Measuring Lentivirus Size and Titer (Particle Concentration) with the Zetasizer Advance Ultra

Introduction

Lentivirus is a popular viral vector used in gene therapy development. It has several advantages over other viral and non-viral vector-based gene therapies, including the ability to transport large or multiple genes, infect both dividing and non-dividing cells, and integrate transgenes into the host cell genome¹.

However, these desirable lentiviral vector properties require a more complex structure compared to other common viral vectors, such as the adeno-associated virus (AAV). Lentivirus is an enveloped, spherical virus, with a diameter between 90-130 nm, and consists of multiple components, including a transgene, nucleocapsid, capsid, envelope, and surface membrane proteins².

The complex structure of lentivirus presents analytical challenges when measuring attributes like size and titer, which are essential for optimizing its stability, efficacy, and storage conditions.

In this application note, we present Zetasizer Ultra size and particle concentration (titer) results for lentivirus samples.

The Zetasizer Ultra Red uses dynamic light scattering (DLS)³ to measure particle size and multi-angle dynamic light scattering (MADLS)⁴ to measure high-resolution size and particle concentration⁵. Particle concentration is calculated from the measured MADLS size, the material scatter rate, material refractive index, dispersant viscosity and refractive index.

Materials and Methods

Ultra-purified lentivirus samples were purchased from Vector Builder produced with the pLV[Exp]-EGFP:T2A:Puro-EF1A>mCherry vector. Size and concentration measurements were performed on neat lentivirus sample by pipetting 20 microliters of freshly thawed sample directly into the ZEN2112 quartz cuvette inside of a BSL-2 hood. Cuvettes were capped and then transported to a Zetasizer Ultra instrument located on an adjacent bench before starting the measurements. All sample handling was performed inside a BSL-2 hood.

Results and Discussion

Single angle back-scatter DLS produced an intensity-weighted size distribution with a single broad size peak at 184 nm, a Z-avg of 152 nm, and a medium polydispersity value of 0.168 (Figure 1). These results indicate that the single-size peak is likely a mixture of multiple components, possibly containing enveloped and non-enveloped lentivirus monomer particles and larger aggregates. For biological samples, a polydispersity index value below 0.1 is considered mostly monodisperse.

Figure 1: Lentivirus DLS particle size distribution (triplicate overlay).

Multi-angle DLS (MADLS) generated a higher resolution intensity-weighted size distribution with two peaks at 157 and 424 nm (Figure 2). The primary peak at 157 nm, is likely a mixture of lentiviral monomers and smaller aggregates, whereas the larger peak at 424 nm contains larger aggregates.

Figure 2: Lentivirus MADLS particle size distribution (triplicate overlay).

Particle concentration was calculated from the MADLS size distribution, producing a particle concentration of 2.1E11 particles/mL for the 154 nm population, and 3.7E9 particles/mL for the 412 nm population. Relative standard deviation (RSD) was calculated for each peak over three repeat measurements. Peak 1 was highly reproducible with a RSD of only 16%. The accuracy and repeatability of the MADLS particle concentration measurement is dependent on the size accuracy and polydispersity of the sample.

Figure 3: Lentivirus MADLS particle concentration distribution (three measurement overlay).

The linearity of the Zetasizer Advance Ultra lentivirus titer measurement was determined by measuring particle concentration over a serial dilution from the undiluted stock sample (Figure 4). A linear fit to the measured versus expected titer results based on dilution from stock produced an R-squared over 0.983, indicating the titer results are reproducible and linear over a range of concentrations.

Figure 4: Zetasizer Advance Ultra lentivirus titer linearity over a serial dilution from stock solution.

Conclusions

The Zetasizer Ultra was successfully able to measure the size, polydispersity, and particle concentration titer of a purified lentivirus sample. The particle concentration results produced by the MADLS measurement of 2.0E11 particles/mL are higher than the infectious titer of >1E9 TU/mL, suggesting that there is significant room for improvement for optimizing lentiviral vector transfection efficiency. These results stress the importance of performing both vector nanoparticle characterization in addition to functional infectious assays.

Want to learn more?

• Learn about optimal storage conditions for lentivirus with Zetasizer and NanoSight: read more
  
• Read how lentivirus thermal stability can be studied using NanoSight and Zetasizer: read more
• Find out how NanoSight Pro is used to measure lentivirus size and titer: read more
  

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References

1. Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases. Viruses. 2021 Aug; 13(8): 1526. doi: 10.3390/v13081526.
2. Production and titration of lentiviral vectors. Curr Protoc Hum Genet. 2007, 12: 12.10. doi: 10.1002/0471142905.hg1210s54.
3. ISO 22412:2017. Particle size analysis — Dynamic light scattering (DLS)
4. Improved component resolution with Multi-Angle DLS (MADLS). 2018 Malvern Panalytical Application Note.
5. Nanoparticle number concentration measurements by multi-angle dynamic light scattering. J Nanopart Res 22, 108 (2020). https://doi.org/10.1007/s11051-020-04840-8