RESUMEN
This paper employs a high-throughput parallel batch (microtiter plate) adsorption screen with sequential salt step increases to rapidly generate protein elution profiles for multiple resins at different pHs using a protein library. The chromatographic set used in this work includes single mode, multimodal anion-exchange (MMA), and multimodal cation-exchange (MMC) resins. The protein library consists of proteins with isoelectric points ranging from 5.1 to 11.4 with varying hydrophobicities as determined by their retention on hydrophobic interaction chromatography. The batch sequential experiments are carried out using one protein at a time with a wide set of resins at multiple pH conditions, thus enabling simple microtiter plate detection. A mathematical formulation is then used to determine the first moment of the distributions from each chromatogram (sequential step elution) generated in the parallel batch experiments. Batch data first moments (expressed in salt concentration) are then compared to results obtained from column linear salt gradient elution, and the techniques are shown to be consistent. In addition, first moment data are used to calculate one-resin separability scores, which are a measure of a resin's ability, at a specified pH, to separate the entire set of proteins in the library from one another. Again, the results from the batch and column experiments are shown to be comparable. The first moment data sets were then employed to calculate the two-resin separability scores, which are a measure of the ability of two resins to synergistically separate the entire set of proteins in the library. Importantly, these results based on the two-resin separability performances derived from the batch and column experiments were again shown to be consistent. This approach for rapidly screening large numbers of chromatographic resins and mobile phase conditions for their elution behavior may prove useful for enabling the rapid discovery of new chromatographic ligands and resins.
Asunto(s)
Interacciones Hidrofóbicas e Hidrofílicas , Proteínas , Cromatografía por Intercambio Iónico/métodos , Concentración de Iones de Hidrógeno , Proteínas/análisis , Proteínas/química , Proteínas/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/métodos , Resinas de Intercambio de Catión/química , Resinas de Intercambio Aniónico/química , Adsorción , Punto IsoeléctricoRESUMEN
In this paper, we examined the chromatographic behavior of a new class of guanidine-based multimodal anion exchange resins. The selectivities and protein recoveries on these resins were first evaluated using linear gradient chromatography with a model acidic protein library at pH 5, 6 and 7. While a single-guanidine based resin exhibited significant recovery issues at high ligand density, a bis-guanidine based resin showed high recoveries of all but two of the proteins evaluated in the study. In addition, the bis-guanidine resin showed a more pH dependent selectivity pattern as compared to the low density single-guanidine resin. The salt elution range for the low density single-guanidine and bis-guanidine resins was also observed to vary from 0.250 to 0.621 M and 0.162 to 0.828 M NaCl, respectively. A QSAR model was then developed to predict the elution behavior of these proteins on the guanidine prototypes at multiple pH with overall training and test scores of 0.88 and 0.85, respectively. In addition, molecular dynamics simulations were performed with these ligands immobilized on a self-assembled monolayer (SAM) to characterize their conformational preferences and to gain insight into the molecular basis of their chromatographic behavior. Finally, a recently developed framework was employed to evaluate the separability of the bis-guanidine resin as well as its orthogonality to the multimodal cation exchanger, Nuvia cPrime. This evaluation was carried out using a second model protein library which included both acidic and basic proteins. The results of this analysis indicated that the bis-guanidine prototype exhibited both higher pair separability (0.73) and pair enhancement (0.42) as compared to the less hydrophobic commercial Nuvia aPrime 4A with pair separability and enhancement factors of 0.57 and 0.22, respectively. The enhanced selectivity and orthogonality of this new multimodal anion exchange ligand may offer potential opportunities for bioprocessing applications.
