Lentiviral vectors analyses using transmission electron microscopy

Lentiviral-based vector characterization

The use of lentiviral-based vectors has exponentially surged (10%) 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, paving the way for lentiviral-based vector products for the treatment of diseases related to dividing and non-dividing cells. Transmission electron microscopy (TEM) is an analytical method that enables the characterization of sample characteristics such as overall morphology, particle integrity, size distribution, packaging analysis, and aggregation of the products containing lentiviral vectors.

What can be monitored in different manufacturing processes by using TEM

The production process of lentiviral-based vectors must be closely monitored to assure the safety and efficacy of the final product. For this reason, the manufacturers' main focus is on 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 when product formulations are modified. The following parameters can be monitored using TEM:

Product clusters/aggregates formed during upstream and downstream processes
Failure to remove host cell debris/impurities
Presence of genomic material contained within the vector
Morphology or integrity variations caused by changes in processing conditions or product formulations

Classifying lentiviral vectors with cryoTEM

Cryogenic transmission electron microscopy (cryoTEM) is a valuable technique for inspecting lentiviral vectors, as the rapid fixation of the particles within a vitreous ice and subsequent imaging under cryogenic conditions preserves the particle structure in a close-to-native state. With this technique, several particle features can be distinguished, including the internal structure of the vectors, providing useful information for particle classification into the following categories:

High density particles
Low density particles
Lentiviral particles exhibiting a visible core
Liposome-like particles
Others depending on the phase of development of sample production

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

Green: Particles without traces of spikes on the surface or cone-shaped cores, displaying neither high nor low internal density
Blue: Particles with traces of spikes on the surface, displaying a distinct outer shell, a low internal density similar to the image background and without a cone-shaped core
Red: Particles with traces of spikes on the surface, displaying a higher internal density than the image background and with little/no clear distinction between the outer membrane and the core
Pink: Particles with traces of spikes on the surface, displaying a visible cone-shaped core with either high or low internal density

Characterize purity, integrity, and stability with nsTEM

Negative stain transmission electron microscopy (nsTEM) is a technique which involves the application of a stain onto a sample, acting as both an embedding and contrast agent. With nsTEM, the characteristic surface structures of lentiviral particles can be distinguished. This allows the lentiviral particles to be differentiated from other membrane structures that often lack surface spikes (see Figure 3). Furthermore, the observation of different morphological features provides useful information on the purity, integrity, and stability of the lentiviral vector product.

Our proprietary VAS software can be used in combination with cryoTEM or nsTEM to quantify relative ratios of cluster/aggregates and individual particles (see Figure 4). Furthermore, the morphological analysis we provide offers useful information that can be correlated with the safety and efficacy of the product. Together, the quantitative and qualitative data can help to guide and inform processing decisions regarding purification and storage.

Several images of lentiviral particles captured with the nsTEM technique.

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

A staple diagram to the left showing cluster/aggregate and individual on the x-axis and particle class distribution on the y-axis. To the right, there is an image of lentiviral vectors.

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

FAQ

Can TEM distinguish a lentiviral vector from other membraneous structures?
The outer membrane of lentiviral vectors has spikes protruding outwards. A typical membranous structure produced along with lentiviral vectors will not have such spikes on their outer surface. These spikes can be visualized by using both nsTEM and cryoTEM.

Can the internal structure of lentiviral vectors be observed with TEM?
The internal structures (e.g. capsid) of lentiviral vectors can be observed with cryoTEM, but not with nsTEM.

From what stage of production do you recommend sending the lentiviral vector sample to Vironova for analysis?
All samples after the first stage of purification can be analyzed by Vironova using TEM.

Will the stain used in nsTEM damage the lentiviral particles?
We have an established protocol which minimizes stain-induced artefacts and assures the integrity of the lentiviral particles.

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Lentiviral vectors