Size Exclusion
BioHPLC Columns
1
SEC Chromatography
Define the separation Goals:
Aggregation or Stability Studies?
What is the molecular weight of your protein of interest?
What are you trying to separate it from?
The answers to these questions help you choose the
appropriate column.
2
Size Exclusion Process
Smaller molecules spend longer
in the pores and elute later.
Larger molecules spend less time
in the pores and elute sooner.
3
Choosing The Right Pore Size
FLOW
Page 4
Choose The Right Pore Size
• Choose a pore size that allows you
to work in the linear portion of the
calibration curve.
• If two molecules have the same
molecular weight but different size
in solution they may be separated
• The calibration curve describes
how different size molecules elute
from the column and can be used to
determine
molecular
weight
equivalents based on solution size
5
Some General Guidelines for SEC
1. SEC will only provide baseline separation of molecules
with more than a 2 fold difference in MW.
2. Sample volume should be limited to below 5% of the total
column volume.
3. When methods are to be validated, test for ruggedness
with several different column lots, mobile phase
preparations, and operators.
6
Column Evaluation and Comparison
Bio-Rad Size Exclusion Standards
1. Thyroglobulin- 670 KD
2. Gamma Globulin – 320KD and 150 KD
3. Ovalbumin – 44.3 KD
4. Myoglobin- 17 KD
5. Vitamin B12 - 1,350 Da
7
Comparison of Agilent And Tosoh
Agilent Bio SEC-5 in 150 mM NAPO4 pH 7.0
Tosoh TSKgel G3000 SWxl in 100 mM NaPO4 + 100 mM Na2SO4, pH 7.0
Rs – 1.30
Agilent
Bio SEC-5, 300Å
N, B12 - 21,481
Rs – 1.47
Tosoh
G3000 SWxl
8
N, B12 - 29,115
Agilent Bio SEC-3
Separation of Bio-Rad Size Exclusion Protein Standard
Agilent Bio SEC-3, 150Å,
7.8x300mm
1
4
5
3
Columns: Bio SEC-3, 150Å, 7.8x300mm
2
& Bio SEC-3 300Å, 7.8x300mm
Agilent Bio SEC-3, 300Å,
7.8x300mm
Buffer: 0.15 M Phosphate, pH 7.0
4
3
5
Flow rate: 1.0 mL/min
1
Detector: 214 nm
2
Injection: 10 µL
Sample:
0
1
2
3
Peak
4
5
6
Protein
7
Min
8
9
10
11
12
SEC-3
150Å
SEC-3
300Å
1
Thyroglobulin
12420
1760
2
γ-Globulin
2860
3650
3
Ovalbumin
6620
11760
4
Myoglobulin
15020
20810
5
Vitamin B12
34370
35460
13
14
15
1) Thyroglobulin, 670 kD
2) γ-Globulin, 158 kD
3) Ovalbumin, 44 kD
4) Myoglobulin, 16.9 kD
5) Vitamin B12, 1355 D.
Total Column Pore Volume Comparison
(A) Agilent Bio SEC-5 in 100 mM NaPO4+100 mM NaSO2 buffer, pH 7.0
(B) Tosoh TSKgel G3000SWxl in 100 mM NaPO4+100 mM NaSO2 buffer, pH 7.0
7,43 mL
N, B12- 21,481
A
6.29 mL
B
10
N, B12- 29,115
Pore Size Choice for Antibody Analysis
Columns:
Eluent:
System:
Detector:
Flow rate:
Sample:
Agilent Bio SEC-3 100Å, 150Å & 300Å 3µm 4.6x300mm
50mM Na2HPO4, 50mM NaH2PO4 + 0.15M NaCl, pH6.8
Agilent 1260 Infinity Bio-Inert LC System
UV@220nm
0.35ml/min
Mouse IgG
2
4
300Å
150Å
1
3
1. Dimer
2. Monomer
3. Monomer Fragment
4. Azide
100Å
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Confidentiality Label
August 29, 2013
Determine Optimal Mobile Phase Conditions
What conditions give you the best results?
