Quick viral vector purity assessment with NanoSight Pro

Introduction

In the development of viral vector formulations, quickly assessing sample purity is essential because it directly affects the vector’s properties and performance. This application note demonstrates how NanoSight Pro, utilizing Nanoparticle Tracking Analysis (NTA) technology, can serve as an efficient alternative to bioassays for rapid and effective characterization of virus purity and heterogeneity.

Purity and heterogeneity

This study presents examples of using NanoSight Pro to characterize lentivirus and MVA formulations. The lentivirus* and MVA* samples were diluted to the optimum for NTA concentration (~3-5E8 particles/ml) in PBS or formulation buffer* and analyzed with NanoSight Pro equipped with a 488 nm laser. 

The acceptable level of purity varies depending on the viral vector platform used and the stage of process optimization. For example, most lentivirus-based formulations aim to achieve a pure and homogeneous solution. Other viral vectors can be more challenging to purify, and for MVA-based formulation a lower level of purity is often accepted (Figure 1). 

Sample purity is reflected in the shape of the particle size distribution profile. For well-purified samples, the size distribution is presented as one peak with the modal size reflecting the expected viral vector size. For less pure samples, the size distributions are less smooth and often contain a wide spread of sizes and additional shoulders in the size distribution curve.

NanoSight Pro enables rapid assessment of sample heterogeneity by directly reporting on particle size, size distribution and titer. The direct imaging of samples allows for immediate evaluation of sample complexity, providing quick insights into formulation purity (Figure 1).

Figure 1: The size distributions typical of lentivirus (blue line) and MVA (green line) are shown for comparison

The expected size of lentivirus particles is around 100 nm, which is clearly visible and confirmed by the main peak in the size distribution of good purity lentivirus sample (Fig 1 blue line). The NanoSight Pro image further supports the uniformity of particles in the sample. However, additional shoulders in the distribution indicate the small presence of larger particles, which could be lentivirus aggregates or other impurities. NanoSight Pro reports sample concentration, which is equivalent to viral titer in a well-purified sample. 

In our example, the total lentivirus sample concentration is 3.6E11 particles/ml, and expected lentivirus titer in the size range of 50-150nm, is 2E11 lentivirus/ml. In contrast, the green line represents an example of unpurified MVA formulation, which exhibits a highly polydisperse size distribution (Fig 1 green line). The NanoSight Pro image highlights the complexity of the sample, showing a wide range of particle sizes. MVA particles are typically around 200 nm in size, but the peak corresponding to the population of this particle size is difficult to distinguish in such a complex mixture. Viral titer in an unpurified sample can be assessed with NanoSight Pro by utilizing fluorescence mode to detect sample subpopulations, specifically tagged viral vectors.

Conclusions

NanoSight Pro provides an effective assessment of viral vector formulation purity in minutes. It does this by instantly reporting viral vector size and viral titer, which it captures in a size distribution curve. This is further validated by the direct NTA sample image. 

Further reading

This application is part of a five-part series. Explore the rest of the series to dive deeper into this topic:

• Viral vector stability and storage assessment with NanoSight Pro 
• Identify viral vectors with NanoSight Pro F-NTA
• Identify VSV-G positive lentivirus with NanoSight F-NTA

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*Lentivirus and MVA samples along with formulation buffers were kindly supplied by one of Malvern Panalytical’ s collaborators.