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Cell expression systems, optimization

Optimization of Cell Expression Systems to Maximize Production... [Pg.48]

Brent Pollock (Biomira Inc.) presented a talk on the rational development of a second-generation production process for recombinant human interleukin-2 that incorporates recent advances in cell expression systems, fermentation optimization, protein extraction, refolding, and purification. A key concept of this talk was to know thy protein. The driving forces for process changes were regulatory concerns/robustness, economics, the ability to use standard equipment, and the elimination of bottlenecks in the process. [Pg.707]

In the next contribution we learn about hands-on experience and recent improvements with different production systems for biopharmaceuticals at Bayer Health-Care. As previously also published in Nature by Heiner Apeler, Head of Expression, an E. coli host/vector system was originally developed for the efficient production of an interleukin-4 variant, but afterwards it was optimized for the expression of other proteins and even Fab fragments. Process development and optimization of the yeast secretory Saccharomyces cerevisiae for expression of a protease inhibitor will also be presented. The focus, however, is on the use of a recently developed mammalian HKBll (hybrid clone of human kidney and B cells) expression system for recombinant human glycoprotein biopharmaceuticals. HKBll is a favorable cell host for the production of human proteins, because it dehvers biopharmaceuticals that are structurally identical to the natural product. The host/vector system supports the production of gram quantities of proteins in a large-scale transient transfection format as well as the development of stable cell fines. These systems together... [Pg.2015]

Stability and Purification of the Recombinant Protein. There are no hard and fast rules specifying, eg, whether a recombinant protein is available in a soluble state in the cell. In some cases, the expression system must be engineered by in vitro mutagenesis to optimize overall yield of the protein. [Pg.237]

The RTS system includes two different technology platforms for cell-free protein expression as well as a number of tools for finding optimal conditions (Scheme 1.1). All expression systems use the T7-polymerase for transcription and an E. coli lyzate with reduced nuclease and protease activity for translation. The conditions are optimized for a coupled transcription/translation reaction so that the DNA can be directly used as the template. [Pg.30]

In addition to screening molecules for intestinal absorption, Caco-2 cells have also been used to study mechanisms of drug transport. For many compounds, intestinal permeation involves a transporter to either aid or limit transepithelial transport. The value of Caco-2 cells in this type of studies is due to the fact that these cells express various membrane transporters relevant to drug absorption.1719-23,28,30 However, when interpreting results of studies that involve carrier-mediated transport, discretion, and scaling factors may be required because of the difference in expression level of transporters between in vitro and in vivo systems.12 Another important consideration in carrier-mediated transport studies is that some transport systems in Caco-2 cells may achieve maximal expression level at different days in culture.17,21,38,74 Thus, validation of Caco-2 cells for mechanistic studies should include the identification of the time for optimal expression of transporters as well as the qualitative evaluation of the transporters to establish that they are representative of the native intestinal transporters. [Pg.171]

As it is imperative that the plant-derived hiopharmaceutical product must be obtained repeatedly and on a consistent basis, a master cell culture bank, seed bank for transgenic plants, or virus seed stock for transient expression systems must be constantly maintained. Storage conditions must therefore he optimized to prevent contamination and ensure viability. Both transgene stability (e.g., reversion to wild type or sequence drift of plant virus expression vectors) and protein expression levels must be monitored in a representative plant of a given bank or stock to minimize any possible variation in expression levels that may affect safety and consistency of the hnal product. A program that monitors lot-to-lot consistency of the hiochemical and biological properties by comparing the product with appropriate in-house reference standards could he implemented as a fundamental component of product development. [Pg.184]

While the initial goal of the discovery process is to express and purify the recombinant protein, without regard to cost and as quickly as possible, ultimately the production of a recombinant protein must be optimized to ensure that it can be produced at a reasonable cost. In most cases expression systems used to produce small quantities of recombinant protein for preliminary evaluation are not suitable to produce large quantities. Therefore, the gene inserted in the initial expression vectors must be systematically modified and recloned into vectors that can produce (1) a high yield of expressed protein, (2) stable host cell transfections, and (3) protein excretion. [Pg.48]

Since constitutive expression in high yields negatively influences cell metabolism with consequences up to and including cell death, the commonly used expression systems are inducible. The goal of inducible overexpression is a yield of soluble target protein between 5 and 50%, typically about 20%, of total cell protein. If expression as soluble protein is deemed impossible, alternatives are (i) expression as inclusion bodies (insoluble protein fraction), (ii) expression as a fusion protein, or (iii) secretion into the medium. For a good ratio of soluble to insoluble recombinant protein (the ratio is hardly ever infinity), expression criteria have to be optimized. [Pg.82]

In summary, it can be concluded that the optimization of the expression of heterologous genes in animal cells deserves careful design. Firstly, the biological properties of the final product and the amount required for its further application (e.g. diagnostics or therapy) have to be considered. These two factors determine the strategy and expression system that best suit the needs of the recombinant product. Both the vector and the target... [Pg.66]

Independent clones isolated by transfection of a given host strain by the same vector can differ in their expression capabilities. Such clonal variation has been observed in most expression systems currently in use, and therefore examination of several independent clones for the desired expression and fermentation characteristics is a routine part of protein yield optimization. However, clonal variation is an ongoing process in all cultures that are propagated and is therefore not eliminated once a pure strain has been isolated. Thus, the properties and genetic background of the production strains have to be monitored constantly. Master cell banks (MCBs) and master working cell banks (MWCBs) are prepared to ensure... [Pg.83]


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