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March 01, 2010 (Vol. 30, No. 5)

Characterizing Viral Titers in Manufacturing

New Instrument Counts and Sizes Viruses and Their Aggregates in Liquid Suspension

  • Understanding Virus Aggregation

    Click Image To Enlarge +
    Figure 3: A highly purified preparation of influenza virus (A) and a sample with a high degree of aggregation following freeze-thawing (B).

    The NanoSight technique measures particle size on a particle-by-particle basis and can generate high-resolution particle-size distributions. The size distribution can be used to estimate relative concentrations of monomeric vs. aggregated material because the technique not only measures particle size but counts the number of particles of a specific size.

    The qualitative aspect of the technique also provides quick insight into the state of aggregation, as can be seen in Figure 3. Figure 3A shows a highly purified preparation of influenza virus, while Figure 3B shows a sample with a high degree of aggregation. Independent of a number distribution, the user can quickly and reliably understand the state of aggregation within a preparation.

    Infectivity assays are unable to distinguish between aggregated and nonaggregated material in viral preparations. A plaque-forming unit may be a single virion or a single aggregate containing many potentially infective viruses. Aggregated viral material may de-aggregate in vivo upon administration and, as such, an infectivity assay may grossly underestimate the infectious viral content within a preparation.

  • Virus-Like Particles

    Virus-like particles (VLPs) are a relatively new area within vaccine development. They have been designed to overcome the problems associated with certain recombinant protein vaccines that have poor immunogenicity resulting from poor presentation of the viral antigens to the immune system. This can be overcome to a certain extent through the addition of adjuvants, but perhaps another more attractive option is available through the creation of VLPs.

    VLPs consist of an assembled structure of viral antigens creating a more authentic structure and conformation of the viral antigen. VLPs have been shown to have significant potential in eliciting a stronger and lengthier immune response than more traditional recombinant protein vaccines.

    In terms of characterizing such structures, infectivity assays cannot be used as VLPs, are devoid of the RNA or DNA required for replication, and hence, they are noninfectious. In addition, they cannot be quantified using qPCR again due to the fact that they do not contain DNA or RNA. The NanoSight technique does not require the particle to be infectious nor contain DNA/RNA and hence represents an attractive method of characterizing these structures.

    The NanoSight technique can be used as a tool to quantify the total viral content and can be used alongside infectivity assays to calculate the relative concentrations of infectious to noninfectious viruses in a live attenuated vaccine. This method is ideally suited to calculate viral titers in inactivated vaccines where infectivity assays cannot be used to characterize the final product.

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