AAV & Gene Therapy

AAV & Gene Therapy Vector Characterization

Empty/full capsid ratio, aggregation profiling, and capsid integrity using orthogonal biophysical methods. SEC-MALS, SV-AUC, mass photometry, and DLS for AAV critical quality attributes.

Get a Characterization Plan AAV Method Guide

Empty/full ratioCapsid aggregationCapsid integrityOrthogonal methods

Typical project details

AAV serotype and genome size
Available volume and estimated titer (vg/mL)
Development stage (research, process development, lot comparability)
Primary CQAs of interest (empty/full ratio, aggregation, identity)
Buffer composition and storage conditions

AAV Characterization Questions?

Empty/full ratioAggregationCapsid profilingFormulation screeningLot comparabilityMethod qualification

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Why It Matters

Why Orthogonal AAV Characterization Matters

AAV gene therapy vectors are large macromolecular assemblies (~3.8 MDa full capsid, ~2.8 MDa empty capsid) with complex quality attribute profiles. Unlike monoclonal antibodies or recombinant proteins, AAV requires specialized analytical approaches that account for its size, heterogeneity, and the critical distinction between genome-containing (full) and empty capsids.

FDA guidance discusses orthogonal empty/full capsid characterization as part of a broader analytical strategy for gene therapy products. SV-AUC is widely considered the gold standard, while SEC-MALS and mass photometry provide complementary mass-based confirmation. Multi-method orthogonal approaches have become the industry standard for reliable AAV characterization across development stages.

AAV Critical Quality Attributes

Empty / Full / Partial Capsid Ratio: Direct impact on potency and immunogenicity. Orthogonal measurement by AUC + SEC-MALS is recommended.
Capsid Aggregation & Fragments: Aggregates reduce effective dose; SEC-MALS and DLS for orthogonal size profiling.
Capsid Population Profiling: Intact capsid mass distribution and heterogeneity assessment by SEC-MALS or mass photometry.
Formulation Stability: Aggregation rates under thermal stress, freeze-thaw, and shear.

FDA GuidanceRecommends orthogonal empty/full capsid quantification for IND submissions

SV-AUCGold standard for empty/full ratio; matrix-free, serotype-independent

SEC-MALSAbsolute mass confirmation of empty, partial, and full capsid populations

Mass PhotometryRapid capsid screening; excellent agreement with AUC within 1–3%

Why Choose Our AAV Characterization Service

Orthogonal Methods In-House

We provide AUC, SEC-MALS, mass photometry, and DLS directly — not through subcontracting. This means faster coordination, consistent data interpretation across methods, and a single point of contact for your empty/full ratio and aggregation profiling.

Stage-Appropriate Analytical Depth

Early process development may only need rapid mass photometry screening. Lot comparability may require full orthogonal AUC + SEC-MALS. We match the method depth to your actual decision need, not a fixed panel.

Research-Phase Focus, Faster Access

Our services are designed for development-stage programs where GMP compliance is not yet required. This means simpler project setup, more flexible scheduling, and data packages focused on internal decision-making rather than regulatory formatting.

Integrated CRO Coordination

When genome titer, capsid protein ID, or infectivity data is needed alongside biophysical characterization, our team can coordinate with qualified partners — delivering a unified data package without requiring you to manage multiple vendors.

CQA 1 · Capsid Composition

AAV Empty / Full Capsid Ratio Determination

The ratio of genome-containing capsids to empty capsids is the most critical AAV CQA, directly affecting potency, dosing, and immunogenicity risk. Orthogonal measurement by at least two independent methods is standard industry practice.

Sedimentation Velocity AUC

Widely considered the gold standard for empty/full capsid ratio. Separates AAV populations by sedimentation coefficient in native buffer without column interaction. Multi-wavelength detection (230/260/280 nm) differentiates empty, partial, and full capsids. Compatible with AAV1–AAV9 and engineered serotypes. Requires ~5 × 10¹¹ vg/mL, ~400 µL/run.

