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Insect cells, protein production

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...
Key words Baculovirus, Insect cells. Protein expression and production. Secreted proteins. [Pg.187]

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. [Pg.200]

At its most fundamental level, the circadian cycle rests on the influence of so-called clock genes . These genes have been studied most extensively in insects but they have also been found in humans. Their protein products enter the cell nucleus and regulate their own transcription. This feedback process is linked to exposure to light and so it is not surprising that visual inputs are important for maintenance of circadian rhythms. However, it is not the reception of specific visual information, transmitted in the optic nerve to the lateral geniculate nucleus (LGN) and visual cortex (i.e. visual discrimination), that is responsible for the rhythm but the more simple, almost subconscious, reception of light. [Pg.478]

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]

Andersen, J.N., "Temperature Effect on Recombinant Protein Production Using a Baculovirus/Insect Cell Expression System", Diploma Thesis, University of Calgary and Technical University of Denmark, 1995. [Pg.391]

Although proteins can be expressed in many heterologous production systems, including bacteria such as Proteus mirabilis [1], fungi such as Pichia pastoris [2, 3] and Aspergillus awamori [4] and insect cells [5, 6], the pharmaceutical industry has narrowed down process development to a small number of platform technologies ... [Pg.267]

Most of the recombinant subunit vaccines tested in the first half of this decade employed gp 120 or gp 160 expressed in yeast, insect or mammalian (mainly CHO) cell lines. Eukaryotic systems facilitate glycosylation of the protein products. Like all subunit vaccines, these stimulate a humoral-based immune response but fail to elicit a strong T-cell response. The failure to elicit a cell-based... [Pg.409]

Host Cell Impurities Various organisms have been used to produce recombinant proteins yeast, bacteria (e.g., E. coli), insect cells, and mammalian cells such as Chinese hamster ovary (CHO) cells. During the purification process, some HCPs can copurify with the protein product. Because of the specificity of the antigen-antibody interaction, an ELISA can be used to detect and quantitate the contaminating HCPs. Detecting host impurities is important for quality process control as well as for product safety issues. The intent is to avoid unsafe levels of residual HCPs which might lead to adverse reactions.11... [Pg.288]

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]

For other production hosts (yeast, insect, and mammalian cells), standard promoter formats have been used in combination with FITP cloning methods to produce vectors for expression screening (see Section 2.3.2). A particularly interesting development is the use of multipromoter plasmids for expression in two or more hosts from a single vector. The construction of a dual E.coli (T7 promoter) and baculovirus transfer vector (polH promoter) for expression in insect cells has been described (Chambers et al., 2004). A three-promoter vector (T7, plO, and hCMV or CAG promoter) is available from Novagen (pTrlEX ) and its use reported for comparing protein expression in E. coli and insect cells (Xu and Jones, 2004). [Pg.27]

The Baculoviridae are a family of large enveloped DNA viruses that are characterised by rod-shaped nucleocapsids and relatively large double stranded DNA genomes. Autographa californica Multicapsid Nuclear Polyhedrosis Virus (AcMNPV) is the baculovirus most currently used as vector for protein production with insect cells. Several reviews are available describing baculovirus structure and its molecular biology [6-8]. [Pg.185]

The industrial application of CLPs and VLPs is in the development phase. This can be expected since the baculovirus-insect cells system has become one of the most popular systems for heterologous protein and CLP/VLP production at laboratory scale. After defining the appropriate particle composition for the viruses of interest, research is now addressing the engineering issues in this system (Scheme 1). [Pg.185]

For the production of CLPs and VLPs with baculovirus infected insect cells the specific proteins that are required for particle formation should be chosen at an early step. The specific particle composition, along with the expression of other non-structural (NS) proteins often has a great impact on particle stability [11] or on particle localisation [12], i.e. - cell-associated or secreted to the supernatant. [Pg.187]

The selection of the monomers to incorporate in the capsid should consider other items along with antigenicity, such as product release by the cells [ 15,24]. Product secretion can have a large impact on overall particle production since baculovirus-infected insect cells often exhibit proteolytic activity, which is mainly intracellular at early times post-infection [25]. The appearance of proteases often coincides with plO and polyhedrin-driven late protein production [26]. [Pg.189]

Optimal conditions for insect cell growth have been extensively studied, but for product expression with a baculovirus infected insect cell the focus should be on the difference in the metabolic requirements of infected vs. uninfected cells, which has been observed to differ after infection. The alanine specific production rate decreases almost four-fold, while phenylalanine specific consumption rate increase 11-fold and glutamine specific consumption decrease [65]. Both an increase [66] and a decrease [67] in glucose consumption rates of insect cells after infection have been reported. This reflects some differences in the media and vectors that were used however, it is normal to expect a higher metabolic burden after infection due to the increase in protein expression rates caused by the infection. This creates a concern about the impact of nutrient limitations on the productivity of the system. [Pg.194]

Insect cells have an optimal cultivation temperature of 27-28°C. However, for protein production this temperature may not always be the most adequate. [Pg.196]

Besides the currently used constant temperature mode it has been reported that temperature oscillation can enhance cell viability of Sf9 insect cells and ba-culovirus production of occlusion bodies (OB) and extracellular virus (ECV) compared with constant temperature in stationary culture and suspension culture, with the optimal oscillation range at 24-28°C [82]. As a curiosity Pham et al. found that, by raising the infection temperature to 30 °C, they more than doubled the protein productivity of human interleukin-2 (IL-2), in insect larvae, Trichoplusia ni [83]. [Pg.196]


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See also in sourсe #XX -- [ Pg.431 ]




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