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.


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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

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Proprietary technology and processes increase the AAV yield in HEK293 serum-free cell suspension systems by more than 10 fold.



Our production process increases suspensive cell production by 10-25 fold.



Unique process development procedures greatly reduce the key impurities (HCD, endotoxin, etc.).



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.

AAV9-yield-in-the-293-suspension-cells-system (1)

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
Comparison of AAV5 yield from different scales

AAV production process using serum free 293 suspension cells (200L)


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)
Analytical Ultracentrifuge (AUC)
Lower HC

Lower HC

Reducing the plasmid DNA residue (Packaged plasmid impurity) significantly by unique molecular modification on plasmid

Sample Relative proportion, %
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
AAV yield and wrong packaging ratios before and after plasmid modification

Reducing the plasmid DNA residue (Packaged plasmid impurity) significantly by unique molecular modification on plasmid

batch capacity of scalable ultracentrifugtion production process
recovery rate and empty vector rate of scalable ultracentrifugation production process

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

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