RESUMO
Application of Raman spectroscopy as a T cell characterization tool supporting cell therapy drug product development has been evaluated. Statistically significant correlations between a set of Raman signals and established flow cytometry markers associated with apoptosis of T cells detected during drug product cryopreservation are presented in this study. Our study results demonstrate the potential of Raman spectroscopy for label-free measurements of T cell characteristics relevant to cell therapy product design and process control.
Assuntos
Preparações Farmacêuticas , Análise Espectral Raman , Apoptose , Morte Celular , Terapia Baseada em Transplante de Células e Tecidos , Projetos Piloto , Análise Espectral Raman/métodos , Linfócitos TRESUMO
The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor that are produced by the Gram-positive bacterium, Bacillus anthracis. Current vaccines against anthrax use PA as their primary component. In this study, we developed a scalable process to produce and purify multi-gram quantities of highly pure, recombinant PA (rPA) from Escherichia coli. The rPA protein was produced in a 50-L fermentor and purified to >99% purity using anion-exchange, hydrophobic interaction, and hydroxyapatite chromatography. The final yield of purified rPA from medium cell density fermentations resulted in approximately 2.7 g of rPA per kg of cell paste (approximately 270 mg/L) of highly pure, biologically active rPA protein. The results presented here exhibit the ability to generate multi-gram quantities of rPA from E. coli that may be used for the development of new anthrax vaccines and anthrax therapeutics.
Assuntos
Antígenos de Bactérias/isolamento & purificação , Bacillus anthracis/genética , Toxinas Bacterianas/genética , Toxinas Bacterianas/isolamento & purificação , Escherichia coli/metabolismo , Antígenos de Bactérias/biossíntese , Toxinas Bacterianas/biossíntese , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Microbiologia Industrial/métodos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
Anthrax is caused by the gram-positive, spore-forming bacterium, Bacillus anthracis. The anthrax toxin consists of three proteins, protective antigen (PA), lethal factor, and edema factor. Current vaccines against anthrax use PA as their primary component since it confers protective immunity. In this work, we expressed soluble, recombinant PA in relatively high amounts in the periplasm of E. coli from shake flasks and bioreactors. The PA protein was purified using Q-Sepharose-HP and hydroxyapatite chromatography, and routinely found to be 96-98% pure. Yields of purified PA varied depending on the method of production; however, medium cell density fermentations resulted in approximately 370 mg/L of highly pure biologically active PA protein. These results exhibit the ability to generate gram quantities of PA from E. coli.