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Integrated AAV Offerings: From Cutting-Edge Products to Advanced Custom Services
Overcoming AAV Technology Challenges with Expertly Tailored Vector Design and Production
AAV Packaging Service
Virus packaging is a complex, labor-intensive, and resource-demanding process, but Biohippo is here to simplify it for you. We offer reliable, affordable, and fully customizable AAV packaging service designed to streamline your workflow. Whether you need assistance starting from vector construction, or packaging your plasmids, or specific quality control (QC) services, our end-to-end solutions save you time and ensure high-quality results. With a focus on precision, scalability, and stringent quality standards, we support your research needs at every step, allowing you to focus on your experiments without the hassle of in-house virus production.
Service Details & Checklist
Plasmid(s) for packaging: We will use customer-provided plasmids for packaging. You may ship your endotoxin-free plasmids in an eppendorf tube to our address. We require at least 0.3mg/1E+13GC virus. If you are unable to provide plasmids in the requested amount, we offer maxiprep service at an additional charge of $200, and we require at least 10μg raw material plasmid from the customer to start with.
Productivity: The productivity is estimated based on the average yield of common transgene vectors of the same serotype using rep-cap plasmid. If the productivity is significantly lower than average due to customer's plasmid vector (e.g. virus genome size exceeds 4.7kb), a scale-up production with additional chrage may be required to deliver the requested amount.
Packaging Method: HEK293T cells will be used for AAV packaging by transient transfection of plasmid.
Purification Method: AAV vector will be purified by two rounds of CsCl, or iodixanol gradient ultracentrifugation, or by affinity chromatography followed by one round of CsCl ultracentrifugation to get rid of host cell contaminate and enrich AAV capsids with full genome.
Titer Measurement: The titer is measured by SYBRGreen qPCR using primers targeting gene-specific sequences. By default, we will measure ITR titer, but please let us know if you would like use other common targets: CMV, EF1a, GFAP, Syn, eGFP, mCherry, WPRE, WPRE3, bGH.
Formulation Buffer: Our default formulation buffer is 1x DPBS plus additional 150mM NaCl and 0.001% Pluronic F-68. Please let us know if you have your own buffer system.
Pricing, Scale, and Turnaround:
|
Scale
|
Titer
|
Volume
|
Price
|
Turnaround
|
|---|---|---|---|---|
|
1e12 GC
|
1e13 GC/ML
|
1 x 100 µl
|
$1,200
|
2 weeks
|
|
2e12 GC
|
1e13 GC/ML
|
2 x 100 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
|
4X 1,000 µl
|
$17,800
|
2-3 weeks
|
|
5e14 GC
|
1e13 GC/ML
|
10X 1,000 µl
|
$42,800
|
3-4 weeks
|
|
1e15 GC
|
1e13 GC/ML
|
20 X 1,000 µl
|
$79,800
|
3-4 weeks
|
|
>1e15 GC
|
TBD
|
TBD
|
TBD
|
TBD
|
Checklist
☐ Please verify that your plasmids have two functional ITRs.
☐ Please ensure AAV genome size between the ITRs is less than 4.7 kb. Sizes larger than 4.7 kb may have low yield.
☐ Will you provide enough endotoxin-free plasmids (at least 0.3mg/1E+13GC)? If not, we offer maxi prep service and will only need 10ug starting raw material from you.
☐ AAV serotype
☐ Titer and Required volume (i.e. Packaging scale)
Additional Quality Control (QC) Services
| QC | Method | Turnaround | Price | Sample Requirement |
|---|---|---|---|---|
| Genome Titration | ddPCR/qPCR | 3-5 days | $200/sample | ~20 μl of samples |
| Capsid Titration | ELISA | 3-5 days | $400/sample | ~20 μl of samples with specific serotypes |
| Full/Empty Ratio | Mass Photometry | 1 day | $800/sample | ~20 μl of samples with titer >5E+11vg/ml |
| Endotoxin Testing | LAL Assay | 3-5 days | $200/sample | ~20 μl of samples |
| Endotoxin Removal | Resin-PMB | 3-5 days | $200/sample | ~20 μl of samples |
| Bioburden Testing | Direct plating | 3-5 days | $200/sample | ~20 μl of samples |
| Mycoplasma Testing | PCR | 3-5 days | $200/sample | ~20 μl of samples |
| Vector Genome Identity | PacBio Seq | 4 weeks | Request | ~100 μl of sample with titer >1E+12vg/ml |
Supplemental Information
AAV Production Systems
AAVone Single-Plasmid System: Streamlines AAV production, reduces costs, and improves consistency. All required genes are assembled in one plasmid.
AAVdual Two-Plasmid System: Offers flexibility by combining the mini-pHelper and pGOI in one plasmid, while the pRep-Cap remains separate.
