By Sadettin Ozturk, Wei-Shou Hu
Edited by means of of the main uncommon pioneers in genetic manipulation and bioprocess expertise, this bestselling reference provides a accomplished review of present phone tradition expertise utilized in the pharmaceutical undefined. Contributions from numerous best researchers show off the significance of gene discovery and genomic know-how improvement within the construction of biotechnology items, tissue engineering, and cell-based cures. supplying specific counsel, they hide the stairs best as much as the production of plausible remedies together with host mobile choice, cloning and gene amplification, bioreactor layout and operation, protein purification, optimization, scale-up, and facility design.
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Additional resources for Cell culture technology for pharmaceutical and cell-based therapies
For production of small quantities of such molecules over a short timeframe, transient expression systems can be the method of choice whereas for clinical or commercial production, stable expression systems capable of continuous product expression over prolonged periods (months) generally are required. This chapter will discuss in detail the steps involved in the process of construction, selection, and screening of stable cell lines for industrial production of biotechnology products (Fig. 1). Transient Expression In transient expression systems or ‘‘transient transfections,’’ DNA is introduced into the cell and maintained=replicated as an extrachromasomal unit.
In many instances, several media exchanges using centrifugation of cells away from spent media and resuspension in fresh media need to be performed in order to remove waste products and maintain an adequate seeding density (3–10 Â 105 cells=mL). It is important that selective pressure be maintained on the gene of interest throughout this process to ensure retention of high-level gene expression. However, the concentration of selective agent and of supplemental growth factors may need to be temporarily modiﬁed during this procedure to ensure success.
In contrast to dominant markers, recessive markers generally restore a critical enzyme or protein to the cell that is required for survival, and therefore require host cells that are mutant or deﬁcient in that activity. Often the activity is required for cellular DNA synthesis. In the nontransfected host cell, the requirement for the activity is replaced by supplementing the media with the metabolic byproduct of the enzyme’s action. Upon withdrawal of these media components cell survival is dependent on acquiring the needed activity through transfection.