Conventional alginate extraction methods from brown seaweed typically rely on harsh chemicals that are not reused, and valuable pigments are lost during this process. This study applied a novel approach utilising reusable natural deep eutectic solvents (DES) in three-phase partitioning (TPP) to simultaneously extract alginate and pigments from Saccharina latissima . The hydrophobic DES effectively released alginate from the algal cell wall in the aqueous phase and served as a solvent for pigment extraction. Computational screening confirmed that all selected DES had an affinity for pigments chlorophyll a and fucoxanthin, while alginate extraction confirmed their role in disrupting the algal cell wall. Extraction conditions were optimised, resulting in an alginate yield of 101.8 ± 3.1 mg/g DW compared to 55.3 ± 14.1 mg/g DW for conventional alkaline extraction. According to physicochemical characterisation through FT-IR and M/G ratio (mannuronic to guluronic) analysis, the extracted alginate was comparable to that obtained via alkaline extraction, exhibiting similar functional groups and M/G ratios. The DES was reused successfully, showing that it could be reused for up to seven extraction cycles, during which pigments accumulated. After the seventh cycle, alginate yield declined, likely due to partial transfer into the DES phase, possibly driven by reverse micelle formation in the system. This study highlights a novel, mild multiproduct approach of a DES-based TPP system, enhancing economic feasibility by employing gentler and quicker extraction conditions. It facilitates the concurrent recovery of alginate and pigments while allowing for the repeated reuse of the DES.

Deep eutectic solvents (DES) have emerged as green alternative extraction solvents. However, challenges in DES recovery and recycling limit their broader application. In this study, a novel thermo-separating aqueous two-phase system (ATPS) was developed for the continuous, cyclic extraction and separation of alginate from Laminaria digitata using DES and temperature-responsive copolymers. The system enables a novel approach by repeatedly reusing both the DES phase and the EOPO copolymer phase five times, thereby reducing waste generation and enhancing process sustainability. This research demonstrated that DES can be efficiently recycled for ten cycles using temperature-responsive ethylene oxide-propylene oxide (EOPO) copolymers, remaining stable yields. For the extraction and subsequent separation of alginate, three different DESs were evaluated, all of which demonstrated to extract and recover alginate. DESs ChCl:Ethylene glycol and ChCl:Urea exhibited a preference for EOPO1000 (66 and 75 % recovery, respectively), whereas Bet:Urea achieved the highest recovery with EOPO3900 (66 % recovery). Subsequent recycling of the recovered DES showed that DES could be recycled for ten cycles, maintaining stable extraction yields between 74 and 86 mg alginate/g DW and alginate recovery yields of 55–65 %. Furthermore, combined DES and EOPO recycling could be performed for up to five cycles while maintaining an alginate recovery yield between 50 and 65 mg/g DW. This thermo-separating ATPS presents a novel, circular and sustainable approach for DES recycling compared to the non-circular conventional alkaline extraction. This proposed method can be applied in a simple and effective manner to both recover and recycle DES.

Integrated Continuous Biomanufacturing reduces manufacturing costs while maintaining product quality. A key contributor to high biopharmaceutical costs, specifically monoclonal antibodies (mAbs), is chromatography. Protein A ligands are usually preferred but still expensive in the manufacturing context, and batch chromatography under-utilizes the columns' capacity, compromising productivity to maintain high yields. Continuous chromatography increases columns' Capacity Utilization (CU) without sacrificing yield or productivity. This work presents the in-silico optimization of a 3 Column Periodic Counter-current Chromatography (3C-PCC) of a capture and polishing step for mAbs from a high titer harvest (cmAb = 5 g/L). The 3C-PCC was modeled and Pareto-fronts for continuous and batch modes were used to optimize the 3C-PCC steps varying the flow rate and percentage of breakthrough achieved in the interconnected loading, maximizing Productivity and CU, for varying concentrations of mAb (batch mode concentration of 5 g/L and continuous mode concentration of 2.5, 5, 7.5, and 10 g/L). The shape of the breakthrough curve significantly impacts the optimization of 3C-PCC. The model output was validated for three different protein A ligands using a pure mAb solution. MAb Select SuRe pcc was selected to continuously capture mAb from a high-titer clarified cell culture supernatant (harvest). The product eluates were pooled and used for continuous polishing using a Cation-Exchange resin (CaptoS ImpAct). Experimental results validated model predictions (<7% deviation in the worst case) and a process with two 3C-PCC in sequence was proposed, with a productivity of approximately 100 mg/mL res/h.

The monoclonal antibody (mAb) industry is becoming increasingly digitalized. Digital twins are becoming increasingly important to test or validate processes before manufacturing. High-Throughput Process Development (HTPD) has been progressively used as a tool for process development and innovation. The combination of High-Throughput Screening with fast computational methods allows to study processes in-silico in a fast and efficient manner. This paper presents a hybrid approach for HTPD where equal importance is given to experimental, computational and decision-making stages. Equilibrium adsorption isotherms of 13 protein A and 16 Cation-Exchange resins were determined with pure mAb. The influence of other components in the clarified cell culture supernatant (harvest) has been under-investigated. This work contributes with a methodology for the study of equilibrium adsorption of mAb in harvest to different protein A resins and compares the adsorption behavior with the pure sample experiments. Column chromatography was modelled using a Lumped Kinetic Model, with an overall mass transfer coefficient parameter (k_ov). The screening results showed that the harvest solution had virtually no influence on the adsorption behavior of mAb to the different protein A resins tested. k_ov was found to have a linear correlation with the sample feed concentration, which is in line with mass transfer theory. The hybrid approach for HTPD presented highlights the roles of the computational, experimental, and decision-making stages in process development, and how it can be implemented to develop a chromatographic process. The proposed white-box digital twin helps to accelerate chromatographic process development.