Input for Purification Protocol
Development
Guidelines for Protein Purification
Sample Preparation before
Chromatography
Chromatography: General
Considerations
1
PURIFICATION STRATEGY
 General approach: Input for Protocol Development
 Guidelines for Protein Purification. Commonly confronted decisions.
Properties of Target Protein
 Sequence of Events: Cell harvesting, Cell Disruption, Extraction and
Clarification, Chromatography
 Sample Preparation before Chromatography: Cell Debris Removal,
Clarification and Concentration: dialysis, filtration, ultrafiltration , others
 FPPLC, columns, resolution, selectivity, efficiency and capacity
 Three Phase Strategy: Linking Chromatography Techniques
2
Applications of Protein Purification
 In vitro Activity assays
 Post-translational modification tests
 Antibody development / production
 N-terminal sequencing
 Protein:protein interaction assays
 Electromobility shift assay (band shift)
 Cell-based activity assays
 Ligand-binding assays
 Mass-spectrometric analysis
 DNA footprinting
 Protein cross-linking studies
 Vaccine development/production
 Probes for protein arrays/chips
 Structural analysis
 Expression library screening
 In vivo activity assay
 Other
For each application you need:
Each purification project must be
adapted to your start material and
your final needs
different quantities
different protein purity
start material is different, etc
different strategy
Don’t waste clear thinking on dirty
or not healthy proteins!!!!
Protein Purification Strategy
Simple Purification
ONE STEP Affinity
70 - 95% Purity
FUSION PROTEIN
For higher purity
Multi-Step Purification
Capture
Intermediate Purification
Polishing
EXPRESSION
NON-FUSION PROTEIN
4
Protein Modifications May Require Further
Purification
Aggregation
COOH
Misfolding, random
disulfide bridges
Glycosylation
Phosphorylation
Acylation
NH2
N or C terminal
heterogeneity
Desamidation of Asp and Glu
Proteolytic cleavage
Oxidation of methionine
5
Protein Purification - Aims
 Satisfactory

expression levels

protein activity

purity

homogeneity

stability
 Economical use of reagents/equipment
• Goal to Success:
 Define the purification objectives
 Selection or optimization of the best source or best expression conditions
 A good understanding of the protein needs
 Selection and optimization of the most appropriate technique for each step
7
Protein Production Pipeline
Target Selection
Target Optimization
Gene Cloning
Selection of Expression Vector
Selection of Expression Host
Expression Analysis
Solubility Analysis
Scaling Up
Fermentation
Purification
Purification Optimization
Characterization
Concentration & Storage
Pharmaceutical Studies
Structural Studies:
Crystallization – NMR- etc
Biochemical Studies
Commonly confronted decisions
 Which is the best natural source?
 How much do we need?
 Active? Which assay?
 Purification grade?
 Which hosts: bacteria, yeast, insect cells or in human cells?
 Which expression vector should be used? Which strain(s)? Intra or extracelular?
 Should the protein be tagged? which affinity tag is the best?
 Which is the best purification strategy?
 Which buffers should I use?
 Optimization of each purification step, where to stop?
 How do I want my sample? Can I concentrate it? How much? Buffer?
 How to keep activity, solubility and homogenicity of my sample?
