Impact of stabilizer and thermal history on the storage stability of freeze-dried pharmaceuticals

Stabilization of labile biopharmaceuticals like proteins in amorphous solids is becoming increasingly important as more and more biologics are introduced. However, the mechanism of protein stabilization in dried glasses is still not completely understood. Since the understanding of this stabilization mechanism would be very useful as a guideline for rational design of stable pharmaceutical products including proteins, the objective of this study is to investigate how formulation and process impact the storage stability of dried proteins, and thus provide guidelines for optimizing the physical stability during storage. ^ First, the impact of protein formulation on the long-term stability was investigated by study of different sucrose/protein mass ratios. Protein-sugar interactions in freeze-dried solids were characterized by BET analysis of gravimetric water sorption isotherms. The extent of protein-sugar interactions in different proteins was then correlated with secondary structure preservation measured by Fourier Transform Infrared (FTIR) spectroscopy. Protein aggregation during long-term storage was quantified using Size Exclusion Chromatography. The global mobility corresponding to a long time scale was studied using a Thermal Activity Monitor (TAM). However, protein stabilization by sugar can NOT be completely explained by dilution effect, global mobility, or FTIR structure throughout the whole range of protein compositions. It was found that fast local dynamics, with a timescale of nanoseconds, measured with neutron backscattering, correlates very well with storage stability of protein formulations. Also, the free volume in the dried solids was estimated from high precision density data obtained from a gas pycnometer, and good coupling between protein stability and free volume was observed. ^ Second, the impact of thermal history on the long-term stability of pharmaceuticals was investigated by heat treatment (annealing) at different conditions. Storage stability of both a small molecule, ethacrynate sodium, and IgG1 protein systems improved upon annealing, and annealed samples actually showed less degradation than the un-treated control samples after storage in spite of some degradation during the heat treatment. The impact of thermal treatment on the enthalpy recovery has been investigated using Differential Scanning Calorimetry (DSC), and the optimum annealing temperature for maximum structural relaxation in the lyophilized amorphous systems was found to be about 15-20 °C below the glass transition temperature for all the systems studied. The global mobility, studied with TAM, was found to decrease significantly upon thermal treatment. However, solid state NMR and neutron backscattering studies of the local dynamics of the protein system showed that annealing does not measurably impact the local motions. Also there is no significant change in protein native structure as measured by solid-state FTIR upon annealing. Given the similar local dynamics and specific surface area, the improved stability upon thermal treatment is mainly a result of decrease in global mobility. These studies help provide an understanding of the critical factors responsible for stabilization of pharmaceuticals by thermal treatment, and provide guidance on how to optimize annealing condition to improve the pharmaceutical stability.