Upstream AAV Characterization With Mass Photometry

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. refeyn.com Refeyn Refeyn 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 press ‘Record’ in AcquireMP to measure the sample. References 1 Refeyn App Note: Upstream AAV characterization with mass photometry. 2024 https://refeyn.com/app-note-upstream-aav-characterization-withmass-photometry 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. refeyn.com Refeyn Refeyn This protocol was created by Généthon.