An Overview on the Recent Advances in Alternative Solvents as Stabilizers of Proteins and Enzymes

Currently, the use of alternative solvents is increasing, namely ionic liquids (ILs) and deep eutectic solvents (DESs) in diverse fields of knowledge, such as biochemistry, chemistry, chemical engineering, biotechnology and biomedicine. Particularly, when compared to traditional solvents, these alternative solvents have great importance for biomolecules due to the enhanced solubility, structure stability and the biological activity of biomolecules, such as protein and enzymes. Thus, in this review article, the recent developments and efforts on the technological developments carried out with ILs and DESs for the stabilization and activation of proteins and enzymes are provided. The most studied IL-and DES-based formulations for proteins and enzymes are discussed and the molecular mechanisms and interactions related to the increased stability promoted by these alternative solvents are disclosed, while emphasizing their main advantagespublishe

Good's Buffer Ionic Liquids as Relevant Phase-Forming Components of Self-Buffered Aqueous Biphasic Systems

A series of new self-buffering ionic liquids (ILs) based on Good's buffers (GBs) anions and the tetrabutylphosphonium cation ([P4444]+) was here synthesized and characterized. The self-buffering behaviour of the GB-ILs was confirmed by measuring their protonation constants by potentiometry. Further, their ability to form aqueous biphasic systems with the biodegradable potassium citrate salt was evaluated, and further investigated for the extraction of proteins, using bovine serum albumin (BSA) as a model protein. If these ionic structures display self-buffering characteristics as well as a low toxicity towards the luminescent bacteria Vibrio fischeri, they were additionally found to be highly effective in the formation of ABS and in the extraction of BSA - extraction efficiencies of 100% to the IL-rich phase obtained in a single-step. The BSA secondary structure in the aqueous IL-rich solutions was evaluated through infrared spectroscopic studies revealing the protein-friendly nature of the synthesized ILs. Dynamic light scattering (DLS), "COnductor-like Screening MOdel for Real Solvents" (COSMO-RS), and molecular docking studies were finally carried out to better understand the main driving forces of the extraction process. The results suggest that van der Waals and hydrogen-bonding interactions are important driving forces of the protein migration towards the GB-IL-rich phase, while the molecular docking investigations demonstrated a stabilizing effect of the studied ILs over the protein.publishe

Opposite effects induced by cholinium-based ionic liquid electrolytes in the formation of aqueous biphasic systems comprising polyethylene glycol and sodium polyacrylate

Cholinium-based ionic liquids ([Ch]-based ILs) were investigated as electrolytes in the formation of aqueous biphasic systems (ABS) composed of polyethylene glycol (PEG) and sodium polyacrylate (NaPA) polymers. Both enhancement and decrease in the liquid-liquid demixing ability induced by electrolytes in PEG-NaPA aqueous biphasic systems were observed. It is shown that the ILs that most extensively partition to the PEG-rich phase tend to act as inorganic salts enhancing the two-phase formation ability, while those that display a more significant partition to the NaPA-rich phase decrease the ABS formation capacity. The gathered results allowed us to confirm the tailoring ability of ILs and to identify, for the first time, opposite effects induced by electrolytes on the PEG-NaPA ABS formation ability. The distribution of the electrolyte ions between the coexisting phases and the polyelectrolyte ion compartmentalization are key factors behind the formation of PEG-NaPA-based ABS.publishe

A simple approach for the determination and characterization of ternary phase diagrams of aqueous two-phase systems composed of water, poly(ethylene) glycol and sodium carbonate

In this work, an experimental protocol to determine liquid-liquid phase diagrams of aqueous two-phase systems (ATPS) in a chemical engineering course is described. Throughout this laboratory set of experiments, students will determine liquid-liquid ternary phase diagrams, comprising the solubility curves, tie-lines, tie-line lengths and critical points, for systems composed of poly(ethylene) glycol, sodium carbonate and water. Furthermore, students will apply the NRTL (Non-Random Two Liquid) model to fit the experimental tie-lines.publishe

Aqueous biphasic systems in the separation of food colorants

Aqueous biphasic systems (ABS) composed of polypropylene glycol and carbohydrates, two benign substances are proposed to separate two food colorants (E122 and E133). ABS are promising extractive platforms, particularly for biomolecules, due to their aqueous and mild nature (pH and temperature), reduced environmental impact and processing costs. Another major aspect considered, particularly useful in downstream processing, is the "tuning" ability for the extraction and purification of these systems by a proper choice of the ABS components. In this work, our intention is to show the concept of ABS as an alternative and volatile organic solvent-free tool to separate two different biomolecules in a simple way, so simple that teachers can effectively adopt it in their classes to explain the concept of bioseparation processes. Informative documents and general information about the preparation of binodal curves and their use in the partition of biomolecules is available in this work to be used by teachers in their classes. In this sense, the students use different carbohydrates to build ABS, then study the partition of two food color dyes (synthetic origin), thus evaluating their ability on the separation of both food colorants. Through these experiments, the students get acquainted with ABS, learn how to determine solubility curves and perform extraction procedures using colorant food additives, that can also be applied in the extraction of various (bio)molecules.publishe

