engleski

Application of Fractional Factorial Design of Experiments to Live Attenuated Rubella Vaccine Potency Assay Robustness Testing

The potency assay for the freeze-dried live attenuated rubella vaccine is a cell culture based biological assay. Susceptible cell culture monolayers prepared in microtiter plates are infected by a series of virus dilutions and occurrence of the cytopathic effect is monitored. As an assay which involves biological material, the rubella potency assay is prone to variations. To revise the standard operating conditions and enhance the flexibility of the assay performance, the robustness testing of the rubella vaccine potency assay was performed. Seven intra-assay operating conditions that could have an effect on the assay performance were identified. The operating conditions involving cell cultures preparation were (1) the cell passage, (2) the age of cells within the passage, and (3) the method of the cell monolayer preparation. The operating conditions involving the virus adsorption to the prepared cell culture monolayers were (4) the duration of the viral adsorption, (5) the temperature and (6) the light regime during the adsorption. Finally, (7) the incubation time of the microtiter plates during the assay was monitored. The values for two experimental levels of the selected seven factors were defined. Fractional factorial design of experiments (DOE) with resolution IV (27-3(IV)) was applied, and the specification for 16 experiments was obtained. The effect of each factor was compared to the overall assay variability obtained from all experiments and to the European Pharmacopoeia potency assay requirements. It was shown that six out of seven factors had no effect on the assay variability. The effect of the remaining factor, the method of cell monolayer preparation, was shown to be very subtle, but noticable, and considerably smaller in comparison to the assay variability requirements proposed by the European Pharmacopoeia. The robustness testing through DOE showed that the rubella vaccine potency assay is a very robust method which can be performed in all of the assay conditions examined in this study. The assay sensitivity and reproducibility could be additionally improved by fixing the assay performance always on a higher level of factor 3 – method of cell monolayer preparation.

Potency assay validation ; Rubella vaccine ; Design of experiments ; Robustness

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