Ion exchange chromatography

Anion (AIEC) – Cation (CIEC)

1
 Ion exchange chromatography (IEC)
 Principles of IEC

 Main stages in Chromatography

 Resolution, efficiency, selectivity and capacity

 Determination of start conditions

 Parameters for absorption optimization

 Parameters for elution optimization

 Troubleshooting

 Examples

 Summary 2
 What is ion exchange chromatography?
 IEC is a form of adsorption chromatography which separates molecules on
the basis of their charge.

 Interactions of positively or negatively charged molecules “binding” to

oppositely charged in the resin. This process is reversible via:

 Salt elution (Competitive ): Increasing the ionic strength of the buffer by

addition of salt elutes the bound molecules, in a selective way , producing a
separation based on charge differences.

 pH elution (charge): By altering the pH of the mobile phase proteins

become uncharged or oppositely charged and will elute according to their
pI values. 3
 Why use ion exchange?

 Useful at all stages of purification and at all scales.

 Controllable

 High selectivity

 High capacity

 Concentrating

 High recovery
4
Basis for selectivity

Some of the charged regions which will influence ion

exchange

• Interaction between opposite charges

• Charged groups on the proteins interact with
charged groups on the ion exchanger. Different
proteins have different charges and interact
differently.
• Anion or cation exchange
• Anion exchange binds negatively charged
(anionic) proteins
• Cation exchange binds positively charged
(cationic) proteins

5
 Main stages in Chromatography
 Equilibrate the gel and the sample to binding conditions

 Apply the sample

 Wash out contaminants

 Elute

 Wash and Regenerate column

 https://www.youtube.com/watch?v=q3fMqgT1do8
6
 Effect of pH on charge

COOH
R NH3+
Hydrogen gained

Low pH COO -
High pH
R NH3+
Positive charge Negative charge
Hydrogen lost COO -
R NH2

7
 Titration curves

+ acid isoelectric point alkaline

excess positive charge balanced positive and negative charge excess negative charge
Charge on protein

COOH
R NH3+
3 10
COO -
R NH2
COO -
NH3+
- R

pH
The overall charge on a protein depends on pH
8
 Controlling selectivity by pH

+
Anion exchanger
Charge on protein

3 10
pH

Cation exchanger
-

Each protein has its own unique net charge versus pH relationship which can
9
be visualized as a titration curve
 Charged groups
 Anion exchangers: If the protein is most stable at a pH above it pI
+
• Diethylaminoethyl (DEAE) -OCH2CH2N (CH2CH3)2
+
• Quaternary aminoethyl (QAE) -OCH2CH2N (C2H5) 2CH2CHOHCH3
+
• Quaternary ammonium (Q) -CH2N (CH3)3

 Cation exchangers: If the protein is most stable at a pH below it pI

–
• Carboxymethyl (CM) -OCH2COO
–
• Sulphopropyl (SP) -CH2CH2CH2SO3
–
• Methylsulphonate (S) -CH2SO3

If stability is high over a wide pH range on both sides of pI, either

type of ion exchanger can be used.
10
Titration curves of ion exchangers

Weak anion
exchanger
Weak cation
exchanger  Weak ion exchangers:
charge varies with pH

 Strong ion exchangers:

Strong anion
exchanger
Strong cation
exchanger
charge does not vary
with pH

11
 Ion exchange chromatography (IEC)
 Principles of IEC

 Main stages in Chromatography

 Resolution, efficiency, selectivity and capacity

 Determination of start conditions

 Parameters for absorption optimization

 Parameters for elution optimization

 Troubleshooting

 Examples

 Summary 12
 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
13
capacity
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)
14
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 can compensate
for low selectivity…
high efficiency
But:
High cost
low efficiency
High Back Pressure
Low flow-rate
High selectivity
high efficiency If selectivity is high, low
efficiency can be tolerated
low efficiency (if large peak volume is
acceptable).
16
 30 µm
SOURCE™
5 µm
15 µm
RPC

