Optimization of Stable IgG Formulation Containing Amino Acids and Trehalose During Freeze-Drying and After Storage: a Central Composite Design.

Abstract

The physical and structural stability of freeze-dried immunoglobulin G (IgG) were examined by applying trehalose and amino acids (glycine, phenylalanine, and serine). The efficacy of amino acids was statistically compared considering their side-chain characteristics. The amount of amino acids (X1) and trehalose (X2) was considered as independent variables. Size exclusion chromatography (SEC-HPLC) was utilized to calculate the soluble aggregates, as dependent variables. The amounts of excipients were optimized through the central composite design (CCD). The beta-sheet conformation of IgG was quantified by Fourier transform infrared spectroscopy (FTIR). Thermal behavior and molecular integrity of IgG were evaluated by differential scanning calorimetry (DSC) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimized formulations of powders were as follows: 24.5mg serine-139.5mg trehalose, 14mg glycine-118mg Trehalose, and 25mg phenylalanine-139.5mg trehalose. The amounts of soluble aggregates after processing were 0, 4.50, and 2.20%, respectively. The corresponding induced aggregates following storage conditions were 1.02, 7.0, and 3.70%. In all preparations, there were no detectable fragments. The native conformation of IgG was well preserved in the presence of amino acids. Excluding the glycine-based sample with minor endotherm at about 45°C, serine and phenylalanine incorporating powders were fully amorphous at examination temperatures. Trehalose was more potent than the amino acids in the stabilization of IgG. Serine was the most effective amino acid; phenylalanine and glycine were the next ones, respectively. Glycine crystallization was assumed to have accounted for low stabilization capability. The statistically synergistic phenomenon was only observed in the co-application of trehalose and phenylalanine. Graphical abstract.