Isolation and quantification of alginate in choline chloride-based deep eutectic solvents.

Extraction of seaweed compounds using Deep Eutectic Solvents (DES) has shown high interest. Quantification, however, is challenging due to interactions with DES components. In this research work, three chemical separation techniques were investigated to isolate and quantify alginate from a set of choline chloride-based DES. While choline chloride served as the hydrogen bond acceptor (HBA); Urea, Ethylene Glycol, Propylene Glycol, Glycerol, Sorbitol, Xylitol and Glucose were used as hydrogen bond donors (HBD). DES containing sodium alginate were subjected to precipitation with sulfuric acid 0.2 M (pH 1.6), ethanol-water mixture (80 % v/v) and calcium chloride (1 % w/v CaCl2·2H2O). Alginate in precipitates was quantified and used to evaluate the performance of each separation technique. The highest recovery yields (51.2 ± 1.3 %) were obtained using the ethanol-water mixture followed by calcium chloride (45.7 ± 1.2 %), except for polyols (e.g. sorbitol). The lowest recovery yields were obtained with acid, with a particularly low recovery yield when urea was used as HBD (9.6 ± 1.3 %). Estimations of ManA/GulA ratios showed lower values for precipitates from DES compared to the ones obtained from water. This research shows ethanolic precipitation as a suitable method for alginate separation from the studied set of choline chloride-based DES.

Real-time detection of mAb aggregates in an integrated downstream process.

The implementation of continuous processing in the biopharmaceutical industry is hindered by the scarcity of process analytical technologies (PAT). To monitor and control a continuous process, PAT tools will be crucial to measure real-time product quality attributes such as protein aggregation. Miniaturizing these analytical techniques can increase measurement speed and enable faster decision-making. A fluorescent dye (FD)-based miniaturized sensor has previously been developed: a zigzag microchannel which mixes two streams under 30 s. Bis-ANS and CCVJ, two established FDs, were employed in this micromixer to detect aggregation of the biopharmaceutical monoclonal antibody (mAb). Both FDs were able to robustly detect aggregation levels starting at 2.5%. However, the real-time measurement provided by the microfluidic sensor still needs to be implemented and assessed in an integrated continuous downstream process. In this work, the micromixer is implemented in a lab-scale integrated system for the purification of mAbs, established in an ÄKTA™ unit. A viral inactivation and two polishing steps were reproduced, sending a sample of the product pool after each phase directly to the microfluidic sensor for aggregate detection. An additional UV sensor was connected after the micromixer and an increase in its signal would indicate that aggregates were present in the sample. The at-line miniaturized PAT tool provides a fast aggregation measurement, under 10 min, enabling better process understanding and control.

Application of a fluorescent dye-based microfluidic sensor for real-time detection of mAb aggregates.

The lack of process analytical technologies able to provide real-time information and process control over a biopharmaceutical process has long impaired the transition to continuous biomanufacturing. For the monoclonal antibody (mAb) production, aggregate formation is a major critical quality attribute (CQA) with several known process parameters (i.e., protein concentration and agitation) influencing this phenomenon. The development of a real-time tool to monitor aggregate formation is then crucial to gain control and achieve a continuous processing. Due to an inherent short operation time, miniaturized biosensors placed after each step can be a powerful solution. In this work, the development of a fluorescent dye-based microfluidic sensor for fast at-line PAT is described, using fluorescent dyes to examine possible mAb size differences. A zigzag microchannel, which provides 90% of mixing efficiency under 30 s, coupled to an UV-Vis detector, and using four FDs, was studied and validated. With different generated mAb aggregation samples, the FDs Bis-ANS and CCVJ were able to robustly detect from, at least, 2.5% to 10% of aggregation. The proposed FD-based micromixer is then ultimately implemented and validated in a lab-scale purification system, demonstrating the potential of a miniaturized biosensor to speed up CQAs measurement in a continuous process.

