Table 1 Summary of AAV biomanufacturing development
From: Advanced biomanufacturing and evaluation of adeno-associated virus
Host cells | Production container | Scale (mL) | AAV serotype | Transfect. reagent | Transfection VCD (c/mL) | DNA:cell (µg:10 6 ) | Productivity (10 10 vg/mL) | Purification | Purify recovery | Desalt/concentrate | Overall recovery |
|---|---|---|---|---|---|---|---|---|---|---|---|
HEK 293F | Shaker flask | 30–60 | AAV-DJ8 | Liposomes | 0.4 × 106 | 1.6:1 | 0.53 ± 0.08 | 10 kDa ultrafilter (UF) | N/A | ||
Spinner flask | 60–100 | AAV-DJ8 | Liposomes | 0.4 × 106 | 1.6:1 | 0.50 ± 0.08 | 10 kDa UF | N/A | |||
VPC | Shaker flask | 30–60 | AAV2 | AAV-MAX | 3 × 106 | 0.5:1 | 7.88 ± 0.39 | Anion IEX (HPQ) | 40–60% | G-25/Vacuum | 30–50% |
HPQ | 40–60% | PES UF | > 50% | ||||||||
Affinity (AVB), Sepharose (IEX) | < 5% | N/A | < 5% | ||||||||
30–60 | AAV5 | AAV-MAX | 3 × 106 | 0.5:1 | 2.97 ± 0.13 | HPQ | 40–60% | G-25/Vacuum | 30–50% | ||
30–60 | AAV-DJ8 | AAV-MAX | 2 × 106 | 0.5:1 | 0.14 ± 0.05 | HPQ | 40–60% | G-25/Vacuum | 30–50% | ||
4 × 106 | 0.5:1 | 0.25 ± 0.13 | |||||||||
3 × 106 | 0.4:1 | 0.25 ± 0.09 | |||||||||
3 × 106 | 0.6:1 | 0.14 ± 0.01 | |||||||||
30–60 | AAV-DJ8 | AAV-MAX | 3 × 106 | 0.5:1 | 2.40 ± 0.06 | Anion IEX (aPrime 4A) | 85–95% | PES UF | > 80% | ||
HPQ | 40–60% | G-25/Vacuum | 27–34% | ||||||||
HPQ | 40–60% | PES UF | 52–64% | ||||||||
AVB, IEX | < 5% | N/A | < 5% | ||||||||
30–60 | AAV-DJ | AAV-MAX | 3 × 106 | 0.5:1 | 5.60 ± 5.14 | HPQ | > 90% | Cellulose UF | 65–91% | ||
Spinner flask | 60–100 | AAV-DJ8 | AAV-MAX | 3 × 106 | 0.5:1 | 0.41 ± 0.20 | HPQ | > 30% | G-25/Vacuum | 10–20% | |
Stirred-tank bioreactor | 1,200 | AAV-DJ | AAV-MAX | 3 × 106 | 0.5:1 | 8.14 ± 1.91 | HPQ | 40–60% | Dialysis/Vacuum | 18–33% | |
1,500–2,000 | AAV-DJ8 | AAV-MAX | 3 × 106 | 0.5:1 | 7.52 ± 0.49 | aPrime 4A | > 85% | G-25/Vacuum | > 80% | ||