PEG Precipitation: A Powerful Tool for Monoclonal Antibody Purification
This alternative purification method to chromatography is readily scalable and fits a fully
disposable downstream process.
Mar 2, 2010
By: Michael Kuczewski, Emily Schirmer, PhD, Blanca Lain, PhD, Gregory ZarbisPapastoitsis, PhD
BioPharm International Supplements
ABSTRACT
Various precipitation techniques have been used in the industrial purification of
proteins for many years. Precipitation processes can be separated into two main
categories: impurity precipitation and product precipitation. Impurity precipitation is
operationally simpler but carryover of the precipitants can challenge subsequent unit
operations. Product precipitation may have a higher risk of damage to the target
molecule, but in addition to purifying the product, product precipitation also enables
buffer exchange and concentration during the resolubilization step. An antibody
precipitation step has been developed using a recombinant antibody produced in
PER.C6 cells and statistical design of experiments to optimize product yield and host
cell protein (HCP) removal. After appropriate precipitation conditions were developed,
two methods to capture the antibody pellet were evaluated: depth filtration and
microfiltration. A wash step was incorporated in both methods to reduce soluble
impurities. The final process resulted in a product yield of 90% and HCP reduction of
approximately 1 LRV.
The method of pellet capture was shown to have a significant
impact on the purity of the redissolved product. The
precipitation step is readily scalable and fits a fully disposable
downstream process.
Efforts are ongoing to identify alternatives to packed bed
chromatography to reduce the time and cost of processing hightiter product streams. Although many efforts focus on
membrane adsorbers that directly replace columns of the same
Percivia LLC
or similar chemistries, some older technologies are beginning to
gain ground in recombinant protein manufacturing. One attractive alternative to
chromatography is precipitation, which has been used in the plasma protein industry for many
years.1 A simple method of precipitation involves titrating the process fluid to the isoelectric
point of the protein that is to be precipitated.2 Lyotropic salts, such as ammonium sulfate, also
have a long history of use in precipitation processes.3 Short-chain fatty acids, such as caprylic
acid, are well known for their ability to precipitate DNA and host cell proteins (HCPs).4
Polyionic species also are useful precipitants for capturing a product of interest or removing
contaminating proteins.5,6
Polyethylene glycol (PEG) has been used for product and impurity precipitation.7,8 It also can
be combined with isoelectric precipitation to improve the efficiency of the separation.9,10
After precipitation, centrifugation or filtration can be used to perform solid–liquid
separation.11 Although centrifugation is a well-established method to achieve this separation,
washing the product pellet to remove impurities could be problematic, and it is not suited to a
single-use process. Filtration—normal- or tangential-flow—requires more development, but
washing the pellet is simpler, and it is readily adaptable to a single-use process.
In the present work, a product precipitation step was developed using PEG to recover a
monoclonal antibody (MAb) from clarified PER.C6 cell culture media. Appropriate
precipitation conditions were identified through the use of full factorial experimental designs.
Two filtration steps were evaluated for the capture and washing of the precipitated product,
and the superior method was scaled-up 10-fold. The total precipitation process resulted in
yields of approximately 90% and HCP reduction of 1 LRV with no significant increase in the
aggregate level of the redissolved MAb. Finally, the impact of the precipitation step on the
subsequent cation exchange (CEX) capture step was investigated.
Materials and Methods Reagents
USP grade salts, Tween 20, hydrochloric acid, acetic acid, and sodium hydroxide were
purchased from JT Baker (Phillipsburg, NJ). PEG was of reagent grade and purchased from
JT Baker or EMD Chemicals (Gibbstown, NJ). All buffers were prepared using MilliQ-grade
water (Millipore, Billerica, MA) and were filtered by 0.22-µm filtration before use.
Feedstock
A human MAb (IgG1, pI = 8.3, 150 kDa) was produced at Percivia, LLC using a PER.C6 cell
line. PER.C6 cells are human embryonic retinal cells immortalized by the adenovirus E1
gene, as described in US patent 5,994,128.12 The cells were cultured in a standard fed-batch
process or the XD process, both using chemically defined media.13,14 The fed-batch media
were clarified by sedimentation and depth filtration, and the XD media were clarified by the
enhanced cell settling (ECS) method followed by depth filtration.15 During ECS, Silica-PEI
resin was used to enhance cell settling and also reduce DNA and HCP.