Insights on the laccase extraction and activity in ionic-liquid-based aqueous biphasic systems

Due to their catalytic properties, selectivity, and efficiency, enzymes are excellent biocatalysts. In particular, laccases are versatile multi-copper oxidases with great interest for a wide plethora of biotechnological and environmental applications. Even though several laccase-catalysed processes have been reported at an industrial level, the high costs of their downstream processing required to provide biocatalysts with high purity levels, stability and activity remains one of the main drawbacks when economically evaluating the overall processes. Aqueous biphasic systems based on ionic liquids (ILs) can be foreseen as a promising alternative approach for the extraction and activity maintenance/improvement of enzymes, essentially due to the designer solvents ability of ionic liquids. However, to take advantage of this feature and to use the full potential of IL-based aqueous biphasic systems, it is necessary to understand the effect of ILs as phase-forming constituents and how they affect the enzymes extraction and activity. In order to overcome the lack of information on this topic in the literature, in this work, IL-based aqueous biphasic systems were investigated to extract and enhance the laccase activity, in order to gather evidences that could be used to improve the enzymes downstream processing. To this end, a wide screening of imidazolium-, pyridinium-, pyrrolidinium-, piperidinium-, tetraalkylphosphonium-, and tetraalkylammonium-based ILs as phase-forming components of ABS was carried out. Furthermore, these ILs were used to create ABS combined with salts, polymers and used as adjuvants in polymer-based ABS. Most ABS comprising ILs revealed to be highly efficient extraction platforms, allowing the complete extraction of laccase for all the conditions tested, and with an enzyme activity enhancement by more than 50%. Overall, the obtained results demonstrate that laccase preferentially partitions to the most hydrophilic phase in ABS comprising ILs, both used as adjuvants or as phase-forming components, corresponding to the phase in which the IL is enriched. Furthermore, the IL chemical structure of the IL plays a significant role in the enzyme activity, where ILs with a higher number of hydroxyl groups seem to be relevant to improve the laccase activity.publishe

Supported ionic liquids as customizable materials to purify immunoglobulin G

Over the past few years, antibodies such as immunoglobulin G, IgG, have increased their market share as alternative therapeutics. However, their production at high purity levels is still costly due to the absence of a cost-effective platform for their recovery and purification from the complex biological media in which they are produced. This work describes, for the first time, that materials modified with ionic liquids (ILs) can be designed for the effective capture and purification of antibodies from complex matrices, allowing both the selective adsorption of IgG or the selective adsorption of other proteins present in the media. The best results correspond to IgG with 59 % of yield and 84 % of purity in the aqueous solution, and IgG with 76 % of yield and 100 % of purity on the surface of one SIL due to the selective adsorption of IgG from human serum. The best conditions and materials were then applied to other IgG-containing matrices, namely rabbit serum and Chinese hamster ovary (CHO) cell culture supernatants, proving the robustness of the developed strategy. Furthermore, it is demonstrated that the secondary structure of IgG is preserved during the purification process and that these antibodies remain biologically active. In summary, it is shown that by only changing the IL chemical structure at the material surface it is possible to selectively adsorb IgG or to adsorb other proteins leaving IgG in solution. These findings prove that SILs are customizable materials with future potential to act in the flow-through or bind-andelute modes. Therefore, SILs can be envisioned as potential chromatographic columns capable of substituting the high-cost commercial chromatographic columns based on biological ligands currently used to purify IgG.publishe

Using three-phase partitioning for the purification and recovery of antibodies from biological media