IEX/HIC/RPC
IEX/RPC

17
 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.
18
19
20
21
22
 MERCK: Fractogel® Tentacle Chromatography Resin
Eshmuno® IEX Family of Chromatography Resin
Synthetic methacrylate based polymeric beads
Excellent pressure stability
High flow rates
M-type beads with a particle size of 40-90 μm
S-type beads with a particle size in the range of 20-40 μm
Tentacles are long, linear polymer chains that carry the functional ligands.
Covalently attached to hydroxyl groups of the Fractogel® matrix.
This configuration provides a high surface area for biomolecules to bind without steric hindrance
Eshmuno® ion exchange resins carry an innovative tentacle structure that is able to bind target
substances much more effectively
Hydrophilic polyvinyl ether base matrix
High binding capacity and excellent pressure-flow behavior
High selectivity
23
 Ion exchange chromatography (IEC)
 Principles of IEC

 Main stages in Chromatography

 Resolution, efficiency, selectivity and capacity

 Determination of start conditions

 Parameters for absorption optimization

 Parameters for elution optimization

 Troubleshooting

 Examples

 Summary 24
 Determination of start conditions
 Literature references/known applications

 Isoelectric point: practical or theoretical (http://web.expasy.org/protparam/)

 Test tube method

 Scouting run at different pH

 pH stability range of target molecule 25

 Determination of start conditions
Test tube method
1) Fill test tubes with 1 ml exchanger resin.

2) Equilibrate resins with buffers of different pH-

values.

3) Add sample in buffers of different pH values.

Mix

4) Analyze the supernatants for protein of interest.

pH 6.0 6.5 7.0 7.5 8.0 In this example, the sample is completely bound at
pH 8.

Conclusion: use an anion exchanger, initial pH 8.

26
 Determination of start conditions
Scouting different pH
mAU M NaCl
50 1
pH scouting for the
40
separation of pancreatin

30 pH 5
Conditions:
pH 6 0.5 A1: 0.05 M 1-methylpiperazine,
0.05 M Bis-Tris, 0.025 M Tris
20
A2: 0.1 M HCl
pH 7 B1: Water
B2: 2 M NaCl
10
pH 8 System: ִKTA™explorer 100
Flow rate: 6 ml/min
pH 9 Column: RESOURCE™ Q, 6 ml
Sample: 2 mg Pancreatin
0 0 Gradient: as depicted
0 20 40 60 80 100 120

Elution volume (ml)

27
Tips on choosing an ion exchange resin:
Choose proper resin
 Cation or Anion:
pI charge characteristics of target and contaminants
pH stability of target
empirical determination

 According to purification step

 Sufficient capacity

 Scale-up potential

 Consistent, reliable supply

28
Functional properties of ion exchangers

• Charged groups
– Type – Selectivity
– Density – Capacity

• Matrix
– Porosity – Capacity, speed
– Particle size – Selectivity, speed, back
pressure
– Chemistry – Useful life, recovery

29
 Parameters for absorption optimization
Adjust start buffer conditions of the sample:
a) pH value b) buffer capacity c) salt concentration
pH value pH value
AU AU

gradient
gradient

volume volume

 To ensure a proper pH value, the sample should be dissolved / equilibrated in buffer A, or adjusted
(very critical for large volume samples): a) dialysis, b) dilution + buffer adjustment, c) buffer exchange
columns or d) ultrafiltration

 Buffering ion concentrations: 20–50mM are usually enough

30
 Load at the higher salt concentration that allow target binding and avoid binding of contaminants
Parameters for absorption optimization
 IEX is a binding technique, independent of sample volume

 Use the higher speed that do not affect considerably the dynamic
capacity of the column

 Sample loads can be increased if resolution is satisfactory or when using

a step elution

 For good resolution use around 20% of column capacity

 Applied sample at the higher salt concentration that allow target binding
and avoid binding of contaminants

 Use additives only if necessary

31
 Parameters for optimization
Buffer components
 The buffering substance should have the same charge
as the charged groups on the stationary phase: TrisHCl, BisTris for AEIX;
Acetate, MES, HEPES, phosphate for CEIX

 The counter-ions (salt ions) used in IEX are almost always Na+ for cation
exchange and Cl– for anion exchange

 Note different salt concentrations for similar elution strength:

 Sulfate (SO3-2)150mM
 Chloride (Cl-1)350mM

 For the adjustment of pH in both the binding buffer and elution buffer, the
same counter ion should be used as in the salt (e.g. HCl for NaCl elution on
anion exchange)

 Buffer contaminants may produce extra peaks. Use highly purified buffer salts
and perform a blank run.
32
 Choice of Buffer
According to: GE Healthcare (Amersham-Biosciences – Pharmacia)

Cation Exchange
Anion Exchange Volatile buffers
33
 Parameters for elution optimization:
Continuous gradient elution
AU %B

