FUSION PROTEINS: APPLICATIONS AND CHALLENGES - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 11

albumin, transferrin, and insulin [104]. Proteins can aggre-
gate even when secreted from CHO cells. This should be
minimized already in the upstream process to reduce losses
in the downstream and to improve formulation. Adding
dexamethasone at nanomolar concentrations increases
glutathione reductase levels, but not protein disulfide-isom-
erase. Thus, a balanced redox condition is established in the
cells, effectively reducing aggregate formation as demon-
strated on the example of an IgG-fusion protein [105].
In addition to cell engineering, the drastic 100-fold
increase of productivity within 20 years can mainly be
attributed to better understanding of cultivation conditions.
This includes media composition, nutrient content, pH,
temperature, the addition of histone deacetylase inhibitors
such as sodium butyrate or valproic acid, a better dissipation
of dissolved oxygen by reactor design, and metabolite
triggered feed strategies [106].

10-50% of the total cell protein containing up to 95% of
a single protein species [109]. Due to their high density IBs
can easily be isolated by sedimentation and often allow an
enrichment of approximately 90%. Functional proteins from
IB require two steps; solubilization under denaturing and
reducing conditions, then refolding by removal of denatur-
ants and reformation of disulfide bridges [110].
In some cases, the fusion partner helps by enabling an
elegant downstream processing route. One striking example
is the utilization of elastin-like peptides (ELP) that facilitate
the purification by temperature dependent aggregation that
allows simple capturing by sedimentation [111]. A typical
antibody-based production, standardized for Fc-based puri-
fication, achieves an expression level of 2-5 g/L and an
overall yield of 70-80%. Unfortunately, nonantibody Fc
fusions do not always achieve such high titers and often
they bind less efficiently to Protein A resins [112]. But
despite of these deviations, most of the platform solutions
derived from antibody processing are also applicable for Fc-
fusion proteins. Protein A and its derivatives are the most
frequently used capture ligand for Fc-based proteins on
chromatography columns. The elution at low pH also inac-
tivates potential viral contaminations if it is below pH 3.7.
But an acidic pH might destabilize some Fc-fusion proteins
and cause aggregation. This aggregation can be overcome by
simultaneously adding a chaotropic agent during elution
[113]. Furthermore, ion exchange chromatography step(s)
and a final virus filtration complete the standard Fc-
dependent purification protocol [114].
If Fab fragments or scFv are fusion partners, also
dedicated affinity matrices such as the kappa light chain
specific Protein L [115], FabSorbent 1 [116]orothernovel
synthetic molecules [117] canbeused.ProteinLwasalso
successfully applied to purify trispecifc antibodies lacking
an Fc part [118]. Even for albumin fusions a platform
technology, Albupure TM was created [119]. Especially for
unconventional fusion proteins such as Amediplase TM
customized affinity matrices have been designed that
enable a scalable manufacturing process [120].
One of the challenges of contemporary downstream
processes is the constantly increasing protein titer resulting
from upstream improvements. Sometimes the column
capacity in affinity chromatography is not sufficient to
capture these high protein concentrations. Therefore, ion
exchange chromatography has been used as alternative. This
might also be generally applicable for fusion proteins lack-
ing domains for which platform technologies exist.
As alternative to classical chromatographic procedures,
aqueous two phase systems (ATPS) have been used recently
for the manufacturing of biopharmaceuticals. The phenom-
enon of ATPS is based on the incompatibility of two
components. The typical pairs are either polymers or salts.
By forcing the (soluble) protein of interest into the upper
phase, simultaneously aggregates can be removed as well.

1.5.2 Downstream Process

The downstream process starts with harvesting the product
of interest. For secreted proteins that are mainly produced by
eukaryotic cells (animal cells or yeast), there are two
alternative procedures; either centrifugation to sediment
the cell mass, or filtration to obtain a cell-free supernatant.
Secreted expression is preferable because in state-of-the-art
serum-free medium only few contaminants besides the
protein of interest are present. A combined unit operation
such as expanded bed absorption (EBA) connects cell
removal and first product capture in a single step. The
protein of interest retained in the cell-free solution is then
separated from other contaminants such as other proteins,
DNA, or viruses by a series of chromatographic polishing
steps. Finally, the isolated protein is pure and can be trans-
ferred to the subsequent formulation step [107].
If the protein is not secreted, a product release step, where
the microbial cell is homogenized by physical or chemical
means, must be included. Most of the employed methods
suffer from a rather high unspecific homogenization. This
means the product of interest is released together with many
contaminants that have to be removed in laborious down-
stream procedures. A selective product release step mini-
mizes contaminants, increases the adsorption capacity of
chromatography and reduces viscosity. Important parame-
ters to consider for the optimal disruption process are
particle size and density, recovery rate, viscosity, processing
time, and scalability. In general, mechanical (e.g., high
pressure, cavitation), chemical (e.g., osmotic shock, buffer
conditions), and genetic (e.g., induced cell lysis) methods
can be distinguished. Ideally, the product is accumulated in a
subcellular compartment or the periplasmatic space that
simplifies specific release [108].
A special case for intracellular accumulation is the
formation of IB. In many cases, IBs can represent