RESUMEN
This study was designed to assess the effects of cooling rate, storage temperature, and formulation composition on trehalose phase distribution and protein stability in frozen solutions. The data demonstrate that faster cooling rates (>100°C/min) result in trehalose crystallization and protein aggregation as determined by Fourier Transform Near-Infrared (FT-NIR) spectroscopy and size-exclusion chromatography, respectively. Conversely, at slower cooling rates (≤1°C/min), trehalose remains predominantly amorphous and there is no effect on protein stability. Evaluation of storage temperatures demonstrates that aggregation increases more rapidly at -14°C compared with higher (-8°C) and lower (-20°C) storage temperatures; however, a relatively higher amount of cumulative aggregation was observed at lower (-20°C) temperature compared with higher storage temperatures (-14°C and -8°C). Further evaluation of the effects of formulation composition suggests that the phase distribution of amorphous and crystallized trehalose dihydrate in frozen solutions depends on the ratio of trehalose to mAb. The results identify an optimal range of trehalose-mAb (w/w) ratio, 0.2-2.4, capable of physically stabilizing mAb formulations during long-term frozen storage-even for fast cooled (>100°C/min) formulations.
Asunto(s)
Proteínas/química , Trehalosa/química , Química Farmacéutica/métodos , Cromatografía en Gel/métodos , Cristalización/métodos , Estabilidad de Medicamentos , Almacenaje de Medicamentos/métodos , Liofilización/métodos , Congelación , Estabilidad Proteica , Soluciones/química , Espectroscopía Infrarroja por Transformada de Fourier/métodos , TemperaturaRESUMEN
Protein aggregation is a common issue encountered during manufacture of biotherapeutics. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the environment (eg, media components) and implementing appropriate strategies to minimize the extent of aggregation. Steps to remove aggregates have been successfully used at a manufacturing scale. Care should be taken when developing a process to monitor the compatibility of the equipment and process with the protein to ensure that potential aggregation is minimized.
Asunto(s)
Química Farmacéutica , Proteínas/química , Técnicas de Cultivo de Célula , Interacciones Hidrofóbicas e Hidrofílicas , Tamaño de la Partícula , Proteínas/aislamiento & purificaciónRESUMEN
The destabilizing effect of a multidose preservative, benzyl alcohol, on IFN-gamma was investigated. Hydrogen-deuterium isotope exchange (HX) detected by mass spectrometry (MS) was used to detect tertiary structure changes and measure global unfolding rates. The experiments showed that tertiary structure changes previously reported using circular dichroism may involve only a limited portion of the protein with the hydrophobic core of the protein remaining intact. Protein unfolding rates measured by hydrogen exchange were very sensitive to benzyl alcohol concentration, and increased markedly when salt was also added. Dynamic light scattering and size-exclusion chromatography showed that a small fraction of the protein formed large aggregates during the first few days. Measurements at longer incubation times (up to 8 days) showed that a significant fraction of protein was trapped in a structure less protected from hydrogen exchange, but not completely unfolded. This fraction of protein may be responsible for the irreversible loss of activity observed in earlier studies.