Table 1.
Step | Premaster virus seed | Master virus seed | Working virus seed |
---|---|---|---|
Nominal vessel volume (L) | 0.25 | 50 | 2000 |
Culture working volume used for seed (L) | 0.03 | 20a | 1600 |
Cell density at infection (cells/mL) | 0.8 × 106 | ||
Multiplicity of infection (VP/cell)b | 6 | ||
Required input (VP) | 1.4 × 108 | 1.9 × 1011 | 7.7 × 1012 |
Required input as proportion of previous output | – | 0.194 | 0.012 |
Predicted usable output (VP)c | 9.9 × 1011 | 6.6 × 1014 | 5.3 × 1016 |
Observed usable output (VP) | 4.5 × 1012 | 2.0 × 1015 d | 1.6 × 1017 d |
Abbreviations: IU, infectious unit; pPCR, quantitative polymerase chain reaction; VPs, viral particles.
On the basis of the assumed total working volume of 40 L at master virus seed stage, with half of this used to provide drug substance for a clinical trial.
On the basis of quantitative PCR; corresponds to 0.1 IU/cell (plausible range, 0.03–0.20 IU/cell) assuming a typical particle:infectivity ratio.
Assumes 1.0 × 1011 VP/mL upstream productivity and 33% recovery by depth filtration and anion‐exchange chromatography.
Values for master virus seed and working virus seed are extrapolated from 3.0 × 1014 VP (measured by qPCR, as the mean of triplicate samples each assayed in triplicate wells on a single plate) obtained in a 3‐L working‐volume scaled‐down model (i.e., 1.0 × 1011 of seed recovered per milliliter of culture).