Assessing AAV capsid content is critical for AAV research, development and production. There is an urgent need for reliable techniques that can characterize AAV samples efficiently and accurately, particularly when AAV sample loading shows a high level of heterogeneity .
A recent study compared two techniques – mass photometry and charge-detection mass spectrometry (CDMS) – for their abilities to distinguish AAV loading distributions .
To gain further insight into this research, we spoke to Eduard Ebberink – a postdoc in Albert Heck’s lab, Utrecht University, and the study’s first author.
Keep reading and register for a free webinar where Eduard will talk about AAV analytics and the power of mass photometry.
Introducing researcher Eduard Ebberink
Eduard Ebberink is a postdoc in the Biomolecular Mass Spectrometry and Proteomics lab at Utrecht University. His work focuses primarily on single-particle mass analysis, such as proteins or AAVs. The two techniques he mainly works with are CDMS and mass photometry.
On LinkedIn: Eduard Ebberink | LinkedIn
The experimental aim: To compare mass photometry and CDMS for AAV mass analysis
To compare mass photometry and CDMS, Eduard and his colleagues used each technique to evaluate AAVs from two different serotypes (AAV8 and AAV2), either empty or packed with a transgene. The AAVs were obtained from different suppliers, who had produced them using different platforms (Fig. 1) . The instruments used were a SamuxMP mass photometer (Refeyn) and an Orbitrap Q Exactive UHMR mass spectrometer (Thermo Fisher Scientific). Eduard explained:
“The idea was to see whether different techniques can be used to characterize the different pools – do we see differences between them or are they all the same? … We could compare the different techniques, the different serotypes, the different empty vs. filled particles and see whether we could notice differences between mass photometry and CDMS. That was the starting point of the project.”
Fig. 1: Study overview. The authors used mass photometry and CDMS to evaluate AAV samples from two different serotypes (AAV8 and AAV2) and three different suppliers. Each serotype was produced via three different production platforms (one insect-cell-based and two human-cell-based platforms); all were either empty or packed with a transgene .
Differences between mass photometry and CDMS
Mass photometry and CDMS are both single-particle techniques that can quantify the mass of individual AAV particles, but they use distinct physical principles.
CDMS is a mass spectrometry variant that is better suited than conventional mass spectrometry for analyzing larger structures. CDMS simultaneously detects ions’ charge as well as their mass-to-charge ratio, facilitating mass analysis. Mass photometry, by contrast, determines a particle’s mass from its induced light scattering.
Additionally, as a form of mass spectrometry, CDMS requires that AAV particles be converted to ions, through nanoelectrospray ionization. Mass photometry, on the other hand, is done in solution and requires no sample manipulation .
Both mass photometry and CDMS confirmed variability in AAV loading
Eduard and his colleagues showed that different production methods generated AAVs with different packaging efficiencies, particularly for AAV pools that were loaded with a transgene .
For the pools of transgene-loaded AAV8 capsids, both mass photometry and CDMS identified populations with partially filled and overfilled capsids (in the AAV8_Vir and AAV8_Vig pools) as well as empty capsids (in the AAV8_Vir and AAV8_Sir pools). Similarly, for the pools of transgene-loaded AAV2 capsids, both techniques showed populations of empty (AAV2_Sir and AAV2_Vig), partially loaded (AAV2_Sir) and overfilled (AAV2_Vir) capsids (Fig. 2) .
Fig. 2 Mass photometry and CDMS analysis of AAV2 capsids loaded with a transgene. Both techniques showed additional populations beyond the expected single loading: Empty, partially loaded and overfilled capsids. Figure adapted from: .
Mass photometry and CDMS are both great methods for AAV mass analysis
The authors concluded that both mass photometry and CDMS are excellent techniques for measuring the DNA packaging of AAVs. Both mass photometry and CDMS displayed similar mass distribution patterns . The authors highlighted that mass photometry has the advantage of being a much simpler and faster technique that can measure the mass of AAVs under physiological conditions. Eduard said:
“Mass photometry is very straightforward and convenient. You just need to dilute your sample in the right concentration and then you can quickly measure your sample in solution. You can also quickly analyze your measurements”.
When asked about CDMS, Eduard noted that it is more accurate than mass photometry in terms of its mass resolution and range but requires “more analysis and perhaps some MS experience to calibrate the instrument before use”. Compared to CDMS, mass photometry has the advantage of measurements occurring in solution, which means that the biochemical properties of the samples are not altered .
By contrast, CDMS involves measurements in harsher conditions (e.g., buffer exchange, electrospray, high voltage transmission, low pressure), which can potentially lead to sample deterioration  . Eduard added:
“Mass photometry happens in solution, whereas CDMS is a gas-phase technique. Therefore, there can be differences as particles might not behave the same way”. In this study, however, both CDMS and mass photometry appear to be in excellent agreement.
Looking ahead – Next steps for Eduard’s research
Eduard plans to continue applying mass photometry in his research. He said his next step is more in-depth analysis of AAVs to find out “what partially filled or overfilled means”. That is, he will investigate whether the extra content is DNA or other contaminants. He also plans on using mass photometry in other contexts – including to explore larger particles as well as protein binding and complex assembly.
Further resources about using mass photometry for AAV analysis:
In this application note, data collected by Pharmaron Gene Therapy show that mass photometry can distinguish populations of AAV capsids with heterogeneous loading of genomic content and quantify the proportion of each population in a sample.
In this webinar, we show data proving the ability of the SamuxMP mass photometer to reliably quantify the empty-full particle ratio for different AAV serotypes and at different purification levels, including benchmark data comparing the mass photometry solution with cryoTEM and analytical ultra-centrifugation.
This blog shows how the SamuxMP mass photometer is an easy-to-use tool that can measure the empty/full ratio of AAV capsids of any serotype in minutes, with no special training or sample preparation required.
 E. H. T. M. Ebberink, A. Ruisinger, M. Nuebel, M. Thomann, and A. J. R. Heck, “Assessing production variability in empty and filled adeno-associated viruses by single molecule mass analyses,” Molecular Therapy - Methods & Clinical Development, vol. 27, pp. 491–501, Dec. 2022, doi: 10.1016/j.omtm.2022.11.003.
 S.-H. Lai, S. Tamara, and A. J. R. Heck, “Single-particle mass analysis of intact ribosomes by mass photometry and Orbitrap-based charge detection mass spectrometry,” iScience, vol. 24, no. 11, p. 103211, Nov. 2021, doi: 10.1016/j.isci.2021.103211.