Protein Purification
Protein Purification
Protein Purification
Protein
Purification
Handbook
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18-1132-29
Edition AC
Handbooks
from Amersham Pharmacia Biotech
Antibody Purification
Recombinant Protein Handbook
Gel Filtration Principles and Methods
Ion Exchange Chromatography Principles and Methods
Hydrophobic Interaction Chromatography Principles and Methods
Affinity Chromatography Principles and Methods
Expanded Bed Adsorption Principles and Methods
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Code
Code
Code
Code
Code
No.
No.
No.
No.
No.
No.
No.
18-1037-46
18-1142-75
18-1022-18
18-1114-21
18-1020-90
18-1022-29
18-1124-26
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Code
Code
Code
No.
No.
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18-1124-19
18-1127-31
18-1100-98
18-1121-86
Code
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Code
Code
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No.
No.
No.
No.
18-1128-62
18-1129-81
18-1128-63
18-1123-93
18-1129-75
Antibody Purification
Handbook
18-1037-46
Protein Purification
HiTrap, Sepharose, STREAMLINE, Sephadex, MonoBeads, Mono Q, Mono S, MiniBeads, RESOURCE, SOURCE,
Superdex, Superose, HisTrap, HiLoad, HiPrep, INdEX, BPG, BioProcess, FineLINE, MabTrap, MAbAssistant, Multiphor,
FPLC, PhastSystem and KTA are trademarks of Amersham Pharmacia Biotech Limited.
Handbook
18-1132-29
Affinity Chromatography
Chromatofocusing
Gel Filtration
Protein
Purification
Handbook
Contents
Introduction........................................................................................................5
Chapter 1
Purification Strategies - A Simple Approach ......................................................7
Preparation ..............................................................................................8
Three Phase Purification Strategy ............................................................8
General Guidelines for Protein Purification ............................................10
Chapter 2
Preparation ......................................................................................................11
Before You Start .................................................................................... 11
Sample Extraction and Clarification ......................................................14
Chapter 3
Three Phase Purification Strategy ....................................................................17
Principles ................................................................................................17
Selection and Combination of Purification Techniques ..........................18
Sample Conditioning ..............................................................................24
Chapter 4
Capture ............................................................................................................27
Chapter 5
Intermediate Purification ..................................................................................35
Chapter 6
Polishing ..........................................................................................................38
Chapter 7
Examples of Protein Purification Strategies ......................................................43
Three step purification of a recombinant enzyme ..................................43
Three step purification of a recombinant antigen binding fragment ......47
Two step purification of a monoclonal antibody ....................................52
One step purification of an integral membrane protein ..........................55
Chapter 8
Storage Conditions ..........................................................................................59
Extraction and Clarification Procedures ................................................ 60
Chapter 9
Principles and Standard Conditions for Purification Techniques ......................71
Ion exchange (IEX) ................................................................................71
Hydrophobic interaction (HIC) ..............................................................77
Affinity (AC) ..........................................................................................83
Gel filtration (GF) ..................................................................................86
Reversed phase (RPC) ............................................................................90
Expanded bed adsorption (EBA) ............................................................93
Introduction
The development of techniques and methods for protein purification has been an
essential pre-requisite for many of the advancements made in biotechnology.
This handbook provides advice and examples for a smooth path to protein
purification. Protein purification varies from simple one-step precipitation
procedures to large scale validated production processes. Often more than one
purification step is necessary to reach the desired purity. The key to successful and
efficient protein purification is to select the most appropriate techniques,
optimise their performance to suit the requirements and combine them in a logical
way to maximise yield and minimise the number of steps required.
Most purification schemes involve some form of chromatography. As a result
chromatography has become an essential tool in every laboratory where protein
purification is needed. Different chromatography techniques with different selectivities can form powerful combinations for the purification of any biomolecule.
The development of recombinant DNA techniques has revolutionised the production of proteins in large quantities. Recombinant proteins are often produced in
forms which facilitate their subsequent chromatographic purification. However,
this has not removed all challenges. Host contaminants are still present and problems related to solubility, structural integrity and biological activity can still
exist.
Although there may appear to be a great number of parameters to consider, with
a few simple guidelines and application of the Three Phase Purification Strategy
the process can be planned and performed simply and easily, with only a basic
knowledge of the details of chromatography techniques.
Advice codes:
general advice for any purification
shortcuts
Chapter 1
Purification Strategies
- a simple approach
Apply a systematic approach to development of a purification strategy.
The first step is to describe the basic scenario for the purification. General
considerations answer questions such as: What is the intended use of the product?
What kind of starting material is available and how should it be handled? What
are the purity issues in relation to the source material and intended use of the
final product? What has to be removed? What must be removed completely?
What will be the final scale of purification? If there is a need for scale-up, what
consequences will this have on the chosen purification techniques? What are the
economical constraints and what resources and equipment are available?
Most purification protocols require more than one step to achieve the desired
level of product purity. This includes any conditioning steps necessary to transfer
the product from one technique into conditions suitable to perform the next
technique. Each step in the process will cause some loss of product. For example,
if a yield of 80% in each step is assumed, this will be reduced to only 20%
overall yield after 8 processing steps as shown in Figure 1. Consequently, to reach
the targets for yield and purity with the minimum number of steps and the
simplest possible design, it is not efficient to add one step to another until purity
requirements have been fulfilled. Occasionally when a sample is readily available
purity can be achieved by simply adding or repeating steps. However, experience
shows that, even for the most challenging applications, high purity and yield can
be achieved efficiently in fewer than four well-chosen and optimised purification
steps. Techniques should be organised in a logical sequence to avoid the need for
conditioning steps and the chromatographic techniques selected appropriately to
use as few purification steps as possible.
Limit the number of steps in a purification procedure
Yield (%)
10
80
95% / step
60
90% / step
40
85% / step
20
80% / step
75% / step
0
1
8 Number of steps
Preparation
The need to obtain a protein, efficiently, economically and in sufficient purity and
quantity, applies to every purification. It is important to set objectives for purity,
quantity and maintenance of biological activity and to define the economical and
time framework for the work. All information concerning properties of the target
protein and contaminants will help during purification development. Some simple
experiments to characterise the sample and target molecule are an excellent investment. Development of fast and reliable analytical assays is essential to
follow the progress of the purification and assess its effectiveness. Sample
preparation and extraction procedures should be developed prior to the first
chromatographic purification step.
With background information, assays and sample preparation procedures in place
the Three Phase Purification Strategy can be considered.
Purity
Polishing
Achieve final
high level purity
Intermediate
purification
Capture
Preparation,
extraction,
clarification
Remove bulk
impurities
Isolate, concentrate
and stabilise
Step
Fig. 2. Preparation and the Three Phase Purification Strategy
KEEP IT SIMPLE!
10
Chapter 2
Preparation
Before You Start
The need to obtain a protein, efficiently, economically and in sufficient purity and
quantity, applies to any purification, from preparation of an enriched protein
extract for biochemical characterisation to large scale production of a therapeutic
recombinant protein. It is important to set objectives for purity and quantity,
maintenance of biological activity and economy in terms of money and time.
Purity requirements must take into consideration the nature of the source
material, the intended use of the final product and any special safety issues. For
example, it is important to differentiate between contaminants which must be
removed and those which can be tolerated. Other factors can also influence the
prioritisation of objectives. High yields are usually a key objective, but may be
less crucial in cases where a sample is readily available or product is required only
in small quantities. Extensive method development may be impossible without
resources such as an KTAdesign chromatography system. Similarly, time
pressure combined with a slow assay turnaround will steer towards less extensive
scouting and optimisation. All information concerning properties of the target
protein and contaminants will help during purification development, allowing
faster and easier technique selection and optimisation, and avoiding conditions
which may inactivate the target protein.
Development of fast and reliable analytical assays is essential to follow the
progress of the purification and assess effectiveness (yield, biological activity,
recovery).
Define objectives
Goal: To set minimum objectives for purity and quantity, maintenance of
biological activity and economy in terms of money and time.
Define purity requirements according to the final use of the product.
Purity requirement examples are shown below.
Extremely high > 99%
High 95- 99 %
Moderate < 95 %
11
Temperature stability
pH stability
Detergent requirement
Protease sensitivity
Redox sensitivity
Molecular weight
Charge properties
Biospecific affinity
Hydrophobicity
13
The importance of a reliable assay for the target protein cannot be overemphasised. When testing chromatographic fractions ensure that the buffers used
for separation do not interfere with the assay. Purity of the target protein is most
often estimated by SDS-PAGE, capillary electrophoresis, reversed phase
chromatography or mass spectrometry. Lowry or Bradford assays are used most
frequently to determine the total protein.
The Bradford assay is particularly suited to samples where there is a high lipid
content which may interfere with the Lowry assay.