What additives may be required to reduce non-specific
interactions?
What is the optimal pH?
What is the optimal flow rate?
12
Mobile Phase Considerations:
• Mobile phase should contain buffer/salt (to overcome ionic interactions).
• Mobile phase should not contain too much buffer/salt (to prevent
hydrophobic interactions).
• Mobile phase should not alter the analyte (cause degradation /
aggregation etc.).
• Mobile phase should be made up fresh and used promptly (bacterial
growth is rapid in dilute buffer stored at room temperature).
• Buffer shelf life < 7 days unless refrigerated.
• Mobile phase should be filtered before use. Particulates may be present
in water (less likely) or in buffer salts (more likely).
13
2
A Note About Pre-Made PBS:
Note: PBS is typically around 10mM phosphate, pH 7.2, 0.8% NaCl
(150mM).
Our preference is to use 150mM phosphate buffer, pH 7.0 to avoid
use of salt (NaCl).
Other possibilities: sodium sulfate instead of sodium chloride (but
remember 0.15M Na2SO4 is twice the ionic strength of 0.15M NaCl).
14
Bio-applications Training Program
13-17 February 2012
100mM Phosphate Buffer
(Mixing NaH2PO4 and Na2HPO4 stock solution)
x mL
0.2M Na2HPO4
4.0
6.15
9.25
13.25
18.75
24.5
30.5
36.0
40.5
43.5
45.75
47.35
y mL
0.2M NaH2PO4
46.0
43.85
40.75
36.75
31.25
25.5
19.5
14.0
9.5
6.5
4.25
2.65
z mL
H2O
50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
200 mL 0.1M
pH, 25°C
5.8
6.0
6.2
6.4
6.6
6.8
7.0
7.2
7.4
7.6
7.8
8.0
Stock Solutions:
0.2M Na2HPO4 = 28.39 g/L Na2HPO4 (anhydrous) or 71.64 g/L Na2HPO4.12H2O
0.2M NaH2PO4 = 31.21 g/L NaH2PO4.2H2O
15
Bio-applications Training Program
13-17 February 2012
RECOMMENDED STARTING CONDITIONS
150 Mm phosphate buffer, pH 7.0
Flow rate of 0.1-1.25 ml/min for 7.8 mm id columns
Isocratic for 30 minutes
Temperature 20-30 C. Higher temperatures can be used, see
manufacturer’s spec.
16
Non-Specific Interaction Example
Columns:
Eluent:
System:
Detector:
Flow rate:
Sample:
Agilent Bio SEC-3 100Å, 150Å & 300Å 3µm 4.6x300mm
50mM Na2HPO4, 50mM NaH2PO4 + 0.15M NaCl, pH6.8
Agilent 1260 Infinity Bio-Inert LC System
UV@220nm
0.35ml/min
Mouse IgG
2
4
300Å
150Å
100Å
17
1
3
1. Dimer
2. Monomer
3. Monomer Fragment
4. Azide
EXAMPLES OF ADDITIVES TO REDUCE
NONSPECIFIC INTERACTIONS
100-150mM NaCl
100-150mM NaSO4
50-100mM urea
Guanidine hydrochloride can also be used
5-10% ethanol
5% DMSO
18
About Particle Size, Dimensions, and Flow Rate
19
2 WAYS TO IMPROVE SEC RESOLUTION
With SEC, there are two ways to improve efficiency/resolution:
1. Increase column length
2. Decrease column particle size
20
Agilent Bio SEC-5
Monoclonal Antibody Aggregation Monitoring
0.020
0.018
0.016
0.014
Columns: Bio SEC-5, 300Å, 7.8x300mm
Buffer: 150 mM PBS, pH 7
Flow Rate: 1.0 mL/min
Sample: Mab
Temperature: Ambient
0.012
MAb Dimer
0.010
AU
Excipients
0.008
0.006
Mab Aggregates
0.004
0.002
0.000
-0.002
-0.004
-0.006
-0.008
1.0
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2.00
3.0
4.0
5.0
6.0
7.0
8.0
Min
9.0
10.0
11.0
12.0
13.0
14.0
15.0
Size Exclusion
Monitoring Aggregation and Impurities of Monoclonal Antibodies
0.035
0.030
0.025
Column: Bio SEC-3 300Å, 7.8x300mm
Mobile phase: 150 mM Phosphate, pH 7
Flow rate: 1.0 mL/min
Temperature: Ambient
Sample: Monoclonal antibody (10 µL, 5 mg/mL)
AU
0.020
0.015
MAb
Aggregates
0.010
MAb
Dimer
Low Molecular
Weight Impurities
Buffer/Excipients
0.005
0.000
-0.005
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
Minutes
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9.00
10.00
11.00
12.00
13.00
14.00
15.00
INCREASE THROUGHPUT/REDUCE TIME
Smaller particle size columns allow you to reduce time and
improve throughput by:
1. allows you to use shorter column lengths without losing
resolution
2. Allows you to also increase flow rate because you have more
resolution room.
