AAV Packaging Service
The challenges associated with generating, controlling, and qualifying AAV vectors are widely acknowledged in the field. Variability in production methods, purification processes, and capsid properties can significantly impact vector performance. For researchers, particularly those new to working with AAVs, accessing a reliable and cost-effective source of high-quality vectors is essential to ensure consistent and dependable tools for their experiments. At AAVnerGene, we prioritize maintaining the highest standards in AAV production and implementing rigorous quality control measures. This commitment ensures that the AAV vectors we deliver are not only reliable but also thoroughly characterized, enabling researchers to focus on their scientific goals without the added burden of vector-related uncertainties.
Short Turnaround
2 weeks for ≤ 5E13GC
Guaranteed Titer
≥1E13GC/mL (qPCR-SYBR)
High Full Particle
≥80% by Mass Photometry
Good Quality
≥95% Purity
Large Capacity
up to 1E17 GC
Multiple Systems
AAVone/AAVdual/AAVtri
Abundant Serotypes
>1000 AAV Capsids
Extensive Experience
>5,000 delivered AAV vectors
Experienced Support
>20 Years AAV Experts
AAV Production Scale, Price and Turnaround
We will package your AAV vector into any serotypes or capsids, followed by ultracentrifugation (iodixanol gradient (one round) or CsCl gradient (two rounds) or AAV-X affinity purification (for production scale ≥1e13 vg). We strongly advise against using unpurified AAVs for any experiments, as the presence of empty AAV particles and residual cellular proteins can significantly impact titration accuracy and transduction efficiency.
Scale | Titer | Volume | Price | Turnaround |
1e12 GC | >1e13 GC/ML | 1 X100 µl | $1,200 | 2 weeks |
2e12 GC | >1e13 GC/ML | 2 X100 µl | $1,400 | 2 weeks |
5e12 GC | >1e13 GC/ML | 2 X 250 µl | $1,600 | 2 weeks |
1e13 GC | >1e13 GC/ML | 2 X 500 µl | $2,400 | 2 weeks |
2e13 GC | >1e13 GC/ML | 2 X 1,000 µl | $4,200 | 2 weeks |
5e13 GC | >1e13 GC/ML | 5 X 1,000 µl | $7,800 | 2-3 weeks |
1e14 GC | >1e13 GC/ML | 10 X 1,000 µl | $11,800 | 2-3 weeks |
2e14 GC | >1e13 GC/ML | 4 X 5,000 µl | $17,800 | 2-3 weeks |
5e14 GC | >1e13 GC/ML | 10 X 5,000 µl | $42,800 | 3-4 weeks |
1e15 GC | >1e13 GC/ML | 20 X 5,000 µl | $79,800 | 3-4 weeks |
>1e15 GC | TBD | TBD | TBD | TBD |
Notes
- Service Pricing: All prices listed are for AAV packaging services only. Customers must provide sufficient amounts of transgene plasmids (0.5 mg for 1e13 GC). We also offer Midiprep or Maxiprep services at an additional cost (see our Gene Synthesis and Cloning services in AAV vector design page).
- Turnaround Time. The stated turnaround times apply solely to standard AAV production and quality control. This timeframe is calculated from the receipt of the plasmid to the shipment of the AAVs. Additional services, such as DNA preparation or extra QC requests, may extend the timeline. We encourage customers to communicate with us to ensure our services align with your project requirements.
- GC (Genome Copy): The titer is measured using gene-specific primers via the qPCR-SYBR method. Titers are based on typical production yields for common AAV serotypes and transfer vectors. Gene-specific titer via the qPCR-TaqMan method and ddPCR method are available upon request. While we typically deliver high titers, in rare cases—due to certain serotypes or transfer vectors—the expected titer may not be reached. In such instances, we reserve the right to discuss options with customers or cancel the order.
- Higher Titers (>2e13 GC/ml): AAV vectors with high titers can be achieved upon request. However, aggregation may occur for certain AAV serotypes, such as AAV2, which can impact titer measurements and overall vector performance. We recommend discussing your specific needs with our team to optimize production and ensure the best possible outcomes for your experiments.
- Special Capsids: For custom capsids, we cannot guarantee titers exceeding 1e13 GC/ml, and will deliver AAVs as produced after one time repeat. Additional costs may be charged based on the yields to achieve 1e13GC/ml.