Asunto(s)
Resinas de Intercambio Aniónico , Proteínas , Cromatografía por Intercambio Iónico , Guanidina/química , Ligandos , Proteínas/análisis , Proteínas/aislamiento & purificaciónRESUMEN
A coarse-grained Brownian dynamics model was used to simulate two proteins of similar sizes inside model membrane pores of varying size and hydrophobicity. The two proteins, which have radii of gyration of approximately 9.5 Å in their native states, are a 36-residue hydrophilic villin head piece (HP-36) and a 40-residue hydrophobic amyloid beta (Aß-40). From calculations of the separation factor, it is found that the two proteins are best separated using a pore radius of 15 Å and that hydrophobic pores select Aß-40 while the hydrophilic pores preferentially pass through HP-36. In addition, it is found that a simple model based on the net hydropathy of a protein is capable of estimating the separation factor trends of other protein pairs. Together, the coarse-grained Brownian dynamics model and the simple model are fast methodologies to guide experimental membrane design and to provide insights on protein structure variations. Graphical Abstract Simulation setup and snapshots of protein in various pore sizes.
Asunto(s)
Péptidos beta-Amiloides/química , Modelos Moleculares , Simulación de Dinámica Molecular , Proteínas de Neurofilamentos/química , Fragmentos de Péptidos/química , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Proteica , Pliegue de ProteínaRESUMEN
A multipath liquid chromatography with mass spectrometry instrument was constructed with the help of restricted access media to online segregate small and large molecules. This liquid chromatography system was custom built with five pumps and three two-position six-port valves to control the flow in a multipath system for the simultaneous analysis of small molecules and proteins. On separate chromatographic channels, small molecules trapped and proteins excluded from the online restricted access media were analyzed downstream using high-efficiency columns and a triple quadrupole mass spectrometer. A model sample, which included five proteins and 22 small molecules with different physicochemical properties, was used to evaluate the system. Following injection, the complete multipath separation and detection was performed in 22 min. Protein exclusion by the restricted access media was not quantitative. Four commercial trap columns were evaluated for their exclusion efficiency toward the proteins. Exclusion efficiency varied from <50% to only a maximum of 75% exclusion across the trap columns tested. An attempt was made to optimize the exclusion efficiency using different flow rates, flow rate gradients, and different additives both in the sample and the mobile phases. Protein exclusion was still erratic and generally nonquantitative.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Proteínas/análisis , Bibliotecas de Moléculas Pequeñas/análisis , Espectrometría de Masas en Tándem/métodos , Automatización , Cromatografía Líquida de Alta Presión/instrumentación , Espectrometría de Masas en Tándem/instrumentaciónRESUMEN
Isoelectric focusing (IEF) is a powerful tool for amphoteric protein separations because of high sensitivity, bio-compatibility, and reduced complexity compared to chromatography or mechanical separation techniques. IEF miniaturization is attractive because it enables rapid analysis, easier adaptation to point of care applications, and smaller sample demands. However, existing small-scale IEF tools have not yet been able to analyze single protein spots from array libraries, which are ubiquitous in many pharmaceutical discovery and screening protocols. Thus, we introduce an in situ, novel, miniaturized protein analysis approach that we have termed surface isoelectric focusing (sIEF). Low volume printed sIEF gels can be run at length scales of â¼300 µm, utilize â¼0.9 ng of protein with voltages below 10 V. Further, the sIEF device platform is so simple that it can be integrated with protein library arrays to reduce cost; devices demonstrate reusability above 50 uses. An acrylamide monomer solution containing broad-range carrier ampholytes was microprinted with a Nano eNablerTM between micropatterned gold electrodes spaced 300 µm apart on a glass slide. The acrylamide gel was polymerized in situ followed by protein loading via printed diffusional exchange. A pH gradient formed via carrier ampholyte stacking when electrodes were energized; the gradient was verified using ratiometric pH-sensitive FITC/TRITC dyes. Green fluorescent protein (GFP) and R-phycoerythrin (R-PE) were utilized both as pI markers and to test sIEF performance as a function of electric field strength and ampholyte concentration. Factors hampering sIEF included cathodic drift and pH gradient compression, but were reduced by co-printing non-ionic Synperonic® F-108 surfactant to reduce protein-gel interactions. sIEF gels achieved protein separations in <10 min yielding bands < 50 µm wide with peak capacities of â¼8 and minimum pI differences from 0.12 to 0.14. This new sIEF technique demonstrated comparable focusing at â¼100 times smaller dimensions than any previous IEF. Further, sample volumes required were reduced four orders of magnitude from 20 µL for slab gel IEF to 0.002 µL for sIEF. In summary, sIEF advantages include smaller volumes, reduced power consumption, and microchip surface accessibility to focused bands along with equivalent separation resolutions to prior IEF tools. These attributes position this new technology for rapid, in situ protein library analysis in clinical and pharmaceutical settings.
