principles and practices for sec, iex for intact protein ... · 3 cv gradient 1 2 57 ©2012 waters...
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UPLC User meeeting April 2012UPLC User meeeting April 2012
Principles and Practices for SEC, IEX for Principles and Practices for SEC, IEX for Intact Protein Analysis by UPLC Intact Protein Analysis by UPLC
[email protected][email protected]
©2012 Waters Corporation 1
AgendaAgendagg
Ion-Exchange Chromatography– Theory and practice
P t i P k Hi R IEX C l – Protein-Pak Hi Res IEX Columns – Method Development Strategies
Size-Exclusion Chromatographyg p y– ACQUITY UPLC for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7 µm ColumnsInsulin Analysis– Insulin Analysis
– Combination of SEC and MS
©2012 Waters Corporation 2
IonIon--Exchange ChromatographyExchange Chromatographyg g p yg g p y
Binds at Low Ionic Strength Elute with Step or Continuous Gradients of Increasing Ionic Gradients of Increasing Ionic Strength
Separations are based on net surface charge on protein with oppositely charged groups on ion-exchanger
©2012 Waters Corporation 3
charged groups on ion exchanger
Proteins elute from column using either a gradient of increasing salt concentration (most common) or changing pH (less common)
Select buffer pHSelect buffer pHIsoelectric Point of a Protein (pI)Isoelectric Point of a Protein (pI)(p )(p )
Isoelectric Isoelectric point (pI)Zero net
charge at this gpH
©2012 Waters Corporation 4
Select buffer pHSelect buffer pHIsoelectric Point of a Protein (pI)Isoelectric Point of a Protein (pI)(p )(p )
pH below pIp pProtein has net +ve charge pH region cation exchange
pH above pIP t i h t h
©2012 Waters Corporation 5
Protein has net -ve chargepH range for anion exchange
Ion Exchange Chromatography Ion Exchange Chromatography BuffersBuffers
Select buffer with pKa near to desired pH
Buffer ions should have same charge as functional groups on packing material (PO4
- for cation, Tris+ for anion)
©2012 Waters Corporation 6
packing material (PO4 for cation, Tris for anion)
Common Customer ConcernsCommon Customer Concerns
Reproducibility between columns
Not getting required resolution from the start
Recovery and carryover
©2012 Waters Corporation 7
ProteinProtein--Pak Hi Res IEXPak Hi Res IEX
©2012 Waters Corporation 8
Attributes of ProteinAttributes of Protein--Pak™ Hi Res Pak™ Hi Res IEX ColumnsIEX Columns
Multi-layered network of ion-exchange groups (SP, CM or Q)
Effective diffusion and binding o Effective diffusion and binding
o High sample loading and resolution
o Minimal non-desired interactions
No MW limitations: non-porous material
QC tested with protein samples for batch-to-batch reproducibility
High chemical stability: hydrophilic, polymer-based IEX particles
– Wide pH range (3- 10)
– high salt concentrations (1M)
St d d ( t 1450 i f CEX d 2175 i f AEX) – Standard pressures (up to 1450 psi for CEX and 2175 psi for AEX)
– Can be cleaned with aggressive washing
eCord enabled for data tracking
©2012 Waters Corporation 9
Strategies to Developing an Strategies to Developing an IonIon--Exchange Protein SeparationExchange Protein Separationg pg p
Selectivity is most conveniently optimized with pH
Retention is optimized by adjustment of ionic strength
Changing buffer and counter ion may improve selectivity
Methods may require adjustment if the temperature is changed Methods may require adjustment if the temperature is changed
©2012 Waters Corporation 10
Effect of pH on SelectivityEffect of pH on Selectivity
