Bioprocess Engineering
47
separation. The major drawback of tradi-
tional electrophoresis is lack of scalability.
Thus, electrophoresis has not made sig-
niFcant inroads into protein downstream
processing, although two research-scale
instruments are commercially available.
Both are based on isoelectric focusing in
free solution and do not need gels or other
supporting matrices. Preparative isoelec-
tric focusing does not require expensive
buffers and is more easily scaled up.
Liquid–liquid extraction originally used
immiscible liquids such as an organic
and an aqueous phase. ±or example, an
aqueous DNA solution is extracted with
phenol and ether to remove lipids and
other cell debris. Extraction is used to
separate inhibitory end products such
as alcohols from fermentation broths, or
to recover antibiotics. However, organic
solvents are not compatible with many
proteins. Instead, protein separation is
accomplished by means of aqueous two-
phase extraction. Typical aqueous phases
contain water-soluble but mutually in-
compatible polymers such as polyethylene
glycol and dextran. The latter polymer, or
any other bottom phase polymer, such as
cellulose derivatives, may be substituted
by highly concentrated salt solutions.
Partitioning of the solute or protein is
described using a partition coefFcient,
K
:
K
=
x
y
(
7
)
where
x
is the concentration of protein
in the lighter liquid phase and
y
is the
concentration of the same protein in the
heavier liquid phase. The value of
K
is gov-
erned by thermodynamics; the logarithm
of
K
is proportional to the difference in
the chemical potentials of the standard
states of heavy and light liquids. ±actors
that affect the partition coefFcient include
temperature, ionic strength, and pH.
Membranes are used to concentrate
products and to clarify solutions; they
are used to separate proteins based on
size. Membranes are used to purify water
through reverse osmosis. ±iltration is
generally governed by Darcy’s law, which
relates the velocity of the liquid through
porous media,
v
, to the pressure drop
1
P
:
v
=
k
1
P
µ
l
(
8
)
where
k
is the permeability constant, which
is a function of the Fltrate,
µ
is the
viscosity of the liquid, and
l
is the bed
or membrane thickness. Darcy’s law holds
for low Reynolds number flow. The major
drawback of membranes is biofouling.
Layers
of
proteins
build
up
on
the
surface and clog the membranes. Cross-
flow or tangential-flow Fltration decreases
fouling, but does not eliminate it.
Other separation processes include crys-
tallization – typically the last step in pro-
ducing highly puriFed products like an-
tibiotics, and drying to remove any sol-
vents. The focus for the future contin-
ues to be combining techniques such
as chromatography with membranes or
chelators with liquid–liquid extraction.
±urthermore, clever methods to combine
fermentation and separation are always
being investigated, since optimization of
both will lead to the most cost-effective
process.
±inally, an essential part of all down-
stream processing is quality control – the
assessment of sterility, absence of ge-
netic materials such as viruses or nu-
cleic acids, and absence of pyrogens, as
well as the purity of the Fnal product
such as the desired protein or antibody.
Various chromatographic, mass spectro-
metric, and electrophoretic processes are
routinely
used
for
quality
assurance.
These techniques, when combined with
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