Release and Stability Testing Programs for a Novel Virus-Like Particle Vaccine - Release testing involves both standard potency assays and unique assays (particle size, NA activity) developed to


Release and Stability Testing Programs for a Novel Virus-Like Particle Vaccine
Release testing involves both standard potency assays and unique assays (particle size, NA activity) developed to ensure the physical, chemical, and biological stability of this type of vaccine.

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Clinical Lot Release Testing

Table 1. Analytical testing of influenza VLP bulk vaccine
Release testing is designed to monitor the safety, identity, purity and potency of the VLP vaccine (Table 1). Many of the methods are typical of those used to release licensed split-virion and whole-virus influenza vaccines and were adapted to the VLP product. Additional methods were developed to specifically characterize the VLPs and to demonstrate removal of host and baculovirus DNA and protein as well as materials used in the manufacturing process.

Single Radial Immunodiffusion (SRID) Potency Assay

Single radial immunodiffusion (SRID) is the standard potency assay used by all manufacturers and appropriate regulatory agencies to determine the concentration of immunologically active HA in all licensed inactivated influenza vaccines.3 The assay requires two standards for each influenza vaccine strain: 1) a reference HA protein whose concentration is determined by an alternative method and 2) a polyclonal antibody source that recognizes the reference HA protein. The method is based on diffusion of the HA protein into an agarose gel containing a prequalified amount of specific antibody, raised against the HA protein, which forms precipitation rings proportional to a fixed concentration of the reference HA. The potency for a vaccine lot is computed by the parallel line bioassay method using the reference standard and test vaccine dose response curves.

Table 2. Comparison of the source of the single radial immunodiffusion (SRID) reagants used by regulatory agencies to release standard inactivated influenza vaccines and the reagants developed by Novavax to determine the potency of the baculovirus-derived VLP product.
Table 2 compares the source of reagents used by regulatory agencies to release standard inactivated influenza vaccines with reagents developed by Novavax to determine the potency of our baculovirus-derived product. Whereas regulatory agencies use HA antigen and corresponding antisera derived from egg-grown influenza virus, Novavax uses HA protein derived from baculovirus and prepares a corresponding antisera to that antigen.

Table 3. Comparison of the HA concentration results for H5N1 VLPs from single radial immunodiffusion (SRID) potency assays using egg-derived and baculovirus-derived reagents. The two different reagents give comparable results for multiple lots of vaccines.
Multiple VLPs have been tested for potency in the SRID assay comparing the egg- and baculovirus-derived reagents, and results for H5N1 pandemic vaccine lots are presented as a representative example in Table 3. Baculovirus- and egg-derived SRID reagents give comparable values for HA concentration for multiple lots of pandemic VLP vaccines.

Table 4. Matched and heterologus strain testing with egg- and baculovirus-derived reagents. The homologous A/H1N1 and B/Florida components of both Novavax (NVAX) virus-like particle and inactivated egg-produced influenza vacccines gave comparable potency values when tested with either baculovirus- (NVAX) or egg-derived (CBER) reagants. However, when the heterologous H3N2 HA potencies were tested, accurate potency measurements required homologous reagants (baculovirus for VLPs, and egg-derived for inactivated egg-produced vaccine).
The 2008–2009 inactivated influenza vaccine used a like H3N2 strain (A/Uruguay instead of the recommended A/Brisbane) because of problems with production of the recommended strain. Because VLP development does not require the growth of the actual influenza virus, the protein coding sequence for the A/Brisbane H3N2 HA was used to generate the baculovirus recombinant and subsequently the purified VLP vaccine containing the recommended HA. Potency of trivalent VLP and inactivated 2008–2009 influenza vaccines was tested with baculovirus- and egg-derived reagents in the SRID assay. Table 4 demonstrates that the homologous A/H1N1 and B/Florida components of both vaccines gave comparable potency values when tested by baculovirus- or egg-derived reagent where the results with homologous reagents (baculovirus-derived for VLP and egg-derived for inactivated egg produced vaccine) were considered the expected value. However, when the heterologous H3N2 HA potencies were determined, it was seen that accurate potency measurements required homologous reagents, i.e., H3N2 HA potency in VLP was accurately measured by baculovirus reagents but not by egg-derived reagents and the opposite was seen with egg-derived vaccine. The table also demonstrates that the source of the antisera was not an issue because baculovirus- and egg-derived antisera could be interchanged with the source of antigen and resulted in accurate HA concentration determinations. However, the source of HA reference protein was crucial; only baculovirus-derived A/Brisbane protein was accurate for measuring VLP H3N2 HA content and the egg-derived A/Uruguay HA protein was necessary to accurately test egg-derived vaccine for this strain.

NA Activity

In conventional inactivated influenza vaccines, no attempt is made to quantify or maintain NA activity. A number of preclinical and clinical studies have pointed to a significant role for NA-inhibiting (NAI) antibodies in protective immunity.4–7 NA-specific responses observed in human and mouse studies are responsible for a reduction of viral replication and disease prevention. In primates, NA is known to induce NAI, virus-neutralizing, and HAI antibodies. Immunity toward NA is also known to affect primary viral outcomes (disease progression and viral titers) and prevention of secondary bacterial infections. Moreover, NA-specific responses protect against lethal doses of highly pathogenic avian influenza by eliciting NAI and virus-neutralizing antibodies. NA-specific responses are also known to have reduced the impact of the 1968 H3N2 pandemic.

VLPs contain quantifiable levels of active NA protein and we have shown that VLP-associated NA is very similar to the NA of the influenza virus. The enzymatic properties, Vmax (maximal velocity) and Km (substrate concentration required to achieve maximal velocity) for VLP and viral NA in H3N2 and H5N1 strains are similar. In addition, VLP NA is found to be inhibited by oseltamivir carboxylate (Tamiflu) at a concentration similar to corresponding virus with a <3-fold difference in IC50. Moreover, in the clinical trials summarized above, we have demonstrated that immunization with VLPs elicits NAI antibodies in addition to HA-specific responses, demonstrating the broader immunological response of this vaccine.

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