Insect Cell Culture

Subunit vaccines based on the surface proteins of vims are also being explored. It has been demonstrated that the two major protective antigens are haemagglutinin (HA) and neuraminidase (NA). The genes for these antigens have been cloned and expressed in baculovims in insect cell culture (84). 

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. 

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 ... 

KUTCHAN, T.M., BOCK, A., DITTRICH, H., Heterologous expression of strictosidine synthase and berberine bridge enzyme in insect cell culture, Phytochemistry, 1994, 35, 353-360. 

Insect cell culture, 5 346 applications, 5 35 It Insect control... 

Insect cell cultures High protein yield, Different culture Preparation of... 

With the widespread availability of cell culture facilities, the reduced costs of media and reagents and above all, the commercialisation of a variety of transfection and expression kits, mammalian cells have now become probably the standard for functional studies of transmembrane transporters. Unsurpassed predictivity of the mammalian models may outweigh the higher costs and the lengthiness of the process, compared with bacterial cultures or Xenopus oocytes. Nevertheless, structural studies may require larger amounts than those easily produced in mammalian cells and the appeal of insect cell cultures for... 

Using large- and small-volume samples, mixed with synthetically produced samples, excellent values were developed. Samples were analyzed for alanine, glucose, glutamine, and leucine in samples removed from an Sf-9 insect cell culture bioreactor. It was seen that purely synthetic standards led to poor predictions of actual runs. The best results were achieved from mixed — actual and synthetic — samples. 

Protocol 1.5 Starting an insect cell culture Materials... 

Insect cell culture at 1-2 x 10 cells/ml EX-CEL 405 serum-free medium for Insect cells containing gentamycin sulphate (50 iJig/ml), amphotericin B (2.5 iJig/ml), and fetal calf serum (10%)... 

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. 

In this paper the fundamental aspects concerning the production of CLPs and VLPs with baculovirus infected insect cells are reviewed. It is not the goal of this communication to review all the aspects of insect cell culture technology, since this would be a task impossible to achieve in a single chapter. The interested reader should refer to the references. This review is structured in four parts ... 

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. 

Sf9 and Sf21 cells can grow either as adherent or as suspension cultures, are easily adapted to most of the common serum free insect cell culture media [52], and it is most often possible to obtain culture titers of viral structural proteins higher than 30 mg/1 [20]. 

Incubation temperature and medium pH are also important regarding proteolytic activity of baculovirus infected insect cell cultures. Cruz et al. [25] have shown that the highest proteolytic activity was obtained at the normal culture conditions, 27 °C and pH 6.5. This could then be considered a drawback when the production of protease sensitive particles Hke HIV-CLPs and HIV-VLPs is envisaged [5]. The pH of Sf9 cells has been reported to reach a minimum of 5.9 in serum-free media under uncontrolled pH conditions in stirred tank reactors... 

Murhammer and Goochee [93, 94] have confirmed the beneficial effect of Pluronic F-68 addition to insect cell cultures and its protective effect upon bubble damage. The use of PF-68 is today ubiquitous in bioprocess development involving insect cell culture technology, although it may conceivably interfere with VLP formation in some instances. For a more detailed study of the effect of bubble in cell damage mechanisms and the mechanism of protection of PF-68 see the review by Chalmers and the references cited therein [95]. 

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...

Walker, G.M., Ozers, M.S., Beebe, D.J., Insect cell culture in microfluidic channels. Biomed. Microdevices 2002, 4(3), 161-166. 

For insect cells, culture medium is buffered with sodium phosphate, and the use of CO2 or pH indicators is not required. 

Insect cell culture was started in 1915 but no cell line capable of replicating indefinitely was established until 1962. In that year Grace showed the ability of the insect cell line Antheraea eucalypti to replicate indefinitely in... 

Agathos SN (1991), Production scale insect cell culture, Biotechnol. Adv. 9 51-68. 

Ferrance JP, Goel A, Ataai MM (1993), Utilization of glucose and amino acids in insect cell cultures quantifying the metabolic flows within the primary pathways and medium development, Biotechnol. Bioeng. 42 697-707. 

Traditionally, from 0.1 to 1 mM of each amino acid is added to the culture medium, including both essential and non-essential amino acids. The concentrations of amino acids added to insect cell culture media are much higher than those found in media for vertebrate cells (Echalier, 1997), probably due to the fact that higher amino acid concentrations are found in insect hemolymph (insect body fluid) in comparison with blood serum. 

Berdad C, Tom R, Kamem A (1993), Growth, nutrient consumption, and end product accumulation in Sf9 and BTI-EAA insect cell cultures insights into growth limitation and metabolism, Biotechnol. Prog. 9 615-624. 

Ikonomou L, Schneider YJ, Agathos SN (2003), Insect cell culture for industrial production of recombinant proteins, Appl. Microbiol. Biotechnol. 62 1-20. 

Maiorella B, Inlow D, Shauger A, Harano D (1998), Large-scale insect cell-culture for recombinant protein production, Bio/Technology 6 1406-1410. 

The polymer must have a high molar mass. PEG at a range of 6-20 kDa is commonly used (Ersson et al., 1998). Precipitation with PEG was used, for instance, by Demi et al. (1999) as one of the purification steps for the isolation of hepatitis B virus surface antigen (HBsAg) from the insect cell culture supernatant of genetically modified DS-2 Drosophila melanoga-ster Schneider-2) cells. 

Electron micrographs illustrating polyhedra produced by nucleopolyhedrovirus in insect cell culture with multiple nucleocapsids per envelope (A), or with single nucleocapsids per envelope (B). 

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