Unlocking AAV gene therapy potential: The role of successful characterization

Adeno-associated viruses (AAVs) are promising delivery tools for gene therapy and are used in many clinical studies. However, less than ten AAV-based therapies have been approved to date. To mitigate the risk of serious side effects and even death, a comprehensive quality control and characterization process is crucial. This process includes identifying features that may influence efficacy and safety but are not yet fully understood.

AAVs have considerable potential for developing new therapies for untreatable genetic diseases, including ocular, neuromuscular, neurodegenerative, cardiovascular, and metabolic disorders and cancer.

Several AAV serotypes exist with different tissue tropisms that allow for targeted delivery of genetic material to specific tissues, such as muscle or brain. While the first targeted therapies have been approved, e.g., for retinal dystrophy and spinal muscular atrophy, several serious and sometimes even fatal side effects, including severe inflammation and liver failure, have hindered the widespread adoption of AAV-based gene therapies.

Similar to other therapeutic biomolecules, such as monoclonal antibodies and antibody-drug conjugates, thorough analytical characterization and process control are essential to ensure efficacy and safety. 

The importance of AAV characterization

Because AAVs are commonly found in humans, they are considered safe to use. However, the viruses can induce an immune response, especially with repeated administration. Often, doses are increased to achieve a therapeutic effect with only one administration. While this can help to avoid triggering the immune response, higher doses can increase the risk of other side effects.

The manufacturing process of AAVs is complex, requiring specialized cell cultures and additional helper genes, usually provided in the form of plasmids. This is because AAVs have been engineered to contain only minimal genetic material that does not allow virus replication in living cells, rendering them non-pathogenic. Instead, new genetic information – mostly single-stranded (ss)DNA – can be packaged into the virus capsids. 

When administered to the patient, the AAV capsids enter the target tissue cells and release the ssDNA, which circularizes to form an episome that is maintained separately from the chromosomes and is stable within the cell for years. The longevity of the introduced therapeutic DNA underscores the critical need for meticulous quality control of AAVs.

AAV capsid filling state: A new critical quality attribute

The high complexity of the production process can lead to the formation of protein aggregates and incorrectly assembled capsids. In addition, the amount of DNA packaged into individual capsids can vary, resulting in AAV subpopulations with no DNA, less DNA, or even more DNA than intended. 

Research has shown that the innate immune response can be triggered by both protein aggregates and different capsid filling states. Also, empty or underfilled AAVs can reduce potency, lowering the desired therapeutic effect. 

Overfilled capsids have only recently been discovered. AAV serotype 5 capsids, for example, can accommodate up to 4.7 kb of DNA, whereas many therapeutic nucleic acid products are smaller, leaving room for packaging additional DNA. This can be an extra, complete, or partial copy of the therapeutic gene or other DNA derived from the production host cell, with unpredictable effects. 

The filling state of AAVs is, therefore, a critical quality attribute because it directly affects the immunogenicity, potency, and potential side effects of the drug.

“We must ensure that the species pattern is consistent between the production batches we deliver to patients. This means that we need to understand and characterize the size distribution of these drug products in a precise and careful way and discard affected lots if they do not meet specifications,“ stated Karl Maluf, Director of the Biophysical Characterization Core at KBI Biopharma, a global CDMO with extensive experience in AAV characterization.

Navigating challenges in AAV gene therapy: The role of characterization

Conventional analytical methods do not permit the identification of differently packaged subgroups of AAV capsids, so they have not been extensively studied until recently. In particular, overfilled capsids have only been detected and characterized in detail by novel technologies. 

Current approaches include SEC-MALS, which combines multi-angle light scattering (MALS) with size exclusion chromatography (SEC). Because the different capsid species have nearly identical hydrodynamic sizes, they cannot be separated by SEC. In principle, estimating the number of differently filled capsids would be possible by assessing their spectroscopic properties, but the MALS analysis currently does not account for partially filled or overfilled capsids.

CsCl density gradient sedimentation equilibrium is frequently used to separate particles based on density, allowing different packaging states to be characterized by their UV absorbance spectra. However, data obtained in the presence of high concentrations of CsCl may not reflect the properties of the final product administered to patients, which is based on a physiological, aqueous solution.