Asunto(s)
Mezclas Anfólitas/química , Focalización Isoeléctrica/métodos , Técnicas Analíticas Microfluídicas/métodos , Proteínas/análisis , Fuerza Protón-Motriz , Acrilamidas/química , Electricidad , Colorantes Fluorescentes/química , Geles , Humanos , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica/instrumentaciónRESUMEN
Fiber-based monoliths for use in liquid chromatographic separations are defined by columns packed with aligned fibers, woven matrices, or contiguous fiber structures capable of achieving rapid separations of proteins, macromolecules, and low molecular weight components. A common denominator and motivating driver for this approach, first initiated 25 years ago, was reducing the cost of bioseparations in a manner that also reduced residence time of retained components while achieving a high ratio of mass to momentum transfer. This type of medium, when packed into a liquid chromatography column, minimized the fraction of stagnant liquid and resulted in a constant plate height for non-adsorbing species. The uncoupling of dispersion from eluent flow rate enabled the surface chemistry of the stationary phase to be considered separately from fluid transport phenomena and pointed to new ways to apply chemistry for the engineering of rapid bioseparations. This paper addresses developments and current research on fiber-based monoliths and explains how the various forms of this type of chromatographic stationary phase have potential to provide new tools for analytical and preparative scale separations. The different stationary phases are discussed, and a model that captures the observed constant plate height as a function of mobile phase velocity is reviewed. Methods that enable hydrodynamically stable fiber columns to be packed and operated over a range of mobile phase flow rates, together with the development of new fiber chemistries, are shown to provide columns that extend the versatility of liquid chromatography using monoliths, particularly at the preparative scale. Graphical Abstract Schematic representation of a sample mixture being separated by a rolled-stationary phase column, resulting separated peaks shown in the chromatogram.
Asunto(s)
Cromatografía Liquida/instrumentación , Proteínas/aislamiento & purificación , Animales , Celulosa/análogos & derivados , Celulosa/química , Cromatografía Liquida/métodos , Diseño de Equipo , Vidrio/química , Humanos , Dispositivos Laboratorio en un Chip , Polímeros/química , Dióxido de Silicio/químicaRESUMEN
To overcome limitations of periodic separations of proteins in batch chromatographic columns Carousel Multi-Column Setup (CMS) has been recently suggested and theoretically analyzed in a previous study (R. Bochenek, W. Marek, W. Piatkowski, D. Antos, J. Chromatogr. A, 1301 (2013) 60-72). In this system, feed and mobile phase streams are subsequently delivered through parallel columns to mimic their countercurrent movement with respect to the fluid flow. All fluxes in the system are synchronized to ensure continuous feed delivery, which however causes reduction in the size of the operating window compared to batchwise-operating systems. In this study to improve the performance of CMS, additional process variables have been considered, such as the flow rate gradient and feed concentration. Though altering both variables allowed improving the separation selectivity and extending the operating window, the feed concentration appeared to be the most influential parameter affecting the process performance. Moreover, a procedure for practical realization of protein separations in CMS has been developed, including hints about the process design, configuration of columns and detectors, and use of pumps. As the case study, the separation of a ternary mixture of proteins, i.e., cytochrome C, lysozyme and immunoglobulin G, on hydrophobic interaction columns was used. A target product was a protein with intermediate adsorption strength that was isolated out of a more and less strongly adsorbed compound.
