AAV Vector Characterization: Analytical Methods for Full, Empty and Partial Capsid Ratio Assessment
Adeno-associated viruses (AAV) have emerged as leading vectors in gene therapy due to their favorable safety profile, efficient gene delivery, and adaptability. These non-pathogenic viruses package therapeutic DNA—up to ~4.7 kb—within a protein capsid, enabling applications in gene replacement, gene silencing, or gene editing.
Following administration, AAV enters cells via receptor-mediated endocytosis and traffic to the nucleus, where the genome is released for expression. Their ability to transduce both dividing and non-dividing cells, along with advances in capsid engineering, makes AAV highly versatile for targeting tissues such as the liver, CNS, retina, and muscle. Clinically approved therapies like Luxturna and Zolgensma further demonstrate the therapeutic value of AAV.
To ensure product quality, we will review key analytical assays for AAV characterization, including established methods such as analytical ultracentrifugation (AUC), transmission electron microscopy (TEM), dynamic light scattering (DLS), and introduce the newer technique of mass photometry (MP). These tools are critical for assessing capsid composition, genome packaging, size distribution, and purity throughout the AAV production process.
Why Capsid Composition Matters: The Full/Empty Ratio as a Critical Quality Attribute
Despite the promise of AAV vectors, manufacturing challenges persist—particularly regarding capsid genome packaging efficiency. AAV preparations typically consist of a heterogeneous mixture of capsid populations (Figure 1):
- Full capsids contain the complete therapeutic genome and represent the pharmacologically active component.
- Empty capsids lack the therapeutic gene. These may be entirely void of DNA or encapsulate non-functional nucleic acid fragments such as host cell DNA, plasmid backbone sequences, or AAV inverted terminal repeats (ITRs).
- Partial capsids contain fragmented or incomplete genomes. These are especially common in vectors utilizing self-complementary AAV (scAAV) designs due to their reduced packaging capacity.
Figure 1. Schematic of different AAV capsid populations
The full/empty ratio—the proportion of fully packaged capsids relative to empty ones—has emerged as a critical quality attribute (CQA) in AAV gene therapy development, manufacturing, and regulatory evaluation. Importantly, this metric also reflects the integrity of the packaged genome and the presence of capsid-associated impurities, both of which directly impact therapeutic performance and patient safety:
- Impact on Gene Therapy Efficacy and Potency
Only full capsids deliver the therapeutic payload, meaning a low full/empty ratio dilutes the effective dose, impacting efficacy and complicating PK/PD modeling. Genome integrity is also vital; fragmented genomes reduce potency and can pose safety risks.
- Safety, Immunogenicity, and Impurity Burden
Empty and partial capsids, though non-functional, are immunogenic, triggering immune responses that limit redosing and cause inflammation. Co-packaged impurities within these non-functional capsids, such as host-cell DNA, can also lead to off-target effects and genotoxicity, necessitating strict regulatory monitoring.
- Product Consistency, Regulatory Compliance, and Mechanistic Clarity
Maintaining a consistent full/empty ratio across batches ensures product comparability, predictable performance, and regulatory compliance, as regulatory bodies like the FDA and EMA recognize this as a CQA. A well-defined empty/full profile also enhances mechanistic understanding, linking therapeutic outcomes directly to functional vectors.
Analytical Strategies for Measuring Full/Empty AAV Capsid Ratios
Given the complexity and heterogeneity of AAV vector preparations, accurate quantification of full, empty, and partial capsids is a crucial aspect of product characterization and release. Multiple analytical methods have been developed to address this need, each offering distinct advantages and limitations in terms of resolution, throughput, regulatory compliance, and ease of integration into GMP workflows. Below are key techniques currently employed in the field:
- Analytical Ultracentrifugation (AUC):
Principle: AUC separates particles based on their mass, size, and shape within a centrifugal field. Due to differences in buoyant density, empty AAV capsids (protein shell only) sediment slower than full capsids (protein shell + DNA genome). Sedimentation Velocity AUC can directly measure the sedimentation rate, producing distinct peaks for empty, partially filled, and full capsids based on their differing sedimentation coefficients (Figure 2).
