No abstract

The impact on immunogenicity of the degree of adsorption of three Streptococcus pneumoniae (Sp) vaccine antigens to aluminum adjuvants was studied. The three antigens evaluated (Sp1, Sp2 and Sp3) were highly adsorbed by aluminum hydroxide adjuvant, but not adsorbed by aluminum phosphate adjuvant. All of the Sp antigens adjuvanted with aluminum hydroxide elicited higher antibody responses in mice than formulations prepared with aluminum phosphate or non-adjuvanted antigen. Varying the percent aluminum-bound Sp antigen in the formulated vaccine affected the observed antibody responses. These observations suggest that the antibody response observed for Sp antigens in this study is stimulated by a depot effect of the antigen bound to an aluminum adjuvant.

This methodology provides a direct measurement of the concentration of a vaccine antigen adsorbed onto an aluminum adjuvant.

The adsorption of three Streptococcus pneumoniae (Sp) vaccine antigens by aluminumcontaining adjuvants was studied. The antigens showed high binding affinity isotherms with aluminum hydroxide adjuvant described by the Langmuir equation but virtually no binding to aluminum phosphate adjuvant. The effects of ionic strength and ethylene glycol were evaluated to determine whether electrostatic or hydrophobic interactions were responsible for the observed binding to aluminum hydroxide, but no significant change in the adsorptive capacity was observed at either high ionic strength nor high concentrations of ethylene glycol for any of the antigens. This indicates that neither electrostatic nor hydrophobic interactions appear to be responsible for the observed binding, which means that ligand exchange may be the primary mechanism for this interaction. Further studies to evaluate the ability to elute a Sp antigen from aluminum hydroxide using fibrinogen (adsorptive coefficient 2.2 mL/µg) as a competitive protein resulted in evidence that Sp antigen follows the trend that proteins with higher adsorptive coefficients are able to displace those with lower adsorptive coefficients. It was also noted that the Sp antigens and α-lactalbumin (adsorptive coefficient 1.8 mL/µg) have similar adsorptive coefficients indicative of high affinity binding isotherms but do not contain phosphate, which has previously been used to explain ligand exchange for such proteins as α-casein and hepatitis B surface antigen (HBsAg). Further investigations using α-lactalbumin as a model protein may elucidate the binding interaction between the antigens in this study and aluminum adjuvants.

A reversed-phase high-performance liquid chromatography method using evaporative light-scattering detection is developed for the determination of residual octylglucoside (OG) levels after a detergent exchange step for in-process samples of a vaccine antigen. The reversed-phase column not only provides separation of the OG but also functions as an extraction column to remove the vaccine antigen from the sample, thereby eliminating off-line sample manipulations. In addition to column selection, the mobile phase is optimized to enhance extraction and separation. The vaccine antigen is irreversibly bound to the column, allowing nonprotein components to interact with the column for separation and elution. The assay is linear over the range of 0.00050-0.050% OG. Precision tested at 0.0010% and 0.0050% OG is 2.9% and 7.2% relative standard deviation, respectively. The limits of quantitation and detection are determined to be 0.00050 and 0.000125% OG, respectively. Accuracy is determined to be 103 and 98%, based on spike recoveries of 0.0010% and 0.0050% OG, respectively.