Lentiviral vector characterization

Use of lentiviral-based vectors has exponentially surged (10.1 %) during the last decade in gene therapy and vaccine products. This is mainly due to their ability to stabilize the gene of interest in the host cell (dividing and non-dividing). This paved a path to use lentiviral-based vector products to treat several diseases related to non-dividing cells. Transmission electron microscopy (TEM) is an analytical method that enables the characterization of sample characteristics such as sample general morphology, particle integrity, size distribution, packaging analysis as well as, aggregation of the products containing lentiviral vectors.

What can be monitored in different manufacturing processes by using TEM

The production process of lentiviral must be closely monitored. For this reason, the main manufacturer's 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.

  • Product clusters/aggregates formed during upstream and downstream
  • Failure to remove host cell debris/impurity
  • Presence of genomic material inside the vector (Packaging analysis)
  • Changes of particle morphology or integrity due to changes in the process
  • Morphology or integrity changes caused by changes in the formulations

CryoTEM will characterize product stability

In general, lentiviral vectors are classified as following depending on the sample:

  • High density particles
  • Low density particles
  • Lentiviral particles exhibiting a visible core
  • Liposomes like particles
  • Others depending on the phase of development of sample production
Figure 1: Representative cryotem images of lentiviral-particles

Figure 1. Representative cryoTEM images of lentiviral particles, original images (left) and the corresponding classified images (right).

  • Green: Particles without a trace of spikes or cone shaped core, and not having either high or low internal density,
  • Blue: Particles having a trace of spikes and displaying a distinct outer shell and less dense interior similar to the image background.
  • Red: Particles having a trace of spikes on the surface and displaying a higher internal density than the background of the image with little/no clear distinction between the outer membrane and the core.
  • Pink: Particles having a trace of spikes and displaying a visible internal core and having either high or low internal density.

Circularity and Size distribution

Circularity analysis provides information about shape of the particles in the sample containing lentiviral vectors. Particle size distribution provides information about size of particles in the sample containing lentiviral vectors.

The degree of circularity and size distribution enables the classification of the lentiviral vector particles in a sample.

Figure 2

Figure 2. The histograms representing circularity distribution (left) and size distribution (right) obtained from semi-automated detection.

Negative staining will characterize the purity, stability, and size distribution

Lentivirus particles and the spikes (see Figure 3) on the surface are characteristic features that help to distinguish lentivirus from other membrane structures which are often lack of spikes on their surfaces.

Figure 3

Figure 3. Representative nsTEM images showing different characteristic morphological structures of lentiviral particles.

The shattered segments of lentivirus membrane with spikes protruding in the border, which indicates a broken lentiviral particle. The lentiviral particles appeared preferentially as single entities, but clusters and aggregates of various sizes were often observed. By using VAS, the relative ratios can be quantified (see Figure 4), leading to an informed process decision. The morphological analysis we provide helps in choosing the right purification /storage process and can be correlated with efficacy and safety of the product.

Figure 4

Figure 4. The histogram representing cluster/aggregate and individual particles distribution obtained from semi-automated detection, see detected image with the detected particles overlayered with red/green circles).  

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.

Download example reports
Electron microscopists working at computers



Contact us

Contact us if you have any questions about the content on this page or anything else related to our electron microscopy services. 

Contact us