RESUMO
A multi-dimensional fractionation and characterization scheme was developed for fast acquisition of the relevant molecular properties for protein separation from crude biological feedstocks by ion-exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), and size-exclusion chromatography. In this approach, the linear IEX isotherm parameters were estimated from multiple linear salt-gradient IEX data, while the nonlinear IEX parameters as well as the HIC isotherm parameters were obtained by the inverse method under column overloading conditions. Collected chromatographic fractions were analyzed by gel electrophoresis for estimation of molecular mass, followed by mass spectrometry for protein identification. The usefulness of the generated molecular properties data for rational decision-making during downstream process development was equally demonstrated. Monoclonal antibody purification from crude hybridoma cell culture supernatant was used as case study. The obtained chromatographic parameters only apply to the employed stationary phases and operating conditions, hence prior high throughput screening of different chromatographic resins and mobile phase conditions is still a prerequisite. Nevertheless, it provides a quick, knowledge-based approach for rationally synthesizing purification cascades prior to more detailed process optimization and evaluation.
Assuntos
Anticorpos Monoclonais/isolamento & purificação , Biotecnologia/métodos , Cromatografia/métodos , Misturas Complexas/química , Proteínas/química , Anticorpos Monoclonais/química , Misturas Complexas/análise , Bases de Dados Factuais , Eletroforese em Gel de Poliacrilamida , Hibridomas , Interações Hidrofóbicas e Hidrofílicas , Modelos Biológicos , Proteínas/análise , Espectrometria de Massas em TandemRESUMO
This work demonstrates that the type of ion-exchanger (anion or cation), the mode of operation (bind-and-elute or flow-through), and the operational pH of ion-exchange chromatography (IEX) can be selected in a fast and rational way by analytical pH-gradient IEX operations, thereby eliminating the need for pH scouting or high-throughput screening. The developed approach was applied for the selection of an IEX process for the capture of a monoclonal antibody (MAb) from hybridoma cell culture supernatant (CCS). It was found within a day that MAb can optimally be captured by bind-and-elute mode cation-exchange chromatography (CEX) at pH 4.5 or anion-exchange chromatography (AEX) at pH 7.2 without lowering the salt concentration in the CCS. The performance of both CEX and AEX was predicted to be equal for this particular MAb capture.
Assuntos
Cromatografia por Troca Iônica/métodos , Concentração de Íons de Hidrogênio , Resinas de Troca Aniônica , Resinas de Troca de Cátion , Eletroforese em Gel de PoliacrilamidaRESUMO
This work demonstrates that a highly linear, controllable and wide-ranged pH-gradient can be generated through an ion-exchange chromatography (IEC) column. Such a pH-gradient anion-exchange chromatography was evaluated with 17 model proteins and found that acidic (pI<6) and basic (pI>8) proteins elute roughly at their pI, whereas neutral proteins (pI 6-8) elute at pH 8-9 regardless their pI values. Because of the flat nature of protein titration curves from pH approximately 6 to approximately 9, neutral proteins indeed exhibit nearly zero net charge at pH approximately 9. The elution-pH in pH-gradient IEC or the titration curve, but not the pI, was identified as the key parameter for pH optimization of preparative IEC in a fast and rational way. The pH-gradient IEC was also applied and found to be an excellent analytical tool for the fractionation of crude protein mixtures.
Assuntos
Cromatografia por Troca Iônica/métodos , Proteínas/química , Força Próton-Motriz , Resinas de Troca Aniônica/química , Proteínas/isolamento & purificação , Reprodutibilidade dos TestesRESUMO
Understanding protein phase behavior is important for purification, storage, and stable formulation of protein drugs in the biopharmaceutical industry. Glycoproteins, such as monoclonal antibodies (MAbs) are the most abundant biopharmaceuticals and probably the most difficult to crystallize among water-soluble proteins. This study explores the possibility of correlating osmotic second virial coefficient (B(22)) with the phase behavior of an intact MAb, which has so far proved impossible to crystallize. The phase diagram of the MAb is presented as a function of the concentration of different classes of precipitants, i.e., NaCl, (NH4)2SO4, and polyethylene glycol. All these precipitants show a similar behavior of decreasing solubility with increasing precipitant concentration. B(22) values were also measured as a function of the concentration of the different precipitants by self-interaction chromatography and correlated with the phase diagrams. Correlating phase diagrams with B(22) data provides useful information not only for a fundamental understanding of the phase behavior of MAbs, but also for understanding the reason why certain proteins are extremely difficult to crystallize. The scaling of the phase diagram in B(22) units also supports the existence of a universal phase diagram of a complex glycoprotein when it is recast in a protein interaction parameter.