Advantages:
Gold Standard: Widely considered the gold standard for empty/full ratio determination due to its fundamental physical separation principle and high accuracy.
High Resolution: Can resolve subtle density differences, allowing differentiation of empty, partially filled, and full capsids.
Limitations:
Low Throughput: Labor-intensive and time-consuming, making it unsuitable for high-throughput screening.
High Cost: Requires specialized, expensive equipment and highly trained personnel.
Sample Volume & Concentration: Requires relatively large sample volumes at specific concentration ranges.
Figure 2. AAV empty and full capsid separation by analytical ultracentrifugation
- Transmission Electron Microscopy (TEM):
Principle: TEM uses a beam of electrons to visualize particles at nanometer resolution. AAV capsids are directly observed, and the presence or absence of electron-dense material (the packaged genome) within the capsid is assessed. Samples are typically negatively stained to enhance contrast. Empty capsids appear translucent, while full capsids appear opaque due to the packaged DNA. Images are captured, and particles are manually or semi-automatically counted and categorized (Figure 3).
Advantages:
Direct Visualization: Provides direct visual evidence of capsid morphology, integrity, and genome packaging.
Qualitative and Quantitative: Offers both qualitative assessment of particle integrity and quantitative determination of empty/full ratios (by counting).
Detection of Aggregates: Excellent for identifying aggregated particles and other morphological impurities.
Limitations:
Low Throughput: Extremely labor-intensive and time-consuming for statistically significant counts.
Sample Preparation Artifacts: Sample preparation can introduce artifacts (e.g., aggregation, damage to capsids, uneven staining).
Cost: Requires expensive equipment and specialized expertise.
Limited Resolution for Partial Fill: Interpretation can be subjective, requiring highly experienced operators. Often it is difficult to definitively distinguish between full and partially filled capsids; thus, the quantitative accuracy is not high.
Figure 3. AAV empty and full capsid captured in TEM
- Dynamic Light Scattering (DLS):
Principle: DLS measures the hydrodynamic size of particles in solution by detecting fluctuations in scattered light due to Brownian motion. While its primary applications are characterizing particle size distribution and assessing aggregation, DLS can, in specific cases, provide an indirect indication of total particle concentration (titer). Multiangle DLS (MADLS) offers improved reproducibility in determining particle concentration and aggregate content compared to single-angle DLS. DLS is barely used alone to estimate the AAV empty/full ratio. It typically requires combination with other techniques, such as Static Light Scattering (SLS) and UV-Vis spectroscopy, for more accurate and robust quantification.
Advantages and limitations:
DLS offers several practical advantages, including its rapid and non-invasive nature, allowing for quick measurements with minimal sample preparation. The equipment is also relatively inexpensive compared to techniques like AUC or TEM, and it requires only small sample volumes. These features make DLS particularly useful for initial screening to quickly assess overall particle size, homogeneity, and the presence of aggregates. However, DLS faces significant disadvantages, primarily its limited resolution and lower accuracy for empty/full ratio determination. It is hard to detect subtle differences in hydrodynamic size between empty and full capsids. Thus, DLS is not recommended for precise empty/full ratio determination due to its limited accuracy.
- Mass Photometry (MP):
MP is a relatively new and rapidly advancing biophysical technique that provides label-free, single-molecule/single-particle mass determination in solution. It was developed in 2018 by a University of Oxford team led by Philipp Kukura and Daniel Cole. The technology was initially demonstrated at the Biophysical Society’s Annual Meeting in San Francisco, where a prototype mass photometer was showcased. The technology has since been commercialized by Refeyn. MP has quickly gained traction in various fields, including gene therapy, due to its unique capabilities and impressive sensitivity. MP bridges a crucial gap between traditional ensemble-averaging techniques (like DLS or AUC) and high-resolution imaging methods (like TEM).
Core Principle: At its heart, MP works by measuring the mass of individual molecules or nanoparticles by quantifying the light scattered when they adsorb onto a precisely engineered glass surface (typically a microscope slide). The process involves:
- Immobilization: A dilute sample of particles flows across a treated glass surface. Individual particles diffuse and transiently adsorb onto the surface.