9
Overview: separation techniques
Technique
Parameter
for separation
Based on
Gel filtration
Size/Shape
MW, Shape, and oligomeric state
of the molecule
Ion exchange/
Hydroxyapatite/
Chromatofocusing
Charge interaction
Asp, Glu, Lys, Arg, His
Hydrophobic/
Reversed phase
Hydrophobic sites
interaction
Trp, Phe, Ile, Leu, Tyr, Pro
Met, Val, Ala
Affinity
Biological function
eg: antibody – antigen
Metal chelate
Affinity for metals
poly His
Covalent
Covalent interaction
Uses SH groups (Cys)
Multimodal
Mixture
Hydrophobic + Ionic Interaction
10
Input for Purification Protocol Development
General Input
Three phase
strategy
Separation technique
knowledge
Sample Specific Input
Purification
protocol
Required purity
and quantity
Physical-chemical
properties of target and
main contaminants
Source material
information
Economy and
resources
Scouting runs and
optimization
11
Yields from Multistep Protein Purifications
Yield (%)
100
80
95% / step
60
90% / step
40
85% / step
80% / step
75% / step
20
0
1
2
3
4
5
6
7
8
Number of steps
12
Use relevant analytical tools
 A rapid and reliable assay for the target protein: biological assay,
enzymatic, SDS-PAGE, Western, etc
 Purity determination
SDS-PAGE, Native, IEF, RPC, analytical GF or EIX, etc
 Total protein determination – Interference of detergents, reducing
agents, sugars, others - ultraviolet absorption, colorimetric method, etc
13
Define Properties of Target Protein (I)
Temperature Stability Need to work rapidly at low temperature
pH Stability Selection of buffers for each step
Organic Solvents Stability Selection of Conditions for RPC
Salt Stability Selection of Conditions for all steps
Co-factors for Stability or Activity Selection of Additives, pH, Salts, Buffers
Protease Sensitivity Fast removal of proteases. Protease Inhibitors
Sensitivity to Metal Ions Need of EDTA or EGTA in Buffers
Define Properties of Target Protein (II)
Redox Sensitivity Need of reducing agents to protect reduce Cys: DTT, DTE or
on the contrary, need to protect disulfide bridges
Molecular Weight/Oligomeric State Selection of Gel Filtration Media / UF
Charge Properties Selection of Ion Exchange Conditions
Biospecific affinity Selection of ligand for Affinity Medium
Post Translational Modifications Selection of Group Specific Affinity: Lectins
Hydrophobicity Solubility prediction - Selection of medium for HIC
Initial Bioinformatics Investigation
Using Bioinformatic Tools to Strategically Design
Expression/Purification Projects
Dr. Nurit Kleinberger-Doron
http://wolfson.huji.ac.il/expression/software.html
16
Bioinformatics Tools-I
Physical and chemical parameters
http://www.expasy.org/tools/protparam.html
Computation of various physical and chemical parameters for a given protein: molecular
weight, theoretical pI, amino acid composition, atomic composition, extinction
coefficient, estimated half-life, instability index, aliphatic index and grand average of
hydropathicity (GRAVY)
http://www.scripps.edu/~cdputnam/protcalc.html
Generates molecular weight information (including scanning mass spectrometry results),
estimated charges (including pI estimation), uv absorption coefficients,
crystallographic solvent content percentage and Vm, and counts atoms and residues
based on the protein sequence
Proteolytic Cleavage
http://www.expasy.org/tools/peptidecutter/
Predicts potential cleavage sites cleaved by proteases or chemicals in a given protein
sequence
http://www.cf.ac.uk/biosi/staff/ehrmann/tools/proteases.index.html
Protease database of E.Coli
17
Bioinformatics Tools-II
Post-translational modification prediction
http://www.expasy.org/tools/#ptm
Prediction of Ser, Thr and Tyr phosphorylation sites in eukaryotic proteins
Prediction of N-acetyltransferase A (NatA) substrates (in yeast and mammalian proteins
Prediction of O-GalNAc (mucin type) glycosylation sites in mammalian protein
Prediction of N-glycosylation sites in human proteins
Prediction of N-terminal myristoylation by neural networks
Recombinant Protein Solubility Prediction : The statistical model predicts protein solubility
assuming the protein is being overexpressed in Escherichia coli.
http://www.biotech.ou.edu/
S-S bonds: Predicts cysteins that are likely to be partners in cysteine bridges
http://clavius.bc.edu/~clotelab/DiANNA/
http://gpcr.biocomp.unibo.it/cgi/predictors/cyspred/pred_cyspredcgi.cgi
FoldIndex© tries to answer to the question: Will this protein fold?
