Impact of Drying Technologies on the Physical Stability and Aerosol Performance of Biological Formulations

Solid-state biological formulations are generally more stable than their liquid counterparts. As a result, drying techniques that incorporate stabilizers, such as sugars, are commonly used to maintain the stability of macromolecules in their dry state. Lyophilization is the most widely used method for producing solid-state biologics. This process employs low temperature and pressure to remove moisture from liquid formulations through sublimation. The resulting formulations are typically reconstituted and administered via the parenteral route. However, lyophilization produces cakes that are not readily dispersible, making them unsuitable for alternative delivery methods such as pulmonary delivery.

To overcome this limitation, other drying techniques, including spray drying and spray-freeze drying, have been explored to produce solid-state biological formulations. The choice of drying method requires careful consideration of its advantages and limitations. While lyophilization effectively preserves the structural integrity of biologics, it is both time-consuming and expensive. Spray drying, on the other hand, is a faster process but may expose sensitive components to high temperatures, potentially compromising their stability. Ongoing research aims to develop more stable drug products by investigating alternative drying techniques, such as spray-freeze drying, supercritical spray drying, and thin-film freeze drying.

Although a well-established drying process can stabilize biologics, it may also introduce additional stress unrelated to formulation. Excipients, such as disaccharides, are incorporated to protect biomolecules from freezing stress during lyophilization, thermal stress during spray drying, or shear stress from atomization in spray drying and spray-freeze drying. The selection of excipients depends not only on the drying technique but also on the intended mode of delivery after drying. For instance, sucrose and trehalose are widely used as stabilizers for biological formulations in lyophilization. Meanwhile, excipients such as mannitol, leucine, and trileucine function as dispersion enhancers in spray drying, improving powder flow and enabling pulmonary delivery of spray-dried formulations.

It is crucial to understand how excipients influence both the physical stability and aerosol performance of dried biological formulations. While an excipient may help stabilize a biomolecule, it may also negatively impact aerosol performance. Striking a balance is essential in addressing the dual impact of excipients on stability and delivery efficiency.

The purpose of Chapter One was to examine the effect of a commonly used excipient, mannitol, on the stability and aerosol performance of spray-dried protein formulations. The study found that protein formulations containing more than 33% mannitol by weight exhibited crystallization tendencies, leading to increased monomer loss over time. Solid-state nuclear magnetic resonance (ssNMR) data further indicated mixing heterogeneity between BSA and mannitol in formulations with high mannitol content. Additionally, fine particle fraction (FPF) decreased over time for BSA:mannitol formulations with ratios of 2:1, 1:2, and 1:5, due to particle agglomeration caused by mannitol crystallization. These findings highlight the significant influence of excipients like mannitol on the aerosol performance and long-term stability of spray-dried protein formulations. This research aims to bridge the knowledge gap between using excipients for stability and achieving optimal aerodynamic performance in spray-dried and spray-freeze-dried biologics. While low amounts of mannitol enhanced protein stabilization, higher amounts reduced protein stability and aerosol performance. Therefore, additional excipients or combinations of excipients was needed to be evaluated to achieve both stability and optimal aerosol performance.

To address this, a combination of trehalose and L-leucine was investigated for its effects on stability and aerosol performance. Trehalose is a well-known stabilizer that plays a crucial role in preventing protein degradation during spray drying and storage. However, the hygroscopic nature of spray-dried trehalose can lead to moisture absorption and subsequent protein instability. Amino acids such as L-leucine have been studied as an excipient that helps mitigate the effects of high humidity on hygroscopic formulations. However, interactions between L-leucine, protein, and trehalose remain less understood. This chapter systematically examined the role of L-leucine and trehalose in enhancing the physical and aerosol stability of spray-dried protein formulations. Results showed that an L-leucine concentration of 46.7% w/w effectively inhibited the crystallization of amorphous trehalose and prevented particle agglomeration, even under high humidity conditions (75% RH).

Beyond protein formulations, this research also sought to apply drying principles to complex systems such as RNA/lipid nanoparticles (RNA/LNPs). This led to the development of lyophilized and spray-freeze-dried RNA/LNPs. The impact of varying sucrose concentrations was evaluated in lyophilized RNA/LNP formulations, while the effects of the spray-freeze drying (SFD) process—including the introduction of annealing during freezing—were assessed for SFD RNA/LNPs. For lyophilized formulations, long-term stability studies demonstrated that they were superior to solution-state formulations. For SFD formulations, introducing annealing and using sucrose at concentrations of 10% w/v or higher proved to be the most effective approach.

Lastly, the final part of this thesis summarizes key conclusions and provides recommendations for future research directions.