Benefiting from a strong academic and industry background in AAV gene therapy, the AAVnerGene team is adept at establishing a dependable platform for AAV characterization and analysis. Our offerings go beyond standard quality control testing, encompassing advanced evaluations like vector genome identity, titration, purity analysis, toxicity testing, scrutiny of endotoxin or mycoplasma levels, assessment of empty/full ratios, and a diverse range of comprehensive analyses. These services cater to the needs of our esteemed clientele.
AAV Genome Quantification
AAV genome quantification is a critical step in gene therapy research and production, as it helps determine the concentration of viral genomes present in a sample. This quantification is essential for ensuring accurate dosing of AAV vectors in experimental and clinical applications. There are several methods commonly used to quantify AAV genomes:
Quantitative PCR (qPCR): qPCR is a widely used method for AAV genome quantification. It involves the amplification of a specific region of the AAV genome using AAV-specific primers. The amount of amplified DNA is measured in real-time, allowing for the quantification of the AAV genome copies in the sample.
Digital PCR (dPCR): dPCR is a precise and absolute quantification method that partitions the sample into thousands of individual reactions. It counts the number of positive reactions and determines the absolute amount of AAV genome copies in the sample. dPCR is particularly useful for low-level AAV genome detection and quantification.
Droplet Digital PCR (ddPCR): ddPCR is a variation of dPCR that partitions the sample into thousands of droplets. Each droplet acts as a separate reaction chamber, allowing for the absolute quantification of AAV genome copies. ddPCR offers high sensitivity and precision in AAV genome quantification.
Next-Generation Sequencing (NGS): NGS can also be used to quantify the AAV genome. It involves the sequencing of the AAV genome within the sample and counting the number of sequencing reads that align to the AAV genome. By comparing the read counts to a standard curve or reference sample, the AAV genome copies can be quantified.
DNA microarray: DNA microarrays can be designed to specifically detect and quantify AAV genomes. The sample DNA is hybridized to the microarray, and the signal intensity of the bound DNA probes is used to estimate the amount of AAV genome present in the sample.
Spectrophotometry: This method involves measuring the absorbance of UV light by AAV DNA at specific wavelengths. The concentration of AAV DNA in the sample can be determined based on its absorbance. However, this method might not be as accurate for samples with contaminants or impurities.
Fluorescence-Based Assays: Some fluorescence-based assays, such as PicoGreen or SYBR Green, use fluorescent dyes that specifically bind to DNA. By measuring the fluorescence signal, the amount of DNA in the sample can be estimated, which indirectly reflects the amount of AAV genomes.
Electron Microscopy: In some cases, especially during process development or quality control, electron microscopy can be used to directly visualize and count individual AAV particles in a sample. This method provides information about particle concentration, but it doesn’t distinguish between empty and full capsids.
AAV Genome Integrity Analysis
Denaturing agarose-gel electrophoresis, southern blot analysis, CE-LIF, and PacBio sequencing, are commonly used for AAV genome integrity analysis. Each method offers its own advantages and considerations, allowing researchers to choose the most suitable technique based on their specific requirements. These methods play a crucial role in assessing the integrity and structural variations of the AAV genome, which is important for ensuring the quality and functionality of AAV vectors used in gene therapy and other applications.
1. Denaturing agarose-gel electrophoresis is a commonly used method for AAV genome integrity analysis. This technique involves denaturing the AAV DNA using heat or chemicals and separating the DNA fragments on an agarose gel based on their size. By comparing the migration patterns of the AAV DNA samples to a molecular weight marker, structural changes or alterations in the AAV genome can be identified
2. Southern blot analysis is another technique used for AAV genome integrity analysis. It involves digesting the AAV DNA with restriction enzymes, separating the DNA fragments by gel electrophoresis, transferring them onto a membrane, and hybridizing the membrane with specific DNA probes that target regions of interest within the AAV genome. This allows for the detection of specific DNA sequences and provides information about the size and integrity of the AAV genome fragments.
3. CE-LIF (Capillary Electrophoresis with Laser-Induced Fluorescence) is a high-resolution analytical technique used for AAV genome integrity analysis. It involves the separation of AAV DNA fragments in a capillary electrophoresis system and their detection using laser-induced fluorescence. By labeling the AAV DNA fragments with fluorescent dyes, their migration patterns can be analyzed, and structural variations or alterations in the AAV genome can be detected with high sensitivity and accuracy.
4. NGS sequencing, is a high-throughput sequencing method that allows for the comprehensive analysis of the AAV genome structure and integrity. It provides long-read sequencing data, which enables the detection of structural variations, such as insertions, deletions, and rearrangements, and the accurate resolution of repeat regions within the AAV genome.
Characterizing AAV genome is a crucial step in gene therapy and related research, and having access to expert advice and services can greatly benefit researchers in this field. If readers have specific inquiries or requests related to the characterization of AAV genome, we encourage you to reach out to our team(Customer@aavnergene.com) for more personalized guidance and support.
