Measuring Lentivirus Size and Titer (Particle Concentration) with the NanoSight Pro

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 to the target cell, 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 NanoSight Pro size and particle concentration (titer) results for lentivirus samples.

The NanoSight Pro uses Nanoparticle Tracking Analysis (NTA)³ to measure both lentivirus high resolution size and absolute particle concentration by simultaneously counting and tracking the motion of each individual lentivirus particle directly. Particle size is determined by quantifying the mean squared displacement of each lentivirus particle and converting to a hydrodynamic size using the Stokes-Einstein equation. The particle-by-particle analysis enables a high resolution and number-weighted particle size distribution. Particle concentration, or viral titer, is calculated by counting the number of viral particles in a known volume. NTA size and concentration measurements occur simultaneously with a measurement time of about five minutes per sample.

Materials and Methods

Ultra-purified lentivirus samples were purchased from Vector Builder produced with the pLV[Exp]-EGFP:T2A:Puro-EF1A>mCherry vector. Samples were diluted 1000x into HBSS buffer immediately prior to analysis using the NanoSight Pro using NS Xplorer software version 1.1. Samples were measured using auto focus and camera setup algorithms. All sample preperation and measurements were performed inside a BSL-2 hood, including operation of the NanoSight Pro instrument. Serial dilutions for the linearity assesment were performed gravimetrically with a calibrated mass balance also inside a BSL-2 hood.

Results and Discussion

The NanoSight Pro produced a lentivirus particle size distribution with peaks at 133 nm, a shoulder peak at 210 nm, and a peak at 370 nm. The peak at 133 nm is the primary Lentivirus peak and can be integrated from 90 to 150 nm to determine the Lentiviral particle titer. The peaks at 210 and 370 nm are likely aggregates that formed during shipment or storage, considering that the samples were ultra-purified and prepared for in-vivo analysis by the vendor before shipping.  

Figure 1: NanoSight Pro lentivirus particle size distribution and lentivirus NTA Movie.

The measured concentration of the 133 nm peak is 5.7E8 lentivirus/mL, which can be multiplied by the dilution factor of 1000 to calculate the stock solution Lentiviral titer of 5.7E11. Compared to the infectious titer of the mCherry vector-containing lentivirus, which is >1E9 TU/mL, the NTA results suggest the majority of Lentiviral particles are non-infectious and significant improvements to lentiviral potency can be made.

Figure 2: NanoSight Pro lentivirus titer linearity over a serial dilution from stock solution.

The lentivirus titer linearity was calculated by measuring the particle concentration at five concentrations in a serial dilution from the lentivirus stock solution. A linear fit to a scatter plot comparing measured concentration to the dilution factor produced an R-squared value of 0.995, showing the robustness of the NanoSight Pro lentivirus concentration measurement.

Conclusions

NanoSight Pro was successfully able to measure the high-resolution size and titer particle concentration of the purified lentivirus sample. The NTA Lentivirus titer particle concentration results show that the particle titer is significantly higher than the infectious titer. These results stress the importance of performing vector nanoparticle characterization in addition to functional infectious assays.

Want to learn more?

• Find out how Zetasizer Advance Ultra is used to measure lentivirus size and titer: read 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

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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 19430:2024(en).  Determination of particle size distribution and number concentration by particle tracking analysis (PTA)