(T1430-09-48) Exploring Mannitol's Influence on Protein Stability: Spray-Dried rHGH Formulations

Purpose: The study investigates the influence of spray drying with mannitol on the stability of recombinant Human Growth Hormone (rHGH). Owing to the vagaries during the spray drying process, excipients are frequently employed to enhance protein stability. The optimal protein-mannitol ratio was found to preserve the structural integrity of both the protein and mannitol crystals under various spray drying conditions. No noticeable differences were observed in the secondary structure and crystallization patterns between the initial samples and those subjected to testing for that optimal ratio.

Methods: The spray drying process was conducted with varying protein-mannitol ratios, with an increasing concentration of mannitol. The parameters used included protein-mannitol ratios of 1:0.5, 1:1.5, 1:4, and 1:6, with a constant feed rate for each batch. The impact of different aspirator rates Inlet temperatures (80°C, 100°C, 120°C) on protein’s α-Helix and the crystallization peaks of the spray-dried powder were evaluated using Circular Dichroism and PXRD, respectively. The batches were subjected to a stability study in a chamber set at 40°C/75%RH for 1 month to assess the impact of crystallization on the protein structure. The effect of the stability study on the α-Helix (%) and the crystallization property of the spray-dried powder was also evaluated. The dimerization of the protein was estimated using SDS PAGE for all samples (both initial and 1-month stability samples). The morphology and particle size of the spray-dried powder were examined using Scanning Electron Microscopy (SEM) for the initial samples.

Results: Aspirator Impact on protein α – Helix: Batches utilizing a 100% aspirator percentage displayed a notably diminished α-helix confirmation. Conversely, batches with aspirator percentages around 85% and 70% exhibited a substantial amount of α-helix, indicating that mannitol stabilizes the protein structure at these lower aspirator percentages. Aspirator Impact on spray dried powder crystallization peaks: Aspirator plays an important role in the fineness of the spray dried particles, increase in aspirator percentage increases the surface area of the spray leading to more fine particles. The crystallization peaks are clearly seen in the batches with high aspirator percentage, but a clear amorphous halo is visible and low aspirator percentage. Aspirator percent has some impact on the crystallization property of the powder. Impact of Inlet Temperature on protein α – Helix: Spray drying temperature has a 10% impact on the protein stabilization which was significant. As the temperature increases the crystalline peaks are more predominant in the PXRD analysis which shows that the protein stabilizing nature of mannitol is weakened which was reflected in the decrease in the α-helix content. Impact of Temperature on spray dried powder crystallization peaks: As the temperature increases the polymorphic nature of mannitol changes as it is influenced by spray drying temperature. The number of crystalline peaks increased in batches with higher inlet temperature and a decrease in α – Helix (%) indicates that the crystallization has an impact on stability of secondary structure. Stability study impact on α – Helix (%): A one-month study conducted at 40°C and 75% relative humidity examined several batches to assess the influence of crystallization on protein secondary structure. The study focused on the ratio of protein to mannitol, with varying ratios analyzed for their impact. The results revealed notable decrease in the α-helix percentage for ratios 1:0.5, 1:1.5 and 1:6. Conversely, the 1:4 ratio displayed an increase or no change in α-helix percentage indicating that 1:4 is the optimal ratio for stabilizing the protein. Stability study impact on crystallization property of spray dried powder. The PXRD study reveals additional crystalline peaks during stability, suggesting potential crystallization or form conversion in the stability chamber, which could lead to a decrease in the α-Helix %. SDS PAGE study: The SDS PAGE study was conducted to further investigate the α-Helix % loss, which could be due to protein fragmentation or aggregation in the 40/75 chamber. The study found clear dimer bands in the stability samples, indicating that mannitol crystallization during stability could reduce the secondary structural stability of the protein by forming dimers. See attachment 3 Scanning electron microscopy (SEM): The morphology of the spray dried powder and particle size estimation are done by SEM for initial samples.

Conclusion: The study found that variations in aspirator settings and temperature during Spray Drying significantly affect the secondary structure of proteins and mannitol crystallization.
The stability study showed no visible differences in the secondary structure of proteins or mannitol crystallization patterns between the initial and stability-tested samples, confirming that the optimal ratio (1:4) effectively maintains the integrity of both the protein structure and mannitol crystallization under different conditions.

References: Altay Benetti A, Bianchera A, Buttini F, Bertocchi L, Bettini R. Mannitol Polymorphs as Carrier in DPIs Formulations: Isolation Characterization and Performance. Pharmaceutics. 2021; 13(8):1113. https://doi.org/10.3390/pharmaceutics13081113
Ohtake, Satoshi, Yoshiko Kita, and Tsutomu Arakawa. "Interactions of formulation excipients with proteins in solution and in the dried state." Advanced drug delivery reviews 63.13 (2011): 1053-1073

Aspirator effect on rHGH secondary structure and crystallization

Stability study effect on rHGH secondary structure and mannitol crystallization peaks

Sodium Dodecyl sulphate – polyacrylamide gel electrophoresis (SDS PAGE) - non-reducing and reducing