http://bip.weizmann.ac.il/fldbin/findex
18
Is the Recombinant Protein Correctly Expressed
SDS-PAGE and
immuno blotting
Analytical GF / DLS
/Native PAGE / IEF
19
Size
Proteolytic cleavage
Presence of impurities
Aggregation
Heterogeneity
Biological activity
Stability at different
 pH
 Ionic strengths
 Protein concentrations
 Detergent concentrations
MS / N-terminal sequencing
Truncated forms
Heterogenous N-terminus
Advantages or Disadvantages of Intra or
Extracellular Expression - I
Intracellular expression
Insoluble in
Cytoplasm
Soluble in
Cytoplasm
Extracellular expression
Cell disruption
Cell wall disruption /
Osmotic Shock
Cell debris removal
Harvest
inclusion
bodies
Culture medium
Periplasmic
space
Recover
Supernatant
Cell removal
Clarification
Clarification
Recover
Clarified
sample
Recover
Clarified
sample
Purification-Chromatography
Advantages or Disadvantages of Intra or
Extracellular Expression - II
Extracellular expression
Intracellular expression
Insoluble in
Cytoplasm
Soluble in
Cytoplasm
Recover
pellet
Recover
Supernatant
High quantities of
almost pure protein
Renaturation
problem
Lower sensitivity to
proteases
Problems with
disulphide bond
formation
Periplasmic
space
Recover supernatant
after cell lysis
Highly impure protein
and lipid concentration
Sensitive to proteases
Sometimes well expressed
Small extraction volume
Problems with disulphide
bond formation
Culture medium
Recover clarified sample
after cell wall lysis and
osmotic shock
Partially pure protein
Relatively low protein
target in small volume
Low lipid conc.
Lower degradation
Correct disulphide
bond formation
Difficult to scale-up
Purification-Chromatography
Recover
Clarified
sample
Partially pure
protein
Very large
volume
Low protein
concentration
Lower degradation
Correct disulphide
bond formation
PURIFICATION STRATEGY
 General approach: Input for Protocol Development
 Guidelines for Protein Purification. Commonly confronted decisions.
Properties of Target Protein
 Sequence of Events: Cell harvesting, Cell Disruption, Extraction and
Clarification, Chromatography
 Sample Preparation before Chromatography: Cell Debris Removal,
Clarification and Concentration: dialysis, filtration, ultrafiltration , others
 FPPLC, columns, resolution, selectivity, efficiency and capacity
 Three Phase Strategy: Linking Chromatography Techniques
22
Extraction and Clarification
 Definition: Primary isolation of target protein from source material.
Removal of debris or other contaminants which are not compatible with
chromatography.
 Goal: Preparation of clarified sample for further purification.
 The chosen technique must be robust and suitable for all scales of
purification.
 Choice of additives and buffers must be carefully
considered before scaling up
 Use additives only if essential for stabilization of
product or improved extraction; select those that
are easily removed.
23
Common Substances Used in Sample Preparation
Tris HCl 20-50mM pH 7.5-8.0 or other buffers (HEPES, Phosphate, etc)
NaCl/KCl 0.3-0.5M (to maintain ionic strength). For soluble proteins NaCl can lower to 50mM. For some insoluble
proteins it can be increase till 1M
Glycerol 5-10% to stabilize prone to aggregate proteins (can be increase till 20%). Increase viscosity and back flow
of columns
 DNaseA 25-50µg/ml (or Benzonase): degrade DNA. Reduce viscosity. Eukaryotic cells could need more Dnase
Lysozyme 0.2mg/ml for wall lysis of bacterial cells
Detergents (NP40, Triton X100, Tween 20, OG, DDM etc) for solubilization of some insoluble proteins or extraction
of membrane proteins. Use only if it does not affect protein stability!!!!
Reducing agents: 1-15mM BME, up to 2mM DTT or DTE, 1-5mM TCEP. Use only for Cys containing proteins without
disulfide bridges (maintain Cys in reduce form). Not all the IMAC columns can be use with all the reducing agents
EDTA 1-10mM Reduce oxidation damage. Chelate metal ions. Metalloprotease inhibitor. Do not use with IMAC.