1
3 4 5 7
2 6

Column volume
 Smaller peaks by increasing the gradient slope.
 Give faster separations and sharper peaks, but peaks will
be eluted closer together.
 Higher selectivity by decreasing the gradient slope.
 But separation times will be longer and there will be
greater peak broadening
 First choice during method development.
 The results obtained can then serve as a base from which
to optimize the separation. 34
 Parameters for elution optimization:
Step gradient elution (stepwise)
High resolution and small peak volumes.
But: Substances can elute together.
Peaks tend to have sharp fronts and pronounced tailing
Reduces the total number of column volumes used for a separation.
This speeds up separation times and reduces buffer consumption

Elution of Elution of Elution of Elution of two target Elution of tightly

unbound first target unwanted molecules together bound molecules
molecules molecule material

AU %B
1 2
3+4 5+6 7

Column volume
The ideal IEX separation for production: target
35
proteins well resolved at short time
Parameters for
elution
optimization:
Step
gradient
elution
(stepwise)

 Conditions are chosen to maximize binding of the target proteins and minimize binding of
contaminants during sample application

 The target protein is then eluted by a single buffer change in an enriched, concentrated form
using conditions that minimize elution of unwanted contaminants

 The advantage of step elution when used at larger scale is that it is often possible to apply a
greater amount of sample, since the molecules which would elute early in a gradient separation,
no longer take up binding capacity on the column.
36
 Parameters for elution optimization:
Complex gradient elution
Choose either highest selectivity or smallest peak volumes

Offers maximum flexibility in terms of combining resolution with speed

AU %B
1
7

3 4 5
2 6

Column volume

Long, shallow gradients when you need maximum separation between peaks
Short, steep gradients where resolution is good enough
Aim: reduced separation time, minimal volume and maintained resolution
37
 First trial, general conditions

Binding buffer: 20-50 mM

Elution buffer: 20-50 mM + 1 M NaCl

Gradient: 0–50 % B in 20 column volumes

Cleaning: 51–100 % in 3–5 column volumes

pH working range: ± 0.5 pH units from the buffer salt pKa

38
 Parameters for elution optimization:
pH value
mAU M NaCl
50 1
pH scouting for the
40
separation of pancreatin

30 pH 5
Conditions:
pH 6 0.5 A1: 0.05 M 1-methylpiperazine,
0.05 M Bis-Tris, 0.025 M Tris
20
A2: 0.1 M HCl
pH 7 B1: Water
B2: 2 M NaCl
10
pH 8 System: ִKTA™explorer 100
Flow rate: 6 ml/min
pH 9 Column: RESOURCE™ Q, 6 ml
Sample: 2 mg Pancreatin
0 0 Gradient: as depicted
0 20 40 60 80 100 120

Elution volume (ml)

39
 Controlling selectivity by pH

+
Anion exchanger
Charge on protein

3 10
pH

Cation exchanger
-

Each protein has its own unique net charge versus pH relationship which can
40
be visualized as a titration curve
Controlling selectivity by pH

41
Controlling selectivity by pH

Agilent 42
Parameters for elution optimization Use high flow rate for
Flow rate high sample throughput
high productivity
0,03

0,02

1800 cm/h
(4.98 ml/min)
0,01  Lower flow rate for
0

4.5
0 4,5

0
maximum resolution
0,03

0,02

900 cm/h 0,01  Select the highest flow

(2.49 ml/min)
0
0 9
rate that maintains
0 9
0,03

0,02
resolution and minimizes
300 cm/h separation time
(0.83 ml/min) 0,01

0
0 27
Conditions
0,03
0 27
Sample: Myoglobin 0.11 mg/ml
Conalbumin 0.34 mg/ml
200 cm/h 0,02
Transferrin 0.29 mg/ml
b-Lactoglobulin B and A 0.51 mg/ml
(0.55 ml/min) 0,01
Volume: 100 µl
Buffer A: 10 mM piperazine pH 6.0
0 Buffer B: 10 mM piperazine + 1 M NaCl pH 6.0
0 40
Gradient: 0 % B for 3 ml
0 40 0-40 % B for 17 ml
Time (min) Column: 100 x 4.6 mm ID
Detection: UV 280 nm
System: FPLC
43
 0,03

