The secret sauce of AAV analytics: success starts with better sample prep
At NanoMosaic, we have developed a fast, precise AAV sample preparation method designed specifically for quantifying full-length AAV genomes. Paired with the Tessie™ nanoneedle detection platform, it gives teams the clarity they need to optimize yield, validate product quality, and drive down manufacturing costs.
- Nuclease treatment to remove unpackaged nucleic acids
- Proteinase K for removal of nucleases and assistance in capsid opening
- Restriction digestion for removal of AAV ITRs (Inverted Terminal Repeats)
- NanoMosaic probe incorporation
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Step 1: Remove free DNA for accurate AAV genome titration
During the upstream manufacturing stage of AAV production, leftover DNA from plasmids and the host cell can inflate your titers. Benzonase is commonly used to remove this unpackaged DNA, but it is hard to deactivate and may persist through heat inactivation or metal chelation.
Our solution is to follow this Benzonase step with Proteinase K digestion to inactivate the Benzonase and assist in lysis of the viral capsids. This ensures the only DNA left for measurement came from previously intact capsids.
Step2: Protect full-length viral genomes during heatinactivation of enzymes
Deactivating Proteinase K usually requires heating to 95°C for at least 5 minutes. However, this can break the full-length AAV genomes if buffer conditions aren’t optimized, leading to an underestimation of true genome titers.
NanoMosaic’s proprietary buffers stabilize genomic AAV DNA during heat exposure, maintaining integrity and ensuring accurate quantification of full-length AAV vectors during inactivation.
Step 3: Eliminate AAV ITRs to avoid self-priming
AAV ITRs(inverted terminal repeats) are essential for replication and packaging into capsids but problematic for PCR analysis. Their hairpin structure enables self-priming, creating misleading signals and preventing accurate quantification.
Our workflow incorporates restriction digestion to remove the 3’-self primed ITR structure to allow accurate quantification of the full-length viral genome content.
Better sample prep means better process decisions
When you know exactly how many full-length genomes you have, everything improves. You can compare upstream production runs with confidence, evaluate purification steps like TFF or chromatography, and ensure dose consistency, backing everything up with reliable, regulatory-grade data.
NanoMosaic’s Tessie™ platform uses nanoneedle-based sensors to directly detect full-length AAV genomes - no fluorescent labels or cleanup steps necessary. It works with crude lysates and scales effortlessly for high-throughput workflows. Paired with our optimized sample prep workflow, it delivers accurate, reliable data at every step of the manufacturing process, helping you improve yield and validate vector quality.
For accurate AAV quantification, sample preparation matters!
Explore More
- Download our white paper on AAV genome quantification
- View our webinar “Redefining AAV Quality Control in Cell and Gene Therapy Development & Manufacturing”
The secret sauce of AAV analytics: success starts with better sample prep
At NanoMosaic, we have developed a fast, precise AAV sample preparation method designed specifically for quantifying full-length AAV genomes. Paired with the Tessie™ nanoneedle detection platform, it gives teams the clarity they need to optimize yield, validate product quality, and drive down manufacturing costs.
- Nuclease treatment to remove unpackaged nucleic acids
- Proteinase K for removal of nucleases and assistance in capsid opening
- Restriction digestion for removal of AAV ITRs (Inverted Terminal Repeats)
- NanoMosaic probe incorporation
Step 1: Remove free DNA for accurate AAV genome titration
During the upstream manufacturing stage of AAV production, leftover DNA from plasmids and the host cell can inflate your titers. Benzonase is commonly used to remove this unpackaged DNA, but it is hard to deactivate and may persist through heat inactivation or metal chelation.
Our solution is to follow this Benzonase step with Proteinase K digestion to inactivate the Benzonase and assist in lysis of the viral capsids. This ensures the only DNA left for measurement came from previously intact capsids.
Step2: Protect full-length viral genomes during heatinactivation of enzymes
Deactivating Proteinase K usually requires heating to 95°C for at least 5 minutes. However, this can break the full-length AAV genomes if buffer conditions aren’t optimized, leading to an underestimation of true genome titers.
NanoMosaic’s proprietary buffers stabilize genomic AAV DNA during heat exposure, maintaining integrity and ensuring accurate quantification of full-length AAV vectors during inactivation.
Step 3: Eliminate AAV ITRs to avoid self-priming
AAV ITRs(inverted terminal repeats) are essential for replication and packaging into capsids but problematic for PCR analysis. Their hairpin structure enables self-priming, creating misleading signals and preventing accurate quantification.
Our workflow incorporates restriction digestion to remove the 3’-self primed ITR structure to allow accurate quantification of the full-length viral genome content.
Better sample prep means better process decisions
When you know exactly how many full-length genomes you have, everything improves. You can compare upstream production runs with confidence, evaluate purification steps like TFF or chromatography, and ensure dose consistency, backing everything up with reliable, regulatory-grade data.
NanoMosaic’s Tessie™ platform uses nanoneedle-based sensors to directly detect full-length AAV genomes - no fluorescent labels or cleanup steps necessary. It works with crude lysates and scales effortlessly for high-throughput workflows. Paired with our optimized sample prep workflow, it delivers accurate, reliable data at every step of the manufacturing process, helping you improve yield and validate vector quality.
For accurate AAV quantification, sample preparation matters!
Explore More
- Download our white paper on AAV genome quantification
- View our webinar “Redefining AAV Quality Control in Cell and Gene Therapy Development & Manufacturing”