Release Testing Of AAV Gene Therapies For Clinical Trials
By Mark Haydock, biologics CMC consultant, Agile Biologics Consulting LLC
Adeno-associated virus (AAV) gene therapies that are designed to deliver a therapeutic transgene to patients are complex products that can be challenging to manufacture. Because of their complexity, a variety of analytical methods are required to ensure that these viral vectors are of high quality and purity, will function as intended, and have batch-to-batch consistency. This requires confirming the vector’s identity, titer, purity, and safety using a variety of analytical methods.
Identity
To confirm the identity of an AAV gene therapy, the capsid identity, serotype, and vector genome identity must each be confirmed. The capsid identity and serotype can be verified using ELISA, western blot, biacore, and liquid chromatography–mass spectrometry (LC-MS). The vector genome identity is usually confirmed with next-generation DNA sequencing.
Titer
There are several different ways of measuring the titer of an AAV gene therapy product. The physical titer is strictly a measurement of how much virus is present. It measures the total number of functional and non-functional viral particles and is usually expressed as viral particles/mL (VP/mL). The physical titer can be measured using enzyme-linked immunosorbent assay (ELISA), biacore, AAV capsid titration, or high-performance liquid chromatography (HPLC). The OD260/280 ratio is also sometimes used because it not only measures the physical concentration of viral particles but is also an indicator of purity since A260 measures DNA concentration and A280 measures protein concentration.
The genomic titer measures the number of viral genomes in each capsid. Genomic titers are often measured after treatment with nucleases so that any residual plasmid and unpackaged DNA have been removed and only viral genomes inside a capsid remain in the sample. Historically, DNA hybridization was used, which compared the signal from the sample to a standard curve. Today, double-drop PCR (ddPCR) is now the most popular way to measure genomic titer.
The functional titer, or infectious titer, measures the viral vector product’s ability to infect cells. The functional titer is measured using cell-based assays and is always lower than the physical titer due to the physical titer measuring functional and non-functional viral vectors.
Effective delivery of a therapeutic transgene to clinical patients depends on using the optimal multiplicity of infection (MOI) for the target cell. Knowing the virus titer is essential to using the correct MOl during clinical studies. Because of the complexity of AAV vectors and the need for a multi-step manufacturing process, AAV vector manufacturers don't always achieve the expected gene expression level in their final vector products. When there are gene expression issues with the product, being able to accurately measure the viral titer during the manufacturing process can help determine whether a gene expression problem occurred during viral packaging, transduction, or vector purification.
Potency
Depending on the viral vector product’s therapeutic indication, a potency assay that measures the product-specific transgene function on its therapeutic target may also be necessary. This is usually a cell-based assay that — depending on the product — can use a variety of reported endpoints using assays such as ddPCR, quantitative polymerase chain reaction (qPCR), ELISA, or flow cytometry. If a product-specific potency assay is required, it must be designed to biologically reflect the product's therapeutic mechanism of action.
Purity
The product purity, capsid content (empty/full ratio), and process-related impurities need to be measured and quantitated for GMP viral vector products. The overall product purity can be determined using sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE), capillary electrophoresis sodium dodecyl sulfate (CE-SDS), or HPLC. Aggregates can be measured using dynamic light scattering (DLS), ultra-centrifugation (AUC), or size exclusion chromatography with multi-angle static light scattering (SEC-MALS).
Historically, the capsid content (empty/full ratio) was determined using analytical AUC and transmission electron microscopy (TEM). However, more groups are now using capillary isoelectric focusing (cIEF) to take advantage of the negative charge of DNA.
Due to their complicated structure and manufacturing processes, viral vectors have many potential process-related impurities. These can include residual host cell proteins and DNA, residual viral DNA, and residual plasmids. Residual host cell protein is usually measured using ELISA. Residual host DNA and plasmid DNA are typically measured by qPCR. PCR also can be used to measure residual viral DNA, but ddPCR is often needed due to the hairpin inverted terminal repeat (ITR) DNA structures in the AAV DNA sequence.
Depending on the product manufacturing process, the final viral vector may also need to be tested for residual benzonase, transfection reagent, tween, affinity purification column ligands, and bovine serum albumin (BSA).
Safety
To reduce the risk of bacterial or fungal contamination, the viral vector bulk is tested for bioburden or sterility. The final viral vector drug product that will be given to patients must always be tested for sterility. Viral vectors must also be tested for endotoxin, mycoplasma, and potential adventitious agents. AAV vector products also need to be tested for replication-competent virus using cell-based infectivity assays to show that the final AAV gene therapy product is not able to replicate on its own.
Compendial Assays
A series of compendial tests also need to be performed on viral vector products. These include visual appearance, pH, osmolality, and measuring the visible and sub-visible particles in the product.
Additional Characterization Assays
Some AAV serotypes can have post-translational modification on their capsid proteins. Depending on the serotype, these can include ubiquitination, phosphorylation, glycosylation, acetylation, and deamidation. While AAV1 historically has shown no post-translational modifications, AAV serotypes 2-10 can all have modifications that differ between the serotypes. Depending on the final AAV gene therapy serotype, additional characterization assays may be needed on each batch to ensure lot-to-lot consistency.
Conclusion
This article provides a high-level overview of the QC testing requirements for GMP AAV viral vectors that will be used for human clinical trials. Due to their complexity, a diverse set of assays are needed to verify the product’s identity, quantity, purity, quality, and safety to ensure they are appropriate for use in patients.
This article was previously published on the Agile Biologics Consulting LLC website. Republished with permission.
About The Author:
Mark Haydock is a biopharmaceutical CMC consultant and founder of Agile Biologics Consulting LLC. Before becoming a consultant, he served as vice president of CMC at Cue Biopharma. He has more than 30 years of experience in biopharmaceutical CMC operations, at Chiron, Aventis, Medarex, Morphotek, Eisai, and Mersana Therapeutics. During his career, Haydock established the CMC departments for three successful VC-funded companies and has supported over 30 products. He holds a B.S. in microbiology from California Polytechnic State University and an M.S. in molecular genetics and cell biology from the University of Chicago.