AAV Characterization: Current Status and Future Advances

Gene therapy is one of the most exciting treatment approaches on the market. It holds significant promise to reduce the number of routine medical interventions otherwise required and even deliver curative, long-acting therapeutic effects. This has been realized in the clinic most recently with Pfizer’s announcement of the treatment of Hemophilia B, a rare inherited bleeding disorder, with fidanacogene elaparvovec.¹

For the successful delivery of functional genes to patients, recombinant adenoviral-associated vectors (AAVs) are the most established and leading platform for rare and severe human diseases. AAV is a non-enveloped, single-stranded DNA virus that belongs to the family Parvoviridae and is a Dependoparvovirus, meaning that it requires a helper virus for efficient production (generally adenovirus or herpes simplex virus). The engineered AAV vector contains an expression cassette up to 4.7 kilobases in size.²

The first AAV gene therapy was approved by the U.S. Food and Drug Association in 2017, and as of 2022 over 200 clinical trials were ongoing across the globe using this vector.² Citeline’s Pharma R&D Annual Review also highlights AAVs as the most popular vector with over 500 projects in the pipeline.³

Why is this the case? Offering low immunogenicity, long-term gene expression, and non-pathogenic behavior, AAVs are ideal for gene therapy, with bioengineering having additionally improved transduction efficiency and their ability to navigate immunological barriers.⁴

Mateusz Imiołek, Principal Scientist at Waters Corporation, comments on the rise of AAVs: “This was evident for me during this year’s American Society for Gene and Cell Therapy (ASGCT) Annual Meeting, where there were at least 50 scientific sessions dedicated to this viral vector, and over one-third of the 1,900 conference proceedings featured 'AAV' in their title. “This shouldn’t come as a surprise considering AAVs are the most mature gene delivery technology, first discovered over 60 years ago.”

Shawn Sternisha, Global Commercial Product Manager for Centrifugation at Beckman Coulter Life Sciences, adds that “Another benefit is that AAVs can be developed in Biosafety Level 1 (BSL1) laboratories. They also exhibit tissue tropism, targeting specific regions of the body, and can enter both dividing and non-dividing cells. Furthermore, they can maintain high levels of gene expression in patients over long periods of time.”

With so many advantages to be had with AAVs, the routine and standardized monitoring of their production remains crucial to ensuring safety and efficacy. Svea Cheeseman, Director of Product Market Management at Refeyn, highlights a key safety risk: “The patient’s immune system may react to the treatment, and this risk is increased if there are impurities in the product (i.e., AAV vector), such as empty or only partially loaded capsids, which also reduce the treatment’s efficacy.”

Analytical characterization

Critical quality attributes (CQAs) that need to be monitored include capsid titer, packaging efficiency (i.e., empty-to-full capsid ratio), viral genome integrity, aggregation content, and other process-related impurities. Not all these methods are high throughput, with some being quite the opposite, in that they can require longer turnaround times, especially methods such as transmission electron microscopy (TEM), which is used to determine whether capsids are empty, partially full, or completely full.

Imiołek comments on the slow progress of such methods, and the impact this has on gene therapy’s eventual cost once on the market: “New therapies are being continuously approved, as we have just seen with Pfizer’s treatment for hemophilia B. What’s surprising is that advances in analytical characterization have not quite followed suit, especially for routine measurements needed for ever-expanding manufacturing that supports clinical trials. Cost-effective manufacturing and efficient analytical workflows are still underdeveloped, which weighs on the final price of current gene therapy treatments.”

Sternisha says there is normally consensus on so-called gold-standard techniques, which have historically provided the most accepted results for a given parameter. Analytical ultracentrifugation (AUC), for example, has been applied for years to measure the encapsidation heterogeneity of samples. This technique provides a relative abundance determination for how many particles are full versus empty or only partially filled.

AUC was first applied to AAVs around 2015 and has since become the standard for assessing particle loading, delivering the highest resolution for quantifying partial capsids. “…[resolution] is where many other techniques fall flat,” mentions Sternisha.

