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Process for animal cell non-serum cluster perfusion culture

A serum-free medium, animal cell technology, applied in the field of cell engineering and biopharmaceuticals, can solve the problem of no report, and achieve the effect of increasing yield, simple method and increasing cell density

Active Publication Date: 2009-09-23
石药集团明复乐药业(广州)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since these problems are not well resolved, there are no reports of actual use in production so far

Method used

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  • Process for animal cell non-serum cluster perfusion culture
  • Process for animal cell non-serum cluster perfusion culture

Examples

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Effect test

Embodiment 1

[0025] Production of Thrombolytic Drug TNK-tPA by Condensed Serum-free Perfusion Culture of CHO Engineering Cell A2 Strain Under Bubble-free Ventilation

[0026] 1. Cell acclimatization: the production cell is the genetically recombined CHO engineered cell A2 cell line, which is cultured in a DMEM / F12 medium supplemented with 5% calf serum in a stirring bottle at a stirring speed of 150 rpm. Then reduce the amount of serum to 3%, and then to 2%, 1%. At this time, add folic acid, ferric citrate, zinc sulfate, copper sulfate, sodium selenite, Vc, VB6, peptone and insulin to the medium . Later, the serum was further reduced to 0.5%, so that the serum was completely eliminated. The acclimatization process is completed when the growth and proliferation speed of the cells are comparable to that when cultured with 5% serum, and then they are frozen.

[0027] 2. Cell clumping: The cells that have adapted to serum-free culture are cultured in three ways: square flask, spinner flask a...

Embodiment 2

[0031] Production of TNK-tPA by Condensed Serum-free Perfusion Culture of CHO Engineering Cell A2 Strain Under Surface Ventilation and Bubble Free Ventilation

[0032] 1. Recovery of acclimated cells: Take out the frozen cell tube from the liquid nitrogen tank, put it in water at about 40°C to melt immediately, centrifuge the cell solution at 1500rpm for 3 minutes, discard the preservation solution, and replace it with DMEM supplemented with 2% calf serum / F12 medium 10ml, cultured in T30 square bottle at 37°C, digested with trypsin after 2 days, gradually amplified at a ratio of 1:3, and then transferred some cells to spinner bottles for 2% serum culture, and some cells were transferred to Stir flask serum-free culture for further expansion.

[0033] 2. Cell clumping: with the method described in (2) of Example 1, the cells cultured with serum will be replaced with serum-free medium after the cells are adhered to the sheet, so that the cells are clumped, and then initially co...

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Abstract

The invention discloses a technique used for the culture of serum-free clustering and perfusion of animal cell, comprising the steps that: (1) engineering cell is domesticated during a stirring culture in a serum-free culture medium; (2) the engineering cell is primarily clustered during a culture in the serum-free culture medium in a rotary bottle, a square bottle and a stirring bottle; (3) the cell is further clustered and cultured in a reactor jar through a combination perfusion system of ultrasonic and settlement; (4) part of cells are drawn irregularly to keep a proper high density of cells in the jar and are applied to large-scale production. The technology of the invention greatly improves the clustering efficiency and interception efficiency of the cells during the perfusion and ensures the ultrasonic perfusion system to fully give play to the double-function of cell clustering and interception, thereby leading the clustering culture of cells to be really applied to actual production, reducing production cost, improving cell density, prolonging production cycle and increasing output.

Description

technical field [0001] The invention relates to the fields of cell engineering and biopharmaceuticals, in particular to a method for serum-free clumping perfusion culture of animal cells. Background technique [0002] At present, in terms of biotechnology pharmaceuticals, the use of large-scale cultivation of animal cells to produce genetically engineered drugs has accounted for 70%. Therefore, how to improve the technology of mass culture of cells is the key to increasing production and reducing costs. One of them is to strive to replace the traditional batch and batch-feeding process with perfusion culture technology, increase the cell density in the reactor, and prolong the culture cycle. For perfusion culture, in addition to effective cell retention equipment, how to increase the volume of cell clusters will also help the separation of cells and medium. The use of microcarrier culture has proven to be a good method. However, microcarriers are expensive and cumbersome ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/06C12N5/02
Inventor 肖成祖杨琴李智黄小乐梁倩茹陈小飞郑敦武陈晓铭
Owner 石药集团明复乐药业(广州)有限公司
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