RESUMEN
Biosurfactants are surface-active molecules produced principally by microorganisms. They are a sustainable alternative to chemically-synthesized surfactants, having the advantages of being non-toxic, highly functional, eco-friendly and biodegradable. However they are currently only used in a few industrial products due to costs associated with production and purification, which exceed those for commodity chemical surfactants. DAMP4, a member of a four-helix bundle biosurfactant protein family, can be produced in soluble form and at high yield in Escherichia coli, and can be recovered using a facile thermal phase-separation approach. As such, it encompasses an interesting synergy of biomolecular and chemical engineering with prospects for low-cost production even for industrial sectors. DAMP4 is highly functional, and due to its extraordinary thermal stability it can be purified in a simple two-step process, in which the combination of high temperature and salt leads to denaturation of all contaminants, whereas DAMP4 stays stable in solution and can be recovered by filtration. This study aimed to characterize and understand the fundamental drivers of DAMP4 stability to guide further process and surfactant design studies. The complementary use of experiments and molecular dynamics simulation revealed a broad pH and temperature tolerance for DAMP4, with a melting point of 122.4 °C, suggesting the hydrophobic core as the major contributor to thermal stability. Simulation of systematically created in silico variants of DAMP4 showed an influence of number and location of hydrophilic mutations in the hydrophobic core on stability, demonstrating a tolerance of up to three mutations before a strong loss in stability occurred. The results suggest a consideration of a balance of stability, functionality and kinetics for new designs according to their application, aiming for maximal functionality but at adequate stability to allow for cost-efficient production using thermal phase separation approaches.
Asunto(s)
Simulación de Dinámica Molecular , Proteínas Recombinantes/química , Tensoactivos/química , Secuencia de Aminoácidos , Rastreo Diferencial de Calorimetría , Dicroismo Circular , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Conformación Proteica , Desnaturalización Proteica , Ingeniería de Proteínas/métodos , Estabilidad Proteica , Proteínas Recombinantes/metabolismo , Homología Estructural de ProteínaRESUMEN
Oxidative stress can lead to T cell hyporesponsiveness. A reducing micromilieu (e.g. provided by dendritic cells) can rescue T cells from such oxidant-induced dysfunction. However, the reducing effects on proteins leading to restored T cell activation remained unknown. One key molecule of T cell activation is the actin-remodeling protein cofilin, which is dephosphorylated on serine 3 upon T cell costimulation and has an essential role in formation of mature immune synapses between T cells and antigen-presenting cells. Cofilin is spatiotemporally regulated; at the plasma membrane, it can be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show by NMR spectroscopy that a reducing milieu led to structural changes in the cofilin molecule predominantly located on the protein surface. They overlapped with the PIP2- but not actin-binding sites. Accordingly, reduction of cofilin had no effect on F-actin binding and depolymerization and did not influence the cofilin phosphorylation state. However, it did prevent inhibition of cofilin activity through PIP2. Therefore, a reducing milieu may generate an additional pool of active cofilin at the plasma membrane. Consistently, in-flow microscopy revealed increased actin dynamics in the immune synapse of untransformed human T cells under reducing conditions. Altogether, we introduce a novel mechanism of redox regulation: reduction of the actin-remodeling protein cofilin renders it insensitive to PIP2 inhibition, resulting in enhanced actin dynamics.
Asunto(s)
Factores Despolimerizantes de la Actina/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Linfocitos T/metabolismo , Factores Despolimerizantes de la Actina/química , Factores Despolimerizantes de la Actina/genética , Actinas/metabolismo , Western Blotting , Línea Celular Tumoral , Células Cultivadas , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Oxidación-Reducción , Fosfatidilinositol 4,5-Difosfato/química , Fosforilación , Polimerizacion , Unión Proteica , Conformación Proteica , Estructura Terciaria de ProteínaRESUMEN
Plasmid DNA (pDNA) is among the promising gene delivery vehicles currently evaluated for gene therapy. The large-scale production of pDNA for pharmaceutical application necessitates purification steps with a high capacity and good separation of RNA from pDNA. Most commonly used process step in the production of biopharmaceutical, namely the divers modes of chromatography, fail as they offer too limited a capacity for the considerably larger pDNA molecules. Alternative separation steps might thus be beneficial. One such separation step, aqueous two-phase extraction (ATPE) has previously been shown to work well for the purification of pDNA. The application of such a process step is however hampered by the large amount of material and time that goes into its development. In this publication, we demonstrate the use of an automatic, miniaturized ATPE screening system to the separation of pDNA from RNA. Two optimization strategies are presented: response surface methodology and genetic algorithms. Using a fully automated optimization strategy, we derived promising conditions that were scale-up tenfold. The resulting purity and recovery surpassed previously published results demonstrating that a complex optimization task such as ATPE demands an appropriately complex optimization routine.