Asunto(s)
Técnicas de Química Analítica/instrumentación , Técnicas de Química Analítica/métodos , Cromatografía Liquida , Proteínas/aislamiento & purificación , Adsorción , Distribución en Contracorriente , Interacciones Hidrofóbicas e HidrofílicasRESUMEN
This study describes the in silico design, surface property analyses, production and chromatographic evaluations of a diverse set of antibody Fab fragment variants. Based on previous findings, we hypothesized that the complementarity-determining regions (CDRs) constitute important binding sites for multimodal chromatographic ligands. Given that antibodies are highly diversified molecules and in particular the CDRs, we set out to examine the generality of this result. For this purpose, four different Fab fragments with different CDRs and/or framework regions of the variable domains were identified and related variants were designed in silico. The four Fab variant libraries were subsequently generated by site-directed mutagenesis and produced by recombinant expression and affinity purification to enable examination of their chromatographic retention behavior. The effects of geometric re-arrangement of the functional moieties on the multimodal resin ligands were also investigated with respect to Fab variant retention profiles by comparing two commercially available multimodal cation-exchange ligands, Capto MMC and Nuvia cPrime, and two novel multimodal ligand prototypes. Interestingly, the chromatographic data demonstrated distinct selectivity trends between the four Fab variant libraries. For three of the Fab libraries, the CDR regions appeared as major binding sites for all multimodal ligands. In contrast, the fourth Fab library displayed a distinctly different chromatographic behavior, where Nuvia cPrime and related multimodal ligand prototypes provided markedly improved selectivity over Capto MMC. Clearly, the results illustrate that the discriminating power of multimodal ligands differs between different Fab fragments. The results are promising indications that multimodal chromatography using the appropriate multimodal ligands can be employed in downstream bioprocessing for challenging selective separation of product related variants.
Asunto(s)
Fragmentos Fab de Inmunoglobulinas/química , Biblioteca de Péptidos , Picratos/inmunología , Animales , Sitios de Unión , Cromatografía Liquida , Regiones Determinantes de Complementariedad/química , Regiones Determinantes de Complementariedad/genética , Simulación por Computador , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Ligandos , Ratones , Mutagénesis Sitio-DirigidaRESUMEN
Superficially porous particles (SPPs) with pore size ranging from 90Å to 120Å have been a great success for the fast separation of small molecules over totally porous particles in recent years. However, for the separation of large biomolecules such as proteins, particles with large pore size (e.g. ≥ 300Å) are needed to allow unrestricted diffusion inside the pores. One early example is the commercial wide pore (300Å) SPPs in 5µm size introduced in 2001. More recently, wide pore SPPs (200Å and 400Å) in smaller particle sizes (3.5-3.6µm) have been developed to meet the need of increasing interest in doing faster analysis of larger therapeutic molecules by biopharmaceutical companies. Those SSPs in the market are mostly synthesized by the laborious layer-by-layer (LBL) method. A one step coating approach would be highly advantageous, offering potential benefits on process time, easier quality control, materials cost, and process simplicity for facile scale-up. A unique one-step coating process for the synthesis of SPPs called the "coacervation method" was developed by Chen and Wei as an improved and optimized process, and has been successfully applied to synthesis of a commercial product, Poroshell 120 particles, for small molecule separation. In this report, we would like to report on the most recent development of the one step coating coacervation method for the synthesis of a series of wide pore SPPs of different particle size, pore size, and shell thickness. The one step coating coacervation method was proven to be a universal method to synthesize SPPs of any particle size and pore size. The effects of pore size (300Å vs. 450Å), shell thickness (0.25µm vs. 0.50µm), and particle size (2.7µm and 3.5µm) on the separation of large proteins, intact and fragmented monoclonal antibodies (mAbs) were studied. Van Deemter studies using proteins were also conducted to compare the mass transfer properties of these particles. It was found that the larger pore size actually had more impact on the performance of mAbs than particle size and shell thickness. The SPPs with larger 3.5µm particle size and larger 450Å pore size showed the best resolution of mAbs and the lowest back pressure. To the best of our knowledge, this is the largest pore size made on SPPs. These results led to the optimal particle design with a particle size of 3.5µm, a thin shell of 0.25µm and a larger pore size of 450Å.
