Evolution and Genetic Engineering - Bioprocess Engineering: Kinetics, Biosystems, Sustainability and Reactor Design - page 727

but also that all posttranslational processing is identical to that in the whole animal. In some
cases, the cells in culture may not do the posttranslation modifications identically to those
done by the same cell while in the body. But for bioreactor processes, mammalian cell tissue
culture provides the product closest to its natural counterpart. Another advantage is that
most proteins of commercial interest are readily excreted.
Slow growth, expensive media, and low protein expression levels all make mammalian
cell tissue culture very expensive. Awide variety of reactor systems can be used with animal
cell cultures. Although many of these can improve efficiency significantly, processes based on
mammalian cells remain very expensive. Several cell lines have been used as hosts for the
production of proteins using recombinant DNA. The most popular hosts are probably lines
of CHO (Chinese hamster ovary) cells.
In addition to the cost of production, mammalian cells face other severe constraints.
Normal cells from animals are capable of dividing only a few times; these cell lines are mortal.
Some cells are immortal or continuous and can divide continuously, just as a bacterium can.
Continuous cell lines are transformed cells. Cancer cells are also transformed (i.e. have lost
the inhibition of cell replication). The theoretical possibility that a cancer promoting
substance could be injected along with the desired product necessitates extreme care in
the purification process. It is particularly important to exclude nucleic acids from the
product. The use of transformed cells also requires cautions to ensure worker safety. Addi-
tionally, the vectors commonly used with mammalian cell cultures have been derived
from primate viruses. Again, there is concern about the reversion of such vectors back to
a form that could be pathogenic in humans.
Most of these vectors cannot give high expression levels of the target protein in common
host cells (usually 5% of total protein). However, higher levels of expression can be

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obtained (e.g. 100 mg/L of secreted, active protein) through amplification of number of

gene copies. It may take 6 months with a CHO cell line to achieve stable high-level expres-
sion. It is often easier to obtain high titers (or product concentration) when producing mono-
clonal antibodies from hybridoma cultures.
While the quality of the protein product may change upon scale-up with any system, this
issue is particularly important with animal cell cultures. This contention is due, in part, to the
fact that animal cell cultures are used primarily because authenticity of the protein product is
a major concern. Since culture conditions (shear, glucose, amino sugars, dissolved oxygen,
etc.) can change upon scale-up, the efficiency of cellular protein-processing can change,
altering the level of posttranslational processing. Furthermore, protein quality may change
with harvest time in batch cultures. This change may be due to alterations in intracellular
machinery, but often it is due to release of proteases and siladase (an enzyme that removes
the silalic acid cap from glycosylated proteins) from dead cells. Also, excessive levels of
protein production may saturate the intracellular protein-processing organelles (i.e. ER
and Golgi), leading to incompletely processed proteins. These problems can significantly
impact process strategy. For example, harvest up to 24-h early may be required to maintain
the silalic acid/protein ratio specified for the product. Early harvest resulted, however, can
result in significant (up to 30%) loss in protein concentration. Strategies to reduce such prob-
lems include selection of cell lines or genetic manipulation of cell lines with reduced levels of
siladase production or enhanced protein-processing capacity. Redesign of medium can be
beneficial; chemicals that inhibit undesirable extracellular enzyme activity can be added or

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