AAV analytics accelerated
Fast, informative AAV analytics for every stage of development
Adeno-associated viruses (AAVs) are the leading viral vectors in clinical gene therapy. However, fast, cost-effective, and accurate characterization of purity – quantifying empty, partially filled, full, and overfilled capsids – is difficult or impossible with traditional methods. Mass photometry (MP) delivers accurate, label-free quantification of all capsid populations, using minimal sample and producing results in minutes.
Key pain points encountered by scientists during process optimization and GMP manufacturing:
Limited upstream insight
Vector genome/ viral particle (VG/VP ratios) – widely used in early development stages – cannot distinguish empty, partially filled, full, and overfilled capsid populations, limiting true process understanding and informed decision making.
Slow, sample-intensive analytics
Time-intensive, sample-heavy analytical methods prevent efficient process optimization – particularly in early development, where AAV material is scarce and costly.
Cost and GMP compatibility constraints
To balance cost-effectiveness, sample economy, and GMP compatibility in both process development and release testing, teams use multiple assays – driving up costs, complexity, and timelines.
Figure 1. Mass photometry can resolve each population in AAV samples (empty, partially filled, full, and overfilled) with high accuracy. Schematic representation of AAV particle populations resolved by mass. The increasing mass gradient (left to right) reflects progressive genome packaging within the capsid, enabling differentiation of particle heterogeneity based on molecular mass
Challenges
Reliable capsid content analysis – how upstream uncertainty threatens quality
One of the greatest challenges in AAV process optimization and production is obtaining early, accurate insight into capsid packaging efficiency in upstream samples. At this stage, reliable capsid profiling is particularly difficult because crude lysates are complex to analyze. As a result, teams typically rely on VG/VP (vector genome/ viral particle) ratios derived from qPCR and ELISA to infer capsid content. Traditional techniques – AUC, TEM, SEC-MALS, and CDMS – often struggle with sample heterogeneity, require extensive preparation, and are impractical for rapid decision-making. As a result, teams typically rely on VP/VG (viral protein/vector gene) ratios derived from ELISA and qPCR to infer capsid content. This indirect approach lacks the resolution to distinguish empty, partially filled, full, and overfilled capsids, increasing the risk of optimizing yield at the expense of product quality. Empty capsids introduced upstream are notoriously difficult and costly to remove during downstream processing.
Mass photometry (MP) addresses this critical gap by enabling direct, label-free quantification of all capsid populations at the upstream stage with minimal sample clarification (taking only 1–3 hours). Find out more from this infographic which outlines how upstream implementation supports better process control, reduces batch variability, and enhances overall product quality. Stereotype-agnostic and fast, MP works with any AAV serotype without assay redevelopment, conserving valuable upstream material and delivering results in minutes.
By giving scientists early visibility into this critical quality attribute (CQA), MP ensures that process optimization decisions are guided not only by yield, but by true product quality – at a stage when corrections are easiest and most cost-effective to make.
Figure 2. Combined with small-scale purification (simple cleanup), MP enables monitoring of AAV purity during upstream optimization. Example shows MP analysis of clarified lysate before purification (A), and after simple sample cleanup for AAV8 (B) and AAV9 (C) serotypes. Data collected by Généthon using a Samux™ mass photometer. To learn more, see Upstream AAV characterization with mass photometry: Application note and Protocol.
Cost- and resource-efficient optimization
Even when an AAV process ultimately achieves strong productivity, high yields, and a high percentage of full capsids, the path to optimization is rarely linear. In practice, reaching the desired performance often requires multiple experimental iterations, spanning both upstream and downstream development, with numerous conditions tested in parallel. These iterations significantly increase time, cost, and material consumption.
As gene therapies move closer to commercialization, the impact of these inefficiencies becomes clear: Every microliter of AAV material has tangible value, and analytical strategies must be designed to preserve it. Analytical methods are needed across the entire optimization workflow up to 180 minutes based on protocol, and must be fast, cost-effective, and information-rich.
MP supports near real-time decision-making during process optimization. It dramatically reduces sample consumption, requiring only 10 µL of sample at 1 × 10¹¹ VP/mL – up to tenfold less material than typical ELISA-based assays. Data acquisition can be completed in under 5 min per sample, and as little as 90 minutes for 24 samples using automated workflows* – all supported by intuitive software and minimal data analysis requirements.
Routine MP offers far more than capsid population quantification alone. In a single, rapid measurement, it enables genome length estimation across differently filled populations within a defined mass range. This breadth of insight allows scientists to assess multiple quality-relevant attributes from a single, rapid measurement.
By delivering rapid, high-value insight while conserving precious material, MP enables scientists to optimize processes faster, reduce development costs, and protect product quality without compromising on analytical depth up to 180 minutes based on protocol.
