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.

Label-free glycoprofiling with multiplex surface plasmon resonance: a tool to quantify sialylation of erythropoietin.

Protein glycosylation is among the most common and well-defined post-translational modifications due to its vital role in protein function. Monitoring variation in glycosylation is necessary for producing more effective therapeutic proteins. Glycans attached to glycoproteins interact highly specific with lectins, natural carbohydrate-binding proteins, which property is used in the current label-free methodology. We have established a lectin microarray for label-free detection of lectin-carbohydrate interactions allowing us to study protein glycosylation directly on unmodified glycoproteins. The method enables simultaneous measurement of up to 96 lectin-carbohydrate interactions on a multiplex surface plasmon resonance imaging platform within 20 min. Specificity determination of lectins succeeded by analysis of neoglycoproteins and enzymatically remodeled glycoproteins to verify carbohydrate binding. We demonstrated the possibilities for glycosylation fingerprinting by comparing different Erythropoietin sources without the need for any sample pretreatment and we were able to accurately quantify relative sialylation levels of Erythropoietin.

High-throughput and multiplexed regeneration buffer scouting for affinity-based interactions.

Affinity-based analyses on biosensors depend partly on regeneration between measurements. Regeneration is performed with a buffer that efficiently breaks all interactions between ligand and analyte while maintaining the active binding site of the ligand. We demonstrated a regeneration buffer scouting using the combination of a continuous flow microspotter with a surface plasmon resonance imaging platform to simultaneously test 48 different regeneration buffers on a single biosensor. Optimal regeneration conditions are found within hours and consume little amounts of buffers, analyte, and ligand. This workflow can be applied to any ligand that is coupled through amine, thiol, or streptavidin immobilization.

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.

Quantitative proteomics reveals distinct differences in the protein content of outer membrane vesicle vaccines.

At present, only vaccines containing outer membrane vesicles (OMV) have successfully stopped Neisseria meningitidis serogroup B epidemics. These vaccines however require detergent-extraction to remove endotoxin, which changes immunogenicity and causes production difficulties. To investigate this in more detail, the protein content of detergent-extracted OMV is compared with two detergent-free alternatives. A novel proteomics strategy has been developed that allows quantitative analysis of many biological replicates despite inherent multiplex restrictions of dimethyl labeling. This enables robust statistical analysis of relative protein abundance. The comparison with detergent-extracted OMV reveales that detergent-free OMV are enriched with membrane (lipo)proteins and contain less cytoplasmic proteins due to a milder purification process. These distinct protein profiles are substantiated with serum blot proteomics, confirming enrichment with immunogenic proteins in both detergent-free alternatives. Therefore, the immunogenic protein content of OMV vaccines depends at least partially on the purification process. This study demonstrates that detergent-free OMV have a preferred composition.

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.

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.

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.

Multi-dimensional fractionation and characterization of crude protein mixtures: toward establishment of a database of protein purification process development parameters.

A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size-exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation.

Alternative affinity tools: more attractive than antibodies?

Antibodies are the most successful affinity tools used today, in both fundamental and applied research (diagnostics, purification and therapeutics). Nonetheless, antibodies do have their limitations, including high production costs and low stability. Alternative affinity tools based on nucleic acids (aptamers), polypeptides (engineered binding proteins) and inorganic matrices (molecular imprinted polymers) have received considerable attention. A major advantage of these alternatives concerns the efficient (microbial) production and in vitro selection procedures. The latter approach allows for the high-throughput optimization of aptamers and engineered binding proteins, e.g. aiming at enhanced chemical and physical stability. This has resulted in a rapid development of the fields of nucleic acid- and protein-based affinity tools and, although they are certainly not as widely used as antibodies, the number of their applications has steadily increased in recent years. In the present review, we compare the properties of the more conventional antibodies with these innovative affinity tools. Recent advances of affinity tool developments are described, both in a medical setting (e.g. diagnostics, therapeutics and drug delivery) and in several niche areas for which antibodies appear to be less attractive. Furthermore, an outlook is provided on anticipated future developments.

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.

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.

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.

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.

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.

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.

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.

Selection of pH-related parameters in ion-exchange chromatography using pH-gradient operations.

This work demonstrates that the type of ion-exchanger (anion or cation), the mode of operation (bind-and-elute or flow-through), and the operational pH of ion-exchange chromatography (IEX) can be selected in a fast and rational way by analytical pH-gradient IEX operations, thereby eliminating the need for pH scouting or high-throughput screening. The developed approach was applied for the selection of an IEX process for the capture of a monoclonal antibody (MAb) from hybridoma cell culture supernatant (CCS). It was found within a day that MAb can optimally be captured by bind-and-elute mode cation-exchange chromatography (CEX) at pH 4.5 or anion-exchange chromatography (AEX) at pH 7.2 without lowering the salt concentration in the CCS. The performance of both CEX and AEX was predicted to be equal for this particular MAb capture.

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.