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Simple Western Quantifies AAV Empty/Full Ratio Content­­

AAV capsid content ratio

Not every AAV capsid is packaged correctly with AAV DNA. An empty AAV capsid impacts the safety and efficacy of your final drug product, but identifying an empty or partially empty AAV capsid is challenging, and current methods are laborious and cannot scale with AAV manufacturing needs to meet demand. 

Simple Western™ provides automated AAV empty/full ratio measurements in 6 hours or less. Just load 3 µL of your AAV sample and Simple Western will perform AAV empty full separation and AAV quantification of AAV DNA and AAV capsid proteins, providing sensitive and reproducible AAV empty/full ratio measurements. Plus, Simple Western works in complex samples like HEK293T whole-cell lysates - no sample cleanup required!

Background on AAV Empty/Full Ratio | Related Simple Western Resources

How Does Empty/Full AAV Quantification Work?

  • AAV capsid protein and DNA components are separated and detected using specific antibodies. 
  • Ratio of AAV capsid protein to DNA correlated with the vendor-supplied % full values.
  • Only 3 µL of AAV sample per well (as low as 7.2 x 107 VP per well).
  • Up to 24 samples per hand-free run. Runtimes <6 hours.
  • The method is unaffected by sample impurities and can be applied directly in crude sample types like HEK293T whole-cell lysates for upstream process development.
Measure AAV Empty/Full Capsid Content Ratio with Simple Western
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Minimal Sample Expenditure

AAV production is challenging and expensive. Simple Western requires as little as 3 μL of starting material and can detect as few as 7.2 x 107 VP/well.

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No Laborious Purification Steps

Simple Western is an immunoassay that is capable of specifically detecting proteins in complex samples like HEK293 whole-cell lysate without sample cleanup.

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Automated and High-Throughput

With Simple Western, 24 samples may be analyzed in just 5 hours, or 96 samples overnight, so you can scale with your AAV manufacturing workflow.

Multi-Attribute AAV Capsid Analysis

AAV Relative Potency.  Simple Western is the preeminent tool for protein expression potency assays. Get analytical-grade relative potency measurements in a platform approach.

AAV Purity. Simple Western has total protein detection that beats SDS-PAGE gel staining like SYPRO Ruby.

AAV Identity. Simple Western uses specific antibodies to provide the identity of your AAV capsid protein.

AAV Capsid Protein Ratio and Titer. Get AAV capsid protein ratio and titer measurements using 500X less AAV protein than Western blot.

Jess Simple Western AAV characterization including AAV viral titer and capsid ratio and viral identification

Empty AAV Capsid Particles Are Problematic Byproducts During AAV Production

AAVs are used as gene delivery vectors in innovative new treatments of severe diseases, with over 200 studies around the world conducting active clinical Phase 1-3 trials. As gene delivery systems, AAVs include a gene of interest encoded in plasmid DNA that can be up to 5kb in length. AAVs can exist as a heterogeneous population, giving a final sample that is, for example, 30% full (including the desired AAV DNA) and 70% empty or partially empty (devoid of the desired AAV DNA). These empty or partially empty AAV capsid particles can impact potency and immunogenicity and thus are unwanted bioprocess contaminants. Traditional analytical tools such as transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and ion-exchange chromatography (IEX) can be used to characterize AAV capsid content but are complex, labor-intensive, and pose challenges in data reproducibility, throughput, and scalability. Therefore, there is a need for better methods and systems for the efficient and sensitive quantification of empty/full status of AAV samples for gene delivery.

Here, we developed new AAV quantification and characterization methods of AAV empty/full ratio in a sample using Simple Western Size and Charge assays with antibodies that target AAV capsid proteins and AAV DNA individually. The results obtained by this method showed a linear correlation between the signal targeting the AAV DNA and the percentage of full AAV capsid particles in each sample, enabling a quantitative assessment of the percentage of empty or partially empty AAV capsid particles in a sample.

A major advantage of this method is that Simple Western is a fully automated immunodetection platform with a throughput of up to 96 samples that can be processed overnight. This automation decreases labor costs and enables faster iterations during in-process development. Equally advantageous is the tiny sample volume requirement of Simple Western (as little as 3 μL sample) that minimizes impact on final product titer and allows for the analysis of as few as 7.2 x 107 VP per well, making Simple Western a scalable solution for AAV quantification in bioprocess workflows.