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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Percentage filled and empty AAV capsids

Adeno Associated Virus (AAV) is one of the most used delivery vectors in gene therapy. 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; namely empty, partially filled, and filled capsids. Empty and partially filled AAVs capsids are considered impurities since they either lack the genomic material or contain only fragments of the transgene. On the other hand, filled AAV capsids are the target of production since the full-length transgene is incorporated.

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 particles can obstruct the transduction of the filled AAV capsids by competing for vector binding sites when administered to the 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 is a critical quality attribute requirement for any AAV vector manufacturing process and to assure the quality of the product. CryoTEM allows the study of AAV samples in cryogenic conditions close to the native structure. This allows the observation of the internal structure of the AAV particles and provides a distinct discrimination between filled and empty capsids (Figure 1).

CryoTEM images of adeno associated virus (AAV).

Figure 1 - CryoTEM images of Adeno associated virus (AAV). The filled/empty particles are identified on the imaged on the left. While a semi-automatic detection and classification is highlighted by the overlaid circles on the right (blue circles correspond to empty particles, green circles correspond to uncertain particles and red circles correspond to filled particles).

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

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

Figure 2 - Graphical representation of the filled and empty analysis. CryoTEM image, the detected AAV particles over layered with red, blue, and green circles. The particle classification is based on each particle internal density analysis.

Validated method for your own product

Through the years Vironova has provided 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 to measure the percentage of filled AAV particles in a specific product. The study is done according to the Vironova’s standard operating procedures (SOP) and the principles of Validation of Analytical Procedures, ICH Q2 (R1). For example, the validation includes tests for:

  • Specificity
  • Dilutional accuracy
  • Repeatability
  • Intermediate precision
  • Linearity
  • Robustness

The validation of this method must be tested for your own product. If you would like to receive more information you can find it here.

Overall morphology of AAV samples

Other type of impurities, apart from the empty and partially filled capsids, which are commonly produced during the manufacturing of AAV vectors. These include host cell proteins, mammalian DNA, and other contaminants, which probably will 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.

An image showing AAV particles using the nsTEM technique.

Figure 3 - nsTEM image of AAV particles. AAV particles appear as bright intact particles exhibiting a well-defined exterior. Another class of particles that appear in the image are internally stained AAV representing broken or incorrectly assembled particles. In the image is also possible to identify the debris present in the background, as well as dimeric AAV particles.

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

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

Figure 4 - Percentage of cluster/aggregates and individual AAV particles are detected. On the right side of the image the detected classes are over laid in the nsTEM image (Yellow corresponds to cluster/aggregates class while red corresponds to the individual AAV particles).

The nsTEM will provide useful information in tracking all the steps of the upstream and downstream processes i.e in the 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 in the acceptance or rejection of the purification method to apply to product purification in function of the nsTEM images provided. Different purification methods were applied to the same AAV sample, the representative images showed the presence of different impurities, after each purification step: large number of debris and proteasomes (A), large number of proteasomes (B), and few number of debris (C).

Download example reports

We have various example reports of different types of analyses available for you to download. The reports give you a deeper insight in what to expect from hiring our electron microscopy services.

The example reports are free and easily accessed by submitting a form. Just click the button below to get to the submission page.

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