you
today we rely heavily on biotechnology
for many of the necessities of life for
the food we eat the clothes we wear
fuels for transportation earth-friendly
plastics pharmaceuticals to treat our
illnesses and dietary supplements to
help us stay healthy many of these
products are produced using three core
biotechnology processes fermentation
recovery and purification fermentation
is basically cell farming we program
cells to produce a product we nurture
them as they grow and reproduce and then
we harvest them in recovery we separate
our product from the cells where they
were housed and then in purification we
go a step further by removing everything
else that's contaminating our product
leaving us with a very pure concentrated
solution in this program we're going to
look at a typical purification process
used in the manufacture of GFP green
fluorescent protein GFP is broadly used
as a biological marker because it's very
well tolerated by most cells and doesn't
interfere with normal cellular function
will examine the technologies equipment
and materials used how to prepare for
the process and how the GFP purification
process is managed step-by-step
there are two main operations used
within purification chromatography and
filtration before we can appreciate
exactly what these process steps are
accomplishing we need to take a closer
look at what our green fluorescent
protein has been through so far
back in recovery our harvested cells
from fermentation were homogenized our
target protein was inside each cell so
to get at it
the cells had to be ruptured homogenous
ation freed a flood of cellular
components including membrane debris
cytoplasm DNA and proteins including our
target protein GFP and they were all
mixed into the buffering solution used
to suspend the cells most of these
solids were then removed through
centrifugation or by centrifuge but the
liquid that remained the clarified
lysate was still rich in biological
products including dissolved chemicals
proteins and other impurities amazingly
through purification we can target a
single protein within this biological
soup
in our GFP purification process we'll be
using multiple types of column
chromatography the column is a cylinder
filled with glass ceramic or polymeric
beads which are engineered to interact
with or bind with molecules based on one
or more physical properties
chromatography relies on differences
each molecule has a unique set of
physical characteristics such as size
charge or extent of interaction with
water chromatography uses these
differences to separate the target
protein from other proteins and
chemicals sometimes size is used to
differentiate some beads have small
holes in them and can temporarily trap
or at least slow down smaller molecules
as they travel through the column of
resin beads while molecules too large to
enter the pores move around the beads
and exit the column first this type of
chromatography is called size exclusion
in the case of charge opposites attract
so a negatively charged chromatography
bead will attract and bind two
positively charged components in the
process stream likewise a positively
charged bead will bind negatively
charged components in the process stream
this charged base chromatography is
called ion exchange chromatography and
then there's water molecules that
readily interact with and dissolve in
water are called hydrophilic
water-loving while those that don't are
called hydrophobic water-hating proteins
contain regions that are hydrophobic and
regions that are hydrophilic because
water tends to form a shield around the
hydrophobic patches within the proteins
they are not exposed to interact with
the resin beads by adding salt to the
protein solution we remove the water
shield exposing these hydrophobic
patches on the protein and resin so they
can interact this is how HIC or
hydrophobic interaction chromatography
works
in our process which uses ion exchange
and hydrophobic interaction
chromatography the chromatography
equipment is housed on a skid to make it
compact and mobile the main part of the
apparatus is a glass column filled with
resin beads but we also have pumps to
move the clarified lysate through the
process a supply hose and port to feed
the column a pre-filter to remove any
remaining particulates usually solid
cell debris that has not previously been
removed before the clarified lysate
enters the column an exit port for the
processed solution and auto switching
valves for directing processed solution
to either waste or collection to help
monitor the chromatography equipment and
the solutions flowing through the unit
during the process a number of sensors
are located along the product flow path
there is an electrical conductivity
sensor at the column Inlet a pressure
sensor just before the pre-filter to
help identify a filter clog a flow meter
to measure the rate of solution movement
through the column and an air sensor to
ensure that no air has entered the flow
path as a solution leaves a column it
passes a UV sensor that reads optical
density a second conductivity sensor and
a pH sensor that measures how acidic or
basic the solution is the conductivity
sensors let us know when a new buffered
solution has filled the column when the
conductivity reading on the exit of the
column matches the reading from the
sensor at the inlet of the column then
we know the new solution has completely
displaced the old one the ultraviolet UV
sensor monitors the concentration of
protein in the product by observing the
optical density of the passing solution
this sensor works hand-in-hand with the
valves on the exit of the column through
the controller program we can set a
protein concentration threshold when the
optical density of the solution leaving
the column is below the threshold
valve directs the flow to waste
when the optical density of the solution
leaving the column is at or above the
threshold
this means that solution contains our
purified product and the solution is
directed to a collection vessel
tff tangential flow filtration is at its
heart a simple process step we're going
to pump a fluid through and across a
special type of filter known as an ultra
filtration membrane the size of the
pores in the filter material determines
what passes through and what's held back
as with almost any filtering process we
can choose what we want to keep the
solution that passes through the
membrane is referred to as the permeate
because the pores of the ultra
filtration membrane are small enough to
keep the product from passing through
the permeate contains no product and is
sent to waste the portion of the feed
stream that does not permeate the
membrane is called the retentate it
contains the retained product and is the
stream we are most interested in what
makes tff different is a core technology
that enables it to be faster more
efficient more flexible and even
self-cleaning
in conventional or terminal filtration a
fluid is pumped directly into a filter
the particles within the stream that
can't fit through the pores of the
filter build up at the filter surface
eventually clogging it in tangential
flow filtration the stream moves across
the filter that is tangential to the
filter instead of directly added the
cross flow current actually picks
material back out of the filter media or
membrane and into the stream this
retained material called retentate is
recirculated to the supply tank and will
continue to loop through the filter for
as long as the process runs will be
using tff for two different tasks within
