Biomedical Engineering Reference
In-Depth Information
Many microbial processes generate IBs that need refold-
ing. For instance, the granulocyte macrophage-colony-stim-
ulating factor (GM-CSF) has been produced in E. coli. This
required a series of washing steps to purify the IBs that are
subsequently refolded at low concentration. When combin-
ing chemical extraction of IBs with EBA labor costs were
fivefold lower and overall costs 50% less [136]. Interest-
ingly, the costs for soluble expression of Heparinise in E. coli
were twice the expenses of insoluble expression. The dif-
ference was primarily dependent on the expression levels
that could be achieved in both approaches. Overall it was
observed that fermentation related costs represent only a
minor fraction, and yield optimization in downstream pro-
cedures has the highest impact on cost savings [137].
When comparing the production costs of tissue plasmin-
ogen activator stimulating factor (tPA) in E. coli and mam-
malian cells it was found out that refolding concentrations of
more than 4mg and refolding yields above 20%would make
the process economically attractive. But it has to be taken
into account that the cost of the competing mammalian
expression is dominated by the cost for serum in the
cultivation medium [138]. This extra cost can nowadays
be neglected since most large-scale mammalian cultivations
are serum free. Particularly, the absence of animal derived
components, minimizes concerns of virus or prion contami-
nations, and improves the attractiveness of microbial fer-
mentation besides
disposables can also be used in microbial processes. In a
case study, the conventional production of an antibody
fragment in E. coli was compared to a process with single
use equipment. This more than 10-year-old study could
demonstrate a 25% advantage of net present value for the
conventional manufacture [142]. However, this gap is con-
stantly narrowing, since particularly in downstream proc-
essing membrane steps can efficiently replace previous
chromatographic column steps. This substitution saves
cost in form of 25% less required volume of the stationary
phase. Consequently 40% less aqueous waste is generated.
Furthermore, labor hours are reduced by 40% because of
obsolete cleaning and validation tasks. Overall the antibody
purification process based on disposable membranes was
50% faster and 23% cheaper [143]. However, it should be
taken into account that smaller and more frequent batches
have higher QC and QA costs.
Obviously, the downstream process is a major cost driver
suffering from low economy of scale effects. Typical chro-
matography steps are volume dependent directly impacting
space requirements. Further complications are the interme-
diate cleaning cycles causing downtimes and cost [144].
1.5.5 Glycosylation
Besides production cost also product quality hast to be taken
into account when selecting an expression host. One element
contributing to product quality is glycosylation, and inter-
estingly about 70% of marketed recombinant proteins are
glycoproteins. Huge variations and deviations from a typical
human glycan pattern can be observed in economically
favorable organisms such as bacteria, yeasts, fungi, insects,
plants, or nonhuman mammalian species [145].
Glycosylation has a huge impact on the therapeutic effect
by improving pharmacokinetics, pharmacodistribution and
the selectivity of binding to receptors. This requires a careful
selection of upstream process parameters including the host
organism [146]. Even the cultivation conditions can affect
the glycosylation pattern. For example, N-glycolylneuraminic
acid (Neu5Gc) does not exist in humans, but is added when
glycoproteins are expressed in Chinese hamster ovary (CHO)
cells. The addition of Neu5Gc is dependent on a number if
cultivation conditions. Neu5Gc content can be significantly
lowered in presence of sodium butyrate, a decrease of
temperature after exponential growth, high carbon dioxide
concentrations and the utilization of sodiumhydroxide for pH
control [147]. From a downstream perspective glycosylation
makes purification more difficult since often heterogeneity
is observed. Since it is not easy to separate glyco-isoforms
by preparative chromatography all efforts are taken to sup-
press heterogeneity already during upstream processes.
Nevertheless, to guarantee a reproducible, homogeneous
drug substance, high resolution analytical tools must be
implemented [148].
its cost advantage and the short
production cycles.
The technological progress of mammalian cell culture
with increasing titers for antibodies currently causes bio-
reactor overcapacity issues. It is anticipated that in 2013
available bioreactor volume will reach 4 million liters
compared to 2.4 million liters in 2007. Overall COG pro-
jections for antibodies and Fc-fusion proteins currently
range from 50 to 100$/g but achieve a median sales price
of 8000$/g. Therefore, royalty payments have a huge impact
on the total COG. A 10-fold increase in titer would decrease
the COG for the drug substance by 85%. For other recom-
binant (fusion) proteins requiring large doses the product
titer is still a major determinant of COG [139]. At an
antibody production below 10 kg/year and a titer around
0.1 g/L the ratio between upstream and downstream costs is
around 50:50. Increasing the output to 100 kg/year at a titer
of 0.5 g/L, upstream costs only represent 20%, indicating
that at high levels, savings by the expression system plays
only a minor role [140].
The aforementioned overcapacity of mammalian cell
culture has been observed for many years and increased
from 23.6% in 2003 to 36.7% in 2007. This is a result of
constantly increasing product titers and simultaneously
expanding the capacity by building new factories [141].
New production installations seem to move away from large
single product units to more flexible multiproduct plants
that nowadays also include disposable equipment. But
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