Comparison of in vitro and in vivo methods to study stability of PLGA microencapsulated tetanus toxoid vaccines

The purpose of this study was to investigate the utility of various in vitr o and in vivo methods to assess the stability of experimental vaccines cont aining tetanus toroid (TT) within PLGA microspheres. In vitro, the breakdow n of the encapsulating polymers into their acid components led to changes i n the structure of TT, as determined by the physico-chemical methods, rende ring it undetectable by capture ELISA and altering its structural integrity . The changes in TT were directly related to increasing acidity of the vacc ine supernate. Purified toroid (not encapsulated) exposed to low pH (2.5) u nderwent similar changes but re-neutralisation of buffer containing free to roid, even after one week at pH 2.5 led to some re-folding of protein as de termined by fluorescence spectroscopy and gel filtration chromatography. Th e microencapsulated vaccines were still able to generate an antibody respon se in mice even after prolonged pre-incubation at 37 degreesC and the appar ent absence of detectable toroid in the vaccine supernate. Electron microsc opy demonstrated differences in the amount of degradation between different formulations of microspheres. Vaccines that had retained their spherical m orphology after incubation in vitro for up to 28 days were able:lo induce p rotective antibodies response equal to that of freshly prepared vaccines, w hich indicates that the toroid within intact microspheres remained immunoge nic. Immunochemical and physico-chemical detection methods, performed on an tigen released from PLGA vaccines in vitro, are valuable in providing infor mation on product characteristics but may not be able to predict effectiven ess and should be used with in vivo methods to evaluate the stability of su ch formulations. (C) 2000 Elsevier Science Ltd. All rights reserved.