- Light Scattering: A focused light beam (e.g., from a laser) illuminates the surface. When a particle binds, it causes a measurable increase in the scattered light intensity.
- Mass-Dependent Signal: Crucially, the intensity of the scattered light signal is directly proportional to the mass of the adsorbed particle. Larger, more massive particles scatter more light, producing a brighter signal.
- Real-Time Detection: A camera captures these scattering events in real-time. By analyzing the intensity fluctuations as particles bind, the instrument can generate a mass histogram (or mass distribution) of the sample.
Figure 4. MP measures the mass of individual molecules
Advantages for AAV Characterization:
The inherent single-particle resolution and label-free nature of MP make it exceptionally well-suited for characterizing complex biological samples like AAV vectors:
- Direct Mass Measurement: MP directly measures the mass of individual AAV capsids, providing unambiguous information about their content, unlike DLS (infers size) or AUC (separates by density).
- High-Resolution Empty/Full/Partial Capsid Measurement: Direct mass measurement allows exquisite resolution between empty, full, and even partially packaged AAV capsid populations, offering nuanced understanding of genome integrity.
- Accurate Titer Measurement: MP quantifies total particle concentration (titer) and specifically, the concentration of functional (full) particles by counting individual particles and correlating their mass.
- Low Sample Volume & Rapid Analysis: MP requires only small sample quantities and delivers results within minutes, ideal for precious samples and high-throughput needs.
- Simple Sample Preparation & Label-Free: Samples require minimal preparation (often just dilution) and no labels, simplifying workflows and preventing artifacts.
- Detection of Aggregates & Impurities: MP can detect and quantify larger species like AAV aggregates or other protein impurities, providing a comprehensive overview of sample heterogeneity.
Limitations:
While MP offers significant advantages for AAV characterization, it also has certain limitations:
- Specialized Equipment Required: MP is a newer technology compared to established methods like AUC or TEM, and its operation requires specialized equipment. As such, it is still undergoing widespread adoption.
- Sensitivity to Surface Adsorption & Buffer Conditions: MP performance is highly influenced by sample interaction with the sensor surface. Buffer composition, pH, ionic strength, and detergents require careful optimization.
- Concentration Dependence & Dynamic Range: MP operates optimally within a specific nanomolar to picomolar concentration range. Overly concentrated samples or aggregates can lead to co-adsorption, complicating analysis.
- Limited Structural Information: MP provides precise mass but no direct structural visualization like TEM. It can’t assess capsid morphology or specific structural defects beyond mass inferences.
MP Analysis of AAV Capsid Populations Throughout Purification Steps
As mentioned, MP is a rapid, label-free technique for quantifying the proportions of empty, partially filled, and full AAV capsids. The method requires only minimal sample handling: typically, 5–20 µL of AAV sample at a concentration of about 1e11 particles/mL is added directly to the measurement buffer, with no need for labeling or extensive preparation. It can deliver results in under 10 minutes.
The provided data illustrates the step-wise purification of AAV, tracking the percentages of empty, partial, and full capsids throughout the process (Figure 5):
Figure 5. MP data from step-wise AAV purification
- Post Lysis: The initial lysate contains 42.8% empty capsids, 1.9% partially filled, and 55.3% full capsids. This indicates a relatively high proportion of full capsids even before purification, but with a significant presence of empty and partial particles.
- Purification Step 1: After the first purification, the proportion of empty capsids increases to 65.3%, while full capsids drop to 25.4%, and ambiguous particles account for 9.3%. At this initial purification step, full capsid enrichment has not yet been achieved.
- Purification Step 2: At this stage, the sample contains 50.5% empty, 9.5% partial, and 40% full capsids. The proportion of full capsids increases compared to the previous step, indicating improved enrichment of full particles.
- Purification Step 3: The process becomes more efficient, with 25.1% empty, 6.4% partial, and a substantial 68.5% full capsids. This demonstrates significant enrichment of full capsids and removal of empty and partial ones.
- Purification Step 4: Here, 28.1% of capsids are empty, 11.9% partial, and 60.0% full. The proportion of full capsids remains high.
