Gene therapies based on AAV application in vivo have proven
powerful to treat and cure genetic diseases, but are prohibitively
expensive. A significant cost driver is the difficulty optimizing
AAV processes. Specifically, there is a lack of analytical techniques
to assess AAV quality during the upstream process – where
AAVs are produced. Instead, the resulting AAV samples need
to undergo lengthy downstream purification and concentration
procedures before they can be characterized – and before a
judgement can be made on the upstream performance. This
slows down process development and increases the associated
costs. It also limits the detection of quality deviations during
upstream processing, as operators are confined to monitoring
parameters like cell density and pH to determine if the process
is functioning correctly.
The main reasons for the lack of analytics in upstream processing
are that most analytical methods, including size-exclusion
chromatography coupled to multi-angle light scattering (SECMALS), analytical ultracentrifugation (AUC) and cryo-electron
microscopy (cryo-EM), either cannot work with the level of
background present in unpurified samples or require a much
higher AAV titer than is available during upstream processing.
The need for a highly concentrated sample is a particular issue
during process optimization, when titers tend to be low.
Fig. 1 Overview of method for efficient, at-line AAV analysis during upstream processing. A clarified lysate from the bioreactor undergoes
a simple, plate-based cleanup followed by empty/partial/full analysis with the SamuxMP mass photometer. The entire process takes around three
hours, from bioreactor to results, and requires just 1.5 mL of cell culture sample. The method is described in detail in the attached Protocol,
which was created by Généthon.
APPLICATION NOTE
Upstream AAV characterization with mass photometry
A simple, plate-based cleanup method for clarified lysates, plus mass photometry, enable analysis of AAV quality during
upstream processing. Taking just 3 hours from bioreactor to results and using only 1.5 mL of cell culture sample, the
approach measures % empty, partially filled and full capsids. Applicable for multiple serotypes, it makes process optimization
faster and more cost-effective than traditional post-purification checkpoint control.
®
In this application note, we offer a solution that overcomes this
analytics challenge. We present a simple, scalable, column-free
cleanup method that takes three hours and renders clarified
lysates measurable by mass photometry (Fig. 1). Mass photometry
is a powerful analytical tool that measures the mass of individual
AAVs in solution. Single-particle resolution allows specialized
mass photometers, the SamuxMP and the SamuxMP Auto, to
provide information about critical quality attributes (CQAs) of
AAV samples, quantifying:
• Empty (E), partially filled (P), full (F) and overfilled capsids1
• Capsid degradation2
• Sample titer1
• Sample purity
• AAV genome length3
Mass photometry is also ideally positioned for at-line, upstream
AAV sample characterization. The measurements take less than
five minutes from sample loading to results and can be applied to
AAVs of any serotype. Importantly, mass photometry uses very
low sample volumes (5–20 µL) and sample concentrations (~1011
particles/mL). As we show, this low sample requirement means
that mass photometry can be applied after a simple cleanup
from a starting volume of just 1.5 mL of cell culture sample.
We first show that our simple cleanup method is sufficient to
enable mass photometry analysis of the proportions of empty,
partially filled and full capsids present in AAV samples of two
different serotypes. We compare the mass photometry results
to those obtained from SEC-MALS analysis. Next, we confirm
that the results obtained after the simple cleanup step are
consistent with those obtained after traditional column-based
purification – using mass photometry as well as AUC.
A simple cleanup method enables mass photometry
analysis
To first verify the need for sample cleanup, the clarified lysate for
an AAV candidate was analyzed directly using mass photometry.
As expected, the high level of impurities present in the sample
at that stage prevented resolution of the AAV capsids (Fig. 2A).
To remove these impurities, a simple, plate-based cleanup
method was developed, which isolates and enriches AAV from
Fig. 2 The simple cleanup method enables AAV capsid analysis with mass photometry. A) Directly after harvest and lysis, AAV capsids could
not be resolved in the mass histogram. B-C) After simple cleanup, populations of empty, partially filled and full capsids could be resolved for AAV
candidates A9, of serotype AAV8 (B) and B1, of serotype AAV9 (C). The blue shading indicates the mass ranges for empty (E), partially filled (P)
and full (F) capsids; the ranges were the same for all measurements shown. These data were collected by Généthon.
Fig. 3 Analysis of upstream AAV samples
after application of the quick cleanup
method. SEC-MALS (orange bars) was
used to quantify the % loaded (partially or
fully loaded) capsids while mass photometry
was used to quantify the proportion of
partially loaded (mid blue) or fully loaded
(dark blue) capsids in each sample. SEC-MALS
data represent AAV loaded with nucleotide
as monitored by UV-vis spectroscopy (see
Methods for details). For both methods, each
bar represents a single measurement. These
data were collected by Généthon.
