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

Chengqian Zhang; Flemming Steen Jørgensen; Marco van de Weert; Simon Bjerregaard; Jukka Rantanen; Mingshi Yang

doi:10.1016/j.ijpharm.2024.124217

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

Int J Pharm. 2024 May 10:659:124217. doi: 10.1016/j.ijpharm.2024.124217. Online ahead of print.

Authors

Chengqian Zhang¹, Flemming Steen Jørgensen², Marco van de Weert¹, Simon Bjerregaard³, Jukka Rantanen¹, Mingshi Yang⁴

Affiliations

¹ Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark.
² Department of Drug Design and Pharmacology, University of Copenhagen, Copenhagen, Denmark.
³ Ferring Pharmaceuticals, Copenhagen, Denmark.
⁴ Department of Pharmacy, University of Copenhagen, Copenhagen, Denmark; Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, China. Electronic address: mingshi.yang@sund.ku.dk.

PMID: 38734275
DOI: 10.1016/j.ijpharm.2024.124217

Abstract

Amino acids (AAs) have been used as excipients in protein formulations both in solid and liquid state products due to their stabilizing effect. However, the mechanisms by which they can stabilize a protein have not been fully elucidated yet. The purpose of this study was to investigate the effect of AAs with distinct physicochemical properties on the stability of a model protein (lysozyme, LZM) during the spray-drying process and subsequent storage. Molecular descriptor based multivariate data analysis was used to select distinct AAs from the group of 20 natural AAs. Then, LZM and the five selected AAs (1:1 wt ratio) were spray-dried (SD). The solid form, residual moisture content (RMC), hygroscopicity, morphology, secondary/tertiary structure and enzymatic activity of LZM were evaluated before and after storage under 40 °C/75 % RH for 30 days. Arginine (Arg), leucine (Leu), glycine (Gly), tryptophan (Trp), aspartic acid (Asp) were selected because of their distinct properties by using principal component analysis (PCA). The SD LZM powders containing Arg, Trp, or Asp were amorphous, while SD LZM powders containing Leu or Gly were crystalline. Recrystallization of Arg, Trp, Asp and polymorph transition of Gly were observed after the storage under accelerated conditions. The morphologies of the SD particles vary upon the different AAs formulated with LZM, implying different drying kinetics of the five model systems. A tertiary structural change of LZM was observed in the SD powder containing Arg, while a decrease in the enzymatic activity of LZM was observed in the powders containing Arg or Asp after the storage. This can be attributed to the extremely basic and acidic conditions that Arg and Asp create, respectively. This study suggests that when AAs are used as stabilizers instead of traditional disaccharides, not only do classic vitrification theory and water replacement theory play a role, but the microenvironmental pH conditions created by basic or acidic AAs in the starting solution or during the storage of solid matter are also crucial for the stability of SD protein products.

Keywords: Microenvironmental pH; Molecular descriptors; Solid form; Stability; Therapeutic protein.