Svea Cheeseman, Refeyn, explains why better efficiency is needed to advance the production of viral vectors for use in gene therapies.
Adeno-associated viruses (AAVs) are a promising, widely used vector for delivering gene therapies. As demand for AAV gene therapies increases, it is becoming essential to maximise the efficiency and accuracy of AAV vector production.
A crucial part of the AAV workflow is the purification of viral vectors. This helps ensure a high concentration of loaded capsids and adequate removal of impurities, such as host cell proteins and DNA. Enabling this requires analytical tools that can determine impurity load and quantify empty versus full capsids. Here, we discuss the importance of impurity and empty (or partially-filled) capsid removal in AAV gene therapy production and discuss the key analytical techniques that can provide information regarding the content distribution of AAV capsids.
The threat of empty capsids
Capsids are the protein shell that would usually enclose the genetic material of a virus. In AAV therapeutics, empty capsids are those that are not packaged with the therapeutic DNA. Although content distribution can vary significantly between vector preparations, up to 95% of capsids produced upstream in cell culture may be empty1.
There are two primary reasons that underpin the importance of removing empty capsids from AAV preparations2. First, empty capsids may have the potential to induce an elevated immune response to high concentrations of viral particles. Second, empty capsids are thought to impair therapeutic potency due to receptor competition. Partially-filled capsids may also be present, and contain packaged process-related impurities or truncated genetic material. As such, characterising the proportions of empty, partially-filled, and full AAV capsids is vital for determining sample quality and estimating efficacy during both process development and for quality control.
Figure 1: Example AAV analytics during a downstream purification process. The ability to quickly and accurately characterise AAV samples at various stages would make it possible to optimise the workflow, helping maximise production efficiency.What makes an ideal AAV analytics tool for process development?
During AAV process development, there are numerous variables that need to be optimised to maximise the number of full capsids while minimising contaminants. An ideal analytical method suitable for in-process development (see Fig 1) would have the following characteristics:
- Ability to identify and quantify AAV capsids: to determine capsid loading (empty, partially-filled, and full).
- Minimal sample volume requirements: to maximise the amount of recoverable material for downstream processing.
- Robustness: to be able to tolerate matrix effects (type of buffer, residual contaminants, ionic strength, etc.)
- Quick turnaround time and high throughput: to minimise process development time and ensure statistical significance.
Technology options for AAV analytics
There are various analytical tools available and in development that can be used for analysing AAV capsids. Often, these tools provide quantification only as averages, rather than data on distributions of differentially loaded capsids and impurities, which would require single-particle resolution.
Methods such as ELISA/qPCR offer well-established, sensitive, and specific analyses that can measure capsid titre versus genome titre, quantifying empty and full capsids3. This approach, however, can be time-consuming, laborious, and require expensive reagents, including serotype-specific antibodies. Moreover, any variability in either step, such as sample dilution for ELISA or replication efficiency in qPCR, will affect the accuracy and reproducibility of the whole analysis2,4.
The current gold standard techniques are cryo transmission electron microscopy (TEM) and analytical ultracentrifugation (AUC), which have emerged as options well-suited for quantifying empty capsid proportions and impurities in AAV yields.
Transmission electron microscopy (TEM)
TEM creates an image with nanometre resolution by using an accelerated, focused electron beam that passes through a thin sample. The electrons pass through to a detector, which enables them to be converted into an image4.
TEM for AAV capsid analysis is typically coupled with negative staining. This provides visualisation of stained, partially stained, and non-stained capsids that relate to empty, partially filled, and full capsids, respectively. However, a common challenge in using TEM in AAV analytics is quantification in non-purified samples since proteins and cell debris can obscure TEM images, leading to inaccurate discernment between full and partially full capsids. TEM is often performed at cryogenic temperatures (cryo-TEM), which is thought to improve the accuracy of quantification by limiting the interference of cell debris and producing more even staining of capsids. Nevertheless, low throughput and long turnaround make it unsuitable for the routine analyses needed in purification and process development2,4.
Figure 2: A typical mass photometry measurement of AAV capsids. Mass photometry clearly resolves empty versus full capsids, showing two distinct peaks: one with lower mass corresponding to empty capsids, and a second with higher mass corresponding to full capsids. Measurements were performed on the Refeyn Samux mass photometer.Analytical ultracentrifugation (AUC)
AUC is the de facto standard method for quantifying empty, partially-filled, and full capsids. It works on the principle that, when centrifugal force is applied to a sample, full capsids will sediment more quickly through solution compared to empty capsids due to the differences in their buoyant densities. AUC offers a highly repeatable option for distinguishing between empty, partially-filled, and full capsids2.
While it may enable greater accuracy compared to TEM, AUC is also costly, slow, and low throughput, requiring prohibitively large amounts (400–500 µL) of purified sample at high concentrations (around 2 × 1012 to 5 × 1012 vg/mL)4. As such, it has limited use as a routine analytic tool and is instead generally used to validate more rapid methods that have lower resolution4.
Novel methods to optimise AAV capsid purification
Due to the limitations of the current gold standard techniques, especially in the context of the routine analysis during process development, there is a need for novel analytical tools that require less sample input and provide higher throughputs with quicker turnaround times.
Several new methods are beginning to emerge and be commercialised. Recently developed antibody-based methods, such as miniaturised ELISAs, may offer rapid, high-throughput analysis involving less sample preparation through the use of automated microfluidic platforms4. However, the requirement for serotype-specific antibodies remains an obstacle for antibody-based analytical tools in AAV analysis.