Ionic Liquid-Assisted Selective Extraction and Partitioning of Biomolecules from Macroalgae.

Macroalgae are a promising feedstock for several industries due to their large content of proteins and carbohydrates and the high biomass productivities. A novel extraction and fractionation concept based on ionic liquids (ILs) using Ulva lactuca as model organism is presented. Biomolecules are first extracted by means of IL-assisted mechanical shear, followed by two-phase partitioning or ultrafiltration in order to fractionate proteins and carbohydrates and to recover the IL. Ethyl methyl imidazolium dibutyl phosphate ([Emim][DBP]) is strongly selective to proteins, leading to extraction yields up to 80.4% for proteins and 30.7% for carbohydrates. The complete process, including extraction and ultrafiltration, allowed protein recovery of up to 64.6 and 15.4% of the carbohydrates in the retentate phase, while a maximum of 85.7% of the IL was recovered in the permeate phase. The native structure of the extracted proteins was preserved during extraction and fractionation as shown by gel electrophoresis. Selective extraction of proteins from macroalgae under non-denaturing conditions using ILs followed by the recovery of IL using ultrafiltration is for the first time reported. The proposed extraction-fractionation approach is simple and can be potentially applied for the biorefinery of macroalgae at the commercial scale.

Design of a microfluidic mixer channel: First steps into creating a fluorescent dye-based biosensor for mAb aggregate detection.

A major challenge in the transition to continuous biomanufacturing is the lack of process analytical technology (PAT) tools which are able to collect real-time information on the process and elicit a response to facilitate control. One of the critical quality attributes (CQAs) of interest during monoclonal antibodies production is aggregate formation. The development of a real-time PAT tool to monitor aggregate formation is then crucial to have immediate feedback and process control. Miniaturized sensors placed after each unit operation can be a powerful solution to speed up an analytical measurement due to their characteristic short reaction time. In this work, a micromixer structure capable of mixing two streams is presented, to be employed in the detection of mAb aggregates using fluorescent dyes. Computational fluid dynamics (CFD) simulations were used to compare the mixing performance of a series of the proposed designs. A final design of a zigzag microchannel with 45° angle was reached and this structure was subsequently fabricated and experimentally validated with colour dyes and, later, with a FITC-IgG molecule. The designed zigzag micromixer presents a mixing index of around 90%, obtained in less than 30 seconds. Therefore, a micromixer channel capable of a fast and efficient mixing is hereby demonstrated, to be used as a real-time PAT tool for a fluorescence based detection of protein aggregation.

Mild acoustic processing of Tisochrysis lutea for multiproduct biorefineries.

Cellular agriculture could represent a more sustainable alternative to current food and nutraceutical production processes. Tisochrysis lutea microalgae represents a rich source of antioxidants and omega-3 fatty acids essential for human health. However, current downstream technologies are limiting its use. The present work investigates mild targeted acoustic treatment of Tisochrysis lutea biomass at different growth stages and acoustic frequencies, intensities and treatment times. Significant differences have been observed in terms of the impact of these variables on the cell disruption and energy requirements. Lower frequencies of 20 kHz required a minimum of 4500 J to disrupt 90% of the cells, while only 1000 J at 1146 kHz. Comparing these results with current industry standards such as bead milling, up to six times less energy use has been identified. These mild biomass processing approaches offer a certain tunability which could suit a wide range of microorganisms with only minor adjustments.

Two-Component Nanoparticle Vaccine Displaying Glycosylated Spike S1 Domain Induces Neutralizing Antibody Response against SARS-CoV-2 Variants.