0.040
0.050
p yp y
α-Chymotrypsinogen 1
Ribonuclease A 2pH 6.6
1 3
AU
0.020
0.030cytochrome c 3
2
0.000
0.010
0.050
1 3
AU
0.020
0.030
0.040
pH 5.0
2
0.000
0.010
Minutes4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00 36.00
©2012 Waters Corporation 11
Column: Protein-Pak Hi Res CM 4.6 x 100 mm column
Fine tune pH Fine tune pH pH Effect on mAb SeparationpH Effect on mAb Separationp pp p
0.006
0.008 pH 6.4
AU
0.000
0.002
0.004
0.006
AU
0 000
0.002
0.004
0.006 pH 6.6
0.000
AU 0.005
0.010 pH 6.8
A
0.000
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
©2012 Waters Corporation 12
Column: Protein-Pak Hi Res CM 4.6 x 100 mm column Gradient: 0.0 –0.10 M NaCl, 20mM Sodium Phosphate in 40 min Flow: 0.5 mL/min
Effect of Salt Gradient Slope Effect of Salt Gradient Slope % 24.00
36.00
48.00
pp
Ovalbumin 1
Myoglobin 20-0.15M NaCl
Elutes in high salt wash step1
3
25
28 00
0.00
12.00
Minutes3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80
Ribonuclease A 3
Cytochrome C 4
Lysozyme 51
3
2
4
%
7.00
14.00
21.00
28.00
0-0.3M NaCl
3
4 5
0.00
Minutes3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80
36.00
48.00
Unbound proteins
1 24 5
%
0.00
12.00
24.00
Minutes0.00 3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80
0- 0.5M NaCl3
©2012 Waters Corporation 13
Minutes
Higher salt gradients result in earlier elution of bound proteins High salt wash may be needed in shallower gradients to elute tightly bound proteins Column: Protein-Pak Hi Res CM 4.6 mm x 100mm
Effect of Salt Gradient Slope:Effect of Salt Gradient Slope:Ovalbumin Variants Ovalbumin Variants
8 10 cv gradient
AU 0.02 1 2
3 4
56
79
10
0.00
AU 0.02
5 cv gradient
3 45
6
7
0.00
1 25 7
3 4
6
AU
0.00
0.02
Min tes4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
3 cv gradient1 2
3 45 7
©2012 Waters Corporation 14
Minutes
Longer gradient: increased resolution, lower sensitivity
Column: Protein-Pak HI Res Q, 4.6 x 100 mm
Effect of Buffer on SelectivityEffect of Buffer on Selectivity
0.025
yy
α-Chymotrypsinogen 1
Ribonuclease A 2
20mM Sodium Phosphate
1 2
3
AU
0.010
0.015
0.020 cytochrome c 32
0.000
0.005
20mM MES (Morpholino ethane13
AU
0 010
0.015
0.020
0.02520mM MES (Morpholino ethaneSulfonic acid)
12
0.000
0.005
0.010
Minutes5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00
©2012 Waters Corporation 15
Buffer can alter selectivity and retention of proteins at same pH (6)
Column: Protein-Pak Hi Res CM 4.6 x 100 mm column
Counter Ion EffectsCounter Ion Effects
Ribonuclease A 1
cytochrome c 2
AU 0.010
0.020 12
3
Aprotinin 3
0.000
0020
NaCl
AU
0 000
0.010
0.020
KCl0.000
Minutes14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00
C t i h l ti it d t ti f t i
©2012 Waters Corporation 16
Counter ion may change selectivity and retention of proteins
Effects tend to be minimal
Temperature EffectsTemperature Effectspp
45 °C
AU 0.005
40 °C
0.000
AU 0.005
35 °C
A
0.000
U 0 005
30 °C
AU
0.000
0.005
KKK
Temperature may effect selectivity and retention
AU
0.000
0.005
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00
KKK
©2012 Waters Corporation 17
p y y Changes may be similar to those observed with pH change Column: ProteinP-ak Hi Res CM 4.6 x 100 mm column
IEX SummaryIEX Summaryyy
Protein-Pak Hi Res IEX column benefits
– Consistent batch-to-batch performance (tested with protein p ( pstandards)
– Minimal column related carryover
– Stable over a wide pH rangeStable over a wide pH range
Method Parameters to optimize areS l ti it i t i tl ti i d ith H– Selectivity is most conveniently optimized with pH