Precipitation Condition Optimization
The conditions used to precipitate the MAb—PEG molecular weight, PEG concentration, and
pH—were optimized by full factorial experimental designs using Minitab software (State
College, PA). The pH of the clarified XD media was adjusted to the desired level with 2-M
Tris in a 15-mL conical tube. The PEG was added as a 40% (w/w) stock solution to the
desired final concentration. The tube was then centrifuged at 1,000g and the supernatant
decanted. Finally, the pellet was redissolved in phosphate-buffered saline (PBS).
Pellet Capture by Depth Filtration or Microfiltration
Depth filtration was performed with various grades of filter media. Millistak+HC D0HC,
C0HC, and X0HC were purchased from Millipore Corp. (Billerica, MA), and ZetaPlus
60SP02A was purchased from Cuno (Meriden, CT). Precipitation was carried out using a
40% (w/w) stock solution of PEG-3350 and the precipitated media was loaded at a feed flux
of 50 L/m2 /h until all of the material was loaded or the transmembrane pressure (TMP) was
15 psid. The filters were then washed with 20–30 L/m2 of 20 mM Tris pH 8.5 + 14.4% (w/w)
PEG-3350. After washing, 80 L/m2 of resolubilization buffer was passed through the filters at
100 L/m2 /h, and the permeate was recirculated through the device at 600 L/m2 /h until the
A280 of the permeate pool was stable indicating complete MAb dissolution. Finally, any
held-up product was recovered with a 20 L/m2 buffer flush and air blowdown of the filter
module. In some tests, the filter media was subsequently washed with 85-mM acetate pH 5.3
followed by 1-M NaCl. Pressure and flow data were collected using a custom engineered
system from ARC Technology Services (Nashua, NH).
Microfiltration was performed with a 0.22-µm hollow fiber membrane from GE Healthcare
Life Sciences (Piscataway, NJ). The PEG-3350 was added as a 40% (w/w) stock solution for
the small-scale experiment and in powder form for the scale-up work. The feed flux was 710
L/m2 /h and the retentate and permeate were unrestricted. The precipitate was first
concentrated 10- to 14-fold and then washed with three diafiltration volumes of 20 mM Tris
pH 8.5 plus 14.4% (w/w) PEG-3350. Finally, the precipitate was redissolved in 85 mM
sodium acetate pH 5.3 or 20 mM Tris plus 50 mM NaCl pH 7.5. Pressure, flow, and
conductivity data were collected using a Slice 200 benchtop system (Sartorius, Gottingen,
Germany) for small-scale testing and a SciPro system (SciLog, Middleton, WI) for the scaleup experiment.
Cation Exchange Chromatography
Toyopearl GigaCap S-650 was procured from Tosoh Bioscience (Montgomeryville, PA) in
the Toyoscreen 5-mL format. This resin has been previously demonstrated as a high capacity
capture step for MAbs.16 The column was equilibrated with 74-mM sodium acetate pH 5.3
and loaded to 90–95 mg-MAb/mL-resin using either clarified media or clarified and PEGtreated material, each adjusted to the same pH and conductivity as the equilibration buffer.
The column was then washed with equilibration buffer and the antibody eluted with 50 mM
sodium acetate pH 5.3 plus 90 mM NaCl.
Analytical Techniques
The MAb concentration in media-containing samples was determined by analytical Protein A
HPLC (Applied Biosystems, Foster City, CA). Aggregate levels were measured by sizeexclusion chromatography (SEC) using a TSKgel G3000SWXL column from Tosoh
Bioscience (Montgomeryville, PA), with peak detection by UV absorbance at 280 nm. HCP
levels were quantified by a PER.C6-specific ELISA from Cygnus Technologies (Southport,
NC). SDS-PAGE was performed with NuPAGE 4–12% Bis-Tris gels and staining was done
with SimplyBlue SafeStain, both from Invitrogen (Carlsbad, CA).