For large scale protein purification the need to assay for target proteins and
critical impurities is often essential. In practice, when a protein is purified for
research purposes, it is too time consuming to identify and set up specific assays
for harmful contaminants. A practical approach is to purify the protein to a
certain level, and then perform SDS-PAGE after a storage period to check for
protease cleavage. Suitable control experiments, included within assays for
bio-activity, will help to indicate if impurities are interfering with results.
14
The need for sample preparation prior to the first chromatographic step is
dependent upon sample type. In some situations samples may be taken directly to
the first capture step. For example cell culture supernatant can be applied directly
to a suitable chromatographic matrix such as Sepharose Fast Flow and may
require only a minor adjustment of the pH or ionic strength. However, it is most
often essential to perform some form of sample extraction and clarification
procedure.
If sample extraction is required the chosen technique must be robust and suitable
for all scales of purification likely to be used. It should be noted that a technique
such as ammonium sulphate precipitation, commonly used in small scale, may be
unsuitable for very large scale preparation. Choice of buffers and additives must
be carefully considered if a purification is to be scaled up. In these cases
inexpensive buffers, such as acetate or citrate, are preferable to the more complex
compositions used in the laboratory. It should also be noted that dialysis and
other common methods used for adjustment of sample conditions are
unsuitable for very large or very small samples.
For repeated purification, use an extraction and clarification technique
that is robust and able to handle sample variability. This ensures a
reproducible product for the next purification step despite variability in
starting material.
Use additives only if essential for stabilisation of product or improved
extraction. Select those which are easily removed. Additives may need to
be removed in an extra purification step.
Use pre-packed columns of Sephadex G-25 gel filtration media, for
rapid sample clean-up at laboratory scale, as shown in Table 2.
Table 2. Pre-packed columns for sample clean-up.
Pre-packed column
Sample volume
loading per run
Sample volume
recovery per run
Code No.
2.5 -15 ml
0.25 - 1.5 ml
0.05 - 0.2 ml
1.5 - 2.5 ml
7.5
1.0
0.2
2.5
17-5087-01
17-1408-01
17-0774-01
17-0851-01
20 ml
2.0 ml
0.3 ml
3.5 ml
Sephadex G-25 gel filtration media are used at laboratory and production scale
for sample preparation and clarification of proteins >5000. Sample volumes of up
to 30%, or in some cases, 40% of the total column volume are loaded. In a single
step, the sample is desalted, exchanged into a new buffer, and low molecular
weight materials are removed. The high volume capacity, relative insensitivity to
sample concentration, and speed of this step enable very large sample volumes to
be processed rapidly and efficiently. Using a high sample volume load results in a
separation with minimal sample dilution (approximately 1:1.4). Chapter 8
contains further details on sample storage, extraction and clarification
procedures.
15
Sephadex G-25 is also used for sample conditioning, e.g. rapid adjustment of pH,
buffer exchange and desalting between purification steps.
Media for consideration:
Sephadex G 25 gel filtration
For fast group separations between high and low molecular weight substances
Typical flow velocity 60 cm/h (Sephadex G-25 Superfine, Sephadex G-25 Fine),
150 cm/h (Sephadex G-25 Medium).
Note:
16
Chapter 3
Purity
Polishing
Achieve final
high level purity
Intermediate
purification
Capture
Preparation,
extraction,
clarification
Remove bulk
impurities
Isolate, concentrate
and stabilise
Step
Fig. 3. Preparation and the Three Phase Purification Strategy.
During the intermediate purification phase the objectives are to remove most of
the bulk impurities, such as other proteins and nucleic acids, endotoxins and
viruses.
In the polishing phase most impurities have already been removed except for trace
amounts or closely related substances. The objective is to achieve final purity.
It should be noted that this Three Phase Strategy does not mean that all strategies
must have three purification steps. For example, capture and intermediate
purification may be achievable in a single step, as may intermediate purification
and polishing. Similarly, purity demands may be so low that a rapid capture step
is sufficient to achieve the desired result, or the purity of the starting material may
be so high that only a polishing step is needed. For purification of therapeutic
proteins a fourth or fifth purification step may be required to fulfil the highest
purity and safety demands.
The optimum selection and combination of purification techniques for Capture,
Intermediate Purification and Polishing is crucial for an efficient purification
process.
Speed
Capacity
Recovery
Technique
Charge
Size
Hydrophobicity
Affinity (AC)
19
Main features
Capture
Intermediate
Polish
Sample Start
condition
Sample End
condition
IEX
high resolution
high capacity
high speed
high ionic
strength or
pH change
concentrated
HIC
good resolution
good capacity
high speed
low ionic
strength
concentrated
AC
high resolution
high capacity
high speed
specific binding
conditions
sample volume
not limiting
specific
elution
conditions
concentrated
GF
high resolution
using Superdex
limited sample
volume (<5% total
column volume)
and flow rate
range
buffer
exchanged (if
required)
diluted
RPC
high resolution
requires organic
solvents
in organic
solvent, risk loss
of biological
activity
concentrated
Table 4. Suitability of purification techniques for the Three Phase Purification Strategy
Capture
GF
desalt mode
GF
desalt mode
AC
IEX
Intermediate
Purification
Polish
GF
GF
GF
desalt mode
HIC
IEX
dilution may
be needed
IEX
HIC
GF
GF
Capture
AC
IEX
Intermediate
Purification
Polishing
GF
or
RPC
GF
or
RPC
IEX
Precipitation
(e.g. in high ionic strength)
HIC
Resolubilise
GF
Treat as for
sample in high salt
concentration
For any capture step, select the technique showing the strongest binding
to the target protein while binding as few of the contaminants as possible,
i.e. the technique with the highest selectivity and/or capacity for the protein of
interest.
21
Capture by IEX
Basic proteins
STREAMLINE SP or SP Sepharose XL
Suggested binding buffer:
20 mM sodium phosphate, pH 7
Suggested elution buffer:
Binding buffer + 0.5 M NaCl
Acidic proteins
STREAMLINE DEAE or Q Sepharose XL
Suggested binding buffer:
50 mM Tris.HCl, pH 8
Suggested elution buffer:
Binding buffer + 0.5 M NaCl
Polishing by GF
Superdex 75 prep grade or Superdex 200 prep grade
Suggested buffer: as required by subsequent use
22
Sample:
Column:
Flow:
Buffer A:
Buffer B.
Gradient:
A280nm
0.5
A280nm
0.5
(3)
Sample:
Column:
Flow rate:
Buffer A:
Buffer B.
Gradient:
10
20
30
Time (min)
0
(3)
10
20
Time (min)
Consider RPC for a polishing step provided that the target protein can
withstand the run conditions.
Reversed phase chromatography (RPC) separates proteins and peptides on the
basis of hydrophobicity. RPC is a high selectivity (high resolution) technique,
requiring the use of organic solvents. The technique is widely used for purity
check analyses when recovery of activity and tertiary structure are not essential.
Since many proteins are denatured by organic solvents, the technique is not
generally recommended for protein purification where recovery of activity and
return to a correct tertiary structure may be compromised. However, in the
polishing phase, when the majority of protein impurities have been removed, RPC
can be excellent, particularly for small target proteins which are often not
denatured by organic solvents.
If a purification is not intended for scale up (i.e. only milligram quantities of
product are needed), use high performance, pre-packed media such as
Sepharose High Performance (IEX, HIC), SOURCE (IEX, HIC),
MonoBeads (IEX), or Superdex (GF) for all steps.
Recommended media for a standard protocol
Purification step
Media
Quantity
Code No.
Capture
Capture
Capture
Capture
Intermediate purification
Polishing
Polishing
Sample clarification/conditioning
Sample clarification/conditioning
Sample clarification/conditioning
STREAMLINE SP
STREAMLINE DEAE
HiPrep 16/10 SP XL
HiPrep 16/10 Q XL
HiPrep 16/10 Phenyl FF (high sub)
HiLoad 16/60 Superdex 75 prep grade
HiLoad 16/60 Superdex 200 prep grade
Pre-packed PD-10 Column
HiTrap Desalting
HiPrep 26/10 Desalting
300 ml
300 ml
1 column
1 column
1 column
1 column
1 column
30 columns
5 columns
1 column
17-0993-01
17-0994-01
17-5093-01
17-5092-01
17-5095-01
17-1068-01
17-1069-01
17-0851-01
17-1408-01
17-5087-01
23
Sample Conditioning
Although additional sample handling between purification steps should be
avoided, it may be necessary to adjust the buffer conditions of an eluted product
(pH, ionic strength and/or buffering ions) to ensure compatibility with the
following purification technique.
Sephadex G-25 is an ideal media for rapid desalting and pH adjustment by buffer
exchange between purification steps. Sample volumes of up to 30%, or in some
cases 40%, of the total column volume are loaded. In a single step, the sample is
desalted, exchanged into a new buffer, and low molecular weight materials are
removed. Figure 7 shows a typical desalt/buffer exchange separation. The high
volume capacity and speed of this step enable very large sample volumes to be
processed rapidly and efficiently. The high sample volume load results in a
separation with minimal sample dilution. Sephadex G-25 is also used for rapid
sample clean-up at laboratory scale.