Vaccines pave the way out of the SARS-CoV-2 pandemic. Besides mRNA and adenoviral vector vaccines, effective protein-based vaccines are needed for immunization against current and emerging variants. We have developed a virus-like particle (VLP)-based vaccine using the baculovirus-insect cell expression system, a robust production platform known for its scalability, low cost, and safety. Baculoviruses were constructed encoding SARS-CoV-2 spike proteins: full-length S, stabilized secreted S, or the S1 domain. Since subunit S only partially protected mice from SARS-CoV-2 challenge, we produced S1 for conjugation to bacteriophage AP205 VLP nanoparticles using tag/catcher technology. The S1 yield in an insect-cell bioreactor was ∼11 mg/liter, and authentic protein folding, efficient glycosylation, partial trimerization, and ACE2 receptor binding was confirmed. Prime-boost immunization of mice with 0.5 µg S1-VLPs showed potent neutralizing antibody responses against Wuhan and UK/B.1.1.7 SARS-CoV-2 variants. This two-component nanoparticle vaccine can now be further developed to help alleviate the burden of COVID-19. IMPORTANCE Vaccination is essential to reduce disease severity and limit the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protein-based vaccines are useful to vaccinate the world population and to boost immunity against emerging variants. Their safety profiles, production costs, and vaccine storage temperatures are advantageous compared to mRNA and adenovirus vector vaccines. Here, we use the versatile and scalable baculovirus expression vector system to generate a two-component nanoparticle vaccine to induce potent neutralizing antibody responses against SARS-CoV-2 variants. These nanoparticle vaccines can be quickly adapted as boosters by simply updating the antigen component.

Multiproduct Microalgae Biorefineries Mediated by Ionic Liquids.

Ionic liquids (ILs) are salts with low melting points that can be used as solvents for mild extraction and selective fractionation of biomolecules (e.g., proteins, carbohydrates, lipids, and pigments), enabling the valorisation of microalgal biomass in a multiproduct biorefinery concept, while maintaining the biomolecules' structural integrity and activity. Aqueous biphasic systems and emulsions stabilised by core-shell particles have been used to fractionate disrupted microalgal biomass into hydrophobic (lipids and pigments) and hydrophilic (proteins and carbohydrates) components. From nondisrupted biomass, the hydrophobic components can be directly extracted using ILs from intact cells, while the most fragile hydrophilic components can be obtained upon further mechanical cell disruption. These multiproduct biorefinery concepts will be discussed in an outlook on future separations using IL-based systems.

Eutectic solvents with tuneable hydrophobicity: lipid dissolution and recovery.

Despite the promising advantages of eutectic solvents, the application of these solvents as an extraction solvent is still limited due to the challenging product recovery. Previously, it was reported that lipids could be recovered from a hydrophobic eutectic solvent with the principle of switchable hydrophobicity. However, this method still involves additional chemicals, such as polymeric amines, water, and CO2, which need to be removed later. In this study, we proposed a different approach by shifting the hydrophobicity spectrum of a semi-hydrophobic solvent. Made of hydrophilic imidazole and hydrophobic hexanoic acid, this combination showed tuneable hydrophobicity when the composition was changed, shown by the change of dipolarity (π*) scale from solvatochromic analysis. At low imidazole content, the solvent was able to dissolve sunflower oil and algae oil, whereas, at high imidazole content, the solvent showed high affinity towards water. By adding imidazole to the solution of oil and the solvent, a phase split was induced between the oil-rich upper phase and the solvent-rich lower phase. With this approach, ∼75% of recovery efficiency was achieved for the two oils, with the purity of ∼100% for sunflower oil and 86% for algae oil.

Design of Value Chains for Microalgal Biorefinery at Industrial Scale: Process Integration and Techno-Economic Analysis.

