Separation of albumin from bovine serum applying ionic-liquid-based aqueous biphasic systems

In this work, the extraction and separation of bovine serum albumin (BSA) from its original matrix, i.e., bovine serum, was performed using a novel ionic-liquid-based aqueous biphasic system (IL-based ABS). To this end, imidazolium-, phosphonium-, and ammonium-based ILs, combined with the anions’ acetate, arginate and derived from Good Buffers, were synthesized, characterized, and applied in the development of ABS with K2HPO4/KH2PO4 buffer aqueous solutions at pH 7. Initial studies with commercial BSA revealed a preferential migration of the protein to the IL-rich phase, with extraction efficiencies of 100% obtained in a single-step. BSA recovery yields ranging between 64.0% and 84.9% were achieved, with the system comprising the IL tetrabutylammonium acetate leading to the maximum recovery yield. With this IL, BSA was directly extracted and separated from bovine serum using the respective ABS. Different serum dilutions were further investigated to improve the separation performance. Under the best identified conditions, BSA can be extracted from bovine serum with a recovery yield of 85.6% and a purity of 61.2%. Moreover, it is shown that the BSA secondary structure is maintained in the extraction process, i.e., after being extracted to the IL-rich phase. Overall, the new ABS herein proposed may be used as an alternative platform for the purification of BSA from serum samples and can be applied to other added-value proteins.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

Cystic Echinococcosis in Spain: Current Situation and Relevance for Other Endemic Areas in Europe

Cystic echinococcosis (CE) remains an important health problem in many regions of the world, both where no control measures have been implemented, and where control programs have been incompletely successful with ensuing re-emergence of the disease. In Spain, official data on CE show an increase in the proportion of intermediate hosts with CE during the last few years, and autochthonous pediatric patients have been reported, a sign of active local transmission of disease. A similar picture emerges from data reported to the European Food Safety Authority by other European countries. Nevertheless, several crucial aspects related to CE that would help better understand and control the disease have not been tackled appropriately, in particular the emergence of infection in specific geographical areas. In this respect, while some data are missing, other data are conflicting because they come from different databases. We review the current situation of CE in Spain compared with areas in which similar problems in the CE field exist, and offer recommendations on how to overcome those limitations. Specifically, we believe that the introduction of national registries for CE with online data entry, following the example set by the European Registry for Alveolar Echinococcosis, would help streamline data collection on CE by eliminating the need for evaluating and integrating data from multiple regions, by avoiding duplication of data from patients who access several different health facilities over time, and by providing much needed clinical and epidemiological data that are currently accessible only to clinicians

Triblock copolymers as versatile constituents of double stimuli-responsive ionic-liquid-based aqueous biphasic systems

This work proposes the use of triblock copolymers as structurally versatile constituents to develop double stimuli-responsive aqueous biphasic systems (ABS) comprising ionic liquids (ILs). Phase diagrams of ABS comprising cholinium acetate ([Ch][C2O2]) and triblock copolymers composed of polyethylene oxide (PEO) and polypropylene oxide (PPO) with adjustable compositions of PEO, 20% (PE 6200) and 40% (PE 6400), were determined by the cloud point titration method at several temperatures (25–45 °C) and pH (5–7). These ABS present, for all the tested pH conditions, a Lower Critical Solution Temperature (LCST)-like behavior. However, only ABS with PE 6400 copolymer exhibit a simultaneous response to both stimuli. Compared to the homologous homopolymer polypropylene glycol (PPG), and despite their lower capacity to form ABS, copolymers with an adequate content of PEO provide a stronger response to the simultaneous action of pH and temperature. The results obtained highlight the value of an appropriate adjustment of the polymer structure/monomer composition in the development of ABS with double response to temperature and pH. The enhanced tailoring ability of the proposed systems is demonstrated with the simultaneous separation of two dyes by the application of the studied stimuli.publishe

Double-Stimuli-Responsive (Temperature and pH) Aqueous Biphasic Systems Comprising Ionic Liquids

Aqueous biphasic systems (ABS) can integrate multiple unit operations and operate under continuous mode, contributing to the development of sustainable separation processes. Encouraged by the designer solvent features of ionic liquids (ILs), we herein propose their use as components of double-stimuli-responsive (temperature- and pH-driven) ABS. Resorting to choline-alkanoate-based ILs as the pH-responsive components and poly(propylene glycol) (PPG 400) as the thermo-responsive component, the ABS ternary phase diagrams are determined at various temperature (25–45 °C) and pH (3–7) conditions. While the liquid–liquid phase diagram response to temperature obeys a lower critical solution temperature-like behavior, the response to pH correlates with the pKa of the IL anion parent acid. The simultaneous responsiveness to temperature and pH is then shown, whose results inspire the development of customizable separation techniques as proved with the simultaneous (one-step) separation of two dyes. By a proper customization of the IL chemical structure and stimuli applied, ABS may be designed to improve the performance and sustainability of separation processes.publishe