*Up to 180 minutes based on protocol
Figure 3. As an at-line tool, MP allows monitoring of sample purity and enrichment of the full AAV population during downstream optimization. It enables testing of each fraction under different conditions such as buffer comparisons. Overlaying the UV signal (measured by UV detector in chromatography system) with the percentage of full AAV (measured by MP) clearly shows the stage at which enrichment occurs.
| AUC | CDMS | MP | SEC-MALS | UV (A260/280) | |
|---|---|---|---|---|---|
|
%
Full particles |
91% |
80% |
91% |
98% |
95% |
|
% Partially filled particles |
6.4% |
15.7% |
3.2% |
— |
— |
|
Molecular
Weight of full
particles (MDa)
|
— |
4.79 |
4.53 |
4.56 |
— |
Table 1. Analysis of the USP AAV8 (Full) standard – comparing MP to complementary analytical techniques for quantifying % full and % partially filled (where available) capsids, and measuring molecular weight (where available). The close agreement of MP with AUC highlights MP’s value as a highly accurate and reliable approach for assessing AAV purity. Adapted from: USP Biologics (2025). Application note. AAV8 Reference Standards: revolutionizing empty/full capsid analysis.
Overcoming the cost and complexity of GMP-ready AAV analytics
Gene therapies are ultimately developed for clinical approval and patient use, yet many analytical methods used in GMP environments today are expensive, complex to use, and slow. These challenges directly contribute to the high cost of gene therapies and slow progression from development to clinic.
MP addresses this gap by delivering reliable, low-cost AAV analytics that support GMP readiness from early development through to QC release testing. Enabled by GMP-ready instrumentation and fully 21 CFR Part 11–compliant software, MP enables consistent, regulatory-aligned characterization of AAV capsid populations using minimal sample volume and streamlined, user-friendly workflows.
MP has demonstrated strong reliability for AAV purity analysis and has been recognized by major regulatory and standards-setting bodies, including the United States Pharmacopeia (USP), British Pharmacopeia, and Chinese National Institute for Food and Drug Control. The USP identified MP as a suitable method for AAV purity assessment, using it alongside established techniques to characterize its full and empty AAV reference standards (Table 1).
By combining regulatory alignment, speed, and affordability, MP enables scalable, GMP-compatible analytics that support both innovation and compliance in gene therapy development.
Mass photometry solutions for AAV vector analytics
Refeyn SamuxMP & SamuxMP Auto
Samux™ mass photometers are optimized for fast, accurate AAV characterization across all development stages.
SamuxMP quickly and accurately measures full-to-empty capsid ratios, as well as partially filled and overfilled capsid populations, across all AAV serotypes. It can also estimate genome length for differently filled populations, estimate capsid titer, and as part of the standard data analysis procedure.
Designed to meet the key challenges of AAV analytics, SamuxMP offers:
Accurate measurement of capsid ratios
Quantifies empty, partially filled, full and overfilled capsids – even upstream with a quick cleanup.
Speed and low sample consumption
Delivers results in 5 min (SamuxMP) or in as little as 90 min for 24 samples (SamuxMP Auto)*. Uses microliter-scale sample volumes at 1 × 10¹¹ VP/mL.
Acceleration of GMP readiness
Reliable AAV analytics from discovery to QC release, with regulatory recognition by USP, BP and NIFDC.
*Up to 180 minutes based on protocol
MassGlass™ UC slides are high-quality measurement surfaces designed for mass photometry.
MassGlass™ UC slides provide an out-of-the-box, ready-to-use solution that delivers reliable and consistent performance for AAV measurements.
Refeyn offers MassGlass™ UC Sample Prep Kits for the Samux™ and Samux™ Auto systems, delivering everything you need for a smooth, ready-to-run workflow and effortless, high-quality AAV measurements.
Key advantages include:
Ready-to-use format
Enables effortless AAV analysis with a true out-of-the-box workflow.
Guaranteed quality
Provides consistently accurate, high-quality AAV data with minimal effort.
Serotype agnostic
Universal compatibility for all AAV serotypes, enabling parallel analysis of multiple AAV serotypes within a single workflow.
Software for data acquisition, analysis, and GMP compliance
Refeyn provides a complete, end-to-end software suite for both process development and GMP-compliant environments.
Designed to streamline data acquisition and interpretation, this software suite enables rapid, high-quality measurement with clear user guidance and powerful analytical tools. Supported by a fully GMP-compliant platform, it empowers researchers working with AAV samples to make fast, confident, and data-driven decisions. Two tailored solutions are available for research and process development:
For research and process development:
AcquireMP – optimized for intuitive, efficient data acquisition
DiscoverMP – advanced analysis and interpretation for R&D workflows
Together, these tools support fast, confident decision-making across development stages.
For 21 CFR Part 11 compliance
Refeyn also offers a complete GMP-compliant software environment aligned with FDA 21 CFR Part 11 and EU GMP Annex 11 requirements.
