18-1128-63 Edition AD
equilibration
sample application
gradient elution
re-equilibration
+
tightly bound molecules elute in high salt wash
1M
Cation
equilibration
sample application
gradient elution
re-equilibration
0 Anion
pH
1M
1020 cv
Abs
2 cv 0 Column volumes [cv] 2 cv
Abs
Abs
Abs
[ammonium sulphate]
Figure 1.
Figure 2.
The net surface charge of proteins varies according to the surrounding pH. IEX can be repeated at different pH values to separate several proteins which have distinctly different charge properties, as shown in Figure 2.
0
Sample Preparation
Samples should be at the same pH and ionic strength as the starting buffer, and free from particulate matter.
Buffer Preparation
If charge characteristics are unknown try these conditions first: Anion Exchange Start buffer (A): Gradient: 20 mM Tris-HCl, pH 7.4 0100% elution buffer in 1020 column volumes
Sample Preparation
Samples should be at the same pH as the starting buffer, in high ionic strength solution and free from particulate matter.
Elution buffer (B): 20 mM Tris-HCl + 1 M NaCl, pH 7.4 Cation Exchange Start buffer (A): 20 mM Na2HPO42H2O, pH 6.8 Elution buffer (B): 20 mM Na2HPO42H2O + 1 M NaCl, pH 6.8 Gradient: 0100%B in 1020 column volumes
Buffer Preparation
Try these conditions first if hydrophobic characteristics are unknown: Start buffer (A): Gradient: 50 mM Na2HPO42H2O pH 7.0 + 1.0 M ammonium sulphate 0100% B in 1020 column volumes Elution buffer (B): 50 mM Na2HPO42H2O pH 7.0
Optimisation Parameters
1. Select medium from a HiTrap HIC Selection Kit or RESOURCE HIC Test Kit. 2. Select optimum gradient to give acceptable resolution. For unknown samples begin with 0100%B (0%B=1 M ammonium sulphate). 3. Select highest flow rate which maintains resolution and minimises separation time. 4. For large scale purifications and capture steps transfer to a step elution. 5. Samples which adsorb strongly to a gel are more easily eluted by changing to a less hydrophobic medium.
Optimisation Parameters
1. Select optimal ion exchanger. 2. Select for optimum pH. 3. Select steepest gradient to give acceptable resolution at selected pH. 4. Select highest flow rate which maintains resolution and minimises separation time. 5. For large scale purifications and capture steps transfer to a step elution to reduce separation times and buffer consumption.
equilibration
gel regeneration
Absorbance
12 cv
x cv
>1 cv
12 cv
Sample Preparation
Samples must be free from particulate matter and contaminants which may bind non-specifically to the column e.g. lipids.
Buffer Preparation
Binding, elution and regeneration buffers are specific to each affinity medium. Follow instructions supplied.
Optimisation Parameters
1. Select correct specificity for target protein. 2. Follow manufacturers recommendations for binding or elution conditions. 3. Select optimum flow rate to achieve efficient binding. 4. Select optimum flow rate for elution to maximise recovery. 5. Select maximum flow rate for column regeneration to minimise run times.
RPC is not recommended for protein purifications if recovery of activity and return to a correct tertiary structure are required, since many proteins are denatured in the presence of organic solvents.
column equilibration 100% B sample application gradient elution clean after gradient re-equilibration
Absorbance
24 cv
540 cv
2 cv
Sample Preparation
Samples must be free from particulate matter. Viscous samples should be diluted. During separation, sample buffer is exchanged with buffer in the column.
Buffer Preparation
Select a buffer in which the purified product should be collected and which is compatible with protein stability and activity. Ionic strength can be up to 150 mM NaCl, to avoid non-specific ionic interactions with the matrix. When working with a new sample try these conditions first: Buffer: 0.5 M Na2HPO42H2O, pH 7.0 + 0.15 M NaCl or select the buffer in which the sample should be eluted for the next step
Sample Preparation
Samples should be free from particulate matter and, when possible, dissolved in the start buffer. If sample is insoluble try 1) 10-30% acetic acid, 2) 70% formic acid, 3) 6 M guanidine-HCl, 4) 100% DMSO (dimethyl sulphoxide), 5) TFA (trifluoroacetic acid). Note that a very hydrophobic peptide dissolved in DMSO may precipitate or bind irreversibly to an RPC matrix. Test first with aliquots of sample.
