Table 2.
In vivo potency (IU/mL) of diphtheria and tetanus toxoid antigens in DTwP vaccines formulated with or without 7.5% (v/v) propylene glycol
| Diphtheria potency (95% CI) | Tetanus potency (95% CI) | |||||||
|---|---|---|---|---|---|---|---|---|
| No PG, no FT | PG, FT | No PG, no FT | PG, FT | |||||
| Time points (months) | 2°–8°C | 22°–25°C | 2°–8°C | 22°–25°C | 2°–8°C | 22°–25°C | 2°–8°C | 22°–25°C |
| 0 | 67.2 (54.5–82.8) | 67.2 (54.5–82.8) | 62.2 (50.7–76.0) | 62.2 (50.7–76.0) | 145.1 (100.1–211.2) | 145.1 (100.1–211.2) | 136.4 (72.7–202.5) | 136.4 (72.7–202.5) |
| 1 | 69.8 (53.7–90.6) | 65.9 (51.6–86.2) | 75.2 (66.8–104.8) | 63.5 (47.7–85.4) | 153.6 (108.3–213.7) | 136.1 (93.9–195.3) | 137.9 (96.7–195.4) | 123.8 (87.5–176.9) |
| 3 | 73.5 (58.8–92.0) | 59.8* (41.8–91.3) | 61.3 (49.2–76.0) | 64.1 (52.4–81.6) | 164.2 (110.6–255.1) | 114.3* (78.6–167.0) | 121.4 (81.6–183.1) | 114.7* (77.4–171.6) |
The vaccines were exposed to freeze-thaw treatment (FT) or not exposure (no FT) and stored at the indicated temperature for 0, 1, or 3 months before testing using in vivo potency assays; IU: international unit; PG: propylene glycol; FT: freeze-thaw.
Diphtheria toxin potency was determined using an in vitro toxin neutralization test. NIH/OlaHsd mice were immunized with 0.5 mL of serial dilutions of test vaccines or reference toxoid.9 Mice were bled 5 weeks after immunization and serial dilutions of heat-inactivated serum were incubated with diphtheria toxin before being added to Vero cell monolayers. After 6 d of incubation (37°C, 5% CO2), cell death was scored by pH change as indicated by a color change in the culture medium and visual inspection of the monolayers, and the potency of the diphtheria toxoid was calculated.
To assess tetanus toxin potency, NIH mice were immunized with serial dilutions of test vaccines or tetanus toxoid reference vaccine. After 4 weeks, the test groups were challenged subcutaneously with 50 LD50 (in 0.5 mL) tetanus toxin per mouse. The control group was challenged with 1 LD50 (in 0.5 mL) tetanus toxin per mouse. All groups were observed daily for mortality for 5 d The potency of the toxoid was calculated from the survival dose‑response curve.
For both the D and T potency assays, the percentage of animals surviving at each dilution was used to calculate the potency compared with the international standard using the Reed-Muench equation and converted to potency IU per mL.
*Potency of sample is below the minimum required potency.