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.
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:
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:
Figure 1. Representative cryoTEM images of lentiviral particles, original images (left) and the corresponding classified images (right).
Circularity and particle size distribution analyses respectively provide information about the shape and size of the lentiviral particles in a sample, which can be utilized to classify the particles into different categories.
Figure 2. Representative histograms showing the circularity distribution (left) and size distribution (right), as determined from semi-automated detection
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.
Figure 3. Representative nsTEM images showing different characteristic morphological structures of lentiviral particles
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.
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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