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.
Through the use of a recombinant baculovirus–insect host cell system, Novavax has developed a platform technology to produce
VLP vaccines to multiple targets including influenza, SARS, and HIV. Influenza VLPs have been biochemically characterized
and GMP-produced materials have been tested in five clinical trials, with a total of more than 5,000 subjects.1
The establishment of a recombinant baculovirus containing the influenza genes for the hemagglutinin (HA) and neuraminidase
proteins (NA) together with the gene for Matrix (M1) protein is the key step in this process.2 The HA and NA genes are obtained from viral RNA of relevant circulating seasonal or pandemic influenza strains by molecular
means from isolated RNA. Alternately, they can be chemically synthesized and then, through a series of cloning steps, placed
in a single plasmid (bacmid) under the control of baculovirus-specific transcription initiation and translation elements along
with a constant M1 gene sequence shown to allow enhanced VLP formation. Transfection of the bacmid into Spodoptera frugiperda (Sf9) insect cells generates baculoviruses that encode and express the three influenza genes. Baculovirus stocks are further
amplified on Sf9 cells to generate master and working virus stocks. Infection of Sf9 cells with the recombinant baculovirus
allows expression of the HA, NA, and M1 proteins which form pleomorphic spherical membrane-containing particles with HA and
NA protein spikes on their surface and an M1 core. An example of an influenza VLP is shown in Figure 1. These particles are
released into the cell culture supernatant and then purified, resulting in the VLP vaccine. The membrane nature of these particles
is different from currently licensed VLP vaccines and presents purification and characterization challenges.
VLP Production and Purification
Production and purification of influenza VLPs has been performed for Phase 2 clinical trial materials. Sf9 insect cells from
a working cell bank are amplified to seed a 100-L disposable wave bioreactor cell culture, which is then infected with a master
baculovirus seed. During harvest, cells are removed by tangential flow filtration (TFF) and the filtrate is concentrated and
diafiltered to remove media components and cell debris.
Figure 2. Purification of virus-like particles (VLPs) using an ion exchange (IEX) column. Host cell contaminants—baculovirus
(BV), DNA, and RNA—bind, whereas VLPs flow through.
VLPs are separated from baculovirus (BV) particles and contaminating RNA and DNA using a flow-through ion exchange (IEX) chromatography
step in which BV, RNA, and DNA are bound to the column while VLP is allowed to flow through (Figure 2). This process step
takes advantage of the charge difference between BV and VLP; BV is highly negatively charged by virtue of its DNA content,
whereas VLP lacks this charge given the absence of DNA. Allowing VLP to be separated from BV without binding to the column
matrix eliminates the need to modify this purification step from season to season in response to changes in the HA protein.
Figure 3. Residual host contaminants are removed by size exclusion chromatography.
Residual BV is inactivated by treatment with beta-propiolactone (BPL) and residual host contaminants are removed by size exclusion
chromatography (SEC), as shown in Figure 3. The VLP preparation is sterile filtered to produce the bulk vaccine.