Results and Discussion Precipitation Conditions
For both molecular weights of PEG, 3,350 and 6,000 Da, the PEG
concentration was the dominant factor in the recovery and purity of the
redissolved MAb. Precipitation with PEG-3350 resulted in the highest
recovery (Figure 1). However, the higher recovery came at the expense of
higher HCP burden in the redissolved MAb. In the case of aggregated
MAb, the levels were not significantly different from the starting material,
but it should be noted that the particular MAb used in this work is not prone
to forming aggregates. The improvement of the HCP reduction with the use
of PEG-6000 was offset by the reduction in product yield. The final
Figure 1
condition selected was 14% (w/v) PEG-3350 (equivalent to 14.4% w/w)
and pH 8.5.
Pellet Capture by Depth Filtration
In the first experiment, ECS-clarified XD media was
precipitated and the pellet was captured by depth filtration
with X0HC media. No substantial increase in the
transmembrane pressure (TMP) was observed during the
loading of 361 g-MAb/m2 (data not shown). After washing,
resolubilization of the immobilized pellet was done with 85
Table 1. Yield and impurity
mM acetate pH 5.3. Even after recirculation for 2 h, the
removal for the PEG
precipitation operation using antibody had not completely redissolved, so 0.1% v/v Tween
depth filtration with X0HC to 20 was added to the resolubilization buffer. After an
additional 30 min of resolubilization, the MAb was fully
recover the product
dissolved.
Mass balance data are summarized in Table 1. The HCP
reduction was lower than in the previous experiment (Figure 1),
where only 5,000 ppm of HCP was in the final pool as compared
to 8,300 ppm in this experiment. Some depth filters, however,
are known to have hydrophobic and anion exchange (AEX)
adsorptive characteristics.17 The precipitated media was loaded at
a relatively high pH (8.5) and low conductivity (<10 mS/cm),
which may have induced binding of acidic proteins on an AEX
matrix. Furthermore, in the presence of PEG, the protein binding Figure 2
capacity of ion-exchange matrices has been shown to increase.18,19 Therefore, it is probable
that some of the HCPs that remained in solution after precipitation bound to the depth filter
media and eluted into the product during resolubilization. This hypothesis also is supported
by the difference in the HCP content of the precipitation supernatant and the depth filter
flow-through (9,900 versus 240 mg) which indicates HCP removal from the solution by the
depth filter. Following immobilization of the antibody onto the depth filter, it was redissolved
at a significantly lower pH (5.3) resulting in the elution of bound HCP, and reducing the
purity of the final product. This phenomenon could be mitigated by using less adsorptive
depth filter media, such as Millistak+HC D0HC/C0HC and ZetaPlus SP filters like 60SP02A,
or by redissolving the product in a low-salt, higher-pH buffer, like 20-mM Tris pH 7.5.
Table 2. Yield
and impurity
removal for the
PEG
precipitation
operation using
depth filtration
with various
filter media to
recover the
product
These strategies were tested by loading precipitated fed-batch media onto
D0HC, C0HC, X0HC, and 60SP02A filters. All filters were washed identically with the
PEG/Tris buffer, but the resolubilization was done with 20 mM Tris pH 7.5 plus Tween 20
for the Millipore filters and 85 mM acetate pH 5.3 plus Tween 20 for the Cuno filter. The
Millipore filters also were stripped with a low pH buffer (85 mM acetate pH 5.3) and high
salt (1 M NaCl) after the product was recovered to determine if any bound HCPs could be
eluted. Figure 2 shows that there was substantial fouling of all filters tested. Because the fedbatch media was not clarified by the ECS method, which has been shown to significantly
reduce DNA levels in XD harvests, there may be more DNA present, which precipitates in
high PEG concentrations.20 The higher DNA content in the precipitate may have reduced
filter capacities, but this has not been investigated. Table 2 shows that each of the
experiments resulted in better HCP reduction (84–88% versus 46%), and even though the
starting HCP burden was higher (49,000 ppm versus 13,000 ppm), the redissolved MAb
pools generally had lower HCP contents (6,000–7,800 ppm versus 8,300 ppm). The use of
low-adsorptive filters and redissolving in a higher pH buffer appear to solve the problem of
HCPs eluting from the depth filter media. SDS-PAGE of the strip fractions revealed that both
HCPs and product were bound to the filters—including the less adsorptive D0HC and C0HC
filters—and confirmed that X0HC media were more adsorptive than D0HC and C0HC media
(Figure 3).
