Moving through three‐dimensional phase diagrams of monoclonal antibodies

Protein phase behavior characterization is a multivariate problem due to the high amount of influencing parameters and the diversity of the proteins. Single influences on the protein are not understood and fundamental knowledge remains to be obtained. For this purpose, a systematic screening method was developed to characterize the influence of fluid phase conditions on the phase behavior of proteins in three‐dimensional phase diagrams. This approach was applied to three monoclonal antibodies to investigate influences of pH, protein and salt concentrations, with five different salts being tested. Although differences exist between the antibodies, this extensive study confirmed the general applicability of the Hofmeister series over the broad parameter range analyzed. The influence of the different salts on the aggregation (crystallization and precipitation) probability was described qualitatively using this Hofmeister series, with a differentiation between crystallization and precipitation being impossible, however. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1103–1113, 2014     

Get closer and make hotspots: liquid–liquid phase separation in plants

Liquid–liquid phase separation (LLPS) facilitates the formation of membraneless compartments in a cell and allows the spatiotemporal organization of biochemical reactions by concentrating macromolecules locally. In plants, LLPS defines cellular reaction hotspots, and stimulus‐responsive LLPS is tightly linked to a variety of cellular and biological functions triggered by exposure to various internal and external stimuli, such as stress responses, hormone signaling, and temperature sensing. Here, we provide an overview of the current understanding of physicochemical forces and molecular factors that drive LLPS in plant cells. We illustrate how the biochemical features of cellular condensates contribute to their biological functions. Additionally, we highlight major challenges for the comprehensive understanding of biological LLPS, especially in view of the dynamic and robust organization of biochemical reactions underlying plastic responses to environmental fluctuations in plants.     

The use of native cation-exchange chromatography to study aggregation and phase separation of monoclonal antibodies

This study introduces a novel analytical approach for studying aggregation and phase separation of monoclonal antibodies (mAbs). The approach is based on using analytical scale cation‐exchange chromatography (CEX) for measuring the loss of soluble monomer in the case of individual and mixed protein solutions. Native CEX outperforms traditional size‐exclusion chromatography in separating complex protein mixtures, offering an easy way to assess mAb aggregation propensity. Different IgG1 and IgG2 molecules were tested individually and in mixtures consisting of up to four protein molecules. Antibody aggregation was induced by four different stress factors: high temperature, low pH, addition of fatty acids, and rigorous agitation. The extent of aggregation was determined from the amount of monomeric protein remaining in solution after stress. Consequently, it was possible to address the role of specific mAb regions in antibody aggregation by co‐incubating Fab and Fc fragments with their respective full‐length molecules. Our results revealed that the relative contribution of Fab and Fc regions in mAb aggregation is strongly dependent on pH and the stress factor applied. In addition, the CEX‐based approach was used to study reversible protein precipitation due to phase separation, which demonstrated its use for a broader range of protein–protein association phenomena. In all cases, the role of Fab and Fc was clearly dissected, providing important information for engineering more stable mAb‐based therapeutics.     

Efficient phase separation and product recovery in organic‐aqueous bioprocessing using supercritical carbon dioxide

Biphasic hydrocarbon functionalizations catalyzed by recombinant microorganisms have been shown to be one of the most promising approaches for replacing common chemical synthesis routes on an industrial scale. However, the formation of stable emulsions complicates downstream processing, especially phase separation. This fact has turned out to be a major hurdle for industrial implementation. To overcome this limitation, we used supercritical carbon dioxide (scCO₂) for both phase separation and product purification. The stable emulsion, originating from a stereospecific epoxidation of styrene to (S)‐styrene oxide, a reaction catalyzed by recombinant Escherichia coli, could be destabilized efficiently and irreversibly, enabling complete phase separation within minutes. By further use of scCO₂ as extraction agent, the product (S)‐styrene oxide could be obtained with a purity of 81% (w/w) in one single extraction step. By combining phase separation and product purification using scCO₂, the number of necessary workup steps can be reduced to one. This efficient and easy to use technique is generally applicable for the workup of biphasic biocatalytic hydrocarbon functionalizations and enables a cost effective downstream processing even on a large scale. Biotechnol. Bioeng. 2010;107:642–651. © 2010 Wiley Periodicals, Inc.     

ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid–liquid phase separation and liquid-to-solid transition

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid–liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis.     

Design and evaluation of a continuous semipartition bioreactor for in situ liquid‐liquid extractive fermentation

Extractive fermentation (or in situ product removal (ISPR)) is an operational method used to combat product inhibition in fermentations. To achieve ISPR, different separation techniques, modes of operation and physical reactor configurations have been proposed. However, the relative paucity of industrial application necessitates continued investigation into reactor systems. This article outlines a bioreactor designed to facilitate in situ product extraction and recovery, through adapting the reaction volume to include a settler and solvent extraction and recycle section. This semipartition bioreactor is proposed as a new mode of operation for continuous liquid‐liquid extractive fermentation. The design is demonstrated as a modified bench‐top fermentation vessel, initially analysed in terms of fluid dynamic studies, in a model two‐liquid phase system. A continuous abiotic simulation of lactic acid (LA) fermentation is then demonstrated. The results show that mixing in the main reaction vessel is unaffected by the inserted settling zone, and that the size of the settling tube effects the maximum volumetric removal rate. In these tests the largest settling tube gave a potential continuous volumetric removal rate of 7.63 ml/min; sufficiently large to allow for continuous product extraction even in a highly productive fermentation. To demonstrate the applicability of the developed reactor, an abiotic simulation of a LA fermentation was performed. LA was added to reactor continuously at a rate of 33ml/h, while continuous in situ extraction removed the LA using 15% trioctylamine in oleyl alcohol. The reactor showed stable LA concentration of 1 g/L, with the balance of the LA successfully extracted and recovered using back extraction. This study demonstrates a potentially useful physical configuration for continuous in situ extraction.     

Myricetin slows liquid–liquid phase separation of Tau and activates ATG5-dependent autophagy to suppress Tau toxicity

Intraneuronal neurofibrillary tangles composed of Tau aggregates have been widely accepted as an important pathological hallmark of Alzheimer''s disease. A current therapeutic avenue for treating Alzheimer''s disease is aimed at inhibiting Tau accumulation with small molecules such as natural flavonoids. Liquid–liquid phase separation (LLPS) of Tau can lead to its aggregation, and Tau aggregates can then be degraded by autophagy. However, it is unclear whether natural flavonoids modulate the formation of phase-separated Tau droplets or promote autophagy and Tau clearance. Here, using confocal microscopy and fluorescence recovery after photobleaching assays, we report that a natural antioxidant flavonoid compound myricetin slows LLPS of full-length human Tau, shifting the equilibrium phase boundary to a higher protein concentration. This natural flavonoid also significantly inhibits pathological phosphorylation and abnormal aggregation of Tau in neuronal cells and blocks mitochondrial damage and apoptosis induced by Tau aggregation. Importantly, using coimmunoprecipitation and Western blotting, we show that treatment of cells with myricetin stabilizes the interaction between Tau and autophagy-related protein 5 (ATG5) to promote clearance of phosphorylated Tau to indirectly limit its aggregation. Consistently, this natural flavonoid inhibits mTOR pathway, activates ATG5-dependent Tau autophagy, and almost completely suppresses Tau toxicity in neuronal cells. Collectively, these results demonstrate how LLPS and abnormal aggregation of Tau are inhibited by natural flavonoids, bridging the gap between Tau LLPS and aggregation in neuronal cells, and also establish that myricetin could act as an ATG5-dependent autophagic activator to ameliorate the pathogenesis of Alzheimer''s disease.     

Small heat shock protein 22 kDa can modulate the aggregation and liquid–liquid phase separation behavior of tau

Alzheimer''s disease is a progressive fatal neurodegenerative disease with no cure or effective treatments. The hallmarks of disease include extracellular plaques and intracellular tangles of aggregated protein. The intracellular tangles consist of the microtubule associated protein tau. Preventing the pathological aggregation of tau may be an important therapeutic approach to treat disease. In this study we show that small heat shock protein 22 kDa (Hsp22) can prevent the aggregation of tau in vitro. Additionally, tau can undergo liquid–liquid phase separation (LLPS) in the presence of crowding reagents which causes it to have an increased aggregation rate. We show that Hsp22 can modulate both the aggregation and LLPS behavior of tau in vitro.     

