The effect of freeze-dried antibody concentrations on its stability in the presence of trehalose and hydroxypropyl-β-cyclodextrin: a Box–Behnken statistical design

Abstract

The present study aimed at preparation and optimization of stable freeze-dried immunoglobulin G (IgG) applying proper amount of antibody with efficient combination of trehalose and hydroxypropyl-β-cyclodextrin (HPβCD). Response surface methodology was employed through a three-factor, three-level Box–Behnken design. Amounts of IgG (X1), trehalose (X2) and HPβCD (X3) were independent variables. Aggregation following process (Y1), after one month at 45 °C (Y2), upon two month at 45 °C (Y3) and beta-sheet content of IgG (Y4) were determined as dependent variables. Results were fitted to quadratic models (except for beta-sheet content), describing the inherent relationship between main factors. Optimized formulation composed of 55.85 mg IgG, 52.51 mg trehalose and 16.01 mg HPβCD was prepared. The calculated responses of the optimized formulation were as follows: Y1 = 0.19%, Y2 = 0.78%, Y3 = 1.88% and Y4 = 68.60%, respectively. The thermal analysis confirmed the amorphous nature of optimum formulation and the integrity of IgG was shown to be favorably preserved. Validation of the optimization study demonstrated high degree of prognostic ability. The DOE study successfully predicted the optimum values of antibody as well as stabilizers for desirable process and storage stabilization of freeze-dried IgG.