AAV Quantification

AAV quantification

High-throughput, low-volume adeno-associated virus titer quantification and empty/full ratio

Adeno-associated virus (AAV) quantification and empty/full ratios are a major challenge for developing and manufacturing gene therapy vectors. AUC, TEM, ELISA or PCR-based meth­ods each give you some of the info on a sample, but none of them deliver results quickly, easily or from low sample volumes. TEM and AUC are gold-standard measurements, but require a lot of time, sample, and specialized equipment. ELISA and qPCR are solid workhorse techniques but take hours and require development and optimization for each AAV vector.

Stunner determines sample titers quickly and easily so it’s possible to check capsid titer and empty/full ratios as often as you want. A read on Stunner before a PCR method takes the guess­work out of choosing a dilution factor or can be a quick check for when the slower PCR run is overkill. For those experiments, a quick check of sample titer on Stunner spots aggregates that will mess up your results.

Using Stunner to judge capsid titers gives a faster and fuller picture of your sample that avoids slow and expensive assays like ELISA.

Figure 1: How Stunner uses DLS, SLS and UV/Vis to determine empty/full ratio, AAV capsid titer and genome titer.

Figure 1: How Stunner uses DLS, SLS and UV/Vis to determine empty/full ratio, AAV capsid titer and genome titer.

The AAV Quant app on Stunner combines dynamic light scattering (DLS), static light scattering (SLS) and UV/Vis techniques to determine titers, total protein, total DNA, and empty/full ratio (Figure 1). DLS is a classic light scattering technique to identify size and size distribution for particles in solution. DLS intensity distributions show the relative inten­sities of light scattered by capsids and the aggre­gates commonly found in AAV samples. The SLS intensity, gathered simultaneously to DLS results, is directly proportional to the particle concentration. However, since full and empty capsids scatter dif­ferent amounts of light at the same concentration, you need more than just DLS and SLS to get titers – you need UV/Vis data too.

UV/Vis spectroscopy determines the total amount of protein and DNA present in a sample, but by it­self cannot tell how much of that protein and DNA is packaged into AAV capsids. For a purified sam­ple, the ratio of protein and DNA present is also related to the empty/full ratio. When combined with DLS & SLS results, Stunner has enough info to calculate total capsid titer, full capsid titer, and the empty/full ratio of an AAV sample (Figure 2).

Figure 2: Stunner reports AAV total capsid titer, full capsid titer, free & aggregated protein, free & aggregated ssDNA, % full, % empty, and empty/full ratio.

Stunner calcu­lates full capsid titers by combining the ratio of ssDNA and pro­tein, determined by UV/Vis absorbance, with the number of intact capsids, determined by DLS and SLS (Figure 3, left). This full capsid titer is equivalent to the total amount of encapsidated ssDNA in a sample, and is not subject to the variation seen in PCR-based methods that is caused by selecting primers that target different parts of the AAV genome. As such, Stunner provides titers across AAV serotypes and different gene inserts without the need for different primers or standard curves.

ELISAs are slow, typically requiring multiple hours and repeated incubation steps. Stunner’s capsid titers are label-free and standard-free and take only 1 hour for 96 samples. When comparing Stunner capsid titers to ELISA, the correlation between the Stun­ner and ELISA capsid titers for empty and full AAVs had R2 values > 0.98 and slopes near 1, showing a high level of agree­ment between these two methods (Figure 3, right).

Figure 3: Stunner full titer of AAV 9 dilution series compared to SYBR Gold encapsidated ssDNA titer (left) and total titer of empty & full AAV9 dilution series comparted to ELISA (right).

Figure 3: Stunner full titer of AAV 9 dilution series compared to SYBR Gold encapsidated ssDNA titer (left) and total titer of empty & full AAV9 dilution series compared to ELISA (right).

Quantification of AAV titers and empty/full ratios doesn’t have to be arduous. While other methods are time-consuming or require significant sample amounts, Stunner speeds things up with high-throughput determi­nation of capsid titer, ssDNA titer, and empty/full ratio from tiny sample volumes. Regardless of whether a vector is in research, development or manufacturing, Stunner gives you the data you need and helps you know your capsid inside out. If you need more information on how Stunner serves up AAV quantification or quality checks, check out this app note that’s all about AAVs.

Stunner delivers:

  • Reagent- and standard-free AAV quantification for titers ≥1E12 vg/mL
  • Tiny 2 µL sample volumes
  • 96 measurements in about 1 hour
  • Accurate and precise full-spectrum UV/Vis absorbance results from 230-750 nm
  • Wide dynamic range from 0.03 to 275 OD
  • Size measurements from 0.3-1000 nm
  • Highly sensitive detection of aggregates, even ones too large or too rare for traditional SEC analysis
  • Fixed pathlength microcuvettes for maximum sensitivity and reproducibility
  • Low maintenance and no clean-up
  • Performance Verification services for USP and Ph. Eur. compliance

Stunner

Want to do a quick check of your AAV titer or empty/full ratio, or see if it is just a blob of aggregates? Stunner adds plate-based, high-throughput UV/Vis to dynamic light scattering for a one-of-a-kind combination. Pairing up these techniques checks titers, empty/full ratio, hydrodynamic size, polydispersity, and detection of aggregates off your to-do list in one shot. Know that you can trust the data and the sample you hand off to the next scientist.

Stunner

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