A280 nm
(mS/cm)
0.25
0.20
10.0
0.15
protein
salts
0.10
5.0
0.05
0.00
0.0
1.0
2.0
(min)
Time
Sample volume
loading per run
Sample volume
recovery per run
Code No.
2.5 -15 ml
0.25 - 1.5 ml
0.05 - 0.2 ml
1.5 - 2.5 ml
7.5
1.0
0.2
2.5
17-5087-01
17-1408-01
17-0774-01
17-0851-01
24
20 ml
2.0 ml
0.3 ml
3.5 ml
25
26
Chapter 4
Capture
Resolution
Speed
Capacity
Recovery
The most common technique for a capture step is ion exchange chromatography
(IEX) which has high binding capacity. IEX media are resistant to harsh cleaning
conditions which may be needed after purification of crude samples. Typically
proteins are eluted from an IEX column using a salt gradient. However, during
method development, a transfer to a step elution will give a simple, robust
separation with a shorter run time and decreased buffer consumption. It is often
possible to use high sample loadings since the focus is not on resolution (high
sample loadings will decrease resolution). High speed and capacity and low buffer
consumption are particularly advantageous for large scale purification, as shown
in Figure 8.
A280 nm
%B
a
100
3.0
EGF
2.0
150
Column:
Sample:
1.0
15
0
0
150
Volume (I)
100
50
Conductivity
Column:
Adsorbent:
Sample:
Buffer A:
Buffer B:
Flow:
Gradient:
Eluate:
Spec. act.
5.0
10.0
15.0
20.0
Volume (l)
Column:
Sample:
Sample volume:
Starting buffer:
Elution buffer:
Flow:
2.0
1.5
1.0
0.5
0.0
0
200
400
600
Volume (m)
For large scale capture, throughput will often be the focus during method
development. It is important to consider all aspects: sample extraction
and clarification, sample loading capacity, flow rate during equilibration, binding,
washing, elution and cleaning, and the need for cleaning-in-place procedures.
In principle, a capture step is designed to maximise capacity and/or speed at the
expense of some resolution. However, there is usually significant resolution and
purification from molecules which have significant physicochemical differences
compared to the target protein. Recovery will be of concern in any preparative
situation, especially for production of a high value product, and it is important to
assay for recovery during optimisation of the capture step. Examples of capture
steps are shown on page 30.
Media for capture steps should offer high speed and high capacity.
Sepharose XL (IEX)
For capture steps handling crude mixtures at laboratory and process scale.
Fast removal and a combination of high capacity and good resolution at high
flow rates are the main characteristics. Recommended flow velocity is
100-500 cm/h.
Particle size: 90 m. Available in pre-packed columns and as bulk media.
29
A 280 nm
A 405 nm
Column:
Sample:
HiTrap Chelating, 1 ml
5 ml cytoplasmic extract containing (His)-tagged
glutathione-S-transferase
Binding buffer: 20 mM phosphate buffer, 0.5 M NaCl,
20 mM imidazole, pH 7.4
Elution buffer: 20 mM phosphate buffer, 0.5 M NaCl,
500 mM imidazole, pH 7.4
Flow:
2 mL/min (312 cm/h)
31
HiTrap column
Code No.
Quantity/
components
Approximate
binding capacity per ml gel
Isolation of
immunoglobulins
IgG classes, fragments
and subclasses
HiTrap rProtein A
17-5079-01
17-5080-01
17-5029-02
5 x 1 ml
1 x 5 ml
2 x 1 ml
human
IgG 50 mg/ml
HiTrap Protein A
17-0402-01
17-0402-03
17-0403-01
5 x 1 ml
2 x 1 ml
1 x 5 ml
human
IgG 20 mg/ml
HiTrap Protein G
17-0404-01
17-0404-03
17-0405-01
5 x 1 ml
2 x 1 ml
1 x 5 ml
human
IgG 25 mg/ml
MAbTrap GII
17-1128-01
HiTrap Protein G
column (1 ml),
accessories, pre-made
buffers for 10 purifications
as above
Mouse recombinant
Single chain antibody
Fragment variable
(ScFv) produced in
E. coli
RPAS Purification
Module
17-1362-01
0.17 mg
ScFv/5 ml
HiTrap IgY
Purification
17-5111-01
1 x 5 ml
IgY 20 mg/ml
IgM
HiTrap IgM
Purification
17-5110-01
5 x 1 ml
IgM 5 mg/ml
32
Approximate
binding capacity per ml gel
Application
HiTrap column
Code No.
Quantity/
components
HiTrap Blue
17-0412-01
17-0413-01
5 x 1 ml
1 x 5 ml
HSA 20 mg/ml
HiTrap Chelating
17-0408-01
17-0409-01
5 x 1 ml
1 x 5 ml
(His)6-tagged
protein (27.6
kD) 12 mg /ml
Histidine-tagged fusion
proteins
HisTrap
17-1880-01
HiTrap Chelating
column (1 ml), accessories, pre-made
buffers
as above
HiTrap
Streptavidin
17-5112-01
5 x 1 ml
biotinylated
BSA 6 mg/ml
Coagulation factors,
lipoprotein lipases,
steroid receptors,
hormones, DNA
binding proteins,
interferon, protein
synthesis factors
HiTrap Heparin
17-0406-01
17-0407-01
5 x 1 ml
1 x 5 ml
ATIII (bovine)
3 mg/ml
HiTrap
NHS-activated
17-0716-01
17-0717-01
5 x 1 ml
1 x 5 ml
ligand specific
33
System:
Column:
Medium:
Sample:
Buffer A:
Buffer B:
Flow:
BioProcess Modular
STREAMLINE 200 (i.d. 200 mm)
STREAMLINE DEAE, 4.7 L
4.7 kg of cells were subjected to osmotic shock and
suspended in a final volume of 180 L 50 mM Tris
buffer, pH 7.4, before application onto the expanded bed.
50 mM Tris buffer, pH 7.4
50 mM Tris, 0.5 M sodium chloride, pH 7.4
400 cm/h during sample application and wash
100 cm/h during elution
A 280 nm
100
1.0
80
2.0
60
40
20
Sample application
100
Washing
Buffer A
200
Elution
Buffer B
260
Volume (L)
Fig. 10. Purification of a recombinant protein Pseudomonas aeruginosa exotoxin A capture step.
34
Chapter 5
Intermediate Purification
Resolution
Speed
Capacity
Recovery
Column:
Medium:
Sample:
A280 nm
0.50
Buffer A:
Buffer B:
Gradient:
Flow:
0.40
0.30
0.20
0.10
Pool
0.00
0.0
2.0
4.0
6.0
8.0
10.0
12.0
Volume (L)
Column:
Buffer A:
Buffer B:
Sample:
Gradient:
Flow:
Annexin V
60
Time (min)
SOURCE (IEX)
For fast, high resolution and high capacity intermediate purification.
SOURCE media are for high throughput, high capacity and high resolution
purification. Frequently, if filtered samples are used, the intermediate purification
step can be combined with the capture step. A flow velocity up to 2000 cm/h is
possible. SOURCE 30 is also a good choice at large scale for intermediate
purification.
Particle size: 15 m. Available in pre-packed columns and as bulk media.
Particle size: 30 m. Available as bulk media.
36
Code No.
17-6001-01
17-1349-01
17-1187-01
37
Chapter 6
Polishing
Resolution
Speed
Capacity
Recovery
38
A280 nm
monomeric
ZZ-Brain IGF
Column:
a
0.01
Sample:
Sample load:
Buffer:
Flow:
0.005
VO
I Fraction 1
XK 16/60 packed
with Superdex 75
prep grade
partly purified
ZZ-brain IGF
1.0 ml
0.3 M ammonium
acetate pH 6.0
0.5 ml/min
(15 cm/h)
Vt
3 I4
4
Time (h)
A280 nm
%B
100
0.10
Sample:
Column:
Buffer A:
Buffer B:
Gradient:
Flow:
80
0.08
60
0.06
0.04
40
0.02
20
0
0
20.0
40.0
0
60.0
Time (min)
Fig 13. Final polishing step of recombinant epidermal growth factor, using reversed phase
chromatography. Method developed on pre-packed RESOURCE RPC and scaled up on
SOURCE 15RPC.
Gel filtration is also the slowest of all chromatography techniques and the size of
the column determines the volume of sample that can be applied. It is therefore
most logical to use gel filtration after techniques which reduce sample volume so
that smaller columns can be used.
39
Superdex (GF)
High productivity gel filtration media for polishing.
Superdex media are high resolving gel filtration media for short run times and
good recovery. Superdex is the first choice at laboratory scale and Superdex prep
grade for large scale applications. Typical flow velocity is up to 75 cm/h.