the purification process concentration
and daya filtration we are processing
purified GFP from a chromatography step
the fluid from the chromatography step
is purified green fluorescent protein
dissolved in a buffering solution in
daya filtration we add new buffer to the
GFP retentate why
displacing the old buffer effectively
exchanging buffer solutions note the GFP
is retained by the membrane if we don't
add a new buffer then we're
concentrating our solution concentration
is simply the removal of water and
buffer components from the feed solution
that results in a more concentrated
solution of green fluorescent protein
from operation to operation or product
to product the purification process can
look quite different it could be as
simple as a single chromatography step
one cycle of tff to concentrate the
product and final filtration or it could
involve several different types of
chromatography daya filtration between
chromatography steps and a final
conventional filtration our green
fluorescent protein process is pretty
typical of the process for bio
pharmaceutical products the clarified
lysate will be pre-filtered go through
an ion exchange chromatography and the
GFP collected and pooled next ammonium
sulfate will be added to make the
solution high salt followed by
hydrophobic interaction chromatography
HIC the GFP is then collected and pooled
again the solution then goes through a
tff ultra filtration step which includes
concentration of the gfp followed by
daya filtration to exchange buffer
solutions and remove salt and the
process finishes up with final
filtration into bulk bottles or bags
it's time to gather everything we'll
need for the purification process and
ensure that the area and equipment are
ready to go the chromatography skid and
tff system are checked for proper
operation process hoses are attached and
examined for leaks we verify that we are
using the correct column resin and that
the resin is properly packed the column
is filled with a storage buffer and the
product path is checked for trapped air
and purged if necessary our raw
materials include the clarified lysate
from the recovery process various buffer
solutions that are tailored to specific
process steps and ammonium sulfate that
we add to one of the buffers to make it
high salt the purification process is
managed through the use of a batch
record there is a separate batch record
for each processing operation these
documents lead the operator through the
process step-by-step with each step
requiring a sign-off and seperate
verification by a second operator the
batch record also includes spaces for
documenting times activities operation
steps and instrument readings before the
process can begin the purification area
must be cleaned disinfected and
organized any unnecessary equipment or
materials are removed all equipment must
be cleaned sanitized and setup as
required by standard operating
procedures all required materials and
documentation must be gathered and
prepared before the process may begin
the purification process begins as the
transfer tank of clarified lysate from
the recovery process is connected to the
inlet pump on the chromatography skid
the first chromatography step in our
green fluorescent protein purification
process is an ion exchange at this point
in the process the pH of the clarified
lysate is about eight point zero which
means that the protein is negatively
charged because it is negatively charged
GFP will bind to the positively charged
anion exchange resin the pump draws the
lysate from the vessel pass the first
conductivity sensor and pressure sensor
and through the 0.45 micron pre filter
the prefilter removes any residual cell
debris or other particulates that may
have contaminated the solution if the
pre-filter begins to clog the pressure
sensor at the inlet side of the filter
will register a rising pressure and the
controller will signal the need for a
filter change after pre filtering and
before the column the lysate passes
through a flow meter and an air sensor
then as the lysate passes over the resin
beads the negatively charged protein
binds to the positively charged beads
the solution leaving the column passes a
UV optical density sensor a conductivity
sensor and a pH sensor the optical
density sensors low readings confirm
that the GFP is not in the solution so
the outlet valve sends the solution to
waste when all the lysate has entered
the column or when the capacity of the
beads to bind the protein has been
reached it's time for illusion illusion
is the release of in this case green
fluorescent protein from the beads by
using a new solution in this case a
buffer that includes NaCl sodium
chloride solution as the new buffer is
pumped through the beads at some point
the GFP no longer
binds to the beads and is released into
the buffer the resulting product stream
is usually referred to as the lu8 the UV
optical density sensor which measures
protein concentration indicates when
product begins eluding from the column
at this point outlet valves are switched
to allow flow of the lu8 the product
stream to a collection vessel when the
UV sensor indicates that all of the gfp
has come off of the chromatography resin
the outlet valves are switched to waste
when all of the lu8 has been collected
and pooled the anion exchange
chromatography step is finished and it's
time for HIC hydrophobic interaction
chromatography hydrophobic interaction
chromatography is based on the principle
that hydrophobic chemicals on the resin
surface will bind to hydrophobic patches
on the GFP protein in order for this to
occur the resin and protein lu8 have to
be in a high salt environment to remove
the water shielding the salt we use is
ammonium sulfate to remove the attached
GFP protein from the HIC column we
simply lower the salt concentration
during Ellucian causing the water
shielding to reform and the GFP protein
detaches from the resin into the
aleutian stream the protein-rich lu8 is
collected and pooled the product is now
ready for the last major step tff at
this point in the process tangential
flow filtration will be used to
concentrate and daya filter the gfp
product stream the lu8
is rich in green fluorescent protein but
it's still too dilute and too high in
salt as the solution moves through the
tff apparatus it leaves that supply tank
is pulled through a pump past a pressure
sensor and then across the filter
membrane everything that passes through
the membrane including the buffer
solution is known as permeate and for
this process is sent to waste the GFP
protein is larger than the pores of the
membrane and therefore is retained the
retained material called retentate is
recirculated to the supply tank
recirculation of the feed continues
until the desired concentration of gfp
is achieved following concentration and
while the protein solution is
recirculating a new solution a storage
buffer is added to the feed in effect
the protein is being washed by the flow
of a new buffer solution
in and the old buffer solution out as
this daya filtration step proceeds the
buffer solution that is being added to
the feed replaces the buffer solution
that the GFP was originally in
effectively removing any remaining salt
as well when this process is complete
the GFP solution is routed through a
point to two micron final filter and
then collected in appropriate containers
usually bottles or bags the purification
process is complete the green
fluorescent protein concentrate can now
move downstream to final fill finish to
be freeze dried and packaged