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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);
grow as a monolayer;
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 when compared to 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 at lab and industrial scale. If grown in roller bottles/other low-volume containers, 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 3.15(b) in conjunction with the use of microcarrier beads. Microcarriers are solid or sometimes porous spherical particles approximately 200 mm 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 surfaces/outer pores.
An overview of the steps normally undertaken during downstream processing is presented in Figure 3.16. Details of the exact steps undertaken during the downstream processing of any specific biopharmaceutical product are usually considered highly confidential by the
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Figure 3.16. Overview of a generalized downstream processing procedure employed to produce a finished product (protein) biopharmaceutical. Quality control also plays a prominent role in downstream processing. QC personnel collect product samples during/after each stage of processing. These samples are analysed to ensure that various in-process specifications are met. In this way, the production process is tightly controlled at each stage
Figure 3.17. A likely purification procedure for tPA produced in recombinant E. coli cells. The heterologous product accumulates intracellularly in the form of inclusion bodies. In this particular procedure, an ultrafiltration step is introduced on several occasions to concentrate the product stream, particularly prior to application to chromatographic columns. Lysine affinity chromatography (Lys-chromatography) is employed as tPA is known to bind immobilized lysine molecules. Adapted with permission from Datar et al. (1993)
manufacturer. Such details are thus rarely made generally available. However, a potential downstream processing procedure for recombinant tPA is presented in Figure 3.17, and other examples are provided at various stages through the remainder of this text.
Downstream processing is normally undertaken under clean room conditions, with the final steps (e.g. sterile filtration and aseptic filling into final product containers) being undertaken under Grade A laminar flow conditions (Figure 3.18).
In general, animal cell culture-derived biopharmaceutical products are secreted into the media (i.e. are produced as extracellular proteins), whereas the product accumulates intracellularly in many recombinant prokaryotic producer cell types. In the case of intracellular proteins, fermentation is followed by harvesting of the cells. This is normally achieved by centrifugation (Figure 3.19) or sometimes filtration. Recovery of cells is followed by their disruption in order to release their intracellular contents, including the protein of interest. A variety of means may be used to disrupt cells. Amongst the most popular microbial cell disrupters are homogenizers. During homogenization, a suspension of cells is forced under high pressure through an orifice of
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Figure 3.18. Photograph illustrating a typical pharmaceutical clean room and some of the equipment usually therein. Note the presence of a curtain of (transparent) heavy-gauge polyethylene strips (most noticeable directly in front of the operator). These strips box off a grade A laminar flow work station. Product filling into final product containers is undertaken within the grade A zone. The filling process is highly automated, requiring no direct contact between the operator and the product. This minimizes the chances of accidental product contamination by production personnel. Photo courtesy of SmithKline Beecham Biological Services s.a., Belgium