Particle size Superdex: 13 m. Available in pre-packed columns.
Particle size Superdex prep grade: 34 m. Available in pre-packed columns and as
bulk media.
MonoBeads (IEX)
Media for polishing at laboratory scale when highest resolution is essential.
These media offer high capacity and high resolution separations at laboratory
scale. Typical flow velocity is 150-600 cm/h.
Particle size: 10 m. Available in pre-packed columns.
40
SOURCE 30 (IEX)
SOURCE 30 media are for high throughput, high capacity and high resolution
purification. However, these media can be an alternative choice for polishing
offering a flow velocity of up to 2000 cm/h at large scale.
Particle size: 30 m. Available as bulk media.
Note:
41
42
Chapter 7
Examples of
Protein Purification Strategies
The Three Phase Purification Strategy has been successfully applied to many
purification schemes from simple laboratory scale purification to large, industrial
scale production. Examples highlighted in this chapter demonstrate applications
in which a standard protocol was applied, i.e. sample extraction and clarification,
capture, intermediate purification and polishing. There are also examples where
strategies were developed requiring even fewer steps, by following the general
guidelines for protein purification given in this handbook and selecting the most
appropriate technique and media to fulfil the purification objectives. In most of
these examples methods were developed using KTAdesign chromatography
systems.
Example 1.
Target Molecule
Deacetoxycephalosporin C synthase (DAOCS), an oxygen-sensitive enzyme.
Source Material
Recombinant protein over-expressed in soluble form in the cytoplasm of E. coli
bacteria.
43
Capture
The capture step focused on the rapid removal of the most harmful contaminants
from the relatively unstable target protein. This, together with the calculated
isoelectric point of DAOCS (pI = 4.8), led to the selection of an anion exchange
purification. A selection of anion exchange columns, including those from HiTrap
IEX Test Kit, were screened to select the optimum medium (results not shown)
before using a larger column for the optimisation of the capture step. Q
Sepharose XL, a high capacity medium, well suited for capture, was chosen.
As shown in Figure 14, optimisation of the capture step allowed the use of a step
elution at high flow rate to speed up the purification. This was particularly
advantageous when working with this potentially unstable sample.
System:
Column:
Sample:
Sample volume:
Buffer A:
Buffer B:
Flow:
mAU
KTAFPLC
HiPrep 16/10 Q XL
Clarified E. coli extract
40 ml
50 mM Tris-HCl, 1 mM EDTA, pH 7.5; 2 mM DTT, 0.2 M benzamidine-HCl, 0.2 mM PMSF
A + 1.0 M NaCl
10 ml/min (300 cm/h)
mS/cm
mS/cm
mAU
400
80
3000
80
300
60
20
100
200
300
ml
40
1000
1000
60
40
20
0
80
3000
2000
40
100
mS/cm
60
2000
200
mAU
0
0
100
200 ml
20
0
0
0
50
100
150
200 ml
Fig. 14. Capture using IEX and optimisation of purification conditions. The elution position of DAOCS is shaded.
44
Intermediate Purification
Hydrophobic interaction chromatography (HIC) was selected because the
separation principle is complementary to ion exchange and because a minimum
amount of sample conditioning was required. Hydrophobic properties are
difficult to predict and it is always recommended to screen different media.
The intermediate purification step was developed by screening pre-packed hydrophobic interaction media (RESOURCE HIC Test Kit) to select the optimum
medium for the separation (results not shown). SOURCE 15ISO was selected on
the basis of the resolution achieved. In this intermediate step, shown in Figure 15,
the maximum possible speed for separation was sacrificed in order to achieve
higher resolution and to allow significant reduction of remaining impurities.
System:
Column:
Sample:
Sample volume:
Buffer A:
Buffer B:
Gradient:
Flow:
KTAFPLC
SOURCE 15ISO, packed in HR 16/10 column
DAOCS pool from HiPrep 16/10 Q XL
40 ml
1.6 M ammonium sulphate, 10% glycerol,
50 mM Tris-HCl, 1 mM EDTA, 2 mM DTT,
0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5
50 mM Tris-HCl, 10% glycerol, 1 mM EDTA, 2 mM DTT,
0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5
016% B in 4 CV, 1624% B in 8 CV, 2435% B in
4 CV, 100% B in 4 CV
5 ml/min (150 cm/h)
A 280 nm
400
300
200
100
0
0
100
200
ml
Polishing
The main goal of the polishing step was to remove aggregates and minor
contaminants and to transfer the purified sample into a buffer suitable for use in
further structural studies. Superdex 75 prep grade, a gel filtration medium giving
high resolution at relatively short separation times, was selected since the
molecular weight of DAOCS (34 500) is within the optimal separation range for
this medium. Figure 16 shows the final purification step.
45
System:
Column:
Sample:
Sample volume:
Buffer:
Flow:
KTAFPLC
HiLoad 16/60 Superdex 75 prep grade
Concentrated DAOCS pool from SOURCE 15ISO
3 ml
100 mM Tris-HCl, 1 mM EDTA, 2 mM DTT,
0.2 mM benzamidine-HCl, 0.2 mM PMSF, pH 7.5
1 ml/min (30 cm/h)
A 280 nm
1000
800
600
400
200
0
0
20
40
60
80
100
ml
Analytical assays
Figure 17 shows the analysis of collected fractions by SDS-PAGE and silver
staining using Multiphor II, following the separation and staining protocols
supplied with the instruments.
Lane 1, 6: LMW Marker Kit
Lane 2: Cell homogenate
Lane 3: DAOCS pool from
Q Sepharose XL
Lane 4: DAOCS pool from
Source 15ISO
Lane 5: DAOCS pool from
Superdex 75 prep grade
Fig. 17. Analysis of purification steps using ExcelGel SDS Gradient 8-18.
The final product was used successfully in X-ray diffraction studies, Figure 18.
Fig. 18. Crystals, diffraction pattern and high resolution electron density map of purified
DAOCS. Figures supplied by Prof. I. Andersson and Dr A. Terwisscha van Scheltinga,
Swedich University of Agricultural Sciences, and Dr K. Valegrd, Uppsala University,
Uppsala, Sweden.
46
Example 2.
Target Molecule
Recombinant antigen binding fragment (Fab) directed against HIV gp-120.
Source Material
The anti-gp 120 Fab was expressed in the periplasm of the E. coli strain BM170
MCT61. E. coli pellets were stored frozen after being harvested and washed once.
47
Column:
Adsorbent:
Sample:
Buffer A:
Buffer B:
Flow:
A280 nm
2.0
1.0
Sample application
50
Washing, Buffer A
100
Elution, Buffer B
Pool
150
5 10 15
Volume (litres)
Intermediate Purification
Hydrophobic interaction chromatography (HIC) was selected because the
separation principle is complementary to ion exchange and because a minimum
amount of sample conditioning was required since the sample was already in a
high salt buffer after elution from STREAMLINE SP.
Hydrophobic properties are difficult to predict and it is always recommended to
screen different media. A HiTrap HIC Test Kit (containing five 1 ml columns
pre-packed with different media suitable for production scale) was used to screen
for the most appropriate medium. Buffer pH was kept at pH 5.0 to further
minimise the need for sample conditioning after capture. Results of the media
screening are shown in Figure 20.
48
System:
Sample:
Columns:
Buffer A:
Buffer B:
Gradient:
Flow:
KTAexplorer
Fab fraction from STREAMLINE SP, 2 ml
HiTrap HIC Test Kit (1 ml columns), Phenyl Sepharose High Performance,
Phenyl Sepharose 6 Fast Flow (low sub), Phenyl Sepharose 6 Fast Flow (high sub),
Butyl Sepharose 4 Fast Flow, Octyl Sepharose 4 Fast Flow
1 ml (NH4)2SO4, 50 mM NaAc, pH 5.0
50 mM NaAc, pH 5.0
20 column volumes
2 ml/min (300 cm/hr)
A280nm
Conductivity (mS/cm)
400
150
Conductivity
300
100
200
Fab
50
100
0
0
0.0
10.0
Time (min)
Fig. 20. HIC media scouting using HiTrap HIC Test Kit.
Phenyl Sepharose 6 Fast Flow (high sub) was selected since the medium showed
excellent selectivity for the target protein thereby removing the bulk
contaminants. Optimisation of elution conditions resulted in a step elution being
used to maximise the throughput and the concentrating effect of the HIC
purification technique. Figure 21 shows the optimised elution and the subsequent
scale up of the intermediate purification step.
