Manufacturing: AAV Analysis

Refeyn discusses the challenges associated with AAV manufacture, the key analytical techniques currently used to facilitate insight into AAV samples and novel techniques striving to overcome current limitations

Svea Cheeseman at Refeyn

The field of gene therapy has made significant progress over the last several decades in the treatment of diseases for which current medical options are limited or ineffective. In its simplest form, gene therapy introduces genetic material into target cells via viral or non-viral vehicles to treat or prevent diseases by correcting or supplementing defective genes.

One of the most common viral vectors used in gene therapy are adeno-associated viruses (AAVs), due to their long-lasting gene expression, specificity and low immunogenicity.1

However, producing a safe and effective AAV-based treatment has its challenges. The formation of empty, partially loaded and overfilled capsids during the production of AVV vectors represents a major challenge, affecting both the immunogenic risk of the patient and the efficacy of the treatment. As the industry continues to advance, the number of clinical trials involving recombinant AAV vectors has dramatically risen, increasing the need for more effective manufacture and quality control.2

Challenges in AAV-based treatments

To comply with regulatory authorities, it’s essential to identify and measure several critical quality attributes (CQAs), including the purity of the final product.

A significant bottleneck in the manufacture of AAVs is the production of a heterogeneous capsid population. AAV capsids may either lack genetic cargo (empty), contain truncated versions of the transgene (partially filled) or encapsulate more than the intended genome size (overfilled), rather than the desired AAV vectors loaded with the intact genome of interest (full).

Dealing with the presence of empty, partially filled and overfilled capsids during AAV manufacturing is a formidable challenge, impeding efforts to optimise product yield and efficacy. Empty capsids, in particular, can range from 20% to over 98% in vector preparations, contributing to variability across production batches, potentially amplifying immune responses and compromising transduction efficiency.3 Therefore, accurately profiling the proportions of empty, partially filled, overfilled and full AAV capsids is essential for the development of proper dosing, patient safety and the optimisation of culture productivity and production consistency.

Analytical techniques for AAV capsid analysis

Due to the structural and qualitative similarities inherent in a heterogeneous capsid population, managing the purification or removal of product impurities whilst simultaneously preserving the potency and yield of full capsids can be extremely challenging. This requires analytical platforms that are highly accurate, require minimal sample volumes and don’t compromise the structural integrity of the capsids. Additionally, for GMP settings, it’s crucial that analytical platforms are scalable, 21CFR11 compliant and can be standardised.

There are several bioanalytical characterisations approaches available for assessing empty/full capsid ratios in manufacturing environments, including:

• Sedimentation velocity analytical ultracentrifugation (SV-AUC)
• ELISA and quantitative PCRs (qPCRs)
• Transmission electron microscopy (TEM)
• Anion exchange chromatography (AEX)
• UV-spectroscopy using 260/280 ratios
• Size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS).

Each technique offers a unique approach to measuring the ratio of AAV capsids and each has advantages and limitations, particularly when applied in GMP contexts (Table 1).

Table 1: Comparison of AAV analytical technologies

Measuring heterogeneous populations

ELISA and qPCR

One of the most common methods for generating data on empty/full capsid ratios is a combination of ELISA and qPCR. By quantifying capsid and genome titres with ELISAs and qPCR, respectively, this approach can estimate the content ratio of a given AAV sample. While this is well established, it is also time-consuming, laborious and dependent on the accuracy and availability of expensive serotype-specific antibodies.4 Additionally, because the results are influenced by the combined variability of the two approaches, the accuracy and reproducibility of this indirect method are limited.

UV-spectroscopy

UV-spectroscopy, on the other hand, is a simple and rapid method that can effectively distinguish between empty and full capsids based on the intrinsic chromophore absorption characteristics of DNA and proteins. However, this method is limited to highly purified, concentrated samples, as the presence of both nucleic acid and protein impurities will affect the results.4 Furthermore, UV spectroscopy lacks the capability to differentiate partially filled capsids and so may overestimate the prevalence of empty capsids.

Transmission electron microscopy

As TEM allows direct visualisation of the particles present in a sample, it has become a well-established method for characterising AAVs. With nanometer resolution, this technique can provide valuable insights when evaluating AAV capsids, including aggregation profile, particle morphology and the presence of host cell debris.5 However, this analytical approach is susceptible to background signals and can’t be used to identify partially filled capsids either. While this technology has been improved with cryo-EM, the method still has low throughput, long turnaround times and requires highly specialised equipment.

Chromatography

Chromatography techniques such as SEC-MALS and AEX offer a straightforward approach to comprehensively characterise AAV capsids. SEC-MALS has been shown to provide detailed characterisation information, including aggregation profile, size distribution, capsid content, capsid molar mass, encapsulated DNA molar mass, and total capsid and vector genome titre.6 However, while chromatographic techniques are often used for empty and full capsid quantitation due to their reproducibility, accuracy and ease of use, neither of these methods has the resolution or sensitivity to detect partial capsids.7

Analytical centrifugation (AUC)

To date, the gold standard for assessing empty, partial, overfilled and full capsids is AUC.8 Based on the differences in buoyant masses, AUC can provide detailed insight into the composition of the encapsulated DNA, the success of the purification, the presence of aggregates and the relative quantity of capsids. However, despite being frequently used in the biopharmaceutical industry, implementing AUC in a GMP setting presents some challenges such as low throughput, high turnaround times, high sample requirements, complex data analysis and the need for specialised training.

