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

Insights into coacervative and dispersive liquid-phase microextraction strategies with hydrophilic media - a review

Since the development of liquid-phase microextraction (LPME), different LPME modes depending on the experimental set-up to carry out the extraction have been described. Dispersive liquid-liquid microextraction (DLLME), in which a small amount of the water-insoluble extraction solvent is dispersed in the sample, is the most successful mode in terms of number of applications reported. Advances within DLLME have been mainly shifted to the incorporation of green, smart and tunable materials as extraction solvents to improve the sustainability and efficiency of the method. In this sense, hydrophilic media represent a promising alternative since the water-miscibility of these substances increases the mass transfer of the analytes to the extraction media, leading to higher extraction efficiencies. Considering the variety of hydrophilic media that have been incorporated in LPME approaches resembling DLLME, this review aims to classify these methods in order to clarify the confusing terminology used for some of the strategies. Hydrophilic media covered in this review comprise surfactants, polar organic solvents, deep eutectic solvents, ionic liquids, water-miscible polymers, and switchable solvents. Different physicochemical mechanisms of phase separation are discussed for each LPME method, including the coacervation phenomena and other driving forces, such as pH, temperature, salting-out effect, metathesis reaction and organic solvents. LPME modes are classified (in cloud-point extraction, coacervative extraction, aqueous biphasic systems, and different DLLME modes depending on the extraction medium) according to both the nature of the water-miscible extraction phase and the driving force of the separation. In addition, the main advances and analytical applications of these methods in the last three years are described.publishe

Aqueous Biphasic Systems Comprising Natural Organic Acid-Derived Ionic Liquids

Despite the progress achieved by aqueous biphasic systems (ABSs) comprising ionic liquids (ILs) in extracting valuable proteins, the quest for bio‐based and protein‐friendly ILs continues. To address this need, this work uses natural organic acids as precursors in the synthesis of four ILs, namely tetrabutylammonium formate ([N4444][HCOO]), tetrabutylammonium acetate ([N4444][CH3COO]), tetrabutylphosphonium formate ([P4444][HCOO]), and tetrabutylphosphonium acetate ([P4444][CH3COO]). It is shown that ABSs can be prepared using all four organic acid‐derived ILs paired with the salts potassium phosphate dibasic (K2HPO4) and tripotassium citrate (C6H5K3O7). According to the ABSs phase diagrams, [P4444]‐based ILs outperform their ammonium congeners in their ability to undergo liquid–liquid demixing in the presence of salts due to their lower hydrogen‐bond acidity. However, deviations to the Hofmeister series were detected in the salts’ effect, which may be related to the high charge density of the studied IL anions. As a proof of concept for their extraction potential, these ABSs were evaluated in extracting human transferrin, allowing extraction efficiencies of 100% and recovery yields ranging between 86 and 100%. To further disclose the molecular‐level mechanisms behind the extraction of human transferrin, molecular docking studies were performed. Overall, the salting‐out exerted by the salt is the main mechanism responsible for the complete extraction of human transferrin toward the IL‐rich phase, whereas the recovery yield and protein‐friendly nature of these systems depend on specific “IL-transferrin” interactions.publishe

SCARB2 mutations as modifiers in Gaucher disease: the wrong enzyme at the wrong place?

Unlike most lysosomal proteins, β-glucocerebrosidase (GCase), the hydrolase defective in Gaucher disease (GD), is delivered to lysosomes through its interaction with the transmembrane protein LIMP2. A few years ago, mutations in its coding gene, SCARB2, were reported to modify the severity of GD phenotype. The existence of a great variety of GD phenotypes is well-known, with numerous patients who carry identical genotypes presenting remarkable phenotypic variability. Over the years, that variability has been attributed to other genetic, epigenetic and/or environmental factors. Still, there is still much to learn on this subject. Recently, an association between Parkinson's disease (PD) and the presence of mutations in the GBA gene has been demonstrated. Moreover, there are also studies suggesting that genetic variants in the SCARB2 gene may also be risk factors for PD. We analysed the SCARB2 gene in the Portuguese cohort of 91 GD patients, having identified 3 different SCARB2 coding variants. Of those, 2 were known polymorphisms with high prevalence in the normal population (p.M159V and p.V396I) and the third was a novel coding variant, p.T398M, present in heterozigousity in a single patient. Our study demonstrated that, at least for the Portuguese population, genetic variability at SCARB2 does not account much to the GD phenotypic spectrum. Nevertheless, in vitro analyses of the novel p.T398M are envisaged, in order to further characterize the effect of this variant on the levels and sub-cellular location of GCase. The clinical presentation of the patient harbouring this coding variant will also be discussed.FCT PTDC/SAU-GMG/102889/2008; SFRH/BD/124372/2016N/

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

Using supported ionic liquids to remove cytostatics from human urine

In 2018, over 18 million new cancer cases emerged [1]. Progresses accomplished in cancer treatment, namely in the development of effective ant-cancer drugs (e.g., cytostatics) allow cancer patients to live better and longer [2]. Nevertheless, cytostatics, as with other drugs, are excreted through urine, reaching wastewater treatment plants (WWTPs), and subsequently contaminating the aquatic environment [3, 4]. A promising technological tool to remove cytostatic drugs from aqueous matrices is represented by the use of supported ionic liquids (SILs). This work is aimed at synthesizing and characterizing several SILs based on quaternary ammonium structures using silica as the support material. The determination of adsorption kinetics and isotherms was then carried out to infer on SILs adsorption capacity for cyclophosphamide (model cytostatic drug). SILs capacity to remove cyclophosphamide from aqueous samples was contingent on the cation structure, opening the way to design effective alternative adsorbents for cytostatics removal.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

Evaluating Self-buffering Ionic Liquids for Biotechnological Applications

A new range of Good’s buffer ionic liquids (GB-ILs), displaying simultaneously the properties of ionic liquids and Good’s buffers, were synthesized by combination of Good’s buffers anions (MOPSO, BES, TAPSO and CAPSO) and tetrabutylammonium, tetrabutylphosphonium and cholinium cations via an acid-base neutralization reaction. The activity and stability of a lipolytic enzyme from Pseudomonas cepacia in aqueous solutions of these buffers were evaluated and the results show their advantage as media for enzymatic reactions when compared to conventional phosphate buffers. Moreover aqueous biphasic systems (ABS) composed by these GB-ILs and potassium citrate were investigated and shown to be highly effective and selective for the partitioning of the lipolytic enzyme into the GB-IL-rich phase. The results allow the development of an efficient and biocompatible process combining the self-buffering and enzyme-stabilizing properties of the GB-ILs in the reaction step, with the advantages of GB-ILs as extraction solvents in ABS