Unlikethe traditional three plasmid system in HEK293 cells, One-Bac 4.0 system is an insect/baculovirus system for AAV production. The production process consists of several key parts: baculovirus plasmid construction, seed virus production, seed virus amplification, seed virus infection of insect cells, and AAV harvest. This system achieves linear amplification of production by infecting insect cells with baculovirus containing the AAV genome, overcoming the bottleneck of large-scale AAV preparation. One-Bac 4.0 system has the potential to benefit more patients due to its high yield compared to other AAV production systems.
Genevoyager has successfully completed the delivery of a number of projects, some of the data are shown below.
● High Safety Profile
The One-Bac 4.0-derived AAVs present low levels of impurities and are free of rcAAV.
Data from second-generation sequencing (illumina) and third-generation sequencing (PacBio) confrm that Genevoyager's One-Bac 4.0-derived AAVs exhibit high purity.
DNA was extracted from the culture supernatant after three rounds of cell culture,and rcAAV was detected by qPCR.The results demonstrated the following:
For the positive control samples(wtAAV2/9)at 100 IU and 10IU,Ct values progressively decreased over the three amplification rounds,indicating successful amplification of rcAAV during culture.
For rAAV2/9 produced using the One-Bac 4.0 system,with inoculation doses of 1E+11vg and 1E+10vg,no significant decrease in Ct values was observed after three rounds.The Ct values remained comparable to the negative control(NC),indicating no detectable rcAAV.
In contrast,for rAAV2/9 produced using the HEK293T system at a dose of 1E+10 vg,Ct values showed a marked decreasing trend over the three amplification rounds,confirming amplification of rcAAV during culture.
● High Yields
The One-Bac 4.0-derived AAVs maintain high yields both before and after purification at a production scale of 50L.
● High Full Capsid Ratio
The One-Bac 4.0 system achieves an impressive full capsid ratio of over 90% in its final product.
|
Manufacturing process |
Product stage |
Purification method |
AUC% empty |
AUC% partial |
AUC% full |
AUC% aggregates |
|
Genevoyager HEK293 |
Final product |
Affinity+Anion Exchange |
7.52 |
0 |
>90 |
0.16 |
|
One-Bac 4.0 |
Final product |
Affinity+Anion Exchange |
1.1 |
7.2 |
>90 |
0 |
● High Infectious Activity
Results from cell and animal experiments demonstrate that One-Bac 4.0-derived AAV exhibits equivalent or higher infectivity compared to HEK293 AAV.
Comparison of firefly luciferase activity in HEK293 cells infected with AAV-Luc produced by the One-Bac 4.0 system and the HEK293 system at the same MOI.
Comparison of transduction efficiency of rAAV9-EGFP and rAAV9-mCherry produced by the One-Bac 4.0 system andthe HEK293system(specific activity:physical titer/infectioustiter,TCID50).
● High Passage Stability of BEV
The Baculovirus Expression Vector (BEV) employed in the One-Bac 4.0 system maintains high stability over multiple passages and during long-term storage at ≤-80℃ for over 24 months,ensuring consistent high yields and robust expression levels of AAV batches at commercial scales.
● High Recovery Rate
With a consistent recovery rate of ≥30% from 5L to 200L scale-up, One-Bac 4.0 system demonstrates good scalability and effciency for large-scale production.
● Low Aggregation at High Concentration
AAVs produced using the One-Bac 4.0 system exhibit low aggregation even at high concentrations. Stability assessments,including ddPCR-GOl and main peak SEC-HPLC,demonstrate that product quality remains stable under 4℃ and withstands multiple freeze-thaw cycles.
|
Stability at 4℃ and 27℃(AAV9) |
|||||
|
Incubation Conditions |
Test Results |
||||
|
Temperature |
Days(D) |
ddPCR-GOI(vg/mL) |
Main peak SEC-HPLC(%) |
DLS |
|
|
Diameter(nm) |
|||||
|
Control group |
0 |
2.76E+14 |
96.18 |
31.76 |
|
|
4℃ |
7 |
2.84E+14 |
95.80 |
33.20 |
|
|
14 |
2.69E+14 |
95.51 |
33.70 |
||
|
27℃ |
3 |
2.86E+14 |
95.91 |
31.11 |
|
|
7 |
2.49E+14 |
95.70 |
31.98 |
||
|
14 |
2.84E+14 |
94.60 |
30.32 |
||
|
Freeze-thaw Stability at-80℃(AAV9) |
|||||
|
Processing Conditions |
Test Results |
||||
|
ddPCR-GOI(vg/mL) |
Main peak SEC-HPLC(%) |
DLS |
|||
|
Diameter(nm) |
|||||
|
Control |
2.76E+14 |
96.18 |
31.76 |
||
|
3 Freeze-thaw cycles |
2.98E+14 |
95.05 |
31.15 |
||
|
5 Freeze-thaw cycles |
2.64E+14 |
94.12 |
32.12 |
||
Moreover,the One-Bac 4.0-derived AAVs are free of residual rcAAVs,exhibit high purity and high expression levels,addressing critical safety and effcacy concerns for gene therapy.
● Improved Accessibility
The One-Bac 4.0 system offers a more cost-effective approach to AAV production by replacing costly GMP plasmids and transfection reagents with a streamlined process for baculovirus vector production, significantly reducing the cost of materials for AAV vector manufacturing.
● Reduced timelines
The establishment of a virus seed bank facilitates a rapid and simplified process for viral infection during production. This feature becomes more prominent as the scale of virus production increases, resulting in reduced timelines for production and delivery.
|
Packaging System
|
Phase Il (up to 50 patients) |
Phase Ⅲ
(up to 200 patients)
|
Commercial Stage
(up to 3,000 patients)
|
|
Genevoyager
One-Bac 4.0 system
|
Fermentation at 500L |
Fermentation at 2,000L |
Fermentation at 5,000L |
|
Purification at 500L |
Purification at 2,000L |
Purification at 5,000L |
|
|
Testing |
Testing |
Testing |
|
|
Estimated duration |
10-12 weeks |
12-14 weeks |
14-15 weeks |
|
HEK293 serum-free
suspension system
|
Preparation of 3 plasmids |
—— |
—— |
|
Fermentation at 500L-1,000L |
—— |
—— |
|
|
Purification at 1,000L |
—— |
—— |
|
|
Testing |
—— |
—— |
|
|
Estimated duration |
18-20 weeks |
Difficult to achieve |
Difficult to achieve |
US: 3675 Market Street, Suite 200, Philadelphia, PA19104 Tel: +1 (215) 205-6963 | +086 027-65023363
E-mail: hui.wang@genevoyager.com
China: No128, Guanggu 7th Rd, East Lake High-tech Development Zone, Wuhan, China Tel: 17720522078
E-mail: marketing@genevoyager.com
US: 3675 Market Street, Suite 200, Philadelphia, PA19104 Tel: +1 (215) 205-6963 | +086 027-65023363
E-mail: hui.wang@genevoyager.com
China: No128, Guanggu 7th Rd, East Lake High-tech Development Zone, Wuhan, China Tel: 17720522078
E-mail: marketing@genevoyager.com