Optimisation Parameters
1. Select a medium which has your target protein close to the middle of its separation range. 2. Select the highest flow rate which maintains resolution and minimises separation time. Lower flow rates improve resolution of high molecular weight components, faster flow rates may improve resolution of low molecular weight components. 3. Determine the maximum sample volume which can be loaded without reducing resolution (sample volume should be 0.55% of total column volume). 4. To further improve resolution increase column length by connecting two columns in series.
Buffer Preparation
Try these conditions first when sample characteristics are unknown: Start buffer (A): Gradient: 0.065% TFA (trifluoroacetic acid) in water 280% elution buffer in 20 column volumes Elution buffer (B): 0.05% TFA in acetonitrile
Group separations
For sample preparation and clarification use Sephadex G-25 for desalting, buffer exchange and removal of lipids and salts from proteins >5000. Gel filtration is also ideal for sample preparation before or between purification steps. Sample volumes of up to 30% 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. Any sample volume can be processed rapidly and efficiently. The high sample volume load gives a low resolution separation but with minimal sample dilution.
Optimisation Parameters
1. Select medium from screening results. 2. Select optimum gradient to give acceptable resolution. For unknown samples begin with 280%B. 3. Select highest flow rate which maintains resolution and minimises separation time. 4. For large scale purifications transfer to a step elution. 5. Samples which adsorb strongly to a gel are more easily eluted by changing to a less hydrophobic medium.
Sample Preparation
AC
Capture
GF
Purity OK or impurities differ in size from or repeat affinity step after target molecule tag cleavage if only tag
removal is required
Intermediate Purification
GF
Purity OK
Polishing
GF
At this stage in a purification gel filtration can be used as the final step if impurities differ in size from the target molecule. However, gel filtration is highly recommended as a final step in all purification strategies to ensure removal of any low molecular weight contaminants.
IEX
GF GF
Purity OK or impurities differ in size from target molecule
Purity OK
GF
Purity OK
Target sensitive to organic solvents
Filter or centrifuge For desalting and buffer exchange use SephadexTM media.
GF
IEX
HIC
GF
RPC
Purity OK
tar
r he
Unknown isoelectric point (pI) or pI below pH 7, use AIEX. pI above 5 or high DNA contamination, use CIEX.
Increase ionic strength of sample before HIC. Peptides or very hydrophobic proteins are not suitable for HIC separations. Go to GF
Evaluate results of each step. Evaluate purification strategy. Use alternative IEX selectivity to improve resolution or recovery. Use alternative ligand.
GF
Ammonium sulphate or other precipitation Screen for HIC medium (to avoid extra desalting step). Adjust (NH4)2 SO4 conc.
mS/cm
GF
Purity OK
HIC
IEX
mAU
mAU 3000
mS/cm
mAU
mS/cm
mAU 1000
Std
80
10.0
2000
60 40 20
protein
salt
Sample Preparation
Buffer exchange HiPrep 26/10 Desalting Mouse plasma (10 ml)
1000
Capture
IEX HiPrep 16/10 Q XL DAOCS enzyme from clarified E. coli extract Step elution
Intermediate Purification
HIC SOURCE 15 ISO in HR 16/10 DAOCS enzyme
Polishing
GF HiLoad 16/60 Superdex 75 prep grade DAOCS enzyme Analysis of fractions from DAOCS purification SDS PAGE, silver stain
0 0 100 200 ml
1.0
2.0
Time (min)
Peptide Purification
AIEX
RPC
Isoelectric point below pH 7.0
Purification Strategies
This selection guide offers general guidelines for protein and peptide purification strategies. Selection of the final strategy will always depend upon specific sample characteristics, the condition of the starting material and the required level of purity.