AAVtri Triple-Plasmid System: Utilizes a modified, smaller adenoviral helper gene plasmid for efficient AAV vector production.
| Production System | Plasmid Name | Plasmid Size (kb) | Package Efficiency |
|---|---|---|---|
| AAVone | pAAVone | 14.0-18.0 | 200%-400% |
| AAVdual | pAAVdual | 9.4-13.4 | 100%-250% |
| AAVtri | mini-pHelper | 8.4 | 100%-250% |
Cell Lines for AAV Production
We offer a variety of cell lines for AAV vector production, available in both adherent and suspension formats. This flexibility allows researchers to select the most suitable system for their production needs, whether for small-scale experiments or large-scale production.
| Cell Type | Cell Line | Description | Advantages | Applications |
|---|---|---|---|---|
| Adherent Cells | HEK 293 | Human embryonic kidney cells expressing adenoviral gene E1, aiding in AAV packaging. | Well-established for AAV production, high transfection efficiency. | Suitable for smaller-scale experiments and adherent culture methods. |
| HEK 293T | HEK 293 derivative expressing SV40 T antigen for increased cell cycling and plasmid amplification. | Improved transfection efficiency and higher vector production. | Ideal for small to medium-scale AAV production in adherent cultures. | |
| Suspension Cells | HEK 293 (Suspension) | Suspension-adapted version of HEK 293 cells derived from adherent cells. | Easy to scale up, better mixing, widely used for GMP-grade AAV production. | Best for large-scale production where suspension culture systems are preferred. |
| HEK 293T (Suspension) | Suspension-adapted HEK 293T cells, retaining high transfection efficiency. | Combines high transfection efficiency with suspension culture benefits for scaling. | Optimal for large-scale production and clinical-grade vector manufacturing. |
By providing both adherent and suspension cell lines, AAVnerGene meets diverse research needs, catering to those who prefer traditional adherent culture methods and those who require scalable, suspension-based systems for larger production volumes.
The combination of suspension HEK 293T cells with our AAVone system enables researchers to achieve crude titers of over 1e15 VG/L for most AAV serotypes. For AAV9 serotype, titers can exceed 3e15 VG/L. Even in HEK 293 cells, the AAVone system can achieve titers around 1.5e15 VG/L with AAV9, demonstrating its robust productivity across different cell lines.
Here is a summary of AAV production efficiency using HEK 293 and HEK 293T cells with different AAV serotypes and transfection reagents:
| Cell Lines | pAAVone Size (kb) | AAV Serotype | AAV Size (kb) | pDNA (µg/1e6 cells) | TR/pDNA (Ratios) | Crude Titer (GC/L) |
|---|---|---|---|---|---|---|
| HEK 293T |
15.2 | AAV9 | 2.2 | 0.25 | PEIMax (2:1) | 3.72E+15 |
| 17.4 | AAV9 | 4.4 | 0.25 | PEIMax (2:1) | 2.26E+15 | |
| 17.6 | AAV9 | 4.6 | 0.25 | PEIMax (2:1) | 1.76E+15 | |
| 17.7 | AAV9 | 4.7 | 0.25 | PEIMax (2:1) | 1.94E+15 | |
| HEK 293 |
15.2 | AAV2 | 2.2 | 0.35 | PEIMax (2:1) | 3.54E+14 |
| 15.2 | AAV6 | 2.2 | 0.35 | PEIMax (2:1) | 2.91E+14 | |
| 15.2 | AAV8 | 2.2 | 0.35 | PEIMax (2:1) | 8.69E+14 | |
| 15.2 | AAV9 | 2.2 | 0.50 | PEIMax (2:1) | 1.55E+15 | |
| 15.2 | AAV2 | 2.2 | 0.375 | PEIMax (2:1) | 4.98E+14 | |
| 15.2 | AAV2 | 2.2 | 0.25 | FectoVIR (1:1) | 4.66E+14 | |
| 15.2 | AAV2 | 2.2 | 0.375 | FectoVIR (2:1) | 4.42E+14 | |
| 15.2 | AAV2 | 2.2 | 0.25 | FectoVIR (1:1) | 5.12E+14 | |
| 15.2 | AAV2 | 2.2 | 0.375 | FectoVIR (2:1) | 6.48E+14 | |
| 15.2 | AAV2 | 2.2 | 0.25 | PEIMax (2:1) | 6.53E+14 | |
| 15.2 | AAV9 | 2.2 | 0.35 | PEIMax (2:1) | 1.53E+15 | |
| 15.2 | AAV9 | 2.2 | 0.35 | PEIMax (2:1) | 1.21E+15 |
By leveraging different cell lines and AAV production systems, we ensure high flexibility and productivity, catering to specific experimental needs and scales.
AAV Services
Custom AAV Vector Design and Cloning
Expert consultation and design of custom AAV vectors tailored to your research needs.
AAV Production Services
Scalable production services from small pilot batches to large-scale bioreactor production.
AAV Purification Services
High-quality purification using state-of-the-art techniques like affinity chromatography.
AAV Quality Control and Characterization
Comprehensive quality control including titration, full/empty ratio, endotoxin, mycoplasma testing, etc.
Preclinical Development Services
In vitro and in vivo studies to evaluate transduction efficiency, tissue tropism, and safety.
Ready-to-Use AAV Vectors
Off-the-shelf AAV vectors for standard applications and proof-of-concept studies.
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