Sucrose or Glucose 25mM Stabilize lysosomal membranes in eukaryotic cells. Reduce protease release.
Protease or Phosphatase inhibitors if necessary
Minimize use of additives: they must be removed in extra purification steps or may interfere
24
with activity assays
Protease Inhibitors
Protease
Inhibitor
Specificity of inhibition
Working
concentration
Antipaindihydrochloride
Papain, Trypsin, Cathepsin A and B
1-100µM
Aprotinin
Benzamidine HCl
Trypsin, Plasmin, Chymotrypsin,
Kallikrein
Serine Proteases
Bestatin
Aminopeptidases
Chymostatin
Chymotrypsin and Cysteine Proteases
10-100µM
Extracts from animal tissues
E-64
Cysteine Proteases
10µM
contain mainly serine-, cysteine-,
EDTA (or EGTA)
Metalloproteases (Calcium)
2-10mM
Leupeptin
Serine and Cysteine Proteases such as
Plasmin, Trypsin, Papain, Cathepsin B
10-100µM
PMSF and AEBSF
Serine Proteases
0.1-1mM
Pepstatin
Phosphoramidon
Aspartic Proteases
Metalloproteinases, specifically,
Thermolysin
1µg/ml
1-10µM
Serine proteases are widely
distributed in most types of cells.
Bacterial extracts typically
2µg/ml
contain serine and
0.5-4µM
metalloproteases.
and metalloproteases. (some also
contain aspartic proteases).
Plant extracts contain large
amounts of serine and cysteine
proteases
Remove proteases early in the first purification step!!: load on capture column
25
immediately after lysis and clarification.
Protease Inhibitor Cocktail Set III (Cat. No. 539134)
MERCK – EMD
Recommended for mammalian cells/tissue
1 ml sufficient for 20 g cells (~1 L). Dilution 1:100 to1:300
EDTA-free (good for His-Tag® protein purification)
Cell Disruption considerations
 Stability of the released protein
 Location of target protein within the cell (membrane, nucleus, mitochondria, etc.)
 Yield and kinetics of the process. Extent of disruption: possible use of marker
substances, measure protein concentration. Balance: volume & lysis efficiency.
 Suggested lysis volume for bacterial cells: 10-20% of original cell culture
 Scale-up
 Consider if protein purification can be performed directly from the cell lysate
without a cell debris clarification step (bed absorption chromatography)
27
Methods to monitor lysis
 Reduction of whole cells: decrease of Abs660nm before and
after treatment.
 Decrease of weight cell pellet after lysis
 Monitor nucleic acid release : Increase in the Abs260nm
during lysis
(This method could be difficult because of the “haze” generated, which can alter absorbance readings. )
 Microscopically : Compare cells before and after treatment
28
CELL DESINTIGRATION AND EXTRACTION: METHODS THAT DO NOT NEED
SPECIAL EQUIPMENT
 Freezing and thawing: Repeated cycles (can denature protein). For cells without a cell wall
(animal cells). Not suitable for large scale. Not reliable method
 Osmotic shock: Transfering cells from a high to a low osmotic pressure. Useful to release
periplasmic proteins from Gram negative bacteria. Not reliable method
 Chaotropic agents (urea, GuHCl): Extremely denaturative. Not suitable for large scale. Use
for extremely insoluble proteins or inclusion bodies
 Detergents (Brij, NP40, DDM, etc.): Anionic and non-ionic detergents permeabilize Gram
negative cells. Can interfere in downstream process. Dissolve membrane-bound proteins.
Use in combination with mechanical methods. Problematic!!! Bacterial Expression Screen - DDM (Dodecyl
Maltoside) lysis - Small Affinity binding http://wolfson.huji.ac.il/purification/TagProteinPurif/DDM_Bacterial_Expr_screen.html
 Organic solvents: Toluene, ether, chloroform, isoamyl alcohol, etc at different
concentration can release different materials from the cell. Extremely denaturative. Use
only for solvent resistent proteins. Not reliable method
 Enzymatic lysis: Lysozyme hydrolyze linkages in the peptido-glycan of bacterial cell walls.