Asunto(s)
Anticuerpos Monoclonales/análisis , Proteínas/análisis , Inmunoglobulina G/análisis , Tamaño de la Partícula , Péptidos/análisis , PorosidadRESUMEN
In this study, a unique set of antibody Fab fragments was designed in silico and produced to examine the relationship between protein surface properties and selectivity in multimodal chromatographic systems. We hypothesized that multimodal ligands containing both hydrophobic and charged moieties would interact strongly with protein surface regions where charged groups and hydrophobic patches were in close spatial proximity. Protein surface property characterization tools were employed to identify the potential multimodal ligand binding regions on the Fab fragment of a humanized antibody and to evaluate the impact of mutations on surface charge and hydrophobicity. Twenty Fab variants were generated by site-directed mutagenesis, recombinant expression, and affinity purification. Column gradient experiments were carried out with the Fab variants in multimodal, cation-exchange, and hydrophobic interaction chromatographic systems. The results clearly indicated that selectivity in the multimodal system was different from the other chromatographic modes examined. Column retention data for the reduced charge Fab variants identified a binding site comprising light chain CDR1 as the main electrostatic interaction site for the multimodal and cation-exchange ligands. Furthermore, the multimodal ligand binding was enhanced by additional hydrophobic contributions as evident from the results obtained with hydrophobic Fab variants. The use of in silico protein surface property analyses combined with molecular biology techniques, protein expression, and chromatographic evaluations represents a previously undescribed and powerful approach for investigating multimodal selectivity with complex biomolecules.
Asunto(s)
Cromatografía de Afinidad/métodos , Cromatografía Liquida/métodos , Biología Computacional , Fragmentos Fab de Inmunoglobulinas/aislamiento & purificación , Fragmentos Fab de Inmunoglobulinas/metabolismo , Biotecnología/métodos , Análisis Mutacional de ADN , Interacciones Hidrofóbicas e Hidrofílicas , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/genética , Mutagénesis Sitio-Dirigida , Tecnología Farmacéutica/métodosRESUMEN
Continuing interest in larger therapeutic molecules by pharmaceutical and biotech companies provides the need for improved tools for examining these molecules both during the discovery phase and later during quality control. To meet this need, larger pore superficially porous particles with appropriate surface properties (Fused-Core(®) particles) have been developed with a pore size of 400 Å, allowing large molecules (<500 kDa) unrestricted access to the bonded phase. In addition, a particle size (3.4 µm) is employed that allows high-efficiency, low-pressure separations suitable for potentially pressure-sensitive proteins. A study of the shell thickness of the new fused-core particles suggests a compromise between a short diffusion path and high efficiency versus adequate retention and mass load tolerance. In addition, superior performance for the reversed-phase separation of proteins requires that specific design properties for the bonded-phase should be incorporated. As a result, columns of the new particles with unique bonded phases show excellent stability and high compatibility with mass spectrometry-suitable mobile phases. This report includes fast separations of intact protein mixtures, as well as examples of very high-resolution separations of larger monoclonal antibody materials and associated variants. Investigations of protein recovery, sample loading and dynamic range for analysis are shown. The advantages of these new 400 Å fused-core particles, specifically designed for protein analysis, over traditional particles for protein separations are demonstrated.
Asunto(s)
Cromatografía Liquida/instrumentación , Microesferas , Proteínas/aislamiento & purificación , Peso Molecular , Porosidad , Proteínas/químicaRESUMEN
A theoretical study has been performed on the effectiveness of isolating a target component out of a multi-component protein mixture using different arrangements of chromatographic columns. Three continuous systems have been considered which were able to perform solvent gradient separations, such as: open loop simulated moving bed, countercurrent solvent gradient purification and carousel multicolumn setup. The performance of the continuous processes was examined with respect to productivity, yield and eluent consumption and compared to a single-column batch system. As a case study separation of a ternary mixture of proteins on HIC media has been selected. Two separation problems have been analyzed referring to the situation when the target component was the most strongly adsorbed as well as when it exhibited intermediate adsorption strength. A mathematical model has been used to simulate the process dynamics and to optimize operating conditions for the separation. The numerical study indicated that batch column arrangements can outperform SMB-based configurations regarding all performance indicators considered, which has been attributed to solvent mixing in the recycled streams and distortion of the gradient shape in SMB units. It has been concluded that the performance of complex multicolumn systems should be verified vs. batch column operations prior to the realization of the separation process.