Adenoviral vector characterization

The TEM technique for adenoviral characterization

Adenovirus-based vectors are the most widely used vectors applied as vaccine products, and gene and cancer therapies, due to their capacity to induce a robust immune response. Today, adenovirus-based vectors account for around 19% of the global market of vaccines.

Transmission electron microscopy, TEM, is a well-described technique used for adenovirus characterization for determining overall sample morphology, integrity, and clustering/aggregation formation.

What can be monitored with TEM during adenovirus vector manufacturing?

It is critical to assure the safety and efficacy of products administered to patients. As such, adenovirus-based vector production processes are closely monitored to guarantee the quality of the final product, and manufacturer’s focus strongly on characterizing properties such as the stability, purity, and integrity of these vectors. TEM analysis provides comparable data when there are changes in the production conditions of upstream and downstream processes, or modifications to product formulations. The following parameters can be monitored using TEM:

• Product clusters/aggregates formed during upstream and downstream processes
• Failure to remove host cell debris/impurities
• Morphology or integrity variations caused by changes in processing conditions or product formulations

Assessing the internal density of adenoviral vectors

Cryogenic transmission electron microscopy (cryoTEM) enables the inspection of adenovirus samples under cryogenic conditions, which preserves the structure of the viral particles close to their native state. Through this technique, the internal density if the studied particles is also observable, which allows one to distinguish the internal vector genome, if it is present (see Figure 1).

Figure 1. CryoTEM image of adenovirus particles displaying variations in their internal density (left). The classification of these particles was performed based on their internal density profile (right).

Our proprietary VAS software  is used in combination with cryoTEM images to accurately provide statistical data on the distinction between Type I and Type II adenovirus particles (see Figure 2).Figure 2. A representative histogram (left) displaying the Type I (red - high density) and Type II (blue - low density) adenovirus vectors particle analysis. The corresponding image (right) shows the detected and classified particles overlaid with red and blue circles.

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Method validation

Throughout the years, Vironova has been providing validation, feasibility, and qualification studies that aim to verify that client-specific analytical procedures using TEM are suitable for measuring certain product attributes. The method validation is product-specific. Further information on how Vironova validates your method of analysis can be found here.

Overall morphology of adenovirus samples

Negative stain transmission electron microscopy (nsTEM) (nsTEM) provides detailed information about overall morphology of the adenovirus samples. The overall morphology includes integrity of the adenovirus particles, presence of debris/impurities and characterization of clusters/aggregations. Figure 3 displays the morphology of the adenovirus particles as well as structural features of the hexons that constitute the adenovirus capsid.

Figure 3. nsTEM images of an adenovirus sample. The appearance of disassembled adenovirus segments, hexons, and fibers  (left), as well as impurities in the background (right) may guide processing decisions regarding purification methods.

The presence of clusters and aggregates can be visualized, and relative ratios can be quantified to aid informed processing decisions (see Figure 4).

Figure 4. A representative histogram (left) of the cluster/aggregates and individual particles analysis. The corresponding images show the detected cluster/aggregates and individual particles overlaid with red and blue outlines respectively (middle and right).

Integrity analysis of adenovirus particles

The integrity of the adenovirus-based vectors can be evaluated using nsTEM and/or the cryoTEM technique (see Figure 5).

Figure 5.  CryoTEM (upper) and nsTEM (bottom) images of adenovirus samples. The images on the right display the particle integrity classification overlay for intact (green) and broken (yellow) particles.

FAQ

Can TEM be used to study aggregation?
Due to technical differences nsTEM is more suitable for aggregation studies. Clustering/aggregation analysis is recommended to be used for comparison studies between samples having similar formulation, to obtain a relative assessment of their cluster/aggregate state. The analysis of an individual sample is also possible although this needs to be optimized by us for your own product. Please contact us for further information.

What is the optimal buffer composition of the sample in terms of sugar, salt, and others?

What level of biosafety is avaiable for sample analysis at Vironova?
We can perform TEM analysis for samples up to biosafety level 2 at our GMP-certified laboratory in Stockholm.

Although Vironova are specialists in GMP-certified analysis, is it possible to perform a non-GMP analysis?
Yes, we provide non-GMP analysis in cases where there is no requirement for GMP compliance.

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