Assuntos
Anticorpos Monoclonais/química , Animais , Anticorpos Monoclonais/isolamento & purificação , Fenômenos Biofísicos , Biofísica , Precipitação Química , Cromatografia em Agarose , Cristalização , Humanos , Técnicas In Vitro , Solubilidade , Soluções , TermodinâmicaRESUMO
Solution conditions under which proteins have a tendency to crystallize correspond to a slightly negative osmotic second virial coefficient (B22). A positive B22 value guarantees no crystallization to occur. On the other hand, a B22 value within the so called "crystallization slot" thermodynamically supports the crystallization processes but does not guarantee successful crystal growth. It is, however, a prerequisite for protein crystallization that the B22 value must be in the slightly negative regime. Self-interaction chromatography (SIC) is designed in this work as an analytical tool for determining B22 in a precise and reproducible way. The methodology was demonstrated in detail in terms of its theoretical basis, experimental methodology, troubleshooting and data analysis for different protein samples and solution conditions. The inherent error limit of SIC is found to be comparatively less than other B22 measurement techniques. The designed experimental approach was applied for mapping crystallization conditions of a model protein, i.e. lysozyme. Good agreement between the obtained lysozyme B22 values and literature values confirms the accuracy of the approach.
Assuntos
Cromatografia em Gel/métodos , Muramidase/química , Cristalização , Reprodutibilidade dos TestesRESUMO
In contrast to performing assays on a substrate using immobilization techniques, wet analysis in nanoliter-sized wells allows quantitative monitoring of enzyme-based reactions by measuring luminescence with time. However, a suitable dispensing method is required to accurately deposit stabilized enzyme solutions into nanoliter wells in such a manner that the enzyme activities are preserved prior to and during measurements. Furthermore, an efficient method is required to add sample liquid to these wells in such a manner that evaporation of assay liquid is completely prevented during sample introduction and monitoring. A powerful methodology is presented in this paper allowing quantitative analysis of enzyme-based reactions in identical nanoliter volumes on-chip. In a first step, picoliter amounts of protein solutions are deposited as uniform dry dots into wells using our reported electrospraying technique (Moerman, R.; Frank, J.; Marijnissen, J. C. M.; Schalkhammer, T. G. M.; van Dedem, G. W. K. Anal. Chem. 2001, 73, 2183-2189.). The silicon chips are then stored at temperatures as low as -80 degrees C. At the time of analysis, a sample solution is slid into the wells using a coverslip. With the edge of the coverslip, sample solution is pushed across the wells at a speed of 1.5-2.5 cm/s to prevent carryover of reagents to neighboring wells. Evaporation of assay liquid from the wells is prevented because the coverslip seals the wells and "bonds" to the chip by adhesion forces. Electrospraying appears to be an excellent method to deposit enzyme solutions containing up to 20% (w/v) of trehalose without being hampered by clogging of the capillary or splashing of droplets. After being sprayed on-chip (silicon nitride), the enzymes pyruvate kinase and lactate dehydrogenase remained stable for a period of 1.5-2 months at a storage temperature of -20 degrees C. The coverslip method completely prevented evaporation for minutes up to hours allowing monitoring of enzyme-based reactions in arrays of nanoliter wells.
Assuntos
L-Lactato Desidrogenase/metabolismo , Nanotecnologia/métodos , Piruvato Quinase/metabolismo , Estabilidade Enzimática , Fluorescência , L-Lactato Desidrogenase/química , Piruvato Quinase/química , TrealoseRESUMO
Conventional enzymatic assays for alcohol dehydrogenase, pyruvate kinase, and enolase performed in 96-well microtiter plates were compared with assays monitored in 25-well nanoarrays. All miniaturized reactions could be performed in maximum volumes of 6.3-8 nL and were read out with a conventional fluorescence microscope system equipped with a scientific grade CCD camera. Substrate and cofactor were already present inside the wells after having been presprayed, or they were applied in solution to the wells of the nanoarray shortly before the assays started. For all of the assays, commercially available enzymes and enzymes present in cell-free extracts were used. Assays carried out in premixed nanoarrays gave results comparable to those performed in presprayed nanoarrays. Enzyme activities determined in nanoarrays by using two different methods were in good agreement with assays performed in microtiter plates. Also, good correspondence was found between expected and observed enzyme levels. In short, enzymatic assays performed in premixed and in particular in presprayed nanoarrays are a promising low-volume and low-reagent- and sample-consuming alternative to current methodology and could find applications in many different areas of analytical chemistry.
