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Cell strain for efficiently expressing recombinant human erythropoietin and production process thereof

A high-efficiency expression and cell line technology, which is applied in the field of high-efficiency expression of recombinant human erythropoietin cell lines and production technology

Active Publication Date: 2020-04-17
BEIJING FOUR RINGS BIOPHARM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The object of the present invention is to provide a cell line and production process for highly expressing recombinant human erythropoietin, so as to solve at least one technical problem in the prior art

Method used

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  • Cell strain for efficiently expressing recombinant human erythropoietin and production process thereof
  • Cell strain for efficiently expressing recombinant human erythropoietin and production process thereof
  • Cell strain for efficiently expressing recombinant human erythropoietin and production process thereof

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0121] Sample Preparation:

[0122] Take out the cryopreserved CHO cells from the liquid nitrogen tank. The CHO cells are purchased from ATCC. After resuscitation, they are inoculated into square flasks for cultivation. Stably grown cells were used as sample cells in this experimental example.

[0123] Scheme 1-1

[0124] S1: The sample cells were cultured in square flasks in F-12K medium containing 10% FBS, and the culture conditions were 37°C, 5% CO 2 Static culture; when the cell confluency reaches 70-80%, digest with trypsin for 30 seconds, add complete medium containing 15% FBS to stop digestion, collect the cells detached from the wall by centrifugation, and inoculate into a medium containing 5% FBS Square flask culture was carried out in F-12K medium at 37°C, 5% CO 2 Static cultivation;

[0125] S2: When the confluence of the cells reaches 70-80%, digest with trypsin for 30 seconds, add complete medium containing 15% FBS to stop the digestion, and collect the cells ...

experiment example 2

[0146] Sample Preparation:

[0147] The cryopreserved CHO cells were taken out from the liquid nitrogen tank, and the CHO cells were purchased from ATCC. After resuscitation, they were inoculated into F-12K medium containing 10% FBS for square bottle culture. The culture conditions were 37°C, 5% CO 2 Static culture; when the cell confluence reached 70-80%, the cells were collected and inoculated into F-12K medium containing 5% FBS for square flask culture. The culture conditions were 37°C, 5% CO 2 Static culture; when the cell confluence reached 70-80%, the cells were collected and inoculated into F-12K medium containing 1% FBS for square flask culture. The culture conditions were 37°C, 5% CO 2 Static culture; and the cells that grow stably in this medium are used as sample cells in this experimental example.

[0148] Scheme 2-1

[0149] S1: Culture the cells to be acclimated in a square flask in F-12K medium containing 1% FBS; when the confluence of the cells reaches 70-80%...

experiment example 3

[0198] This experimental example tests the methods or steps in the third round of domestication.

[0199] Sample Preparation:

[0200] D1: Take out the frozen CHO cells from the liquid nitrogen tank. The CHO cells are purchased from ATCC. After resuscitation, they are inoculated into F-12K medium containing 10% FBS for square bottle culture. The culture conditions are 37°C, 5% CO 2 Static culture; when the cell confluence reached 70-80%, the cells were collected and inoculated into F-12K medium containing 5% FBS for square flask culture. The culture conditions were 37°C, 5% CO 2 Static culture; when the cell confluence reached 70-80%, the cells were collected and inoculated into F-12K medium containing 1% FBS for square flask culture. The culture conditions were 37°C, 5% CO 2 Static culture; when the cell confluency reaches 70-80%, digest with trypsin for 30 seconds, add complete medium containing 15% FBS to stop the digestion, centrifuge to collect the cells detached from t...

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Abstract

The invention provides a cell strain for efficiently expressing recombinant human erythropoietin and a production process thereof. The cell strain is a CHO cell, the CHO cell comprises at least one expression vector, the expression vector enables the CHO cell to express recombinant human erythropoietin under a suitable culture condition, and the cell concentration in a unit culture solution is increased by domesticating the CHO cell, so that the yield of EPO is increased. The production process comprises the steps of cell strain recovery, reactor culture, protein purification and the like, andby optimizing the culture and purification processes, the growth state of cells in a suspension culture environment is improved, and the expression quantity of EPO and the purification yield of EPO are increased.

Description

technical field [0001] The invention relates to the field of biopharmaceuticals, in particular to a cell line and a production process for highly expressing recombinant human erythropoietin. Background technique [0002] Erythropoietin (Erythropoietin, EPO) was first discovered in 1906. It is a glycoprotein mainly produced by the kidney, with a molecular weight of about 35KD. It plays an important regulatory role in the proliferation, differentiation and maturation of progenitor cells. [0003] Before the birth of genetic recombination technology, EPO was mainly extracted from the urine of anemia patients and sheep blood. The physical, chemical and biological properties were difficult to determine, and the yield was low and unstable, making it impossible to apply on a large scale. With the development of technology, DNA recombination technology can be used to construct EPO gene into Chinese hamster ovary cells (CHO cells) to form CHO cells that can express recombinant human...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N5/071C12N15/85C12P21/02C07K14/505C07K1/14C07K1/36C07K1/34C07K1/22C07K1/20C07K1/18
CPCC07K14/505C12N5/0682C12N15/85C12N2500/32C12N2500/35C12N2500/38C12N2500/90C12N2501/90C12N2501/91C12N2509/00C12N2510/02C12N2800/107
Inventor 甘富韩明娣桑建彬杨宋
Owner BEIJING FOUR RINGS BIOPHARM
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