High-officient production of recombinant adenovirus carrier

A recombinant adenovirus and vector technology, which is applied in the field of cell engineering, can solve the problems of inability to culture cell sampling and observation, difficulties in harvesting recombinant adenovirus vectors and scale-up of production, and achieve the effects of tight connection, convenient harvesting, and easy scale-up

Inactive Publication Date: 2006-05-31
INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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  • Application Information

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Problems solved by technology

However, this production process has the disadvantages that it is impossible to sample and observe the cultured cells, and it is difficult to harvest the recombinant adenovirus vector and scale up the production scale.

Method used

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  • High-officient production of recombinant adenovirus carrier
  • High-officient production of recombinant adenovirus carrier
  • High-officient production of recombinant adenovirus carrier

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1, expand pAd-TH-GFP in 250ml stirring type cell culture flask

[0026] (1) Carrier-free immobilized culture of HEK cells:

[0027]HEK cells (purchased from Invitrogen, USA) cultured in 100ml tissue culture flasks were digested with 0.25% (w / v) trypsin, and treated with 1% (v / v) calf serum, 4mM glutamine DMEM / F12 medium (DMEM:F12 (1:1, v / v)) was dispersed and the concentration of the cell suspension was adjusted to 4×10 5 cells / ml, inoculate a stirred cell culture flask with a working volume of 250ml at 250ml / bottle, at 37°C, 5% CO 2 Suspension culture under culture conditions.

[0028] On the 1st to 3rd day of culture, the stirring speed was set at 25-50 r / min, so that the cells aggregated with each other to form a spherical cell mass. From the 2nd to 3rd day of culture, place the stirred cell culture bottle in a clean workbench for 1 to 2 minutes every day, so that the cell mass settles at the bottom of the bottle, and replace with fresh medium according...

Embodiment 2

[0035] Example 2, Propagation of pAd-TH-GFP in a 5L Stirred Tank Bioreactor

[0036] (1) Preparation of HEK seed cells.

[0037] HEK cells were adjusted to a cell suspension concentration of 3.5×10 with DMEM / F12 medium containing 1% (v / v) calf serum and 4 mM glutamine. 5 cells / ml, inoculate 250ml / bottle into a stirred cell culture flask with a working volume of 250ml, and culture HEK cells according to the above-mentioned carrier-free immobilized culture method.

[0038] After culturing for 8 days, replace 90% of the culture volume with Ca-free culture medium containing 500 IU heparin and 1% (v / v) calf serum. 2+ DMEM medium, set the stirring speed to 60r / min, 37°C, 5% CO 2 Suspension culture under culture conditions for 24 hours to loosen and disaggregate HEK cell clusters, and serve as seed cells for carrier-free immobilized culture of HEK cells and expansion of pAd-TH-GFP in a 5L stirred tank bioreactor.

[0039] (2) Carrier-free immobilized culture of HEK cells in a 5L s...

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Abstract

The invention is about a method that can produce recombination adenovirus vector highly, and belongs to the field of cell engineering technology. The cell, which puts the HEK cell as the recombination adenovirus vector, can produce the dissociative cell into cell mass under the Suspension Culture, or depolymerization cell mass as dissociative cell, using the free settling of cell mass or rotary filter interception cell mass and replacing substrate or affusing substrate continuously, it can recombinate the adenovirus infection HEK cell mass and culture it to amplify in the cell. Its advantages are:óƒThe activity of the cell is greater or equal to 90%, the consistency of live cell is greater or equal to 1í‡10 to the power 7 cells/ml.óIt uses the recombination adenovirus vector to infect HEK cell mass directly and enlarge breed in the cell, and can carry into execution substrate replacing or continuous perfusion with the free settling of HEK cell mass or intraoral rotary filter.ó�The titer of pAd-TH-GFP can reach 1-5í‡10 to the power 13 GTU/ml, the product capacity of 5L stirred tank bioreactor can reach 1-2í‡10 to the power 17 GTU/ml, the gain of recombination adenovirus vector is convenient.óœThe producing process is tight and unhindered, and the size can be enlarged easily.

Description

technical field [0001] The invention relates to a method for efficiently producing recombinant adenovirus vectors, more specifically, a process method for efficiently producing recombinant adenovirus vectors by using carrier-free immobilized culture of HEK cells, which belongs to the technical field of cell engineering. Background technique [0002] Recombinant adenoviral vectors are viral vectors for effectively introducing therapeutic genes into target cells and / or target tissues in gene therapy research and clinical applications. The production of the recombinant adenoviral vector is usually achieved by infecting human embryonic kidney cells (humanembryonic kidney cells, HEK cells) and making them proliferate in large quantities in the HEK cells. Due to the large dosage and high quality requirements of recombinant adenoviral vectors required for gene therapy, the production of recombinant adenoviral vectors has become one of the key links in the in-depth research and appl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/861C12N5/10
Inventor 陈昭烈刘红刘兴茂吴本传黄培堂李世崇倪小平王启伟
Owner INST OF BIOENG ACAD OF MILITARY MEDICAL SCI OF THE CHINESE
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