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

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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

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

Purification of immunoglobulin Y from egg yolk using thermoresponsive aqueous micellar two-phase systems comprising ionic liquids

Immunoglobulin Y (IgY) represents an important class of antibodies, being present in egg yolk, with relevant medical applications and involving non-invasive methods of extraction. However, due to the complexity of egg yolk, the purification levels required for use in most medical applications demands the application of multi-step and cost-intensive techniques. Therefore, it is of upmost importance to develop a biocompatible and cost-effective downstream process to purify IgY from egg yolk. In this work, IgY was purified from the egg yolk water-soluble protein fraction (WSPF) by the application of thermoresponsive aqueous micellar two-phase systems (AMTPS) composed of the non-ionic surfactant Triton X-114 and surface-active ionic liquids (SAILs) as co-surfactants. The best thermoresponsive systems allowed, in a single step, to recover IgY in the surfactant-poor phase with a purity level of 69%, and up to 73% if consecutive extraction cycles are performed, while maintaining the structural integrity of the antibodies. Considering these results, a downstream process was designed and proposed, consisting of four main steps: (i) recovery of the WSPF; (ii) purification of IgY by applying AMTPS; (iii) isolation of the IgY from the main solvents using an ultrafiltration step; (iv) recycling of the AMTPS phase-forming components by precipitating the contaminant proteins.publishe

Sustainable liquids support as a media for biocatalytic reactions using aqueous biphasic systems

Over the last few years a significant interest in the application of biocatalyst in several industries as an alternative to the commonly used chemical biocatalyst has emerged, since enzymes, such as laccase, allow for an improved, sustainable and biodegradable catalytic process partially due to their high specificity to its substrates. Nevertheless, its application can still be considered a costly process thus to overcome this shortcoming emerges the need for the enzyme reutilization employed in the catalytic reaction 1. One of the alternatives is to implement aqueous biphasic systems (ABS), which are a biocompatible liquid-liquid extraction as a liquid support. Moreover, the introduction of ionic liquids (ILs) in ABS have shown promising results in the system overall performance and could potentially allow for an improved biocatalyst performance. Therefore, our goal was to form an ABS composed of cholinium-based ILs and polypropylene glycol (PPG 400) as novel liquid supports for enzymes, using laccase as models in order to evaluated the degradation of the textile dye Remazol Brilliant Blue R (RBBR) as well as the capacity to reutilized the biocatalyst for further applications.publishe

Aqueous two-phase systems comprising ionic liquids for the extraction of recombinant proteins

The progress made on the use of new biopharmaceuticals has a high influence on the global health, and since the approval of humulin (recombinant insulin) by FDA, new protein-based pharmaceuticals have been introduced in the market such as the interferon alfa 2b (IFNα2b), which has been used in in the treatment of oncological diseases for more than 30 years. Nowadays, the (bio)pharmaceutical industry is committed to improve the quality of their products while endeavoring a reduction of its production costs. Currently, the downstream processing of biopharmaceuticals is dominated by chromatography and, despite being highly effective, is responsible for their current high-cost [1]. Accordingly, there is a crucial need to develop effective alternative purification platforms. In this way, aqueous two-phase systems (ATPS) emerge. Due to their high-water content, ATPS may be considered a biocompatible technique for the purification of (bio)molecules. Additionally, the use of ionic liquids (ILs) in low concentrations as adjuvants in ATPS affords an enhanced performance in what concerns to (bio)molecules selectivity. In this work it is shown the use of polymer-polymer ATPS comprising ILs as adjuvants to purify recombinant proteins. Several variables were studied in the extraction of the target proteins, namely pH, polymer molecular weight, and the effect of different families of ILs and their concentration. Overall, imidazolium-based ILs seem responsible for an improved ATPS performance towards a higher purity, and is additionally shown that electrostatic interactions between the negatively charged protein and ILs cations may account for this behavior.publishe

Downstream processing of recombinant proteins using alternative purification platforms based on ionic liquids

