The TEM technique for adenovirus characterization

Adenovirus-based vectors are the most widely used vectors applied as gene therapy, cancer therapy and vaccine products, due to their capacity to induce a robust immune response. Based on that fact adenovirus-based vectors represent 19% of the total number of vaccines.

TEM is a well described technique used for adenovirus characterization in terms of overall sample morphology, integrity, and aggregation/clustering processes.

What is monitored using TEM in the adenovirus manufacturing

The production process of adenovirus must be closely monitored to assure that the final product is safe and efficient when administered to the patient. For this reason, the main manufacturers focus is guaranteeing that these vectors are well characterized in terms of stability, purity, and integrity. TEM analysis provides comparable data when production conditions are changed in upstream and downstream processes or product formulations. The following parameters can be monitored using TEM:

  • Product clusters/aggregates formed during upstream and downstream
  • Failure to remove host cell debris/impurity
  • Changes of particle morphology or integrity due to changes in the process
  • Morphology or integrity changes caused by changes in the formulations

Assessing the internal density of adenovirus

CryoTEM allows the study of adenovirus samples in cryogenic conditions preserving close to the native structure of the viral particles. The presence of the vector genome will contribute to the internal density of the particles, see Figure 1, meaning that the internal structures of the virus can be observed using CryoTEM.

Image of adenovirus using the CryoTEM technique

Figure 1 – CryoTEM images of adenovirus samples the classification on the right was performed having in account the internal density profile of each particle.

A graph showing high density versus low density of adenovirus particles.

Figure 2 - Graphical representation of the high density (Type I) versus low density (Type II) analysis. CryoTEM image, the detection of adenovirus particles is overlay in the image with red and blue circles.

This method can be validated for your own product

Through the years Vironova has been providing validation, feasibility, and qualification studies. 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 adenovirus samples

Negative stained TEM (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. Presence of clusters and aggregates can be visualized, and relative ratios can be quantified, leading to an informative process decision (Figure 4).

Figure 3 shows nsTEM image of an adenovirus sample.

Figure 3 - nsTEM image of an adenovirus sample. The visualization of the disassembly of adenovirus segments, hexons and fibers (left imaged) as the presence of impurities in the samples, will be helping in the choice of the right purification method in the lab.

Figure 4 shows 3 different images in one. The first is a graphical comparison, the last two nsTEM images of adenovirus.

Figure 4 - Graphical representation of the individual versus cluster/aggregates analysis. In the nsTEM image (right), the detected cluster/aggregates and the individual adenovirus particles are overlayered with red and blue circles, respectively.

Integrity analysis of the adenovirus particles

The integrity of the adenovirus particles products can be evaluated using nsTEM and/or the cryoTEM technique, see Figure 5.

Figure 5 shows both cryoTEM and nsTEM images of adenovirus samples.

Figure 5 - CryoTEM (upper) and nsTEM (bottom) images of adenovirus samples. The image on the right has the classification overlaid having in account the integrity of the adenovirus particles (green circles intact particles and yellow circles broken particles).


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