RESUMEN
Changes in the measurements of a macromolecular biopharmaceutical's physical form are often used to predict changes in the drug's long-term stability. These can in turn be used as important markers of changes to a drug's efficacy and safety. Such stability estimates traditionally require human judgment and are frequently tentative. We introduce methods for developing mathematical models that predict a drug's long-term storage stability profile from measurements of short-term physical form and behavior. We measured the long-term (2 year) chemical and colloidal stability of Granulocyte Colony Stimulating Factor (GCSF) in 16 different liquid formulations. Shortly after formulations were placed on stability, we also employed various spectroscopic techniques to characterize the short-term thermal unfolding response of GCSF in the 16 formulations. The short-term data were processed using several data reduction methods, including reduction to spectra at low temperature, to melt curves, and to transition temperatures. Least squares fitting was used to predict the long-term stability measurements from the reduced short-term spectroscopic measurements. On the basis of the cross-validation and a permutation test, many of the long-term stability predictions have less than 1% probability of occurring by chance.
Asunto(s)
Química Farmacéutica/métodos , Factor Estimulante de Colonias de Granulocitos/química , Factores Inmunológicos/química , Modelos Moleculares , Fenómenos Químicos , Dicroismo Circular , Coloides , Estabilidad de Medicamentos , Excipientes/química , Factor Estimulante de Colonias de Granulocitos/genética , Factor Estimulante de Colonias de Granulocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Factores Inmunológicos/genética , Factores Inmunológicos/metabolismo , Análisis de los Mínimos Cuadrados , Nefelometría y Turbidimetría , Estabilidad Proteica , Estructura Secundaria de Proteína , Desplegamiento Proteico , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia , Temperatura de TransiciónRESUMEN
Microcalorimetry is frequently used as a high throughput predictive method in order to screen different formulations for their storage stability. However, the predictive power of measuring unfolding temperatures (Tm), although studied for the stability of proteins under stress, has not been investigated systematically for long term stability of pharmaceutical proteins yet. In this study, the microcalorimetric Tm is evaluated as a predictive tool for long term stability of 24 liquid formulations of Granulocyte Colony Stimulating Factor. Those were tested with respect to the effect of different buffer salts in different concentrations, different pH values, and the effect of two excipients: polysorbate 80 and hydroxy propyl-ß-Cyclodextrin. Formulations were first ranked based on the measured Tm. The same formulations were then ranked based on a long term stability study at 2-8 °C for a period of up to 24 months. For this study, standard analytical methods were used to assess both physical and chemical stabilities of the formulations on long term. Microcalorimetric Tm based ranking was then compared with the long term stability ranking. Determining Tm turned out to be a successful predictive tool to select good formulations and exclude bad ones with an acceptable low degree of error. In particular, physical long term stability at a storage temperature of 2-8 °C was better predicted by just measuring Tm than by conducting stress studies at elevated temperatures.