AAV Analysis
AAV analysis is essential to ensure product quality, safety, and efficacy across both research and clinical development. AAVnerGene delivers a comprehensive suite of AAV analysis services, built on a foundation of deep expertise from both academic research and industrial application. Our platform covers every critical aspect of vector characterization, including AAV capsid structure and AAV purity, AAV full-to-empty particle ratio, AAV physical titer and AAV infectious titer, AAV genome integrity, AAV neutralizing antibody (NAb) profiling, and AAV biosafety assessment. By combining advanced analytical tools with proven scientific insight, we provide high-resolution, reliable data to support the development, consistency, and therapeutic performance of AAV products.
AAV Characterization Services at AAVnerGene
| QC | Method | Turnaround | Price | Sample Requirement | |
| AAV Physical Titers | AAV Genomic Titer | qPCR | 1-2 day | $50/sample (Free in AAV Packaging Services) | *Primer/probe sequences; ~20 μl of samples |
| ddPCR | 3-5 day | $200/sample | *Primer/probe sequences; ~20 μl of samples | ||
| AAV Capsid Titer | ELISA | 3-5 day | $400/sample | *Serotypes and buffers; ~20 μl of samples | |
| AAV Infection Titer | AAV-Q TCID50 | 2-3 Weeks | $5000/sample | ~20 μl of samples | |
| AAV Purity Analysis | SDS-PAGE | 3-5 day | $200/sample | ~20 μl of sample with titer >1E+12 vg/ml | |
| AAV Full/Empty Ratio | Genomic Titer/Capsid Titer | 1-2 weeks | Request | ~20 μl of samples | |
| Mass Photometry | 1-2 day | $800/sample | 20 μl of sample with titer >5+11 vg/ml | ||
| CD-MS | 2-3 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml | ||
| AAV Genomic Identity/Integrity | TapeStation | 1-2 day | $200/sample | ~100 μl of sample with titer >1E+12vg/ml | |
| CD-MS | 2-3 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml | ||
| Nanopore Sequencing | 1-2 weeks | Starting from $600 | ~100 μl of sample with titer >1E+12vg/ml | ||
| PacBio Seq | 4 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml | ||
| AAV Genomic Impurity | qPCR | 3-5 day | $50/Target | ~20 μl of samples | |
| ddPCR | 3-5 day | $200/Target | ~20 μl of samples | ||
| Nanopore Seq | 1-2 weeks | Starting from $600 | ~100 μl of sample with titer >1E+12vg/ml | ||
| PacBio Seq | 3-4 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml | ||
| AAV Biosafety | Endotoxin | LAL assay | 3-5 day | $200/sample | ~20 μl of samples |
| Endotoxin Removal | Resin-PMB | 3-5 day | $200/sample | ~20 μl of samples | |
| Bioburden Testing | Direct plating | 3-5 day | $200/sample | ~20 μl of samples | |
| Mycoplasma Testing | PCR | 3-5 day | $200/sample | ~20 μl of samples | |
| Sterility test | Innoculation | 2-3 weeks | $200/sample | ~20 μl of samples | |
| rcAAV | ddPCR | 3-5 day | $200/Target | ~20 μl of samples | |
| AAV-Q rcAAV (Cell based) | 3-4 weeks | $5000/sample | ~20 μl of samples | ||
AAV Genome Analysis
AAV Genome Analysis is critical for ensuring the integrity, identity, and functionality of the packaged vector genome. Comprehensive genome characterization includes assessing genome size, sequence accuracy, structural integrity (e.g., ITR integrity and recombination events) and genomic impurities. Techniques such as qPCR and ddPCR are widely used to quantify viral genome titers, while next-generation sequencing (NGS) and long-read sequencing (e.g., PacBio, Oxford Nanopore) enable detailed analysis of genome composition, transgene integrity, and potential contaminating sequences. Additionally, Bioanalyzer or TapeStation systems provide sizing and purity information. These analyses are essential for confirming product quality, batch-to-batch consistency, and regulatory compliance during AAV vector development and manufacturing.
AAV Genomic Titer
Accurate measurement of the vector genome (VG) copy number is fundamental for dose determination and lot-to-lot consistency. This is typically performed using:
Quantitative PCR (qPCR): Detects a specific region within the transgene.
Droplet Digital PCR (ddPCR): Provides absolute quantification without the need for standard curves and is less sensitive to impurities.
Genome Identity and Sequence Integrity
To ensure that the correct therapeutic gene is packaged and intact:
Sanger sequencing or Next-Generation Sequencing (NGS) confirms the presence and accuracy of the gene cassette.
ITR sequencing checks for mutations or deletions in the inverted terminal repeats (ITRs), which are essential for genome replication and packaging.
Restriction enzyme digestion or Southern blot can be used to validate the expected genome size and structure.