- Transgene Size: If the transgene size exceeds 4.7 kb, an additional service fee ranging from 50% to 100% may apply, depending on the yield and complexity of the vector production process.
- Toxic Genes: Certain genes can be toxic to cells, which may result in extremely low yields during AAV production. We’ll discuss with customer how to process the production.
- Purification Methods: Our standard purification method is iodixanol, but we can also use CsCl (two rounds) or AAV-X resin (for production scale ≥1e13 GC) if preferred by the customer. We strongly advise against using unpurified AAVs for any experiments, as the presence of empty AAV particles and residual cellular proteins can significantly impact titration accuracy and transduction efficiency.
- Buffer System: Our standard buffer is 0.001% F-68/DPBS with 150 mM NaCl. If you have a preferred buffer system, please inform us.
- Aliquot Services: AAV vectors are relatively stable compared to other viral vectors, making them a reliable choice for gene delivery. However, proper handling and storage are still essential to maintain their potency and effectiveness over time. To preserve the quality and effectiveness of your AAV vectors, we offer aliquot services. Please contact us before placing your order for AAV packaging to discuss your aliquot needs and ensure optimal handling and storage of your vectors.
Checklist
Before proceeding with AAV packaging, please thoroughly review your AAV vector design and experimental plan to ensure accuracy and optimal results.
- ITR integrity: Confirm that your transgene plasmids have two functional ITRs by performing digestion with SmaI/AhdI or by using AAV-ITR sequencing or whole plasmid sequencing. ITR130 or ITR119 is able to package AAVs.
- AAV genome size: Confirm your AAV vectors size between two ITRS (including ITRs) is less than 4.7kb. AAV size between 4.7-5.2kb are still packageable, but have low yield and high partial genomic containing particles. AAV genome size less than 2.5 would package both monomer and dimmer genomes. scAAV can only package a half size of regular AAV genome, which is around 2.5kb in total.
- Amount of AAV vectors: Plan according you experiment design and produce at least 2X AAV vectors. Usually, our titer is measured using gene specific primers with SYBR method. Titer with ddPCR method may be lower than SYBR method.
- AAV Serotypes and variants: Select the AAV variants you want to use in your experiments. If you are unsure about which AAV variant to use for your experiments, please consult with us.
- Control AAV vectors: Control AAV vectors play a crucial role in experimental design and data interpretation. Control vectors are used as references to assess the effects of the experimental AAV vectors and to distinguish between specific effects and background noise. They provide a baseline for comparison and help ensure the accuracy and reliability of experimental results.
- Empty AAV vectors: Do you need the byproduct-empty AAV or truly-empty AAV vectors without any genetic materials as control?
- Purification methods: Our regular purification method is one round of Iodixanol. We can also use two rounds of CsCl ultracentrifuge and AAVx resin to purify the AAV vectors if customer prefers.
- Buffer: Our buffer is 0.001% F-68/DPBS with additional 150mM NaCl. Please let us know if you have your own buffer system.
- Additional Tests: Check whether you need additional tests besides titers, such as genome integrity, purity, endotoxin, sterility, mycoplasma, AAV capsid titer, empty/full ratio, plasmid related impurities, etc.
Quality control (QC) tests are crucial for ensuring the consistency, safety, and efficacy of AAV vectors used in experiments and therapeutic applications. Comprehensive QC testing ensures that the AAV vectors meet stringent regulatory standards and perform as expected in both preclinical and clinical settings. Here’s an overview of the key QC tests performed for AAV vectors provided by AAVnerGene.
Standard QC
For each AAV produced by AAVnerGene, the standard QC is genomic titer measured by gene-specific primers with qPCR-SYBR method and full/empty ratio qualified by mass photometry. To include the full/empty ratio results in your COA, an extra cost is required.
