Effect of formulation variables on adsorption of DTP-JE antigens on adjuvants

Abstract

Aluminium hydroxide gel (AH) and aluminium phosphate gel (AP) were individually adsorbed with diphtheria toxoid (DT), tetanus toxoid (TT) or JE antigen at different conditions: temperature 37 ํC and 9 ํC; pH 6.0 to 7.4 in 0.01 M phosphate buffer and centrifuged to precipitate the adsorbed adjuvant at 3,000 rpm. The extent of adsorption of adsorbed single antigen AH suggested that DT and JE favored to adsorb at 9 ํC than 37 ํC, whereas TT preferred at 37 ํC. In addition, a gradual decrease in an adsorption of all antigens was observed as the pH was raised. For AP, poor adsorption of all antigens was noted. At 9 ํC, JE displayed a higher degree of adsorption, while DT and TT favored at physiological temperature. The calculated quantity of AH used in combined preparations were less than that of AP. Therefore, AH was chosen to adsorb at 9 ํC, pH 6.5. The surface charge in deionized water of AH and JE were positive while AP, DT and TT were negativeand PT was slightly negative or neutral charge. The antigens possibly adsorbed on AH and AP by electrostatic force and ligand exchange mechanism. The particle size of adsorbed AH and AP were larger than blank adjuvants. Morphology of AH and AP by SEM were spherical complex particle and aggregate irregular shape, respectively. Adsorbed AH could be observed by SEM, while adsorbed AP showed three-dimensional surface morphology from AFM. The principle peak of IR spectra and X-ray diffraction bands of adsorbed AH and AP indicated that the adsorption had no effect on their chemical structures. The antigen contents combined preparations after storage at 2-8 ํC for 3 months and analyzed by ELISA showed more DT, TT and JE content from formulation of separate adsorption than that of competitive method. Mixing time affected the antigen content of DT, TT and JE whereas adsorption process and mixing time did not affect PT content.