Insect cell-based systems

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  

Baculovirus/insect cell-based systems are cited as having a number of advantages, including  

However, a number of disadvantages are also associated with this production system, including  

---

Site-directed mutagenesis has been done using mammalian and insect cell-based systems, and models have been in existence for some time. More recent modeling work - has been done, with an emphasis on docking of inhibitors in SRS-1. 

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. 

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... 

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. 

We have demonstrated that this insect cell-free protein synthesis system is one of the most effective protein synthesis systems among those based on animal extracts (2). Furthermore, it has the potential to perform eukaryote-specific protein modifications such as protein W-myristoylation and prenylation (3, 4). Thus, we expect that the insect cell-free protein synthesis system will be a useful method for target protein production in the reverse chemical genetics era, as well as for postgenomic studies. In this chapter, we describe standard protocols to synthesize proteins of interest using the insect cell-free protein synthesis system. 

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,... 

An interesting and more recent expression system for the development of cell-based assays is based on BacMams. These recombinant baculoviruses containing mammalian cell-active expression cassettes seem to be an efficient strategy to speed up assay development. These viruses are produced in insect cells and transiently express but do not replicate in transduced mammalian cells. The expression level can be well controlled by titrating the amount of virus. Highly reproducible transient expression levels, which are a prerequisite to use transient transfection for HTS, might thus be an attractive alternative for some approaches. 

---