Amino acids as stabilizers for lysozyme during the spray-drying process and storage

Area of Science:

• Protein formulation and stabilization
• Pharmaceutical excipients
• Biophysical chemistry

Background:

• Amino acids (AAs) are utilized as excipients in protein formulations for their stabilizing properties.
• The precise mechanisms of AA-mediated protein stabilization remain incompletely understood.

Purpose of the Study:

• To investigate the impact of distinct amino acids (AAs) on the stability of a model protein, lysozyme (LZM), during spray-drying and storage.
• To elucidate the role of AA physicochemical properties and microenvironmental pH in protein stabilization.

Main Methods:

• Selection of five distinct AAs (Arginine, Leucine, Glycine, Tryptophan, Aspartic acid) using multivariate data analysis.
• Spray-drying of LZM with selected AAs (1:1 wt ratio).
• Evaluation of solid form, residual moisture, hygroscopicity, morphology, secondary/tertiary structure, and enzymatic activity before and after accelerated storage (40°C/75% RH for 30 days).

Main Results:

• Spray-dried LZM with Arginine, Tryptophan, or Aspartic acid formed amorphous powders, while Leucine or Glycine resulted in crystalline powders.
• Recrystallization and polymorph transitions were observed after accelerated storage.
• Tertiary structural changes and decreased enzymatic activity were noted for LZM formulated with Arginine or Aspartic acid, linked to pH effects.

Conclusions:

• Amino acid selection significantly influences the solid-state properties and stability of spray-dried proteins.
• Beyond vitrification and water replacement theories, microenvironmental pH generated by basic or acidic AAs is critical for protein stability in solid formulations.
• This highlights the importance of considering AA physicochemical properties for optimizing protein stabilization strategies.