Critical Factors in IEX resolution 1800 cm/h

(4.98 ml/min)
0,02

0,01

0
0 4,5

 Shape and volume of the gradient

0,03

900 cm/h 0,02

mAU
50 M NaCl (2.49 ml/min)
0,01

 Effect of flow rate on resolution 40 0

0 9

0,03
300 cm/h

 Effect of pH
30

(0.83 ml/min) 0,02

20 0,01

 Effect of different salts

0
0 27
10

0,03

200 cm/h
(0.55 ml/min) 0,02

 Use of additives (detergents, ligands, co-Elution volume (ml)

0
0 20 40 60 80 100 120

0,01

factors, etc
0 40

 Effect of temperature
 Column length (volume)
 Type of ligand (strong, weak, mixed)
 Degree of substitution
 Particle size of matrix (efficiency)
 Supplier
44
 CRITICAL FACTORS
Selecting media Selecting elution conditions
Type of ligand Shape and volume of the gradient
Degree of substitution Effect of flow rate on resolution
Type of Matrix: Particle size
Effect of pH

Type and column length

Selecting adsorption condition
Use of additives
Effect of salt concentration
Unexpected results
Effect of pH
Poor resolution
Column Volume
Elution too early or too late
Effect of temperature
Precipitation of protein
Effect of different salts
Poor recovery
45
 Ion exchange chromatography (IEC)
 Principles of IEC

 Main stages in Chromatography

 Resolution, efficiency, selectivity and capacity

 Determination of start conditions

 Parameters for absorption optimization

 Parameters for elution optimization

 Troubleshooting

 Examples

 Summary 46
 RAS - OPTIMIZATION 1st STEP
Be Un Wa 2 4 5 6 7 8 9 M 10 11 12 13 14 15 16 17 18 19 20 22 24 M 26 28 29 31 33 34 36

RAS

Repurification: F4 and
fractions13-16

Pool for GF: 2-12

500ml culture after lysis and sonication. DE-Sepharose FF 100x10mm (~8ml) in 50mM TrisHCl pH8.0 buffer
+ additives.
WASH: 5cv 0M NaCl ELUTION: gradient 15cv 0-0.15M NaCl + 5cv 0.15-1M NaCl
 RAS - OPTIMIZATION 2nd STEP

Repurification of RAS in Q-Sepharose FF column 100x10mm (~8ml) in 25mM TrisHCl pH7.9

buffer + additives. Step gradient from 20 to 160mM NaCl
 RAS - OPTIMIZATION 2nd STEP

400-1000mM

160-400mM

RAS

160mM
140mM
120mM
100mM
40mM 80mM
20mM 60mM
 RAS - CAPTURE - Anion Exchange

BeUnWa 2 4 6 8 10 12 M R 14 15 16 17 18 19 21 23 25 27 M R 28 30 32 34 36 38 40

RAS

500ml culture after lysis and sonication. Q-Sepharose FF 100x16mm (~20ml) in 25mM TrisHCl pH8.0 buffer + additives.
WASH: 7cv 70mM NaCl ELUTION: gradient 10cv 70-200mM NaCl + 5cv 0.2-1M NaCl
RAS - CAPTURE - Anion Exchange

RAS
 RAS - POLISH - Size Exclusion
6 7 8 R MW 17 18 bef aft ultr 19 20 21 22 23 MW R 25 27 29 31 32 33 34 35

RAS

60 OD280nm (8ml) RAS after Q-Seph. - Load Sephacryl S100 920x26cm -

Flow 2.5ml/min - Pool RAS after GF: 36.8 OD280nm
 15 16 17 18 19 20 21 22 23 24 25 26 27
Bef. Unb. 1 6 8 10 12 14 16 18 20 22 24
Load 90ml crude on 3ml Cation Exchange EMD-SO3
(M)
Buffer A: 20mM NaPO4 pH7.0
Buffer B: A + 1M NaCl
FractSO3M3ml01:1_UV1_280nm FractSO3M3ml01:1_UV2_260nm FractSO3M3ml01:1_Cond FractSO3M3ml01:1_Conc FractSO3M3ml01:1_Fractions
FractSO3M3ml01:1_Logbook

%B Western
100
PAGE-SDS Coomasie staining

80

5cv 75%B + 10cv 100%B

60

40

5cv 40%B + 5cv 50%B

20 5cv 25%B + 5cv 30%B

10cv10%B + 6cv 20%B

Pool: 24-26

0
F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
80 100 120 140 160 180 200 ml
 5 2 6 8 9 11 13 14 15 16 17 20
Load 150ml crude on 2.2ml Cation Exchange EMD-
SO3 (M)
Buffer A: 20mM NaPO4 pH7.0
Buffer B: A + 1M NaCl

