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European Biopharmaceutical Review

Cell Culture Considerations

Recombinant protein technologies are clearly essential for the manufacture of biological therapeutics. Additionally, they are equally important in the discovery of novel protein inhibitors, where they are used to validate targets, pan, and screen libraries in the optimisation of binding affinities, crystallographic studies, and the selection of final candidates for properties such as specificity. Although the underlying technologies between the generation of proteins as drugs and as reagents are similar, the practice is different. Generating proteins as reagents usually only requires, at maximum, a few tens of milligrams and, sometimes, only micrograms, but these reagents are quickly required in a matter of weeks. Furthermore, multiple proteins are frequently needed to support a single project. These can be variants of the target itself as well as a number of homologous proteins or protein complexes.

The first choice to express any protein, if it can be achieved, would always be a microbial host, in particular Escherichia coli, as the cells are easy, fast, and cheap to culture. However, many targets for biological drugs will be post-translationally modified, and E coli is very limited in this respect. Additionally, many proteins will not express in a soluble form in E coli. As a consequence, higher eukaryotic hosts are frequently used, the two most common being baculovirus-infected insect cells and various different mammalian cell hosts – most commonly Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. Both insect and mammalian systems are used most simply when the cells are maintained in suspension culture in protein-free medium. Both insect and mammalian cells are able to generate a full range of post-translational modification, with the one exception being that N-linked glycosylation in insect cells is less complicated, thereby restricted to simpler, mostly mannose structures unless the cell has been modified with additional enzymes to create mammalian-type structures in a system known as SweetBac. Culturing cells in suspension enables a much greater cell density to be achieved and, therefore, higher expression per volume of cell culture medium. The use of protein-free medium greatly facilitates purification if the protein is secreted.

The focus of this article is on the provision of proteins as reagents using eukaryotic expression systems, in particular, transient expression in mammalian cell lines and baculovirus infection of insect cells, and has recently been reviewed (1).

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Dr Mark Abbott is the founder and CEO at Peak Proteins. He obtained his PhD in membrane protein biochemistry from the University of Nottingham, UK. Mark then worked for five years as an MRC postdoctoral fellow studying lectin recognition of oligosaccharide structures. This was followed by a 20-year pharmaceutical career at AstraZeneca, latterly as a Principal Scientist, leading teams and projects undertaking both small molecule and biologics drug discovery. Mark has 29 peer reviewed publications.
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