Asunto(s)
Fraccionamiento Químico/métodos , ADN/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Plásmidos/aislamiento & purificación , Contaminación de ADNRESUMEN
Optimization of protein refolding parameters by automated, miniaturized, and parallelized high throughput screening is a powerful approach to meet the demand for fast process development with low material consumption. In this study, we validated methods applicable on a standard liquid handling robot for screening of refolding process parameters by dilution of denatured lysozyme in refolding buffer systems. Different approaches for the estimation of protein solubility and folding were validated concerning resolution and compatibility with the robotic system and with the complex buffer and protein structure composition. We established an indirect method to assess soluble lysozyme concentration independent of matrix effects and protein structure varieties by automated separation of aggregated protein, resolubilization, and measurement of absorption at 280 nm. Using this nonspecific solubility assays the correlation between favorable parameters for high active and soluble lysozyme yields were evaluated. An overlap of good refolding buffer compositions was found provided that the redox environment was controlled with redox reagents. In addition, the need to control unfolding conditions like time, temperature, lysozyme, and dithiothreitol concentration was pointed out as different feedstocks resulted in different refolding yields.
Asunto(s)
Automatización/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Muramidasa/química , Animales , Pollos , Oxidación-Reducción , Conformación Proteica , Pliegue de Proteína , Replegamiento Proteico , Solubilidad , TemperaturaRESUMEN
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-PO(4) 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-PO(4) 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.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Fraccionamiento Químico/métodos , Ensayos Analíticos de Alto Rendimiento , Polietilenglicoles/química , Proteínas Recombinantes/aislamiento & purificación , Soluciones/químicaRESUMEN
The development and manufacturing of safe and effective vaccines relies essentially on the availability of robust and precise analytical techniques. Virus-like particles (VLPs) have emerged as an important and valuable class of vaccines for the containment of infectious diseases. VLPs are produced by recombinant protein expression followed by purification procedures to minimize the levels of process- and product-related impurities. The control of these impurities is necessary during process development and manufacturing. Especially monitoring of the VLP size distribution is important for the characterization of the final vaccine product. Currently used methods require long analysis times and tailor-made assays. In this work, we present a size-exclusion ultra-high performance liquid chromatography (SE-UHPLC) method to characterize VLPs and quantify aggregates within 3.1min per sample applying interlaced injections. Four analytical SEC columns were evaluated for the analysis of human B19 parvo-VLPs and murine polyoma-VLPs. The optimized method was successfully used for the characterization of five recombinant protein-based VLPs including human papillomavirus (HPV) VLPs, human enterovirus 71 (EV71) VLPs, and chimeric hepatitis B core antigen (HBcAg) VLPs pointing out the generic applicability of the assay. Measurements were supported by transmission electron microscopy and dynamic light scattering. It was demonstrated that the iSE-UHPLC method provides a rapid, precise and robust tool for the characterization of VLPs. Two case studies on purification tools for VLP aggregates and storage conditions of HPV VLPs highlight the relevance of the analytical method for high-throughput process development and process monitoring of virus-like particles.
Asunto(s)
Cromatografía en Gel , Ensayos Analíticos de Alto Rendimiento , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Cromatografía Líquida de Alta Presión , Enterovirus Humano A/inmunología , Antígenos del Núcleo de la Hepatitis B/inmunología , Parvovirus B19 Humano/aislamiento & purificación , Poliomavirus/aislamiento & purificación , Proteínas Recombinantes/inmunologíaRESUMEN
Hydrophobic interaction chromatography (HIC) is one of the most frequently used purification methods in downstream processing of biopharmaceuticals. During HIC, salts are the governing additives contributing to binding strength, binding capacity, and protein solubility in the liquid phase. A relatively recent approach to increase the dynamic binding capacity (DBC) of HIC adsorbers is the use of salt mixtures. By mixing chaotropic with kosmotropic salts, the DBC can strongly be influenced. For salt mixtures with a higher proportion of chaotropic than kosmotropic salt, higher DBCs were achieved compared with single salt approaches. By measuring the surface tensions of the protein salt solutions, the cavity theory-proposed by Melander and Horváth-that higher surface tensions lead to higher DBCs, was found to be invalid for salt mixtures. Aggregation temperatures of lysozyme in the salt mixtures, as a degree of hydrophobic forces, were correlated to the DBCs. Measuring the aggregation temperatures has proven to be a fast analytical methodology to estimate the hydrophobic interactions and thus can be used as a measure for an increase or decrease in the DBCs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:346-354, 2016.