FractSO3M2ml002:1_UV1_280nm FractSO3M2ml002:1_Cond FractSO3M2ml002:1_Conc FractSO3M2ml002:1_Fractions FractSO3M2ml002:1_Inject

FractSO3M2ml002:1_Logbook

%B
100 PAGE-SDS Coomasie staining

80

60
Load x 2 150ml sup GS3 on 2.2ml Fractogel SO3(M) ~2.2ml

15cv 0%B + 15cv 20%B + 4cv 25%B + 5cv 30%B + 7cv 45%B + 4cv 50%B + 6cv 75%B + 8cv 100%B

40

20

Pool: 16-18

0 F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Waste
0 50 100 150 ml
 Bef Unb 5 6 8 9 10 11 13 14 15 16 17 20

Load after Fract SO3 on 1ml Cation Exchange Mono S

Buffer A: 20mM NaPO4/Citrate pH6.0 + 0.15M NaCl
Buffer B: 20mM NaPO4/Citrate pH6.0 + 1M NaCl

PAGE-SDS Coomasie staining

FractSO3M2ml002:1_UV1_280nm FractSO3M2ml002:1_Cond FractSO3M2ml002:1_Conc FractSO3M2ml002:1_Fractions FractSO3M2ml002:1_Inject
FractSO3M2ml002:1_Logbook

%B
100

80

60
Load x 2 150ml sup GS3 on 2.2ml Fractogel SO3(M) ~2.2ml

15cv 0%B + 15cv 20%B + 4cv 25%B + 5cv 30%B + 7cv 45%B + 4cv 50%B + 6cv 75%B + 8cv 100%B

40

20

Pool: 16-18

0 F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Waste
0 50 100 150 ml
 Bef Unb 2 4 6 8 10 11 13 15 16 17 18 20 22 24 26 28 30 32

Upscale: Load 500ml crude

7.8ml Cation Exchange EMD-SO3 (M)
Buffer A: 20mM NaPO4 pH7.0
Buffer B: A + 1M NaCl

FractSO3M8ml001:1_UV1_280nm FractSO3M8ml001:1_UV3_220nm FractSO3M8ml001:1_Cond FractSO3M8ml001:1_Conc FractSO3M8ml001:1_Fractions

FractSO3M8ml001:1_Inject FractSO3M8ml001:1_Logbook

%B
100 PAGE-SDS Coomasie staining

80

Load 500ml Sup. Heparanase GS3 on Fractogel SO3 (M) 100x10mm

60

40

10cv30%B + 7cv 45%B + 2cv 50%B + 10cv 50-75%B + 4cv 75%B + 5cv 100%B
20

------------POOL: 19-29--------

0 F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 37 38 39 40 41
0 50 100 150 200 250 300 350 ml
 Bef Unb 5 6 7 9 11 12 13 14 15 16 23 25
Load Pool after Fract SO3 on
1ml Cation Exchange Mono S
Buffer A: 20mM NaPO4/Citrate pH6.0 + 0.15M NaCl
Buffer B: 20mM NaPO4/Citrate pH6.0 + 1M NaCl

MonoS1ml001:1_UV1_280nm MonoS1ml001:1_UV2_260nm MonoS1ml001:1_Cond MonoS1ml001:1_Conc MonoS1ml001:1_Fractions

MonoS1ml001:1_Inject MonoS1ml001:1_Logbook
PAGE-SDS Coomasie staining
%B
100

80 Load Heparanase GS3 after FractSO3(M) dil.1:4 on MonoS 1ml

Wash: ~30cv 0%B + 18cv 12%B. Eluton: 30cv 12-30%B + 5cv 30%B + 7cv 30-100%B
60

40

20
POOL I: 6-12

POOL II (repurification): 5 + 13/17

0 F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
260 280 300 320 340 ml
 Fractions 6-12 after Mono S concentrated with
Centriplus 30000 to 3.3 OD280nm/ml and load on 3ug BSA
1ul 3.3 OD280nm/ml
Superose 12 anal. Column 300x10mm

Superose12AnalA001:1_UV3_220nm Superose12AnalA001:1_Fractions Superose12AnalA001:1_Inject Superose12AnalA001:1_Logbook

