RESUMEN
A chromatographic system was adapted to allow monitoring of eluent of preparative column via absorbance and with the chromatographic analysis of the target macromolecule on the same chromatographic system. The proposed approach was tested on important macromolecules, such as monoclonal antibodies, monoclonal antibody aggregates and plasmid DNA (pDNA). A frontal analysis was made on the preparative column, while a chromatographic on-line analysis was performed by sequentially injecting the preparative column outlet on a convection-based analytical column, operating on the same chromatographic system. Cation and/or anion exchangers were used as the chromatographic media (along with a protein A), depending on the sample to be purified. The method was found to be robust and reproducible. To adjust the limit of detection, an algorithm varying the number of injections was used, enabling accurate monitoring of an early breakthrough for concentrations below 1% of the feed concentration. The accuracy varies according to the applied flow rate, but it is typically in the range of few percent, or even below. Due to its simplicity and flexibility, the proposed method can be easily adapted to a pharmaceutical environment.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Algoritmos , Anticuerpos Monoclonales/aislamiento & purificación , ADN/análisis , Sistemas en Línea , Concentración Osmolar , Plásmidos/genética , ARN/análisis , Proteína Estafilocócica A/análisis , Factores de TiempoRESUMEN
A fully continuous, downstream process represents one of the most interesting novel purification approaches in the biosimilars industry, because it would enhance the production output while reducing the costs of complex biopharmaceuticals. Since it generally involves several chromatographic steps, the selection of appropriate chromatographic columns is of utmost importance. In this study we compared several commercially available ion-exchange-membrane adsorbers (NatriFlo®, Sartobind® and Mustang®) for the removal of deoxyribonucleic acid (DNA), host cell proteins (HCPs) and monoclonal antibody aggregates in flow-through mode. Design of Experiments (DoEs) was employed to determine the optimal pH and conductivity conditions. We demonstrated that all the anion-exchange-membrane adsorbers were capable of removing DNA and HCPs from monoclonal antibody mixtures below the required threshold across a wide range of sample pH and conductivity values, and that the HCPs' normalized outlet concentration increases almost linearly with the loading, being independent of the HCPs' concentration. No significant differences in the profile of the adsorbed HCPs with respect to the membrane adsorbers were observed, based on 2D electrophoresis analysis data, although they exhibited different binding capacities. Cation-exchange-membrane adsorbers were also tested for the removal of aggregates. The Yamamoto model was used to determine the number of binding sites and estimate the conductivity range for efficient removal of aggregates, while maintaining a high monoclonal antibody recovery. However, the obtained range had to be further fine-tuned experimentally, due to displacement phenomena. Differences in the trends of binding-site number with a change in the pH value for the tested cation-exchange adsorbers indicate slightly different adsorption mechanisms. To obtain optimal process performance, adjustments to the pH and the conductivity were required between the anion- and cation-exchange steps.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Membranas Artificiales , Adsorción , Cationes , ADN/aislamiento & purificación , Conductividad Eléctrica , Concentración de Iones de Hidrógeno , Intercambio Iónico , Agregado de Proteínas , Sales (Química)/químicaRESUMEN
ZnO nanoparticles are utilized in an ever growing number of products and can, therefore, be readily encountered in our everyday life. Human beings' outermost tissues consist of different epithelia and are, therefore, the most exposed to materials from the environment. In this paper, Caco-2 and Calu-3 cell lines were used, having been previously broadly applied for in vitro modelling of intestinal and respiratory epithelia, respectively. The toxicity of synthesized micro-, submicro- and nanoparticulate ZnO on these epithelia was measured and compared to the efficacy of the same ZnO particles as antibacterial agents. An approximately four-fold excess in antibacterial activity of ZnO nanoparticles over ZnO granulate was observed. The results of this paper reveal a sharp distinction between toxic nanoparticulate ZnO and safe ZnO particles of larger sizes in intestinal and airway in vitro epithelial models. In contrast, ZnO of larger particle sizes had only modestly lower antibacterial activity, which can be compensated for with higher dosing. These results show that nanoparticulate ZnO requires critical in vivo assessment before application.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Nanopartículas/química , Óxido de Zinc/química , Óxido de Zinc/farmacología , Antibacterianos/efectos adversos , Células CACO-2 , Línea Celular , Supervivencia Celular/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Humanos , Nanopartículas/efectos adversos , Óxido de Zinc/efectos adversosRESUMEN
Different studies of long-term chondrocytes viability have shown a gradual reduction as a function of time and ambient temperature. The aim of our in vitro study was to establish chondrocyte postmortem viability curves for 4°C, 11°C, 23°C, 35°C during 63 days after the donors' death. Osteochondral cylinders were procured from the knees of 16 male donors (20-47 years), stored in preservation media that was not changed, and analyzed in 3-day intervals using a confocal laser scanning microscope. A significant influence of time on viability was found from Day 9 (p = 0.0029) and onwards (p < 0.0001). The lowest overall chondrocyte viability was at 35°C, followed by 4°C (p < 0.0001). The conditions used in this in vitro analysis suggest that similar viabilities may occur while in situ in the decedent. Further studies of chondrocyte viability from individuals with known postmortem intervals may show premise to help evaluate time since death in the late postmortem interval.