AUC for AAV Details → AUC Platform →

SEC-MALS

Absolute molar mass resolves empty (~2.8 MDa) and full (~3.8 MDa) AAV capsids by mass, not elution position. SEC separates capsids from aggregates before MALS detection. Combined UV (A260/A280) provides confirmatory empty/full estimation. Widely adopted across the gene therapy industry for orthogonal capsid characterization.

SEC-MALS Details →

Mass Photometry

Single-molecule mass measurement resolves empty, partial, and full capsids in minutes using 5–20 µL of AAV. Published studies (Wörner et al., Mol. Ther. Methods Clin. Dev. 2022) have reported good agreement with AUC for empty/full ratios across multiple AAV serotypes. Ideal for rapid at-line screening during process development.

Mass Photometry Details →

CQA 2 · Aggregation & Size

AAV Capsid Aggregation and Fragment Analysis

AAV aggregates reduce transduction efficiency and increase immunogenicity risk. Capsid fragments and partially assembled species must be monitored for lot consistency and formulation development.

SEC-MALS for Aggregate Profiling

SEC separates monomeric AAV capsids from higher-order aggregates (dimer, trimer, multimer) and fragments. MALS provides absolute mass for each peak. Available column pore sizes (100 Å, 300 Å, 500 Å) selected based on capsid size. DLS add-on provides hydrodynamic radius for orthogonal size confirmation. Typical injection: 50–100 µL at 0.5–5 × 10¹² vg/mL.

SEC-MALS Details →

DLS for Capsid Size & Polydispersity

Rapid hydrodynamic radius (Rh), polydispersity index, and large aggregate detection. Highly sensitive to trace HMW aggregates. Standard for AAV formulation screening, thermal stability, and freeze-thaw comparability. Complements SEC-MALS for comprehensive size characterization. Minimal volume: ~20 µL at ≥5 × 10¹¹ vg/mL.

DLS Details →

Complementary Analytics

Broader AAV Characterization Capabilities

A comprehensive AAV characterization program may extend beyond biophysical methods to include additional analytical dimensions:

Genome TiterddPCR, qPCR — absolute genome copy quantification per lot

Capsid Protein IDLC-MS/MS — VP1:VP2:VP3 stoichiometry, PTM mapping

Infectious TiterTCID50, cell-based transduction — functional potency

Genome IntegrityNGS, alkaline gel — fragmentation, packaging consistency

This page focuses on biophysical characterization Our team can help coordinate complementary analyses through our broader CRO network as needed.

What You Receive

Deliverables for AAV Characterization Studies

Empty/full capsid ratio reportOrthogonal report with AUC and SEC-MALS data, confidence intervals, and population distribution.

Aggregation profileSEC-MALS and DLS aggregate quantification, size distribution, and PDI.

Capsid mass distributionMass photometry or SEC-MALS intact capsid mass and heterogeneity assessment.

Formulation stability comparisonAggregation rates and capsid integrity under thermal, freeze-thaw, and shear stress.

Lot/process comparability summarySide-by-side comparison of CQAs across production conditions.

Study reportComplete analytical report with methods, data, and interpretation for internal decision-making.

Sample Requirements

Sample Requirements for AAV Characterization

Exact requirements vary by method, but general guidelines are:

Purified AAV at ≥5 × 10¹¹ vg/mL preferred
Volume: 100–500 µL per method; ~1–2 mL for orthogonal package
Buffer composition and formulation details
Serotype and genome size for data interpretation
Any existing titer, purity, or batch records

Method Guide

AAV Characterization: Which Technique for Which CQA

CQA	Primary Method	Orthogonal Method	Typical Output
Empty / Full Capsid Ratio	SV-AUC	SEC-MALS + Mass Photometry	% empty, partial, full with orthogonal concordance
Aggregate Content	SEC-MALS	DLS	% aggregate, Rh, PDI
Capsid Identification	SEC-MALS (mass)	Mass Photometry	Intact capsid mass; serotype-specific mass fingerprint
Formulation Stability	DLS + SEC-MALS	AUC (stress)	Aggregation rate, size evolution over time

AAV Workflow

AAV Biophysical Characterization Workflow

Capsid CompositionAUC + SEC-MALSOrthogonal empty/full ratio with mass confirmation

