An intrinsic fluorescence method for the determination of protein concentration in vaccines containing aluminum salt adjuvants.

Determination of protein concentration in vaccines containing aluminum salt adjuvant typically necessitates desorption of the protein prior to analysis. Here we describe a method based on the intrinsic fluorescence of tyrosine and tryptophan that requires no desorption of proteins. Adjuvanted formulations of three model Bordetella pertussis antigens were excited at 280 nm and their emission spectra collected from 290 to 400 nm. Emission spectra of protein antigens in the presence of aluminum salt adjuvants were able to be detected, the effects of adjuvants on the spectra were analyzed, and linear regressions were calculated. The fluorescence method proved to be very sensitive with a limit of quantification between 0.4 and 4.4 µg/mL and limit of linearity between 100 and 200 µg/mL, across the formulations tested. The fluorescence method was found to be influenced by adjuvant presence, type of adjuvant, adjuvant concentration, buffer and pH conditions. The method also demonstrated ability to monitor the percent adsorption of antigens to the adjuvants. Furthermore, intrinsic fluorescence showed good correlation with micro-Kjeldahl elemental assay in quantifying protein concentration. Being a non-invasive, quick and sensitive method, intrinsic fluorescence has the potential to be utilized as a high throughput tool for vaccine development and conceivably implemented in-line, using in-line fluorimeters, to monitor antigen concentration during formulation processing.

Biophysical Characterization and Thermal Stability of Pneumococcal Histidine Triad Protein D in the Presence of Zinc and Manganese.

The pneumococcal histidine triad protein D (PhtD) is believed to play a central role in pneumococcal metal ion homeostasis and has been proposed as a promising vaccine candidate against pneumococcal disease. To investigate for potential stabilizers, a panel of physiologically relevant metals was screened using the thermal shift assay and it was found that only Zn2+ and Mn2+ were able to increase PhtD melting temperature. Differential scanning calorimetry analysis revealed a sequential unfolding of PhtD and the presence of at least 3 independent folding domains that can be stabilized by Zn2+ and Mn2+. UV spectroscopy and fluorescence quenching studies showed significant Zn2+-induced tertiary structure changes in PhtD characterized by decreased accessibility of inner tryptophan residues to the aqueous solvent. Isothermal titration calorimetry data show no apparent binding to Mn2+ but revealed a Zn2+:PhtD exothermic interaction stoichiometry of 3:1 with strong enthalpic contribution, suggesting that 3 of the 5 histidine triads are accessible binding sites for Zn2+. Only Zn+2, but not Mn+2, was able to increase the thermal stability of PhtD in the presence of aluminum hydroxide adjuvant, making it a promising stabilizer excipient candidate in vaccine products containing PhtD.