49
System:
Sample:
Columns:
Buffer A:
Buffer B:
Gradient:
Flow:
KTAexplorer
Fab fraction from STREAMLINE SP, 80 ml
Phenyl Sepharose 6 Fast Flow (high sub)
in XK 16/20 (10 cm bed height)
1 M (NH4)2SO4, 50 mM NaAc, pH 5.0
50 mM NaAc, pH 5.0
Step gradient to 50% B
5 ml/min
System:
Sample:
Columns:
Buffer A:
Buffer B:
Gradient:
Flow:
Conductivity
(mS/cm)
A280 nm
Conductivity
(mS/cm)
A280 nm
KTAexplorer
Fab fraction from STREAMLINE SP, 800 ml
Phenyl Sepharose 6 Fast Flow (high sub)
in XK 50/20
1 M (NH4)2SO4, 50 mM NaAc, pH 5.0
50 mM NaAc, pH 5.0
Step gradient to 50% B
Equilibration: 100 ml/min
Loading and elution: 50 ml/min
b)
2.00
a)
2.00
100
100
1.00
1.00
50
50
0
0
50
100
150
200
250
Volume (ml)
500
1000
1500
2000
2500
Volume (ml)
Polishing
Gel filtration was investigated as the natural first choice for a final polishing step
to remove trace contaminants and transfer the sample to a suitable storage
conditions. However, in this example, gel filtration could not resolve a
contaminant (Mr 52 000) from the Fab fragment (Mr 50 000) (results not
shown).
As an alternative another cation exchanger SOURCE 15S was used. In contrast to
the cation exchange step at the capture step, the polishing cation exchange step
was performed using a shallow gradient elution on a medium with a small,
uniform size (SOURCE 15S) to give a high resolution result, as shown in
Figure 22.
50
System:
Sample:
Columns:
Buffer A:
Buffer B:
Gradient:
Flow:
KTAexplorer
Fab fraction from HIC separation
15 ml eluate diluted 7.5/100
RESOURCE S 6 ml
50 mM NaAc, pH 4.5
50 mM NaAc, pH 4.5, 1M NaCl
50 column volumes
18.3 ml/min
Conductivity
(mS/cm)
A280 nm
100
80.0
80
60.0
60
40.0
Active
Fab
40
20.0
20
100
200
Volume (ml)
Analytical assays
Collected fractions were separated by SDS-PAGE and stained by Coomassie
using PhastSystem, following the separation and staining protocols supplied with
the instrument.
Fab was measured by a goat-anti-human IgG Fab ELISA, an anti-gp120 ELISA
and an in vitro assay which measured the inhibition of HIV-1 infection of T-cells.
Nucleic acid was routinely monitored by measuring A260/A280. The correlation of
a high A260/A280 ratio (>1) with the presence of DNA was verified for
selected samples by agarose gel electrophoresis and ethidium bromide staining.
Endotoxin determination employed a kinetic chromogenic Limulus assay
(Coamatic, Chromogenix AB, Mlndal, Sweden).
51
Example 3.
Target Molecule
Mouse monoclonal IgG1 antibodies.
Source Material
Cell culture supernatant.
Capture
Affinity or ion exchange chromatography are particularly suitable for samples
such as cell culture supernatants as they are binding techniques which concentrate
the target protein and significantly reduce sample volume. For monoclonal antibody purification capture of the target protein can be achieved by using a highly
selective affinity chromatography medium. In this example a HiTrap rProtein A
column was used.
Although general standard protocols were supplied with this pre-packed columns,
it was decided to further optimise the binding and elution conditions for the
specific target molecule.
Most mouse monoclonal antibodies of the IgG1 sub-class require high salt
concentrations to bind to immobilised Protein A, therefore a salt concentration
was selected which gave the largest elution peak area and absence of antibodies in
the flow-through. Results from the scouting for optimal binding conditions are
shown in Figure 23. Scouting for the optimum elution pH also helped to improve
antibody recovery.
Optimisation of binding and elution conditions gave a well resolved peak
containing IgG1, as shown in Figure 24.
52
System:
Sample:
Column:
Binding buffer:
Elution buffer:
Flow:
KTAFPLC
Cell culture supernatant containing monoclonal IgG1, 90 ml
HiTrap rProtein A, 1 ml
100 mM sodium phosphate, 0-3.5 M sodium chloride pH 7.4
100 mM sodium citrate pH 3
1 ml/min
A 280 nm
1200
900
0.0 M NaCl
600
0.5 M NaCl
1.5 M NaCl
300
2.5 M NaCl
3.5 M NaCl
0
120
125
130
135
140
145
150
155
ml
System:
KTAFPLC
Sample:
Cell culture supernatant containing monoclonal IgG1, 100 ml
Column:
HiTrap rProtein A, 1 ml
Binding buffer: 100 mM sodium phosphate, 2.5 M sodium chloride pH, 7.4
Elution buffer: 100 mM sodium citrate, pH 4.5
Flow:
1 ml/min
A 280 nm
2200
1800
Sample
application
1400
Wash with
binding buffer
Elution
IgG1 peak
collected in
Superloop
1000
600
200
90
120
ml
53
Intermediate Purification
No intermediate purification was required as the high selectivity of the capture
step also removed contaminating proteins and low-molecular substances giving a
highly efficient purification.
Polishing
In most antibody preparations there is a possibility that IgG aggregates and/or
dimers are present. It was therefore essential to include a gel filtration polishing
step, despite the high degree of purity achieved during capture. The polishing step
removes low or trace levels of contaminants. Superdex 200 prep grade gel
filtration media was selected as it has the most suitable molecular weight
separation range for IgG antibodies. Figure 25 shows the final purification step.
System:
Sample:
KTAFPLC
Fraction from HiTrap rProtein A column containing
monoclonal IgG1 (3 ml)
HiLoad 16/60 Superdex 200 prep grade
50 mM sodium phosphate, 0.15 M sodium chloride, pH 7.4
1 ml/min
Column:
Buffer:
Flow:
A 280 nm
300
250
A280
200
150
Conductivity
100
50
0
0
50
100
150
ml
Fig. 25. Gel filtration on HiLoad 16/60 Superdex 200 prep grade.
Analytical assay
Collected fractions were separated by SDS-PAGE and silver stained using
PhastSystem, following the separation and staining protocols supplied with the
instrument.
Lane 1. LMW-standard
Lane 2. Starting material
(diluted 2 x)
Lane 3. Eluted IgG1 peak from
HiTrap rProtein A
column (diluted 10 x)
Lane 4. Flow through,
HiTrap rProtein A
Lane 5. Eluted IgG1 peak from
HiLoad Superdex 200
prep grade (diluted 6 x)
Lane 6. LMW-standard
Gel:
1015 % SDS-PAGE
PhastGel
System: PhastSystem
Lane 1
Example 4.
Target Molecule
Histidine-tagged cytochrome bo 3 ubiquinol oxidase from E. coli.
Source Material
The histidine-tagged cytochrome bo 3 ubiquinol oxidase accumulated in the
membrane of E. coli.
55
Membrane Solubilisation
Membrane pellets were thawed, ice cold 1% dodecyl--D-maltoside (a non-ionic
detergent) in 20 mM Tris-HCl, pH 7.5, 300 mM NaCl, 5 mM imidazole was
added. The solution was stirred on ice for 30 min.
Insoluble material was removed by centrifugation.
The presence of non-ionic detergent avoided denaturing conditions and
interference with purification steps whilst maintaining membrane protein
solubility.
Capture
Due to the instability of membrane proteins and their tendency to associate it is
often essential to use fast purification protocols at low temperatures. Attachment
of a histidine tag allowed the use of a HiTrap Chelating column giving a highly
selective affinity chromatography capture step, shown in Figure 26. This
technique also removed contaminating proteins, DNA, lipids and low-molecular
substances and allowed equilibration of detergent-protein complexes with the
detergent solution. The technique was unaffected by the presence of the non-ionic
detergent. Buffers and separation procedure followed the recommendations provided with the HiTrap Chelating column.
System:
Column:
Sample:
Binding buffer:
Elution buffer:
Gradient:
Temperature:
Flow:
KTAFPLC
HiTrap Chelating, 1 ml
Detergent extracts of E. coli membranes
20 mM Tris-HCl, pH 7.5, 5 mM imidazole,
0.03% dodecyl--D-maltoside, 300 mM NaCl
20 mM Tris-HCl, pH 7.5, 500 mM imidazole,
0.03% dodecyl--D-maltoside, 300 mM NaCl
060%, 20 column volumes
+5 C
1 ml/min
A 280 nm
%B
100
1500
80
Fraction 2
1000
60
Fraction 1
40
500
20
0
0
0
10
20
30 ml
56
Analytical assay
Lane 1: Low Molecular Weight
Calibration kit
(LMW) 14 400, 20 100,
30 000, 43 000, 67 000,
94 000
Lane 2: Detergent extract of
Escherichia coli membranes
Lane 3: Flow-through material
Lane 4: Fraction 1 from HiTrap
Chelating 1 ml
Lane 5: Fraction 2 from HiTrap
Chelating 1 ml
Lane 1
Fig. 27. SDS electrophoresis on PhastSystem using PhastGel 825%, silver staining.