Asunto(s)
Sales (Química)/química , Sales (Química)/metabolismo , Tensión Superficial , Temperatura , Adsorción , Interacciones Hidrofóbicas e Hidrofílicas , Muramidasa/química , Muramidasa/metabolismo , Espectrofotometría UltravioletaRESUMEN
High-throughput screening (HTS) technology is gaining increasing importance in downstream process development of cell-based products. The development of such HTS-technologies, however, is highly dependent on the availability of robust, accurate, and sensitive high-throughput cell quantification methods. In this article, we compare state-of-the-art cell quantification methods with focus on their applicability in HTS-platforms for downstream processing of cell-based products. Sensitivity, dynamic range, and precision were evaluated for four methods that differ in their respective mechanism. In addition, we evaluated the performance of these methods over a range of buffer compositions, medium densities, and viscosities, representing conditions found in many downstream processing methods. We found that CellTiter-Glo™ and flow cytometry are excellent tools for high-throughput cell quantification. Both methods have broad working ranges (3-4 log) and performed well over a wide range of buffer compositions. In comparison, CyQuant® Direct and CellTracker™ had smaller working ranges and were more sensitive to changes in buffer composition. For fast and sensitive quantification of a single cell type, CellTiter-Glo™ performed best, while for more complex cell mixtures flow cytometry is the method of choice. Our analysis will facilitate the selection of the most suitable method for a specific application and provides a benchmark for future HTS development in downstream processing of cell-based products.
Asunto(s)
Citometría de Flujo/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Proliferación Celular , Supervivencia Celular , Células HL-60 , HumanosRESUMEN
As the clinical development of cell-based therapeutics has evolved immensely within the past years, downstream processing strategies become more relevant than ever. Aqueous two-phase systems (ATPS) enable the label-free, scalable, and cost-effective separation of cells, making them a promising tool for downstream processing of cell-based therapeutics. Here, we report the development of an automated robotic screening that enables high-throughput cell partitioning analysis in ATPS. We demonstrate that this setup enables fast and systematic investigation of factors influencing cell partitioning. Moreover, we examined and optimized separation conditions for the differentiable promyelocytic cell line HL-60 and used a counter-current distribution-model to investigate optimal separation conditions for a multi-stage purification process. Finally, we show that the separation of CD11b-positive and CD11b-negative HL-60 cells is possible after partial DMSO-mediated differentiation towards the granulocytic lineage. The modeling data indicate that complete peak separation is possible with 30 transfers, and >93% of CD11b-positive HL-60 cells can be recovered with >99% purity. The here described screening platform facilitates faster, cheaper, and more directed downstream process development for cell-based therapeutics and presents a powerful tool for translational research.
Asunto(s)
Separación Celular/métodos , Células/química , Separación Celular/instrumentación , Tratamiento Basado en Trasplante de Células y Tejidos , Células/citología , Humanos , Polietilenglicoles/químicaRESUMEN
Recombinant protein-based virus-like particles (VLPs) are steadily gaining in importance as innovative vaccines against cancer and infectious diseases. Multiple VLPs are currently evaluated in clinical phases requiring a straightforward and rational process design. To date, there is no generic platform process available for the purification of VLPs. In order to accelerate and simplify VLP downstream processing, there is a demand for novel development approaches, technologies, and purification tools. Membrane adsorbers have been identified as promising stationary phases for the processing of bionanoparticles due to their large pore sizes. In this work, we present the potential of two strategies for designing VLP processes following the basic tenet of 'quality by design': High-throughput experimentation and process modeling of an anion-exchange membrane capture step. Automated membrane screenings allowed the identification of optimal VLP binding conditions yielding a dynamic binding capacity of 5.7 mg/mL for human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A mechanistic approach was implemented for radial ion-exchange membrane chromatography using the lumped-rate model and stoichiometric displacement model for the in silico optimization of a VLP capture step. For the first time, process modeling enabled the in silico design of a selective, robust and scalable process with minimal experimental effort for a complex VLP feedstock. The optimized anion-exchange membrane chromatography process resulted in a protein purity of 81.5%, a DNA clearance of 99.2%, and a VLP recovery of 59%.
