RESUMEN
Displacement chromatography can be used for the purification of many types of chemicals and biochemicals. In the constant-pattern mode, this method can be used for concentrating and separating various mixtures, leading to higher yields and sorbent productivities than elution chromatography. There are, however, no commercial-scale applications of displacement chromatography. One major barrier is the difficulty in specifying the design variables for reaching a constant-pattern state. A second barrier is that productivity is limited when the feed mixture contains a minority component. In this study, a constant-pattern design method is developed to overcome the first barrier. Theoretical analyses and strategically chosen combinations of key dimensionless groups are used for reducing the multi-dimensional parameter space into a two-dimensional space. Systematic rate model simulations are used to find a general correlation, which divides the two-dimensional space into a transient region and a constant-pattern region. A predictive design method based on the general correlation is developed to find the designs for achieving required product purities and yields with the highest productivities. The design method was shown to increase significantly the productivities or yields for the separations of three binary mixtures. To overcome the second barrier, a multi-zone displacement chromatography method is presented for dividing the separation tasks in multiple zones. For a specific ternary mixture containing a minority component, the sorbent productivity for a two-zone design was 680 times higher than that of a single-column design. This method can be used for developing efficient displacement chromatography processes for purifying a wide range of complex mixtures.
Asunto(s)
CromatografíaRESUMEN
In downstream processing, large-scale chromatography plays an important role. For its development, screening experiments followed by pilot-plant chromatography are mandatory steps. Here we describe fast, simple, and inexpensive methods for establishing a preparative chromatography for the separation of complex protein mixtures, based on sample displacement batch chromatography. The methods are demonstrated by anion-exchange chromatography of a human plasma protein fraction (Cohn IV-4), including the screening step and upscaling of the chromatography by a factor of one hundred. The results of the screening experiments and the preparative chromatography are monitored by SDS-PAGE electrophoresis. In summary, we provide a protocol, which should be easily adaptable for the chromatographic large-scale purification of other proteins, in the laboratory as well as in the manufacturing of biopharmaceuticals. These protocols cover the initial piloting steps for establishing a large-scale sample batch chromatography. The results from the piloting steps may also be applied for packed columns for performing simulated-moving-bed (SMB) chromatography rather than batch chromatography.
Asunto(s)
Albúmina Sérica Humana/química , Albúmina Sérica Humana/aislamiento & purificación , Seroglobulinas/química , Seroglobulinas/aislamiento & purificación , Cromatografía por Intercambio Iónico , HumanosRESUMEN
Native forms of therapeutic monoclonal antibodies (mAbs) coexist with various acidic and basic charge variants throughout process development and into drug product formulation. During downstream purification, a product's charge variant composition is controlled, as necessary, primarily through peak fractionation and pooling of elution fractions using cation-exchange chromatography (CEX). This can be a cumbersome process with poor resolution and it may result in a significant reduction in product yield. In the present work, separation and enrichment of the native form of a mAb and of basic and acidic variants is achieved using self-displacement chromatography in a multi-column continuous chromatography set-up. Basic mAb variants are more strongly retained in CEX owing to their higher charge, and can displace the native and the acidic variants. Similarly, the native variant can displace the acidic variants if the amount loaded exceeds the total resin capacity. To this end, we utilized a three-column continuous system to consecutively displace acidic, native and basic charge variants of a therapeutic mAb in the order of increasing binding strength during product loading. Using our optimized operating parameters, we were able to enrich the native variant from 65% to 90% while loading above the capacity of the column, with a process yield of above 90%. This method and approach will help to control and reduce in particular the charged variant heterogeneity, and, in general, aid in the separation of charged proteins at preparative scale.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/aislamiento & purificación , Cationes/química , Cromatografía por Intercambio Iónico/métodos , Anticuerpos Monoclonales/análisis , HumanosRESUMEN
A constant-pattern design method for separating ternary mixtures using ligand-assisted displacement chromatography was developed for non-ideal systems. The general correlation for the minimum column length required to achieve the constant-pattern state for binary separations from our previous study was extended to ternary separations. Additionally, an equation for the yield of a target component as a function of key dimensionless groups was derived based on the constant-pattern mass transfer zone lengths. The column length and operating velocity solved from the two equations ensured the yields and the constant-pattern state for the target components. A selectivity weighted composition factor was developed to allow the design method to specify a minimum target yield for one or multiple components. The design method was verified using simulations and experiments for different targeted yields (70-95%), ligand concentrations (0.03-0.06 M), and feed compositions (1/12-5/6). The targeted yields were achieved or exceeded in all cases tested. The minimum column length required to achieve a constant pattern-state and the productivity of LAD are limited by the lowest selectivity or by a minority component with a low concentration in the feed, even when it does not have the lowest selectivity. Sacrificing the yields of minor components can increase the total productivity significantly. The productivities achieved using this design method are 839 times higher than literature results for ternary separations with the same purity and similar yields.
