High temperature is known to cause some instability in polysaccharide-protein conjugated vaccines and studies under stress conditions may be useful in determining whether short-term accidental exposure to undesired conditions can compromise product quality. In this study, we examined the structural stability of three industrial batches of Brazilian Meningococcal C conjugate bulk (MPCT) incubated at 4, 37, and 55 °C for 5 weeks. The effect of exposure to the storage temperatures was monitored by HPLC-SEC, CZE, CD and NMR techniques. The immunological significance of any physicochemical changes observed in MPCT was determined by SBA and ELISA assays of serum from immunized mice. Fluorescence emission spectra at 4 and 37 °C were similar among all samples and compatible with the native fold of the carrier protein. Fluorescence spectra of MPCT stored at 55 °C decreased in intensity and had a significant red-shift, indicating conformational changes. Far-UV CD spectra revealed a trend toward loss of structural conformation as storage temperature was increased to 55 °C. The NMR data showed modified signal intensity of the aromatic and aliphatic residues, mainly for samples incubated at 55 °C, suggesting a partial loss of tertiary structure. About 50% free saccharide content was found in bulks stored at 55 °C, but no difference was observed in the IgG or SBA titers. The present study showed physicochemical methods alone are insufficient to predict the biological activity of a MPCT conjugate vaccine without extensive validation against immunological data. However, they provide a sensitive means of detecting changes induced in a vaccine exposed to adverse environmental condition.
The fragmentation patterns of the precursor ions were compatibles with amino acid cleavages [M+H-H2O] + ; [M+H-NH3 ] + ; [M+H-HCO2H] + and [M+H-CH3CO2H]+. In order to perform SRM studies the transitions 148.1®84.1 and 134.1®74.0 were selected for glutamic and aspartic acids, respectively. The area obtained for the MS/MS peaks selected for glutamic acid was proportional to its concentration. The standard curve was linear in the interval of 2.0-100.0 pmol/ µL (R2> 0.99; SD <10;Bias % < 5). Conclusion: The results so far obtained suggest that LCMS/MS can be used to quantify amino acids targets with accuracy and precision. Subsequent experiments will be carry out with hydrolyzed MTT and its activated form.
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