Assuntos
Enzimas/análise , Análise Serial de Proteínas/métodos , Álcool Desidrogenase/análise , Animais , Nanotecnologia , Fosfopiruvato Hidratase/análise , Piruvato Quinase/análiseRESUMO
We have developed a straightforward coverslip method that combines rapid sample introduction into arrays of 19.8-55.0-microm-deep microwells (prefilled with reactants) on a chip with complete sealing of these wells to avoid evaporation during optical detection. We used coverslips of 300-1500-microm-thick poly(methyl methacrylate) (PMMA) containing arrays of holes with diameters of 200 and 300 microm, and 800-microm-thick quartz slides containing arrays of holes with diameters of 100 and 200 microm. A coverslip was placed on top of a chip, thereby positioning the holes between the wells. A defined amount of aqueous sample solution was pulled between the cover and silicon nitride coated chip by means of capillary forces, resulting in entrapment of air in all wells. By partly or completely positioning the holes over the wells, air could escape through the holes, resulting in complete filling of the wells with the surrounding liquid within 0.1-0.2 s. Immediately after filling, the slide was rapidly shifted back to its original position and was pressed onto the chip for 5-30 s with a force of 1-2 kg cm(-2), resulting in complete sealing of the wells for minutes up to one-half hour. Because the well volumes of an array are identical and the wells are completely filled, reproducible assay volumes and flat menisci are obtained, making this method very suitable for quantitative analysis.
RESUMO
Biotechnology today is a well-established paradigm in many areas of human endeavor, such as the pharmaceutical industry, agriculture, management of the environment and many others. Meanwhile, biology is undergoing a spectacular transition: whereas systematic biology was replaced gradually by molecular biology, the latter is rapidly being transformed into a new systematic era in which entire genomes are being charted by ever more sophisticated analytical techniques. In the wake of this onslaught of data, new fields are germinating, such as bioinformatics in an attempt to find answers to fundamental questions, answers that may be hidden in the massive amounts of data already available today.
Assuntos
Enzimas/análise , Microquímica/instrumentação , Microquímica/métodos , Miniaturização/instrumentação , Miniaturização/métodos , Transdutores , Biopolímeros/análise , Biopolímeros/metabolismo , Biotecnologia/instrumentação , Biotecnologia/métodos , Técnicas de Química Analítica/instrumentação , Técnicas de Química Analítica/métodos , Enzimas/metabolismo , Desenho de Equipamento , Análise de Injeção de Fluxo/instrumentação , Análise de Injeção de Fluxo/métodosRESUMO
In this contribution, a capillary electrophoresis microdevice with an integrated on-chip contactless four-electrode conductivity detector is presented. A 6-cm-long, 70-microm-wide, and 20-microm-deep channel was etched in a glass substrate that was bonded to a second glass substrate in order to form a sealed channel. Four contactless electrodes (metal electrodes covered by 30-nm silicon carbide) were deposited and patterned on the second glass substrate for on-chip conductivity detection. Contactless conductivity detection was performed in either a two- or a four-electrode configuration. Experimental results confirmed the improved characteristics of the four-electrode configuration over the classical two-electrode detection setup. The four-electrode configuration allows for sensitive detection for varying carrier-electrolyte background conductivity without the need for adjustment of the measurement frequency. Reproducible electrophoretic separations of three inorganic cations (K+, Na+, Li+) and six organic acids are presented. Detection as low as 5 microM for potassium was demonstrated.
RESUMO
Microfluidic devices are a promising new tool for studying and optimizing (bio)chemical reactions and analyses. Many (bio)chemical reactions require accurate temperature control, such as for example thermocycling for PCR. Here, a new integrated temperature control system for microfluidic devices is presented, using chemical and physical processes to locally regulate temperature. In demonstration experiments, the evaporation of acetone was used as an endothermic process to cool a microchannel. Additionally, heating of a microchannel was achieved by dissolution of concentrated sulfuric acid in water as an exothermic process. Localization of the contact area of two flows in a microfluidic channel allows control of the position and the magnitude of the thermal effect.
Assuntos
Microquímica/métodos , Microfluídica/instrumentação , Desenho de Equipamento , Microquímica/instrumentação , Oligonucleotídeos/análise , TemperaturaRESUMO
In this article we present a novel device, the BioScope, which allows elucidation of in vivo kinetics of microbial metabolism via perturbation experiments. The perturbations are carried out according to the continuous-flow method. The BioScope consists of oxygen permeable silicon tubing, connected to the fermentor, through which the broth flows at constant velocity. The tubing has a special geometry (serpentine channel) to ensure plug flow. After leaving the fermentor, the broth is mixed with a small flow of perturbing agent. This represents the start of the perturbation. The broth is sampled at different locations along the tubing, corresponding to different incubation times. The maximal incubation time is 69 s; the minimally possible time interval between the samples is 3-4 s. Compared to conventional approaches, in which the perturbation is carried out in the fermentor, the BioScope offers a number of advantages. (1) A large number of different perturbation experiments can be carried out on the same day, because the physiological state of the fermentor is not perturbed. (2) In vivo kinetics during fed-batch experiments and in large-scale reactors can be investigated. (3) All metabolites of interest can be measured using samples obtained in a single experiment, because the volume of the samples is unlimited. (4) The amount of perturbing agent spent is minimal, because only a small volume of broth is perturbed. (5) The system is completely automated. Several system properties, including plug-flow characteristics, mixing, oxygen and carbon dioxide transfer rates, the quenching time, and the reproducibility have been explored, with satisfactory results. Responses of several glycolytic intermediates in Saccharomyces cerevisiae to a glucose pulse, measured using a conventional approach are compared to results obtained with the BioScope. The agreement between the results demonstrates that the BioScope is indeed a promising device for studying in vivo kinetics.
