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Culture systems insect cells

Nguyen B, Jarnagin K, Williams S, Chan H, Barnett J (1993), Fed-batch culture of insect cells a method to increase the yield of recombinant human nerve growth factor (rhNGF) in the baculovirus expression system, J. Biotechnol. 31 205-217. [Pg.473]

Insect and Mammalian Cell Cultures. Animal cell cultures are the systems with highest similarity to human cells with respect to the pattern and capacity of posttranslational modifications. However, their cultivation is more complicated and costly and usually yields lower product titers. Among the known systems, insect cells transformed by baculovirus vectors have reached a comparable popularity as Pichia among yeasts because they are considered to be more stress resistant, easier to handle, and more productive compared with mammalian systems and are thus frequently employed for high-throughput protein expression. The highest reported... [Pg.10]

Gas-liquid-biosolid Bubble-free membrane gassing gas diffuses into the media without bubbles. Used for shear-sensitive animal cell cultures (ex insect cells) and for systems containing serum that are prone to foaming. Use 10 to 25 m /rrf for volume < 150 L. Enzyme membrane reactor Power 10 kW/m maximum volume 0.5 m . Membrane allows diffusion of gas into the liquid without having to use bubbles. [Pg.278]

A scale of label incorporation attributed to Sir Derek Barton says that 1% is excellent, 0.1% is good, 0.01% is positive and 0.001% or less is dubious, probably negative. Especially using C (which is followed by NMR spectroscopy) it is necessary to obtain at least 1% incorporation for successful deduction of the biosynthesis. Better incorporation of the intermediate can be achieved with micro-organisms, tissue culture of insect cells, or excised glands (see coccineUines, Chapter 3), or by having a cell-free system of partially purified enzymes. Alternatively,... [Pg.70]

Andersen et al. (1996) and Andersen (1995) have studied the effect of temperature on the recombinant protein production using a baulovinis/insect cell expression system. In Tables 17.15, 17.16, 17.17, 17.18 and 17.19 we reproduce the growth data obtained in spinner flasks (batch cultures) using Bombyx mori (Bm5) cells adapted to serum-free media (Ex-Cell 400). The working volume was 125 ml and samples were taken twice daily. The cultures were carried out at six different incubation temperatures (22, 26,28, 30 and 32 TT). [Pg.348]

Insect cell systems represent multiple advantages compared with mammalian cell cultures (1) they are easier to handle (Table 2.1) (2) cultivation media are usually cheaper (3) they need only minimum safety precautions, as baculovirus is harmless for humans (4) they provide most higher eukaryotic posttranslational modifications and heterologous eukaryotic proteins are usually obtained in their native conformation (5) the baculovirus system is easily scalable to the bioreactor scale. However, because of the viral nature of the system, continuous fermentation for transient expression is not possible - the cells finally die. [Pg.48]

A plasmid-based transient expression system (InsectDirect system from EMD Biosciences Inc., USA www.emdbiosciences.com) will most probably greatly facilitate parallelization and automation for insect cell cultures. It generally gives lower yields, since expression is driven by an early baculoviral promoter, but it is possible to evaluate protein activity and expression level 24 h after transfection. It is also scalable to 1 L volume. The two main disadvantages, namely the large amount of transfection agent required and the limitation in scalability, can probably be overcome in future. [Pg.49]

A wide range of proteins have been produced at laboratory scale in recombinant insect cell culture systems. The approach generally entails the infection of cultured insect cells with an engineered baculovirus (viral family that naturally infect insects) carrying the gene coding for the desired protein placed under the influence of a powerful viral promoter. Amongst the systems most commonly employed are ... [Pg.118]

Members of the Caliciviridae family can hardly be examined in cell culture or animal models. Therefore, so-called virus-Hke particles (VLP) are employed in current experiments. These particles are expressed recombinantly in insect cells using a baculovirus system and do not carry infectious viral RNA [70-72]. It has been shown by single particle tracking studies that VLPs are internalized into the cells in a similar fashion to native viruses [73]. VLPs are believed to present identical molecular recognition elements to the outside world as do native viruses. [Pg.193]

Insect cells in culture are also hosts for recombinant protein production. Production of recombinant proteins in the baculovirus expression vector system is the most common system. Titers of recombinant protein as high as 11 g/L have been obtained. [Pg.619]

Weber, W, Weber, E., Geisse, S. and Memmert, K. (2002). Optimisation of protein expression and establishment of the Wave Bioreactor system for Baculovirus/insect cell culture. Cytotechnology 38, 77-85. [Pg.43]

In this part the application of mathematical models to CLP and VLP production with baculovirus infected insect cell cultures is discussed. Special emphasis on model evaluation is made along with the definition of directions in future process development research with this system. [Pg.186]

