(M1130-05-28) Understanding the Impact of L-leucine on Stability and Inhalation Potential of Dry Powder Protein Formulations

Purpose: The last decade has seen notable progress in the development of biotherapeutic agents. While liquid formulations have traditionally been predominant, there is a growing interest in solid-state alternatives due to their improved physico-chemical stability. Spray drying is a widely employed method for manufacturing solid-state protein therapeutics intended for pulmonary delivery. Common stabilizers like trehalose play a crucial role in stabilizing proteins against degradation during the spray drying process. However, the hygroscopic nature of these spray-dried sugars poses a risk of moisture absorption and consequent instability. Amino acids such as L-Leucine have garnered attention as dispersibility enhancers and protectors against high humidity conditions, although their interactions with proteins remain less explored. This study aims to investigate the role of L-leucine in spray-dried protein formulations.

Methods: Bovine serum albumin (BSA) was selected as the model protein, with trehalose as the stabilizer and L-leucine as the dispersion enhancer. Formulations were prepared with varying ratios of trehalose and L-leucine while keeping the protein ratio constant (Table 1). Spray drying was conducted using a BUCHI B-290 spray dryer, with inlet temperature set at 120°C and outlet temperature between 60°C to 70°C. Physical stability was measured by size exclusion chromatography, and protein instability assessed by percent loss of monomer peak area. Aerosol performance was evaluated using a Next Generation Impactor (NGI), with FPF determined for particles below 5 µm. Increased moisture conditions were tested at 75% RH for one week. Scanning electron microscopy (SEM) was employed to understand the surface morphology of spray-dried protein formulations. Statistical analysis utilized unpaired t-tests or one-way ANOVA with Tukey-Kramer post hoc tests via GraphPad Prism Software, with significance set at p < 0.05.

Results: Figure 1a illustrates a substantial decrease in percent monomer content over a 90-day period for formulations containing 100% BSA and B:L – 3:7 (p < 0.001), indicating diminished stability, whereas formulations with added trehalose maintained stability over the same duration. Notably, trehalose supplementation significantly bolstered stability, with higher concentrations showing further improvement. This observed enhancement can be attributed to trehalose's stabilizing effect, effectively preserving the protein structure. Conversely, the increased monomer loss in formulations with higher L-leucine content (B:L – 3:7) may be attributed to L-leucine crystallization and the absence of trehalose, suggesting that L-leucine alone does not stabilize the protein. Turning to aerosol performance (Figures 2A and 2B), formulations stored below 20% relative humidity for up to 90 days showed no significant differences (Fig. 1b). However, under 75% relative humidity conditions, formulations with high trehalose ratios exhibited a sharp decline in fine particle fraction (FPF) and signs of agglomeration (Fig.1c). Agglomeration of spray-dried protein formulations was also evident in the SEM images as indicated by figure.3. Increasing the L-leucine ratio resulted in a notable increase in percent FPF, indicating enhanced aerosol performance. This beneficial effect was attributed to the anti-hygroscopic properties of L-leucine, which counteract agglomeration induced by trehalose, thereby facilitating improved dispersion and delivery of the protein formulation via inhalation.

Conclusion: In conclusion, addition of L-leucine led to a decrease in protein stability owing to its propensity for crystallization. However, the increase in L-leucine content improved the aerosol performance of formulations stored at a high relative humidity of 75%. This enhancement may be attributed to the migration of L-leucine to the particle surface, a phenomenon dependent on the L-leucine concentration in the formulation. Thus, this study highlights the importance of stability and aerosolization regarding the use of L-leucine in protein formulations

References: Vehring, Reinhard. "Pharmaceutical particle engineering via spray drying." Pharmaceutical research 25.5 (2008): 999-1022.
Lechanteur, Anna, and Brigitte Evrard. "Influence of composition and spray-drying process parameters on carrier-free DPI properties and behaviors in the lung: A review." Pharmaceutics 12.1 (2020): 55.

Acknowledgements: The work was supported by the National Institute of Allergy and Infectious Diseases of the National Institute of Health under Award Number R01AI146160. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Health.


Table 1. Formulation details


Figure 1. In vitro aerosol performance of spray-dried protein powders; a) Impact of increasing ratios of leucine on the FPF of spray-dried protein formulations; b) FPF of the formulations immediately post spray drying and on 90-day storage at RH less than 20%; c) FPF of the formulations at RH less than 20% and at 75 % (mean ± SD;*, p < 0.05; **, p< 0.01; ***,p < 0.001; ****, p< 0.0001; ns no significant difference)


Figure 2. Scanning electron micrographs of spray-dried BSA: trehalose: leucine formulation on storage at a relative humidity of: A) less than 20 and B) greater than 75% for 1 Week; 1or 1a) 100% BSA; 2 or 2a) B:T – 3:7; 3 or 3a)B: E (T:L) - 3:7 (9:1); 4 or 4a) B: E (T:L) - 3:7 (6:1); 5 or 5a) B: E (T:L) - 3:7 (4:1); 6 or 6a) B: E (T:L) - 3:7 (2:1); 7 or 7a) B: E (T:L) - 3:7 (1:2); 8 or 8a) B:L – 3:7.