Antibodies, in particular immunoglobulin G (IgG), are one of the biopharmaceutical industry highflyers, with relevance for the treatment of several diseases. However, the recovery of antibodies from complex biological media with high quality and purity is difficult and requires multi-step and expensive approaches. Herein, we propose a cost-effective approach using three-phase partitioning (TPP) systems based on polyethylene glycol (PEG)-salt aqueous biphasic systems (ABS) with ionic liquids (ILs) as adjuvants for the purification and recovery of IgG antibodies from three biological media, i.e., human serum, and serum-containing and serum-free Chinese hamster ovary (CHO) cell culture supernatants. The economic analysis of the developed process was carried out. The results obtained using PEG-salt ABS without ILs and human serum show that IgG could be recovered either at the interphase of the TPP or in the top phase, depending on the molecular weight of the PEG. The system composed of PEG with a molecular weight of 1000 g/mol is the PEG-salt system enabling the highest purity of human polyclonal IgG at the interphase (80.7 %, with a recovery yield of 65.8 %). Still, by adding 1 wt% of the ILs tetra(n-butyl)ammonium bromide ([N4444]Br) and 1-butyl-3-methylimidazolium chloride ([C4mim]Cl), it is possible to compete or even outperform the PEG 1000-salt system with no IL regarding purity/recovery performance and production costs under given operation conditions. The best systems were then applied for the purification and recovery of monoclonal antibodies from serum-containing and serum-free culture supernatants. Improved recovery of monoclonal antibodies from serum-free Chinese hamster ovary (CHO) cell culture supernatants with a reduction of the host cell proteins (HCPs) content are obtained by introducing ILs as adjuvants in PEG-salt systems, with [N4444]Br providing the lowest production costs. Overall, TPP systems were shown to be not only cost-effective, but also robust and flexible routes to purify and recover IgG from complex biological matrices as shown here with human serum, serum-containing and serum-free cell culture supernatants.publishe

Sustainable strategies based on glycine–betaine analogue ionic liquids for the recovery of monoclonal antibodies from cell culture supernatants

Monoclonal antibodies (mAbs) are of crucial interest for therapeutic purposes, particularly in vaccination and immunization, and in the treatment of life-threatening diseases. However, their downstream processing from the complex cell culture media in which they are produced still requires multiple steps, making mAbs extremely high-cost products. Therefore, the development of cost-effective, sustainable and biocompatible purification strategies for mAbs is in high demand to decrease the associated economic, environmental and health burdens. Herein, novel aqueous biphasic systems (ABS) composed of glycine–betaine analogue ionic liquids (AGB-ILs) and K2HPO4/KH2PO4 at pH 7.0, the respective three-phase partitioning (TPP) systems, and hybrid processes combined with ultrafiltration were investigated and compared in terms of performance as alternative strategies for the purification and recovery of anti-human interleukin-8 (anti-IL-8) mAbs, which are specific therapeutics in the treatment of inflammatory diseases, from Chinese Hamster Ovary (CHO) cell culture supernatants. With the studied ABS, mAbs preferentially partition to the IL-rich phase, with recovery yields up to 100% and purification factors up to 1.6. The best systems were optimized in what concerns the IL concentration, allowing to take advantage of IL-based three-phase partitioning approaches where a precipitate enriched in mAbs is obtained at the ABS interface, yielding 41.0% of IgG with a purification factor of 2.7 (purity of 60.9%). Hybrid processes combining the two previous techniques and an ultrafiltration step were finally applied, allowing the recovery of mAbs from the different fractions in an appropriate buffer solution for further biopharmaceutical formulations, while allowing the simultaneous IL removal and reuse. The best results were obtained with the hybrid process combining TPP and ultrafiltration, allowing to obtain mAbs with a purity higher than 60%. The recyclability of the IL was additionally demonstrated, revealing no losses in the purification and recovery performance of these systems for mAbs. The biological activity of anti-IL-8 mAbs is maintained after the several purification and recovery steps, indicating that the novel ABS, three-phase partitioning and hybrid processes comprising AGB-ILs are promising and sustainable strategies in mAbs downstream processing.publishe

Monoclonal antibodies as therapeutic agents for inflammatory diseases

Inflammation is a physiological process caused when an agent (chemical, biological or physical) transcends the primary defense barrier of an organism, playing a central role in the fight against those pathogens, setting a series of biological reactions to restore the integrity of such organism. Uncontrolled amplification of these events may lead to undesirable pathological manifestations such as cancer, diabetes, and cardiovascular, neurological, and chronic inflammatory diseases. Monoclonal antibodies (mAbs) were first described in 1975, and since then they have proven to be relevant therapeutic agents in a myriad of diseases. The US Food and Drug Administration (FDA) has already approved more than 90 mAbs for the treatment of several diseases, from which approximately 46% were specifically approved for the treatment of inflammatory diseases, for instance rheumatoid arthritis, Crohn's disease, ulcerative colitis, psoriasis, psoriatic arthritis and palmoplantar pustulosis. This chapter aims to provide an overview on the inflammation process and main biochemical mechanisms, a vision on the current state of the art of the mAbs-based biopharmaceuticals market, and describes the mAbs products already approved by regulatory agencies as powerful therapeutic agents for inflammatory diseases, while highlighting the advantages of these biopharmaceuticals and fomenting their widespread use as recurrent therapies.publishe