Everything you need to know about analysing AAVs with mass photometry

Updated: Aug 18

Ask any AAV researcher – figuring out how many of your capsids are filled and how many are empty is a long and laborious process. There is a clear need to overcome the drawbacks of the current gold-standard analytical methods so users can rapidly and cost-effectively characterise AAVs. The SamuxMP is an easy-to-use tool based on mass photometry, an award-winning technology that can measure the empty/full ratio of AAV capsids of any serotype in minutes, with no special training or sample preparation required. Keep reading to learn how the SamuxMP can streamline your AAV characterisation process.


Gareth Rogers, Ph.D. and Svea Cheeseman Ph.D. are Refeyn’s Product Managers for the SamuxMP. They tell us more about how this mass photometer can help you measure your AAV samples with unparalleled ease.


Gareth: Since launching the SamuxMP, there has been lots of excitement around the instrument and the benefits mass photometry is bringing to AAV workflows. We wanted to answer some of the most common questions we get.


Svea: The SamuxMP is really bringing a change to the AAV analytical workflows - making the empty-full AAV analysis much simpler, faster and more convenient. We will address the key questions below, and if you’d like to see some data, look at the webinars and the AAV brochure linked at the end of the text.






Why should I be aware of the SamuxMP?


The SamuxMP is an analytical instrument which has been designed to analyse AAV samples. It precisely determines the empty/full capsid ratio in samples of any AAV serotype. It uses very small sample volumes and only takes minutes to complete a measurement, saving valuable time and resources.



How does the SamuxMP tell the difference between empty and full AAV capsids?


The SamuxMP uses mass photometry to distinguish between empty and full AAV capsids. Mass photometry relies on light scattering from individual particles as they land on coverslip glass to measure their masses. You can learn how mass photometry works here.


In AAV assembly, when the DNA is packaged inside the rigid capsid, the shape and volume of the capsid are unchanged, but the virion’s mass increases. The SamuxMP is optimised to measure the differences between the masses of empty, full, partially filled and over-filled capsids. As you can see from the graph below, empty and full capsids appear as two distinct peaks on the mass histogram during data analysis.


Figure 1. Mass photometry characterisation of empty and full AAV capsids. The mass histogram shows two distinct peaks: the peak with lower mass corresponds to empty AAV capsids, and the peak with higher mass corresponds to full AAV capsids.



What advantages does the SamuxMP have over the current gold standard technologies for AAV characterisation?


The SamuxMP offers several advantages over traditional AAV characterisation techniques, which include transmission electron microscopy (TEM) and analytical ultracentrifugation (AUC). Although they are the gold-standard techniques in AAV characterisation, there are drawbacks to using these methods: the high costs of purchasing and running the instruments, complexity of operating the instruments, and significant time needed to deliver results.


The SamuxMP is optimised for measuring AAVs and is easy to operate. It requires very little of your sample, so you have plenty of virus left. Each mass photometry measurement takes mere minutes to deliver an empty/full capsid ratio.



What training is required to use the SamuxMP?


Training lab personnel on specialised equipment takes time that could be spent on optimising your products. So, we designed the SamuxMP so that anyone with basic lab skills can use it. That means you only need to be able to pipet the sample. Then, the custom software makes data collection and analysis simple. The SamuxMP allows everyone in your lab to get right to analysing AAV samples.



Does the SamuxMP work on my AAV serotype?


The SamuxMP works with a variety of AAV serotypes, both natural and hybrid. We have measured samples of empty capsids of AAV5, AAV6, AAV8 and AAV-DJ in our labs, and observed an expected 3.7 MDa peak for each sample. You can see the data in our AAVs brochure (linked below).



What concentration does my virus need to be?


The optimum AAV concentration for the SamuxMP is 10^11 particles/mL. You only need 10–20 µL of sample at this concentration to accurately measure the empty/full AAV capsid ratio, leaving the rest of your valuable sample for your experiments.



Do I need to label my virus capsids?


For analysis of AAV samples with the SamuxMP, it is not necessary to label or modify the virus. Mass photometry is a label-free technique, which is one of the reasons it is so easy and quick to use. Just add your sample to the appropriate buffer and measure!



Do I need special buffers for running my sample?

The SamuxMP works well with buffers typically used in AAV purifications. Detergents should also not pose a problem, as the noise they generate in mass photometry experiments is typically far below the mass of AAV capsids.



How do I make figures to show my data?

The software package that comes with the SamuxMP was designed by scientists, for scientists. It lets you acquire and visualise your data with ease, and you can automatically analyse and calculate empty/full AAV ratios for your measurements as a batch. You can even generate and export figures directly from your results and prepare great figures for your publications.


In the screenshots below, you can see what our analysis software, DiscoverMP looks like. In the first image, you can see the clearly distinct peaks in the mass histogram from empty and full AAV capsids. The second image shows how you can analyse your data in batches in DiscoverMP, so you can compare the populations in different samples.



Figure 2. Screenshots of data analysis in DiscoverMP. The top screenshot shows the view in DiscoverMP as a researcher collects data from an AAV population, with a mass histogram clearly displaying distinct peaks corresponding to empty and full AAV capsids. The lower screenshot shows what it looks like to analyse data from multiple samples at once using DiscoverMP.



How do I contact you for more information or a demo?

If you would like to know more about the SamuxMP or the technology itself, there are several options:


To ask questions, get in touch with our mass photometry experts.

To see the SamuxMP in action, register for a remote demo! Delivered online with time for questions!

To try the SamuxMP out with your samples, send us a request!


....And don’t forget to register for the upcoming webinars!



 


Further resources:



Brochure: Mass photometry of AAVs

In this brochure, we show data from the SamuxMP empty/full capsid ratios for AAV samples of different serotypes. We also provide benchmarking of mass photometry to analytical ultra-centrifugation and cryoTEM, showing that results from these techniques are closely aligned to the mass photometry output.



Webinar: Are Your AAVs Filled? Determine Rapidly with Mass Photometry

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.



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