57
58
Chapter 8
2.0
2.3-3.5
3.0-5.0
3.0-6.0
4.0-6.0
6.8-8.8
7.0-8.5
8.5-10.0
7.0-12.0
7.9
8.0-9.5
8.5-10.5
8.5
Formic acid
Pyridine/formic acid
Trimethylamine/formic acid
Pyridine/acetic acid
Trimethylamine/acetic acid
Trimethylamine/HCl
Ammonia/formic acid
Ammonia/acetic acid
Trimethylamine/carbonate
Ammonium bicarbonate
Ammonium carbonate/ammonia
Ethanolamine/HCl
Ammonium carbonate
H+
HCOOHCOOCH3COOCH3COOClHCOOCH3COOCO32HCO3CO32ClCO32-
3.75
3.75;
3.75;
4.76;
4.76;
9.25
3.75;
4.76;
6.50;
6.50;
6.50;
10.0
6.50;
5.25
9.25
5.25
9.25
9.25
9.25
9.25
9.25
9.25
9.25
60
Typical conditions
Protein source
Comment
Gentle
2 volumes water to
1 volume packed
pre-washed cells
erythrocytes, E.coli
periplasm: intracellular
proteins
Enzymatic digestion
bacteria: intracellular
proteins
Hand homogenisation
follow equipment
instructions
liver tissue
Mincing (grinding)
"
muscle
Moderate
follow equipment
instructions
"
Vigorous
Ultrasonication
or
bead milling
follow equipment
instructions
cell suspensions:
intracellular proteins in
cytoplasm, periplasm,
inclusion bodies
Manton-Gaulin
homogeniser
follow equipment
instructions
cell suspensions
French press
follow equipment
instructions
Fractional precipitation
extracellular: secreted
recombinant proteins,
monoclonal antibodies,
cell lysates
Blade homogeniser
precipitates must be
resolubilised
Details from Protein Purification, Principles and Practice, R.K. Scopes and other sources.
61
Purpose
Buffer components
Tris
20 mM, pH 7.4
NaCl
100 mM
EDTA
10 mM
Sucrose or glucose
25 mM
Detergents
lonic or non-ionic detergents
See Table 10
1 g/ml
Protease inhibitors*
PMSF
0.5 - 1 mM
Inhibits
serine proteases
APMSF
0.4 - 4 mM
serine proteases
Benzamidine-HCl
0.2 mM
serine proteases
Pepstatin
1 M
aspartic proteases
Leupeptin
10 - 100 M
Chymostatin
10 - 100 M
chymotrypsin, papain,
cysteine proteases
Antipain-HCl
1 - 100 M
EDTA
2 - 10 mM
EGTA
2 - 10 mM
Reducing agents
1,4 dithiothreitol, DTT
1 - 10 mM
1 - 10 mM
Mercaptoethanol
0.05%
Others
Glycerol
5 - 10%
"
"
for stabilisation, up to 50% can
be used if required
62
Detergents
Non-ionic detergents are used most commonly for extraction and purification of
integral membrane proteins. Selection of the most suitable detergent is often a
case of trial and error. The detergent should be used at concentrations near or
above its critical micelle concentration, i.e. the concentration at which detergent
monomers begin to associate with each other. This concentration is dependent
upon the type of detergent and the experimental conditions. Examples of ionic
and non-ionic detergents are shown in Table 10. Adjustment of the detergent
concentration necessary for optimum results is often a balance between the
activity and yield of the protein. During purification procedures it may be
possible to reduce the concentration of detergent compared to that used for
extraction. However, some level of detergent is usually essential throughout
purification procedures to maintain solubility. Detergents can be exchanged by
adsorption techniques (Ref: Phenyl Sepharose mediated detergent exchange
chromatography: its application to exchange of detergents bound to membrane
protein. Biochemistry 23, 1984, 6121-6126, Robinson N.C., Wiginton D.,
Talbert L.)
Table 10. Examples of ionic and non-ionic detergents.
Sodium dodecyl sulphate
0.1 - 0.5%
Triton X-100
0.1 %
NP-40
0.05 - 2%
"
Dodecyl D-maltoside
1%
"
Octyl D-glucoside
1 - 1.5%
"
For further information on detergents: Protein Purification, Principles, High Resolution Methods and
Applications, J-C. Janson and L. Rydn, 1998, 2nd ed. Wiley VCH.
Sample Clarification
Centrifugation
Use before first chromatographic step
Removes lipids and particulate matter
For small sample volumes and those which adsorb non-specifically to filters:
Centrifuge at 10000g for 15 minutes
For cell homogenates:
Centrifuge at 40 000-50 000g for 30 minutes
63
Ultrafiltration
Use before first chromatographic step
Removes salts, concentrates sample
Ultrafiltration membranes are available with different cut off limits for separation
of molecules from Mr1000 up to 300000. The process is slower than gel
filtration and membranes may clog.
Check the recovery of the target protein in a test run.
Some proteins may adsorb non-specifically to filter surfaces.
Filtration
Use before first chromatographic step
Removes particulate matter
Suitable for small sample volumes.
For sample preparation before chromatography select filter size according
to the bead size of the chromatographic medium.
Filter size
1 m
0.45 m
0.22 m
Check the recovery of the target protein in a test run. Some proteins may
adsorb non-specifically to filter surfaces.
64
Fig 28. Typical elution profile for sample desalting and buffer exchange.
Methodology
Select a pre-packed desalting column from the table below or pack a column.
Pre-packed column
Sample volume
loading per run
Sample volume
recovery per run
Code No.
2.5 -15 ml
0.25 - 1.5 ml
0.05 - 0.2 ml
1.5 - 2.5 ml
7.5
1.0
0.2
2.5
17-5087-01
17-1408-01
17-0774-01
17-0851-01
20 ml
2.0 ml
0.3 ml
3.5 ml
Column packing
The following guidelines apply at all scales of operation:
Column dimensions = typically 10 - 20 cm bed height.
Quantity of gel = five times volume of sample.
For column packing Sephadex G-25 is available in a range of bead sizes
(Superfine, Fine, Medium and Coarse). Changes in bead size alter flow rates and
sample volumes which can be applied (see Figure 29). For laboratory scale
separations use Sephadex G-25 Fine with an average bed height of 15 cm.
Individual product packing instructions contain more detailed information on
packing Sephadex G-25.
200
% of column volume
100
Superfine
Fine
Medium
Coarse
Fig. 29. Sephadex G-25: sample volume and flow rate varies with bead size.
65
Fractional precipitation
For extraction and clarification at laboratory scale
Partially purifies sample, may also concentrate
Use before the first chromatographic step
Most precipitation techniques are not suitable for large scale
preparations.
Precipitation techniques are affected by temperature, pH and sample
concentration. These parameters must be controlled to ensure reproducible
results. Precipitation can be used in three different ways, as shown in Figure 30.
Clarification
Bulk proteins and
particulate matter
precipitated
Supernatant
Extraction Clarification
Concentration
Target protein precipitated
with proteins of similar
solubility
Extraction Clarification
Bulk proteins and
particulate matter
precipitated
Resolubilise
pellet*
Concentration
Target protein
precipitated
Chromatography
Remember: if precipitating agent is
incompatible with next purification
step, use Sephadex G-25 for desalting
and buffer exchange
Resolubilise
pellet*
*Remember: not all proteins are easy to
resolubilise, yield may be reduced
Precipitation techniques are reviewed in Table 11 and the two common methods
are described in more detail.
66
Sample type
Comment
Ammonium sulphate
as described
>1mg/ml proteins
especially immunoglobulins
stabilizes proteins, no
denaturation, supernatant can go directly
to HIC
Dextran sulphate
as described
precipitates lipoprotein
Polyvinylpyrrolidine
"
alternative to dextran
sulphate
Polyethylene glycol
(PEG, M.W. >4000)
up to 20% wt/vol
plasma proteins
Acetone
up to 80% vol/vol at 0 C
Polyethyleneimine
0.1% w/v
Protamine sulphate
1%
Streptomycin sulphate
1%
precipitates aggregated
nucleoproteins
"
precipitation of nucleic
acids
Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer.,
Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and L.
