Mammalian cells protein production

Zhu, J.W. (2012) Mammalian cell protein expression for biopharmaceu-tical production. Biotechnol. Adv., 30, 1158-1170. 

Westaway, E. G., 1973, Proteins specified by group B togaviruses in mammalian cells during productive infections, Virology 51 454. 

If the hGH is exported to the culture medium the product can easily be collected by removal of the cells from the culture medium by centrifiigation. Purification of hGH from the culture medium is faciUtated by low amounts of contaminating proteins present. In fact, it has been shown that hGH can be purified on a laboratory scale by a single purification step on a reversed-phase hplc column (43). Mammalian cells growing in tissue culture have also been used as hosts to produce hGH, which is exported into the culture media (44). 

Insect Cells. In this system the cDNA is inserted into the genome of an insect vims, baculovims. Insect cells, or Hve insect larvae, are then infected with the vims. In this way advantage is taken of the vims s natural machinery for repHcation utilizing the insect cell. This is one of the best systems available for high level production of native protein having post-translational modifications similar to those seen in mammalian cells. Disadvantages of this system include lytic—batch variations, comparatively slow growth, and cosdy scale-up. 

MammaBan. For mammalian proteins, mammalian cells offer the most natural host for expression. Problems of incorrect processing and post-translational modification are avoided using these cells. Mammalian cells are usually grown in continuous cell culture, reducing the variabiUty in results (see Cell CULTURE technology). Moderate-level production of native protein is possible. The procedure, however, is slow and very cosdy, and the level of protein expression is low. Thus large-scale production of proteins in mammalian cells is not practical. When low quantities of protein are sufficient, this system offers the several advantages described. 

Nutritional Requirements. The nutrient requirements of mammalian cells are many, varied, and complex. In addition to typical metaboHc requirements such as sugars, amino acids (qv), vitamins (qv), and minerals, cells also need growth factors and other proteins. Some of the proteins are not consumed, but play a catalytic role in the cell growth process. Historically, fetal calf semm of 1—20 vol % of the medium has been used as a rich source of all these complex protein requirements. However, the composition of semm varies from lot to lot, introducing significant variabiUty in manufacture of products from the mammalian cells. 

Product formation kinetics in mammalian cells has been studied extensively for hybridomas. Most monoclonal antibodies are produced at an enhanced rate during the Gq phase of the cell cycle (8—10). A model for antibody production based on this cell cycle dependence and traditional Monod kinetics for cell growth has been proposed (11). However, it is not clear if this cell cycle dependence carries over to recombinant CHO cells. In fact it has been reported that dihydrofolate reductase, the gene for which is co-amplified with the gene for the recombinant protein in CHO cells, synthesis is associated with the S phase of the cell cycle (12). Hence it is possible that the product formation kinetics in recombinant CHO cells is different from that of hybridomas. 

Normal circulating levels of tPA are low, so that to accomplish this dramatic clot breakdown one would need the amount of tPA contained in 50,000 liters of blood. This is clearly not practical. Instead, the molecule has been cloned and expressed in mammalian cells so that it can be produced in quantity. Using cells from mammals, rather than bacteria, results in a product molecule that has the same folding, internal bonding, and coat of sugar residues as the natural protein. 

Mammalian cells Get export of proteins Get desired post-translational modifications and products not likely to be immunogenic to humans Good expression systems available Large-scale growth of animal cells costly Great care needed to avoid contamination of cultures... 

Factor IX Replacement Hemophilia B therapy may include recombinant (produced via transfection of mammalian cells with the human factor IX gene) or plasma-derived (concentrate from pooled plasma) factor IX (see Table 64-2). Guidelines for choosing the factor-concentrate formulation for hemophilia B are similar to the guidelines for hemophilia A. However, older-generation factor IX concentrates containing other vitamin K-dependent proteins (e.g., factors II, VII, and IX), called prothrombin complex concentrates (PCCs), have been associated with thrombogenic side effects. Consequently, these products are not first-line treatment for hemophilia B.11... 

Geisse, S. and Henke, M. (2005) Large-scale transient transfection of mammalian cells a newly emerging attractive option for recombinant protein production. Journal of Structural and Functional Genomics, 6 (2-3), 165-170. 

For these proteins, mammalian cells proved better hosts, as they could process the protein with intracellular machinery similar to that in humans. However, large-scale production of proteins in cell culture was problematic. Mammalian cells had to grow attached to a solid surface, such as glass in roller bottle culture. While the Federal Drug Administration (FDA) had approved some processes for vaccine production that used cell cultures, it required that these cells be normal. Normal mammalian cells can divide only a few times before they stop growing, making scale-up to large volumes difficult. 

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