23
Fast SEC
1.5ml/min
1.0 ml/min
monomer
Column: Agilent Bio SEC-3, 7.8 x150mm
Sample: mAb (2mg/ml)
Injection: 5ul
Flow rate: 1.0 and 1.5ml/min (56 bar , 75 bar)
Eluent : 150mM sodium phosphate
Detection: 220nm
dimer
monomer
dimer
Flow Rate
Resolution
Monomer/Dimer
Monomer
Efficiency
Percentage
Dimer
1.0ml/min
1.58
3,684
0.65
1.5ml/min
1.31
2,574
0.70
4 Minutes
Fast SEC
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Fast SEC
2.0ml/min
1.5ml/min
1.0 ml/min
Column: Agilent Bio SEC-3, 7.8 x150mm
Sample: mAb (2mg/ml)
Injection: 5ul
Flow rate: 1.0, 1.5 and 2ml/min (56 bar , 75 bar, 105 bar)
Eluent: 150mM sodium phosphate + 100mM Na-sulfate
Detection: 220nm
monomer
dimer
Flow Rate
Resolution
Monomer/Dimer
Monomer
Efficiency
Percentage
Dimer
1.0ml/min
1.53
3,510
0.64
1.5ml/min
1.43
2,502
0. 47
2.0ml/min
1.13
1,917
0.64
4 Minutes
Fast SEC
25
August 29, 2013
• 5µm Particle
• 100Å, 150Å, 300Å, 500Å, 1000Å,
2000Å pore sizes
• High stability and long lifetime
• Great reproducibility
• IDs: 4.6, 7.8 (larger are currently
custom)
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• Unique, 3µm particle
• 100Å, 150Å, 300Å pore sizes
• Highest resolution
• Highest efficiency
• Faster SEC separations
• IDs: 4.6, 7.8 (larger are currently
custom)
27
Consistent, accurate results with reproducible
separation of IgG monomer & aggregates
IgG Inj.
No.
1st
10th
20th
30th
40th
50th
Technology: Small particle SEC Column
Bio SEC-3, 7.8x 300 mm ID column
Result: Consistent Quantitation
28
Aggs
Dimer
Monomer
% Ratio
6.906
17.178
75.916
RS Factor
-
0.72
1.63
% Ratio
6.628
17.045
76.326
RS Factor
-
0.73
1.63
% Ratio
6.336
16.773
76.892
RS Factor
-
0.73
1.63
% Ratio
6.145
16.661
77.194
RS Factor
-
0.74
1.63
% Ratio
6.004
16.610
77.386
RS Factor
-
0.74
1.63
% Ratio
6.345
16.768
76.892
RS Factor
-
0.74
1.63
Overlays of injections from 1, 10, 20,
30, 40, and 50th injection visually
show the consistency of the results.
The table shows the consistent ratios
of monomer, dimer and aggregate
SEC CONCLUSION
Use what works best and helps you achieve your separation
goal!
29
Questions?
30