- AAV Bulk Drug Substance: In the final product, 27.0% of capsids are empty, 6.9% partial, and 66.1% are full. This indicates that the purification process successfully enriches full capsids, yielding a product with a high proportion of therapeutically relevant particles.
As demonstrated, MP enables comprehensive monitoring of AAV capsid composition and determination of the full/empty ratio throughout purification. It also facilitates viral titer calculation, a capability not offered by AUC. Notably, MP’s full/empty ratio measurements have shown good comparability to AUC. For the AAV-Bulk Drug Substance sample, a final product achieving 65.1% full capsids from 1.4e13 particles was obtained, with MP and AUC showing remarkable agreement in full/empty ratio (66.1% vs 64.8% from AUC analysis) in Table 1. This consistency between MP and AUC full capsid percentages demonstrates MP’s capability to provide accurate, high-throughput estimations of vector purity and concentration. Unlike AUC, which typically requires more sample volume, longer analysis times, and specialized technical expertise, MP offers a rapid, low-volume, and user-friendly alternative, making it highly suitable for in-process monitoring or lot release support during AAV production.
Table 1. Titer, full/partial/empty capsid measurements with MP
Summary:
PackGene offers a comprehensive suite of analytical services for AAV characterization, including AUC, TEM, DLS, and MP, etc. As demonstrated in the provided data, MP delivers rapid and accurate quantification of empty, partial, and full capsids, as well as total particle titer, with results that closely match those from AUC—the industry gold standard. Each method has its unique strengths: AUC provides regulatory-accepted, high-resolution quantification; TEM offers direct visualization of capsid morphology; DLS enables quick assessment of particle size and aggregation; and MP allows for high-throughput, precise determination of capsid composition and titer with minimal sample preparation. The comparison is shown in Table 2.
Table 2. Comparison of AAV composition analytical methods
Researchers are encouraged to select the analytical technique that best aligns with their study goals and the specific stage of their AAV development process, whether for early screening, process optimization, or final product quality control. By leveraging PackGene’s full range of testing options, clients can ensure robust, reliable, and regulatory-compliant characterization of their AAV products throughout the entire development pipeline.
Reference:
FDA. (2020). Chemistry, Manufacturing, and Control (CMC) Information for Human Gene Therapy Investigational New Drug Applications (INDs). Guidance for Industry. U.S. Department of Health and Human Services, Center for Biologics Evaluation and Research (CBER). https://www.fda.gov/media/113760/download
EMA. (2010). Reflection Paper on Quality, Non-Clinical and Clinical Issues Related to the Development of Recombinant Adeno-Associated Viral Vectors. EMEA/CHMP/GTWP/587488/2007. https://www.ema.europa.eu/en/quality-non-clinical-clinical-issues-relating-specifically-recombinant-adeno-associated-viral-vectors-scientific-guideline
Wagner, C. (2023). Quantification of Empty, Partially Filled and Full Adeno-Associated Virus Vectors Using Mass Photometry. Int J Mol Sci, 24(13), 11033. doi: 10.3390/ijms241311033
Kontogiannis, T. (2024). Characterization of AAV vectors: A review of analytical techniques and critical quality attributes. Mol Ther Methods Clin Dev, 32(3), 101309. doi: 10.1016/j.omtm.2024.101309.
Wu, D. (2022). Rapid characterization of adeno-associated virus (AAV) gene therapy vectors by mass photometry. Gene Ther, 29(12), 691-697. doi: 10.1038/s41434-021-00311-4.
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PackGene Biotech is a world-leading CRO and CDMO, excelling in AAV vectors, mRNA, plasmid DNA, and lentiviral vector solutions. Our comprehensive offerings span from vector design and construction to AAV, lentivirus, and mRNA services. With a sharp focus on early-stage drug discovery, preclinical development, and cell and gene therapy trials, we deliver cost-effective, dependable, and scalable production solutions. Leveraging our groundbreaking π-alpha 293 AAV high-yield platform, we amplify AAV production by up to 10-fold, yielding up to 1e+17vg per batch to meet diverse commercial and clinical project needs. Moreover, our tailored mRNA and LNP products and services cater to every stage of drug and vaccine development, from research to GMP production, providing a seamless, end-to-end solution.
