As a rich and handsome in the cell world, CHO cells were worth more than $50 billion as early as 8 years ago. Most successful people have a history of blood and tears behind them, and they are no exception.
Long long ago, they are also just obscure ovarian cells in a species called Chinese hamsters.
So, how did Diao Si open the road to counterattack?
Let's turn the clock back to 1919, the E.T. of Peking Union Medical College Hospital. Dr. Hsieh couldn't find a laboratory mouse for a while during his research on pneumococcal infection, so he sent someone to the outskirts of Beijing to catch many Chinese hamsters everywhere for experiments. At that time, no one could have imagined that such a operation, which now seems magical, would take Chinese hamsters to a "point of no return" where they were domesticated into experimental animals. We also don't know if the group of hamsters who wandered around the outskirts of Beijing will regret not planning the course of action well when they recall it...
In 1924, two researchers working at Concord, Jocelyn Smyly and Charles Young, discovered that Chinese hamsters were easily infected with the parasite Leishmania and caused Black fever, so that Chinese hamster babies were "reduced" to a powerful tool for researchers to study various infectious diseases.
Over time, the role of Chinese hamsters in the medical community has become obvious to all. In 1928, still at Union Medical College, a researcher named Marshall Hertig brought 150 Chinese hamsters to Harvard Medical School, hoping to build them into a strain. He didn't succeed.
Until 1943, modern genetics pioneer Guido Pontecorvo (yes, the Ponte who discovered the exact cycle of fungi) looked at the chromosomes of Chinese hamsters under a low-power microscope and found only 14 (actually 2n = 22), less than other commonly used laboratory mice (40 in mice, 42 in rats), which gave Chinese hamsters another life-giving business - for chromosomal research.
Fast forward to December 1948, one dark winter night, Dr. Robert Briggs Watson, who was in China participating in the Rockefeller Foundation's research program on malaria medicine in Asia, was instructed by Victor Schwentker, the largest supplier of laboratory animals in the northern United States, to take 20 Chinese hamsters donated by Professor Hu Zhengxiang, transported through the flying wars to San Francisco, USA, and finally transferred to New York to successfully hand over to Victor In the hands of Schwentker.
After two years of specialized domestication by Victor Schwentker, Chinese hamsters were finally domesticated into a strain of laboratory animals. In another episode, George Yerganian of Harvard University used a better microscope to find that Chinese hamsters have 22 chromosomes, not the 14 previously reported by Pontecorvo, and even then the little hamsters have an absolute advantage for chromosome research.
In the process of raising Chinese hamsters for a long time, Yerganian explored a special breeding method for its characteristics and made this set public. But perhaps because these little hamsters are not used to eating "Western food", or perhaps because the above feeding methods may not be applicable to all Chinese hamster populations. For a long time, Yerganian became the only supplier of this strain in the United States, and a single supply limited the large-scale use of Chinese hamsters.
In 1957, Dr. Theodore T. Puck of the University of Colorado Medical Center and his colleague Fa-Ten Kao got a female Chinese hamster at the Boston Cancer Research Center (you would never think that this hamster was put in a basket by a middle-aged woman and arrived at the laboratory by 2 days by train, which is a wonderful operation) and successfully isolated the well-known CHO-K1 cell line. Due to the rapid suspension growth and high protein expression of this cell, CHO cells began to gain universal application in scientific research and industry.
In 1983, Dr. Theodore T. Puck founded Cytogen Research and Development, Inc., a company that provides Chinese hamsters to research institutions free of charge.
In 1984, Genentech achieved the first recombinant Chinese hamster ovary cells to express tissue plasminogen activator (t-PA) and was successfully approved for marketing in 1987, which was a landmark event in the production of protein drugs by mammalian cell expression systems.
Subsequently, many foreign protein genes were successively transfected into mammalian cells, and some valuable proteins were continuously expressed, including coagulation factors, erythropoietin (EPO), immune globulin, urokinase, hepatitis B surface antigen (HBsAg) and monoclonal antibodies, which greatly promoted the development of the biopharmaceutical industry.
At the same time, with the popularization of CHO cells in the laboratory, scientists have successfully isolated different subtypes of CHO cell lines, such as CHO-S, CHO DXB11, CHO DG44, CHO-M and GS knockout CHO cells that have received sustained attention in recent years (such as CHOZN of Merck/Sigma Aldrich, CHO GS Xceed of Lonza, CHO cells knocked out by Horizon with rAAV technology).
