developing sec methods for proteins and modified proteins · 2012-10-29 · ©2012 waters...

©2012 Waters Corporation 1

Developing SEC Methods for Proteins and Modified Proteins

Dr. Stephan Koza, Principal Applications Chemist Waters CorporationMilford, Massachusetts


Monoclonal Antibodies Antibody Conjugates Fc Fusion Proteins Synthetic Oligonucleotides Protein Subunit Vaccines Recombinant Proteins and Peptides Synthetic Peptides

Common SEC applications:Biotherapeutics Types


Agenda

Size-Exclusion Chromatography– Theory and Practice SEC– ACQUITY UPLC for SEC

o ACQUITY BEH125 SEC, 1.7um Columns o ACQUITY BEH200 SEC, 1.7 µm Columnso ACQUITY BEH450 SEC, 2.5 µm Columns

– Factors Influencing Component Resolution

– Ways to Maximize SEC Column Life


Size Exclusion Separation of Proteins


Higher Resolution Improved Sensitivity Increased Sample Throughput Decreased Mobile Phase Use Capital Expense for UHPLC System Analyst Training

SE-UHPLC vs. SE-HPLC


Why UPLC?Why UPLC?


Requires Columns and Instrumentation to Minimize Band Spreading

Broad BandBroad PeakLess SensitivityLess Resolving Power

HPLC

Advantages of UPLC Technologyfor SEC Separations

Narrow PeakIncreased SensitivityIncreased Resolving Power

Waters UPLC®

Technology


UPLC-SEC vs HPLC-SECof mAb Monomer and Aggregates

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

Minutes2.00 4.00 6.00 8.00 10.00

2.26 % Aggregate

ACQUITY BEH200 SEC, 1.7 µm4.6 x 300mm

8.008.00

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

0.055

0.060

0.065

0.070

Minutes5.00 10.00 15.00 20.00 25.00 30.00

2.24 %Aggregate

HPLC 100% Silica-Diol

SEC 250Å 5µm7.8 x 300 mm

30.0030.00

©2012 Waters Corporation 9- -

AU

0.00

0.50

1.00

1.50

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm

BioSuite125 UHR SEC 4.6 x 300mm

A2

14

AU

0.00

0.50

1.00

1.50

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

AU

0.00

0.50

1.00

1.50

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

125Å , 1.7um4.6 x 300mm

125Å , 4um4.6 x 300mm

A2

14

Improved SE-UHPLC Resolution of Proteins and Peptides (125Å Pore-Size, Aqueous)

Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min

BEH125 column provides increased resolution throughout the lower end of the peptide mass range (132 29,000).


Improved mAb Separation Improved mAb Separation (200(200--250Å Pore250Å Pore--Size)Size)

250Å, 4µm

200Å,1.7µmLMW peaksmAb

mAb dimer

Humanized monoclonal antibody biotherapeutic Conditions: 25 mM Sodium Phosphate, 0.15 M Sodium Chloride Flow rates and Injection volumes scaled for column dimensions


Agenda






Calibration Curves of ACQUITY UPLC BEH450, BEH200, and BEH125 SEC Columns


BEH SEC Particle Overview

The packing material is based on our patented Bridged Ethyl Hybrid base particle and effective diol bonding, which provide a stable chemistry with minimal secondary interactions.


BEHBEH200 SEC, 1.7um Batch-to-Batch Reproducibility


Agenda






AU

-0.026

-0.024

-0.022

AU

0.000

0.002

0.004

0.006

AU

-0.044

-0.042

-0.040

Minutes4.00 5.00 6.00

Minutes6.00 7.00 8.00 9.00

125Å, 1.7 µm (300mm)

Control Control

Sample 1 Sample 1

Sample 2 Sample 2

Fragment

Effect of Pore Size: Insulin

Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm

The HMW and insulin fragments are better resolved on the 125Å pore diameter column as compared to the 200Å pore diameter

200Å, 1. 7µm (300mm)


