RESUMEN
This work aimed to quantify growth and biochemical parameters (viable cell density, Xv; cell viability, CV; glucose, lactate, glutamine, glutamate, ammonium, and potassium concentrations) in upstream stages to obtain rabies virus-like particles (rabies VLP) from insect cell-baculovirus system using on-line and off-line Raman spectra to calibrate global models with minimal experimental data. Five cultivations in bioreactor were performed. The first one comprised the growth of uninfected Spodoptera frugiperda (Sf9) cells, the second and third runs to obtain recombinant baculovirus (rBV) bearing Rabies G glycoprotein and matrix protein, respectively. The fourth one involved the generation of rabies VLP from rBVs and the last one was a repetition of the third one with cell inoculum infected by rBV. The spectra were acquired through a Raman spectrometer with a 785-nm laser source. The fitted Partial Least Square models for nutrients and metabolites were comparable with those previously reported for mammalian cell lines (Relative error < 15 %). However, the use of this chemometrics approach for Xv and CV was not as accurate as it was for other parameters. The findings from this work established the basis for bioprocess Raman spectroscopical monitoring using insect cells for VLP manufacturing, which are gaining ground in the pharmaceutical industry.
Asunto(s)
Virus de la Rabia , Rabia , Animales , Virus de la Rabia/genética , Espectrometría Raman , Línea Celular , Reactores Biológicos , Baculoviridae , Proteínas Recombinantes , Insectos , Spodoptera , MamíferosRESUMEN
Most rabies vaccines are based on inactivated virus, which production process demands a high level of biosafety structures. In the past decades, recombinant rabies virus glycoprotein (RVGP) produced in several expression systems has been extensively studied to be used as an alternative vaccine. The immunogenic characteristics of this protein depend on its correct conformation, which is present only after the correct post-translational modifications, typically performed by animal cells. The main challenge of using this protein as a vaccine candidate is to keep its trimeric conformation after the purification process. We describe here a new immunoaffinity chromatography method using a monoclonal antibody for RVGP Site II for purification of recombinant rabies virus glycoprotein expressed on the membrane of Drosophila melanogaster S2 cells. RVGP recovery achieved at least 93%, and characterization analysis showed that the main antigenic proprieties were preserved after purification.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Glicoproteínas/aislamiento & purificación , Virus de la Rabia/aislamiento & purificación , Proteínas Virales/aislamiento & purificación , Animales , Línea Celular , Drosophila melanogaster/citología , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Humanos , Virus de la Rabia/química , Virus de la Rabia/patogenicidad , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Virales/biosíntesis , Proteínas Virales/genéticaRESUMEN
Monitoring mammalian cell culture with UVvis spectroscopy has not been widely explored. The aim of this work was to calibrate Partial Least Squares (PLS) models from off-line UVvis spectral data in order to predict some nutrients and metabolites, as well as viable cell concentrations for mammalian cell bioprocess using phenol red in culture medium. The BHK-21 cell line was used as a mammalian cell model. Spectra of samples taken from batches performed at different dissolved oxygen concentrations (10, 30, 50, and 70% air saturation), in two bioreactor configurations and with two strategies to control pH were used to calibrate and validate PLS models. Glutamine, glutamate, glucose, and lactate concentrations were suitably predicted by means of this strategy. Especially for glutamine and glucose concentrations, the prediction error averages were lower than 0.5060.10 mM and 2.2160.16 mM, respectively. These values are comparable with those previously reported using near infrared and Raman spectroscopy in conjunction with PLS. However, viable cell concentration models need to be improved. The present work allows for UVvis at-line sensor development, decrease cost related to nutrients and metabolite quantifications and establishment of fed-batch feeding schemes.
Asunto(s)
Reactores Biológicos , Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Espectrofotometría Ultravioleta/métodos , Animales , Calibración , Línea Celular , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Análisis de los Mínimos Cuadrados , Análisis Multivariante , Oxígeno/análisis , Oxígeno/metabolismo , Bicarbonato de Sodio/análisis , Bicarbonato de Sodio/metabolismoRESUMEN
S2 cell populations (S2AcRVGP2K and S2MtRVGP-Hy) were selected after transfection of gene expression vectors carrying the cDNA encoding the rabies virus glycoprotein (RVGP) gene under the control of the constitutive (actin) or inductive (metallothionein) promoters. These cell populations were cultivated in a 1 L bioreactor mimicking a large scale bioprocess. Cell cultures were carried out at 90 rpm and monitored/controlled for temperature (28 °C) and dissolved oxygen (10 or 50% air saturation). Cell growth attained ∼1.53 × 107 cells/mL after 34 days of cultivation. The constitutive synthesis of RVGP in S2AcRVGP2K cells led to values of 0.76 μg/107 cells at day 4 of culture. The RVGP synthesis in S2MtRVGP-Hy cell fraction increased upon CuSO4 induction attaining specific productivities of 1.52 μg/107 cells at days 45. RVGP values in supernatant as a result of cell lysis were always very low (<0.2 μg/mL) indicating good integrity of cells in culture. Overall the RVGP productivity was of 1.53 mg/L. Our data showed an important influence of dissolved oxygen on RVGP synthesis allowing a higher and sustained productivity by S2MtRVGP-Hy cells when cultivated with a DO of 10% air saturation. The RVGP productivity in bioreactors shown here mirrors those previously observed for T-flasks and shaker bottles and allow the preparation of the large RVGP quantities required for studies of structure and function.