PAGE-SDS
mAU kDa 158 67 43 32 13.7 Coomasie staining
5.0

Superose 12 ana. 300x10mm - 20mM Citr./PO4 pH 6.0 + 0.35M NaCl

0.0 Heparanase GS3 after FractSO3 & MonoS 01

4ml 0.047OD280/ml concentr. (30000) X 80 to 55 ul 3.2OD280/ml

Load 10ul 3.2OD280nm/ml

-5.0

-10.0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Waste
0.0 5.0 10.0 15.0 20.0 25.0 30.0 ml
 Crude Supern. + 130mMNaCl
Fractogel EMD SO3 (M) 10x1cm=7.8ml
A: 20mM NaPO4 pH7.0
B: A + 1MNaCl
Column: Res 15S 100x10mm ~7.8ml
A: 20mM MES pH6.0
B: A + 1MNaCl
 1st column (capture): Fractogel EMD SO3 (M) 10x1cm ~7.8 ml
Buffer A: 20mM NaPO4 pH 7.0 - Bufer B: A + 1M NaCL

%B
100

8 10 12 14 16 18 MW 20 22 24 26 29 30 33 34

80

60

40

20 POOL: 10-27
Pool 10-27

0
F3 F4 1 F2 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 Waste
 2nd column (interm purific): Source 15S 10x1cm ~7.8 ml
Buffer A: 20mM MES pH 6.0 - Bufer B: A + 1M NaCL
Bef Unb 6 11 13 17 18 19 20 21 MW 28 31 35 40

%B
100

16-21
80

60

40

20

Pool 16-21

0
F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 Waste
550 600 650 700 750 ml
 3rd column (polish): Fractogel EMD SO3 (M) 20x0.5cm ~4.0 ml
Buffer A: 20mM NaPO4 pH 7.0 - Bufer B: A + 1M NaCL
2 7 14 15 16 17 18 19 20 MW 21 22 23 24

%B
100

80

60

40

Load dil. 1:3 + 2cv 35%B + 3cv 45%B + 5cv 45-70%B + 1cv 70%B + 1cv 755B + 2cv 80%B + 0.5cv

Pool 15-20 for Crystal and 21-22 for repurification

20

Pool 15-20

0
F3 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
90.0 100.0 110.0 120.0 130.0 140.0 150.0 160.0 ml
U I CL Ni FLAG
Expression and purification of active PKB kinase
in E. coli
Shoshana Klein, Inbal Linchevski, Tamar Geiger ,Mario Lebendiker, Anna Itkin,
Karin Assayag and Alexander Levitzki

Double tag strategy: His6 at the N-terminus and FLAG at the C-terminus. The
protein was purified by nickel affinity chromatography, followed by passage
over an anti-FLAG column. The final purification step, anion exchange over a
MonoQ column (10µ), separated phosphorylated from unphosphorylated
MonoQ1ml006:1_UV1_280nm MonoQ1ml006:1_Conc MonoQ1ml006:1_Fractions MonoQ1ml006:1_Inject
14 .protein 19
MonoQ1ml006:1_Logbook
23
%B
100

80

60

40

20

monoQ
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34
0.0 10.0 20.0 30.0 40.0 50.0 ml

U I CL Ni FLAG 14 19 23 27

P-T308-PKB
PKB
GST

65
Surface charge change of the membrane protein Emr-E in the presence of a ligand. Anion exchange
analysis.
M. Shoskin from Sh. Schuldiner lab.
22cEmrE TPP AnEx001:1_UV1_280nm 22cEmrE TPP AnEx001:1_Conc AnExHiTRAPref22CMHsecrun001:1_UV1_280nm

mAU

K22CEmrE with TPP+ K22CEmrE as is

20.0

15.0

10.0

5.0

0.0
20.0 25.0 30.0 35.0 40.0 ml
66
The DNA Damage Response Mediator MDC1 Directly
Interacts with the Anaphase-promoting
Complex/Cyclosome
Gideon Coster et al. The J. of Biol. Chem.282 (44): 32053–32064, ( 2007)

