Such destabilization is usually rationally attributed to the shearing stress in the nozzle, thermal stress within drying, and surface adsorption of protein at the air-liquid interfaces during atomization . molecules from molecular degradation and improved immediate and accelerated stability of spry-dried antibodies. Validation of the optimization study indicated high degree of prognostic ability of response surface methodology in preparation of stable spray-dried IgG. Graphical abstract Graphical abstract Open in a separate window Spray drying of IgG in the presence of Trehalose, Cysteine and Tween 20. strong class=”kwd-title” Keywords: Spray-drying, IgG, Molecular stability, Experimental design, Box-Behnken, Aerosol delivery Background Antibody-based drugs are regarded as major influential components in the treatment of cancers, autoimmune, and inflammatory diseases . Considering about 50 approved monoclonal antibodies, Omalizumab, Bevacizumab, Palivizumab, and Cetuximab are administered in respiratory diseases and more than 9 ZK-261991 molecules are at different ZK-261991 stages of clinical trials . Spray-drying is an emerging technology for the processing of antibody dry powders . However, real antibody solutions have been shown to become substantially aggregated during this process [4, 5]. Such destabilization is usually rationally attributed to the shearing stress in the nozzle, thermal stress within drying, and surface adsorption of protein at the air-liquid interfaces during atomization . Incorporation of appropriate excipients is usually therefore critical for preserving protein stability. Trehalose and Tween 20 are applied as excipients in preventing proteins against destabilization during the spray-drying process . Trehalose is one of the most encouraging inhibitors of antibody aggregation with high glass transition heat, low hygroscopicity, and strong water replacement efficacy . Surfactants such as Tween 20 have also been shown to occupy the airCliquid interface in competition with protein molecules, thus avoiding subsequent protein unfolding and aggregation [9, 10]. Although polysorbates were repeatedly ZK-261991 shown to stabilize numerous proteins against surface denaturation within spray-drying, auto-xidation of poly-oxy ethylene groups at high temperatures is considered as a major challenge in Tween-containing formulations after storage [11, 12]. Our previous investigation launched Cysteine as an appropriate excipient in IgG formulation regarding antibody stability as well as its aerodynamic behavior . In the current study, the combination of Cysteine, Trehalose, and Tween 20 was applied to not only enhance the molecular and thermodynamic stability of IgG, but Rabbit Polyclonal to OR2I1 also ZK-261991 to ZK-261991 examine whether the Cysteineas an anti-oxidantcould protect Tween 20 from auto-oxidation following storage. One particular feature of this study is usually a statistical comparison between Cysteine, Trehalose, and Tween 20 as stabilizing brokers for the spray-dried IgG formulation. The other objective was to probe the existing interactions between immunoglobulin G and sugar, and amino acid and surfactant. To achieve this purpose, a Box-Behnken experimental design was applied to optimize the best combination of the aforementioned additives in spray-dried IgG formulation as a model antibody. The evaluated responses were yield of process, beta sheet content of antibody, and amount of induced aggregation following process and upon storage. Subsequently, optimized formulation was characterized in terms of surface morphology and amorphous/crystalline pattern for further aerosol delivery. Methods Materials L-Cysteine, Tween 20, Phosphoric acid, and KBr were purchased from Sigma (Germany); Trehalose dihydrate, Sodium sulfate, and Disodium hydrogen phosphate were provided by Merck (Germany); Human IgG with molecular excess weight of about 150?kDa was supplied by Kedrion (Italy); antibody answer was dialyzed with deionized water (bag cut off: 8 KDa). Box-Behnken experimental design A three-factor, three-level Box-Behnken design was employed for the optimization process using the statistical software Design Expert 6.0.10 (Stat-Ease Inc., USA). The non-linear quadratic model generated by the design was: Yi =?b0 +?b1X1 +?b2X2 +?b3X3 +?b12X1X2 +?b13X1X3 +?b23X2X3 +?b11X12 +?b22X22 +?b33X32 1 In which Yi is the measured response of each dependent variable; b0 is the intercept; b1 to b33 are the regression coefficients of the factors; and X1, X2, and X3 are the coded levels of impartial variables. The term X1 X2, X1 X3, X2 X3 and Xi 2 ( em i /em ?=?1, 2, or 3) exhibit the interaction and the quadratic terms respectively. A description of the impartial and dependent variables is usually given in Table?1. The models were evaluated in terms of.