Canadian scientists are relying on advanced centrifuge technology to improve the accuracy of adeno-associated virus (AAV) characterization. The technique has applications in gene therapy and new vaccine development.

The team, from the University of Lethbridge, worked with a Beckman Coulter multi-wavelength analytical ultracentrifugation (AUC) instrument to measure twenty-six wavelengths of ultraviolet radiation, allowing them to more accurately determine how much of an AAV sample could have a therapeutic benefit.

“Analytical centrifugation is not a new technique—it’s considered a gold standard [for AAV characterization],” says Amy Henrickson, a student and research associate at the University of Lethbridge. “But, with Beckman’s multi-wavelength [instrument], you can [theoretically, at least] measure up to 100 wavelengths at a time.”

As Henrickson explains, AUC relies on a large centrifuge, equipped with a sample cell that has windows on the top and bottom. A laser beam is shone through the window onto the sample and a spectrometer collects spectral information—traditionally, for AAV analysis, at three separate wavelengths, 230, 260, or 280 nm.

Broad analysis

By analyzing size, shape, sedimentation, and diffusion coefficients of the sample, Henrickson says AUC can identify contaminants, the percentage of capsids filled with therapeutically active viral genome, and other critical quality information.

“This method is strong because it gives a full picture of the sample, and all you need to do is dilute it, you don’t need to fix it to a plate or use dye,” she points out.

Although traditional AUC is more comprehensive than PCR, ELISA, or other analytical techniques, it has the disadvantages of low throughput and a slow processing time of six to eight hours. Also, Henrickson explains, using individual wavelengths tends to overestimate the number of filled capsids.

The new technique, she says, prevents overestimation by using a wider range of wavelengths. The team is also working on a novel method called analytical buoyant density equilibrium, which they claim can increase throughput and reduce the amount of sample required by 20- to 40-fold.

“We use a shorter pathlength than typical AUC and a density gradient that creates bands or peaks—which increases throughput and decreases the amount of sample needed,” Henrickson explains.

Both techniques, she continues, can be applied to different sub-varieties of AAV, as well as other gene therapeutics, such as bacteriophage therapies. Henrickson has also published work on using the first technique with the lipid nanoparticles used in mRNA vaccine production.

The team is writing a paper on both methods and hopes the research will be published early this year.

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