmaceutical used to treat Acute lymphoblastic leukemia (ALL), Oncaspar, is based on the enzyme L-asparaginase (ASNase), and has annual sales of ca. USD $100 million. In addition to other sources, genetically modified Bacillus subtilis is regarded as one of the most promising hosts for the ASNase production. The Aliivibrio fischeri ASNase type II, which has anti-tumour activity due its higher specific affinity for L-asparagine, expressed in B. subtillis is located in the periplasm. Therefore, cell lysis is required for the ASNase recovery. Nevertheless, typical cell lysis approaches, e.g. chemical methods with surfactants lead to some biocompatibility concerns and the need of extra purification steps. To overcome this drawback, in this work, ultrasound sonication (USS) conditions were studied to develop a greener and more biocompatible method for ASNase recovery from B. subtilis cell lysis. The USS cell lysis was optimized regarding the amplitude of USS pulse, number of lysis cycles and mass of cells/volume of solvent ratio. The identification and quantification of ASNase and major impurities present in the cell extract after lysis were investigated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and size exclusion high-performance liquid chromatography (SE-HPLC). ASNase activity was determined by monitoring the hydrolysis of the substrate, L-asparagine. The results obtained show that the ideal conditions for B. subtilis cell lysis are an amplitude of USS pulse of 60%, 40 cycles of lysis and 10 mL of phosphatebuffered saline (PBS) per 1 g of cells. Overall, an optimized sustainable B. subtilis cell lysis method was developed, avoiding the use of surfactants and with low energy consumption.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