Other standard methods, such as DNA gels, can indicate packaging states, but the resulting data are difficult to interpret. Capillary electrophoresis (CE) has the disadvantage of concentrating peaks, promoting aggregation, and potentially affecting result quality. 

Digital droplet PCR (ddPCR), another commonly used technique, measures the total amount of DNA present, which provides valuable information on the overall process but does not allow the separation and analysis of capsid species.

Mass spectrometry is expensive and still too technically challenging to obtain useful data on the packaging states of viral capsids. The recently developed mass photometry technique has promising features but currently uses very low sample concentrations, so the information obtained may not reflect the material properties at the concentrations used in the administered product.

“We have established many of these procedures in our lab and use them for orthogonal result validation. However, what is needed now is a method to resolve and identify the viral species within the actual AAV product. We seek to use techniques that can separate and characterize different capsid filling states and analyze AAVs in an aqueous solution, at the correct concentration, representing the product that will be administered to patients as closely as possible,” explained Maluf.

Methods at the forefront of AAV characterization to advance gene therapy

Sedimentation Velocity Analytical Ultracentrifugation (SV-AUC) has emerged as the new gold standard in AAV characterization because it combines two essential features. First, the AAV samples are hydrodynamically separated in an analytical centrifuge according to their molar mass and shape, giving rise to individual peaks with different capsid-DNA ratios. 

Second, the enhanced absorbance-optical detection capabilities of the Beckman Optima Analytical Ultracentrifuge allow characterization and quantification of the protein and nucleic components within the different fractions and calculation of the genome-to-capsid-ratio for each resolvable species. In addition to the sedimentation coefficients observed for individual peaks, these ratios can be used as quality control parameters for each production batch. 

In a recent case study, the team at KBI Biopharma demonstrated the use of the SV-AUC method to assess the heterogeneity of commercially sourced AAVs. The analysis identified two main peaks (3 and 4). The features of peak 3 were consistent with a correctly filled AAV capsid, while peak 4 contained more ssDNA than expected, corresponding to an overfilled virus. Smaller peaks were identified as partially filled (peak 2) and empty capsids (peak 1).

Results of an SV-AUC analysis performed at KBI Biopharma using commercially sourced AAV preparations with an intended packaged genome size of 2.5 kb. Four peaks could be separated based on their sedimentation coefficient, representing capsids with different sedimentation coefficients.
First graph: Absorbance measurements for each peak were conducted at several wavelengths.
Second graph: By calculating the protein and DNA amounts present in each peak, capsid species could be identified as predominantly empty (1), partially filled (2), correctly filled (3), and overfilled (4) with DNA.

KBI Biopharma leverages decades of experience with SV-AUC to establish state-of-the-art, current Good Manufacturing Practice (cGMP)-certified AAV quality control procedures. In this context, certification involves more than cGMP-compliant software and the expertise to apply it to data analysis. 

“Developing an AUC method that meets the objectives of a cGMP validation required by the regulatory bodies remains a demanding task due to the complex hardware of the AUC instrument and the steps required for assay setup. Despite these challenges, the type of resolution that AUC can provide with respect to distinguishing AAV species is currently unmatched by other methods and will continue to drive demand for the method in a well-controlled, validated manner,” Maluf emphasized.

KBI Biopharma’s analytical expertise in AAV characterization spans a broad range of techniques, including SEC-MALS, CE, ddPCR, and CsCl density gradients. In addition, KBI Biopharma has recently established nano-differential scanning fluorimetry (nano-DSF), a method used to characterize AAV serotypes for different tissues, and is currently implementing mass photometry as a novel approach to determining the differently filled AAV capsid species.

This comprehensive AAV characterization portfolio will help improve lot-to-lot reproducibility and patient safety, paving the way for new, successful AAV-based gene therapies.

Learn how KBI Biopharma’s advanced solutions and analytical expertise in AAV characterization can support your gene therapy program!

Images Courtesy: KBI Biopharma