Method for increasing culture density of CHO cells

A cell culture and density technology, applied in the field of biomedicine, can solve the problems of reduced cell viability protein production, influence of CHO cell density, unfavorable cell growth, etc., to achieve the effect of improving recombinant protein production, reducing production costs, and improving culture density.

Pending Publication Date: 2020-06-30
SHANGHAI SINOMAB BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lactic acid will cause the pH of the medium to drop, and the addition of alkaline substances to adjust the pH value in the bioreactor will lead to an increase in osmotic pressure, which will adversely affect the growth of cells, and ultimately lead to a decrease in cell viability and protein production , so it is a difficult problem to solve the effect of lactic acid accumulation on CHO cell density in the bioreactor

Method used

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  • Method for increasing culture density of CHO cells
  • Method for increasing culture density of CHO cells
  • Method for increasing culture density of CHO cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1, the construction of expression cell line

[0018] The CHO cell line overexpressing human lactate dehydrogenase C was constructed according to "Construction of CHO cell line overexpressing human LDH-C and its effect on lactate metabolism and cell apoptosis" (Soochow University master's degree thesis, author Jing Yu, 2014 2010), the human LDH-C gene (NM_002301.4) was cloned, the eukaryotic expression vector pcDNA3.1 / hLDH-C was constructed, and transfected with CHO-dhfr - Cells (ATCC number CRL-9096), screened for cells stably expressing human LDH-C, named CHO / hLDH-C.

[0019] The CHO cell lines CHO / cLDH-C and CHO / cLDH-Cm overexpressing hamster (Cricetulus griseus) lactate dehydrogenase C (cLDH-C) and its mutant (cLDH-Cm) were constructed in a similar manner to the above, That is, clone the hamster LDH-C gene (NCBI number NM_001244050.1) and the self-designed mutant gene (SEQ ID NO: 2), and add HindIII (AAGCTT), XhoI (CTCGAG) restriction site, respectively ...

Embodiment 2

[0021] Embodiment 2, the measurement of lactate dehydrogenase expression level and enzyme activity, specific activity

[0022] 1. Measurement method

[0023] The cells screened in Example 1 were monoclonalized, and 10 monoclonals were selected for each, and amplified and cultured to 2×10 6 Cell / mL density, then extract the crude enzyme, measure the total protein, enzyme activity, and specific activity. The specific process is as follows:

[0024] (1) Preparation of crude enzyme extract

[0025] Take 1×10 7 Cells were centrifuged at 1000 g for 5 min, and the supernatant was removed. Mitochondria were extracted using the Mitochondria Isolation Kit (Cat. No.: 89874) from ThermoFisher, and 1 mM PMSF (protease inhibitor) was added, and the cells were sonicated with a sonicator at 30% W. Centrifuge at 1000 g for 5 min at 4°C, and the supernatant is the crude enzyme extract. Quantitative determination of total protein in crude enzyme extracts was performed using the BCA protein ...

Embodiment 3

[0045] Example 3, Determination of CHO cell expression level, enzyme activity and specific activity of overexpressing lactate dehydrogenase

[0046] The original CHO-dhfr - Cells and CHO / hLDH-C cells, CHO / cLDH-C cells, CHO / cLDH-Cm cells transfected with exogenous lactate dehydrogenase gene in 2×10 6 The concentration of cells / mL was inoculated in a 1 L bioreactor, using ThermoFisher Scientific Inc. CD OptiCHO Medium (product number 12681-029), adding L-glutamine (product number 25030) to a final concentration of 5 mM, as a basal medium , the initial volume is 60% of the working volume of the bioreactor, and the fed-batch cell culture process is started. During the cell culture process, glucose was supplemented daily to keep the residual sugar in the tank at 2.5-3.5 g / L, the dissolved oxygen was controlled at 40-60%, and the temperature was 37°C. The feed medium was replenished daily from 3 days after inoculation. The formulation of the feed was shown in the 300S medium descr...

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Abstract

The invention provides a method for increasing culture density of CHO cells. An exogenous lactate dehydrogenase gene is transferred in CHO cells, and is expressed in a fermentation process, so that the lactic acid generated in the culture process can be reduced, and the culture density of CHO cells in a bioreactor is increased.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and more specifically, the invention discloses a method for increasing the culture density of CHO cells. Background technique [0002] CHO (Chinese Hamster Ovary) cell is a Chinese hamster ovary fibroblast cell line established by Puck in 1957. Up to now, CHO cell has become the most important expression or production system of biotechnology drugs. With the development of serum-free suspension culture technology, genetic engineering technology, bioreactor design amplification and intensification technology, large-scale high-density fed-batch and continuous perfusion culture technology, the CHO cell system is widely used in antibodies, gene recombinant protein drugs, Research and development and industrial production of biotechnology products such as virus vaccines. CHO cells are currently the preferred system for the production of recombinant glycosyl proteins, because they have accurate pos...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
CPCC12N5/0682C12N9/0006C12Y101/01027C12Y101/01028C12N2510/02
Inventor 郭清城徐进杨立敏
Owner SHANGHAI SINOMAB BIOTECHNOLOGY CO LTD
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