– Retention is optimized by adjustment of ionic strength
– Changing buffer and counter ion may improve selectivity
– Methods may require adjustment if the temperature is changed
©2012 Waters Corporation 18
AgendaAgendagg
Ion-Exchange Chromatography– Theory and practice
P t i P k Hi R IEX C l – Protein-Pak Hi Res IEX Columns – Method Development Strategies
Size-Exclusion Chromatographyg p y– ACQUITY UPLC for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns o ACQUITY BEH125 SEC, 1.7 µm ColumnsInsulin Analysis– Insulin Analysis
– Combination of SEC and MS
©2012 Waters Corporation 19
Common Customer ConcernsCommon Customer Concerns
Column-to-column reproducibility
– Changes in retention timeChanges in retention time
– Changes in spacing between peaks
– Changes in resolution
Column lifetime
– Peak shape deteriorates over time
– Increased pressureIncreased pressure
– Changes in resolution
Tailing of specific proteins
Resolution
Throughput
©2012 Waters Corporation 20
UPLCUPLC--SEC vs HPLCSEC vs HPLC--SECSECof mAb monomer and aggregatesof mAb monomer and aggregatesgg ggg g
0.065
0.070
0.065
0.070
HPLC 100% ACQUITY BEH200
0.045
0.050
0.055
0.060
0.045
0.050
0.055
0.060Silica-Diol
SEC 250Å 5µm7.8 x 300 mm
ACQUITY BEH200 SEC, 1.7 µm4.6 x 300mm
AU
0.030
0.035
0.040
0.045
AU
0.030
0.035
0.040
0.015
0.020
0.025
0.015
0.020
0.025
2.26 % Aggregate 2.24 %
Aggregate
0.000
0.005
0.010
0.000
0.005
0.010
©2012 Waters Corporation 21
Minutes2.00 4.00 6.00 8.00 10.00
Minutes5.00 10.00 15.00 20.00 25.00 30.00
8.00 30.008.00 30.00
Effect of LC System Dispersion onEffect of LC System Dispersion onBEH200 SEC mAb SeparationBEH200 SEC mAb Separationpp
Larger system dispersion decreases component resolution
0.20
0.30 BEH200 SEC 1.7umColumn (4.6 x 300mm)
HPLC System
USP Res= 1.37
AU
0 00
0.10
0.00
0.20
0.25 Waters ACQUITY UPLC SystemUSP Res= 2.37 BEH200 SEC 1.7um
AU
0.05
0.10
0.15 Column (4.6 x 300mm)
©2012 Waters Corporation 22
0.00
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
ACQUITY BEH200 SEC 1.7 µm ACQUITY BEH200 SEC 1.7 µm ColumnsColumns
Application Areas
– Determination of protein molecular weightDetermination of protein molecular weight
– Molecular weight range of 10,000 to 450,000 Daltons
– Determination of size heterogeneity in a protein sample
– Quantitation of protein aggregates primarily in therapeutic monoclonal antibodies.
©2012 Waters Corporation 23
BEH OverviewBEH Overview
The packing material is based on our patented Bridged Ethyl Hybrid base particle and effective diol bonding, which provide a y p g, pstable chemistry with minimal secondary interactions.
©2012 Waters Corporation 24
BEH200BEH200 SEC, SEC, 1.7um1.7umColumn Batch Test Column Batch Test
0.22
Analyte pI MW
1. Thyroglobulin, 3 mg/mL 4.6 669,000
0.16
0.18
0.20
2
4
y g , g/ ,
2. IgG, 2 mg/mL (Vicam) 6.7 150,000
3. BSA, 5 mg/mL 4.6 66,400
U
0.12
0.14 35
4. Myoglobin, 2 mg/mL 6.8, 7.2 17,000
5. Uracil, 0.1 mg/mL N/A 112
A
0.06
0.08
0.10
1
0.02
0.04
0.06
©2012 Waters Corporation 25
0.00
Minutes0.00 0.50 1.00 1.50 2.00 2.50 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
BEH200 SEC, 1.7um BEH200 SEC, 1.7um BatchBatch--toto--Batch ReproducibilityBatch Reproducibilityp yp y
AU
0 00
0.05Batch 1, Column 1
0.00
AU
0 00
0.05Batch 1, Column 2
0.00
AU
0.00
0.05 Batch 1, Column 3
AU
0.00
0.05 Batch 2, Column 1
AU
0.00
0.05 Batch 2, Column 2
©2012 Waters Corporation 26
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
What’s new ? What’s new ?