The objective of this work was to identify industrial scenarios for the most promising microalgal biorefinery value chains on the basis of product selection, yields, and techno-economic performance, using biological characteristics of algae species. The development, value creation, and validation of several new processing routes with applications in food, aquafeeds and non-food products were particularly considered in this work. The techno-economic performance of various single product value chains (SP) and multiproduct value chains (MP) was evaluated for four industrial microalgal strains. Cost-revenue optimization was done for a 10 kton microalgal dry weight y-1 simulated biorefinery plant, using flow sheeting software for equipment sizing, mass and energy flow modeling, and subsequent techno-economic evaluation. Data on yield, material and energy consumption were based on pre- and pilot size production plants (TRL 5-6). Revenue optimization was accomplished by first analyzing the performance of single product value chains of the microalgal strains. Subsequently, a strategy was developed to exploit almost all biomass based on the most promising microalgal strains. The cultivation costs are most of the time the major costs of the value chains. For the single product value chains common process bottlenecks are low product yields, especially for soluble proteins where only a small fraction of the biomass is leading to economic value. The biorefinery costs (excluding cultivation) vary significantly for various species, due to the species-specific operating conditions as well as differences in product yields. For the evaluated single product value chain scenarios the costs for utilities and other inputs were in general the highest contributing expenses. A biorefinery approach significantly increases the biomass utilization potential to marketable products from 7-28% to more than 97%. Although the cascading approach increases the total production costs of the multiproduct value chains significantly, this is more than compensated by the increased overall biomass revenue. For all selected multiproduct chains there is a significant potential to become profitable at a relevant industrial scale of 10 kton per year. Additional insights in the product functionality, quality, and their market size are needed to narrow down the wide range of foreseen product revenues and resulting profits.

Mild Fractionation of Hydrophilic and Hydrophobic Components From Neochloris oleoabundans Using Ionic Liquids.

Microalgae are a promising source for proteins, lipids, and carbohydrates for the food/feed and biofuel industry. To make microalgae production economically feasible, it is necessary to optimally use all produced compounds keeping full functionality. Therefore, biorefining of microalgae is the key to lower the cost of algal products using mild and effective processing techniques. In this article, we have tested the feasibility of aqueous solutions of imidazolium and phosponium ionic liquids to selectively milk the hydrophobic lipids from Neochloris oleoabundans biomass out of intact cells and recover after cell disruption the hydrophilic fraction containing proteins and carbohydrates. The results showed that the ionic liquid tributylmethylphosphonium methylsulfate (TBP SO4; Cyphos 108) is able to permeabilize fresh intact cells of N. oleoabundans for extracting 68% of total lipids out of the cells, whereas, after cell disruption, 80% of total proteins, and 77% of total carbohydrates could be obtained in aqueous buffers. This concept kept the recovered proteins in their native form without interacting with the ionic liquids that will denature the proteins. Selective biorefinery of different components from microalgae using ionic liquid TBP SO4 explains the novelty of this concept.

From Current Algae Products to Future Biorefinery Practices: A Review.

Microalgae are considered to be one of the most promising next generation bio-based/food feedstocks with a unique lipid composition, high protein content, and an almost unlimited amount of other bio-active molecules. High-value components such as the soluble proteins, (poly) unsaturated fatty acids, pigments, and carbohydrates can be used as an important ingredient for several markets, such as the food/feed/chemical/cosmetics and health industries. Although cultivation costs have decreased significantly in the last few decades, large microalgae production processes become economically viable if all complex compounds are optimally valorized in their functional state. To isolate these functional compounds from the biomass, cost-effective, mild, and energy-efficient biorefinery techniques need to be developed and applied. In this review we describe current microalgae biorefinery strategies and the derived products, followed by new technological developments and an outlook toward future products and the biorefinery philosophy.

Integrated Product Recovery Will Boost Industrial Cyanobacterial Processes.

Cyanobacteria promise to be an important industrial platform for the production of a variety of biobased products such as fuels, plastics, and isoprenoids. Recent advances in synthetic biology have resulted in various cyanobacterial strain improvements. Nevertheless, these new strains are still hampered by product inhibition, resulting in low volumetric productivities, product concentrations, and yields on light. To circumvent these issues, continuous product recovery will need to be applied, resulting in economically viable industrial processes. Optimal product recovery strategies can be developed by considering biological and separation process constraints as well as photobioreactor design. Integrated product recovery will be indispensable to bring the cyanobacterial cell factory to industrial scale.