Pellet Capture by Microfiltration
Although the HCP burden of the redissolved MAb was reduced by using a buffer
with a higher pH and less adsorptive depth filter media, the capacities of the
depth filters were low for fed-batch media (<400 g-MAb/m2 ). Microfiltration
(tangential flow filtration, MF TFF) was tested with the aim of improving
capacity with the added benefit of being able to use any buffer for resolubilization
because of the low binding characteristic of the hollow fiber. The hydraulic
performance of the MF TFF concentration and washing of fed-batch precipitate is
shown in Figure 4. The permeate flux was about 100 L/m2 /h and the TMP was
between 1.0 and 2.5 psid for the entire operation. The mass loading of 475 gFigure 3
MAb/m2 was better than that achieved in any of the depth filtration operations.
The product recovery and HCP reduction were both around 90%, slightly better than that
achieved in depth filtration (Table 3).
The resolubilization was much simpler and faster than for the depth
filtration; rather than recirculating buffer through the device, the buffer
was pumped through the inside of the lumens into the retentate vessel with
the permeate closed and allowed to mix for about 60 min. This was
sufficient to redissolve the antibody, and no excipients were needed.
Because of the difficulty in resolubilization with depth filtration, the
product was redissolved at or near the concentration in the feed media.
The final pool from the MF TFF process was nearly two-fold more
concentrated than the starting material.
Figure 4
Table 3. Yield and impurity
Because the retentate and permeate were open to atmospheric removal for the PEG
pressure, minimal instrumentation was required: a crossflow precipitation operation using
pump, one pressure sensor, and a transfer pump for the
microfiltration to recover the
diafiltration. The combination of high capacity, high
product at bench scale
recovery and HCP clearance, and operational simplicity make MF TFF a preferable option
for pellet capture as compared with depth filtration. The precipitation and MF TFF step was
scaled-up 10-fold to a 0.12 m2 hollow fiber device, and the performance was comparable to
the small scale (Table 4).
Cation Exchange Chromatography
Table 4. Yield and impurity
removal for the PEG
precipitation operation using
microfiltration to recover the
product at pilot scale
High capacity cation exchange (CEX) chromatography was
evaluated with feeds pretreated with or without PEG
precipitation. The precipitation step did not have any
significant impact on the step yield or the percentage
reduction of HCPs, but the eluate resulting from the
precipitated load material had seven-fold less HCP (Table 5).
Conclusions
Precipitation has long been
used in the plasma protein
industry to purify proteins at large scales. The technique has
been adapted here to the initial MAb purification from
clarified fed-batch and XD media in a scalable manner. Two Table 5. Yield and HCP
single-use filtration steps have been developed to capture and removal for GigaCap S-650
wash the precipitated product, eliminating the need for
loaded with precipitated
centrifugation. It was shown that the precipitation operation (+PEG) and nonprecipitated (–
did not negatively affect the yield of the CEX capture step, PEG) antibody
and it reduced the HCP content of the eluate by a factor of seven.
The ability to reduce the impurity burden so far upstream in the purification train is key to
truncating the downstream process or replacing traditional chromatography with other singleuse technologies. Lower impurity burdens can improve the loading capacity of flow-through
membrane adsorbers and possibly virus filters, which are generally very expensive items in a
process.
An added benefit is the ability to redissolve the antibody in a buffer that facilitates the
subsequent unit operation. For example, cell culture media typically requires extensive
titration and dilution or a UF–DF step to prepare for capture chromatography with a cation
exchanger. Here, the precipitated antibody can be dissolved in equilibration buffer at high
concentration, thus shortening the processing time. This can be important for products that do
not tolerate long exposure to low pH/conductivity conditions. In the case of this particular
antibody, the clarified media requires a more than two-fold dilution to be loaded onto a CEX
column, whereas the redissolved MAb could be loaded directly at nearly two-fold the
concentration of the unadjusted media. This is at least a four-fold reduction in the load
volume, which can result in substantial time savings for modern, high-capacity CEX resins.