AAV Genome Integrity
Partial, truncated, or rearranged genomes can result from recombination events or packaging errors, potentially reducing efficacy or increasing toxicity. Tools include:
Capillary electrophoresis systems (e.g., Agilent TapeStation, Bioanalyzer) to visualize genome length distribution.
Long-read sequencing platforms (e.g., PacBio, Oxford Nanopore) to analyze full-length genomes and detect rearrangements or fusions.
AAV Genome Impurity
Impurities such as plasmid backbone, helper virus sequences, or unintended DNA fragments can raise safety concerns. These are detected by:
qPCR/ddPCR assays specific for plasmid or helper sequences.
NGS to comprehensively survey off-target and contaminant sequences.
AAV capsid Analysis
AAV Capsid Titer
AAV capsid titer refers to the total number of viral particles—both full (genome-containing) and empty (genome-lacking)—in an AAV preparation. It provides a critical measure of overall particle count, regardless of whether the particles are functional or carry a transgene.
AAV capsid titer, also known as AAV concentration or physical titer, is a critical parameter directly linked to dosage, ensuring the safe and effective performance of AAV-based therapies. ELISA (Enzyme-Linked Immunosorbent Assay) is a widely adopted technique for quantifying the total capsid concentration in AAV preparations. This method utilizes specific antibodies to capture and detect AAV capsids, providing a reliable and cost-effective solution for capsid titration.
ELISA is a popular choice among researchers and manufacturers due to its high sensitivity, reproducibility, and accessibility. While it measures the total capsid concentration and does not differentiate between full (genome-containing) and empty capsids, it remains an essential tool for AAV development and quality control. For a more comprehensive analysis, ELISA can be combined with other methods, such as qPCR (to measure genome copies) or MP, CD-MS or AUC (to distinguish full and empty capsids), offering a complete picture of AAV particle composition.
Identity and Stoichiometry
AAV capsid protein composition is a critical quality attribute of AAV vectors that requires thorough characterization. A typical intact AAV capsid consists of 60 copies of viral proteins (VPs), traditionally assumed to follow a VP1:VP2:VP3 ratio of approximately 1:1:10. However, capsid assembly is inherently stochastic, and stoichiometry can vary depending on serotype and production platform. These variations in VP ratios can significantly impact vector infectivity. For example, vectors with a low VP1 and/or VP2 ratio have been linked to reduced potency, while those with reduced VP3 levels demonstrated enhanced gene transfer efficiency in vivo. Common methods for capsid protein analysis and serotype confirmation include SDS-PAGE and ELISA.
Traditional techniques such as SDS-PAGE are limited in their ability to differentiate capsid proteins from different AAV serotypes due to the minimal molecular weight differences among VP1, VP2, and VP3 proteins across serotypes. Western blotting, though widely used, is time-consuming and often suffers from antibody cross-reactivity, as AAV capsid proteins share high sequence homology. In contrast, liquid chromatography–mass spectrometry (LC-MS) offers a highly sensitive and robust alternative, capable of rapidly analyzing the intact masses of VP proteins and identifying the specific AAV serotype(s) present. Accurate measurement of all three VP proteins is essential, as a single VP mass may not be sufficient to distinguish between certain serotypes.
An alternative approach for rapid serotype identification is differential scanning fluorimetry, which leverages differences in capsid thermal stability. Each AAV serotype displays a distinct melting temperature (Tm), enabling serotype discrimination. However, this method may be less effective when serotypes exhibit similar thermostability profiles.
AAV NAbs
Another critical consideration in AAV capsid characterization is the presence of pre-existing neutralizing antibodies (NAbs) against AAVs. These antibodies, commonly found in human and non-human primate populations due to natural exposure to wild-type AAVs, can bind to the viral capsid and inhibit transduction efficiency by blocking cell entry or promoting vector clearance. NAb titers vary widely depending on the serotype and geographic population, and even low levels of NAbs can significantly reduce gene transfer efficacy, particularly for systemic delivery. Therefore, evaluating NAb responses is essential for patient eligibility screening, serotype selection, and designing effective dosing strategies. In vitro transduction inhibition assays using cell-based models remain the gold standard for measuring NAbs, offering functional insights into the impact of patient serum on AAV infectivity.
In addition to traditional cell-based transduction inhibition assays, ELISA-based methods have emerged as an increasingly popular approach for evaluating AAV NAb levels. While ELISA does not directly assess functional neutralization, it provides a high-throughput, quantitative means to detect anti-capsid antibody binding, offering valuable insight into pre-existing humoral immunity. When combined with transduction assays, ELISA can support a more comprehensive understanding of NAb profiles across different AAV serotypes.
AAV Vector Analysis
AAV Infectious Titer
AAV infectious Titer (AAV potency assay) are crucial for quantifying the functional viral particles in your AAV preparations. Functional titration measures the ability of AAVs to transduce cells and express the transgene, which is a more relevant parameter than AAV genomic titer and capsid titer when assessing viral potency.