1.5 mL of cell culture using POROSTM AAVX AAV-specific resin
(Fig. 1). The method begins with lysis and centrifugation, then
the clarified lysate undergoes capture, wash and elution. The
entire method takes three hours and can be applied to tens of
samples in parallel on the plate. It is described in detail in the
attached Protocol.
Following the simple, 3-hour cleanup, 14 AAV candidates were
analyzed by mass photometry – which took 5 min per sample
including analysis time. Representative mass photometry data
are shown for AAV candidates of serotype AAV8 (Fig. 2B) or
AAV9 (Fig. 2C). Empty, partially filled and full AAV capsids were
clearly resolved, confirming that the simple cleanup method is
sufficient to enable empty/partial/full analysis with the SamuxMP.
Mass photometry vs. SEC-MALS
SEC-MALS was used to validate the mass photometry
measurements after the simple cleanup. However, while SEC-
Fig. 4 Results from mass photometry after simple cleanup align
with those after capture chromatography, with AUC and mass
photometry measurements showing consistent agreement. Three
AAV8 samples were prepared for analysis using either the simple
cleanup method (SC) or capture chromatography (CC). The samples
were analyzed by mass photometry in both cases (blue bars) and by
AUC for the capture chromatography samples only (grey bars) to
measure the proportions of empty, partially filled and full capsids.
Each bar represents a single measurement. These data were collected
by Généthon.
MALS can be used to measure the E/F ratio, it cannot differentiate
partially filled vs. full AAVs. Although mass photometry and
SEC-MALS generally agreed, SEC-MALS consistently returned
slightly lower measurements for the total percentage of loaded
AAVs (Fig. 3). An explanation for this trend is that SEC-MALS
accuracy depends on sample homogeneity, as SEC-MALS
detectors (UV absorbance or differential refractive index) need
different fitting constants to quantify partially filled or full capsids.
The presence of a mixture of both species can thus bias the
SEC-MALS measurement, requiring post-run manual correction.4
Mass photometry, by contrast, records single-particle counts,
directly quantifying distinct AAV populations.5
Compared to mass photometry, SEC-MALS also has practical
drawbacks. First, it requires more sample than mass photometry,
with the precise amount depending on factors such as process
settings, column type and serotype. For the data presented here,
50 µL at 1011 VP/mL was used, but the literature reports the need
for up to 40 µL at 2x1013 vg/mL.4, 6, 7 Also, the measurements
take longer (>15 min per sample for SEC-MALS vs. 1–2 min
for mass photometry).
Cleanup does not alter capsid loading
To compare our simple cleanup method with a traditional columnbased capture, we applied capture chromatography to three
samples and then measured them with mass photometry and
AUC. Compared to the simple cleanup, capture chromatography
requires over 200 times more cell culture sample (400 mL vs.
1.5 mL). It also takes 8 times longer (24 h vs. 3 h), and only one
sample can be purified on the column at a time, rather than many
in parallel. Notably, unlike mass photometry and SEC-MALS,
AUC could not be performed after the simple cleanup because
it requires much greater sample volume and concentration than
is available after the simple cleanup.
Mass photometry measurements from after the simple cleanup
and after capture chromatography were comparable (Fig. 4).
In addition, there was agreement between AUC and mass
photometry results for the samples from capture chromatography
(Fig. 4). Overall, the results confirmed that the simple cleanup
method does not change the proportions of empty, partially
filled and full capsids present in the sample, and that the samples
obtained after simple cleanup or capture chromatography are
equally representative of the sample’s loading status.
Discussion
A lack of analytical techniques for assessing AAV quality during
upstream processing hampers AAV processing and drives up the
cost of AAV-based gene therapies. The introduction of effective
and efficient upstream analytics would, however, speed up AAV
development and production and reduce costs.
We have addressed this challenge by presenting a simple, threehour, scalable, serotype-agnostic cleanup method that, combined
with mass photometry, enables upstream characterization of
AAV samples. Notably, this method uses as little as 1.5 mL of
cell culture and goes from bioreactor to results in 3 hours. As
Mass
photometry SEC-MALS AUC
Purification
requirement
Simple cleanup
(3 h)
Simple cleanup
(3 h)
Traditional
column-based
purification (24 h)
Time per run* 1–2 min > 15 min > 6 h
Sample
consumption
5–20 µL @
~1011 VP/mL
≥50 µL @
~1011 VP/mL
500 µL @
~1012 VP/mL
Resolution Empty, Partial,
Full Empty, Loaded* Empty, Partial,
Full
Ease of use Training in <1
day
Expert user
required
Expert user
required
Table 1 A comparison of mass photometry, AUC and SEC-MALS
for AAV analysis. The information summarizes the attributes of the
three methods highlighted in this application note.