Additional QC
Additional QC services listed above can be provided upon request. More information can be found in AAV Characterization page.
| QC | Method | Turnaround | Price | Sample Requirement |
| Genome Titration | qPCR | 1-2 day | $50/sample (Free in AAV Packaging Services) | *Primer/probe sequences; ~20 μl of samples |
| Genome Titration | ddPCR | 3-5 day | $200/sample | *Primer/probe sequences; ~20 μl of samples |
| Capsid Titration | ELISA | 3-5 day | $400/sample | *Serotypes and buffers; ~20 μl of samples |
| Purity Analysis | SDS-PAGE | 3-5 day | $200/sample | ~20μl of sample with titer >1E+12 vg/ml |
| Full/Empty Ratio | AUC | 3-5 day | $2,000/sample | ~400μl of sample with titer >1E+12 vg/ml |
| Full/Empty Ratio | Mass Photometry | 1-2 day | $800/sample | 20μl of sample with titer >5+11 vg/ml |
| 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 |
| Vector Genome Identity | TapeStation | 1-2 day | $200/sample | ~100 μl of sample with titer >1E+12vg/ml |
| Vector Genome Identity | PacBio Seq | 4 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml |
| Vector Genome Identity | CD-MS | 4 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml |
| DNA Impurity | qPCR/ddPCR | 3-5 day | 200/Target | ~20 μl of samples |
| Library Complexity | NGS | 1-2 weeks | Request | ~20 μl of samples |
| Barcode Analysis | NGS | 1-2 weeks | Request | fresh or frozen tissues/cells |
| AAV Cellular Tropism | scRNA-AAVseq | 3-4 weeks | Request | Fresh tissues/cells |
AAVnerGene offers a variety of AAV production systems (AAVone, AAVdual, and AAVtri) for AAV production, catering to different experimental needs and preferences. Each of these systems likely has its own advantages and considerations, providing researchers with options to choose the most suitable method for their specific gene delivery experiments.
| Production System | Plasmid Name | Plasmid Size(kb) | Number of plasmids | Package efficiency |
| AAVone System | pAAVone | 14.0-18.0 | 1 | 200%-400% |
| AAVdual System | pAAVdual | 9.4-13.4 | 2 | 100%~250% |
| pRCap | 7.5 | |||
| AAVtri System | mini-pHelper-1.0 | 8.4 | 3 | 100%~250% |
| pRCap | 7.5 | |||
| pAAVtri | 4.0~8.0 | |||
| Regular tri-plasmid System | pHelper | 11.6 | 3 | 100% |
| pRCap | 7.5 | |||
| pAAVtri | 4.0~8.0 |
AAVone Single-Plasmid System: In this system, all the Ad helper genes (E2A, E4orf6 and VA RNA), AAV helper genes (Rep and Cap), and AAV vector genome are assembled into one plasmid and AAV vectors are simply generated by transfection one pAAVone plasmid into host cells. AAVone is designed to streamline AAV production process, increase AAV yield, improve product consistency and reduce the cost and labor, especially for GMP grade AAV production.
AAVdual Two-Plasmid System: The AAVdual system combine the mini-pHelper and pGOI in one pAAVdual plasmid, and remain the pRCap alone. This system offers a flexible and efficient approach to generate AAV vectors, as users only need put the GOI into our pAAVdual backbone and package AAV with different serotypes.
AAVtri Three-Plasmid System: Three plasmid co-transfection approach is a well-established method for AAV vector production. It typically involves using three separate plasmids: one containing the gene of interest (pAAVtri-GOI), one with the AAV rep and cap genes (pRCap) required for packaging, and a third with the adenoviral helper (pHelper) genes to facilitate AAV replication and packaging. This system streamlines the traditional triple plasmid approach by using our mini-pHelper plasmid. The “mini-pHelper” refers to a modified smaller version (8.4kb) of the adenoviral helper genes that’s more efficient and easier to work with.
AAVnerGene offers a range of cell lines for AAV vector production, both adherent and suspension, which allows researchers to choose the most suitable system for their production needs. Here’s a summary of the cell lines you provide:
Adherent Cells:
HEK 293 Cells: These are a widely used cell line that originated from human embryonic kidney cells. They are commonly used for AAV vector production due to their ability to express the adenoviral gene E1, which aids in the packaging of AAV vectors.
HEK 293T Cells: HEK 293T cells are a derivative of HEK 293 cells that express the SV40 T antigen. This results in increased cell cycling and higher plasmid amplification upon transfection, leading to improved transfection efficiency and higher vector production.
Suspension Cells:
- HEK 293 Cells (Suspension): These are suspension-adapted HEK 293 cells derived from adherent HEK 293 cells. Suspension culture systems offer advantages for large-scale production due to ease of scaling and better mixing compared to adherent cultures. HEK 293 cells in suspension culture have become a widely used platform for the production of GMP (Good Manufacturing Practice) grade AAV vectors.
