Simple Western Rapidly Quantifies the Content Ratio in AAV Samples

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 the Simple Western Empty/Full AAV Assay Work?

The Simple Western method automatically separates AAV samples by Size or Charge followed by specific and sensitive detection using anti-DNA and anti-VP1/2/3 antibodies directly in the capillary for quantitative and reproducible measurement of these central AAV components.

For each antibody, we identify the range in which the signal intensity is linearly related to the amount of sample loaded, resulting in a rapid and sensitive assay for accurately quantifying the ratio of % full AAVs to total AAVs in a sample, called the Content Ratio.

Measure AAV Empty/Full Capsid Content Ratio with Simple Western

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.

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.

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.

A Multi-Attribute Method for Viral Vector Characterization

Identity. Simple Western uses specific antibodies to provide the identity of AAVs in upstream and downstream workflows. Download our App Note to learn how Simple Western measures AAV identity.

Process-related impurities. Simple Western has Total Protein Detection that is comparable to the most sensitive SDS-PAGE gel staining techniques like SYPRO Ruby. Download our App Note to learn how Simple Western detects impurities in AAV manufacturing with leading sensitivity.

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

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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.

AAV Capsid Content App Note

More AAV Characterization Assays

Related Simple Western Resources

Adeno-Associated Virus (AAV)
Title	Key Data Sets	Take-Home Message
The New Gold Standard for Protein Expression Potency Measurements	(1) Relative protein expression potency measurements between AAV serotypes using parallel line analysis (PLA) (2) Absolute protein expression measurements using a recombinant standard curve (3) Quantification of AAV VP1/VP2/VP3 capsid proteins in transduced cells as a surrogate for AAV infectivity	Simple Western provides analytical-grade protein expression quantification for relative potency assays in a multi-attribute platform approach. AAVs can be detected in transduced cells as a surrogate for infectivity.
Do Your AAVs Contain DNA - Rapid and Sensitive AAV Empty/Full Ratio Assay with Simple Western	(1) Empty/full content ratio of AAVs and assay linearity, reproducibility, and specificity (2) Platform method approach for multiple AAV serotypes (3) Empty/full measurements of intact AAVs using Simple Western Charge as an orthogonal method	Simple Western provides automated empty/full AAV capsid content ratio measurements with high sensitivity, reproducibility, and scalability. Simple Western is a multi-attribute platform method to characterize multiple AAV serotypes in complex sample types.
AAV2 VP Protein Standards and Their Use In Quantifying Capsid Protein Ratio by Western Blot and Simple Western	(1) Establishment of an AAV2 VP1, VP2, and VP3 Protein Standard (PROGEN) (2) Analysis of VP1, VP2, and VP3 for capsid protein ratio and titer measurements (3) Demonstration of Simple Western reproducibility and linearity and comparison to traditional Western blot	PROGEN’s recombinant AAV2 VP protein standards are useful for accurate and sensitive analysis of VP content and VP ratios of AAVs. Simple Western removes the variability of traditional Western blot, plus advantages like automation, short time to results, tiny sample volume requirements, and high sensitivity.
Concentrating on AAV Impurities with Ultrasensitive Total Protein Detection on Simple Western	(1) 5X biotin labeling reagent increases DnaK detection sensitivity (2) RNase A spiked into purified AAV sample and analyzed with 1X and 5X total protein labeling reagent on Jess (3) Multi-attribute identity and purity measurements of AAVs using RePlex (4) Comparison of 5X biotin labeling reagent to SYPRO Ruby	The 5X biotin labeling reagent provides ultrasensitive and reproducible total protein measurements in the upper picogram range, beating SDS-PAGE staining techniques like SYPRO Ruby and silver stain, which require lower nanogram levels for reliable detection.
Simple Western Analysis of Adeno-Associated Virus (AAV) Proteins	(1) Identification of AAV2 purification using VP-specific antibodies (2) Range of detection of the anti-VP1/VP2/VP3 antibody (3) Immunoassay and total protein analysis of AAV during purification from HEK293 whole-cell lysate	Simple Western is a powerful analytical tool throughout the AAV product development pipeline, providing purity and identity measurements to monitor and characterize AAV capsids during product purification from HEK293 cells.
The Simple Western Assay for Residual Benzonase Quantification	(1) Recombinant standard curve for Benzonase quantification (2) Calibration of the Benzonase assay with QC samples (% recovery) (3) Quantification of residual Benzonase in HEK293T whole-cell lysates (<1 ng/mL) and % recovery	Simple Western overcomes the challenges of traditional ELISA without compromising sensitivity and reduces hands-on time to just 30 minutes for sample preparation and plate loading, making it possible to integrate at-line or near-line and achieve fast, actionable results.

Lentivirus
Title	Key Datasets	Take-Home Message
Lentiviral Vector Analysis Made Simple	(1) p24 titer measurements and cross-reactivity with other lentiviral proteins (2) Lentiviral empty/full capsid content and stability assays (3) 5X total protein labeling reagent versus anti-HEK293T HCP antibody (4) Lentiviral transduction of iPSC-derived macrophages (customer data)	Simple Western is an automated multi-attribute platform for scalable at-line lentiviral analytics, including p24 titer, identity, purity, stability, capsid content ratio, and transduction measurements.

Regenerative Medicine
Title	Key Datasets	Take-Home Message
Simple Western Unravels the Signaling Web in Stem Cells for Regenerative Medicine	(1) Quantification of stemness biomarkers (Nanog, OCT3/4, SOX2) in naïve iPSCs and iPSC-derived endoderm, ectoderm, and mesoderm cells (2) Multiplex analysis of total and phospho-protein isoforms (Akt and Smad1/5) plus total protein normalization in naïve iPSCs and iPSC-derived mesodermal cells in a single run using Stellar on Jess, RePlex on Abby, and Peggy Sue	Using as little as 3 μL of starting material, Simple Western assays generate protein expression profiles of stem cells with flexible multiplex strategies and unprecedented sensitivity and reproducibility, revealing new insight into intracellular signaling cascades in response to iPSC differentiation.

Do Your AAVs Contain DNA?

Simple Western Quantifies AAV Empty/Full Ratio Content