Used
for pretreatment of cells in combination with mechanical methods.
29
Yeast cell walls can be hydrolyzed with snail gut enzymes and glucanases
CELL DESINTIGRATION AND EXTRACTION: METHODS THAT NEED SPECIAL
EQUIPMENT
 Combine with chemical treatment: lysozyme, detergents, Dnase, etc.
 Mixers and blenders: Useful for animal and plant tissues (Warring-blender)
 Coarse grinding Grinding with a pestle and mortar of frozen mycelium. Fine grinding in a
bed mill: Useful for yeast, larger cells, algae and filamentous fungi. Use of different glass
beads (Bead-beater)
 Homogenization: Animal cells. Piston/plunger device. Wheaton-Dounce homogenizer
 Sonication: Bacterial cells disrupted by high frequency sound and share forces. Low scale.
Very vigorous process. Heat generation. Not reliable method
 High pressure lysis: Pumping cell suspension through a narrow orifice at high pressure.
Mainly for bacterial cells. Very reliable and efficient method. French-press, Microfluidizer,
Avestin,
etc: medium scale (20-100ml). Microfluidizer, Maunton-Gaulin: For larger volumes
30
Cell Lysis
Equipment in LSI
HTP – Low scale:
Bacterial Expression Screen - DDM (Dodecyl
Maltoside) lysis - Small Affinity binding
http://wolfson.huji.ac.il/purification/TagProteinPur
if/DDM_Bacterial_Expr_screen.html
As French-press but for medium/larger volumes
For bacterial and yeast cells
High speed
Other applications.
Avestin Emulsiflex C3
Microfluidizer
Microfluidizer
low volume
benchtop machine
One Shot Model31
Sample Preparation before Chromatography: Cell Debris
Removal, Protein Clarification and Concentration
 Centrifugation For small sample volumes 15min 10000g .
For very turbid cell homogenates: 30min 50000g
 Filtration before loading in chromatographic column
Pore size filter: 1 μm for particle size of chromatographic medium 90 μm and upward
Pore size filter: 0.45 μm for particle size of chromatographic medium 30 or 34 μm
Pore size filter: 0.22 μm for particle size of chromatographic medium 3, 10, 15 μm
 Filtration large scale, Normal or Dead end: Hollow-fiber. Plates. Spiral Cartridge
 TFF: Tangential Flow Filtration or Cross flow
 Expanded Bed Adsorption
 Fractional Precipitation
 Ultrafiltration Membranes
32
Membrane-Based Systems
Pressure-driven processes, such as ultrafiltration (UF), microfiltration, virus filtration, and
nanofiltration . Or electric field (electroultrafiltration, EUF)
They are mainly used for protein concentration and buffer exchange in preference to SEC on an
industrial scale.
There are charge membranes that can use as IEX, RPC,
Affinity, HIC (Pall, Mustang, etc)
Another emerging technology in membrane separation
processes is high-performance tangential flow filtration
(HPTFF).
Tangential or cross flow and Normal or dead end filtration
TFF: Tangential or cross flow filtration
Merck
Prep/Scale filter modules
Labscale™ Benchtop
TFF System with
Pellicon XL module
ProFlux® M12 Benchtop
TFF system with
spiral wound modules
Fully automated 80 m2 Pellicon system for
concentration and diafiltration
Pellicon cassettes
Large-scale spiral wound34UF/DF
system
TFF: Tangential or cross
flow filtration
Merck
35
Normal / Cross Flow Filtration / Ultrafiltration
GE Healthcare
Normal flow filtration products
Cross flow filtration products
Syringe
filters
Bottle-Top
Filters
Membrane filtration Filter capsules
capsules
ÄKTAcrossflow™
Kvick Cassette family
Fully automated filtration system for cross
flow membrane screening, process
development, and small scale processing.