- HEK293one(Suspension): Our proprietary high-productivity HEK293 subclone optimized for suspension culture, designed to maximize AAV vector yield and efficiency.
- HEK 293T Cells (Suspension): These are suspension-adapted HEK 293T cells derived from adherent HEK 293T cells. The benefits of HEK 293T cells, including their high transfection efficiency, are likely retained in the suspension-adapted version.
By offering both adherent and suspension cell lines, AAVnerGene addresses the diverse requirements of researchers who may have different preferences and needs for AAV vector production. Adherent cells are often suitable for smaller-scale experiments or when adherent culture methods are preferred, while suspension cells are advantageous for larger-scale production intended for research or potential clinical applications.
AAVone Productivity in Suspension HEK 293 Cells
By combination suspension HEK 293 with our AAVone system, researchers are able to get >1e12 VG/ml crude titers for most AAV serotypes. For AAV9, you may be able to get >5e12 VG/ml crude titers.
AAVnerGene provides iodixanol-based density gradients, cesium chloride (CsCl) based density gradients and AAVx resin methods for AAV purification. The purification of AAV vectors is a critical step in their production for both research and clinical applications. Different methods are employed to achieve high-quality and pure AAV vector preparations.
- Iodixanol Gradients: Iodixanol is used to create a density gradient similar to CsCl gradients. The gradient composition and steps are carefully designed to separate contaminants from impure AAV preparations. Different iodixanol concentrations help separate empty capsids and contaminants from genome-containing virions. The 15% iodixanol step has 1M NaCl to destabilize ionic interactions between macromolecules. The 40% and 25% steps are used to remove contaminants with lower densities, including empty capsids. The 60% step serves as a cushion for genome-containing virions. Phenol red is often added to visualize the distinct steps in the gradient. One of the limitations of using iodixanol gradients for AAV purification is the inability to visually observe distinct bands in the gradient as you would with CsCl gradients. The lack of visible bands can make it more challenging to precisely identify and collect specific fractions during the purification process.
At AAVnerGene, we measure each fragment with mass photometry and collect the AAVs only from those with high full particle ratio.
CsCl Gradients: CsCl gradients have been a conventional method for purifying AAV vectors. This technique relies on the differences in density among AAV particles, empty capsids, and contaminants. By subjecting the AAV mixture to ultracentrifugation in a CsCl density gradient, the components separate into distinct bands based on their density. This visual distinction of distinct bands aids in identifying and collecting the desired AAV particles and separating them from contaminants.
Affinity Chromatography with AAVx Resin (for production scale ≥ 1e13 vg; for scale < 1e13 vg, additional 50% fee will be applied) : AAVx resin is a purification technology using affinity chromatography that exploits the binding reactivity of certain AAV serotypes to the resin. This resin facilitates one-step purification of AAV vectors from crude material. It offers several advantages:
- High Purity and Yield: AAVx resin allows for efficient purification, resulting in high purity and yield of AAV vectors.
- High Specificity and Capacity: It exhibits high affinity and specificity for a broad range of AAV serotypes, including AAV1, AAV2, AAV5, AAV8, and AAV9.
- Scalability: AAVx resin can be scaled up easily, making it suitable for both research and production applications.
- Non-Animal Derived: The resin being non-animal derived is significant, especially for processes intended for clinical applications.
Choosing the right purification method depends on various factors, including the specific AAV serotype, the desired purity level, the intended application (research or clinical), and the available resources. The CsCl and iodixanol gradient methods are traditional but effective approaches, while the AAVx resin offers the advantages of simplicity and scalability. Overall, the purification methods you’ve described highlight the ongoing efforts in the field of AAV vector production to optimize purity, yield, and safety while streamlining the process for various applications.
| Method | Description | Advantages | Disadvantages |
|---|---|---|---|
| CsCl Gradient Centrifugation | Uses cesium chloride gradients to separate AAV particles based on density. | High purity and effective removal of empty capsids. | Time-consuming, labor-intensive, and requires ultracentrifugation. |
| Iodixanol Gradient Centrifugation | Separates AAV particles using iodixanol density gradients. | Less toxic than CsCl, preserves particle integrity. | Requires ultracentrifugation, moderate scalability. |
| Affinity Chromatography | Uses specific ligands or antibodies to selectively bind AAV particles. | High specificity and purity, efficient. | High cost of ligands/antibodies, potential for low binding capacity. |
There are hundreds of AAV capsids published, including well known AAV serotypes (AAV1-13), engineered capsids and other native capsids isolated from human, monkey or other species. These versatile AAV serotypes provide us different tropism for specific cells in vitro and in vivo. For your specific purpose, the researcher needs to choose the best AAV capsids to performer for your projects.
