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.
Free Webinar
Topic: Current Challenges and New Approaches to Developing rAAV Manufacturing Processes for Large-scale GMP Production
Time: 7/02/2024 2:00 pm EST/ 11:00am PST
Efficiency
Proprietary technology and processes increase the AAV yield in HEK293 serum-free cell suspension systems by more than 10 fold.
Productivity
Our production process increases suspensive cell production by 10-25 fold.
Quality
Unique process development procedures greatly reduce the key impurities (HCD, endotoxin, etc.).
Viability
Key technology innovations reduce empty capsids rate and increase infection titer.
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 (patent pending).
Based on DOE experimental optimization of key process parameters, the total AAV yield is increased by more than 10 times
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)
Actual AAV production up to 7.3E+16 vg (total yield) for 200L production system
TEM-Empty capsids<5%
Lower HC
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