Asunto(s)
Cartílago Articular/citología , Condrocitos/fisiología , Articulación de la Rodilla/citología , Adulto , Análisis de Varianza , Supervivencia Celular , Células Cultivadas , Humanos , Masculino , Microscopía Confocal , Persona de Mediana Edad , Cambios Post Mortem , Manejo de Especímenes/métodos , Temperatura , Factores de Tiempo , Adulto JovenRESUMEN
Appropriate assessment of transepithelial permeability in vitro is needed to estimate and model trans-mucosal bioavailability to achieve oral delivery of protein biopharmaceuticals. The Caco-2 cell-based intestinal epithelium model is widely used for this purpose for low molecular mass drugs. The aim of this study was to test the suitability of the Caco-2 model for assessing enhanced transepithelial permeability to proteins. Four unrelated proteins were chosen to test the permeability of Caco-2 monolayers. It was found that proteins could cross the epithelium model, in spite of their size. All tested proteins had similar very low apparent permeability coefficients (Papp) of around 4×10(-10)cm/s. Protein stability over three-hour exposure to Caco-2 was also confirmed. Their crossing rate in a cell-free setup was also measured, to determine the upper limit of permeability to proteins. An epithelium permeability enhancer N-decyl-ß-d-maltopyranoside (MP C10) was used to demonstrate accelerated permeability conditions. Papp values could be increased dose dependently up to about 1×10(-7)cm/s, close to the level in the cell-free setup, indicating distinctive potential of the model. This along with enhancing effect of known specific route permeators suggests involvement of the paracellular route in protein transport. Our results thus indicate that the Caco-2 model is a suitable tool for in vitro assessment of enhanced permeability to proteins.
Asunto(s)
Mucosa Intestinal/metabolismo , Maltosa/análogos & derivados , Proteínas/farmacocinética , Disponibilidad Biológica , Células CACO-2 , Humanos , Mucosa Intestinal/citología , Maltosa/farmacología , Modelos Biológicos , Permeabilidad/efectos de los fármacos , Proteínas/farmacologíaRESUMEN
In modern production of protein biopharmaceuticals, a good screening and selection method of high-producing clones can dramatically influence the whole production process and lead to lower production costs. We have created a rapid, simple, and inexpensive method for selecting high-producing clones in the yeast Pichia pastoris that is based on the beta-lactamase reporter system. By integrating the reporter gene and the gene of interest into the same genome locus, it was possible to use beta-lactamase activity as a measure of the expression level of the protein of interest. A novel expression vector with two independent expression cassettes was designed and tested using green fluorescent protein (GFP) as a model. The first cassette contained the GFP gene under the control of a strong, inducible AOX1 promoter, while the second cassette consisted of the beta-lactamase reporter gene under the control of a weak constitutive YPT1 promotor. High-producing GFP clones were selected directly on the plates based on the color change after hydrolysis of the beta-lactamase substrate added to the medium. beta-lactamase activity was found to positively correlate with GFP fluorescence. The reporter system described is widely applicable-it can be easily applied to other, also pharmaceutically relevant proteins and to other yeast expression systems, such as Saccharomyces cerevisiae and Hansenula polymorpha.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Genes Reporteros , Pichia/genética , Selección Genética , beta-Lactamasas/genética , Células Clonales , Genes Fúngicos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Pichia/metabolismo , Plásmidos , Regiones Promotoras Genéticas , Sensibilidad y Especificidad , beta-Lactamasas/metabolismoRESUMEN
The aim of this study was to be able to reversibly bind histidine-rich proteins to the surface of maghemite magnetic nanoparticles via coordinative bonding using Zn ions as the anchoring points. We showed that in order to adsorb Zn ions on the maghemite, the surface of the latter needs to be modified. As silica is known to strongly adsorb zinc ions, we chose to modify the maghemite nanoparticles with a nanometre-thick silica layer. This layer appeared to be thin enough for the maghemite nanoparticles to preserve their superparamagnetic nature. As a model the histidine-rich protein bovine serum albumin (BSA) was used. The release of the BSA bound to Zn-decorated silica-coated maghemite nanoparticles was analysed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrated that the bonding of the BSA to such modified magnetic nanoparticles is highly reversible and can be controlled by an appropriate change of the external conditions, such as a pH decrease or the presence/supply of other chelating compounds.