AAVnerGene has such an extensive collection of over 1000 different AAV capsids available for research and applications. This diversity provides researchers with a remarkable resource for selecting the most suitable AAV vectors for their specific projects.
- Commonly used AAV serotypes, including AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAV12, AAV13, AAV-DJ, AAVrh.10, AAVrh.74, AAV2-Retro.
- Tissue specific AAV capsids:
- Muscle: AAVmyo, AAVmyo2, AAVmyo3, myoAAV-1A, myoAAV-2A, myoAAV-3A, myoAAV-4A, AAV587MTP, AAVM41, AAV9.45, AAV9.61
- Lung: AAV1, AAV2-ESGHGYF, AAV2-ESGHGYF-Y730F-T491V, AAV2-PRSADLA, AAV2-PRSTSDP, AAV2.5T, AAV2H22, AAV4, AAV5, AAV5-PK2, AAV6.2, AAV6.2FF, AAV8-Y733F, AAV9.452sub.LUNG1, HAE1, HAE2
- Retina: AAV2-HBKO, AAV2-7M8, AAV2.GL, AAV2.NN, AAV44.9, AAV44.9(E531D), AAV8BP2, AAV6-K531E-R576Q-K493S-K459S, AAV9.GL, AAV9.NN ,ShH10
- Liver: AAVhum.8, AAV-L2-10, AAV3B-DE5, AAV-KP1, AAV-DJ, AAV-DJ-K137R/T251A/S503A, AAV3-S663V-T492V, AAVrh10, Spark100, AAVrh74, AAV-SL65
- Crossing blood brain barrier(BBB): AAV-PHP.B, AAV-PHP.eB, AAV-PHP.S, AAV.CAP-B10, AAV-F, AAV-S, AAV.Cap-Mac, AAV-PHP.Cs, AAV-PHP.C2, AAV 9P31, AAV-MaCPNS1 ,AAV-MaCPNS2 ,AAV.CPP.16, AAV.CPP.21, AAV9.HR, AAV-B1, AAV/Olig001 ,AAV-AS , AAV-BR1, AAV-ShH10, AAV-ShH10Y445F, AAV Retrograde, AAV1/2 hybrid, AAV.GTX, AAV-Cap-B22, PHP.B1, PHP.B2, PHP.B3, AAV1RX, AAV-True Type (AAV-TT), AAV-PHP.V ,AAV-PHP.C , AAV-PHP.N, AAV9_A2, AAV1_P5, VCAP-101, d VCAP-102, AAV8-THR.
- For researchers who have identified specific capsid names or sequences, AAVnerGene offers capsid cloning services and packaged into AAV vectors. For the low productivity capsids, AAVnerGene offers AAVone single-plasmid system to increase AAV productivity.
- For researchers who are unsure about which capsid to choose for their specific research goals, AAVnerGene offers AAV Serotype Testing Kits, AAV Capsid Barcode Kits, and DNA Barcode Technology, presumably tools that help researchers assess the transduction efficiency and tropism of different capsids.
- For researchers who want to discovery novel AAV capsids, AAVnerGen offers premade AAV capsid Libraries, ATHENA-II Capsid Platform and AAV Capsid Evolution Services to help researchers evolve their our capsids.
- For AAV gene therapy drug developers, AAVnerGene offers different tissue-targeting capsids that performance well in NHPs, such as AAV-ShD, AAV(BBB), AAV(lung), AAV(Liver), AAV(Retina), AAV(Heart), AAV(Kidney), AAV(myo), AAV(Tcells), et al.. Please contact us for more information.
AAVnerGene is committed to providing comprehensive support to researchers at every stage of their projects involving AAV capsids. Whether researchers are utilizing established capsids, exploring new ones, or seeking assistance with various AAV-related services, AAVnerGene is well-equipped to cater to your needs.