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• the immunogenicity of orally administered vaccines can vary widely;
• the stability of antigens in the digestive tract varies widely;
• the genetics of many potential systems remain poorly characterized, leading to inefficient transformation systems and low expression levels.
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 (a viral family that naturally infects 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:
• the silkworm virus Bombyx mori nuclear polyhedrovirus (BmNPV), in conjunction with cultured silkworm cells (i.e. Bombyx mori cells) or;
• the virus Autographa californica nuclear polyhedrovirus (AcNPV), in conjunction with cultured armyworm cells (Spodoptera frugiperda cells).
Baculovirus/insect cell-based systems are cited as having a number of advantages, including:
• high-level intracellular recombinant protein expression. The use of powerful viral promoters such as promoters derived from the viral polyhedrin or P10 genes can drive recombinant protein expression levels to 30-50% of total intracellular protein;
• insect cells can be cultured more rapidly and using less expensive media than mammalian cell lines;
• human pathogens (e.g. HIV) do not generally infect insect cell lines.
However, a number of disadvantages are also associated with this production system, including:
• targeted extracellular recombinant production generally results in low-level extracellular accumulation of the desired protein (often in the mg/l range). Extracellular production simplifies subsequent downstream processing, as discussed later in this chapter;
• post-translational modifications, in particular glycosylation patterns, can be incomplete and/ or can differ very significantly from the patterns associated with native human glycoproteins.
Therapeutic proteins successfully produced on a laboratory scale in insect cell lines include hepatitis B surface antigen, interferon-g and tissue plasminogen activator (tPA). To date, no therapeutic product produced by such means has been approved for human use. Two veterinary vaccines, however, have; ‘Bayovac CSF E2’ and ‘Porcilis Pesti’ are both subunit vaccines (Chapter 10) containing the E2 surface antigen protein of classical swine fever virus as active ingredient. The vaccines are administered to pigs in order to immunize against classical swine fever and an overview of their manufacture is provided in Figure 3.9.
An alternative insect cell-based system used to achieve recombinant protein production entails the use of live insects. Most commonly, live caterpillars or silkworms are injected with the engineered baculovirus vector, effectively turning the whole insect into a live bioreactor. One veterinary biopharmaceutical, Vibragen Omega, is manufactured using this approach and an overview of its manufacture is outlined in Figure 3.10. Briefly, whole live silkworms are introduced into pre-sterilized cabinets and reared on laboratory media. After 2 days, each silkworm is innoculated with engineered virus, using an automatic microdispenser. This engineered silkworm polyhedrosis virus harbours a copy of cDNA coding for feline interferon-
o. During the subsequent 5 days of rearing, a viral infection is established and hence recombinant protein synthesis occurs within the silkworms. After acid extraction, neutralization and clarification, the recombinant product is purified chromatographically. A two-step affinity procedure using blue sepharose dye affinity and copper chelate sepharose chromatography is employed (see later in this chapter). After a gel-filtration step, excipients (sorbitol and gelatin) are added and the product is freeze-dried after filling into glass vials.
PRODUCTION OF FINAL PRODUCT
This section briefly overviews how biopharmaceutical substances are produced in a biopharmaceutical/biotech manufacturing facility. As the vast bulk of biopharmaceuticals are proteins synthesized in recombinant prokaryotic (e.g. E. coli) or eukaryotic (e.g. mammalian cells) production systems, attention will focus specifically upon these.
As defined in this text, the production process is deemed to commence when a single vial of the working cell bank system (see later) is taken from storage, and the cells therein cultured in order to initiate the biosynthesis of a batch of product (Figure 3.11). The production process is deemed to be complete only when the final product is filled in its final containers and those containers have been labelled and placed in their final product packaging.
Production can be divided into ‘upstream’ and ‘downstream’ processing (Figure 3.11). Upstream processing refers to the initial fermentation process, which results in the initial generation of product. Downstream processing refers to the actual purification of the protein