separate intact cells and some cell
debris/lysates from the rest of the
components in the feed stream. Either
the retained cells or the clarified filtrate
can be the product stream. Membrane
pore size cutoffs used for this type of
separation are typically in the range
of 0.05 μm to 1.0 μm.
Ultrafiltration (UF) is one of the most
widely used forms of TFF and is used
to separate proteins from buffer
components for buffer exchange,
desalting, or concentration.
Depending on the protein to be
retained, membrane NMWLs in the
range of 1 kD to 1000 kD are used.
Two types of UF are Virus filtration
(VF) and High Performance tangential
flow filtration (HPTFF). For VF,
membrane NMWL ratings range from
100 kD to 500 kD, or up to 0.05 μm.
This process type is used to separate
virus particles from proteins or from
smaller media components, as either a
virus reduction step or a virus harvest
step. HPTFF is a high resolution
process where protein-protein
separations can be carried out on the
basis of both size and charge, resulting
in product yields and purification
factors similar to chromatography.
Membrane NMWLs used for HPTFF
are in the range of 10 kD to 300 kD.
Reverse Osmosis (RO) and
Nanofiltration (NF) are types of TFF
where very tight membranes are used
to separate salts and small molecules
with molecular masses typically lower
than 1500 Daltons from water or
other solvents. Membranes with
NMWLs of 1 kD and lower are used.
Finally, Diafiltration (DF) is a TFF
process that can be performed in
combination with any of the other
categories of separation to enhance
either product yield or purity. During
DF, buffer is introduced into the
recycle tank while filtrate is removed
from the unit operation. In processes
where the product is in the retentate,
diafiltration washes components out
of the product pool into the filtrate,
thereby exchanging buffers and
reducing the concentration of
undesirable species. When the
product is in the filtrate, diafiltration
washes it through the membrane into
a collection vessel.
Microfiltration Virus
Filtration
High-Performance
Filtration
Ultrafiltration
TFF
Nanofiltration/
Reverse Osmosis
Components retained
by membrane Intact cells
Cell debris Viruses Proteins Proteins
Antibiotics
Sugars
Salts
membrane membrane membrane membrane membrane membrane membrane membrane membrane membrane
membrane membrane membrane membrane
Colloidal material
Proteins
Proteins
Small Peptides
(Salts)
Components passed Viruses
Salts
Salts
Salts
Water
through membrane
Proteins
Salts
Approximate membrane
0.05 μm – 1μm
100 kD – 0.05 μm
10 kD – 300 kD
1 kD – 1000 kD
<1 kD
cutoff range
Figure 2. Subdivisions of tangential flow filtration processes
The remainder of this document will
focus on the development of
concentration and diafiltration steps for
protein processing.
TFF Basics
In a TFF unit operation, a pump is
used to generate flow of the feed
stream through the channel between
two membrane surfaces. A schematic
of a simple TFF system is shown in
figure 3. During each pass of fluid
over the surface of the membrane, the
applied pressure forces a portion of
the fluid through the membrane and
into the filtrate stream. The result is a
gradient in the feedstock concentration
from the bulk conditions at the center
of the channel to the more
concentrated wall conditions at the
membrane surface. There is also a
Diafiltration Retentate Valve to
Buffer Return Apply Pressure
Retentate
Pressure
Feed
TankFeed
Pressure
Stream
Filtration Filtrate
Module
Feed
Pump
Figure 3. Schematic of a simple TFF system
Membrane
concentration gradient along the
length of the feed channel from the
inlet to the outlet (retentate) as
progressively more fluid passes to the
filtrate side. Figure 4 illustrates the
flows and forces described above
with the parameters defined as:
QF: feed flow rate [L h-1]
QR: retentate flow rate [L h-1]
Qf: filtrate flow rate [L h-1]
Cb: component concentration in the
bulk solution [g L-1]
Cw: component concentration at the
membrane surface [g L-1]
Cf: component concentration in the
filtrate stream [g L-1]
TMP: applied pressure across the