Chromatographic parameter determination for complex biological feedstocks.

The application of mechanistic models for chromatography requires accurate model parameters. Especially for complex feedstocks such as a clarified cell harvest, this can still be an obstacle limiting the use of mechanistic models. Another commonly encountered obstacle is a limited amount of sample material and time to determine all needed parameters. Therefore, this study aimed at implementing an approach on a robotic liquid handling system that starts directly with a complex feedstock containing a monoclonal antibody. The approach was tested by comparing independent experimental data sets with predictions generated by the mechanistic model using all parameters determined in this study. An excellent agreement between prediction and experimental data was found verifying the approach. Thus, it can be concluded that RoboColumns with a bed volume of 200 µL can well be used to determine isotherm parameters for predictions of larger scale columns. Overall, this approach offers a new way to determine crucial model input parameters for mechanistic modelling of chromatography for complex biological feedstocks. © 2018 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:1006-1018, 2018.

Rapid screening of IgG quality attributes - effects on Fc receptor binding.

The interactions of therapeutic antibodies with fragment crystallizable γ (Fcγ) receptors and neonatal Fc receptors (FcRn) are measured in vitro as indicators of antibody functional performance. Antibodies are anchored to immune cells through the Fc tail, and these interactions are important for the efficacy and safety of therapeutic antibodies. High-throughput binding studies on each of the human Fcγ receptor classes (FcγRI, FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb) as well as FcRn have been developed and performed with human IgG after stress-induced modifications to identify potential impact in vivo. Interestingly, we found that asparagine deamidation (D-N) reduced the binding of IgG to the low-affinity Fcγ receptors (FcγRIIa, FcγRIIb, FcγRIIIa, and FcγRIIIb), while FcγRI and FcRn binding was not impacted. Deglycosylation completely inhibited binding to all Fcγ receptors, but showed no impact on binding to FcRn. On the other hand, afucosylation only impacted binding to FcγRIIIa and FcγRIIIb. Methionine oxidation at levels below 7%, multiple freeze/thaw cycles and short-term thermal/shake stress did not influence binding to any of the Fc receptors. The presence of high molecular weight species, or aggregates, disturbed measurements in these binding assays; up to 5% of aggregates in IgG samples changed the binding and kinetics to each of the Fc receptors. Overall, the screening assays described in this manuscript prove that rapid and multiplexed binding assays may be a valuable tool for lead optimization, process development, in-process controls, and biosimilarity assessment of IgGs during development and manufacturing of therapeutic IgGs.

Rapid Buffer and Ligand Screening for Affinity Chromatography by Multiplexed Surface Plasmon Resonance Imaging.

Protein purifications are often based on the principle of affinity chromatography, where the protein of interest selectively binds to an immobilized ligand. The development of affinity purification requires selecting proper wash and elution conditions. In recent years, miniaturization of the purification process is applied to speed up the development (e.g., microtiterplates, robocolumns). The application of surface plasmon resonance imaging (SPRi) as a tool to simultaneously screen many buffer conditions for wash and elution steps in an affinity-based purification process is studied. Additionally, the protein A ligand stability after exposure to harsh cleaning conditions often limits the reuse of resins and is determined at lab scale. The SPRi technology to screen ligand life-time with respect to alkali stability is used. It is also demonstrated that SPRi can successfully be applied in screening experiments for process developments in a miniaturized approach. The amount of resin, protein and buffer in these studies is reduced 30-300-fold compared to 1 mL column scale, and approximately 10-1000-fold compared to filter plate experiments. The overall development time can be decreased from several months towards days. The multiplexed SPRi can be applied in screening affinity chromatography conditions in early stage development for ligand development and recombinant protein production.

Mild and Selective Protein Release of Cell Wall Deficient Microalgae with Pulsed Electric Field.