“AUC is also conducted under native biological conditions—without the need for any matrices or standards.” However, users should bear in mind that this method can be difficult to use where timely tracking of manufacturing processes is critical or deployment in a quality control-regulated laboratory is required. In such a situation, alternative techniques might be preferable, he suggests.

Single-particle mass measurement and imaging methods, such as mass photometry or adaptation of mass spectrometry methods in the form of charge detection mass spectrometry (CDMS), and light scattering characterization methods (multiangle light scattering, MALS, and dynamic light scattering) are also useful and increasingly reliable. Information about sample constituents can also be derived with size exclusion chromatography (SEC) coupled to MALS. “In this way, multiple critical quality attributes can be simultaneously determined during a single analytical run,” Imiołek remarks.

On the genome analysis side of things, PCR methods are heavily used for quantifying viral genomes, while ELISA is common for quantifying capsids/particles. Newer developments in next-generation sequencing and optimized genome integrity analyses are available, using liquid chromatography methods with ion pairing reverse phase and anion exchange chromatography. Cell-based assays are also widely used for assessing functionality. “There are several new techniques, but the most common ones are still those that have been well-established,” Sternisha points out.

Newer methods

Imiołek has been most recently investigating the use of SEC for the characterization of AAVs. “We are seeing the adaptation of historical techniques for characterization of AAVs. SEC, for example, had become a very established method in the protein biopharmaceutical industry to help guide the development of a high number of antibody therapeutics.” Additional method development projects, validation studies, and novel consumables have been required in order to translate SEC's separation potential to AAVs. Most especially, low adsorption wide-pore columns have been essential, which was demonstrated in a recent peer-reviewed paper.⁵ Similarly, to efficiently couple it to MALS, dedicated data analysis modules have needed to be developed.

“Our technology—mass photometry—is an ideal tool for characterizing AAV samples because it is fast, easy to use, and consumes very little sample,” Cheeseman comments. “It also offers equivalent or better performance than other techniques in terms of resolving and quantifying empty/partially filled and full capsids. The way mass photometry works is part of what makes it well suited to AAV analysis—it measures the mass of individual particles (e.g., AAV capsids) in solution based on the interference between the light scattered by the capsid and the light reflected by the glass slide the sample is sitting on. It reports the mass distribution of the sample—in the case of AAVs, this means it can identify and quantify empty vs. partially filled vs. filled or even overfilled capsids.”

Sternisha suspects that there will be an increased focus on downstream purification by eliminating ‘partials’, ‘empties’, and any other undesired capsid fragments, trying to achieve the highest percentage fill. “Characterization efforts will support this, ensuring the product's exact identity is known. The FDA is already here, pushing for multiple orthogonal analytical approaches, especially high-resolution, to ensure reproducibility and accuracy.”

“We expect the field of AAV characterization to mature quickly in the near future. At that time, mass photometry will be an essential part of the AAV characterization toolkit,” Cheeseman adds.

“There is also a need for easier, faster, more comprehensive methods that provide multiple CQAs in a single run,” concludes Sternisha, looking toward the horizon of AAV characterization and what might lie ahead. “As AAV products are considerably costly, novel characterization methods and innovations building on existing technology will likely also focus on low sample consumption.”

References

1. Pfizer May 31, 2024 Press Release. Pfizer Receives Positive CHMP Opinion for its One-Time Gene Therapy for Adults with Hemophilia B.

2. Aponte-Ubillus JJ, Barajas D, Peltier J, Bardliving C, Shamlou P, Gold D. Molecular design for recombinant adeno-associated virus (rAAV) vector production. Appl Microbiol Biotechnol. 2018 Feb;102(3):1045-1054. 

3. Citeline Report. Pharma R&D Annual Review 2024. 

4. Wagner C, Innthaler B, Lemmerer M, Pletzenauer R, Birner-Gruenberger R. Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors. Int J Mol Sci. 2022 Oct 22;23(21):12715. 

5. M. Imiołek, S. Fekete, L. Kizekai, B. Addepalli, M. Lauber. Fast and efficient size exclusion chromatography of adeno associated viral vectors with 2.5 micrometer particle low adsorption columns, J. Chromatogr. A., 1714 (2024), 464587.