Enable automation at very small scale, with
capacity ranging from liters down to 25 ml.
Hollow fiber ultrafiltration
cartridges
Available with ten
different molecular weight
Membrane surface area
from 50 cm² to 2.5 m²
MW cutoffs (5k, 10k select,
10k, 30k, 50k, and 100k)
36
Dialysis
 Time
 Temperature
 Solvent
 Volume
 Cut-off
A process of separating molecules in solution by the
difference in their rate of diffusion
Uses of dialysis
• To remove unwanted small molecules from a
protein solution
- DNA
- salts
- high CMC detergents
- small proteins
• For buffer exchange
• “Desalting”
The dialysis membrane
• Molecular weight cutoff (MWCO)- the average pores size
MW>MWCO - molecule will cross membrane
MW<MWCO – molecule will not cross membrane
• MW<<MWCO cross membrane faster than MW<MWCO
MWCO=10 kDa
MWCO=10 kDa
20 kDa
8kDa
20 kDa
Slow & ineffective
8kDa
Fast
Dialysis General Considerations
•
•
•
•
•
Example:
• For 10ml sample of 1M in 10L buffer – sample
will reach to 1mM at equilibrium (~4h)
• Same sample in 1L – 10mM after 4h
Then replace buffer 1L – 0.1mM after 4h.
Concentration.
Protocol:
Choose the membrane due to protein size.
The “old” membranes are with cut-off of 13 kDa
Load the sample into dialysis tubing (wash membrane and check for holes).
Place sample into an external chamber of dialysis buffer (with gentle stirring of the
buffer).
• Dialyze for 2-4 hours
• Change the dialysis buffer and dialyze for another 2-4 hours
• Change the dialysis buffer and dialyze for 2 hours - ON.
Time
Buffer volume
Time
Types of membrane
• There are more then 30 types of synthetic and
natural dialysis membranes
Cellulose
Polysulfone
Polyethylene
Polypropylene
Polyvinylidene fluoride
Membrane with different cut-off
Ultrafiltration
 A process that uses semi-permeable membranes to
separate molecules on the basis of size.
Concentrate
Ultrafiltrate
or Retentate
 It is particularly appropriate for concentration,
partial purification or for buffer exchange.
 Is a gentle and non denaturing method.
 The ultrafiltrate is cleared of macromolecules which are significantly larger than the cutoff of the filter
 The buffer concentration in the ultrafiltrate will be exactly the same as in the concentrate
 Do not replace GF, although the principles are the same: separation according to ratio of the molecule
 Proteins with MW lower than the cut-off, will be retained in the concentrate if they aggregate, or are
part of a complex
 Cut-off at least two or three times of the protein size
 Some proteins can stick to the membranes
Ultra Centrifugal Devices
Amicon / Millipore - Merck
Protein Concentration | 2012
43
Ultrafiltration devices VIVASPIN
Selecting Hollow Fiber Cartridges and Systems
According to GE Healthcare
Ultrafiltration or dialysis
• Protein Desalting or Buffer Exchange
• The protein solution may be purified from low MW materials , like salts, low
MW reagents, etc
• Multiple solvent exchanges, will progressively purify macromolecules from
contaminating solutes.
• Ultrafiltration is faster than dialysis and requires less buffer
• Protein will be concentrated during ultrafiltration
• Diafiltration: Microsolutes are removed most efficiently by adding buffer to
the solution being ultrafiltered at a rate equal to the speed of filtration.
“How can I maximize recovery using
Ultrafiltration?” Merck

Pick an appropriate NMWL:
Example: For a 60 kDa protein: two potential membrane choices are 10 kDa or 30 kDa NMWL
 Pick devices with low non-specific binding
 Check the chemical compatibility of your device
 Devices can be use many times (Check before- Don’t spin to dryness)
 Use an invert spin for small volumes
 Use devices with vertical membrane panels
 Ensure the protein is soluble at the desired final concentration
 Allows simultaneous concentrating and desalting
 Requires much less buffer volumes than dialysis
 Allows multiple sample processing
 Easy to use and relatively fast (if buffer is not viscous)
Expanded Bed Adsorption Chromatography
Protein capture to resins without clarification (HIC, IEX and AC)
48
Sample Preparation: Fractional Precipitation
 Ammonium Sulphate (salting-out): Stabilizes proteins. Non denaturative.
