Full
Outline
To develop a cell-based reporter gene assay (RGA) for bioactivity determination of anti-EpCAM+CD3 bispecific antibody (BsAb).
A HcT116-Luc cell line stably expressing Luciferase (Luc) was generated as the target cell, and a HuT78 cell line served as effector cell. The RGA was established by optimizing the initial concentration of BsAb, incubation time, cell seeding time, the ratio of effector cell to target cell, and cell seeding density. The specificity, accuracy, precision, linearity, subculture stability and robustness of RGA were verified according to the guideline of International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) Q2 and Pharmacopoeia of the People's Republic of China (ChP, 2015).
The parameters of RGA were developed and confirmed after optimization as follows: The ratio of effector cell to target cell was 10∶1; After mixing effector cell and target cell, the mixed cells were seeded in 96-well plate with 1.1×104 cells·well-1, which was cell seeding density; The initial concentration of BsAb, 3 000 ng·mL-1, was serially diluted by 1∶3.5 for 9 gradients, and there were 10 detected concentrations in all; The serially diluted samples were incubated for 72 h with the seeding cells. After the incubation, the Luc intensity was measured, and the dose-response curve was fitted in a 4-parameter mode to calculate the relative bioactivity of sample. The accuracy, precision and linearity verification were performed by 8 potency concentrations, which were 50%, 70%, 80%, 100%, 120%, 130%, 150% and 200% potency concentrations. The recovery rate range of relative bioactivities of 8 potency concentrations was 95.48%~107.37%, and the coefficient of variation (CV) ranged 4.80%~9.97%, which was <10%. The measured potency to expected potency was evaluated by linear regressing model analysis (r2>0.99). The Luciferase was stably expressed in HcT116-Luc cell between 14 and 33 passage.
Based on passage cells, the RGA has been developed as a simple and convenient procedure for determination of bioactivity of anti-EpCAM+CD3 BsAb, and its specificity, precision, accuracy and robustness met the application requirements.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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