Hydrophobic benzyl amines as supports for liquid‐phase C‐terminal amidated peptide synthesis: application to the preparation of ABT‐510

C‐terminal amidation is one of the most common modification of peptides and frequently found in bioactive peptides. However, the C‐terminal modification must be creative, because current chemical synthetic techniques of peptides are dominated by the use of C‐terminal protecting supports. Therefore, it must be carried out after the removal of such supports, complicating reaction work‐up and product isolation. In this context, hydrophobic benzyl amines were successfully added to the growing toolbox of soluble tag‐assisted liquid‐phase peptide synthesis as supports, leading to the total synthesis of ABT‐510 ( 2 ). Although an ethyl amide‐forming type was used in the present work, different types of hydrophobic benzyl amines could also be simply designed and prepared through versatile reductive aminations in one step. The standard acidic treatment used in the final deprotection step for peptide synthesis gave the desired C‐terminal secondary amidated peptide with no epimerization. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Structural biology of RNA-binding proteins in the context of phase separation: What NMR and EPR can bring?

Liquid–liquid phase separation of RNA-binding proteins underlies the formation of membraneless organelles, whose composition is dynamic and whose existence may be transient. These organelles are involved in regulation of RNA processing and translation and, if they behave abnormally, in pathologies. Because disorder phenomena are essential in their formation and dynamics, established methodology is insufficient for characterizing their structure. In this review, we consider the current and potential contribution of NMR and EPR spectroscopy to the understanding of structure and dynamics of phase-separating RNA-binding proteins in, both, their dispersed and condensed state in vitro. We discuss which experiments are applicable under what conditions and which information can be obtained from them. Because for these phenomena, the accessible information depends crucially on metastable phase equilibria, we also consider aspects of sample preparation for NMR and EPR experiments.     

Evaluation of a novel methacrylate‐based protein a resin for the purification of immunoglobulins and Fc‐fusion proteins

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc‐fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline‐tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab‐scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014     

Application of an aqueous two‐phase systems high‐throughput screening method to evaluate mAb HCP separation

Aqueous two‐phase systems (ATPSs) as separation technique have regained substantial interest from the biotech industry. Biopharmaceutical companies faced with increasing product titers and stiffening economic competition reconsider ATPS as an alternative to chromatography. As the implementation of an ATPS is material, time, and labor intensive, a miniaturized and automated screening process would be beneficial. In this article such a method, its statistical evaluation, and its application to a biopharmaceutical separation task are shown. To speed up early stage ATPS profiling an automated application of the cloud‐point method for binodal determination was developed. PEG4000–PO₄ binodals were measured automatically and manually and were found to be identical within the experimental error. The ATPS screening procedure was applied to a model system and an industrial separation task. PEG4000–PO₄ systems at a protein concentration of 0.75 mg/mL were used. The influence of pH, NaCl addition, and tie line length was investigated. Lysozyme as model protein, two monoclonal antibodies, and a host cell protein pool were used. The method was found to yield partition coefficients identical to manually determined values for lysozyme. The monoclonal antibodies were shifted from the bottom into the upper phase by addition of NaCl. This shift occurred at lower NaCl concentration when the pH of the system was closer to the pI of the distributed protein. Addition of NaCl, increase in PEG4000 concentration and pH led to significant loss of the mAb due to precipitation. Capacity limitations of these systems were thus demonstrated. The chosen model systems allowed a reduction of up to 50% HCP with a recovery of greater than 95% of the target proteins. As these values might not be industrially relevant when compared to current chromatographic procedures, the developed screening procedure allows a fast evaluation of more suitable and optimized ATPS system for a given task. Biotechnol. Bioeng. 2011; 108:69–81. © 2010 Wiley Periodicals, Inc.