Adeno-Associated Virus (AAV) is a prominent vehicle for delivering gene therapies. These viruses are modified to be non-pathogenic, serving as delivery systems that carry therapeutic genes into target cells. For these treatments to be both safe and effective, the AAV product must undergo analysis and characterization. This process ensures each batch of the therapy meets established standards before it can be used.
Key Quality Attributes of AAV Vectors
To ensure the consistency and effectiveness of AAV-based medicines, scientists measure several key properties known as Critical Quality Attributes (CQAs). These are quantifiable characteristics that help confirm the product’s identity, purity, and overall quality. The primary attributes measured are:
- Quantity: The concentration, or titer, of the AAV product.
- Capsid Content: The ratio of “full” capsids containing the therapeutic gene to “empty” ones. A high percentage of empty capsids can reduce effectiveness and may trigger an unwanted immune response.
- Purity: The absence of contaminants, such as proteins and other materials left over from the manufacturing process.
- Identity: Verification that the AAV contains the correct genetic material and that the protein capsid is the intended type for targeting specific cells.
Measuring AAV Quantity and Capsid Content
To determine the amount of AAV in a sample, one method is quantitative polymerase chain reaction (qPCR) or its more refined version, droplet digital PCR (ddPCR). These methods work by amplifying and counting the number of viral genomes, which are the DNA strands containing the therapeutic gene. This provides a direct measure of the gene-carrying AAV particles.
Another technique for measuring quantity is the enzyme-linked immunosorbent assay, or ELISA. Unlike PCR-based methods, an ELISA uses antibodies that bind to the AAV’s outer protein shell. This approach quantifies the total number of viral capsids, regardless of whether they contain a gene. Comparing results from both qPCR/ddPCR and ELISA gives a preliminary idea of the full-to-empty capsid ratio.
Transmission electron microscopy (TEM) offers a direct visual method for distinguishing between full and empty capsids. Using TEM, scientists can capture images of individual AAV particles at high magnification, allowing them to literally see which capsids are full and which appear hollow or empty.
For a more quantitative assessment of capsid content, analytical ultracentrifugation (AUC) is frequently used. This technique subjects the AAV sample to immense centrifugal forces, causing particles to separate based on their mass and density. Since capsids full of genetic material are heavier than empty ones, they move at different rates, allowing for precise measurement of the proportion of full, partially full, and empty particles in the sample.
Assessing Purity and Identity
Confirming the purity of an AAV preparation involves identifying and quantifying any unwanted substances. A standard method for this is sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which separates proteins based on their size. The AAV’s three main capsid proteins—VP1, VP2, and VP3—should appear as distinct bands, while any other bands indicate protein impurities.
For a more sensitive analysis, high-performance liquid chromatography (HPLC) is used. In this method, the AAV sample is passed through a column under high pressure, which separates the AAV particles from very small amounts of process-related impurities. Different forms of HPLC, such as size-exclusion or ion-exchange chromatography, can be used to detect and quantify aggregates or other contaminants with high precision.
Verifying the vector’s identity means ensuring both the genetic payload and the protein capsid are correct. Next-generation sequencing (NGS) is used to confirm the identity of the genetic material. This technology sequences the entire DNA packaged inside the AAV, confirming it is the correct therapeutic sequence and is free of mutations that could affect its function.
To confirm the protein shell’s identity, scientists use mass spectrometry (MS). This technique measures the mass of the capsid proteins, which can confirm that the correct AAV serotype was produced. MS can also detect any post-translational modifications to the proteins that might impact the vector’s stability or performance.
Determining Potency and Function
After analyzing physical and chemical attributes, tests are needed to confirm the AAV works as intended. These biological assays measure the vector’s potency, its ability to produce a therapeutic effect. Physical characterization alone cannot guarantee that the vector is biologically active.
Potency is assessed using cell-based assays. Scientists expose target cells to the AAV product to measure if the vector can enter cells, deliver its genetic payload, and facilitate gene expression. This is often quantified by measuring the amount of therapeutic protein produced by the cells.
These functional tests are a direct measure of the product’s therapeutic activity. The results connect the vector’s physical attributes to its biological purpose, which is a culminating step in the quality control process.