Useful before HIC or to concentrate proteins before GF
 Dextran Sulphate or Polyvinylpyrrolidine: Precipitates lipoproteins
 Polyethylene glycol - PEG > 4000 up to 20%w/v: Non-denaturative.
Supernatan can be used directly to IEX or AC
 Acetone/Ethanol: Up to 80%. Useful for peptide or protein concentration.
Highly denaturative.
 Polyethyleneimine 0.1%, Protamine Sulphate or Streptomycin Sulph. 1% :
Removal of nucleic acids. Precipitation of nucleoproteins. Can precipitate
negatively charge proteins
PURIFICATION STRATEGY
 General approach: Input for Protocol Development
 Guidelines for Protein Purification. Commonly confronted decisions.
Properties of Target Protein
 Sequence of Events: Cell harvesting, Cell Disruption, Extraction and
Clarification, Chromatography
 Sample Preparation before Chromatography: Cell Debris Removal,
Clarification and Concentration: dialysis, filtration, ultrafiltration , others
 FPPLC, columns, resolution, selectivity, efficiency and capacity
 Three Phase Strategy: Linking Chromatography Techniques
50
GE Healthcare Chromatography Columns
Gravitation or centrifugation
Disposable plastic columns
Thermo, BioRad, etc
Prepacked Tricorn™
high-performance
columns
HiTrap columns 1 & 5ml
HiScreen columns
HiScale
AxiChrom column
XK columns 1.6 & 2.6 cm
ReadyToProcess columns
prepacked
Magnetic separation
Traditional purification
Centrifuge to
pellet sample
Magnetic bead purification
Careful removal of
supernatant required
to avoid sample loss
Supernatant can be
easily removed with
no sample loss
52
Resolution
 Is a measure of the relative
separation between two peaks
 It shows if further optimization
is necessary
 A complete resolve peak is not
equivalent to a pure substance
 Resolution is proportional to:
selectivity
efficiency
capacity
53
Resolution depends on efficiency and
selectivity
Efficiency is a measure of peak width (ability to elute
narrow, symmetrical peaks)
High efficiency
Related to the zone broadening on the column
(longitudinal diffusion of the molecules)
Low efficiency
Expressed as the number of theoretical plates for the
column under specified experimental conditions.
Highest efficiency is achieved by:
 Using small uniform bead sizes with uniform size distribution (reduce diffusion)
 Good experimental technique (uniform packing, air bubbles, etc)
54
Resolution depends on efficiency and
selectivity
Selectivity is the ability of the
system to separate peaks
(distance between two peaks)
High selectivity
Selectivity depends:
1) IEX & HIC: nature and number of
Low selectivity
ligands and experimental conditions
like pH, ionic strength, etc
2) GF: fractionation range
Good selectivity is more important than
high efficiency for a good resolution
Low selectivity
high efficiency
low efficiency
High selectivity
high efficiency
low efficiency
High efficiency can compensate
for low selectivity…
But:
High cost
High Back Pressure
Low flow-rate
If selectivity is high, low
efficiency can be tolerated
(if large peak volume is
acceptable).
56
SOURCE™
30 µm
5 µm
15 µm
RPC
IEX/HIC/RPC
IEX/RPC
57
Types of capacity
• Total ionic capacity (e.g. 3.5 mM/ml)
• Available capacity (e.g. 25 mg HSA/ml)
Varies with running conditions: pH, sample, ionic strength, etc
• Dynamic capacity (e.g 25 mg HSA/ml, 300 cm/h)
flow rate dependent
also varies with pH, sample, ionic strength
58
Capacity
 Available capacity is the amount of protein that can be bound under defined
experimental conditions
 Dynamic capacity is available capacity at a defined flow rate.
