Overcoming the Limitations of Current LVV Titer Measurement
As described in Part One of this blog series, quantification of LVV particles is time consuming and often laborious. To overcome some of the challenges associated with virus titer determination, Sartorius developed the Virus Counter Plus platform. The platform consists of an instrument, reagents and consumables, and software that work together to provide a user-friendly experience, enabling quicker, more robust titer data acquisition with less hands-on time.
This instrument uses the Virotag® VSVG assay to target the glycoprotein (G protein) of vesicular stomatitis virus (VSV), which is crucial for successful LVV transduction. Highly specific fluorescently labeled anti-VSV-G antibodies label VSV-G pseudotyped LVVs. The virus particles in suspension are directly quantified with high precision via optical detection (Figure 1).
Figure 1: Titer Evaluation Process with the Virus Counter Plus System
Mix virus samples with Virotag reagents
Add labeled virus samples and incubate (dark, room temp)
Place plate in Virus Counter Plus, and initiate walkaway analysis
Review and analyze data in the Virus Counter Plus application
The process is easy to implement, too. Diluted LVV samples are simply mixed with the Virotag VSVG reagent containing a primary antibody stain composed of a monoclonal antibody conjugated with fluorophores in a stabilizing buffer solution. Diluted negative controls and blanks composed of antibody stain in sample diluent are also prepared in the same manner. All three sample types are loaded onto a 96-well plate and incubated in the dark for a minimum of 30 minutes at room temperature. The plates are then loaded into the Virus Counter Plus system for analysis at 4°C, with the blanks analyzed before the actual samples and negative controls. Results are obtained in as little as two to at most 24 hours depending on the number of samples.
The Virus Counter Plus platform with the Virotag VSVG assay can provide physical titer data within a few hours. With any new analytical method, however, it is important to assess performance under actual process conditions. Given the significant potential benefits of rapid LVV titer determination, we elected to conduct a study comparing the data obtained using the Virus Counter Plus to those generated using standard quantitative polymerase chain reaction (qPCR) and p24 enzyme-linked immunosorbent assay (ELISA) techniques for physical titer and a transduction unit (TU) assay for functional titer.
Four runs of ABL’s LVV production process were performed starting with cell growth and transection (lentiviral packaging, envelope, and GFP expression plasmids) in a 20 L bioreactor followed by harvest (two days post-transfection), nuclease treatment, chromatography, sterile filtration, tangential-flow filtration, and a second sterile filtration.
The Virotag VSVG assay was run for the same samples that were tested for physical and functional titer. The results were compared to align the results from the different assays. Descriptions of our results and information on the predictive capabilities of these comparisons are described in Part Three.
Authors: Poorni Adikaram, PhD, Tyler Frazier and Timothy Fouts, PhD, CSO at ABL Inc. and Rebecca Montange, PhD and Brandon Harrell at Sartorius
Learn more about the limitations of current LVV measurements in Part One or find the results of a recent study assessing the correlation of titer data obtained using new and standard analytical methods in Part Three.
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