Protein purification
From Wikipedia, the free encyclopedia
Jump to: navigation, search
Protein purification is a series of processes intended to isolate a single type of protein from
a complex mixture. Protein purification is vital for the characterization of the function,
structure and interactions of the protein of interest. The starting material is usually a
biological tissue or a microbial culture. The various steps in the purification process may free
the protein from a matrix that confines it, separate the protein and non-protein parts of the
mixture, and finally separate the desired protein from all other proteins. Separation of one
protein from all others is typically the most laborious aspect of protein purification.
Separation steps may exploit differences in (for example) protein size, physico-chemical
properties, binding affinity and biological activity.
Purpose
Purification may be preparative or analytical. Preparative purifications aim to produce a
relatively large quantity of purified proteins for subsequent use. Examples include the
preparation of commercial products such as enzymes (e.g. lactase), nutritional proteins (e.g.
soy protein isolate), and certain biopharmaceuticals (e.g. insulin). Analytical purification
produces a relatively small amount of a protein for a variety of research or analytical
purposes, including identification, quantification, and studies of the protein's structure, posttranslational modifications and function. Pepsin and urease were the first proteins purified to
the point that they could be crystallized.[1]
Strategies
Recombinant bacteria can be grown in a flask containing growth media.
Choice of a starting material is key to the design of a purification process. In a plant or
animal, a particular protein usually isn't distributed homogeneously throughout the body;
different organs or tissues have higher or lower concentrations of the protein. Use of only the
tissues or organs with the highest concentration decreases the volumes needed to produce a
given amount of purified protein. If the protein is present in low abundance, or if it has a high
value, scientists may use recombinant DNA technology to develop cells that will produce
large quantities of the desired protein (this is known as an expression system). Recombinant
expression allows the protein to be tagged, e.g. by a His-tag, to facilitate purification, which
means that the purification can be done in fewer steps. In addition, recombinant expression
usually starts with a higher fraction of the desired protein than is present in a natural source.
An analytical purification generally utilizes three properties to separate proteins. First,
proteins may be purified according to their isoelectric points by running them through a pH
graded gel or an ion exchange column. Second, proteins can be separated according to their
size or molecular weight via size exclusion chromatography or by SDS-PAGE (sodium
dodecyl sulfate-polyacrylamide gel electrophoresis) analysis. Proteins are often purified by
using 2D-PAGE and are then analysed by peptide mass fingerprinting to establish the protein
identity. This is very useful for scientific purposes and the detection limits for protein are
nowadays very low and nanogram amounts of protein are sufficient for their analysis.
Thirdly, proteins may be separated by polarity/hydrophobicity via high performance liquid
chromatography or reversed-phase chromatography.
Evaluating purification yield
The most general method to monitor the purification process is by running a SDS-PAGE of
the different steps. This method only gives a rough measure of the amounts of different
proteins in the mixture, and it is not able to distinguish between proteins with similar
apparent molecular weight.
If the protein has a distinguishing spectroscopic feature or an enzymatic activity, this property
can be used to detect and quantify the specific protein, and thus to select the fractions of the
separation, that contains the protein. If antibodies against the protein are available then
western blotting and ELISA can specifically detect and quantify the amount of desired
protein. Some proteins function as receptors and can be detected during purification steps by
a ligand binding assay, often using a radioactive ligand.
In order to evaluate the process of multistep purification, the amount of the specific protein
has to be compared to the amount of total protein. The latter can be determined by the
Bradford total protein assay or by absorbance of light at 280 nm, however some reagents used
during the purification process may interfere with the quantification. For example, imidazole
(commonly used for purification of polyhistidine-tagged recombinant proteins) is an amino
acid analogue and at low concentrations will interfere with the bicinchoninic acid (BCA)
assay for total protein quantification. Impurities in low-grade imidazole will also absorb at
280 nm, resulting in an inaccurate reading of protein concentration from UV absorbance.
Another method to be considered is Surface Plasmon Resonance (SPR). SPR can detect
binding of label free molecules on the surface of a chip. If the desired protein is an antibody,
binding can be translated directly to the activity of the protein. One can express the active
concentration of the protein as the percent of the total protein. SPR can be a powerful method
for quickly determining protein activity and overall yield. It is a powerful technology that
requires an instrument to perform.