*SEC-MALS does not distinguish partially loaded capsids from
fully loaded capsids, so they are quantified together.
Methods
• The simple cleanup procedure for cell culture samples was
performed as described in the attached Protocol. After
completion of the final elution steps, the following steps
were included to enable SEC-MALS analysis:
1. Transfer of the neutralized eluate to Nanosep MG
0.22 μm tubes (Pall DPTFE04C34) for 0.22 μm
filtration.
2. Centrifugation of the tubes for 1 min at 5000 rpm.
3. Removal of the filter from the Nanosep tube.
• Mass photometry measurements were performed on a
Samux® mass photometer (Refeyn Ltd.) with MassGlass® UC
sample carrier slides, following the manufacturer’s
recommended protocol. Samples were prepared by diluting
AAV to 1011 VP/mL in 1x PBS. Calibrations were performed
using the MassFerence® P2 calibrant (Refeyn Ltd.). Data
were acquired and analyzed using integrated software tools
AcquireMP and DiscoverMP (Refeyn Ltd.)
• SEC-MALS was performed using an Agilent 1290 HPLC
system (Agilent Technologies) coupled to DAWN light
scattering and OptiLab refractive index detectors (Wyatt
Technology Co.) equipped with a SEC 4.6*300 mm column.
Samples were filtered with a 0.22 µm filter before injecting
40 µL of AAV (2x1012 vg/mL) with a mobile phase of 20
mM phosphate with NaCl 0.001% pluronic pH 7.4, 150
mM NaCl, pH 7.4. to maintain AAV stability during analysis.
Elution profiles were monitored at 260 and 280 nm with light
scattering to determine molar mass and aggregation state.
• Sedimentation velocity AUC was performed using an
Optima ultacentrifuge (Beckman Instruments). 400 µL of
AAV sample (1012 pp/mL) was loaded into a sample cell,
which was loaded into a An50-Ti rotor and spun at 16
krpm at 20°C for several hours. AUC data were analyzed
using SedFIT.
References
1
Wagner et al., Int J Mol Sci. 2023
https://doi.org/10.3390/ijms241311033
2
Ebberink et al., Mol Ther Methods Clin Dev. 2024
https://doi.org/10.1016/j.omtm.2024.101293
3 Hiemenz et al. Mol Ther Methods Clin Dev. 2023
https://doi.org/10.1016/j.omtm.2023.101162
4 McIntosh et al., Sci Rep. 2021
https://doi.org/10.1038/s41598-021-82599-1
5
Wu et al., Cells. 2023
https://doi.org/10.3390/cells12182264
6
Gimpel et al., Mol Ther Methods Clin Dev. 2021
https://doi.org/10.1016/j.omtm.2021.02.010
7 Chen & Purchel, App Note: Quantifying quality attributes of
AAV gene therapy vectors by SEC-UV-MALS-dRI.
https://www.cellandgene.com/doc/quantifying-quality-attributesof-aav-gene-therapy-vectors-by-sec-uv-mals-dri-0001
Unit 9, Trade City, Sandy Lane West, Oxford OX4 6FF, United Kingdom
©2025 Refeyn Ltd
Refeyn, MassGlass, MassFerence and Samux are registered trademarks of Refeyn Ltd.
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we have shown, results for the analysis of the % empty/partial/
full AAVs obtained after simple cleanup are equivalent to those
obtained after capture chromatography (Fig. 4). Taking a fraction
of the time required for the purification (3 h vs. 24 h), the simple
cleanup represents an efficient option for processing samples
prior to rapid analytics to inform process development.
Integral to our approach is AAV sample characterization by
mass photometry. Compared to SEC-MALS and AUC, mass
photometry has important advantages that make it ideal in the
upstream context (Table 1). Unlike mass photometry, AUC
is unsuitable for use after simple cleanup because its sample
concentration requirements can only be met after traditional,
column-based purification. In contrast to mass photometry,
SEC-MALS cannot resolve partially filled vs. full AAV capsids.
Mass photometry has the further advantages of being faster
and easier to use, and consuming less sample than the other
methods (Table 1).
Overall, the results demonstrate the value of mass photometry
as a tool for at-line, upstream analysis of AAV samples, when
combined with a straightforward cleanup method we described.
