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Recombinant Protein Production using CHO Media Systems

A significant percentage of today’s biopharmaceutical development utilizes recombinant proteins. These important biological molecules are produced with mammalian protein expression systems and preferred for the ability to produce complex proteins and posttranslational modifications (PTM) such as glycosylation and phosphorylation. 

Among the protein expression systems, CHO cells are involved in >70% of recombinant biopharmaceutical proteins. The CHO-GS cell line is the prevalent protein expression system worldwide for therapeutic antibodies.

CHO cell protein production laboratories are predominantly using serum-free CHO media. Read more about why to culture cells using serum-free media.

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Chinese Hamster Ovarian Cells (CHO)

The Chinese Hamster belongs to a family of rodents, native to the desert of northern China. The hamster was introduced in the lab in 1919 for typing pneumococci. In the early 1920s they gained reputation and became valuable tools in epidemiological research, because they were known as carriers of the deadly parasite Leishmania causing Black fever. It was thought in the past the hamsters would be used as agents of biological warfare by infecting them with deadly diseases and parachuting them over land. Instead, Chinese Hamsters are today credited with saving thousands of lives from illnesses such as cancer every day. We know the CHO cells as the most commonly used protein expression system.

Why CHO cells are frequently used for mammalian protein expression: 

  • CHO cells are excellent in protein folding of complex proteins including their posttranslational modification (PTM)
  • CHO cells can grow very well in serum-free cell culture medium which eliminates serum as a potential source for infectious agents, providing more safety 
  • CHO cells grow mostly in suspension, allowing volumetric scalability and resulting in high cell densities 
  • CHO cells protein expression systems facilitate genetic manipulation (dihydro-folate-reductase activity—DHFR or GS deficiency) 
  • CHO cells have a high protein yield. Typically, 2–6 g/L can be achieved (GS Xceed Expression System provides the highest yield) 
  • CHO cells in serum-free media provide easy downstream processing 
  • CHO cells can be transfected transient or stable to express proteins 
  • CHO cells have a long history of regulatory acceptance 
    • In the case of monoclonal antibodies, Chinese Hamster Ovary (CHO) and NS0 (mouse myeloma) cells are the most commonly used lines 
    • CHO cells used in >70% of current approved recombinant proteins

CHO protein expression systems

When working with CHO cells for protein expression, two gene selection systems are most often used: DHFR/MTX and GS/MSX

How the DHFR system works

The diagram to the left shows how the DHFR enzyme catalyzes the conversion of folate to tetrahydrofolate. This precursor is necessary for de-novo synthesis of purines and pyrimidines (Goeddel, 1990), allowing cells to replicate. 
 
When the protein expression system in CHO cells has the DHFR deletion, researchers transfect CHO cells with recombinant DNA consisting of the gene of interest closely linked to the gene for DHFR. The methotrexate (MTX) selection system is used to select CHO cells producing the protein of interest.  MTX, a drug similar to folate, binds to DHFR, thereby inhibiting the production of tetrahydrofolate, which is necessary for the de-novo synthesis of purines and pyrimidines. 
  
During the gene amplification process, CHO cells are cultured in increasingly higher levels of MTX. CHO cells that have increased copies of the DHFR gene, combined with the gene of interest, will be selected. CHO cells with insufficient levels of DHFR are deprived of nucleoside precursors (hypoxanthine and thymidine) and die. Once selected, transfected cell lines derived from the CHO DHFR negative host do not require MTX in the culture medium.


How the GS system works

The diagram to the right shows how the GS enzyme catalyzes the production of glutamine from glutamate and ammonia, allowing cells to replicate.

When the protein expression system in CHO cells has the GS deletion, researchers transfect CHO cells with recombinant DNA consisting of the gene of interest closely linked to the gene for GS. The methionine sulphoximine (MSX) selection system is used to select CHO cells producing the protein of interest. MSX, a drug similar to glutamate, binds to GS, thereby inhibiting the production of glutamine, which is necessary for cell to growth.

During the gene amplification process, CHO cells are cultured in high levels of MSX. CHO cells that have increased copies of the GS gene, with the gene of interest, are selected. CHO cells with insufficient levels of GS will die.

Once selected, transfected cell lines derived from the CHO GS negative host do not require MSX in the culture medium.

Explore our CHO media portfolio

TheraPRO® CHO Media System

An advanced formulated, TheraPRO® CHO Media System, accelerates process development and fast-tracks products to market with a drastically simplified system
  • Chemically defined, non-animal origin, serum free
  • Achieves high titers and product quality in CHO-GS and CHO-K1 cell lines
  • Demonstrates scalability and consistency across various scales
  • Available in a liquid solution, and as medium and feed in powder format

PowerCHO® 2 Media System

PowerCHO® 2 media is designed for high-density CHO cultures, ensuring consistent performance, streamlined downstream purification, and regulatory compliance.
  • Chemically defined, non-animal origin, serum free
  • Achieves high cell density and protein titer in various CHO cell lines (CHO DG44, K1, DXB11) in suspension
  • Demonstrates scalability and consistency across various scales.
     

ProCHO® 5 Media System

ProCHO® 5 media supports high cell density of CHO cells, enhancing the performance and efficiency of commercial manufacturing.
  • Non-chemically defined, serum-free, non-animal origin
  • Achieves high titer, consistent product quality, and improved scalability in various CHO cell lines (CHO DG44, K1, DXB11)
  • Simple formulation to support adaptation and growth in suspension culture

     

Explore our manufacturing capabilities

Verviers, Belgium

  • Operating since 1994, production and distribution site 
  • 30,000 shipments a year
  • Both media and liquid manufacturing
    • 10,000L liquid batches
    • 1,000Kg powder batches
  • ISO 9001:2015
  • ISO13485:2016 certified
  • 21 CFR 820 compliant
Verviers, Belgium Location

Walkersville, USA

  • Operating since 1947, production and distribution site 
  • 64,800 shipments a year 
  • Both media and liquid manufacturing 
    • 10,000L liquid batches
    • 7,000kg powder batches
  • ISO 9001:2015 certified
  • ISO 13485: 2016 certified

Walkersville, USA Location

References

Butler M and Spearman M. The choice of mammalian cell host and possibilities for glycosylation engineering. Curr Opin Biotechnol 2014 Dec:30:107-12

Goeddel DV. Systems for heterologous gene expression. Methods Enzymol 1990:185:3-7