Asunto(s)
Cromatografía por Intercambio Iónico , Simulación por Computador , Modelos Biológicos , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Virología/métodos , Animales , Proteínas Recombinantes/genética , Células Sf9RESUMEN
The demand for vaccines against untreated diseases has enforced the research and development of virus-like particle (VLP) based vaccine candidates in recent years. Significant progress has been made in increasing VLP titres during upstream processing in bacteria, yeast and insect cells. Considering downstream processing, the separation of host cell impurities is predominantly achieved by time-intensive ultracentrifugation processes or numerous chromatography and filtration steps. In this work, we evaluate the potential of an alternative separation technology for VLPs: aqueous two-phase extraction (ATPE). The benefits of ATPE have been demonstrated for various biomolecules, but capacity and separation efficiency were observed to be low for large biomolecules such as VLPs or viruses. Both performance parameters were examined in detail in a case study on human B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A solubility-guided approach enabled the design of polyethylene (PEG) salt aqueous two-phase systems with a high capacity of up to 4.1mg/mL VLPs. Unique separation efficiencies were obtained by varying the molecular weight of PEG, the pH value and by using neutral salt additives. Further improvement of the separation of host cell impurities was achieved by multi-stage ATPE on a centrifugal partition chromatography (CPC) device in 500mL scale. While single-stage ATPE enabled a DNA clearance of 99.6%, multi-stage ATPE improved the separation of host cell proteins (HCPs). The HPLC purity ranged from 16.8% (100% VLP recovery) for the single-stage ATPE to 69.1% (40.1% VLP recovery) for the multi-stage ATPE. An alternative two-step downstream process is presented removing the ATPS forming polymer, cell debris and 99.77% DNA with a HPLC purity of 90.6% and a VLP recovery of 63.9%.
Asunto(s)
Vacunas de Partículas Similares a Virus/análisis , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Virología/métodos , Animales , Proteínas de la Cápside/análisis , Centrifugación , Cromatografía Líquida de Alta Presión , ADN/aislamiento & purificación , Humanos , Concentración de Iones de Hidrógeno , Parvovirus B19 Humano/metabolismo , Polietilenglicoles/química , Células Sf9/citología , Células Sf9/metabolismo , Cloruro de Sodio/química , Solubilidad , Spodoptera , Vacunas de Partículas Similares a Virus/metabolismoRESUMEN
Knowledge of protein phase behavior is essential for downstream process design in the biopharmaceutical industry. Proteins can either be soluble, crystalline or precipitated. Additionally liquid-liquid phase separation, gelation and skin formation can occur. A method to generate phase diagrams in high throughput on an automated liquid handling station in microbatch scale was developed. For lysozyme from chicken egg white, human lysozyme, glucose oxidase and glucose isomerase phase diagrams were generated at four different pH values pH 3, 5, 7 and 9. Sodium chloride, ammonium sulfate, polyethylene glycol 300 and polyethylene glycol 1000 were used as precipitants. Crystallizing conditions could be found for lysozyme from chicken egg white using sodium chloride, for human lysozyme using sodium chloride or ammonium sulfate and glucose isomerase using ammonium sulfate. PEG caused destabilization of human lysozyme and glucose oxidase solutions or a balance of stabilizing and destabilizing effects for glucose isomerase near the isoelectric point. This work presents a systematic generation and extensive study of phase diagrams of proteins. Thus, it adds to the general understanding of protein behavior in liquid formulation and presents a convenient methodology applicable to any protein solution.