A modified TCID50 assay is currently used to determine the infectious titer of AAV using HeLaRC32 cells. In this system, the Rep and Cap proteins expressed by HeLaRC32 cells, along with the presence of wild-type adenovirus 5 (Ad5), enable the replication of AAV genomes within the cells. Real-time PCR is then employed to sensitively and quantitatively detect positive genome replication, providing an accurate measure of infectious titer.
At AAVnerGene, we have developed AAV-Q TCID50 assay, with the following enhancements:
- HEK293RR cell line stably express Rep and AAVR are utilized to enable accurate infectious titer estimation across various AAV serotypes.
- Replication-deficient adenovirus 5 (Rd-Ad5) is used replace wild-type Ad5, minimizing complications related to Ad5 replication and reduced cell cytopathy.
AAV Full-to-Empty Ratio
The full-to-empty ratio—the proportion of AAV particles containing vector genomes (“full”) versus those lacking genomic material (“empty”)—is a critical parameter in AAV vector characterization. This ratio directly impacts vector potency, dosing accuracy, and product safety. A high proportion of empty capsids can dilute the effective dose and may trigger unwanted immune responses. Conversely, an accurate measurement of full capsids ensures consistency in therapeutic efficacy and supports regulatory compliance.
AAVnerGene offers services such as Analytical Ultracentrifugation Mass Photometry, and CD-MS for AAV analysis. Researchers looking for comprehensive and reliable AAV analysis methods can certainly reach out to our team (Customer@aavnergene.com) for support and expertise. Our wide range of technologies will undoubtedly assist researchers in ensuring the quality and integrity of their AAV vectors.
AAV Biodistribution
AAV biodistribution analysis is a critical component of preclinical and clinical gene therapy development. It assesses how AAV vectors distribute across various tissues and organs following administration, helping determine the efficiency, specificity, and safety of gene delivery.
This analysis provides insights into:
Target organ transduction (e.g., CNS, liver, muscle, or retina)
Off-target expression
Persistence and clearance kinetics
Potential systemic exposure or germline transmission risks
Common methodologies include:
qPCR or ddPCR to quantify vector genome copies in tissues.
RT-qPCR or RNA-seq to measure transgene expression.
Immunohistochemistry (IHC) and in situ hybridization (ISH) for spatial localization of capsid or transgene.
In vivo imaging (e.g., bioluminescence or PET) for real-time tracking.
By understanding AAV biodistribution, researchers can optimize vector design, select appropriate routes of administration, ensure safety, and support regulatory filings for investigational gene therapies.
AAV biosafety
Regular Biosafety Tests
There are several other quality control (QC) tests commonly performed to assess the purity, safety, and efficacy of AAV preparations. Some of these tests include:
- Endotoxin testing: Endotoxins are bacterial lipopolysaccharides that can be present in AAV preparations if the production process involves bacterial contamination. Endotoxin testing is performed using methods such as Limulus amebocyte lysate (LAL) assay to detect and quantify the levels of endotoxins in the AAV sample.
- Mycoplasma testing: Mycoplasma contamination can occur during the production process and can affect the quality and safety of AAV preparations. Mycoplasma testing involves methods such as PCR-based assays or culture-based methods to detect the presence of mycoplasma contamination in the AAV sample.
- Bioburden testing: Bioburden testing is performed to assess the level of microbial contamination in the AAV sample. It involves methods such as microbial enumeration or total viable count to quantify the number of viable microorganisms present in the AAV preparation.
- Sterility testing: Sterility testing is conducted to confirm the absence of viable microorganisms in the AAV product. It involves culturing the AAV sample under appropriate conditions to detect any microbial growth.
These tests are important for ensuring the safety and quality of AAV preparations for use in gene therapy applications. Performing comprehensive QC testing helps to identify any potential contaminants or impurities that may impact the efficacy and safety of the AAV product.
rcAAV Tests
AAV continues to lead the field of in vivo gene delivery, vector safety and regulatory compliance have never been more critical. Among the key concerns during AAV vector production is the potential generation of replication-competent AAV (rcAAV) — a rare but significant contaminant that can compromise therapeutic safety, product quality, and clinical trial outcomes. The Food and Drug Administration (FDA) requires that AAV products be screened for rcAAVs to ensure the safety of gene therapy products.
At AAVnerGene, we take a proactive approach, to minimizing and detecting rcAAV to ensure that every AAV product meets the highest safety standards. We have successfully developed the AAV-Q rcAAV assay with the following enhancements:
- HEK293R cell line stably expressing AAVR is utilized to accurate rcAAV titration across various AAV serotypes.
- Replication-deficient adenovirus 5 (Rd-Ad5) is used replace wild-type Ad5, minimizing complications related to Ad5 replication and reduced cell cytopathy.