Impact of Protein Internal Dynamics, Process and Formulation Variables on Freeze-Dried Protein Quality Attributes

Lyophilization has been used to improve the quality of pharmaceutical proteins when sufficient stability in the aqueous state cannot be achieved. However, due to inherent challenges in both the freeze-drying process and formulation of proteins for freeze drying, efficient delivery of drug products with uniformly improved stability is not trivial. The overall objective of this work was to investigate freeze-dried protein quality attributes affected by stress factors arising from process and formulation. The natural variation in ice nucleation temperature (Tn) necessitates the control of ice nucleation to ensure uniformity in product quality across a batch. Further, it is less well understood what critical factors are attributed to protein stability after ice nucleation. Freezing parameters including Tn, shelf ramp rate and isothermal hold after ice nucleation were studied. Controlling ice nucleation at a higher temperature was found to not only improve average protein stability, but also improved batch uniformity on in-process stability. Additionally, the findings suggested that a long residence time in the freeze-concentrate, at a temperature above Tg’ of the formulation can result in significant protein aggregation. Further, to address whether there is a shelf location-dependent mass flow resistance (Rp) during primary drying, differences in Rp between center and edge vials were estimated from correlations that were established between Tn, SSA of the freeze-dried solid, and Rp. The findings provide insights into developing methods to accurately measure Rp in individual vials. Lastly, formulation variables can greatly impact stability during process and storage. Storage below Tg slows molecular mobility of the system on a pharmaceutical relevant timescale and reduces degradation. However, physical and chemical degradation still occurs below Tg; other motions, such as internal dynamics within a protein molecule, have been proposed to exist in solid proteins and potentially impacts long-term stability of lyophilized formulations. Solid-state hydrogen/deuterium (H/D) exchange with FTIR spectroscopy was employed to investigate this phenomenon and its potential impact on protein stability in the solid state. The studies demonstrated that the better stability in the rHSA:sucrose formulation correlated with lower extent of H/D exchange, which provides a measure of structure and/or dynamics in the protein formulation