Methods to characterization AAV Capsid
There are several methods available for the characterization of AAV capsids. Here are some commonly used techniques:
- Electron Microscopy (EM): EM allows for the direct visualization of AAV capsids at high resolution. It provides information about the size, shape, and morphology of the capsids. Cryo-electron microscopy (cryo-EM) is particularly useful as it allows for the visualization of AAV capsids in their native state.
- Dynamic Light Scattering (DLS): DLS measures the size distribution of particles in solution. It can be used to determine the hydrodynamic diameter of AAV capsids, which provides information about their size and stability.
- Atomic Force Microscopy (AFM): AFM is a high-resolution imaging technique that can be used to study the topography and structural details of AAV capsids. It can provide information about the surface features, such as spikes or pores, present on the capsids.
- Gel Electrophoresis: Gel electrophoresis techniques, such as native-PAGE (polyacrylamide gel electrophoresis) and SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), can be used to analyze the protein composition and purity of AAV capsids. These techniques separate the capsid proteins based on their size and charge.
- Mass Spectrometry (MS): MS is a powerful technique for the identification and quantification of proteins in AAV capsids. It can provide information about the capsid protein composition, post-translational modifications, and potential impurities.
- Western blotting: Western blotting allows for the detection and quantification of specific capsid proteins using specific antibodies. It can be used to assess the expression levels and integrity of capsid proteins.
- Dynamic Light Scattering (DLS): DLS measures the size distribution of AAV particles in solution, providing information about particle size and aggregation state.
- Zeta Potential Measurement: Zeta potential is a measure of the surface charge of particles, including AAV capsids. It can provide insights into capsid stability and interactions in solution.
- Sedimentation Velocity Analysis: This technique involves ultracentrifugation to separate particles based on their sedimentation rates. It can provide information about capsid size and shape.
- Size Exclusion Chromatography (SEC): SEC separates particles based on size. It can help assess the purity and aggregation state of AAV capsids.
- ELISA (Enzyme-Linked Immunosorbent Assay): ELISA is a widely used technique for quantifying the total capsid concentration in AAV preparations. It uses specific antibodies to capture and detect the capsids.
- Thermal Stability Analysis: Differential scanning calorimetry (DSC) or thermal shift assays can assess capsid stability under different temperature conditions.
Characterizing AAV capsids is a crucial step in gene therapy and related research, and having access to expert advice and services can greatly benefit researchers in this field. If readers have specific inquiries or requests related to the characterization of AAV capsids, we encourage you to reach out to our team(Customer@aavnergene.com) for more personalized guidance and support.
Analyzing the ratio of empty to full AAV particles, also known as the empty/full ratio, is an important aspect of AAV vector characterization. The ratio can impact the efficacy and safety of gene therapy applications.
Here are some common methods used for empty/full ratio analysis:
- Electron Microscopy (EM): Transmission electron microscopy (TEM) or cryo-electron microscopy (cryo-EM) can directly visualize and distinguish empty and full AAV particles based on their internal contents. This method provides qualitative information about the ratio.
- Gradient Centrifugation: Density gradient centrifugation using cesium chloride (CsCl) or iodixanol can separate AAV particles based on their density. Empty and full capsids have different densities, allowing for quantification of their ratio.
- Cryo-Electron Tomography (Cryo-ET): Cryo-ET can provide three-dimensional reconstructions of individual AAV particles, allowing for visualization of the genome within the capsid and determination of their full or empty status.
- Analytical Ultracentrifugation(AUC): Analytical ultracentrifugation can separate particles based on their sedimentation rate. Empty and full AAV particles might sediment differently, allowing for ratio determination.
- SDS-PAGE: This technique can be used to quantify the presence of capsid proteins, helping to estimate the number of AAV particles and differentiate between full and empty ones.
- Differential Scanning Calorimetry (DSC): DSC measures the heat absorbed or released during thermal denaturation of AAV particles. Differences in thermal stability between empty and full particles can help determine their ratio.
- Analytical Size Exclusion Chromatography (SEC): SEC can separate AAV particles based on their size and structure. Differences in elution profiles can provide insights into the empty/full ratio.
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.
|Genome Titration||ddPCR/qPCR||3-5 day||$200/sample||*Primer/probe sequences; ~20μl of samples|
|Capsid Titration||ELISA||3-5 day||$400/sample||*Serotypes and buffers; ~20μl of samples|
|Purity Analysis||SDS-PAGE||3-5 day||$200/sample||~20μl of sample with titer >1E+12vg/ml|
|Full/Empty Ratio||AUC||3-5 day||$2000/sample||~400μl of sample with titer >1E+12vg/ml|
|Endotoxin||LAL assay||3-5 day||$200/sample||~20μl of samples|
|Endotoxin Removal||Resin-PMB||3-5 day||$200/sample||~20μl of samples|
|Bioburden Testing||Direct plating||3-5 day||$200/sample||~20μl of samples|
|Mycoplasma Testing||PCR||3-5 day||$200/sample||~20μl of samples|
|Vector Genome Identity||PacBio Seq||4 weeks||Request||~100μl of sample with titer >1E+12vg/ml|
* it is better if customers can provide the range of titers.
If customers have specific inquiries or requests related to the characterization of AAV genome, we encourage you to reach out to our team(Customer@aavnergene.com) for more personalized guidance and support.