Rydn, 1998, 2nd ed. Wiley VCH and other sources
67
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
113
144
176
208
242
277
314
351
390
430
472
516
561
608
657
708
761
85
115
146
179
212
246
282
319
358
397
439
481
526
572
621
671
723
10
57
86
117
149
182
216
251
287
325
364
405
447
491
537
584
634
685
15
28
58
88
119
151
185
219
255
293
331
371
413
456
501
548
596
647
20
29
59
89
121
154
188
223
260
298
337
378
421
465
511
559
609
29
60
91
123
157
191
228
265
304
344
386
429
475
522
571
30
61
92
125
160
195
232
270
309
351
393
438
485
533
30
62
94
128
163
199
236
275
316
358
402
447
495
31
63
96
130
166
202
241
281
322
365
410
457
31
64
98
132
169
206
245
286
329
373
419
32
65
99
135
172
210
250
292
335
381
33
66
101
138
175
215
256
298
343
33
67
103
140
179
219
261
305
34
69
105
143
183
224
267
34
70
107
146
186
228
35
72
110
149
190
36
73
112
152
37
75
114
37
76
38
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
68
Removal/comment
Urea
2-8M
Guanidine hydrochloride
3-8M
Triton X-100
2%
Sarcosyl
1.5%
N-octyl glucoside
2%
0.1 - 0.5%
alkaline pH
> pH 9, NaOH
Details taken from Protein Purification, Principles and Practice, R.K. Scopes. 1994, Springer.,
Protein Purification, Principles, High Resolution Methods and Applications, J-C. Janson and
L. Rydn, 1998, 2nd ed. Wiley VCH and other sources
69
70
Chapter 9
sample
application
gradient
elution
wash
re-equilibration
1-4 cv
[NaCl]
10-20 cv
2 cv
2 cv
0
Column volumes [cv]
The net surface charge of proteins varies according to the surrounding pH. When
above its isoelectric point (pI) a protein will bind to an anion exchanger, when
below its pI a protein will behind to a cation exchanger. Typically IEX is used to
bind the target molecule, but it can also be used to bind impurities if required.
IEX can be repeated at different pH values to separate several proteins which
have distinctly different charge properties, as shown in Figure 32. This can be
used to advantage during a multi-step purification, as shown in the example on
page 24.
71
Selectivity
pH of mobile phase
Abs
Abs
Abs
Abs
cation
pH
anion
Abs
Abs
Abs
Abs
72
73
Group
separations
Preparative
separation
highest capacity,
industrial production
mg - kg
mg and more
mg and more
mg and less
Fig. 33. Ion exchange media selection guide. (Code no. 18-1127-31)
Crude material or
production scale
g - kg
Partially purified or
lab scale
Preparative
separation
Analytical or
semi-preparative
STREAMLINE
Sepharose XL
SOURCE 30
SOURCE 15
Mono Beads
Mini Beads
DEAE, SP
Q, SP
Q, SP
Q, SP, DEAE, CM
Q, SP
Q, S
Q, S
Q, S
high speed
high capacity
ideal for scale up
low-medium pressure systems
extreme resolution
micropurification and analysis
upper-medium pressure systems
Resolution
Media selection
Parameters such as scale of purification, resolution required, speed of separation,
sample stability and media binding capacity, should be considered when selecting
a chromatographic medium. Figure 33 on page 75 shows a guide to selecting ion
exchange media.
Sample Preparation
Correct sample preparation ensures good resolution and extends the life of the
column. To ensure efficient binding during sample application samples should be
at the same pH and ionic strength as the starting buffer. Samples must be free
from particulate matter, particularly when working with bead sizes of 34 m or
less (see page 65 for details of sample clarification procedures).
Column Preparation
Pre-packed columns
To increase speed and efficiency in method development, use small pre-packed
columns for media scouting and method optimisation. HiTrap IEX Test Kit is
ideal for this type of work, as shown in Figure 34.
Sample:
nm
Columns:
Buffer A:
Buffer B:
Flow:
Ribonuclease A,
human apo-transferrin, -lactalbumin
HiTrap IEX Test Kit
20 mM piperazine, pH 9.7
20 mM piperazine, 1 M NaCl pH 9.7
2 ml/min (310 cm/h)
Fig. 34. Media selection using 2 columns from HiTrap IEX Test Kit.
Using pre-packed columns at any scale will ensure reproducible results and high
performance.
Column packing
Buffer Preparation
Buffering ions should have the same charge as the selected medium, with a pKa
within 0.6 pH units of the working pH. Buffer concentration should be sufficient to
maintain buffering capacity and constant pH during sample application and while
an increase in salt concentration is applied.
When working with a sample of unknown charge characteristics,
try these conditions first:
Anion Exchange
Gradient: 0-100% elution buffer B in 10 - 20 column volumes
Start buffer A: 20 mM Tris-HCl, pH 8.0
Elution buffer B: 20 mM Tris-HCl + 1 M NaCl, pH 8.0
Cation Exchange
Gradient: 0-100% elution buffer B in 10 - 20 column volumes
Start buffer A: 20 mM Na2HPO4.2H2O, pH 6.8
Elution buffer B: 20 mM Na2HPO4.2H2O + 1 M NaCl, pH 6.8
Further information
Ion Exchange Chromatography: Principles and Methods Code no. 18-1114-21.
76
equilibration
sample
application
gradient
elution
re-equilibration
[ammonium sulphate]
1M
2 cv
2 cv
0
Column volumes [cv]
77
Media selection
In HIC the characteristics of the chromatographic matrix as well as the
hydrophobic ligand affect the selectivity of the medium. This should be
considered, together with parameters such as sample solubility, required
resolution, scale of purification and availability of the medium at the scale
intended. Figure 37 on page 81 shows a guide to selecting HIC media.
Sample Preparation
Correct sample preparation ensures good resolution and extends the life of the
column. To ensure efficient binding during sample application samples should be
at the same pH as the starting buffer and in high ionic strength solution (e.g.
1.5 M ammonium sulphate or 4 M NaCl). Samples must be free from particulate
matter, particularly when working with bead sizes of 34 m or less (see page 65
for details of sample clarification procedures).
78
Batch
separations
Preparative
separation
mg and kg
g and more,
high speed and capacity
mg and more
mg and less
Crude material or
production scale
gkg
Partially purified
or lab scale
Preparative
separations
Analytical or
semi-preparative
Sepharose FF
Sepharose HP
SOURCE 15
Superose
butyl
octyl
phenyl
phenyl
ethyl
isopropyl
phenyl
phenyl
high speed
high capacity
ideal for scale up
79
Increasing resolution
Column Preparation
Pre-packed columns
To increase speed and efficiency in method development use small pre-packed
columns for media scouting and method optimisation. HiTrap HIC Test Kit and
RESOURCE HIC Test Kit are ideal for this work. Using pre-packed columns at
any scale will ensure reproducible results and high performance. Figure 38 shows
an example of media screening with HiTrap HIC Test Kit
A280nm
Conductivity (mS/cm)
400
System:
Sample:
Columns:
150
Conductivity
300
100
200
Fab
Buffer A:
Buffer B:
Gradient:
Flow:
KTAexplorer
Fab fraction from STREAMLINE SP, 2 ml
HiTrap HIC Test Kit (1 ml columns),
Phenyl Sepharose High Performance,
Phenyl Sepharose 6 Fast Flow (low sub),
Phenyl Sepharose 6 (high sub),
Butyl Sepharose 4 Fast Flow, Octyl Sepharose 4 Fast Flow
1 ml (NH4)2SO4, 50 mM NaAc, pH 5.0
50 mM NaAc, pH 5.0
20 column volumes
2 ml/min (300 cm/hr)
50
100
0
0
0.0
10.0
Time (min)
Column packing
The following guidelines apply at all scales of operation:
Column dimensions =
typically 5 - 15 cm bed height.
Quantity of gel =
estimate amount of gel required to bind the sample,
use five times this amount to pack a column.
See individual product packing instructions for more detailed information on a
specific medium.
Buffer Preparation
Buffering ion selection is not critical for hydrophobic interaction. Select a pH
compatible with protein stability and activity. Buffer concentration must be
sufficient to maintain pH during sample application and changes in salt
concentration.
When working with a sample of unknown hydrophobic characteristics,
try these conditions first:
Gradient: 0-100% elution buffer B in 10 - 20 column volumes
Start buffer A: 50 mM sodium phosphate pH 7.0 + 1 - 1.5 M ammonium
sulphate
Elution buffer B: 50 mM sodium phosphate pH 7.0
80
81
Further information
Hydrophobic Interaction Chromatography: Principles and Methods
Code no. 18-1020-90
82
UV
equilib
ration
adsorption of
sample and
elution of
unbound material
begin sample
application
1-2 cv
wash
away
unbound
material
elute
bound
protein(s)
gel regeneration
change to
elution buffer
x cv
1-2 cv
>1
cv
1-2 cv
83
Media selection
Parameters such as scale of purification and commercial availability of affinity
matrices should be considered when selecting affinity media. To save time and
ensure reproducibility, use prepacked columns for method development or small
scale purification. HiTrap affinity columns are ideal for this work. Table 6 on
page 34 shows examples of prepacked affinity columns. Specific affinity media are
prepared by coupling a ligand to a selected gel matrix, following recommended
coupling procedures.
Further details on other affinity media are available in the Affinity
Chromatography Product Profile (Code No. 18-1121-86).
Sample Preparation
Correct sample preparation ensures efficient binding and extends the life of a
column. Removal of contaminants which may bind non-specifically to the
column, such as lipids, is crucial. Stringent washing procedures may damage the
ligand of an affinity medium, destroying the binding capacity of the column.