Methods of protein purification
The methods used in protein purification can roughly be divided into analytical and
preparative methods. The distinction is not exact, but the deciding factor is the amount of
protein that can practically be purified with that method. Analytical methods aim to detect
and identify a protein in a mixture, whereas preparative methods aim to produce large
quantities of the protein for other purposes, such as structural biology or industrial use. In
general, the preparative methods can be used in analytical applications, but not the other way
around.
Extraction
Depending on the source, the protein has to be brought into solution by breaking the tissue or
cells containing it. There are several methods to achieve this: Repeated freezing and thawing,
sonication, homogenization by high pressure, filtration, or permeabilization by organic
solvents. The method of choice depends on how fragile the protein is and how sturdy the cells
are. After this extraction process soluble proteins will be in the solvent, and can be separated
from cell membranes, DNA etc. by centrifugation. The extraction process also extracts
proteases, which will start digesting the proteins in the solution. If the protein is sensitive to
proteolysis, it is usually desirable to proceed quickly, and keep the extract cooled, to slow
down proteolysis.
Precipitation and differential solubilization
Main article: Ammonium sulfate precipitation
In bulk protein purification, a common first step to isolate proteins is precipitation with
ammonium sulfate (NH4)2SO4. This is performed by adding increasing amounts of
ammonium sulfate and collecting the different fractions of precipitate protein. Ammonium
sulphate can be removed by dialysis.The hydrophobic groups on the proteins gets exposed to
the atmosphere and it attracts other protein hydrophobic groups and gets aggregated. Protein
precipitated will be large enough to be visible. One advantage of this method is that it can be
performed inexpensively with very large volumes.
The first proteins to be purified are water-soluble proteins. Purification of integral membrane
proteins requires disruption of the cell membrane in order to isolate any one particular protein
from others that are in the same membrane compartment. Sometimes a particular membrane
fraction can be isolated first, such as isolating mitochondria from cells before purifying a
protein located in a mitochondrial membrane. A detergent such as sodium dodecyl sulfate
(SDS) can be used to dissolve cell membranes and keep membrane proteins in solution
during purification; however, because SDS causes denaturation, milder detergents such as
Triton X-100 or CHAPS can be used to retain the protein's native conformation during
complete purification.
Ultracentrifugation
Main article: Ultracentrifuge
Centrifugation is a process that uses centrifugal force to separate mixtures of particles of
varying masses or densities suspended in a liquid. When a vessel (typically a tube or bottle)
containing a mixture of proteins or other particulate matter, such as bacterial cells, is rotated
at high speeds, the inertia of each particle yields an outward force proportional to its mass.
The tendency of a given particle to move through the liquid because of this force is offset by
the resistance the liquid exerts on the particle. The net effect of "spinning" the sample in a
centrifuge is that massive, small, and dense particles move outward faster than less massive
particles or particles with more "drag" in the liquid. When suspensions of particles are "spun"
in a centrifuge, a "pellet" may form at the bottom of the vessel that is enriched for the most
massive particles with low drag in the liquid.
Non-compacted particles remain mostly in the liquid called "supernatant" and can be
removed from the vessel thereby separating the supernatant from the pellet. The rate of
centrifugation is determined by the angular acceleration applied to the sample, typically
measured in comparison to the g. If samples are centrifuged long enough, the particles in the
vessel will reach equilibrium wherein the particles accumulate specifically at a point in the
vessel where their buoyant density is balanced with centrifugal force. Such an "equilibrium"
centrifugation can allow extensive purification of a given particle.
Sucrose gradient centrifugation — a linear concentration gradient of sugar (typically sucrose,
glycerol, or a silica based density gradient media, like Percoll) is generated in a tube such that
the highest concentration is on the bottom and lowest on top. Percoll is a trademark owned by
GE Healthcare companies. A protein sample is then layered on top of the gradient and spun at
high speeds in an ultracentrifuge. This causes heavy macromolecules to migrate towards the
bottom of the tube faster than lighter material. During centrifugation in the absence of
sucrose, as particles move farther and farther from the center of rotation, they experience
more and more centrifugal force (the further they move, the faster they move). The problem
with this is that the useful separation range of within the vessel is restricted to a small
observable window. Spinning a sample twice as long doesn't mean the particle of interest will
go twice as far, in fact, it will go significantly further. However, when the proteins are
moving through a sucrose gradient, they encounter liquid of increasing density and viscosity.
