Table 2.
Summary of VC enrichment via AEX
| Sample | Relative proportion, % |
Fold VC enrichment | Relative ratio (ECs:VCs) | Relative ratio (heavier:intermediate capsids) | ||
|---|---|---|---|---|---|---|
| ECs |
VCs |
|||||
| Lighter capsids | Intermediate population | Heavier capsids | ||||
| AAV5-gfpa,b | ||||||
| 66S | 79S | 95S | – | – | – | |
| Affinity-purified AAV5 | 90.91 | 3.43 | 5.66 | – | 10:1 | 1.6:1 |
| EC peak fraction | 95.6 | 4.4 | ND | – | – | – |
| VC peak fraction | 19.81 | 14.69 | 65.50 (89S) | 8.8× | 1:4 | 4.4:1 |
| AAV8-gfpa,b | ||||||
| 63S | 74S | 84S | – | – | – | |
| Affinity-purified AAV8 | 62.56 | 2.03 | 35.41 | – | 1.7:1 | 17.4:1 |
| EC peak fraction | 96.6 | ND | 3.4 | – | – | – |
| VC peak fraction | 3.13c | 4.22 | 92.65 | 2.6× | 1:31 | 22:1 |
| AAV6-gfpd | ||||||
| Affinity-purified AAV6 | 63.17 | 36.83 | – | 1.7:1 | – | |
| EC peak fraction | 93.28 | 6.72 | – | – | – | |
| VC peak fraction | 5.36 | 94.64 | 2.6× | 1:18 | – | |
| AAV6-cas9d | ||||||
| Affinity-purified AAV6 | 60.59 | 39.41 | – | 1.5:1 | – | |
| EC peak fraction | 95.72 | 4.38 | – | – | – | |
| VC peak fraction | 4.54 | 95.46 | 2.4× | 1:21 | – | |
| AAV9-gfpd | ||||||
| Affinity-purified AAV9 | 67.63 | 32.37 | – | 2.1:1 | – | |
| EC peak fraction | 94.96 | 5.04 | – | – | – | |
| VC peak fraction | 5.28 | 94.72 | 2.9× | 1:18 | – | |
AEX, anion-exchange chromatography; EC, empty capsid; VC, vector capsid; ND, not detected.
Average value for multiple runs (n = 3), relative SD < ± 5%.
Values reported from sv-AUC analyses for determination of the relative proportion of ECs and VCs are based on 260 nm signal data.
No visible peak of AAV8 63S was observed in AUC profiles of VC fraction (Figures 4E and 5A). In the sample with a very low UV signal, a small peak at 63S was observed (e.g., Figure 5A, lot #2). Although not clear if it was the noise of the UV detection, as the absorbance was below 0.001, calculations were made based on the UV signal peak area at this 63S position so as not to exclude the possibility of the 63S population present in trace amount.
Values reported from the optical-density method for determination of the relative proportion of ECs and VCs.