By providing critical sample quality insights at a much earlier stage
than is typically feasible, this approach offers a much-needed
means to improve AAV process development – and accelerate
the production of AAV-based therapies.
Refeyn thanks Généthon for their
collaboration in the creation of this
application note.
Reagents and materials for simple cleanup
• AcroPrep 96-well 1 mL 1.2 μm plate (Pall 8130)
• 2 mL 96-well collection plate (Corning 3961)
• POROSTM CaptureSelectTM AAVX Affinity resin (ThermoFisher
A36741), prepared as slurry following manufacturer’s instructions
• PBS 1X buffer
• 1 M Tris (pH 8)
• 100 mM Sodium Acetate (pH 3)
• AAV cell culture
• Detergent for lysis
• 50 mL conical tube
Equipment for simple cleanup
• Orbital shaker
• Vacuum pump with manifold for multi-well filtration plates
• Benchtop centrifuge
• For storage: -80°C freezer and 4°C refrigerator
Procedure for simple cleanup
Harvest and addition of resin
1. Harvest 1.5 mL of cell culture from the bioreactor.
2. Add 0.5% v/v of detergent and incubate for 2.5 h at 37°C
under agitation.
3. Spin down the cell debris at 1000g for 10 minutes at room
temperature.
4. Collect the supernatant.
5. (optional) If the samples are stored at -80°C, thaw overnight
at 4°C.
6. Deposit 50 μL of POROSTM CaptureSelectTM AAVX Affinity
resin slurry (25 μL of resin) per well in the AcroPrep 96-well
filter plate.
PROTOCOL
Simple cleanup for upstream AAV characterization with mass photometry
Some existing technologies AAV analytics require concentrated and highly purified samples. However, mass photometry
can be applied to AAV cell culture samples after a simple, plate-based cleanup.1
The method, which takes three hours and
can be applied to tens of samples in parallel, is described here.
®
All subsequent steps are to be performed at room temperature.
Washing
1. Place a 96-well collection plate in the vacuum manifold and
aspirate the filter plate until all liquid is removed.
2. Wash by adding 600 μL of PBS buffer per well. Shake for
5 min at 200–250 rpm.
3. Aspirate to remove all liquid.
4. Deposit 750 μL of sample per well according to the plate
layout.
5. Incubate for 30 min without shaking.
6. Aspirate the flow-through until the well is empty.
If the vacuum well becomes blocked, resuspend with 500 μL PBS
and transfer to an empty well.
Omit steps 7–9 if the sample titer is known to be > 1 x 1011 vg/mL.
7. Deposit a further 750 μL of sample per well according to
the plate layout.
8. Incubate for 1 h without shaking.
9. Aspirate the flow-through until the well is empty.
The following wash steps (10–12) are to be repeated, so they are
performed a total of 3 times.
10. Wash by adding 800 μL of PBS per well.
11. Incubate for 5 min without shaking.
12. Aspirate until all liquid is removed. Remember to empty the
excess liquid from the collection plate.
Elution
1. Add 20 μL of 1 M Tris (pH 8) directly into each well of a new
collection plate. This will neutralize the eluate.
2. Replace the collection plate from the previous step with the
one containing 1 M Tris (pH 8).
3. Add 100 μL of 100 mM Sodium Acetate (pH 3) as eluate.
4. Incubate for 15 min without shaking, then aspirate (for long
enough to collect the entire volume).
Storage
1. Store samples at 4°C if analysis is to be performed within
the week, or at -20°C or -80°C for longer-term storage.
Analysis by mass photometry
Analyze samples following the approach described in detail in the
latest Refeyn SamuxMP Mass Photometer User Manual (Experimental
Guidelines). In brief:
1. Switch on the mass photometer one hour prior to beginning
measurements.
2. Launch the acquisition software AcquireMP and the analysis
software DiscoverMP.
3. Prepare and mount the MassGlass® UC sample carrier.
4. Add 10 μL of buffer to one well and focus the instrument
using the droplet-dilution button on AcquireMP.
5. Add 10 μL of MassFerence® P2 to the focused well from
Step 4, mix 3x, close the lid and press ‘Record’ in AcquireMP
to measure the calibrant.
Calibration is not necessary before every measurement, but should
be performed twice a day.
6. Move to the next well and repeat Step 4.
7. Dilute the sample, if necessary, to 2x higher than the
concentration to be measured (e.g. 2x1011 particles/mL).
8. Add 10 μL of the sample to the focused well from Step
6, mix 3x, close the lid and