Pulsed electric field (PEF) is considered to be a very promising technology for mild cell disruption. The application of PEF for microalgae that have a rigid cell wall, however, is hampered by the presence of that rigid outer cell wall. A cell wall free mutant of C. reinhardtii was used to mimic pretreated microalgae with removed cell wall, to investigate the possibility of using PEF for protein release from microalgae. A complete release of hydrophilic proteins from the cell wall free mutants was observed whereas PEF treatment on the cell wall containing species resulted in substantially lower protein yields. Additional experiments showed that even at low energy input (0.05 kWh/kgbiomass), still about 70% of the proteins could be released with respect to bead beating as reference. These released proteins were water-soluble while the hydrophobic chlorophyll remained mainly entrapped in cell particles. SEM-analysis of these cell particles showed that PEF only opened the cells, instead of completely fragmenting them into smaller particles. These results indicate that PEF is an energy-efficient cell disruption method for selective release of water-soluble proteins, after the microalgal outer cell wall is removed. Enzymatic pretreatment to degrade the cell walls before PEF treatment was shown to be an efficient method to remove the cell wall.

Optimization of biopharmaceutical downstream processes supported by mechanistic models and artificial neural networks.

Downstream process development is a major area of importance within the field of bioengineering. During the design of such a downstream process, important decisions have to be made regarding the type of unit operations as well as their sequence and their operating conditions. Current computational approaches addressing these issues either show a high level of simplification or struggle with computational speed. Therefore, this article presents a new approach that combines detailed mechanistic models and speed-enhancing artificial neural networks. This approach was able to simultaneously optimize a process with three different chromatographic columns toward yield with a minimum purity of 99.9%. The addition of artificial neural networks greatly accelerated this optimization. Due to high computational speed, the approach is easily extendable to include more unit operations. Therefore, it can be of great help in the acceleration of downstream process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:696-707, 2017.

Characterization of low affinity Fcγ receptor biotinylation under controlled reaction conditions by mass spectrometry and ligand binding analysis.

Chemical protein biotinylation and streptavidin or anti-biotin-based capture is regularly used for proteins as a more controlled alternative to direct coupling of the protein on a biosensor surface. On biotinylation an interaction site of interest may be blocked by the biotin groups, diminishing apparent activity of the protein. Minimal biotinylation can circumvent the loss of apparent activity, but still a binding site of interest can be blocked when labeling an amino acid involved in the binding. Here, we describe reaction condition optimization studies for minimal labeling. We have chosen low affinity Fcγ receptors as model compounds as these proteins contain many lysines in their active binding site and as such provide an interesting system for a minimal labeling approach. We were able to identify the most critical parameters (protein:biotin ratio and incubation pH) for a minimal labeling approach in which the proteins of choice remain most active toward analyte binding. Localization of biotinylation by mass spectrometric peptide mapping on minimally labeled material was correlated to protein activity in binding assays. We show that only aiming at minimal labeling is not sufficient to maintain an active protein. Careful fine-tuning of critical parameters is important to reduce biotinylation in a protein binding site.

3D-liquid chromatography as a complex mixture characterization tool for knowledge-based downstream process development.

Knowledge-based development of chromatographic separation processes requires efficient techniques to determine the physicochemical properties of the product and the impurities to be removed. These characterization techniques are usually divided into approaches that determine molecular properties, such as charge, hydrophobicity and size, or molecular interactions with auxiliary materials, commonly in the form of adsorption isotherms. In this study we demonstrate the application of a three-dimensional liquid chromatography approach to a clarified cell homogenate containing a therapeutic enzyme. Each separation dimension determines a molecular property relevant to the chromatographic behavior of each component. Matching of the peaks across the different separation dimensions and against a high-resolution reference chromatogram allows to assign the determined parameters to pseudo-components, allowing to determine the most promising technique for the removal of each impurity. More detailed process design using mechanistic models requires isotherm parameters. For this purpose, the second dimension consists of multiple linear gradient separations on columns in a high-throughput screening compatible format, that allow regression of isotherm parameters with an average standard error of 8%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1283-1291, 2016.