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Analysis of biomolecules bySEC and Ion-Exchange UPLC
Anders Feldthus Waters Nordic
©2011 Waters Corporation 1
Anders Feldthus, Waters Nordic
Waters Commitment
To develop, commercialize and market columns that when used on Waters ACQUITY UPLC® systems, give the speed, sensitivity, resolution and method reproducibility that has not been previously resolution, and method reproducibility that has not been previously achieved for the characterization of biological macromolecules with traditional HPLC.
©2011 Waters Corporation 2
Agendag
Ion-Exchange Chromatography— Theory and practice Theory and practice — Protein-Pak Hi Res IEX Columns — Method Development Strategies
o Auto•Blend Plus Technology and ACQUITY UPLC H-Class Bio SystemSystem
Size-Exclusion Chromatography— Theory and practice— ACQUITY UPLC for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns — Factors Influencing Component Resolution— Ways to Maximize SEC Column LifeWays to Maximize SEC Column Life
©2011 Waters Corporation 3
Ion-Exchange Chromatographyg g p y
Na +
Cl -Na +Cl -
Anion-Exchange
Na +
Na +Cl -
Cl
Na +
Cl -Na +Cl
Cl -
Anion-Exchangeg
Particle
Cl -Na +
Cl -
Na +
Na +Cl -Cl -
Cl -
gParticle
Elute with Step or Continuous
Na +Na +
Cl -
Binds at Low Ionic StrengthElute with Step or Continuous
Gradients of Increasing Ionic StrengthSeparations are based on net surface charge on protein with oppositely charged groups on ion-exchanger
©2011 Waters Corporation 4
Proteins elute from column using either a gradient of increasing salt concentration (most common) or changing pH (less common)
Ion-Exchange Gradient: Starting pointg p
Sample High Equilibration Injection Gradient Salt Re-Equilibration
~ 5 cv
Volume Wash
unbound proteinsproteins
10-20 cv
5-10 cv 5-10 cv
©2011 Waters Corporation 5
Typical linear gradient for ion-exchange chromatography
Ion-Exchangeg
Advantages Limitations
S ti b h d Moderate resolution
Concentrating technique
Separation by charge and charge distribution; insensitive to other properties
— Can load large volumes of a dilute sample For salt gradients, time-
consuming pH optimization required for best resolution
Non-denaturing protein elution techniques
required for best resolution.
Fractions may need to be desalted prior to the next
Preferred first step when a cost-effective affinity purification is not available
desalted prior to the next purification or characterization step (e.g., Mass Spectrometry)
©2011 Waters Corporation 6
Protein Isoelectric Points and IEX
ParticleSupport
ParticleSupport
©2011 Waters Corporation 7
Strong versus Weak Ion Exchangersg g
Strong and weak DO NOT indicate how tightly the protein is bound
A t h i l i i d hil k h ’ A strong exchanger is always ionized while a weak exchanger’s ionization varies with pH
— Weak Cation Exchange column- pka 3.5-4.5
— Weak Anion Exchange column – pka ~9
Strong exchangers are recommended where there is a need to run at H ( id i )extreme pH (wider operating range)
The ion exchange capacity of a weak ion exchanger varies with pH
— Sample loading (binding) capacity can vary with pH due to loss of charge from the exchanger
©2011 Waters Corporation 8
Selectivity differences exist between weak and strong exchangers— Binding constants are a function of the IEX functional group
Agendag
Ion-Exchange Chromatography— Theory and practice y p
— Protein-Pak Hi Res IEX Columns
— Method Development Strategies
o Auto•Blend Plus Technology and ACQUITY UPLC H-Class Bio gy QSystem
Size-Exclusion Chromatography— Theory and practice
— ACQUITY UPLC for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns — Factors Influencing Component Resolution
©2011 Waters Corporation 9
Protein-Pak Hi Res IEX
©2011 Waters Corporation 10
Attributes of Protein-Pak™ Hi Res IEX 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
©2011 Waters Corporation 11
Batch to Batch Reproducibility:Protein-Pak Hi Res Q Column
0.035
Q
Std Dev %RSD1 - Myoglobin 0.06 1.282 T f i 0 03 0 53
Retention Time
0.025
0.0301
2 3
2 - apo-Transferrrin 0.03 0.533 - Trypsin Inhibitor 0.03 0.27
AU
0.015
0.020 Batch A
0 005
0.010
Batch C
Batch B
0.000
0.005
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00
Batch C
©2011 Waters Corporation 12
Minutes
Reproducible retention time observed for multiple batches
Carryover
0.16
y
1st Repeat Gradient
2nd Repeat Gradient
0.12
0.14
Gradient Gradient
AU
0 06
0.08
0.10
0.02
0.04
0.06
Expected Elution Points
0.00
Minutes0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00 110.00 120.00 130.00 140.00 150.00 160.00
©2011 Waters Corporation 13
No detectable carryover of lysozyme (100 µg injection)
Ion-Exchange Analysis of Antibodies on Weak Cation Exchange Column
AU0.020