Asunto(s)
Técnicas de Química Analítica/métodos , Cromatografía , Metales de Tierras Raras/aislamiento & purificación , Técnicas de Química Analítica/instrumentación , Ligandos , Peso MolecularRESUMEN
Ligand-assisted displacement chromatography (LAD) has been developed for separating rare earth elements since the 1950's. Isotachic displacement trains, which are similar to those in conventional displacement chromatography, were reported previously. However, there has been no general theory delineating the conditions required to form constant-pattern displacement trains for non-ideal systems (or systems with significant mass transfer resistance). The constant-pattern state is critical for obtaining pure products with high yield and high productivity. Without theoretical guidance, all the previous studies found the constant-pattern state by experimental trial and error, which was time consuming and costly. In this study, an efficient rate model and simulations of LAD were developed and verified with experimental data for non-ideal systems. Verified simulations were used to understand the mechanisms of separations and the transition from the transient state to the constant-pattern state. The key dimensionless factors affecting the transition for binary non-ideal systems were identified. Dimensionless groups were developed to reduce the number of variables. Simulations were used to find the transition points fromthe transient state to the constant-pattern state, which indicates the minimum dimensionless column lengths in the multi-parameter space. Strategic combination of the key dimensionless groups allows the minimum dimensionless column lengths to correlate with the combined groups in a two-dimensional diagram (or a map). The correlation curve divides the multi-dimensional space into the transient region and the constant-pattern region. The correlation was further verified with five sets of experiments. It can be used to find, without process simulations or experiments, the minimum column lengths for developing constant-pattern isotachic trains for non-ideal systems, which is useful for designing efficient ligand-assisted displacement chromatography at any scale.
Asunto(s)
Cromatografía por Intercambio Iónico , Metales de Tierras Raras/análisis , Concentración de Iones de Hidrógeno , Ligandos , Metales de Tierras Raras/aislamiento & purificación , Modelos Teóricos , Peso Molecular , Neodimio/análisis , Neodimio/aislamiento & purificación , Praseodimio/análisis , Praseodimio/aislamiento & purificaciónRESUMEN
The experiences in the production and application of polymethacrylate-based monolithic supports, since their development almost thirty years ago, are presented. The main driving force for the development of new chromatographic supports was the necessity for the isolation and separation of physiologically active biopolymers and their use for therapeutic purposes. For this sake, a development of a method for fast separation, preventing denaturation and preserving their biological activity was necessary. Development of polysaccharide-based supports, followed by the introduction of polymer-based chromatographic media, is shortly described. This development was followed by the advances in monolithic media that are now used for both large- and small-scale separation of biopolymers and nanoparticles. Finally, a short overview is given about the applications of monoliths for sample displacement chromatography, resulting in isolation of physiologically active biomolecules, such as proteins, protein complexes, and nucleic acid, as well as high-throughput sample preparation for proteomic investigations.
