developing sec methods for proteins and modified proteins · 2012-10-29 · ©2012 waters...
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©2012 Waters Corporation 1
Developing SEC Methods for Proteins and Modified Proteins
Dr. Stephan Koza, Principal Applications Chemist Waters CorporationMilford, Massachusetts
©2012 Waters Corporation 2
Monoclonal Antibodies Antibody Conjugates Fc Fusion Proteins Synthetic Oligonucleotides Protein Subunit Vaccines Recombinant Proteins and Peptides Synthetic Peptides
Common SEC applications:Biotherapeutics Types
©2012 Waters Corporation 3
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
©2012 Waters Corporation 4
Size Exclusion Separation of Proteins
©2012 Waters Corporation 5
Higher Resolution Improved Sensitivity Increased Sample Throughput Decreased Mobile Phase Use Capital Expense for UHPLC System Analyst Training
SE-UHPLC vs. SE-HPLC
©2012 Waters Corporation 6
Why UPLC?Why UPLC?
©2012 Waters Corporation 7
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
©2012 Waters Corporation 8
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).
©2012 Waters Corporation 10
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
©2012 Waters Corporation 11
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
©2012 Waters Corporation 12
Calibration Curves of ACQUITY UPLC BEH450, BEH200, and BEH125 SEC Columns
©2012 Waters Corporation 13
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.
©2012 Waters Corporation 14
BEHBEH200 SEC, 1.7um Batch-to-Batch Reproducibility
©2012 Waters Corporation 15
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
©2012 Waters Corporation 16
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)
©2012 Waters Corporation 17
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
©2012 Waters Corporation 18
AU
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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
©2012 Waters Corporation 19
AU
0.00
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1.00
1.10
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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
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1.00
1.10
1.20
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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
©2012 Waters Corporation 20
Pump
Autosampler
Detector
Col
um
nC
olu
mn
11 12
13
1
2
3
4
Critical fittings and components
©2012 Waters Corporation 21
Sources of Band Spreading –Improper Column Connection
Band SpreadingDead / Void Volume
Proper
ImproperResulting
Peak Shape
PackedBedOf
Particles
No Dead
Volume
©2012 Waters Corporation 22
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
©2012 Waters Corporation 23
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
©2012 Waters Corporation 24
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
©2012 Waters Corporation 25
BEH200 SEC, 1.7um Guard (4.6 x 30mm) Extends UPLC SEC Column Life
©2012 Waters Corporation 26
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
©2012 Waters Corporation 27
BEH200, SEC, 1.7um4.6 x 150 mm
QC Protein Standards Mix onBacterial Contaminated, BEH200 SEC Column
©2012 Waters Corporation 28
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.
©2012 Waters Corporation 29
Acknowledgements
Paula Hong Kenneth Fountain Ed Bouvier Bill Warren