After years of love and killing with researchers, CHO has finally reached the peak of "rat" life.
At present, Chinese hamster ovary (CHO) cells are the preferred host cells for the production of protein drugs, because CHO cells have the following advantages compared with other systems: (1) CHO cells have accurate processing and modification functions for proteins, so the biological activity of the proteins they express is closer to that of natural proteins; (2) CHO cells have relatively strong ability to tolerate shear force and osmotic pressure, and can choose adherent culture or suspension culture according to culture requirements; (3) The cells after the integration of foreign genes are stable, and the recombinant genes can be efficiently expanded and expressed; (4) The expressed target protein can be transported from intracellular to extracellular, and CHO cells only express a small amount of endogenous protein, which is conducive to the extraction of the target protein.
To date, the FDA (US Food and Drug Administration) and EMA (European Medicines Agency) have approved more than 70 therapeutic mAbs, of which 39 are produced by CHO cells (see Table 1), in addition to hundreds of mAbs in clinical stages.
Pro-Health's XtenCHOTM high-density transient expression system
Introduction: XtenCHOTM high-density transient expression system is a set of high-efficiency transient expression system of proteins and antibodies independently developed by Atagenix based on CHO-K1, which not only has a high expression amount (generally 200-400mg/L, and the expression of some antibodies is as high as 1g/L), but also has a simple process, which is suitable for small-volume high-throughput screening of humanized antibodies and large-volume scale-up production.
Recombinant proteins are widely used in antigen preparation, protein interaction, enzymatic analysis, drug target research, etc., and selecting the appropriate recombinant protein expression method is crucial to obtain the required quantity and quality of recombinant protein in a timely manner. Conventional recombinant protein expression system is not unfamiliar to us, mainly including prokaryotic expression system, mammalian cell expression system, yeast expression system, Bacillus subtilis expression system, insect baculovirus expression system, each with its own advantages and disadvantages, among which the mammalian cell expression system is more concerned because the protein it expresses is closer to the natural state and the easiest to retain biological activity, but the expression amount is usually a big problem.
In clinical and laboratory research, it is often required to produce a certain amount of recombinant protein in a short period of time. The production process of recombinant protein by stably expressed cell lines is cumbersome and long-term; Transient gene expression technology can express recombinant proteins in a short period of time, so it is widely used. With the development of biomedical research technology, the rapid production of milligram-level recombinant protein transient expression systems is widely used in new drug screening and preclinical research.
1 Introduction to the XtenCHOTM High-Density Transient Expression System
XtenCHOTM high-density transient expression system is a set of high-efficiency transient expression systems for proteins and antibodies independently developed by Atagenix, mainly including the following components:
① XtenCHOTM cell line； ② ATGfect solution； ③ Basic expression medium/ATGfeed medium。 The system not only has a high expression (generally 200-400mg/L, and the expression of some antibodies is as high as 1g/L), but also has a simple process, which is suitable for small-volume high-throughput screening of humanized antibodies and large-volume scale-up production.
2 Advantages of XtenCHOTM high-density transient expression system
(1) XtenCHOTM cells are a genetically modified recombinant CHO cell line developed by Atagenix, which can increase the number of expression vector copies transfected into cells by using vectors containing supporting elements, and prolong the residence time of free plasmids in cells, so that the target gene carried by the vector can obtain high level and continuous expression.
(2) XtenCHOTM high-density transient expression system improves the conventional transfection method of CHO, adopts a novel high-density transfection method and special cell culture mode, improves the viability rate of transfected cells, and extends the survival time of transfected cells from 6-7 days to 10-14 days, further improving the yield of target protein.
(3) Compared with the commonly used cationic liposome transfection reagents, commercial CHO transient medium and feed media, the transfection reeagent, expression medium and feeding medium of XtenCHOTM high-density transient expression system are greatly reduced, more suitable for industrial production, and more cost-effective in the transient expression production of large-scale recombinant proteins.