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Biopolímeros , Cromatografía , Enzimas Inmovilizadas , Ácidos Polimetacrílicos , Biopolímeros/análisis , Biopolímeros/química , Biopolímeros/aislamiento & purificación , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , ProteómicaRESUMEN
Affinity chromatography based on amino acids as interacting ligands was already indicated as an alternative compared to ion exchange or hydrophobic interaction for plasmid DNA purification. Understanding the recognition mechanisms occurring between histidine-based ligands and nucleic acids enables more efficient purification of a DNA vaccine, as the binding and elution conditions can be adjusted in order to enhance the purification performance. Decreasing pH to slightly acidic conditions increases the positive charge of histidine ligand, what influences the type of interaction between chromatographic support and analytes. This was proven in this work, where hydrophobic effects established in the presence of ammonium sulfate were affected at pH 5.0 in comparison to pH 8.0, while electrostatic and cation-π interactions were intensified. Histidine ligand at pH 5.0 interacts with phosphate groups or aromatic rings of plasmid DNA. Due to different responses of RNA and pDNA on mobile phase changes, the elution order between RNA and pDNA was changed with mobile phase pH decrease from 8.0 to 5.0. The phenomenon was more evident with L-histidine ligand due to more hydrophilic character, leading to an improved selectivity of L-histidine-modified chromatographic monolith, allowing the product recovery with 99% of purity (RNA removal). With the 1-benzyl- L-histidine ligand, stronger and less selective interactions with the nucleic acids were observed due to the additional hydrophobicity associated with the phenyl aromatic ring. Optimization of sample displacement chromatography parameters (especially (NH4 )2 SO4 concentration) at slightly acidic pH enabled excellent isolation of pDNA, by the removal of RNA in a negative mode, with binding capacities above 1.5 mg pDNA per mL of chromatographic support.
Asunto(s)
Cromatografía de Afinidad/métodos , Histidina/química , Proteínas Oncogénicas Virales/genética , Proteínas E7 de Papillomavirus/genética , Vacunas contra Papillomavirus/aislamiento & purificación , Plásmidos/aislamiento & purificación , Proteínas Represoras/genética , Vacunas de ADN/aislamiento & purificación , ADN/aislamiento & purificación , Histidina/metabolismo , Ligandos , Vacunas contra Papillomavirus/genética , Plásmidos/genética , Vacunas de ADN/genéticaRESUMEN
A model based on mass transfer resistances and acid-base equilibriums at the liquid-liquid interface was developed for the pH-zone refining mode when it is used in countercurrent chromatography (CCC). The binary separation of catharanthine and vindoline, two alkaloids used as starting material for the semi-synthesis of chemotherapy drugs, was chosen for the model validation. Toluene/CH3CN/water (4/1/5, v/v/v) was selected as biphasic solvent system. First, hydrodynamics and mass transfer were studied by using chemical tracers. Trypan blue only present in the aqueous phase allowed the determination of the parameters τextra and Pe for hydrodynamic characterization whereas acetone, which partitioned between the two phases, allowed the determination of the transfer parameter k0a. It was shown that mass transfer was improved by increasing both flow rate and rotational speed, which is consistent with the observed mobile phase dispersion. Then, the different transfer parameters of the model (i.e. the local transfer coefficient for the different species involved in the process) were determined by fitting experimental concentration profiles. The model accurately predicted both equilibrium and dynamics factors (i.e. local mass transfer coefficients and acid-base equilibrium constant) variation with the CCC operating conditions (cell number, flow rate, rotational speed and thus stationary phase retention). The initial hypotheses (the acid-base reactions occurs instantaneously at the interface and the process is mainly governed by mass transfer) are thus validated. Finally, the model was used as a tool for catharanthine and vindoline separation prediction in the whole experimental domain that corresponded to a flow rate between 20 and 60 mL/min and rotational speeds from 900 and 2100 rotation per minutes.
