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Cell Line Development
Published in Wei-Shou Hu, Cell Culture Bioprocess Engineering, 2020
Recombinant proteins are produced in mammalian cells through a transient expression of the gene of interest (GOI), or by establishing a cell line that produces the GOI product permanently (Panels 6.2–6.4). Transient expression is frequently used for the generation of small quantities of proteins (up to a few grams) for protein characterization or toxicity testing. First, cells are transfected with a high concentration of plasmids that include the GOI. A large number of plasmids are taken up by cells into the cytoplasm. A fraction of those plasmids then enter the nucleus, where the GOI is transcribed into mRNA. The mRNA molecules are exported back to the cytosol, translated into proteins, and secreted into the medium. Due to the large number of plasmids transfected and the strong promoter used, a large amount of product is produced. The plasmids in the cell are diluted as the cells divide and degraded over time. Thus, the transient production of the protein is usually limited to a few days. Nevertheless, the process is easily carried out (requiring only plasmid preparation) and is frequently used to produce a small quantity of proteins for characterization and testing.
Expression of Cloned Proteins in Mammalian Cells: Regulation of Cell-Associated Parameters
Published in Anthony S. Lubiniecki, Large-Scale Mammalian Cell Culture Technology, 2018
Plate systems, in which the cells can be grown attached to a surface are in many respects the most convenient. The most extensively used are tissue culture dishes, but the same principles apply from the microtiter plates at the small scale to roller bottles and multisurface propagators, having surfaces of up to 0.5 m2/unit, for larger volumes. These systems are convenient and, although labor-intensive, require a minimum of engineering expertise and sophisticated equipment, compared to perfusion or fermentation-type systems. They are, however, relatively inefficient and are useful only for supplying limited quantities of material or for research studies. With recombinant cells, for example, these systems may be the most convenient for producing proteins from transient expression systems. Tissue culture plates are also most often used for the screening studies required for medium optimization since a very large number of conditions must be tested.
Plant-Based Production of Biosimilar Drug Products
Published in Laszlo Endrenyi, Paul Jules Declerck, Shein-Chung Chow, Biosimilar Drug Product Development, 2017
Kenny K. Y. So, Michael R. Marit, Michael D. McLean, J. Christopher Hall
Agroinfiltration can be performed at laboratory scale as spot infiltrations by simply forcing a suspension of Agrobacterium cells into a leaf through its abaxial (under) side using a needle-less syringe (Garabagi et al., 2012b). The technique was first used as part of a rapid screening method to predict whether T-DNA constructs would be successful at directing expression of proteins of interest in stable transgenic plants (Schob et al., 1997). However, it was soon realized that transient expression could rapidly provide large amounts of proteins of interest (Fischer et al., 1999). In our experience, transient expression generally results in 10 to 100 times better expression of proteins of interest compared with expression of the same proteins by stable transgenic plant lines. In published literature, expression differences upwards of 1000-fold in favor of the transient system have been documented (Nausch et al., 2012).
Evaluation of different vector design strategies for the expression of recombinant monoclonal antibody in CHO cells
Published in Preparative Biochemistry & Biotechnology, 2018
Hadi Bayat, Saghar Hoseinzadeh, Eśhagh Pourmaleki, Roshanak Ahani, Azam Rahimpour
Transient expression enables rapid analysis of protein expression in mammalian cells.[15] Therefore, the efficiency of each vector system in expression of the IgG1 antibody was initially analyzed in transient transfection. CHO-K1 cells were transfected using different vector systems in triplicates and antibody expression was evaluated 72 hr post-transfection using a human IgG1-specific ELISA assay (Figure 2). The dual-promoter vector system yielded the highest expression level with titers at 530 ng/ml; which was 1.3- and 1.8-fold higher than the titer achieved in the bicistronic and dual-vector systems, respectively.