Samples must be free from particulate matter (see Chapter 8 for details of sample
clarification procedures).
Column Preparation
Pre-packed columns
Pre-packed columns ensure reproducible results and highest performance.
Column packing
The following guidelines apply at all scales of operation:
Column dimensions =
short and wide.
Quantity of gel =
calculate according to known binding capacity of
medium, use 2-5 times excess capacity.
See individual product packing instructions for more detailed information on a
specific medium.
84
Buffer Preparation
Binding, elution and regeneration buffers are specific to each affinity medium.
Follow instructions supplied with the medium or column.
1. Select the correct specificity for the target protein. Follow the manufacturer's
instructions for binding or elution conditions and check recommended flow
rates for the specific medium.
2. Select optimum flow rate to achieve efficient binding
3. Select optimum flow rate for elution to maximise recovery.
4. Select maximum flow rate for column regeneration to minimise run times.
Further information
Affinity Chromatography: Principles and Methods Code no. 18-1022-29.
85
UV
sample
injection
volume
intermediate
molecular weight
equilibration
1 cv
Column Volumes [cv]
86
87
er
th
ar
Group separation
Desalting
Fractionation
Preparative
/Macro fractionation
(1-500,000 kD)
Preparative
& analytical
(0.1-5,000 kD)
Preparative
(0.5-5,000 kD)
Analytical
(0.1-5,000 kD)
Wide Mw range
(1-5,000 kD)
High
selectivity
(0.1-600 kD)
Wide Mw range
( 1-5,000 kD)
High
selectivity
(0.5-600 kD)
Peptides/small proteins
Proteins
Small particles
Virus
Purification of
macromolecules
Fractionation of
macromolecules
Large proteins
Proteins
Small proteins
Intermediate
fractionation range
Wide fractionation
range
Intermediate
fractionation range
Wide fractionation
range
Proteins
DNA-fragment
Group
separation
Small peptides
Macro molecules
Proteins
separation
Preparative
Analytical separation
Semi-preparative
separation
Small proteins
Polynucleotides
Peptides
Polynucleotides
Proteins
DNA-fragment
Analytical separation
Preparative
Small proteins
Semi-preparative
Peptides
Desalting
Sephadex
Macromolecule
separation
Product line
covering wide
fractionation range
Sephacryl
High recovery
Wide Mw
fractionation
range
Superose
High recovery
High stability
High selectivity
Superdex
St
Sephadex G-10
Sephadex G-25 SF
Sephadex G-25 F
Sephadex G-25 M
Sephadex G-50 F
Sephacryl S-1000 SF
Sephacryl S-500 HR
Sephacryl S-400 HR
Sephacryl S-300 HR
Sephacryl S-200 HR
Sephacryl S-100 HR
Superose 12
Superose 6
Superdex 200
Superdex 75
Superdex Peptide
10
10
10
Exclusion limit
Exclusion limit
Exclusion limit
10
10
10
10
High resolution
Media selection
Parameters such as molecular weight of target proteins and contaminants, resolution required, scale of purification should be considered when selecting gel filtration media. Figure 42 on page 89 shows a guide to selecting of GF media.
Sample Preparation.
Correct sample preparation ensures good resolution and extends the life of the
column. Sample buffer composition does not directly affect resolution. During
separation the sample buffer is exchanged with buffer in the column. Viscous
samples, which could cause an increase in back pressure and affect column
packing, should be diluted. Samples must be free from particulate matter,
particularly when working with bead sizes of 34 m or less (see page 65 for
details of sample clarification procedures)
Column Preparation
Pre-packed columns
Pre-packed columns ensure reproducible results and highest performance.
Column packing
In gel filtration good column packing is essential. The resolution between two
separated zones increases as the square root of column length. The following
guidelines apply:
Column dimensions: =
Bed volume =
Buffer Preparation
Selection of buffering ion does not directly affect resolution. Select a buffer in
which the purified product should be collected and which is compatible with
protein stability and activity.
Buffer concentration must be sufficient to maintain buffering capacity and
constant pH.
Ionic strength can be up to 150 mM NaCl in the buffer, to avoid non-specific
ionic interactions with the matrix (shown by delays in peak elution).
When working with a new sample try these conditions first
Buffer: 50 mM sodium phosphate, pH 7.0 + 0.15 M NaCl
or select the buffer in which the sample should be eluted for the next step
88
Further information
Gel Filtration: Principles and Methods Code no. 18-1022-18.
89
sample
application
column
equilibration
gradient
elution
clean after
gradient
re-equilibration
100% B
2-4 cv
5-40 cv
5 cv
0
2 cv
Column Volumes [cv]
RPC is often used in the final polishing of oligonucleotides and peptides and is
ideal for analytical separations, such as peptide mapping.
RPC is not recommended for protein purification if recovery of activity and
return to a correct tertiary structure are required, since many proteins are
denatured in the presence of organic solvents.
90
Media selection
In RPC the chromatographic medium as well as the hydrophobic ligand affect
selectivity. Screening of different RPC media is recommended.
Sample Preparation
Samples should be free from particulate matter and, when possible, dissolved in
the start buffer.
Column Preparation
Reversed phase columns should be conditioned for first time use, after long term
storage or when changing buffer systems.
Buffer Preparation
Try these conditions first when sample characteristics are unknown:
Gradient: 2-80% elution buffer B in 20 column volumes
Start buffer A: 0.065% TFA (trifluoroacetic acid) in water
Elution buffer B: 0.05% TFA in acetonitrile
91
Method Development
1. Select medium from screening results.
2. Select gradient to give acceptable resolution. For unknown samples begin
0-100%B.
3. Select the highest flow rate which maintains resolution and minimises
separation time.
4. For large scale purification transfer to a step elution.
5. Samples which adsorb strongly to a gel are more easily eluted from a less
hydrophobic medium.
Further information
Visit www.apbiotech.com
92
equilibration
Begin
sample
application
adsorption
of sample
and elution
of unbound
material
Begin
wash with
start buffer
Sample volumes
wash
away
unbound
material
elute
bound
protein(s)
column
wash
Change to
elution
buffer
Volume
93
Sample Preparation
STREAMLINE is able to handle crude, particulate feedstock, reducing the need
for significant sample preparation steps. Adjustment of pH or ionic strength may
be required according to the separation principle being used (IEX, AC, HIC)
Column Preparation
For preliminary method scouting STREAMLINE media is used in packed bed
mode in an XK 16 or XK 26 chromatography column. When used in expanded
bed mode the media must be packed in specially designed STREAMLINE columns, following the manufacturer's instructions.
Buffer Preparation
Buffer preparation will depend upon the chosen separation principle.
Method Development
1. Select suitable ligand to bind the target protein.
2. Scout for optimal binding and elution conditions using clarified material in a
packed column (0.02 - 0.15 litres bed volume of media). Gradient elutions may
be used during scouting, but the goal is to develop a step elution.
3. Optimise binding, elution, wash and cleaning-in-place procedures using
unclarified sample in expanded mode at small scale (0.02 - 0.15 litres bed
volume of media)
4. Begin scale up process at pilot scale (0.2 - 0.9 litres bed volume of media)
5. Full scale production (up to several hundred litres bed volume of media)
94
Further Information
Expanded Bed Adsorption: Principles and Methods Code No. 18-1124-26
BioProcess
95
96
October 2001
Handbooks
from Amersham Pharmacia Biotech
Antibody Purification
Recombinant Protein Handbook
Gel Filtration Principles and Methods
Ion Exchange Chromatography Principles and Methods
Hydrophobic Interaction Chromatography Principles and Methods
Affinity Chromatography Principles and Methods
Expanded Bed Adsorption Principles and Methods
Code
Code
Code
Code
Code
Code
Code
No.
No.
No.
No.
No.
No.
No.
18-1037-46
18-1142-75
18-1022-18
18-1114-21
18-1020-90
18-1022-29
18-1124-26
Code
Code
Code
Code
No.
No.
No.
No.
18-1124-19
18-1127-31
18-1100-98
18-1121-86
Code
Code
Code
Code
Code
No.
No.
No.
No.
No.
18-1128-62
18-1129-81
18-1128-63
18-1123-93
18-1129-75
Antibody Purification
Handbook
18-1037-46
Protein Purification
HiTrap, Sepharose, STREAMLINE, Sephadex, MonoBeads, Mono Q, Mono S, MiniBeads, RESOURCE, SOURCE,
Superdex, Superose, HisTrap, HiLoad, HiPrep, INdEX, BPG, BioProcess, FineLINE, MabTrap, MAbAssistant, Multiphor,
FPLC, PhastSystem and KTA are trademarks of Amersham Pharmacia Biotech Limited.
Handbook
18-1132-29
Affinity Chromatography
Chromatofocusing
Gel Filtration
www.chromatography.amershambiosciences.com