Analysis of vaccine: Structure, storage, moisture, and classification by infrared technology

Aims of this thesis
The research described in this thesis is to extend our knowledge on the properties of vaccines and antigens using fast and cheap infrared techniques. FTIR-ATR spectroscopy is applied to elucidate the structural changes when antigen is adsorbed by aluminium hydroxide and when it is subsequently released. The structural stability during the storage and heating process is investigated as well. The alterations of other parameters, such as adsorption, release, and pH are also investigated during the storage. FT-NIR is applied as a fast and non-destructive method to determine the residual moisture content in lyophilized vaccines and to classify and identify different vaccine products.

Organization of this thesisIn Chapter I, the background of the project and the introductions to infrared spectroscopy as well as chemometrics are given respectively. In Chapter II, the current techniques for protein structure analysis have been discussed, especially the FTIR-ATR method. In Chapter III, the structural changes of two model antigens are investigated when they are adsorbed by aluminium hydroxide and the structures refold to their native state when they are released from adjuvant. The mechanism of the adjuvant enhancing the immune response is discussed in this chapter too. Chapter IV describes the structural stability of adsorbed model antigens in thermal and aging process in comparison to the antigens in solutions. The results indicate that aluminium hydroxide can protect the structure of antigens against aggregation at high temperature and/or after long period storage. It is more pronounced for BSA. Further study is required to confirm if the protection is protein specific. Chapter V gives the alterations of model vaccines during storage at two different temperatures. It demonstrates that the change of adsorption capacity by aluminium hydroxide during the storage is protein specific. The release of adsorbed protein from adjuvant decreases as a function of storing time. The pH and NIR absorbance also change. The aging effects undergo faster modification at 37 ºC than 4 ºC during the storage. The rotation effects when preparing vaccines are also discussed in this chapter as well. NIR method for determination of residual moisture content of lyophilized allergen vaccines is  introduced in Chapter VI. Five different allergen vaccines are analyzed. Acceptable calibration model (RMESEP 10.20 μg H2O / vial with 2 PLS factors) is obtained for multi-products data. The models based on product-specific data are slightly better (Appendix B). However, a general multiproducts model is more attractive since one model can predict all samples. The reference data which are measured by Karl Fischer titration are presented in Appendix A. NIR spectroscopy is applied to classify and identify five different allergen vaccines (the information of samples in Appendix C) at different concentrations of antigens and different batches in Chapter VII. The results show that NIR spectroscopy can be used to classify different allergen vaccines at highest antigen concentration (100,000 SQ) without api. The highest concentration products can be separated from other low concentrations. The discrimination of different batches may also be possible but need further experiments to confirm this. All SIMCA results are given in Appendix C to Appendix S.