Cdc27 and-H2AX bind the same site on MDC1. A,MDC1co-purifies

with both Cdc27 and-H2AX on an anion exchange column. Protein
extract from HeLa S3 IR-treated cells was loaded onto a Q-Sepharose
FF column. Bound proteins were eluted with a two-step NaCl gradient.
Chromatogram depicts detected absorbance at 280 nm as a general
indicator of protein content (upper panel). Salt gradient is
superimposed on chromatogram (upper panel, bold). Collected
fractions were concentrated by acetone precipitation and analyzed by
Western blotting using the indicated antibodies (lower panel). B, the
phosphopeptide that corresponds to the C terminus of -H2AX
abolishes the interaction between MDC1 and the APC/C. GST
pulldown assays were performed with GST-tBRCT and -H2AX
peptide or its unphosphorylated derivate together with protein extracts
from HeLa S3 cells, irradiated with 15 gray, and left for recovery for 1
h before protein extraction.
Bound proteins were visualized by Western blotting against Cdc27. C,
the phosphopeptide corresponding to the C terminus of Cdc27
competes with the binding of -H2AX peptide to MDC1. Peptide
pulldown assays were performed with streptavidin beads, the
biotinylated -H2AX peptide, and GST-tBRCT. Phospho- Cdc27
peptide or its unphosphorylated derivate were added to the reaction
where indicated. Bound proteins were visualized by Coomassie Blue
staining. D, a model for the interactions involving the tBRCT domain
of MDC1 and the APC/C or -H2AX with postulated functions.
67
 CIP (Cleaning in Place) Protocols
According to: GE Healthcare (Amersham-Biosciences – Pharmacia)

Remove ionically bound proteins by washing the column with 0.5 bed volumes of a 2 M
NaCl solution, contact time 10-15minutes, reversed flow direction.

Remove precipitated proteins, hydrophobically bound proteins and lipoproteins by washing

the column with 1 M NaOH solution at a linear flow rate, contact time 1-2 hours,
reversed flow direction. Wash with at least 3 bed volumes of starting buffer

Remove strongly hydrophobically bound proteins, lipoproteins and lipids by washing the
column with 4 bed volumes of up to 70% ethanol or 30% isopropanol, reversed flow
direction. Apply increasing gradients to avoid air bubble formation when using high
concentrations of organic solvents.

Alternatively, wash the column with 2 bed volumes of detergent in a basic or acidic
solution. Use, for example, 0.1-0.5% non-ionic detergent in 0.1 M acetic acid. Wash at
a low linear flow rate, contact time 1-2 hours, reversed flow direction. After treatment
with detergent always remove residual detergent by washing with 5 bed volumes of
70% ethanol.
 Troubleshooting - I
 Some of the protein do not bind or elutes before starting salt gradient

 Increase column volume

 Reduce ionic strength of sample by desalting, or dilution with start buffer.

 Increase buffer pH (for anion exch), or decrease buffer pH (for cation exch).

 Consider possibility of protein precipitation or aggregation: use advices for

prone to aggregate proteins (like use of detergents, additives, low temp, work quickly, change buffers
conditions, etc. or improve expression system)

 Protein(s) of interest eluting in more than one peak of the gradient

 Consider possibility of protein precipitation or aggregation, # oligomeric

69
concentrations, complexes, # post-translational modifications, etc
 Troubleshooting - II

 Protein(s) of interest eluting late in gradient

Proteins are binding too strongly. Increase ionic strength of gradient.

Decrease buffer pH for anion exchanger, or increase buffer pH for a
cation exchanger.

 Protein(s) of interest eluting too early in gradient

Proteins are not binding strongly. Decrease ionic strength of gradient.

Increase buffer pH for anion exchanger, or decrease buffer pH for a cation
exchanger.
70
 Troubleshooting - III
 Proteins(s) of interest not sufficiently resolved

Change gradient, use more resolutive columns, change pH buffer, reduce

flow-rate, increase column volume, change salt type

 Low recovery of activity, but normal recovery of protein.

Protein may be unstable or inactive in the buffer. Determine the pH and

salt stability of the protein.

 Protein yield lower than expected.

Protein may have been degraded by proteases. Or adsorbed to filter. Or

sample precipitates. Or hydrophobic (sticky) protein.
71
 Summary
 Charge-charge interactions of biomolecules to the resin

 Elution by increasing ionic strength or change in pH

 Binding and elution conditions must to be established

 Requires careful consideration of buffer composition including buffering

species, salts and additives

 A technique with very high resolution potential, optimal for every stage

 Many variables to change

 Scale-up is easy and predictable

 Complementary to GF, HIC, affinity 72