BEH 125 SEC UPLC Column– 15 cm/30 cm/Guard15 cm/30 cm/Guard
– Launched on January 10th
– Linear range from 1.000 to 80.000 Dalton
Reference Description
186006504 ACQUITY UPLC BEH125 SEC 1.7µm 4.6x30 Gd
186006505 ACQUITY UPLC BEH125 SEC 1.7µm 4.6x150mm
186006506 ACQUITY UPLC BEH125 SEC 1.7µm 4.6x300mm
©2012 Waters Corporation 27
Calibration Curves for Calibration Curves for ACQUITYACQUITY UPLCUPLCBEH200BEH200 and and BEH125BEH125, SEC, 1.7 , SEC, 1.7 μmμm ColumnsColumns
BEH200, SEC, 1.7um
Thyroglobulin (~ 669,000 Da)
IgG (~ 150,000 Da)
Rnase A (~ 13,700 Da)
Conalbumin (~ 75,000 Da)
Ovalbumin dimer(~ 88,000 Da)
O lb i ( 44 000 D )
Aprotinin (~ 6,500 Da)
Ovalbumin (~ 44,000 Da)
BEH125, SEC, 1.7um Uracil (~ 112 Da)
Angiotensin II (~ 1,045 Da)
©2012 Waters Corporation 28
Resolution of Proteins and Peptides Resolution of Proteins and Peptides pp
1.50ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm1.501.50ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm
AU
0 00
0.50
1.00
AU
0 00
0.50
1.00
AU
0 00
0.50
1.00
0.00
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
0.80
1.00BioSuite125 UHR SEC 4.6 x 300mm
A2
14
0.00
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
0.00
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
0.80
1.00
0.80
1.00BioSuite125 UHR SEC 4.6 x 300mm
A2
14
AU
0.00
0.20
0.40
0.60
AU
0.00
0.20
0.40
0.60
AU
0.00
0.20
0.40
0.60
- -
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
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
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
Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min
©2012 Waters Corporation 29
- BEH125 column provides increased resolution throughout the lower end of the
peptide mass range (132 29,000).
Insulin Analyses by TraditionalInsulin Analyses by TraditionalHPLCHPLC--SEC vs UPLCSEC vs UPLC--SECSEC
Waters Alliance HPLCSystemInsulin HMWP SEC 10 μm(7.8 x 300mm)
Waters ACQUITY UPLCSystemBEH125, SEC, 1.7 μm (4.6 x 300mm)
©2012 Waters Corporation 30
Column Stability for Insulin Analysis Column Stability for Insulin Analysis
Retention Time USP Resolution
y yy y
3
4
5
6
ime
n)
3.0
4.0
5.0
utio
n
0
1
2
3
Ret
entio
n T
(mi n
0.0
1.0
2.0
USP
Res
olu
0 100 200 300 400 500 600 700 800 900
Injection Number
Over 800 injections the retention time of the insulin monomer peak
©2012 Waters Corporation 31
and the resolution between insulin monomer and dimer peaks are maintained.
What about SEC – MS ?
©2012 Waters Corporation 32
SECSEC--MSMSHumanized Monoclonal AntibodyHumanized Monoclonal Antibodyyy
UV @ 2801 2 2
1.4e-2
1.6e-2
1.8e-2
2: Diode Array 280 0.0500Da
Range: 6.757e-125.27
1UV @ 280
AU
4.0e-3
6.0e-3
8.0e-3
1.0e-2
1.2e-2
16.58
2
3
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 17000.0
2.0e-313.22
19.50
1: TOF MS ES+ TIC
7.58e619.493
TIC
%
15.35
16.62
1 2
Scan500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
4
23.7425.06
©2012 Waters Corporation 33
MS: Xevo G2 Q Tof Conditions: 100mM Ammonium Formate, Flow rate: 0.15 mL/min on BEH200 15 cm Post UV detection additive: ACN, 0.8% Formic acid
Extracted SpectrumExtracted SpectrumHerceptin 50%ACN, .4% FA_100mm Amm Form_0.15 mL/min_40CV_AutoQua
1007Oct11_PH_SEC_BEH200_Ext_T_ACN_pt8FA_2_5 907 (15.351) Sm (SG, 10x5.00); Sb (15,2.00 ); Cm (891:932) 1: TOF MS ES+
4.34e33448.14043025.98782907.2991
2797.63792601.3782
2471.37262353.8545
3530.13483706.4951
3801.68433901.6064
3905.8140
pp
