AAV Plasmids
What are AAV Plasmids?
The AAV (Adeno-Associated Virus), a minute virus prevalent in biomanufacturing, is favored for its limited replication and reduced human immunogenicity. Producing high-caliber viral vectors for gene transfer demands an intricate biomanufacturing process, encompassing plasmid DNA manufacturing and other intricate steps.
AAV Plasmid Production Workflow
- Cell Culture: AAV DNA vector construction and plasmid expansion. Preparation and amplification of plasmids encoding all the factors necessary to generate recombinant AAV particles.
- Adapting culture conditions: Optimize suspension or adherent culture systems, including 3D or 2D formats, depending on process requirements.
- AAV Vector production: Introduce AAV vector DNA into cells transiently or establish stable cell lines for 2-3 days of replication and expression.
- AAV Recovery: Filter and centrifuge the cell culture medium to remove debris, concentrating AAV vectors. Tangential flow filtration (TFF) can be used for further purification. Capture and recovery of AAV particles from cell culture media or lysate follow standard techniques used for recombinant proteins including precipitation, centrifugation, chromatography, and tangential flow filtration.
- AAV Purification: Use ultracentrifugation and chromatography, such as affinity or ion exchange, to achieve high-purity AAV vector preparations. Maintain quality control throughout the process.
- AAV Titration: AAV vector quantitative and qualitative analysis to identify the highest titer can be performed following standard techniques including real-time PCR.
- Transduction of mammalian cells: The purified AAV particles can be used for transduction of target mammalian cell lines, including IPSCs, 3D organoids and CAR-T cell therapy.
- Clone Screening & Selection: Clonal screening is required to identify successfully transduced clones. High-value clone selection and pool enrichment can be performed following high-throughput automated methods
- Resolving heterogeneity & Single Cell isolation: Clonal pool heterogeneity is resolved by single-cell isolation techniques together with an image-based analysis, ensuring proof of monoclonality of the established AAV-transduced cell line.
- Expansion & Characterization: Downstream characterization of the target cell line can be performed by high-content imaging automated solutions and ELISA assay.
Innovative solutions to optimize your AAV plasmid production workflow
At Molecular Devices, we understand the intricacies of AAV Plasmid production. Our suite of advanced technological solutions is meticulously designed to support and optimize each phase of your workflow. With a focus on precision, efficiency, and scalability, we are dedicated to driving excellence in every step of your AAV Plasmid production process, ensuring your enterprise achieves industry-leading results.
QPix™ Microbial Colony Pickers
Application in AAV Workflow:
- Used in the AAV DNA Vector Construction and Plasmid Expansion phase.
- Efficiently picks colonies containing the desired plasmid constructs.
- Can be used to screen for colonies with specific phenotypes, ensuring the right plasmid construct is selected.
Features:
- Picks up to 3,000 colonies per hour.
- Supports a variety of microorganisms and selection modalities.
- Ensures sterility with UV light sanitization and pin washing.
- Offers multiple imaging modes for precise colony selection.
- Provides automated plating and streaking capabilities.
- Scalable automation options for varying throughput needs.
ClonePix™ Mammalian Colony Picker
Application in AAV Workflow:
- Used in the Clone Screening & Selection phase.
- Screens and selects high-value clones based on protein productivity and other attributes.
Features:
- Automated system for selecting high-value clones.
- Increases the probability of finding optimal cell lines.
- Offers multiple detection methods including fluorescence.
- Maintains sterility with UV light and pin washing.
- Uses semi-solid media for discrete colony formation.
CloneSelect™ Imager FL
Application in AAV Workflow:
- Used in the Expansion & Characterization phase.
- Provides imaging and analysis of mammalian cells, ensuring monoclonality.
- Can be used to verify gene edits.
Features:
- Offers both white light and fluorescent imaging.
- Generates automatic confluence analysis and monoclonality assurance.
- Rapid acquisition times with the ability to image a 96-well plate in under two minutes.
- Compatible with various cell types including CHO, HEK, and iPSCs.
- Provides high-resolution images and intelligent analysis.
DispenCell™ Single-Cell Dispenser
Application in AAV Workflow:
- Used in the Resolving heterogeneity & Single Cell isolation phase.
- Dispenses single cells ensuring monoclonality of the established AAV-transduced cell line.
Features:
- Provides immediate and traceable proof of clonality.
- Gentle handling preserves cell viability.
- Compact design fits in various laboratory settings.
- Uses a disposable tip to prevent cross-contamination.
- Intuitive interface with no need for calibration.