Formulation approach for the development of a stable, lyophilized formaldehyde-containing vaccine.

Formaldehyde has been used in the inactivation of a number of viral and bacterial toxins used in vaccines. In some cases, a small amount of formaldehyde may be necessary in order to prevent reversion back to the toxic state during storage. When a lyophilized preparation is required, care must be taken to ensure that formaldehyde is not lost during the process in order to ensure safety of the product. A design of experiments (DOEs) approach was taken to devise a stable, lyophilized, vaccine formulation. A formaldehyde-inactivated bacterial toxin was used as a model antigen. Entrapment of formaldehyde in an amorphous matrix and/or interactions with amorphous components was found to be required for complete recovery of formaldehyde during lyophilization. In formulations consisting of sucrose and citrate, formaldehyde could be recovered across a wide range of excipient concentrations. Stability of the antigen was dependent on formaldehyde concentration, with antigen stability decreasing with increasing formaldehyde concentration. This is in contrast to the risk of reversion which increases with decreasing concentrations of formaldehyde. Finally, variations in temperatures during annealing, primary drying, and secondary drying had no impact on formaldehyde recovery.

Influence of protein conformation and adjuvant aggregation on the effectiveness of aluminum hydroxide adjuvant in a model alkaline phosphatase vaccine.

The mechanism(s) of the enhancement of the immune response by addition of aluminum salt adjuvants to parenterally administered protein-based vaccines is still the subject of debate. It has been hypothesized, however, that destabilization of the antigen structure on the surface of the adjuvant may be important for eliciting immune response. Also, it has been suggested that immune response to adjuvanted vaccines is reduced if the adjuvant particles become aggregated before administration because of processing steps such as freeze-drying. In this study, we tested these hypotheses and examined the immune response in a murine model to various liquid, freeze-dried, and spray freeze-dried formulations of a model vaccine, bovine intestinal alkaline phosphatase adsorbed on aluminum hydroxide. Enzymatic activity of the alkaline phosphatase was used as a sensitive indicator of intact native antigen structure. By manipulating the secondary drying temperature during lyophilization, vaccines were produced with varying levels of alkaline phosphatase enzymatic activity and varying degrees of adjuvant aggregation, as assessed by particle size distribution. Anti-alkaline phosphatase titers observed in immunized mice were independent of both the antigen's retained enzymatic activity and the vaccine formulation's mean particle diameter.

Influence of particle size and antigen binding on effectiveness of aluminum salt adjuvants in a model lysozyme vaccine.

It has been suggested that agglomeration of aluminum salt adjuvant particles during freezing and drying can cause loss of immunogenicity of vaccines formulated with such adjuvants. In this study, we tested this hypothesis and examined the immune response in a murine model to various liquid, freeze-thawed, and lyophilized vaccine formulations, using lysozyme as a model antigen. The various processing techniques and excipient levels resulted in a wide range of particle size distributions (PSDs) and antigen-adjuvant binding levels. Anti-lysozyme titers were independent of the PSD for vaccines adjuvanted with either aluminum hydroxide or aluminum phosphate and also were unaffected by the level of antigen binding to the adjuvant.

Inhibition of aggregation of aluminum hydroxide adjuvant during freezing and drying.

Aluminum-salt adjuvants are widely used to increase immunogenicity of recombinant protein vaccines. However, when vaccines formulated with these adjuvants are frozen or lyophilized, losses of efficacy are often reported. This loss of potency is usually attributed to the aggregation of adjuvant particles during processing. In this study, we examine the aggregation behavior of Alhydrogel, a commercial aluminum hydroxide adjuvant, during freeze-thawing and freeze-drying. By cooling Alhydrogel formulations at faster rates or by the addition of sufficient amounts of a glass forming excipient such as trehalose, aggregation of Alhydrogel, can be prevented or minimized. We propose that freeze-concentration of buffer salts induces modifications in adjuvant surface chemistry and crystallinity, which in turn favor aggregation. These modifications, and the resulting aggregation of Alhydrogel particles can be minimized through choice of buffer ions, or kinetically inhibited by rapidly forming a glassy state during freezing.