Assuntos
Reatores Biológicos , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Glucose/metabolismo , Membranas Artificiais , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Desenho de Equipamento , Fermentação , Glicólise , Oxigênio/metabolismo , Tamanho da Amostra , Sensibilidade e EspecificidadeRESUMO
Nearly all analyses by capillary electrophoresis (CE) are performed using optical detection, utilizing either absorbance or (laser-induced) fluorescence. Though adequate for many analytical problems, in a large number of cases, e.g., involving non-UV-absorbing compounds, these optical detection methods fall short. Indirect optical detection can then still provide an acceptable means of detection, however, with a strongly reduced sensitivity. During the past few years, contactless conductivity detection (CCD) has been presented as a valuable extension to optical detection techniques. It has been demonstrated that with CCD detection limits comparable, or even superior, to (indirect) optical detection can be obtained. Additionally, construction of the CCD around the CE capillary is straightforward and robust operation is easily obtained. Unfortunately, in the literature a large variety of designs and operating conditions for CCD were described. In this contribution, several important parameters of CCD are identified and their influence on, e.g., detectability and peak shape is described. An optimized setup based on a well-defined detection cell with three detection electrodes is presented. Additionally, simple and commercially available read-out electronics are described. The performance of the CCD-CE system was demonstrated for the analysis of peptides. Detection limits at the microM level were obtained in combination with good peak shapes and an overall good performance and stability.
Assuntos
Condutividade Elétrica , Eletroforese Capilar/instrumentação , Soluções Tampão , Eletrônica , Eletroforese Capilar/normas , Desenho de Equipamento , Metais/isolamento & purificação , Peptídeos/isolamento & purificaçãoRESUMO
At present, the main limitation for the further adoption of capillary electrochromatography (CEC) in the (routine) laboratory is caused by the lack of reproducible and stable columns. The main source of column instability is concentrated in the frits needed to retain the packed bed inside the CEC capillary. The sintering process used to prepare the frits can be rather problematic and irreproducible, particularly for small stationary phase particles and wide column diameters. Since the (surface) composition of the frits is different from the bulk stationary phase packing, different electroosmotic flow (EOF) velocities are generated. This effect is assumed to be primarily responsible for rapid column destruction. In this contribution, a novel approach for the preparation of fritless CEC capillaries is presented and evaluated. Using 5 microm Hypersil ODS particles, separation efficiencies in the range of 130,000-200,000 plates/m were obtained. In a 100 microm inner diameter packed column, electrical currents up to 50 microA could be tolerated without negative effects such as bubble formation. The prepared CEC columns were found to be stable and could easily be operated continuously for several days without column damage. An additional advantage of the proposed tapering approach is that application of pressure on the in- and outlet vial during separation was not required to prevent bubble formation.
Assuntos
Eletroforese Capilar/instrumentação , Calibragem , Cromatografia Líquida de Alta Pressão , Eletroforese Capilar/normas , Desenho de Equipamento , Hidrocarbonetos Aromáticos/isolamento & purificação , Reprodutibilidade dos Testes , Esteroides/isolamento & purificaçãoRESUMO
In this contribution, the role of bioaffinity interactions on electrokinetically controlled microfabricated devices is reviewed. Interesting applications reported in the literature include enzymatic assays, where enzyme and enzyme inhibition kinetics were studied, often in combination with electrophoretic separation. Attention is paid towards developments that could lead to implementation of electrokinetically controlled microdevices in high-throughput screening. Furthermore, enzyme-facilitated detection in combination with electrophoretic separation on microdevices is discussed. Various types of immunoassays have been implemented on the microchip format. The selectivity of antibody-antigen interaction has been exploited for the detection of analytes in complex sample matrices as required, for example, in clinical chemistry. Binding kinetics as well as stoichiometry were studied in chip-based assays. Automated mixing protocols as well as the demonstration of a parallel immunoassay allow implementation of microdevices in high-throughput screening. Furthermore, demonstration of immunoassays on cheap polymeric microdevices opens the way towards the fabrication of disposable devices, a requirement for commercialization and therefore for application in routine analyses.