Technology Platforms
π-Alpha™ 293 AAV High-yield Production Platform
PackGene’s proprietary π-Alpha™ 293 AAV High-yield Platform uses uniquely designed RC plasmid in the triple-plasmid transfection system to increase AAV production by 3 to 8 times for various AAV serotypes. This technology is paired with both in-process upstream and downstream QbD optimizations to increase total AAV yield up to 10 fold. A single batch of AAV production delivers up to 1E+17vg virus particles, which is enough to meet the needs of most clinical and commercial level of AAV production.
Advantages：
- Efficiency: Proprietary technology and processes increase the AAV yield in a HEK293 serum-free cell suspension system by more than 10 fold.
- The production process increases the suspensive cells production by 10-25 times.
- Quality: Unique process development greatly reduces the key impurities (HCD, endotoxin, etc.).
- Fidelity: Key technology innovations reduce empty capsids rate and increase infection titer.
Intellectual property:
PackGene holds 6 patents for AAV-associated technologies with an additional 2 patents under review.
High-yield RC plasmid increases AAV yield by 3-8 times by adding non-coding regulatory elements to the Rep-Cap plasmid (The patent is pending).
DOE transfection conditions
DOE Engineering Parameters Ambr250 Reactor (Sartorius)
DOE Lysis and harvest conditions
Cell density, total plasmid, plasmid ratio, transfection reagent ratio
Stirring Speed, pH, Dissolved Oxygen, Temperature
Stirring speed, Density, Time, Temperature
AAV production process using serum free 293 suspension cells (200L)
Conclusion
The pilot process development strategy and parameters for the small batches successfully guide the 200L scale-up AAV production process using serum-free 293 suspension cells.
Actual AAV production up to 7.3E+16 vg (total yield) for 200L production system
TEM-Empty capsids＜5%
Analytical Ultracentrifuge (AUC)
Lower HCD
Reducing the plasmid DNA residue (Packaged plasmid impurity) significantly by unique molecular modification on plasmid
| Sample | Relative proportion, % | ||
| ECs | VCs | ||
| Lighter capsids | Intermediate population | Heavier capsids | |
| AAV5-gfp a,b | |||
| 66S | 79S | 95S | |
| Affinity-purified AAV5 | 90.91 | 3.43 | 5.66 |
| EC peak fraction | 95.6 | 4.4 | ND |
| VC peak fraction | 19.81 | 14.69 | 65.50(89S) |
| AAV8-gfp a,b | |||
| 63S | 74S | 84S | |
| Affinity-purified AAV8 | 62.56 | 2.03 | 35.41 |
| EC peak fraction | 96.6 | ND | 3.4 |
| VC peak fraction | 3.13c | 4.22 | 92.65 |
| AAV6-gfp d | |||
| Affinity-purified AAV6 | 63.17 | 36.83 | |
| EC peak fraction | 93.28 | 6.72 | |
| VC peak fraction | 5.36 | 94.64 | |
| AAV6-cas9 d | |||
| Affinity-purified AAV6 | 60.59 | 39.41 | |
| EC peak fraction | 95.72 | 4.38 | |
| VC peak fraction | 4.54 | 95.46 | |
| AAV9-gfp d | |||
| Affinity-purified AAV9 | 67.63 | 32.37 | |
| EC peak fraction | 94.96 | 5.04 | |
| VC peak fraction | 5.28 | 94.72 | |
Reducing the plasmid DNA residue (Packaged plasmid impurity) significantly by unique molecular modification on plasmid
Scalable process by ultracentrifugation allows 200~500L batch production
| Characters | Descriptions |
| High yield | Design of RC plasmid increases AAV yield by 3-8 times |
| CPP optimization increases total AAV yields by 10 times | |
| Improved scalability | Stable scale-up process: from 125 mL to 200 L (suspension) |
| Scalable 200 L suspension process: 7.3E+16 vg yeild, >30% recovery rate | |
| Improved biosafety | HCD residue: 30~90 ng/1E13 vg (suspension) |
| Optimal full capsid ratio at harvest (20~66%) and at final (75~94%) | |
| Novel plasmid design decrease 90% encapsidated plasmid impurity |
π-Alpha™ 293 AAV High-yield Production Platform
