Vironova are experts in transmission electron microscopy (TEM). This technique is used for adeno associated virus characterization in terms of percentage of filled and empty capsids, overall sample morphology, integrity, and aggregation/clustering processes.

We recommend employing TEM analysis in the early stage of your product development to make informed decisions at the right time.

On this page you will find valuable information about characterization of AAV vectors. At the bottom of the page you will find answers to frequently asked questions (FAQs). If you have any further questions, make sure to contact us and one of our electron microscopy experts will help you.


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Using TEM when analyzing AAVs

TEM is a well-described technique used for AAV characterization in terms of percentage of filled and empty capsids, overall sample morphology, integrity, and aggregation/clustering processes.

Percentage filled and empty AAV capsids

Adeno-associated virus (AAV) is one of the most commonly used delivery vectors in gene therapy. An AAV is formed by a closed virus capsid encapsulating a single-stranded DNA. Three types of capsids are produced during the production of AAV vectors; empty, partially filled, and filled capsids. Empty and partially filled AAV capsids are considered impurities as they either lack genomic material or contain only fragments of the transgene that give the vector its functionality.

On the other hand, the filled AAV capsids are the target of production since the full-length transgene is incorporated. The presence of impurities will affect the efficacy and safety of AAV vector products since they increase the risk of immune reaction to the product. Furthermore, the presence of empty or partially filled capsids can obstruct the transduction of the filled AAV capsids by competing for vector binding sites when administered to patients. Therefore, it is essential to determine the amount of these impurities (partially filled and empty AAV capsids), as well as the desired filled AAV capsids. The ratio of filled and empty capsids is a critical quality attribute requirement for any AAV vector manufacturing process. CryoTEM allows the inspection of AAV samples in cryogenic conditions to study their structure close to their native state. This allows the observation of the internal structure of the AAV particles and provides a distinct discrimination between filled and empty capsids (Figure 1). 

Our proprietary VAS software is used in combination with cryoTEM images to accurately provide statistical data on the distinction between filled, partially filled (uncertain) and empty AAV capsids, see Figure 2.

Result of a filled and empty analysis of AAVs

Figure 1 - CryoTEM images of adeno-associated virus (AAV) displaying the filled and empty particles (left). The detected and classified particles are overlaid with red/blue/green circles (right) and correspond to filled, empty, and uncertain particles, respectively.

A graphical representation of a filled and empty analysis using CryoTEM.

Figure 2 - A representative histogram (left) displaying the percentages of filled, empty and uncertain AAV particles, as determined from semi-automated detection. Particle classification is based on internal density analysis of each particle. The corresponding image (right) shows the detected and classified particles overlaid with red/blue/green circles.

Validated method for your own product

Throughout the years Vironova has been providing validation, feasibility, and qualification studies for determining filled and empty AAV ratios for different companies in the gene therapy field. In a validation plan we specify the design and acceptance criteria for a validation study, aiming to verify that the client-specific analytical procedure using cryoTEM is suitable for measuring the percentage of filled AAV particles in a specific product. The study is done according to Vironova’s standard operating procedures (SOPs) and the principles of Validation of Analytical Procedures, ICH Q2 (R1). For example, the validation study includes tests for specificity, dilutional accuracy, repeatability, intermediate precision, linearity, and robustness. The validation of this method must be tested for your own product.

Overall morphology of AAV samples

Other types of impurities apart from the empty and partially filled capsids, which are commonly produced during the manufacturing of AAV vectors, include host cell proteins, mammalian DNA, and other contaminants. These contaminants may influence the immune response to the product. Negative stain TEM (nsTEM) provides detailed information about overall morphology of the AAV samples. The overall morphology includes integrity of the AAV particles, presence of debris/impurities and characterization of clusters/aggregations. Some example images are provided below in Figure 3.

Aggregation is a severe problem during the purification of AAV vectors. There is no accurate method to fully characterize this phenomenon. However, nsTEM can be used as an orthogonal technique to study the aggregation of AAV vectors. We offer this service (Figure 4) on demand.. Please contact us for further information.

An image showing AAV particles using the nsTEM technique.

Figure 3 - nsTEM image of AAV particles. The intact AAV particles appear as brightparticles exhibiting a well-defined exterior. Internally stained AAV particles are  broken or incorrectly assembled particles. In the image it is also possible to identify the debris present in the background, as well as dimeric AAV particles.

Image and graph showing particle class distribution in precentage between cluster(aggregates and individual AAV particles.

Figure 4 - A representative histogram (left) displaying the percentage of individual and cluster/aggregate AAV particles, as determined from semi-automated detection. The corresponding image (right) shows the detected and classified particles overlaid with red circles and yellow outlines.

nsTEM provides useful information in tracking all the steps of the upstream and downstream processes  in the monitoring and screening of purification steps. As illustrated in Figure 5, the nsTEM images allow the user to make an informed decision about the various purification conditions.

An illustration of the purification process using the nsTEM technique.

Figure 5 - Schematic illustration of the use of nsTEM images in the acceptance or rejection of a purification method for  product purification. Different purification methods were applied to the same AAV sample. Various types and proportions of impurities are observed after each purification step: (A) significant amount of debris and proteasomes, (B) a large amount of proteasomes, and (C) minimal debris.

We recommend employing TEM analysis in the early stages of your product development to help make timely and well-informed decisions.

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