 Both capacities depend upon:
The chosen experimental conditions: pH, ionic strength of the buffer, the nature of
the counter-ion, the flow rate and the temperature.
The properties of the protein (molecular size, charge/pH relationship).
Presence of contaminants
The properties of the resin (small molecules that enter the porus matrix will have an
higher capacity).
 Macroporus and highly substituted with many pores to increase surface area
 Non-porus matrices have considerable lower capacity, but higher efficiency due to
shorter diffusion distances.
59
Packed bed of porous particles
Two types of void volume exist!
Interparticle void volume
(preferential flow path)
Intraparticle void volume
(contains majority of
binding sites: > 90 %)
For very big molecules there is a low binding capacity if porous are not big enough, (behave
like nonporous particles) giving wider peak & lower resolution
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60
Three Phase Strategy
achieve final purity,
remove trace impurities,
structural variants,
aggregates etc.
purity
remove bulk
impurities
isolate product,
concentrate, stabilize
polishing
intermediate
purification
capture
step
61
Selection and combination of purification
techniques
 Every technique offers a balance
between resolution, capacity, speed
and recovery
 So, resins should be selected to meet
the objectives of the purification
step
 GOAL: Fastest route to get a product
of required purity
62
Which type of chromatography resin provides the desired
performance?
Objective: High resolution
Small, uniformly sized beads
(e.g., 8-40 μm bead diameter)
Objective: Speed
Large, rigid and uniformly sized beads
provide the highest speed (e.g., 50100 μm, highly cross-linked agarose)
Objective: High binding capacity
Porous beads with high ligand
density and directed ligand
coupling
Objective: High recovery
Recovery is mostly dependent on buffer
conditions and on how peaks are cut
63
For Efficient Purification Strategies
Resolution
Polishing
Intermediate Purification
Speed
Capture
Capacity
Recovery
64
Capture
 GOAL:Initial purification
 Rapid isolation,
of the target molecule from clarified source material.
and concentration (volume reduction) of the target protein
 BONUS: Concentration (smaller
 OPTIMIZATION: Speed
 Most suitable
and faster columns). Stabilization (removal of proteases)
and Capacity: Use Macroporus and Highly Substituted matrix
techniques: IEX / HIC / (Industry)
or Affinity /IMAC/ IEX / HIC (Academics)
 Maximize
Resolution
binding of the target proteins and
minimize binding of contaminants during loading
 Maximize
protein purity during wash & elution
 Higher speed
that do not affect
Speed
considerably the dynamic capacity of the column
Capacity
Recovery
65
Intermediate Purification
Goal: Removal
Focus mainly
of major impurities
on resolution
Continuous gradient or
Most suitable
multi-step elution
techniques: IEX / HIC or expensive affinity
For good resolution
use around 20% of column capacity
Resolution
with HIC or IEX
Use
a different technique (EIX, HIC, GF, Affinity),
Or change the selectivity
(same IEX at different pH,
different ligands or salts concentr for HIC, etc.):
Selectivity optimization
Increase efficiency
Capacity
Speed
by using non-porous smaller beads
Recovery
66
Polishing

Final removal of trace contaminants, or separation of closely related substances, like
structural variants of the target protein and aggregates.

End product of required high level purity and homogenicity (oligomeric conformation,
post-translational modificatons, phosphorilation, etc)

Suitable techniques: GF/IEX/HIC (RPC for suitable proteins)
Resolution
Process Step
Capture
Speed
Polishing
Particle Size
Capacity
Recovery
Intermediate
~90
~30
~10-15
67
Three Phase Strategy
Bead size of
chromatographic
matrix
Polishing
Intermediate
purification
Capture
Step
68
Three Phase Strategy
Flow rate
Polishing
Intermediate
purification
Capture
Step
69
Three Phase Strategy
RESOLUTION
Polishing
Intermediate
purification
Capture
Step
70