3 XtenCHOTM high-density transient expression system test results
3.1 Comparison with other commercial expression systems
One antibody was expressed simultaneously using the XtenCHOTM high-density transient expression system and two other commercial CHO transient expression systems, CHO Expression System 1 and CHO Expression System 2, and the transfected cell density and viability monitoring and antibody yield were shown in Figures 1 and 2:
Fig. 1 Monitoring of cell density activity after transfection of different expression systems
Fig. 2 Comparison of the yield of the same antibody expressed by different expression systems
3.2 Expression tests for different therapeutic recombinant antibodies
Four typical therapeutic recombinant antibody sequences were selected and expressed by the XtenCHOTM high-density transient expression system. Antibody yield is shown in Figure 3:
Figure 3 Yield assay of XtenCHOTM system expressing different antibodies
Ab1: Pembrolizumad, Ab2: Utomilumab, Ab3: PF, Ab4: Claudiximab
Pro-Health Biotech uses its own high-density recombinant antibody expression system XtenCHOTM to achieve full-length and multi-form recombinant antibody expression, and uses self-developed cell lines and transfected cell lines to achieve gram-level production of recombinant antibodies. At the same time, it has a dedicated main line of endotoxin control and removal, which can achieve pharmaceutical-grade endotoxicity control (<0.1EU/mg), and can provide a series of high-quality drug control antibodies for scientific researchers to purchase and use.
| Specificity Target | Name | Catalog | Species | identification | Source |
| IL-6R | Tocilizumab | ATAD00661 | Humanized | IgG1 | CHO cells |
| ANGPTL3 | Evinacumab ( Ivesumab ) | ATAD00368 | Homo sapiens | IgG4-kappa | CHO cells |
| C5 | Eculizumab (eculizumab) | ATAD00006 | Humanized | IgG2-G4-kappa | CHO cells |
| C5 | Ravulizumab | ATAD00472 | Humanized | IgG2-G4-kappa | CHO cells |
| CALCA /CALCB | Eptinezumab | ATAD00452 | Humanized | IgG1-kappa | CHO cells |
| CD19 | Tafasitamab | ATAD00363 | Humanized | IgG1-G2-kappa | CHO cells |
| CD19 | Inebilizumab | ATAD00387 | Humanized | IgG1-kappa | CHO cells |
| CD22 | Inotuzumab ( Olintuzumab ) | ATAD00166 | Humanized | IgG4-kappa | CHO cells |
| CD3 | Teplizumab | ATAD00665 | Humanized | CHO cells | |
| CD3E | Otelixizumab ( Oxixiizumab ) | ATAD00147 | Chimeric,Humanized | IgG1-lambda | CHO cells |
| CD52 | Alemtuzumab ( Alemtuzumab ) | ATAD00001 | Humanized | IgG1-kappa | CHO cells |
| CTLA4/CD152 | Tremelimumab | ATAD00156 | Homo sapiens | IgG2-kappa | CHO cells |
| EGFR/ERBB1 | Necitumumab | ATAD00193 | Homo sapiens | IgG1-kappa | CHO cells |
| ERBB2 | Trastuzumab (trastuzumab) | ATAD00686 | Humanized | Human IgG1 | CHO cells |
| ERBB2/EGFR2/CD340 | Pertuzumab | ATAD00025 | Humanized | IgG1-kappa | CHO cells |
| IGF1R/CD221 | Ganitumab ( ganitumab ) | ATAD00096 | Homo sapiens | IgG1-kappa | CHO cells |
| IGHE | Tanezumab ( Tanezumab ) | ATAD00142 | Humanized | IgG1-nd | CHO cells |
| IL12B | Ustekinumab (ustekinumab) | ATAD00100 | Homo sapiens | IgG1-kappa | CHO cells |
| IL17A | Secukinumab ( Secukinumab ) | ATAD00222 | Homo sapiens | IgG1-kappa | CHO cells |
| IL1B | Canakinumab ( Canakinumab ) | ATAD00155 | Homo sapiens | IgG1-kappa | CHO cells |
Resources:
Rawley JD. Theodore T. Puck (September 24, 1916–November 6, 2005). American Journal HumanGenetics 2006;78(3): 365–366.
Kim JY et al. CHO cells inbiotechnology for production of recombinant proteins: current state and furtherpotential. Applied Microbiology and Biotechnology 2012;93(3): 917–930.
Urlaub G; Chasin LA. Isolation of Chinesehamster cell mutants deficient in dihydrofolate reductase activity[J]. Proceedings of the National Academy of Sciences U.S.A., 1980-07, 77 (7):4216-4220.
Landauer K, Woischnigg H, Hepp N et al (2011)Development of a chemically defined CHO medium by engineering based on a feed solution. BMC Proc 5(Suppl 8):P41
CHO MEDIA PLATFORM FACILITATES INTEGRATED CELLLINE DEVELOPMENT AND MEDIA OPTIMIZATION, IrvineScientic Poster in ESACT 2017