Biomedical Engineering Reference
In-Depth Information
preferred option for mammalian cell culture. The integration
can occur at multiple sites leading to gene duplication and
hence higher expression levels [88]. In microbial cells, a
similar effect can be achieved with high copy number
plasmids. Molecular biology strategies to optimize recom-
binant protein expression can target transcriptional and
translational regulation. On transcriptional level, promoters
and terminators play key roles. For the translation reaction,
the binding of ribosomes to mRNA during the initiation and
their release at termination are very important as well as the
stability of the mRNA in the cytoplasm. Folding of the
nascent polypeptide chain can be enhanced by co-expression
of chaperones [89]. Further optimization can focus at the
selective knock out of proteases that might otherwise destroy
the protein product [87].
When working with E. coli, we should not forget that
post-translational modifications are missing and in some
cases the amino terminus might contain an extra methionine
residue that is not properly removed during translation [90].
The fermentation of E. coli cells depends on media
composition (e.g., carbon source, antibiotic selection) and
cultivation conditions (temperature and oxygen content).
One drawback of high-density culture is the accumulation
of acetate that limits proliferation. This problem can be
solved by a number of different approaches such as intro-
ducing the foreign enzyme acetolactate synthase into E. coli
that generates a less toxic by-product [91]. Alternatively,
glucose uptake can be limited by knocking out the ptsG gene
or the whole phosphotransferase system (PTS) [92].
The attractiveness of E. coli is based on the fast, cheap,
and simple expression of proteins that can be targeted to the
cytoplasm, the periplasm or the cell culture supernatant by
secretion. But often intracellular expression of eukaryotic
proteins in bacteria results in inclusion bodies (IB) that
require refolding approaches during downstream process-
ing. The formation of IBs is mostly caused by an overload of
the folding machinery in the bacterial cell that leads to
exposure of hydrophobic residues triggering aggregation. In
addition, E. coli lacks the ability to form disulfide bridges in
its reducing environment in the cytoplasm [93].
Particularly, when combining protein toxins and a target-
ing moiety, the expression level might be significantly
lowered due to the translational inhibition by the toxin.
One approach to circumvent this difficulty is to express
the toxin fusion in form of IB that cannot harm the host cell
because of its unfolded, nonactive state [94]. Also secretion
to the culture supernatant to keep the toxin away from its
target, the ribosomes, has been accomplished [95]. A third
variant of producing toxin fusions is the expression in the
presence of an inhibitor that is removed during the down-
stream processing [96]. Another possibility is to express
both parts of a fusion protein separately, then fusing them in
a later trans-splicing step with the help of split-inteins [97].
Recently,
successfully manufactured with high density bacterial fer-
mentation under GMP conditions at a yield of 40% and 97%
purity [98].
CHO cells came into the focus of manufacturing after
Genentech's market approval of Activase
1
, the recombinant
tissue plasminogen activator (tPA), in 1987. Currently, CHO
cells are the industry standard for glycosylated complex
proteins and benefit from three accomplishments: serum-
free production is possible and easy, cell engineering
achieves high titers, and high-density large-scale fed-batch
cultivation is well established. Cell engineering addresses
the parameters expression level, duplication time and stabil-
ity of the cell, control of proliferation and viability, reduc-
tion of toxic metabolites, increasing secretion capacity, and
modulation of post-translational modifications [99].
The process starts with expression vector design. Strong
viral promoters, the elimination of cryptic splice sites and
the increase of G/C content by codon optimization are tools
to improve expression levels. A further trick is the insertion
of an intron to enhance mRNA export and stability by
splicing. The gene silencing effect at the genomic insertion
site can be avoided by flanking the coding sequence with
DNA elements that can block the formation of hetero-
chromatin [100].
Usually, DNA constructs are integrated randomly into the
genome by homologous recombination. Besides the gene of
interest, the vector also transfers a selection markers; either
dihydrofolate reductase (DHFR) or glutamine synthesis
(GS). Cultivating the cells in a medium without the respec-
tive metabolites lets only transformed cells survive. Further-
more, genes become amplified when cells are exposed to
increasing concentrations of the inhibitor methotrexate
(MTX) and methionine sulphoximide (MSX) for DHFR
and GS, respectively. High producers with good duplication
rates are cloned and a master cell bank is created [101].
The growth and productivity of cells is mainly dependent
on the metabolism. Therefore, attempts have been under-
taken to reduce waste metabolites, thus making the carbon
metabolism more efficient. Other strategies involve extend-
ing the cell's lifespan by activating anti-apoptosis genes or
engineering cell cycle control. Further improvements were
achieved by addressing the secretion apparatus and over
expressing chaperones [102]. Mutant CHO cell lines can
also be useful for the expression of toxic compounds.
Usually, immunotoxins are produced as IB in E. coli to
prevent cell death. Good secretion levels of a scFv-diptheria
toxin fusion could be obtained in an ADP-ribosylation
insensitive CHO mutant. However, the glycosylation
decreased toxicity. Eliminating the glycosylation sites by
mutagenesis improved toxicity 12-fold over the identical
molecule prepared from E. coli periplasm [103].
In serum-free media nonanimal protein (Sericin) and
nonprotein (phosphatidic acid) substitutes enable cultiva-
tion. Previously, the most important serum components were
it was shown that an immunotoxin can be
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