A properly designed sucrose gradient will counteract the increasing centrifugal force so the
particles move in close proportion to the time they have been in the centrifugal field. Samples
separated by these gradients are referred to as "rate zonal" centrifugations. After separating
the protein/particles, the gradient is then fractionated and collected.
Chromatographic methods
Chromatographic equipment. Here set up for a size exclusion chromatography. The buffer is
pumped through the column (right) by a computer controlled device.
Usually a protein purification protocol contains one or more chromatographic steps. The
basic procedure in chromatography is to flow the solution containing the protein through a
column packed with various materials. Different proteins interact differently with the column
material, and can thus be separated by the time required to pass the column, or the conditions
required to elute the protein from the column. Usually proteins are detected as they are
coming off the column by their absorbance at 280 nm. Many different chromatographic
methods exist:
Size exclusion chromatography
Main article: Gel permeation chromatography
Chromatography can be used to separate protein in solution or denaturing conditions by using
porous gels. This technique is known as size exclusion chromatography. The principle is that
smaller molecules have to traverse a larger volume in a porous matrix. Consequentially,
proteins of a certain range in size will require a variable volume of eluent (solvent) before
being collected at the other end of the column of gel.
In the context of protein purification, the eluent is usually pooled in different test tubes. All
test tubes containing no measurable trace of the protein to purify are discarded. The
remaining solution is thus made of the protein to purify and any other similarly-sized
proteins.
Separation based on charge or hydrophobicity
Hydrophobic Interaction Chromatography
Resin used in the column are amphiphiles with both hydrophobic and hydrophilic regions.
The hydrophobic part of the resin attracts hydrophobic region on the proteins. The greater the
hydrophobic region on the protein the stronger the attraction between the gel and that
particular protein.
Ion exchange chromatography
Main article: Ion exchange chromatography
Ion exchange chromatography separates compounds according to the nature and degree of
their ionic charge. The column to be used is selected according to its type and strength of
charge. Anion exchange resins have a positive charge and are used to retain and separate
negatively charged compounds, while cation exchange resins have a negative charge and are
used to separate positively charged molecules.
Before the separation begins a buffer is pumped through the column to equilibrate the
opposing charged ions. Upon injection of the sample, solute molecules will exchange with
the buffer ions as each competes for the binding sites on the resin. The length of retention for
each solute depends upon the strength of its charge. The most weakly charged compounds
will elute first, followed by those with successively stronger charges. Because of the nature of
the separating mechanism, pH, buffer type, buffer concentration, and temperature all play
important roles in controlling the separation.
Ion exchange chromatography is a very powerful tool for use in protein purification and is
frequently used in both analytical and preparative separations.
Nickel-affinity column. The resin is blue since it has bound nickel.
Affinity chromatography
Main article: Affinity chromatography
Affinity Chromatography is a separation technique based upon molecular conformation,
which frequently utilizes application specific resins. These resins have ligands attached to
their surfaces which are specific for the compounds to be separated. Most frequently, these
ligands function in a fashion similar to that of antibody-antigen interactions. This "lock and
key" fit between the ligand and its target compound makes it highly specific, frequently
generating a single peak, while all else in the sample is unretained.
Many membrane proteins are glycoproteins and can be purified by lectin affinity
chromatography. Detergent-solubilized proteins can be allowed to bind to a chromatography
resin that has been modified to have a covalently attached lectin. Proteins that do not bind to
the lectin are washed away and then specifically bound glycoproteins can be eluted by adding
a high concentration of a sugar that competes with the bound glycoproteins at the lectin
binding site. Some lectins have high affinity binding to oligosaccharides of glycoproteins that
is hard to compete with sugars, and bound glycoproteins need to be released by denaturing
the lectin.