Protein Purity
For every technique used there is a balance between:
Resolution
RPC
Purity OK
RPC
Media selection. Screen for optimum pH and suitable selectivity polymeric matrix pH 112 silica matrix pH < 7.
Polishing
CIEX
Intermediate purification
RPC
highly purified
Synthetic peptides
product from cleavage, de-protection or ether precipitation
Speed
Capacity
RPC
Use polymeric matrix or C18 silica matrix. C8 or C4 if peptide is very hydrophobic.
Natural peptides
products from, for example, cleaned tissue extracts
Use different selectivity, mobile phase or pH on RPC for purity check or removal of minor contaminants if required. Analyse with MS.
Recovery
sample suitable for chromatography
a St
rt
r he
Hydrophobic peptide
Step
Note: In this model capacity refers to amount of target molecule bound per unit volume of gel. Capacity can also refer to sample size, both absolute volume and sample concentration, that can be handled in a single step.
RPC
recycling of product improves purity but reduces yield
Use silica or polymeric matrix at low pH. Very acidic (~25%)
RPC
Glossary of Terms
Sample Characteristics include: Size, charge, hydrophobicity, affinity for a specific ligand, isoelectric point (pI), pH and temperature stability. Sample Preparation: Clarification before first chromatographic separation step. May include extraction and/or concentration procedures.
Resolution
RPC
Purity OK
RPC
Try alternative matrices or conditions. Screen for optimum pH and suitable selectivity polymeric matrix pH 112 silica matrix pH < 7.
RPC
Use polymeric matrix at high pH.
mAU % cond.
mAU 40.0 80 % cond.
Examples of approximate purity level requirements: Extremely high >99% therapeutic use or in vivo studies High 9599% x-ray crystallography and most physico-chemical characterisation methods Moderate <95% Antigen for antibody production
2. Develop analytical assays fast detection of protein activity and recovery to work efficiently 3. Define sample characteristics to simplify technique selection and optimisation 4. Minimise sample handling at every stage avoid lengthy procedures which risk losing activity or reducing recovery
Speed
Capacity
80
20.0 60
Recovery
Resolution
60 40 20 0 10.0 20.0 ml
0.0 40
20
0.0
50
100
150
ml
5. Minimise the use of additives additives may need to be removed in an extra purification step or may interfere with assays 6. Remove damaging contaminants early for example, proteases 7. Use a different technique at each step take advantage of sample characteristics which can be used for separation (size, charge, hydrophobicity, ligand specificity) 8. Minimise number of chromatographic steps extra steps reduce yield and increase time, combine steps logically KEEP IT SIMPLE!
Speed
Capacity
Recovery Resolution
RPC
Speed
Capacity
RPC
Resolution OK
Recovery
Reduction in number of fragments per analysis needed High molecular weight target fragments > M r 3 0005 000
RPC
IEX
Ion exchange chromatography (IEX) Anion exchange chromatography (AIEX) Cation exchange chromatography (CIEX) Affinity chromatography (AC)
HIC
Hydrophobic interaction chromatography (HIC) Fractionation by gel filtration (GF) Reversed phase chromatography (RPC) Sample clean-up by gel filtration (GF)
GF
RPC
AC
RPC
tar
r he
mAU
%B
EBA Expanded bed adsorption is a technique in which target proteins are purified from crude, particulate sample, combining sample preparation and capture in a single step. It is most suitable for large scale purification and therefore not shown in this guide.
GF
80 40.0 60
GF Superdex Peptide
20.0
215 nm
40
0.0
254 nm 280 nm
20
0 0 200 400 ml
Further Information
Selection Guides: to select the correct chromatographic medium for a separation step. Handbooks: to learn more about the details of each chromatographic technique including applications and trouble shooting.
Gel Filtration
Hydrophobic Interaction & Reversed Phase Ion Exchange Applications Interactive learning
Validated Manufacture Regulatory support Capture to Polishing High Productivity Sanitization & CIP/Scalability
www.chromatography.amershambiosciences.com
or
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