Evolving technology has helped advance the field of gene therapy, and today, more and more biopharmaceutical innovators are using groundbreaking approaches to treat diseases. Adeno-associated virus (AAV) vectors have emerged as a leading gene-delivery tool[1], with several AAV-based therapeutics approved by the U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA). However, there are a number of complexities in AAV development and manufacturing to address. Challenges such as process- and product-related impurities can arise during production, which may affect the critical quality attributes (CQAs, which include identity, potency, purity and safety) and stability of the final product. That’s why precise analysis is essential in measuring product quality attributes during process development and manufacturing, before the product reaches patients.
“For AAV, the virus capsid encloses a specific target gene, or the modified genome, that we want to deliver as a therapeutic dose to patients,” says Kelly Rios, Ph.D., an Analytical Development Scientist with viral vector CDMO at MilliporeSigma. “We need to know its exact identity, so that we know exactly what we’re providing to patients.” Otherwise, Rios says process-related impurities may deliver host-cell DNA; or a mutation or truncation in the DNA could result in a partially full or empty capsid, falling short of the therapeutic dose.
Thanks to advancements in next-generation sequencing (NGS), long-read and short-read sequencing can be applied to AAV vector characterization at multiple points during process development and production. Rios shared the following four applications for NGS and also highlighted the methods her team at MilliporeSigma uses to conduct certain testing.
- Plasmid identity testing/raw material testing
Plasmids are an essential starting material for AAV viral vector generation. Typically, three plasmids are co-transfected for production/manufacturing. Those include a plasmid containing the gene of interest (GOI), a helper plasmid and a rep/cap plasmid that is unique to the AAV serotype. It’s important to make sure that the sequences of these starting materials are correct and free of mutations or insertions. Sequence each to verify that each construct contains the expected sequences without any changes from the original design.
Method: Use long-read sequencing for plasmid testing. - Gene-of-Interest identity testing
GOI identity testing is essential for several reasons. Primarily, it can inform that the correct genome is packaged into the recombinant AAV capsids during production. In addition, identity testing is necessary for lot release to comply with regulatory guidelines and ensure product efficacy and safety. The consensus sequence from the sequenced vector genome must match the expected reference sequence, including the therapeutic gene sequence and the flanking inverted terminal repeat regions (ITRs), with appropriate depth of coverage. Variant calling from the aligned reads allows for mutations or variants to be detected.
Method: Full inverted terminal repeat regions (ITR) coverage with long-read technology systems; short-read technology can also be used and is recommended for identity testing of AAV drug products. It’s important to note that long-read sequencing allows for sequencing through the entire viral genome in a single read, resolving those difficult to sequence regions, and examining genomic integrity, whereas coverage of inverted terminal repeat regions with short-read sequencing is challenging because of high guanine-cytosine (GC)-content, repetitive sequence and secondary structure [2]. - Residual DNA impurity testing
Mispackaging of residual DNA into the recombinant AAV particles can happen during production, meaning that host cell DNA, residual transgene plasmid DNA and residual helper plasmid DNA can infiltrate the gene delivery tool. Further, incomplete genomes—such as partial or truncated genomes—may be packaged into particles. These types of impurities can contribute to increased risk of genotoxicity[3] and/or reduced therapeutic effects. Residual DNA impurity testing characterizes and quantifies these contaminants/impurities. Identifying these during process development can aid in designing vectors and process improvement and is also essential for meeting regulatory standards.
Method: Short- and long-read sequencing platforms can be used. The reads can be bioinformatically mapped to the expected GOI reference (identity test), and also mapped to the potential contaminants/impurity sequences. - Genome integrity
Empty and partial capsids are considered impurities because they lack the genomic material or only contain genome fragments. These capsids pose a challenge during production due to their similarity to the desired full capsids, the product with the GOI. Sequencing can assess the size distribution of the viral genomic DNA, determining if it’s full-length vs truncated. This could also identify possible truncation hotspots for viral vector design.
Method: Use long-read sequencing technology systems.
Next-generation sequencing allows for critical insights into gene therapy products, which can help streamline and accelerate everything from process development and production to regulatory approval. At MilliporeSigma’s viral vector CDMO facility, the process development and analytical (PAD) team is comprised of experts in AAV sample handling, DNA extraction, library preparation, sequencing and bioinformatic analyses work with gene therapy innovators to generate and process data across short-read and long-read sequencing platforms while also supporting their program from development through commercialization. “We have the expertise and technology to help you deliver effective gene products to your patients and support you in delivering the treatments they need,” says Rios.
Learn more about MilliporeSigma’s viral vector CDMO services.
MilliporeSigma is the U.S. and Canada Life Science business of Merck KGaA, Darmstadt, Germany.
References:
[1] Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019 May;18(5). https://pmc.ncbi.nlm.nih.gov/articles/PMC6927556/
[2] BioPhorum. J. Dean, et al. Recommendations for establishing a next-generation sequencing method in a GMP setting to confirm identity of a recombinant AAV (rAAV) gene therapy drug product. 2024 June. https://doi.org/10.46220/2024ATMP001
[3] Suoranta, et al. Strategies to Improve Safety Profile of AAV Vectors (Mini Review). Front. Mol. Med. 2022 October. https://doi.org/10.3389/fmmed.2022.1054069
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