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In vitro biological activity determination method of human insulin and its analogs or conjugates

A technology of human insulin and determination method, which is applied in the direction of color/spectral characteristic measurement, analysis through chemical reaction of materials, and material analysis through observation of the influence on chemical indicators, etc., which can solve the problems of ineffective counting and repeatability, Fragile and other issues

Active Publication Date: 2019-08-09
CHONGQING PEG BIO BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the fact that primary mature adipocytes are easily broken, cannot be effectively counted and have poor repeatability, this method has only been reported in the literature of Novo Nordisk for the quality research of insulin detemir

Method used

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  • In vitro biological activity determination method of human insulin and its analogs or conjugates
  • In vitro biological activity determination method of human insulin and its analogs or conjugates
  • In vitro biological activity determination method of human insulin and its analogs or conjugates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Comparison of Three Determination Methods for In vitro Biological Potency Determination of Recombinant Human Regular Insulin

[0022] Using 3T3-L1 preadipocytes (purchased from Shanghai Cell Bank, Chinese Academy of Sciences) and male rat preadipocytes (purchased from male rats from the Animal Institute of Daping Hospital, Third Military Medical University) to measure the activity of recombinant human insulin, recombinant human Ordinary insulin standard (from China National Institutes for Food and Drug Control, each labeled content is 20mg / bottle 28.3IU / mg).

[0023] Rat preadipocyte GOD-POD method

[0024] (1) Take 90g-120g male rats fed with common food, decapitate them after ether anesthesia, soak them in 75% alcohol for complete disinfection, and cut out the abdominal groove, epididymis, and fat pads around the kidneys under aseptic conditions and put them in a petri dish In PBS, use surgical scissors to remove blood vessels and lymph nodes as much as pos...

Embodiment 2

[0048] Example 2 Glucose oxidase method (GOD-POD method) and 3H-glucose radiation assay experiments in rat prelipid adipocytes to determine the biological potency of recombinant human insulin standard and insulin detemir

[0049] The method in Example 1 was used to induce preadipocytes in male rats, and after the cells were induced into 80% adipocytes, they were used for the following experiments.

[0050] Glucose oxidase method (POD method): Add 1.0 mg / ml glucose and 0.8 µCi / well 3H-Glucose to DMEM glucose-free medium as cell maintenance solution. Add 180µl of cell maintenance solution to each well of the adipocytes after discarding the cell fluid, and dilute the recombinant human insulin standard (from China National Institutes for Food and Drug Control, 20mg / tube 28.3IU / mg) to 5μg / ml; Insulin tephra (from Novo Nordisk NovoPing 300 units / 3ml / bottle) was diluted to 20 μg / ml and started to be diluted 3 times, making eight dilutions, adding 20 μl of sample to each well, and rea...

Embodiment 3

[0056] Example 3 Determination of rapid-acting insulin, long-acting insulin and modified conjugate (PEG-Lispro) in vitro activity of rat preadipocytes

[0057] Utilize the method for inducing rat preadipocytes in Example 1 to measure insulin aspart (derived from Novo Nordisk Novo Sharp 300 units / 3ml / branch), insulin lispro (derived from Lilly Humalog 300 units) / 3ml / bottle), ordinary human insulin (from China Food and Drug Control Institute), insulin glargine (from Sanofi-Aventis Lantus 300 units / 3ml / bottle), insulin detemir (from The in vitro biological activity of Novo Nordisk Novoping 300 units / 3ml / branch) and PEG-Lispro (made by the company according to the patent EP2476430 A1), the half-effective response dose (EC50) after 5 repeated tests, the results are as follows Figure 5 .

[0058] After 5 repeated determinations of the recombinant human insulin potency standard product, the half-effective dose EC50 average value was 17.754 ng / ml, and the recombinant human insulin ...

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Abstract

The invention relates to a human insulin and analog including rapid-acting insulin (e.g. Insulinaspart, InsulinLispro, and InsulinGlulisine), long-acting insulin (e.g. Insulinglargine, InsulinDetemir, and InsulinDegludec), and modified conjugate (e.g. PEG-Insulin and PEG conjugatedinsulin lispro ), and also relates to the biological value determination method for the cell culture in vitro of PEG-Insulin and application thereof. The external biological activity determination method for human insulin and analog or conjugate is induced in vitro into adipocyte by using preadipocyte of male rat, and measure the glucose consumption after insulin reacting in the adipocyte nutrient solution by using glucose oxidase method to calculate the corresponding insulin biological value.

Description

technical field [0001] The invention belongs to the technical field of human insulin and analogues or conjugates in vitro biological potency determination and detection technology, including fast-acting insulins such as insulin aspart (Insulin aspart), insulin lispro (Insulin Lispro), insulin glulisine (Insulin Glulisine), long-acting insulin Effective insulin such as insulin glargine (Insulin glargine), insulin detemir (Insulin Detemir), insulin degludec (Insulin Degludec) and modified conjugates such as pegylated insulin (such as PEG-Lispro, PEG-Insulin) and PEG- Insulin (PEG conjugated des B30-insulin) in vitro biopotency determination method and its application. Background technique [0002] Insulin is a protein hormone secreted by pancreatic β cells stimulated by endogenous or exogenous substances. Insulin is the only hormone that lowers blood sugar in the body. Insulin mainly promotes the uptake and utilization of glucose by liver cells, muscle cells, and adipocytes, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/31G01N21/78
Inventor 范开何跃
Owner CHONGQING PEG BIO BIOTECH CO LTD
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