Asunto(s)
Isomerasas Aldosa-Cetosa/química , Sulfato de Amonio/química , Glucosa Oxidasa/química , Muramidasa/química , Polietilenglicoles/química , Cloruro de Sodio/química , Precipitación Química , Cristalización , Concentración de Iones de Hidrógeno , SolucionesRESUMEN
Protein drugs continue to grow both in medicinal importance as in scale of their production. This furthers the interest in separation technologies that have the potential to replace chromatographic steps in a protein purification process. Two such unit operations that are employed in large scale in the chemical industry are extraction and precipitation. Their usefulness for the purification of proteins has been demonstrated, but the integration of such unit operations in a way that generate an output stream of high protein concentration and low process related impurities was missing. In this work, we employ centrifugal partitioning chromatography ('CPC') in combination with precipitation of the protein of interest to purify a cell culture supernatant of a monoclonal antibody producing cell line. Centrifugal partitioning chromatography was used as means of multi-step extraction using aqueous two-phase systems and was able to remove up to 88.2% of host cell protein ('HCP'). The following PEG driven precipitation and resolubilization of the protein of interest was use to condition the CPC output stream to suit subsequent chromatographic steps, to increase mAb concentration, remove the phase forming polymer, further improve HCP clearance, and integrate a low pH hold step for viral clearance. The entire process reduced HCP content by 99.4% while recovering 93% of the protein of interest. High throughput screening techniques were extensively employed during the development of the process.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía Liquida/métodos , Automatización , Línea Celular , Centrifugación , Solubilidad , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
BACKGROUND: Molecular Dynamics (MD) simulations are a promising tool to generate molecular understanding of processes related to the purification of proteins. Polyethylene glycols (PEG) of various length are commonly used in the production and purification of proteins. The molecular mechanisms behind PEG driven precipitation, aqueous two-phase formation or the effects of PEGylation are however still poorly understood. RESULTS: In this paper, we ran MD simulations of single PEG molecules of variable length in explicitly simulated water. The resulting structures are in good agreement with experimentally determined 3D structures of PEG. The increase in surface hydrophobicity of PEG of longer chain length could be explained on an atomic scale. PEG-water interactions as well as aqueous two-phase formation in the presence of PO4 were found to be correlated to PEG surface hydrophobicity. CONCLUSIONS: We were able to show that the taken MD simulation approach is capable of generating both structural data as well as molecule descriptors in agreement with experimental data. Thus, we are confident of having a good in silico representation of PEG.
RESUMEN
High throughput process development (HTPD), the application of miniaturized and automated experiments for the development of protein production and purification processes, has been established over the last decade and has found its way into most process development group in the pharmaceutical industry. The first installment of the HTPD meeting in 2010 had been a huge success in bringing together people from academia and industry dedicated to this topic. With its second occasion, the scope of the meeting was broadened while the quality of the contributions and the balance between academic and industrial topics was kept at the excellently high quality underlining this meetings importance and singular position for the field of HTPD.
Asunto(s)
Biotecnología/métodos , Ensayos Analíticos de Alto Rendimiento/métodosRESUMEN
Aqueous two-phase systems have been demonstrated to be a possible alternative to chromatographic separations during the industrial purification of proteins. While convenient high throughput screening methods were shown to drastically reduce experimental effort for the evaluations of ATPS as a unit operation, the selection of which phases to investigate is currently guided largely by prior knowledge. Correlations between protein descriptors and distribution were found, but the general applicability of such correlations especially under conditions of high protein load, is questionable, as currently no correlations take the saturation of the phases with protein into account. In this manuscript, we demonstrate how precipitation experiments using the phase forming components can guide the selection of both system type and tieline length for the purification of monoclonal antibodies. Phase selection and process development time can thus be significantly reduced, as all the necessary precipitation, binodal, and tieline experiments can be conducted within one day. Good qualitative correlations between precipitation data and both distribution and recovery of the target molecule were found. Most promising systems were selected for upscale to a 500mL CPC. Influence of operation condition on the column and on HCP clearance was investigated. An increase in HCP clearance of more than threefold compared to batch extractions was observed. The importance of load protein concentration underlined the value of using a screening approach that incorporated target protein solubility data.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Centrifugación/métodos , Cromatografía Liquida/métodos , Extracción Líquido-Líquido/métodos , Animales , Anticuerpos Monoclonales/química , Células CHO , Precipitación Química , Cricetinae , Cricetulus , Ensayos Analíticos de Alto Rendimiento , SolubilidadRESUMEN
In process development and during commercial production of monoclonal antibodies (mAb) the monitoring of aggregate levels is obligatory. The standard assay for mAb aggregate quantification is based on size exclusion chromatography (SEC) performed on a HPLC system. Advantages hereof are high precision and simplicity, however, standard SEC methodology is very time consuming. With an average throughput of usually two samples per hour, it neither fits to high throughput process development (HTPD), nor is it applicable for purification process monitoring. We present a comparison of three different SEC columns for mAb-aggregate quantification addressing throughput, resolution, and reproducibility. A short column (150 mm) with sub-two micron particles was shown to generate high resolution (~1.5) and precision (coefficient of variation (cv)<1) with an assay time below 6 min. This column type was then used to combine interlaced sample injections with parallelization of two columns aiming for an absolute minimal assay time. By doing so, both lag times before and after the peaks of interest were successfully eliminated resulting in an assay time below 2 min. It was demonstrated that determined aggregate levels and precision of the throughput optimized SEC assay were equal to those of a single injection based assay. Hence, the presented methodology of parallel interlaced SEC (PI-SEC) represents a valuable tool addressing HTPD and process monitoring.