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

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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.


BioPharm International Supplements


Particle Size

The immunologic advantage of VLPs results from their particle nature, which allows efficient uptake, processing, and presentation by dendritic cells. VLPs are membrane particles with a diameter of 141–192 nm containing the influenza HA and NA proteins as surface spikes. Measurement of VLP particle size by a Malvern Zetasizer is an important release criteria to demonstrate consistency of the manufacturing process and ensures the absence of aggregation.

Purity Assessment

The goal of the purity assessment is to determine the levels of baculovirus (BV) and Sf9 cell DNA and proteins associated with each lot of VLP vaccine. These are determined by specific assays, as summarized in Table 1.

There are two sources for BV and Sf9 preparations in VLP vaccine. Although the current purification process for VLPs completely inactivates BV, it does not completely remove BV particles. Because VLPs bud from the insect cells, there is a likelihood that they acquire cellular and BV proteins during this process. Thus, determining the true host and BV protein contaminants in VLP vaccine is a considerable challenge.


Figure 4. Identification of proteins in virus-like particle influenza vaccines. This SDS-PAGE band profile of the B/Florida/4/06 VLPs, obtained by LC–MS-MS, shows that the proteins detected were predominantly the expected HA, NA, and M1 proteins, along with two baculovirus proteins (the gp64 envelope protein and the p39 major capsid protein).
Multiple batches of a single type of VLP must be analyzed to determine potential BV and Sf9 contamination. Two orthogonal approaches are used to determine VLP purity and the identity of BV and Sf9 proteins. SDS–PAGE followed by Coomassie staining allows us to visualize the proteins in each VLP batch. Scanning the stained sample lane and determining the percentage of each specific protein band allows the percentage of HA, NA, and M1 proteins to be determined and enables us to assign a relative purity value to the VLP (Table 3). LC–MS-MS of either the whole VLP sample or specific bands isolated from gels has been used to identify the BV and Sf9 proteins associated with different VLP lots. A typical VLP protein band profile is shown in Figure 4. The proteins detected were predominantly the expected influenza HA, NA, and M1 proteins engineered into the B/Florida VLP. Baculovirus gp64 envelope protein and p39 major capsid protein were identified through SDS–PAGE analysis of this vaccine along with viral ubiquitin detected by MS analysis. Sf9 proteins detected by MS analysis included tubulin, actin, Hsp70 chaperone, and small amounts of several housekeeping proteins.


Table 5. Comparison of VLP purity determined by SDS–PAGE and BV ELISA
ELISA assays have been developed as a second method to measure the concentration of BV and Sf9 proteins in VLP lots. As seen in Table 5, the purity of VLP lots based on the baculovirus ELISA is generally in good agreement with the values seen using the SDS-PAGE method.


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