Innovations in AAV analysis techniques

The existing methods for AAV characterisation and quantification lack the scalability and sensitivity needed to meet the current and future demands of gene therapy. While AUC can provide all the information needed, it is time-consuming and requires a high concentration and volume of samples. Due to these limitations, there is a need for novel analytical tools that require less sample input and provide easier analysis with quicker turnaround times.

Several new methods are now emerging and undergoing commercialisation. Recently developed antibody-based methods, such as miniaturised ELISAs, promise to improve throughput and turnaround time through the use of automated microfluidic platforms.4 However, further method development is required to establish accuracy and reliability compared to traditional ELISAs.4 Other alternatives include charge-detection mass spectroscopy (CDMS) and capillary isoelectric focusing (cIEF), which both have comparable resolution to AUC and have been shown to be capable of determining empty, partially filled and full capsid ratios. However, these technologies are still immature and require robust benchmarking against current industry standards.

A relatively new, but rapidly maturing technique, mass photometry, quantifies samples at the single-particle level by measuring the light scattered by individual biomolecules in solution. As a single-molecule technique, it provides highly detailed information on the heterogeneity of a sample, capturing measurements from thousands of molecules every minute. In the case of AAVs, it can accurately identify and quantify the relative concentrations of empty, partially filled, overfilled and full capsids, in addition to estimating the size of encapsulated genomes and facilitating titre measurements.9,10

Importantly, this technique has also been validated using the gold standard AUC, providing comparable data and accuracy, but with a 72-fold speed increase (five minutes for a single run versus six hours for AUC) and requiring 1/600th of the sample volume.9, 10 It has also been shown to produce results similar to other established techniques such as TEM and AEX.7

Crucially, for applications in the gene therapy space, mass photometry can be automated with robotics and adapted for GMP environments with specialist software, facilitating analysis of large sample numbers while adhering to strict regulatory requirements. This combination of sensitivity, scalability, speed and ease of use makes it well suited as one of a new generation of methods for assessing purity, composition and homogeneity of AAV preparations in manufacturing.

The future of analytics in AAV manufacturing

With a continuously increasing number of programmes reaching the clinical phase and commercialisation, AAV development has gained a prime role in the future of gene therapy. For this reason, AAV production for pharmaceutical purposes requires upscaling and tight control of the quality and consistency of the products. Achieving this requires analytical tools and methods that offer rapid and accurate assessment of sample purity and homogeneity at increasing scale.

As discussed, current popular analytical methods for characterising AAV preparations tend to be slow, require large amounts of sample or sample preparation, or have poor resolution. A new generation of methods is needed to address these current challenges. Thankfully, there is a concerted effort in the industry to advance analytical technologies to help maximise the effectiveness and commercial viability of gene therapies.

While AUC remains the gold standard for AAV analysis in terms of accuracy, reproducibility and scope of information, it could be supplemented with quicker and easier technologies that are gaining traction. Mass photometry is one technology that has matured rapidly and can obtain accurate and comparable data quickly and with minimal sample consumption. These attributes, combined with the technology’s ease of use and small footprint, make mass photometry systems an attractive option for the rapid characterisation of viral vectors in industrial and GMP applications. Continuing innovation in new technologies and approaches will substantially improve the efficiency and accuracy of AAV capsid production.

As AAV production becomes more reliable and scalable, the full potential of gene therapies can be harnessed, building a path towards a brighter future in healthcare and profoundly impacting countless lives.

References

1. Li C et al (2020), 'Engineering adeno-associated virus vectors for gene therapy,' Nature Reviews Genetics, 21(4): Article 4.
2. Kuzmin D A et al (2021), 'The clinical landscape for AAV gene therapies,' Nature Reviews Drug Discovery, 20(3): pp173–174.
3. Schnödt M et al (2017), 'Improving the Quality of Adeno-Associated Viral Vector Preparations: The Challenge of Product-Related Impurities,' Human Gene Therapy Methods, 28(3): pp101–108.
4. Gimpel A L et al (2021), 'Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies,' Molecular Therapy -Methods & Clinical Development, 20: pp740–754.
5. Dobnik D et al (2019), 'Accurate Quantification and Characterization of Adeno-Associated Viral Vectors,' Frontiers in Microbiology,10: pp1570.
6. McIntosh N L et al (2021), 'Comprehensive characterization and quantification of adeno associated vectors by size exclusion chromatography and multi angle light scattering,' Scientific Reports, 11: pp3012.
7. Richter K et al (2023), 'Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques,' European Journal of Pharmaceutics and Biopharmaceutics, 189: pp68–83.
8. Werle A K et al (2021), 'Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors,' Molecular Therapy – Methods & Clinical Development, 23: pp254–262.
9. Wagner C et al (2023), 'Quantification of Empty, Partially Filled and Full Adeno-Associated Virus Vectors Using Mass Photometry,' International Journal of Molecular Sciences, 24: pp11033.
10. Wu D et al (2022), 'Rapid characterization of adeno associated virus (AAV) gene therapy vectors by mass photometry,' Gene Therapy, 29: pp691–697.