As indicated earlier, insect cells have been cultured either as attached or suspension cultures. For scale-up purposes, systems where cells grow attached to surfaces are less suitable because of the fundamental limitation in surface area upon further increase in reactor volume (surface-to-volume ratio decreases as volume increases) unless microcarriers are utilised, which increases complexity and cost. In contrast, suspension systems are easily scalable and therefore their use is widespread since insect cells, like Sf9, have already been well adapted to suspension systems. [Pg.195]

Baculovirus binding to insect cells is receptor-mediated [60]. From the work of Dee and Shuler it seems that the post-attachment steps occur much faster than attachment and thus the limiting step is the rate of attachment [61]. Another interesting result from the Dee and Shuler report that could have a great impact on bioreaction operational parameter specification is the observation that even in attached cultures, i.e. with baculovirus transport limited by diffusion, the rate of the diffusion of the baculovirus is 10-fold higher than the rate of attachment. This suggests that the overall rate of infection is, in suspension systems fike in stirred tank reactors, independent... [Pg.200]

Figure 3.9. Generalized overview of the industrial-scale manufacture of recombinant E2 classical swine fever-based vaccine, using insect cell culture production systems. Clean (uninfected) cells are initially cultured in 500-1000 litre bioreactors for several days, followed by viral addition. Upon product recovery, viral inactivating agents such as /i-propiolactone or 2-bromoethyl-iminebromide are added in order to destroy any free viral particles in the product stream. No chromatographic purification is generally undertaken as the product is substantially pure the cell culture media is protein-free and the recombinant product is the only protein exported in any quantity by the producer cells. Excipients added can include liquid paraffin and polysorbate 80 (required to generate an emulsion). Thiomersal may also be added as a preservative. The final product generally displays a shelf-life of 18 months when stored refrigerated... Figure 3.9. Generalized overview of the industrial-scale manufacture of recombinant E2 classical swine fever-based vaccine, using insect cell culture production systems. Clean (uninfected) cells are initially cultured in 500-1000 litre bioreactors for several days, followed by viral addition. Upon product recovery, viral inactivating agents such as /i-propiolactone or 2-bromoethyl-iminebromide are added in order to destroy any free viral particles in the product stream. No chromatographic purification is generally undertaken as the product is substantially pure the cell culture media is protein-free and the recombinant product is the only protein exported in any quantity by the producer cells. Excipients added can include liquid paraffin and polysorbate 80 (required to generate an emulsion). Thiomersal may also be added as a preservative. The final product generally displays a shelf-life of 18 months when stored refrigerated...
The Transdirect insect cell is a newly developed in vitro translation system for mRNA templates, which utilizes an extract from cultured Spodoptera fru iperda 21 (S 21) insect cells. An expression vector, pTDl, which includes a 5 -imtranslated region (UTR) sequence from a baculovirus polyhedrin gene as a translational enhancer, was also developed to obtain maximum performance from the insect cell-free protein synthesis system. This combination of insect cell extract and expression vector results in protein productivity of about 50 pg per mL of the translation reaction mixture. This is the highest protein productivity yet noted among commercialized cell-free protein synthesis systems based on animal extracts. [Pg.97]

The basic process technology in vaccine production consists of fermentation for the production of antigen, purification of antigen, and formulation of the final vaccine, In bacterial fermentation, technology is well established. For viral vaccines, cell culture is the standard procedure. Different variations of cell line and process system are in use. For most of the live viral vaccine and other subunit vaccines, production is by direct infection of a cell substrate with the virus. Alternatively, some subunit viral vaccines can be generated by rDNA techniques and expressed in a continuous cell line or insect cells. [Pg.1661]

There exist a variety of vectors for cloning into eukaryotic systems, ranging from yeast (Saccharomyces as well as Pichia) through insect cells (Baculovims) and plants (Ti plasmid from Agrobacterium tumefaciens) to mammalian cells (transfected by viral or mammalian vectors). As expression in eukaryotic hosts is less efficient than bacterial expression in terms of yield and time and more complicated in terms of vector structure and culture conditions, such eukaryotic expression systems are only used for genes whose proteins require posttranslational modification which is not possible in bacteria. Yeast is the preferred option as a relatively easily culturable single-cell system but posttranslational modification capabilities is limited. The additional complexity can be circumvented in part by exploiting the ability of eukaryotic vectors to act as shuttle vectors, which can be shuttled between two evolutionarily different hosts. Thus, eukaryotic vectors can be replicated and analyzed in bacteria and transfected into eukaryotic cells for expression of the recombinant product. [Pg.80]

It is worth emphasizing that all biopharmaceuticals mentioned here are produced from mammalian cell culture. The protein production system based on insect cells known as BEVS (baculovirus expression vector system) is widely employed for the expression of a wide range of proteins, but, due to regulatory issues, biopharmaceuticals produced by insect cells are not yet in the market. However, some of them are being evaluated,... [Pg.6]


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