AAV Vector Characterization with Quantitative TEM

Validated method for precise quantification of full, partial & empty capsids

Accurate characterization of AAV vector content is a critical quality attribute in gene therapy development and manufacturing. In this study, we validated Quantitative Transmission Electron Microscopy (QuTEM) as a robust method for single-particle classification of full, partially filled, and empty capsids, demonstrating strong concordance with established orthogonal techniques.

In collaboration with Viralgen, our recent publication demonstrates how QuTEM can precisely quantify capsid populations and aligns closely with orthogonal techniques including AUC, Mass Photometry, and SEC-HPLC.

QuTEM extends conventional transmission electron microscopy into a quantitative analytical method.

Rather than relying solely on visual inspection, QuTEM measures internal density values at the single-particle level. Each capsid is analyzed and classified based on its internal electron density, enabling objective differentiation between:

Empty capsids
Partially filled capsids
Full genome-containing capsids

Click here to read our article: Orthogonal approaches to AAV vector characterization: Validating quantitative TEM for partially filled particles

From Image to Quantitative classification

WorkFlow

Validated automated workflow

Step 1 – High-Quality TEM Imaging
Native-state capsids are imaged with optimized contrast and controlled background conditions.

Step 2 – Automated Particle Segmentation
Individual particles are automatically detected using our image analysis software Gridsee.

Step 3 – Internal Density Quantification
The images are normalized, with background removal, and the pixel intensity distributions are measured.

Step 4 – Statistical Classification
Density histograms resolve distinct peaks corresponding to empty, partial and full populations.

Method Highlight

Background normalization

Example AAV density profiles for an empty (blue), partial (yellow), and full (red) particle are shown.

Acquired raw images have an uneven background and need to be normalized. An arbitrary value is picked to normalize the background for each of the detected particles. Subsequent internal density measurement is then performed, and each particle’s mean internal density is plotted in a histogram. Gaussian curves are then fitted underneath to calculate peak estimates and vector content of the sample.

Key strengths

Accurate classification of full, partial,
and empty capsids

Consistent across ssAAV and scAAV

Direct visualization of the specimen with
single-particle resolution

Practical impact

Supports process development and comparability studies

Strengthens quality control and regulatory documentation

Complements established methods
such as SV-AUC and Mass Photometry

Orthogonal validation

Benchmarked against established techniques

The comparability study evaluated two different AAV serotypes and included both single-stranded (ssDNA) and self-complementary (scDNA) vectors, ensuring that QuTEM’s performance could be assessed across diverse vector formats.

The results showed that the percentage of empty, partially filled, and full capsids measured by QuTEM aligned closely with established analytical techniques, including Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) and Mass Photometry (MP).