Asunto(s)
Distribución en Contracorriente/métodos , Hidrodinámica , Concentración de Iones de Hidrógeno , Solventes , Vinblastina/análogos & derivados , Vinblastina/aislamiento & purificación , Alcaloides de la Vinca/aislamiento & purificaciónRESUMEN
The chromatographic separation of two proteins into a displacement train of two adjoined rectangular bands was accomplished using a novel method for hydrophobic charge induction chromatography (HCIC) which employs a self-sharpening pH front as the displacer. This method exploits the fact that protein elution in HCIC is promoted by a pH change, but is relatively independent of salt effects, so that a retained pH front can be used in place of a traditional displacer in displacement chromatography. The retained pH front was produced using the two adsorbed buffering species tricine and acetic acid. The separation of lysozyme and α-chymotrypsinogen A into adjoined, rectangular bands was accomplished with overall recoveries based on the total mass injected greater than 90 and 70%, respectively. The addition of urea to the buffer system increased the sharpness of the pH front by 36% while the yields of lysozyme and α-chymotrypsinogen A based on the total mass eluted increased from 76% to 99% and from 37% to 85%, respectively, when the purities of both proteins in their product fractions were fixed at 85%. The results demonstrate that the method developed in this study is a useful variant of HCIC and is also a useful alternative to other displacement chromatography methods.
Asunto(s)
Cromatografía Liquida , Interacciones Hidrofóbicas e Hidrofílicas , Extracción Líquido-Líquido/métodos , Proteínas/química , Adsorción , Quimotripsinógeno/análisis , Quimotripsinógeno/aislamiento & purificación , Concentración de Iones de Hidrógeno , Muramidasa/análisis , Muramidasa/aislamiento & purificación , Proteínas/aislamiento & purificación , Fuerza Protón-MotrizRESUMEN
Hydrophobic interaction chromatography (HIC) offers an orthogonal selectivity to ion exchange chromatography and the combination of the two processes can provide a potential cost-effective alternative to protein A chromatography in industrial antibody purification. However, the application of HIC is limited by its close dependence on high concentrations of kosmotropic salts to achieve desired separation. These salts can cause antibody precipitation and induce the corrosion of manufacturing facilities. Here, we report a new strategy of salt-independent HIC, which can capture antibody at the physiological salt concentration and allow the recovery of bound proteins through cyclodextrin (CD)-based displacement elution. Hydrophobicity-intensified HIC media with different coupling amount of phenyl ligands were prepared and assessed for their antibody binding capacity and selectivity. ß-CD was investigated for its supermolecular interaction with phenyl ligands and elution capacity as a displacer. The results clarified a nearly linear correlation between binding capacity of human immunoglobulin G (IgG) and phenyl coupling density in the range of 44-159 µmol/mL. The host-guest interaction between ß-CD and the phenyl ligands revealed a modest binding strength (Ka=4.1×10(3) M(-1)), and 15 mM ß-CD solution showed a general effectiveness as displacement eluent for these HIC media, with IgG recovery varying with the ligand density. This strategy allowed the direct purification of human IgG from serum with satisfactory purity. The whole procedure of this method, including loading and elution, can be performed under physiological conditions. We expect such a salt-independent mode of HIC could be used as a capture or intermediate step in industrial antibody purification.
Asunto(s)
Ciclodextrinas/química , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina G/aislamiento & purificación , Cromatografía/métodos , Humanos , Ligandos , Sales (Química)/químicaRESUMEN
SIRT6 is a histone deacetylase that has been proposed as a potential therapeutic target for metabolic disorders and the prevention of age-associated diseases. We have previously reported on the identification of quercetin and vitexin as SIRT6 inhibitors, and studied structurally related flavonoids including luteolin, kaempferol, apigenin and naringenin. It was determined that the SIRT6 protein remained active after immobilization and that a single frontal displacement could correctly predict the functional activity of the immobilized enzyme. The previous study generated a preliminary pharmacophore for the quercetin binding site on SIRT6, containing 3 hydrogen bond donors and one hydrogen bond acceptor. In this study, we have generated a refined pharmacophore with an additional twelve quercetin analogs. The resulting model had a positive linear behavior between the experimental elution time verses the fit values obtained from the model with a correlation coefficient of 0.8456.