Size Exclusion BioHPLC Columns

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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.

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Size Exclusion Process

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Larger molecules spend less time in the pores and elute sooner.

Smaller molecules spend longer in the pores and elute later.

Choosing The Right Pore Size

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FLOW

Choose The Right Pore Size

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• 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

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.

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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

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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 – 1.47N, B12 - 29,115

N, B12 - 21,481Agilent Bio SEC-5, 300Å

Tosoh G3000 SWxl

Rs – 1.30

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Columns: Bio SEC-3, 150Å, 7.8x300mm

& Bio SEC-3 300Å, 7.8x300mm

Buffer: 0.15 M Phosphate, pH 7.0

Flow rate: 1.0 mL/min

Detector: 214 nm

Injection: 10 µL

Sample:

1) Thyroglobulin, 670 kD

2) γ-Globulin, 158 kD

3) Ovalbumin, 44 kD

4) Myoglobulin, 16.9 kD

5) Vitamin B12, 1355 D.

Peak

Protein

SEC-3 150Å

SEC-3 300Å

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Thyroglobulin

12420

1760

2

γ-Globulin

2860

3650

3

Ovalbumin

6620

11760

4

Myoglobulin

15020

20810

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Vitamin B12

34370

35460

Min 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

1

2

3 4

5

1 2

3 4 5

Agilent Bio SEC-3, 150Å, 7.8x300mm

Agilent Bio SEC-3, 300Å, 7.8x300mm

Agilent Bio SEC-3 Separation of Bio-Rad Size Exclusion Protein Standard

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

A

B

7,43 mL

6.29 mL

N, B12- 29,115

N, B12- 21,481

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Pore Size Choice for Antibody Analysis

August 29, 2013 Confidentiality Label

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Columns: Agilent Bio SEC-3 100Å, 150Å & 300Å 3µm 4.6x300mm Eluent: 50mM Na2HPO4, 50mM NaH2PO4 + 0.15M NaCl, pH6.8 System: Agilent 1260 Infinity Bio-Inert LC System Detector: UV@220nm Flow rate: 0.35ml/min Sample: Mouse IgG

300Å

150Å

100Å

1

2

3

4

1. Dimer 2. Monomer 3. Monomer Fragment 4. Azide

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?

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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).

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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).

13-17 February 2012 Bio-applications Training Program

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100mM Phosphate Buffer (Mixing NaH2PO4 and Na2HPO4 stock solution)

x mL 0.2M Na2HPO4

y mL 0.2M NaH2PO4

z mL H2O

200 mL 0.1M pH, 25°C

4.0 46.0 50.0 5.8 6.15 43.85 50.0 6.0 9.25 40.75 50.0 6.2

13.25 36.75 50.0 6.4 18.75 31.25 50.0 6.6 24.5 25.5 50.0 6.8 30.5 19.5 50.0 7.0 36.0 14.0 50.0 7.2 40.5 9.5 50.0 7.4 43.5 6.5 50.0 7.6

45.75 4.25 50.0 7.8 47.35 2.65 50.0 8.0

13-17 February 2012 Bio-applications Training Program

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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

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.

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Non-Specific Interaction Example

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Columns: Agilent Bio SEC-3 100Å, 150Å & 300Å 3µm 4.6x300mm Eluent: 50mM Na2HPO4, 50mM NaH2PO4 + 0.15M NaCl, pH6.8 System: Agilent 1260 Infinity Bio-Inert LC System Detector: UV@220nm Flow rate: 0.35ml/min Sample: Mouse IgG

300Å

150Å

100Å

1

2

3

4

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

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About Particle Size, Dimensions, and Flow Rate

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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

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Agilent Bio SEC-5 Monoclonal Antibody Aggregation Monitoring

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AU

-0.008

-0.006

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

Min 1.0 2.00 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0

Excipients

MAb Dimer

Mab Aggregates

Columns: Bio SEC-5, 300Å, 7.8x300mm Buffer: 150 mM PBS, pH 7 Flow Rate: 1.0 mL/min Sample: Mab Temperature: Ambient

Size Exclusion Monitoring Aggregation and Impurities of Monoclonal Antibodies

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AU

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

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)

Buffer/Excipients MAb

Dimer MAb

Aggregates

Low Molecular Weight Impurities

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.

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Fast SEC

Fast SEC

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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

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

monomer dimer

monomer dimer

1.5ml/min 1.0 ml/min

Fast SEC

August 29, 2013

Fast SEC

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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

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

monomer dimer

2.0ml/min 1.5ml/min 1.0 ml/min

• 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)

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Consistent, accurate results with reproducible separation of IgG monomer & aggregates

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IgG Inj. No. Aggs Dimer Monomer

1st % Ratio 6.906 17.178 75.916

RS Factor - 0.72 1.63

10th % Ratio 6.628 17.045 76.326

RS Factor - 0.73 1.63

20th % Ratio 6.336 16.773 76.892

RS Factor - 0.73 1.63

30th % Ratio 6.145 16.661 77.194

RS Factor - 0.74 1.63

40th % Ratio 6.004 16.610 77.386

RS Factor - 0.74 1.63

50th % 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 Technology: Small particle SEC Column

Bio SEC-3, 7.8x 300 mm ID column Result: Consistent Quantitation

SEC CONCLUSION

Use what works best and helps you achieve your separation goal!

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Questions?

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