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Analysis method for evaluating in-vitro activity of thrombopoietin receptor stimulant

A thrombopoietin and receptor agonist technology, applied in the field of protein medicine, bioengineering and the field, can solve the problems of low safety and inaccurate test results, and achieve the effect of improving stability and reliable data results.

Active Publication Date: 2014-12-03
BEIJING TIDE PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008]In addition, the inventors found that the combined use of a new type of chromogenic agent-CCK-8 (cell counting 8) for activity evaluation can further solve the existing problems in the prior art. Organic solvents, low safety, and inaccurate test results

Method used

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  • Analysis method for evaluating in-vitro activity of thrombopoietin receptor stimulant
  • Analysis method for evaluating in-vitro activity of thrombopoietin receptor stimulant
  • Analysis method for evaluating in-vitro activity of thrombopoietin receptor stimulant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 In vitro activity evaluation of recombinant human thrombopoietin mimetic peptide-Fc fusion protein (TMP-Fc) (5-fold dilution gradient)

[0070] (1) Cell recovery and passage

[0071] Take the cell line out of the liquid nitrogen tank, quickly put it into a 37°C water bath, and shake it gently to make it melt quickly. Transfer it to a centrifuge tube in an ultra-clean bench, add 10% FBS+1640 medium to resuspend the cells. After centrifugation at 800rpm for 5min, discard the supernatant, add cell culture medium and growth factor recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) at a final concentration of 10ng / ml, mix well by pipetting, and transfer to a cell culture flask. The cell name, operation date, and cell generation are marked on the bottle wall.

[0072] The cells in the logarithmic growth phase were pipetted and mixed, and 100 μl of cell suspension was taken to determine the concentration of viable cells, cell survival rat...

Embodiment 2

[0087] Example 2 In vitro activity evaluation of recombinant human thrombopoietin mimetic peptide-Fc fusion protein (TMP-Fc) (10-fold dilution gradient)

[0088] Cell recovery and subculture, cell viability evaluation, sample detection, color development method, and data processing detailed steps are shown in Case 1. The sample preparation is 10-fold serial dilution, and 9 concentrations of sample solutions are prepared.

[0089] The result is as image 3 As shown, in the range of TMP-Fc concentration from 0.0005ng / ml to 5000ng / ml, MO7e cell proliferation and the logarithm of TMP-Fc concentration with base 10 showed a good linear relationship, R 2 =0.999. The half-effective concentration of TMP-Fc was 3.0 ng / ml.

Embodiment 3

[0090] Embodiment three romigrastim in vitro activity evaluation

[0091] See Case 1 for detailed steps of cell recovery and passage, cell viability evaluation, sample detection, color development method, and data processing.

[0092] Sample preparation: take a romigrastim sample, dilute the protein concentration to 0.05 mg / ml with 10% FBS+1640 medium, and use 10-fold serial dilution to prepare sample solutions with 9 concentrations.

[0093] The result is as Figure 4 As shown, in the range of romigrastim concentration of 0.0005ng / ml to 5000ng / ml, the MO7e cell proliferation and the logarithm of the romigrastim concentration present a good linear relationship, R 2 =0.995. The half-effective concentration of romigrastim is 3.0 ng / ml.

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Abstract

The invention discloses an analysis method for evaluating in-vitro activity of a thrombopoietin (TPO) receptor stimulant. According to the method, the binding activity of a sample and a TPO receptor and the activity of the thrombopoietin are reflected by evaluating the proliferation-promoting development capacity of the sample on cells. By adopting the method, on one hand, the problem that BaF3-hMpl cell system in the prior art is great in establishing difficulty, great in time consumption and instable in detection result can be solved; on the other hand, the problems that by adopting a suspension cell MTT method, the color developing time is long, an insoluble formazan product is generated, the result reproductivity is poor and the accuracy is low can be solved; meanwhile, a four-parameter data processing method which is better than survival rate evaluation indexes is adopted, so that the activity value of the sample can be directly defined.

Description

technical field [0001] The invention belongs to the field of protein medicine bioengineering and technology, and specifically relates to a novel analysis method for evaluating the in vitro activity of thrombopoietin receptor (TPO receptor) agonists by using suspension cell MO7e. Background technique [0002] Thrombopoietin receptor (TPO receptor) agonists specifically bind to TPO receptor c-Mpl, promote the proliferation and development of megakaryocytes, and then produce platelets. They are usually used to treat thrombocytopenia or thrombocytopenic purpura caused by chemotherapy. Such biologics include recombinant human thrombopoietin (rhTPO), recombinant human megakaryocyte growth and development factor modified with polyethylene glycol (PEG-rhMG-DF), TPO peptide mimetics, and TPO non-peptide mimetics thing. All of them can bind and activate TPO receptor (Mpl) to exert biological effects. [0003] The first generation of TPO receptor agonists is represented by recombinan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/53
CPCG01N33/6863
Inventor 常翠云贾慧齐连权刘福星崔颖刘利波
Owner BEIJING TIDE PHARMA
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