Mammalian cell culture systems

Mammalian cell culture is more technically complex and more expensive than microbial cell fermentation. Therefore, it is usually only used in the manufacture of therapeutic proteins that show extensive and essential post-translational modifications. In practice, this usually refers to glycosylation, and the use of animal cell culture would be appropriate where the carbohydrate content and pattern are essential to the protein's biological activity, its stability or serum half-life. Therapeutic proteins falling into this category include EPO (Chapter 10), the gonadotrophins (Chapter 11), some cytokines (Chapters 8-10) and intact monoclonal antibodies (Chapter 13).

The culture of animal cells differs from that of microbial cells in several generalized respects, which include:

• they require more complex media;

• extended duration of fermentation due to slow growth of animal cells;

• they are more fragile than microbial cells due to the absence of an outer cell wall.

Basic animal cell culture media, such as Dulbecco's modified Eagle's medium, generally contain:

• carbon source (often glucose);

• antibiotics (e.g. penicillin or streptomycin);

• supplemental serum;

• buffering agent (often CO2 based).

Antibiotics are required to prevent microbial growth consequent to accidental microbial contamination. Supplemental serum (often bovine or foetal calf serum, or synthetic serum composed of a mixture of growth factors, hormones and metabolites typically found in serum) is required as a source of often ill-defined growth factors required by some animal cell lines.

The media constituents, several of which are heat labile, are generally dissolved in purified water and filter sterilized into the pre-sterile animal cell reactor. Reactor design (and operation) differs somewhat from microbial fermentations mainly with a view to minimizing damage to the more fragile cells during cell culture (Figure 5.9). Although the generalized reactor design presented in Figure 5.9 is commonly employed on an industrial scale, alternative reactor configurations are also available. These include hollow-fibre systems and the classical roller bottle systems. Roller bottles are still used in the industrial production of some vaccines, some EPO products and growth-hormone-based products. Roller bottles are cylindrical bottles that are partially filled with media, placed on their side and mechanically rolled during cell culture. This system is gentle on the cells, and the rolling action ensures homogeneity in the culture media and efficient oxygen transfer. The major disadvantage associated with applying roller bottle technology on an industrial scale is that many thousands of bottles are required to produce a single batch of product.

Different animal cell types display different properties pertinent to their successful culture. Those used to manufacture biopharmaceuticals are invariably continuous (transformed) cell lines. Such cells will grow relatively vigorously and easily in submerged culture systems, be they roller bottle or bioreactor based.

Unlike transformed cell lines, non-continuous cell lines generally:

• display anchorage dependence (i.e. will only grow and divide when attached to a solid substratum; continuous cell lines will grow in free suspension);

• exhibit contact inhibition (physical contact between individual cells inhibits further division);

• display a finite lifespan, i.e. die, generally after 50-100 cell divisions, even when cultured under ideal conditions;

• display longer population doubling times and grow to lower cell densities than continuous cell lines;

• usually have more complex media requirements.

Many of these properties would obviously limit applicability of non-continuous cell lines in the industrial-scale production of recombinant proteins. However, such cell types are routinely cultured for research purposes, toxicity testing, etc.

The anchorage-dependent growth properties of such non-continuous cell lines impacts upon how they are cultured, both on laboratory and industrial scales. If grown in roller bottles/other low-volume containers, then cells grow attached to the internal walls of the vessel. Large-scale culture can be undertaken in submerged-type vessels, such as that described in Figure 5.9b in conjunction with the use of microcarrier beads. Microcarriers are solid or sometimes porous spherical particles approximately 200 |im in diameter manufactured from such materials as collagen, dextran or plastic. They display densities slightly greater than water, such that gentle mixing within the animal cell bioreactor is sufficient to maintain the beads in suspension and evenly distributed throughout the media. Anchorage-dependent cells can attach to and grow on the beads' outer surface/outer pores.

Overall, therefore, the routine manufacture of a biopharmaceutical product is initiated by large-scale culture of its producing cell line (upstream processing). Subsequent to this, the product is recovered, purified and formulated into final product format. These latter operations are collectively termed downstream processing and are described in Chapter 6.

Diabetes 2

Diabetes 2

Diabetes is a disease that affects the way your body uses food. Normally, your body converts sugars, starches and other foods into a form of sugar called glucose. Your body uses glucose for fuel. The cells receive the glucose through the bloodstream. They then use insulin a hormone made by the pancreas to absorb the glucose, convert it into energy, and either use it or store it for later use. Learn more...

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