Optimizing Lentivirus Storage Conditions using Zetasizer Advance Ultra and 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 complexity of the lentiviral structure creates analytical challenges to measure attributes such as size and titer to optimize stability, efficacy, and storage conditions. Performing Lentiviral transfection and functional tittering assays after different storage conditions can produce wildly different results, indicating that colloidal and morphological changes to the lentivirus structure have significant impact on its efficacy as a gene therapy vector.

In this application note, we present Zetasizer Ultra and NanoSight Pro size, polydispersity, and viral particle titer results for lentivirus stored at room temperature, on ice, in high salt buffer, and after three freeze-thaw cycles.

NanoSight utilizes Nanoparticle Tracking Analysis (NTA)³ to measure nanoparticle size and concentration. Particle size is determined by quantifying the mean squared displacement of multiple individual particles. Viral particle titer is calculated by counting the number of viral particles in a known volume. NTA size and concentration measurements occur simultaneously and require about five minutes per sample.

The Zetasizer Ultra Red uses dynamic light scattering (DLS)⁴ to measure particle size and multi-angle dynamic light scattering (MADLS)⁵ to measure high-resolution size and particle concentration⁶. Viral particle titer 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.

Zetasizer Ultra 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. Serial dilutions were performed gravimetrically using a mass balance in the 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.

NanoSight Pro measurements were performed by diluting samples 1000x into HBSS buffer immediately prior to analysis using the NanoSight Pro using NS Xplorer software version 1.1. Samples were measured using auto camera setup. All sample preperation and measurements were performed inside a BSL-2 hood, including the operation of the NanoSight Pro instrument.

Lentivirus stored at room temperature in HBSS Buffer was measured at time points 0, 1, 4, 24, and 48 hours. Lentivirus stored at 4 °C in HBSS Buffer was measured at time points 0, 2, 3, and 8 days. Lentivirus in HBSS Buffer was measured after 0, 1, 2, and 3 freeze-thaw cycles. Lentivirus in a high salt buffer 5 M NaCl high salt buffer at 4 °C was measured at time points 0, 1, 3, and 8 days.

Results and Discussion

Freshly thawed lentivirus size was measured using both Zetasizer Advance Ultra and NanoSight Pro (Figure 1). These results highlight the relative strengths and weaknesses of both technologies for measuring nanoparticle size.

NTA is a count-based technology that produces high-resolution, number-weighted particle size distributions of a highly diluted sample. Single angle DLS is an ensemble technique that produces intensity-weighted, and low-resolution particle size of a neat sample. The NTA results are smaller on average, with higher weighting to the more abundant and smaller lentivirus peak at 123 nm and a proportionally smaller aggregate peak at 300 nm. Whereas the broader DLS peak at 186 nm is weighted more towards the larger aggregates that scatter more light. DLS has the sensitivity to detect trace amounts of aggregates at 495 nm that are too low by number to be detected by NTA.

When used together, DLS and NTA technologies can orthogonally provide a complete analytical characterization of nanoparticle size.

Figure 1: Zetasizer Advance Ultra and NanoSight Pro particle size distribution results for freshly thawed lentivirus.

Room temperature stability results showed a significant change to the lentivirus colloidal stability after just 1 hour of storage at room temperature (Figure 2). Both Zetasizer and NanoSight results produced significant changes to the lentivirus sample for measured size and concentration over one hour after thawing.

Figure 2: Lentivirus room temperature stability results with Zetasizer Advance ultra and NanoSight Pro.

Lentivirus stored on ice at 4 °C in HBSS buffer showed improved stability over storage at room temperature (Figure 3). However, even in this more stable storage condition, significant changes were observed for both technologies between one and two days. Zetasizer results showed a significant reduction in Lentiviral titer from zero to two days. Whereas, NanoSight Pro results showed an increase in total viral particle titer from days zero, one to three. Both of these changes indicate sample instability after one day of storage. Alternatively, proper sample mixing could be equally as important to optimize, as too gentle mixing may not break up reversible aggregates, and too violent mixing may cause instability.

Figure 3: Lentivirus stability results at 4 °C in HBSS buffer with Zetasizer Advance Ultra and NanoSight Pro.

The effect of freeze-thaw cycles on Lentivirus stability was determined by measuring with Zetasizer and NanoSight Pro (Figure 4). Zetasizer intensity-weighted results showed a significant decrease in viral particle titer from 0 to 1 freeze-thaw cycles, and a significant increase in Z-avg size from 1, 2, and 3 freeze-thaw cycles. NanoSight Pro number-weighted size and concentration results were less sensitive to changes between lentivirus samples, indicating that the lentivirus monomers were more resistant to instability over freeze-thaw cycles. 

It should be noted that the samples were shipped and received frozen from the vendor and immediately stored under liquid nitrogen. For the freeze-thaw study, samples were thawed and aliquoted before the “0 freeze-thaw time point”.

Figure 4: Lentivirus stability results over 3 freeze-thaw cycles in HBSS buffer with Zetasizer Advance Ultra and NanoSight Pro.

The stability of lentivirus in a high salt condition was studied by storing thawed lentivirus in a 5 M NaCl solution at 4 °C (Figure 5). Zetasizer Z-avg results produced a large increase in average size from 156 nm to 1.4 microns over two days. NanoSight Pro results produced a smaller but steady decrease in particle size from 120 nm to 105 nm over 8 days.

The differences in the size and concentration results from these two technologies are likely due to the native weighting of the results from the two different measurement principles. DLS has inherently low resolution and an intensity-weighted result and will be more sensitive to aggregates. NTA is number-weighted and high resolution, and will be far less sensitive to changes due to aggregation. These differences stress the importance of orthogonal characterization.

Additionally, although the high salt conditions were identical, the measured concentrations were 1000x different between the two technologies. The highly concentrated lentivirus sample measured undiluted by the Zetasizer may have a different stability profile than the sample which was diluted 1000x for the NanoSight Pro.

Figure 5: Lentivirus stability results in high salt 5M NaCl buffer with Zetasizer Advance Ultra and NanoSight Pro.

Conclusions

Both the Zetasizer Ultra and NanoSight Pro were successfully able to measure lentivirus over a range of storage conditions. The results suggest that lentivirus samples should be stored frozen, thawed, and then stored on ice for less than one day before being analyzed. Additionally, optimizing the mixing process of the thawed lentivirus aliquot may be an important factor that influences infectious titer and particle colloidal stability.

The results stress the importance of performing both vector nanoparticle characterization using multiple, orthogonal technologies 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
• Read how lentivirus thermal stability can be studied using NanoSight and Zetasizer: read more
  
• Learn 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 19430:2024(en). Determination of particle size distribution and number concentration by particle tracking analysis (PTA).
4. ISO 22412:2017. Particle size analysis — Dynamic light scattering (DLS)
5. Improved component resolution with Multi-Angle DLS (MADLS). 2018 Malvern Panalytical Application Note.
6. 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.