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High-yield reactor for protein production, and production method and application thereof

A bioreactor and reactor technology, applied in the direction of bioreactor/fermenter combination, specific-purpose bioreactor/fermenter, biochemical equipment and methods, etc., can solve the high time and capital cost, can not be retained Target protein, low single-batch protein yield, etc., to reduce abnormal structural changes, increase operational flexibility, and improve product yield and purity.

Inactive Publication Date: 2013-09-18
WUXI BIOLOGICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The conventionally used hollow fiber column has a diameter of 1 mm and a pore size of 0.2 μm, but while retaining cells, it cannot retain the target protein
[0006] Therefore, even if ATF perfusion equipment (Refine Technology Company, USA) is used as a technical module, there are still some significant problems in industrialization, such as the extremely high consumption of medium and the preference of cells for growth rather than protein expression. Disposable production equipment is limited by the low single-batch protein output, which still requires high time and capital costs

Method used

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  • High-yield reactor for protein production, and production method and application thereof
  • High-yield reactor for protein production, and production method and application thereof
  • High-yield reactor for protein production, and production method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Using the above-mentioned high-yield reactor to carry out the production method of monoclonal antibody, including steps:

[0057] (1) Connect the bioreactor 1 with the ATF perfusion device 2 of the corresponding model to complete the off-line sterilization or in-situ sterilization procedure;

[0058] (2) Using the growth medium, seed the cells at 0.3×10 6 ~1×10 6 Inoculate bioreactor 1 to the working volume at the density of cells / mL, control temperature (36-37°C), pH (6.8-7.2), dissolved oxygen concentration (40%-60%), and tank pressure (2-8psi), every 24-hour sampling to detect cell number and biochemical indicators;

[0059] Among them, the biochemical indicators include: pH, O 2 Dissolved partial pressure, CO 2 Partial pressure of dissolution, glutamine content, glutamic acid content, glucose content, lactic acid content, NH 4 + Level, Na + Level, K + Level, Ca + level, osmotic pressure, etc.;

[0060] (3) When the living cell density increased to 3×10 6...

Embodiment 2

[0069] According to the general fed-batch process, although CHO cells for the production of anti-CD20 antibodies can grow to 7×10 6 The living cell density of cells / mL, but the expression level of the target protein anti-CD20 antibody is only 0.25 g / L.

[0070] However, according to the production method of Example 1, the CHO cells for the production of anti-CD20 antibodies have reached the highest cell growth density nearly 10 times that of the general fed-batch process and 8 times the target protein yield (as shown in Figure 5).

Embodiment 3

[0072] According to the production method of Example 1, the CHO cells for the production of Humira monoclonal antibody reached a maximum cell growth density of 140 × 10 in just 11 days. 6 The growth of cells / mL and the yield of target protein (Humira monoclonal antibody) of nearly 9 g / L (as shown in Figure 6).

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Abstract

The invention discloses a high-yield reactor, and a production method and an application thereof. The high-yield reactor comprises a bioreactor and an ATF perfusion apparatus connected with the bioreactor. The protein production method using the high-yield reactor comprises the following steps: 1, connecting the ATF perfusion apparatus with the bioreactor, and completing an offline disinfection or online disinfection program; 2, inoculating seed cells to the bioreactor to a work volume according to the density, and sampling and detecting the number and biochemical indexes of the cells each 24h; 3, starting the ATF apparatus program for perfusion culture when the density of living cells increases to a certain density; and 4, collecting a culture solution in the bioreactor or filtered through hollow fibers in the ATF perfusion apparatus, and purifying to obtain required protein products. The high-yield reactor has the advantages of great shortening of the technological exploitation time of monoclonal antibody or fusion protein expression, production cost reduction, benefiting for the project registering and reporting and the project period shortening, and acceleration of the accomplishment application speeds of the biopharmacy industry.

Description

technical field [0001] The present invention relates to a high-yield reactor for protein production (including monoclonal antibody or monoclonal antibody-like protein), in particular to a novel high-yield reactor for protein production and its production method and application. Background technique [0002] At present, domestic and foreign biopharmaceutical industries use mammalian engineering cell lines to express and produce monoclonal antibodies or fusion proteins, mainly using large-scale batch fed-batch culture. [0003] Among them, batch fed-batch feeding of mammalian cells is a routine method, which requires long-term process exploration and a large number of condition verification and repetition in research and development. In cGMP (Current Good Manufacture Practices) production, it often requires repeated verification and huge volume. Advanced cell culture equipment and complex supporting systems. That is, the traditional fed-batch culture of mammalian cells has so...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12M3/00C12M1/36C12M1/02C12P21/00C12P21/08
Inventor 李锦才李振东
Owner WUXI BIOLOGICS CO LTD
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