It includes:
AcquireMP – secure data acquisition
EvaluateMPS – analysis within a GMP-compliant environment
ManageMP – system configuration, user management, and full audit trails
Together, these software packages provide a robust, validated framework for regulated environments – ensuring data integrity, traceability, and compliance from acquisition through to analysis.
Mass photometry in the AAV literature
Resources
USP AAV standards to support quality testing and characterization
Learn about USP AAV reference standards and Pharmaron’s use of mass photometry to streamline AAV development, optimize capsid purification, and complement other analytical techniques in large-scale manufacturing.
Upstream AAV characterization with mass photometry
Check out this app note to learn a rapid, plate-based lysate cleanup and mass photometry workflow that delivers 3-hour AAV quality results—measuring empty, partial, and full capsids for smarter upstream decisions.
Comparing analytical approaches for AAV characterization
Explore this article to understand key AAV characterization methods—covering strengths and trade-offs across EM, AUC, CDMS, qPCR-ELISA, SEC-MALS and mass photometry—to support confident assessment of critical quality attributes.
Streamlining AAV analytics in gene therapy manufacturing workflows
Read this whitepaper to learn how Refeyn’s SamuxMP software package for GMP-regulated environments complies with GMP manufacturing regulations.
Frequently asked questions
Why is measuring the empty/partially filled/full/overfilled capsid ratio important in AAV production?
The empty/partially filled/full/overfilled capsid ratio is a Critical Quality Attribute (CQA) that reflects vector quality and potency because full capsids contain therapeutic DNA while empty ones do not.
How quickly can mass photometry analyze AAV samples?
Mass photometry measurements take one minute. An experiment can be prepared, collected and analyzed in under 5 minutes. With automated mass photometry (the SamuxMP Auto) 24 samples can be measured in as little as 90 minutes.
How much sample is needed for mass photometry AAV analysis?
Mass photometry requires very small sample volumes (typically ~10 µL) and can work with capsid titers around 10¹¹ Viral particles/mL or lower, preserving valuable material.
Is mass photometry suitable for all AAV serotypes?
Yes, mass photometry is serotype-agnostic, meaning it can measure AAV capsids of any serotype without protocol changes.
Is mass photometry suitable for both development and GMP environment?
Yes, mass photometry is widely used in process development, and it is compatible with GMP environments, supported by regulatory recognition and software that enables consistent quality control workflows. To learn more, read our white paper, AAV analytics in GMP-regulated environments.
How does mass photometry compare to other analytical methods?
Compared to techniques like AUC, TEM, or CDMS, mass photometry is faster, uses less sample, simpler to operate, cheaper and provides comparable quantification of capsid populations. To learn more, read our article, Comparing analytical approaches for AAV characterization (2026) – Refeyn
Can mass photometry estimate AAV genome size?
Mass photometry can estimate the relative genome length within capsids by resolving mass differences between partially filled, full, and overfilled AAV particles. To learn more, read our app note, Measuring AAV genome length with mass photometry.
Has mass photometry been recognized by regulatory standards bodies?
Yes, mass photometry has been acknowledged by organizations such as the United States Pharmacopeia (USP), British Pharmacopoeia (BP) and National Institute for Food and Drug Control in China (NIFDC) as a valid method for AAV empty/full capsid characterization. The US National Institute of Standards and Technology (NIST) has also assessed MP, among other orthogonal methods, as a method for measuring AAV capsid titer.
Is mass photometry automated, or does it require hands-on operation?
Mass photometry systems are available in both manual (SamuxMP) and fully automated (SamuxMP Auto) formats. Manual instruments require only minimal pipetting, while fully autonomous systems can analyze up to 24 samples in as little as 90 minutes, enabling unattended, high-throughput operation.
What other AAV parameters can be measured using mass photometry?
Besides analyzing the purity of AAV samples, mass photometry enables estimation of genome length within differently filled populations, capsid titer estimation, and detection of aggregates up to a defined molecular weight range.
How does MP compare to other technologies, such as AUC and CDMS?
Mass photometry shows excellent agreement with established orthogonal methods such as AUC and CDMS. This has been demonstrated in multiple peer-reviewed publications and in data generated as part of documentation aligned with the United States Pharmacopeia (USP) standard, confirming that MP delivers results consistent with mentioned techniques. To learn more, read our article, Comparing analytical approaches for AAV characterization.
Can MP also measure the empty/full (E/F) ratio of adenoviruses?
Standard mass photometry is not suitable for large viral vectors such as adenoviruses and lentiviruses. However, this capability is enabled by macro mass photometry, implemented in the KaritroMP instrument, which is specifically designed to analyze larger particles. You can learn more about lentivirus analytics with macro mass photometry, or read our app note to learn about analysis of adenovirus full/empty ratios.