AggregationSEC-MALS + DLSAggregate quantification + hydrodynamic size

Capsid IdentityMass PhotometryMass fingerprint and heterogeneity assessment

StabilityDLS + SEC-MALSThermal, freeze-thaw, shear stress

ComparabilityMulti-methodLot-to-lot, process change, formulation

ReportIntegratedData package with methods and interpretation

Complementary CQA support: Genome titer (ddPCR/qPCR) · Capsid protein identification (LC-MS/MS) · Infectious titer (TCID50) — coordinated through CRO network. Ask about full AAV characterization

Frequently Asked Questions About AAV Characterization

What is the recommended approach for measuring AAV empty/full capsid ratio?

Industry practice, supported by the 2025 NIST interlaboratory study, recommends orthogonal measurement by at least two independent methods. SV-AUC is widely considered the gold standard. SEC-MALS provides absolute mass confirmation. Mass photometry offers a rapid orthogonal screening option with excellent method agreement. This multi-method approach is aligned with the NIST 2025 interlaboratory study recommendations and current industry best practice.

How much AAV sample is needed for biophysical characterization?

AUC typically needs ~400 µL at ≥5 × 10¹¹ vg/mL; SEC-MALS requires 50–100 µL; mass photometry needs only 5–20 µL. For a comprehensive orthogonal package, ~1–2 mL of purified AAV at ≥5 × 10¹¹ vg/mL is recommended.

Why can't a single method provide reliable empty/full capsid data?

Each method has inherent limitations: AUC can be affected by rotor speed and acquisition time; SEC-MALS separates by hydrodynamic volume and may co-elute species; mass photometry requires clean samples. Combining methods based on different physical principles (sedimentation, light scattering, single-molecule mass) provides orthogonal confidence. This approach is recommended by FDA guidance and standard practice across the gene therapy industry.

Do you provide GMP-compliant AAV lot release testing?

Our services focus on biophysical characterization for research and development-stage programs. For GMP-compliant lot release testing, we recommend engaging a qualified CDMO or GMP testing laboratory. Our data supports internal decision-making and process development.

Can your methods distinguish partial capsids from empty or full capsids?

SV-AUC with multi-wavelength detection can resolve partial capsids based on sedimentation coefficient differences. SEC-MALS provides absolute mass confirmation, and mass photometry at single-molecule resolution can distinguish empty, partial, and full capsid populations in a single measurement. The ability to resolve partials depends on sample quality, concentration, and the specific method chosen.

Can you compare AAV samples from different purification conditions?

Yes — this is one of the most common applications of our AAV characterization service. SEC-MALS and DLS can directly compare capsid aggregation, empty/full ratio, and size distribution across purification conditions (e.g., different columns, buffers, gradients). AUC provides the most definitive empty/full ratio comparison for side-by-side evaluation of process changes.

Key Literature

AAV Characterization References

Kumar, M. et al. (2025). Interlaboratory Measurement of Adeno-Associated Virus: Comparative Quantification of Full and Empty Capsids. Human Gene Therapy. 36(1-2):36–44. DOI: 10.1089/hum.2024.124

Wörner, T.P. et al. (2022). Mass photometry as a robust method for characterizing AAV critical quality attributes. Mol. Ther. Methods Clin. Dev. 26:166–176. DOI: 10.1016/j.omtm.2022.06.007

Burnham, B. et al. (2015). Analytical ultracentrifugation as an approach to determine rAAV empty/full capsid ratios. Hum. Gene Ther. Methods. 26(5):171–179. DOI: 10.1089/hgtb.2015.050

FDA (2020): CMC Information for Human Gene Therapy INDs. FDA Guidance. Link

Sommer, J.M. et al. (2003). Quantification of AAV particles and empty capsids by SEC-MALS. Mol. Ther. 7(S1):S257. DOI: 10.1016/S1525-0016(03)50038-2

FDA (2020): Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy IND Applications. FDA Guidance for Industry. FDA Guidance

Discuss Your AAV Program

We design fit-for-purpose AAV characterization plans matched to your serotype, development stage, and CQA priorities. Contact our team to discuss your program.

Discuss Your AAV Project