Remarkable advances in the treatment of chronic diseases have been achieved with biopharmaceutical-based therapies, a sector that is currently dominated by therapeutic proteins with over 200 protein drugs in the market1. Improvements in the biopharmaceuticals upstream stage have resulted in high yields 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 costs2. Aiming at finding new cost-effective, efficient and sustainable technologies for proteins purification, novel polymer-polymer aqueous biphasic systems (ABS) using ionic liquids (ILs) as adjuvants were investigated in the downstream of interferon alfa 2b (IFNα2b) from Escherichia coli BL21 cultures. The phase diagrams of ABS composed by polyethylene glycol (PEG) and polypropylene glycol (PPG), with ILs as adjuvants, were determined at 25 °C. Chloride-based ILs combined with cholinium, imidazolium, pyrrolidinium, piperidinium, tetralkylammonium and tetralkylphosphonium cations were investigated. The use of ILs allows the selective removal of the protein impurities present in the culture medium. In summary, the application of ILs allowed to develop effective IFNα2b purification platforms from E. coli based on polymer-polymer-ABS, having significant benefits in terms of economic, biocompatible and environmental issues. References: 1Walsh G, Nat Biotechnol, 2018, 36, 1136-1145; 2Azevedo AM, Rosa PAJ, Ferreira IF, et al. Trends Biotechnol, 2009, 27, 240-247.publishe

Maternal smoking in pregnancy and blood pressure during childhood and adolescence: a meta-analysis

rterial hypertension during childhood or adolescence is rising, and smoking during pregnancy may constitute a modifiable risk factor. This study aims to evaluate the effect of maternal smoking during pregnancy on diastolic (DBP) and systolic blood pressure (SBP) in childhood and adolescence. A bibliographic search was conducted in PubMed, Embase, and CENTRAL databases in March 2022. Meta-analysis was performed with the difference in mean-adjusted SBP/DBP of children and adolescents aged 3–17 years, according to maternal smoking/non-smoking in pregnancy. A random effects model was applied; a leave-one-out analysis and meta-analysis by subgroups were performed. A modified Newcastle–Ottawa scale was used to assess the quality of the studies. Evidence levels were rated using the GRADE system. Fifteen studies were included in the meta-analysis; all of them evaluated the mean-adjusted SBP difference in children or adolescents (N = 73,448), and 6 also that of DBP (N = 31,459). Results showed that maternal smoking during pregnancy significantly increased SBP (β = 0.31 mmHg 95% CI 0.14–0.49). A greater increase in mean-adjusted SBP was observed in those studies that completed the recruitment before 1990, were conducted in non-European countries, used standard mercury or manual sphygmomanometry, adjusted for birth weight, and were in the lowest quality subgroup. No significant association was found for DBP. The GRADE level of evidence was low for SBP and very low for DBP. Conclusion: Smoking in pregnancy might increase SBP in childhood and adolescence. Due to the low level of evidence, solid inferences cannot be drawn about the clinical relevance of these findings.S

Improving the purification of recombinant proteins by the use of ionic liquids as adjuvants in aqueous two-phase systems

The development of new drugs has a high impact on the global health, and since the approval of humulin (recombinant insulin) by FDA, several protein-based biopharmaceuticals have been introduced in the market. These products are usually attained through biotechnological processes using engineered biological sources and have led to remarkable advances in the treatment of distinct human diseases, as for example the case of interferon alfa 2b (IFNα2b) which has been used in in the treatment of oncological diseases for more than 30 years [1]. Currently, the (bio)pharmaceutical industry is committed to improve the quality of their products while endeavouring a reduction of its production costs, which is intimately related to the necessity of improving their downstream processes, since this field is mainly dominated by chromatographic techniques [2] and are responsible for their current high-cost. Therefore, envisaging to develop new, cost-effective, efficient and sustainable techniques for proteins purification, polymer-polymer aqueous two-phase systems (ATPS) with ionic liquids (ILs) as adjuvants are herein proposed for the extraction and purification of IFNα2b from Escherichia coli BL21 cultures. The recombinant protein was produced in the form of inclusion bodies, followed by a dialysis step in order to solubilize the target biomolecule in a specific buffer, with the goal of maintaining the biomolecule stability. Afterwards, several polymers where studied as phase-forming components of ATPS, namely polyethylene glycol (PEG), polypropylene glycol (PPG), as well as Dextran. Furthermore, several ILs belonging to the cholinium, imidazolium, tetralkylammonium and tetralkylphosphonium families, were tested as co-adjuvants for ATPS formation in order to extract and purify of IFNα2b.publishe