Freeze-dried immunoglobulin G (IgG) incorporating trehalose and human serum albumin (HSA) was statistically evaluated regarding the existence of synergism between additives on the stability profile. The levels of HSA (X1) and trehalose (X2) were independent variables. Aggregation following the process (Y1), after 2 and 3 months at 40°C (Y2) and (Y3), respectively, along with the rate constant of aggregation (Y4) were dependent variables. Aggregation and beta-sheet conformation were quantified through size-exclusion chromatography (SEC-HPLC) and Fourier transform infrared spectroscopy (FTIR). Central composite design (CCD) suggested the best formulation. The integrity and thermodynamic stability of optimized formulation were investigated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and differential scanning calorimetry (DSC). The calculated responses were Y1, 0-0.90%; Y2, 0.4-4.3%; Y3, 2.10-13.46%; and Y4, 0.16-0.69 1/month. The optimized formulation had 10 mg IgG, 86 mg trehalose, and 1 mg HSA with observed responses of Y1, 0.01%; Y2, 0.51%; Y3, 3.08%; and Y4, 0.33 1/month. The models were statistically well-fitted. The optimized formulation was amorphous during freeze-drying (FD), and no fragmentation was observed. Trehalose and HSA demonstrated statistical synergism. CCD was successfully employed to recommend the best ratio of stabilizers and achieve the maximum stabilization of IgG as a model freeze-dried antibody.
Keywords: central composite design; freeze-drying; human serum albumin; immunoglobulin G; stability; trehalose.