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Simple Western Rapidly Quantifies the Empty/Full Content Ratio in AAV Samples

­­Measuring empty and full adeno-associated viral vectors (AAVs) is a major challenge in developing gene therapies for which current methods are limited. Now, there is a novel method for quantifying the DNA content of AAV particles with Simple Western™, a next-generation biomolecular analytical platform from ProteinSimple, a Bio-Techne brand. 

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Jess Instrument with AAV cell Hero Image

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

Simple Western Overcomes the Limitations of Traditional Methods

Minimal sample expenditure for analysis. AAVs are challenging and expensive to manufacture. Simple Western requires as little as 3 μL of starting material and can detect as few as 7.2 x 107 viral particles (VP) per well.

Process many samples quickly and without user intervention. With Simple Western, 24 samples may be analyzed in just 5 hours, or 96 samples overnight, providing the high throughput you need to scale with your AAV manufacturing workflows.


Eliminate the need for laborious sample purification steps. Simple Western is an immunoassay that is capable of specifically detecting proteins in crude, partially purified, and purified samples alike. This contrasts with other methods for assessing capsid content like icIEF or CE-SDS, which require purified or partially purified samples.

Multi-attribute analysis. Simple Western provides flexible multiplex chemiluminescence and NIR/IR fluorescence detection with industry-leading sensitivity, which enables simultaneous Total Protein Detection for multi-attribute analysis of process-related impurities combined with an identity assay.

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

Viral titer. Simple Western is highly quantitative, unlike traditional Western blots. Check out this high-impact publication to see how Simple Western measures AAV titer: Self-attenuating adenovirus enables production of recombinant adeno-associated virus for high manufacturing yield without contamination. Su, W. et al. Nat. Commun. 13, 1182 (2022).

Functional potency. Check out this high-impact publication to see Simple Western inaction to measure AAV potency in gene therapy to treat Duchenne muscular dystrophy: Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy. Moretti, A. et al. Nat. Med. 26, 207–214 (2020).

VP1:VP2:VP3 stoichiometry. Simple Western reproducibly quantifies viral capsid protein stoichiometry using ~500X less protein and an order of magnitude less AAV particles than Western blot! To learn how Simple Western and PROGEN's VP Protein Standards and Antibodies team up to quantify capsid protein ratio, download our App Note or watch the On-Demand Webinar.

Empty AAV Particles are Unwanted Bioprocess Impurities for Gene Therapies

AAVs are frequently used as viral vectors in gene therapies to address human 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 plasmid DNA) and 70% empty or partially empty (devoid of the desired plasmid DNA). These empty or partially empty AAV viral vectors 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 exists a need for better methods and systems for the efficient and sensitive quantification of empty/full status of AAV samples to be used for gene delivery.

Here, we developed new methods for the quantitative characterization of the full and empty viral capsid content in a sample using Simple Western Size and Charge assays with antibodies that target the viral capsid and the nucleic acid content individually. The results obtained by this method showed a linear correlation between the signal targeting the nucleic acid content and the percentage of full AAV capsids in each sample, enabling a quantitative assessment of the percentage of empty or partially empty viral capsids 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 not only decreases labor costs but also 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 bioprocess manufacturing workflows.