Intact IgG MW 148 221
m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800
%
0
2353.5356 4118.3599
7Oct11 PH SEC BEH200 Ext T ACN pt8FA 2 5 982 (16.619) Sm (SG, 10x5.00); Cm (969:996) 1: TOF MS ES+
MW 148,221Peak 1
%
100_ _ _ _ _ _ _p _ _ ( ) ( , ); ( )
1.97e3
2652.85842648.5669
2460.41241251.3574
2801.4185
2968.89673154.9690
3370.08893616.4006
ClipMW 100,764Peak 2
100
m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800
0
7Oct11_PH_SEC_BEH200_Ext_T_ACN_pt8FA_2_5 1152 (19.493) Sm (SG, 10x5.00); Cm (1116:1184) 1: TOF MS ES+ 1.22e41537.7053
1489.6832
1478 2386
1643.7091
1702.38311765 3279
Peak 2
%
1478.2386 1765.3279
1985.9363
2056.27692166.3523 2382.8904
2647.5525
Low MW SpeciesPeak 3
©2012 Waters Corporation 34
m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800
0
Deconvoluted molecular weight determined using MaxEnt1
Summary: Waters Summary: Waters ACQUITYACQUITYUPLCUPLC SEC System SolutionSEC System Solutionyy
New SEC column chemistries based on BEH particlesp
True UPLC separation
Application benefits
– Reduced secondary interaction– Reduced secondary interaction
– Improved physical and chemical column lifetime
– Improved column-to-column reproducibility
I d l i– Improved resolution
– Improved throughput
Synergistic combination of UPLC system and column
Higher throughput compared to traditional HPLC
©2012 Waters Corporation 35
MultiMulti--Mode ChromatographyMode ChromatographyAutomated with ACQUITY UPLC HAutomated with ACQUITY UPLC H--Class Bio Class Bio Mouse Ascites FluidMouse Ascites Fluid
0.24
0.26
0.28
0.30
SEC
ACQUITY UPLC BEH200 AU
0.10
0.12
0.14
0.16
0.18
0.20
0.22 ACQUITY UPLC BEH200 4.6x150mm0.5mL/min20mM Na phosphate, pH 6.8 150mM NaCl
0.00
0.02
0.04
0.06
0.08
Minutes0.00 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00
0.070
0.075
0.080
0.085
0.090
0.095
0.100 Cation Exchange
0.032
0.034
0.036
0.038
0.040
0.042
0.044
0.046
0.048
Anion Exchange
Protein-Pak Hi Res SP 4.6x100mm
Protein-Pak Hi Q4.6x100mm0.5mL/min
AU
0 020
0.025
0.030
0.035
0.040
0.045
0.050
0.055
0.060
0.065
AU
0.010
0.012
0.014
0.016
0.018
0.020
0.022
0.024
0.026
0.028
0.030
4.6x100mm0.5mL/min20mM phosphate pH 60-250mM NaCl20mins
20mM TrispH 7.50-250mM NaCl20mins
©2012 Waters Corporation 36
-0.005
0.000
0.005
0.010
0.015
0.020
Minutes0.00 0.50 1.00 1.50 2.00 2.50 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 10.50 11.00 11.50 12.00 12.50 13.00 13.50 14.00
-0.002
0.000
0.002
0.004
0.006
0.008
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 13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00
Interesting Application Notes/postersInteresting Application Notes/posterson IEX and SECon IEX and SEC
IEX Method Development of a Monoclonal Antibody and Its Charge Variants 720003836en on www.waters.comg
Method Development for Size-Exclusion Chromatography of Monoclonal Antibodies and Higher Order Aggregates 720004076en on www waters com720004076en on www.waters.com
Multi-Mode Analytical Separations of Proteins 720003854en on www.waters.com
Improving the Lifetime of UPLC Size-Exclusion Chromatography Columns Using Short Guard Columns 720004034en on www.waters.com
Technology Brief with SEC and IEX guidelines 720004182en on www.waters.com
©2012 Waters Corporation 37
Technology Brief on SEC with MS 720004018en on www.waters.com
Looking for more info ?Looking for more info ?
www.waters.com/biosep
gg
www.waters.com/biopharm
p
©2012 Waters Corporation 38