Minutes4.00 5.00 6.00 7.00

Minutes2.00 2.50 3.00 3.50

Rs= 3.37

Rs= 2.63

Rs= 2.63

Rs= 1.97

Rs= 1.52

Rs= 1.55

300 mm 150 mm

Effect of Column Length : Insulin


AU

0.00

0.10

0.20

0.30

AU

0.00

0.05

0.10

0.15

0.20

0.25

AU

0.00

0.05

0.10

0.15

Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00

0.2 mL/minRs= 2.4~1500 psi

0.4 mL/minRs= 1.8~3000 psi

0.8 mL/minRs= 1.3~6000 psi

IgG dimer

Effect of Flow Rate on RsEffect of Flow Rate on Rs

Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm Column: BEH200 SEC 1.7 µm, 4.6 x 150mm


AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

Minutes

3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

AU

0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

Minutes

3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00

3.7815

3.0235

2.6550

USPInjection Volume

3.7815

3.0235

2.6550

USP Res

Injection Volume

AU0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.80

1.90

Minutes3.50 4.0

04.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00

AU0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.80

1.90

Minutes3.50 4.0

04.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50

AU0.00

0.10

0.20

0.30

0.40

0.50

0.60

0.70

0.80

0.90

1.00

1.10

1.20

1.30

1.40

1.50

1.60

1.70

1.80

1.90

Minutes3.50 4.0

04.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00 10.50 11.00

3.231.25

3.150.625

3.262.5

3.2610

USP Res

Concentration

(mg/ mL)

3.231.25

3.150.625

3.262.5

3.2610

USP Res

Concentration

(mg/ mL)

Effect of Sample Load :Myoglobin

Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min, sample 2 mg/mL, Column: BEH125 1.7µm SEC , 4.6 x 300 mm column

Myoglobin: 5 mg/mL (volume load, and 20 uL injection volume (concentration) Increased injection volumes can result in a significant loss of resolution in UPLC-SEC

analyses.

Effect of Volume Load Effect of Concentration


Pump

Autosampler

Detector

Col

um

nC

olu

mn

11 12

13

1

2

3

4

Critical fittings and components


Sources of Band Spreading –Improper Column Connection

Band SpreadingDead / Void Volume

Proper

ImproperResulting

Peak Shape

PackedBedOf

Particles

No Dead

Volume


AU

0.0000

0.0005

0.0010

0.0015

0.0020

0.0025

0.0030

0.0035

0.0040

0.0045

0.0050

Minutes4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00

Effect of Optimizing Low Dispersion ConnectionsOn two, BEH200 SEC, 1.7um (4.6 x 150m Columns)

Peak 1

Peak 2Peak 3


Details: Fitting Tolerance

1.22% LMW11.13% LMW1

0.51% HMW0.50% HMW

0.33% LMW20.33% LMW2

Void Gap = 0.6 mmVolume = 1.2 uL

UV

(280

nm

)

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

Minutes4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00


Agenda






BEH200 SEC, 1.7um Guard (4.6 x 30mm) Extends UPLC SEC Column Life


ACQUITY UPLC BEH SEC, Care and Use:(Ways to extend column life) Preparation of SEC Mobile Phase and Needle Wash

– Pre filter through <0 .2 um filter (i.e, Don’t inject particulates)– Use high purity water– Replace mobile phases weekly and do not “top off”

Ramp up and down flow to column over 1min to minimize “bed shock”

Attention to SEC Eluent Inlet Filters– Use titanium, NOT stainless steel– Inlet filters can be major source of bacterial contamination

o Consider occasional sinker replacement or 70% alcohol “pull through” to prevent problems

Column Storage Considerations- Overnight: Continuously flush with the mobile phase at 10% of the maximum recommended flow rate- Extended: Store in the HPLC grade water with 10% methanol


BEH200, SEC, 1.7um4.6 x 150 mm

QC Protein Standards Mix onBacterial Contaminated, BEH200 SEC Column


Conclusions

SE-UHPLC can provide significant performance benefits over SE-HPLC.

SEC Component Rs affected by several controlable variables.

Documenting system configuration and careful assembly is critical for successful method transfer.


Acknowledgements

Paula Hong Kenneth Fountain Ed Bouvier Bill Warren

developing sec methods for proteins and modified proteins · 2012-10-29 · ©2012 waters...

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