A deep dive into the benefits of viral vectors

Breaking the vector characterisation bottleneck with macro mass photometry.

Viral vectors play a pivotal role in the advancement of vaccines and cell and gene therapies (CGTs), serving as versatile therapeutic delivery vehicles. Vector characterisation is an important analytical step of the therapeutic production pipeline, but can be a longwinded process, requiring extensive biological (e.g., PCR), cell-based (e.g., infectivity assays), and physicochemical (e.g., analytical ultracentrifugation (AUC)) testing.

Limitations of characterisation techniques

Conventional characterisation techniques bring limitations: Cell-based vector analysis takes days to perform, while more rapid approaches, like nanoparticle tracking analysis (NTA), provide limited characterisation data. Along with a paucity of in-process analysis tools, these obstacles create a significant bottleneck at the vector characterisation stage for vaccine and CGT programs.

In light of the demand for new innovations to improve the accuracy and speed of vector analysis, macro mass photometry has emerged as a promising solution. Using light scattering to analyse particles, this novel method unlocks a host of valuable vector characterization capabilities, including rapid and accurate determination of full/empty capsid ratios, sample purity and more.

Understanding macro mass photometry

Macro mass photometry interrogates two parameters in parallel: particle scattering contrast (a proxy for mass) and size (diameter). During a typical run, a droplet of sample on a carrier slide is illuminated from below and imaged while being moved vertically (Figure 1).

Each analysis returns size and contrast measurements for every particle, making it possible to determine the size-contrast distribution. This enables users to characterise multiple populations within a sample and assess sample purity and stability.

Developed by Refeyn Ltd., macro mass photometry is a rapid, reliable and convenient approach to inform process development and optimisation for vaccine and CGT development. Macro mass photometry is performed in an all-in-one benchtop instrument that delivers results in several minutes. In the analysis of vectors, the technology can be leveraged to inform:

  • Relative population counts (correlating with physical and infectious titer)
  • Particle morphology (size distribution)
  • Sample purity
  • Stability and degradation

Determining AdV sample purity

Adenovirus (AdV) characterisation can be challenging due to the coexistence of various particle types in the process. Before downstream purification, a typical AdV sample will contain full AdVs, empty capsids, helper viruses, and fragments. Full AdVs are functional and therefore desired, while empty capsids and fragments can decrease therapeutic efficacy. Furthermore, helper viruses can be immunogenic and pose safety concerns.

Since molecular analysis techniques struggle to discriminate between these populations, AUC has become the benchmark for determining empty and full AdVs by their density, but the approach lacks scalability. Macro mass photometry can efficiently distinguish between particles in a sample, providing convenient means for quantifying impurities and monitoring the process during manufacturing, saving time and resources (Figure 2).

Functional LVV identIfication

Following a lentiviral vector (LVV) production process, the product containing infectious (functional) LVVs commonly retains a significant proportion of non-infectious vectors missing key genetic or protein components. Macro mass photometry can be leveraged to rapidly assess the amount of LVV present in a sample and distinguish LVV populations based on their functionality.

The study data shown (Figure 3) demonstrates a positive correlation between the percentage counts (determined by macro mass photometry) and physical titer measurements (determined by PERT assay). The percentage counts are also found to correlate positively with results from an infectivity assay, with the exception of measurements of the non-infectious samples as macro mass photometry is insensitive to infectivity. Overall, the results demonstrate the valuable utility of macro mass photometry in characterising LVV samples.

What are viral vectors?

Viral vectors are designed to deliver genetic material into cells and are one of the most effective methods of gene therapy. Viruses have evolved to develop mechanisms that insert their genomes inside the cells they infect. Modified viruses are used as viral vectors (or ‘carriers’) in gene therapy, protecting the new gene from degradation while delivering it to the ‘gene cassette’ in target cells.

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