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To study the bioactivity determination method of human insulin from transgenic cell using in-cell Western technology.
Using CHO-INSR B1284 cells as the target cell, different concentrations of human insulin were used as the stimulants. In-cell Western was used to measure the phosphorylation of human insulin receptor, and the bioactivity of human insulin was determined. According to the 2020 edition 9401 biological products bioactivity/potency determination method verification guiding principles in the General Rules of the fourth part of Chinese Pharmacopoeia, the established method was validated in terms of specificity, precision and durability. The national standard of recombinant human insulin was used as the benchmark to evaluate the relative potency of human insulin drug substance and other insulin analogues.
Different concentrations of human insulin indicated effect on CHO-INSR B1284 cells in a dose-dependent manner. The samples of five potent concentrations were detected for 8 times. The geometric means of the relative potency were (63.76±4.38)%, (78.01±5.97)%, (99.52±4.62)%, (122.84±7.49)% and (151.71±8.81)%, respectively. The relative bias of five potency level were within ±5%, the corresponding geometric coefficient of variations (GCV,%) were lower than 11%, and the upper confidence limit of GCV were all lower than 20%. The bioactivity of human insulin and insulin analogues can be effectively detected using this method.
The in vitro bioactivity determination of human insulin using the In-cell Western was of good specificity, accuracy and precision, which can be used as a routine bioassay method of human insulin.
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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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