A serotype-independent option is dynamic light scattering (DLS), which estimates capsid titre by measuring light scattered by solutes over time. While potentially more accurate than ELISA, it is still a relatively low-resolution method with sensitivity dependent on the optical properties of the sample4.
Other alternatives include charge-detection mass spectroscopy (CDMS) and capillary isoelectric focusing, which do not suffer from the same limitations as serotype-dependent methods and have high resolution4. CDMS measures the charge and charge-to-mass ratio of individual ions, and has been shown to be able to measure empty, partially-filled, and full capsid ratios, like AUC5. Capillary isoelectric focusing takes advantage of the differences in isoelectric points of capsids to determine empty, partially-filled, and full states4. Moreover, they have been shown to match the resolution of AUC with lower turnaround times. However, these technologies are still under development and they require robust benchmarking against the current gold standards.
Another relative newcomer is mass photometry, which is a bioanalytical technique that quantifies samples at the single-particle level. It measures molecular mass by measuring the light scattered by individual biomolecules in solution. As empty capsids have a lower mass than full capsids, the technique can be used to distinguish between the two types of capsids and quantify their relative concentrations in solution (Fig 2). Studies have also shown it can resolve partially packaged capsid impurities and provide information on both sample heterogeneity and the presence of aggregates6.
Benchmarking mass photometry against cryoTEM and AUC. The three analytical techniques were used to measure the percentage of full capsids in four AAV samples containing varying proportions of empty versus full capsids. Data were provided by the Cell and Gene Therapy Catapult (London, UK). The mass photometry measurements were performed on the Refeyn Samux mass photometer.Any novel techniques the industry looks to employ must be validated against the current gold standards to benchmark their performance. Recent studies have highlighted that mass photometry provides equivalent results to cryoTEM and AUC when analysing samples with different proportions of empty/full capsids (Table 1).
A key benefit of technologies like cryoTEM and AUC over antibody-based tools is their ability to deliver reliable results across AAV serotypes, without the need for specific antibodies. Similarly, as mass photometry does not rely on antibodies for quantification of empty/full capsids, it can provide serotype-independent AAV analysis. Indeed, mass photometry has shown consistent results across serotypes, seen as a single, symmetric peak at the expected mass for empty capsids (Fig 3).
The method offers several distinct advantages over cryoTEM and AUC when it comes to routine analysis, process development, and monitoring downstream purification. First, mass photometry enables the quantification of empty and full capsids with very small sample volumes (~20 µL) at low concentrations (1011 particles/mL), minimising the loss of unrecoverable material during early production stages. Second, mass photometry equipment can fit on a benchtop and run analyses in a matter of minutes. Finally, taking measurements requires minimal sample preparation and training, and is inexpensive to run. As such, it could be an ideal solution for those looking for routine or even real-time monitoring.
Figure 3: Mass photometry measurements of empty AAV capsids of four different serotypes reveal a single, symmetric peak at the expected mass indicating consistent results across serotypes.Summary
Quantifying empty and full capsids in AAV manufacturing is essential to ensuring the safety and quality of gene therapy products. The rising demand on production of these therapeutics necessitates technological solutions that increase throughput, reduce turnaround times, and minimise sample use. While there are various options for AAV analytics, the current gold standards are cryoTEM and AUC. However, there are caveats to their use, and new technologies and methods are now emerging that look to address the existing challenges of throughput, cost, and sample volume.
Mass photometry is one such technology, which is serotype-independent, emerging as a promising tool that has potential to help optimise gene therapy manufacturing workflows for both routine and real-time process monitoring of AAV content ratio. A shift in the industry towards novel technologies like this could help address the increasing demand for AAV gene therapies through improving efficiency and accuracy of AAV capsid production.
DDW Volume 24 – Issue 1, Winter 2022/2023
References:
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- Werle AK, Powers TW, Zobel JF, et al. Comparison of analytical techniques to quantitate the capsid content of adeno-associated viral vectors. Mol Ther – Methods Clin Dev. 2021;23:254-262. doi:10.1016/j.omtm.2021.08.009
- Penaud-Budloo M, François A, Clément N, et al. Pharmacology of Recombinant Adeno-associated Virus Production. Mol Ther Methods Clin Dev. 2018 Jan 8;8:166-180. doi: 10.1016/j.omtm.2018.01.002
- Gimpel AL, Katsikis G, Sha S, et al. Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies. Mol Ther – Methods Clin Dev. 2021;20:740-754. doi:10.1016/J.OMTM.2021.02.010
- Pierson EE, Keifer DZ, Asokan A, Jarrold MF. Resolving Adeno-Associated Viral Particle Diversity With Charge Detection Mass Spectrometry. Anal Chem. 2016 Jul 5;88(13):6718-25. doi: 10.1021/acs.analchem.6b00883
- Wu D, Hwang P, Li T, Piszczek G. Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry. Gene Ther 2021. Published online January 20, 2022:1-7. doi:10.1038/s41434-021-00311-4
About the author:
Svea Cheeseman is Director of Product Management for Cell and Gene Therapy at Refeyn. She holds a PhD in single-molecule biophysics and has over five years’ experience in the biopharma industry. Cheeseman is interested in pushing the boundaries of biomolecular analysis to eventually enable real-time release of biomolecular drugs.