Metal binding
Main article: Polyhistidine-tag
A common technique involves engineering a sequence of 6 to 8 histidines into the N- or Cterminal of the protein. The polyhistidine binds strongly to divalent metal ions such as nickel
and cobalt. The protein can be passed through a column containing immobilized nickel ions,
which binds the polyhistidine tag. All untagged proteins pass through the column. The
protein can be eluted with imidazole, which competes with the polyhistidine tag for binding
to the column, or by a decrease in pH (typically to 4.5), which decreases the affinity of the
tag for the resin. While this procedure is generally used for the purification of recombinant
proteins with an engineered affinity tag (such as a 6xHis tag or Clontech's HAT tag), it can
also be used for natural proteins with an inherent affinity for divalent cations.
Immunoaffinity chromatography
A HPLC. From left to right: A pumping device generating a gradient of two different
solvents, a steel enforced column and an apparatus for measuring the absorbance.
Main article: Immunoaffinity chromatography
Immunoaffinity chromatography uses the specific binding of an antibody to the target protein
to selectively purify the protein. The procedure involves immobilizing an antibody to a
column material, which then selectively binds the protein, while everything else flows
through. The protein can be eluted by changing the pH or the salinity. Because this method
does not involve engineering in a tag, it can be used for proteins from natural sources.[2]
Purification of a tagged protein
Another way to tag proteins is to engineer an antigen peptide tag onto the protein, and then
purify the protein on a column or by incubating with a loose resin that is coated with an
immobilized antibody. This particular procedure is known as immunoprecipitation.
Immunoprecipitation is quite capable of generating an extremely specific interaction which
usually results in binding only the desired protein. The purified tagged proteins can then
easily be separated from the other proteins in solution and later eluted back into clean
solution.
When the tags are not needed anymore, they can be cleaved off by a protease. This often
involves engineering a protease cleavage site between the tag and the protein.
HPLC
Main article: High performance liquid chromatography
High performance liquid chromatography or high pressure liquid chromatography is a form
of chromatography applying high pressure to drive the solutes through the column faster.
This means that the diffusion is limited and the resolution is improved. The most common
form is "reversed phase" hplc, where the column material is hydrophobic. The proteins are
eluted by a gradient of increasing amounts of an organic solvent, such as acetonitrile. The
proteins elute according to their hydrophobicity. After purification by HPLC the protein is in
a solution that only contains volatile compounds, and can easily be lyophilized.[3] HPLC
purification frequently results in denaturation of the purified proteins and is thus not
applicable to proteins that do not spontaneously refold.
Concentration of the purified protein
A selectively permeable membrane can be mounted in a centrifuge tube. The buffer is forced
through the membrane by centrifugation, leaving the protein in the upper chamber.
At the end of a protein purification, the protein often has to be concentrated. Different
methods exist.
Lyophilization
If the solution doesn't contain any other soluble component than the protein in question the
protein can be lyophilized (dried). This is commonly done after an HPLC run. This simply
removes all volatile components, leaving the proteins behind.
Ultrafiltration
Ultrafiltration concentrates a protein solution using selective permeable membranes. The
function of the membrane is to let the water and small molecules pass through while retaining
the protein. The solution is forced against the membrane by mechanical pump, gas pressure,
or centrifugation.
Analytical
Denaturing-Condition Electrophoresis
Gel electrophoresis is a common laboratory technique that can be used both as preparative
and analytical method. The principle of electrophoresis relies on the movement of a charged
ion in an electric field. In practice, the proteins are denatured in a solution containing a
detergent (SDS). In these conditions, the proteins are unfolded and coated with negatively
charged detergent molecules. The proteins in SDS-PAGE are separated on the sole basis of
their size.
In analytical methods, the protein migrate as bands based on size. Each band can be detected
using stains such as Coomassie blue dye or silver stain. Preparative methods to purify large
amounts of protein, require the extraction of the protein from the electrophoretic gel. This
extraction may involve excision of the gel containing a band, or eluting the band directly off
the gel as it runs off the end of the gel.
In the context of a purification strategy, denaturing condition electrophoresis provides an
improved resolution over size exclusion chromatography, but does not scale to large quantity
of proteins in a sample as well as the late chromatography columns.
Non-Denaturing-Condition Electrophoresis
An important non-denaturing electrophoretic procedure for isolating bioactive
metalloproteins in complex protein mixtures is termed 'quantitative native continuous
polyacrylamide gel electrophoresis (QPNC-PAGE).