Advances towards the development of a sustainable economy must address the exploitation of bio-based products combined with the use of greener solvents and manufacturing processes that can preserve natural resources and the environment. To address the growing demand for proteins, their production must focus on non-animal sources, such as vegetal biomass and respective residues/waste, and on the use of sustainable solvents and processes for their recovery. This review provides an overview of the advances achieved in the separation and purification processes of proteins from vegetable biomass and respective residues using ionic liquids (ILs) and deep eutectic solvents (DESs) as alternative greener solvents. It begins with an overview of the ability of ILs and DESs to stabilize proteins, followed by the assessment of the extraction and separation of biomass-derived proteins assisted by ILs and DESs. Different types of non-animal biomass and respective residues are considered as protein resources, i.e., algae, plants (e.g., aloe vera and holy basil), cereals (e.g., wheat and oat), fruits (e.g., papaya, pineapple, pomegranate, and seabuckthorn berries), and vegetables (e.g., spinach, radish, and ginger). Several IL- and DES-based approaches are discussed, comprising (i) conventional solid–liquid extraction (SLE), (ii) ultrasound-assisted extraction (UAE), (iii) microwave-assisted extraction (MAE), and (iv) aqueous biphasic systems (ABSs). Finally, the economic and environmental challenges of using such alternative solvents in industrial applications are discussed, including technoeconomic analysis and life cycle assessment.

Ribulose-1,5-biphosphate carboxylase/oxygenase (RuBisCO) is the most abundant protein on the planet, being present in plants, algae and various species of bacteria, with application in the pharmaceutical, chemical, cosmetic and food industries. However, current extraction methods of RuBisCO do not allow high yields of extraction. Therefore, the development of an efficient and selective RuBisCOs’ extraction method is required. In this work, aqueous solutions of biocompatible ionic liquids (ILs), i.e., ILs derived from choline and analogues of glycine-betaine, were applied in the RuBisCO’s extraction from spinach leaves. Three commercial imidazolium-based ILs were also investigated for comparison purposes. To optimize RuBisCO’s extraction conditions, response surface methodology was applied. Under optimum extraction conditions, extraction yields of 10.92 and 10.57 mg of RuBisCO/g of biomass were obtained with the ILs cholinium acetate ([Ch][Ac]) and cholinium chloride ([Ch]Cl), respectively. Circular dichroism (CD) spectroscopy results show that the secondary structure of RuBisCO is better preserved in the IL solutions when compared to the commonly used extraction solvent. The obtained results indicate that cholinium-based ILs are a promising and viable alternative for the extraction of RuBisCO from vegetable biomass.

Background: Infliximab (IFX) and Adalimumab (ADA) are effective drugs in the treatment of inflammatory bowel disease (IBD). Studies suggest that therapeutic drug monitoring (TDM) may allow optimization of drug treatment, but it remains unclear if this strategy is associated with greater therapeutic efficacy and better outcomes. Methods: We compared clinical-based adjustment (CB) with drug-monitoring adjustment (DM) in patients with IBD under IFX and ADA on several outcomes including hospitalization, surgery, clinical remissio and therapeutic failure at 48 weeks of treatment. We also determined the number of therapeutic adjustments, C-reactive protein (CRP) and faecal calprotectin (FC) values (negative <0.5 mg/dl and <50 ug/ml) at 48 weeks of treatment. Clinical remission was defined as absence of hospitalization, surgery and failure or switch of anti-TNF at 48 weeks of treatment. Results: 117 patients were included: 98 with Crohn's disease (CD) and 19 with Ulcerative Colitis (UC). 54.7% were male with a mean age of 29.1 years (range 7–65). 117 were allocated to the DM group and 101 to the CB group. Therapeutic escalation was more frequent in the DM group (47.0% vs 10.9%, p<0.001). DM was associated with longer time to relapse (8.74±42 vs 6.00±3.1 months, p=0.045) and, in patients with positive baseline CRP, with higher probability of clinical remission (p=0.05). The number of patients with negative FC was higher in DM group (77.3% vs 54.2%, p=0.029). There was a trend for higher therapeutic failure in the CB group (5.9% vs 1.7%, p=0.097). There were no differences in the rates of surgery (5.0% vs 6.8%, p=0.385), Hospitalization (6.9% vs 6.8%, p=0.593) or clinical remission (73.5% vs 78.2%, p=0.258). The prevalence of infratherapeutic IFX serum levels (IFX 10) was 33.6% and 17.7%. In the DM group, escalation occurred mainly due to Infratherapeutic serum levels and not due to positive antibodies (p<0.001 and p=0.175). Clinical remission (78.7% vs 60.5%, p=0.036) was more common in patients with higher drug serum levels (7.12±7.34 vs 4.56±3.81, p=0.018). Conclusions: Our results suggest some benefit from DM-based management. The high rates of clinical remission found in both cohorts (73.5% vs 78.2%) may have partly been responsible for the absence of a significant difference between both cohorts. However, the rates of escalation were 4 times higher in the DM group. We hypothesize that longer follow-ups would allow us to reach a difference between both cohorts.

Minicircle DNA (mcDNA) is a non-viral vector known for its superior therapeutic potential and safety, making it a promising biopharmaceutical platform for gene therapy, DNA vaccination, and molecular biology applications. However, its structural similarity to parental plasmid (PP), the presence of isoforms (e.g., open circular mcDNA and supercoiled mcDNA), and host-derived impurities pose significant challenges for accurate detection and quantification, which are key steps for effective quality control. This work reports, for the first time, the development and validation of an anion-exchange chromatographic method for quantifying p53-mcDNA using a customized TSKgel® DNA-NPR column. Chromatographic parameters, including pH, elution gradient, gradient time, and flow rate, were optimized. Real samples from complex Escherichia coli lysates containing p53-mcDNA, p53-PP, miniplasmid (mP), and RNA were used, demonstrating applicability for process monitoring. The method demonstrated consistent linearity (R² = 0.9994) over the 10-100 µg/mL range for total DNA, with accuracy meeting FDA guidelines. The method was validated in terms of selectivity, linearity, accuracy, precision, and sensitivity over the concentration range of 10-100 µg/mL, with the lower limit of quantification (LLOQ) and detection (LOD) established at 10 µg/mL and 1 µg/mL, respectively. Intra- and inter-day precision studies confirmed reproducibility, with coefficients of variation below 6%. Overall, the chromatographic method here developed and proposed effectively separates p53-mcDNA from its parental plasmid and may support process monitoring during upstream and downstream operations.