An in vitro relative potency (IVRP) assay has been developed as an alternative to the mouse potency assay used to release Merck's human papillomavirus (HPV) vaccine, Gardasil ® , for early phase clinical trials. The mouse potency assay is a classical, in vivo assay, which requires 4-6 weeks to complete and exhibits variability on the order of 40% relative standard deviation (RSD). The IVRP assay is a sandwich-type immunoassay that is used to measure relative antigenicity of the vaccine product. The IVRP assay can be completed in three days, has a variability of approximately 10% RSD and does not require the sacrifice of live animals. Because antigen detection is achieved using H16.V5, a neutralizing monoclonal antibody, which binds to a clinically-relevant epitope, the relative antigenicity measured by the IVRP assay is believed to be a good predictor of in vivo potency.In this study, the relationship between immunogenicity, as measured by the mouse potency assay and antigenicity as measured by the IVRP assay, is demonstrated. Freshly manufactured and aged samples produced using two different manufacturing processes were tested using both methods. The results demonstrate that there is an inverse correlation between the IVRP and mouse potency assays. Additionally, clinical results indicate IVRP is predictive of human immunogenicity. Thus, antigenicity, as defined by the H16.V5 epitope, can be used as a surrogate for immunogenicity and the IVRP assay is suitable for use as the sole potency test for Gardasil samples.

The thermostability of GARDASIL (Merck & Co., Inc, Whitehouse Station, NJ, USA), a developmental vaccine against human papillomavirus (HPV), was evaluated using an enzyme immunoassay, referred to as the in vitro relative potency (IVRP) assay and differential scanning calorimetry (DSC). Gardasil samples were stored at temperatures ranging from 4 to 42 degrees C and tested for IVRP at various time points. Extrapolation of the IVRP results indicates GARDASIL is extremely stable. The half-life of the vaccine is estimated to be 130 months or longer at temperatures up to 25 degrees C. At 37 degrees C, the half-life is predicted to be 18 months and at 42 degrees C, the half-life is predicted to be approximately three months. Differential scanning calorimetry (DSC) analysis was used to evaluate the process of protein denaturation during a rapid temperature increase (as opposed to long-term storage at a specific temperature). Differences were seen among the DSC profiles of the four HPV types tested. This indicates that small differences in the amino acid structure can have a significant effect on the intermolecular contacts that stabilize the L1 proteins and the VLP assembly. For the Gardasil samples evaluated here, DSC results demonstrated the relative overall structural stability of the VLPs, but were not predictive of the excellent long-term stability observed with the IVRP assay.

A novel one-step method for determining kinetic rates and equilibrium binding affinities, termed analyte gradient-surface plasmon resonance (AG-SPR) is described. A gradient maker or HPLC pump system is used to produce a gradient so that, under continuous-flow conditions, the concentration of analyte passing over the sensor surface increases linearly with time. The rate at which analyte binds to the immobilized receptors is measured by monitoring the change in the surface plasmon resonance minimum as the analyte concentration increases. Kinetic rates are determined by fitting the data to a modified version of the previously described two-compartment model (Schuck, P.; Minton, A. P. Anal. Biochem. 1996, 240, 262-272). Numerical simulations indicate that AG-SPR results in accurate estimates of both kinetic rates and equilibrium affinities regardless of the intrinsic kinetics of the interaction and can be used for systems under mass transport limitations. Simulations also indicate that AG-SPR can be used to characterize interactions that do not obey pseudo-first-order kinetics due to the presence of a heterogeneous receptor population. Experimentally, the interaction of cytochrome c with cytochrome b5 immobilized on a negatively charged monolayer has been characterized by AG-SPR, and both the specific and the nonspecific interactions were quantitatively analyzed. This new technique is advantageous over traditional SPR methods because it eliminates the need for surface regeneration and is significantly faster than traditional titration experiments.

Compared with biologics, vaccine potency assays represent a special challenge due to their unique compositions, multivalency, long life cycles and global distribution. Historically, vaccines were released using in vivo potency assays requiring immunization of dozens of animals. Modern vaccines use a variety of newer analytical tools including biochemical, cell-based and immunochemical methods to measure potency. The choice of analytics largely depends on the mechanism of action and ability to ensure lot-to-lot consistency. Live vaccines often require cell-based assays to ensure infectivity, whereas recombinant vaccine potency can be reliably monitored with immunoassays. Several case studies are presented to demonstrate the relationship between mechanism of action and potency assay. A high-level decision tree is presented to assist with assay selection.

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