Research On Pivotal Preparation Technology Of Freeze-drying Dos Age Form Of Haemophilus Influenzae Type B Conjugated Vaccine

As known, inoculating Haemophilus influenzae type b (Hib) conjugated vaccine was the most effective method to prevent invasive Hib diseases, such as children’s infectious meningitis and pneumonia. For now, not any kinds of Hib conjugated vaccines are produced, with the activation of CDAP (1-cyano-4-dimethylamino pyridinium), which was conjugated with TT (tetanus toxoid) by ADH linker. At the same time, the freeze-drying dosage form of Hib conjugated vaccine was still not producted by other domestic manufacturer, therefore, it is important to develop freeze-drying type of Hib conjugated vaccine. So in this study, both reponse surface design and orthogonal analysis were utilized to optimize the production technology of the raw Hib-TT bonder, and the new freeze-drying type of Hib conjugtaed vaccine was studied. In parallel, to quantitatively examine the purified Hib polysaccharide, the new ELISA method was developed with high stability and sensitivity.First section:The preparation of the stoste of Hib polysaccharide-TT conjugated vaccineIn this research, the purified Hib polysaccharide firstly was activated by CDAP, then with ADH linker, the activated polysaccharide could be covalently bound with TT, finally, the original polysaccharide-TT bonder was purified through Sepharose 4FF gel chromatograhic colunm. In the process of activation and conjugation, by the use of reponse surface design and orthogonal analysis, the key experimental steps were optimized. After optimization, the binding rate between Hib polysaccharide and TT increased obviously, and further test indicated that the immunogenicity, the content of polysaccharide and high polymer combination of original polysaccharide-TT vaccine was in accordance with the requirement of WHO. Second section:The study on the freeze-drying dosage of Hib conjugated vaccine and the test of the content of polysaccharide by ELISA methodIn this study, different protective agents, such as lactose, trehalose, dextran-40 and mannitol, were utilized during the research on the freeze-drying Hib conjugated vaccine. The experimental data showed that when dextran-40 and lactose were both used as the protective agent, the prepared freeze-drying Hib conjugated vaccine had the best shape and highest immunogenicity. After checking the freeze-drying vaccine product, its immunogenicity, the content of polysaccharide and high polymer combination was in accordance with the requirement of WHO. Compared with other dosages of Hib vaccines, the new freeze-drying type of Hib conjugated vaccine was more easily transported and preserved, which made a good basis for developing DTaP-sIPV-Hib conjugated vaccine in the future.Meanwhile, a new ELISA technology was designed to quantitatively test the antigen content of Hib polysaccharide. Compared with original methods, ELISA method had better precision, stability and sensitivity. In conclusion, not like chemical methods, the new ELISA method for Hib polysaccharide testing, had higher sensitivity, the result of which could not be influenced by impurity substance. Therefore, with the help of new ELISA technology, increasing the output of Hib polysaccharide and decreasing production cost of Hib conjugated vaccine will be realized in the future.