0.030
0.040
gA
0.000
0.010
0.020
Humanized IgG1
AU0.010
0 0 0
Humanized IgG2
0.000
U
0.010
0.015
AU
0.000
0.005
Minutes
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
Chimeric IgG1
©2011 Waters Corporation 14
Minutes
Column: Protein-Pak Hi Res CM, 4.6 x 100mm
Confirmation of c-terminal Lysine Variants of mAb
0 00 Untreated mAb
AU 0.002
0.003
0.004 Untreated mAb
KKK
0.000
0.001
0.008
AU
0.002
0.004
0.006 mAB treated with carboxypeptidase B, 30 °C, 20 minutes
0.000
Minutes24.00 26.00 28.00 30.00 32.00 34.00 36.00 38.00 40.00 42.00 44.00 46.00 48.00 50.00 52.00 54.00 56.00 58.00
©2011 Waters Corporation 15
IEX can be used to confirm the presence of mAb lysine variantsColumn: Protein-Pak Hi Res CM, 4.6 x 100mm
Agendag
Ion-Exchange Chromatography— Theory and practice y p
— Protein-Pak Hi Res IEX Columns
— Method Development Strategies
o Auto•Blend Plus Technology and ACQUITY UPLC H-Class Bio gy QSystem
Size-Exclusion Chromatography— Theory and practice
— ACQUITY UPLC for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns — Factors Influencing Component Resolution— Ways to Maximize SEC Column Life
©2011 Waters Corporation 16
Strategies to Developing an Ion-Exchange Protein Separationg p
Retention is optimized by adjustment of ionic strength
Selectivity is most conveniently optimized with pH
Changing buffer and counter ion may improve selectivity
Methods may require adjustment if the temperature is changedMethods may require adjustment if the temperature is changed
©2011 Waters Corporation 17
Affect of Salt Gradient Slope % 24.00
36.00
48.00
p
0-0.15M NaCl
Elutes in high salt wash step1
3
25 Ovalbumin 1
Myoglobin 2
28 00
0.00
12.00
Minutes3.10 6.20 9.30 12.40 15.50 18.60 21.70 24.80
1
3
2
4 Ribonuclease A 3
Cytochrome C 4
Lysozyme 5
%
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
©2011 Waters Corporation 18
Minutes
Higher salt gradients result in earlier elution of bound proteinsHigh salt wash may be needed in shallower gradients to elute tightly bound proteinsColumn: Protein-Pak Hi Res CM 4.6 mm x 100mm
Affect of Salt Gradient Slope: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
©2011 Waters Corporation 19
Minutes
Longer gradient: increased resolution, lower sensitivity
Column: Protein-Pak HI Res Q, 4.6 x 100 mm
Strategies to Developing an Ion-Exchange Protein Separationg p
Retention is optimized by adjustment of ionic strengthRetention is optimized by adjustment of ionic strength
Selectivity is most conveniently optimized with pH
Changing buffer and counter ion may improve selectivity
Methods may require adjustment if the temperature is changed
©2011 Waters Corporation 20
Effect of pH on Selectivity
0.040
0.050
p y
pH 6.6 α-Chymotrypsinogen 1
Ribonuclease A 2
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
©2011 Waters Corporation 21
Column: Protein-Pak Hi Res CM 4.6 x 100 mm column
Strategies to Developing an Ion-Exchange Protein Separationg p
Retention is optimized by adjustment of ionic strength
Selectivity is most conveniently optimized with pH
Changing buffer and counter ion may improve selectivity
Methods may require adjustment if the temperature is changedMethods may require adjustment if the temperature is changed
©2011 Waters Corporation 22
Effect of Buffer on Selectivity
0.025
y
20mM Sodium Phosphate α-Chymotrypsinogen 1
Ribonuclease A 21 2
3
AU
0.010
0.015
0.020 cytochrome c 32
0.000
0.005
20 M MES1
3
AU
0 010
0.015
0.020
0.025 20mM MESMorpholino 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
©2011 Waters Corporation 23
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 Effects
NaClRibonuclease A 1
cytochrome c 21
2
AU 0.010
0.020Aprotinin 3
13
0.000
0.020KCl
AU
0.000
0.010
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
©2011 Waters Corporation 24
Counter ion may change selectivity and retention of proteins
Effects tend to be minimal
Strategies to Developing an Ion-Exchange Protein Separationg p
Retention is optimized by adjustment of ionic strength
Selectivity is most conveniently optimized with pH
Changing buffer and counter ion may improve selectivity
Methods may require adjustment if the temperature is changedMethods may require adjustment if the temperature is changed
©2011 Waters Corporation 25
Temperature Effectsp
45 °C
AU 0.005
40 °C
0.000
AU 0.005
35 °C
0.000
AU 0.005
30 °C
A
0.000
U 0 005 KKK
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
©2011 Waters Corporation 26
Temperature may effect selectivity and retentionChanges may be similar to those observed with pH changeColumn: ProteinP-ak Hi Res CM 4.6 x 100 mm column
Waters Auto•Blend Plus™ Technologygy
12.5% 100mM NaH 2PO 4
12.5% 100mM Na 2HPO 4
25mM Sodium Phosphate, 150mM NaCl pH 6.8
15% 1000 mM NaCl
60% H20NaCl
©2011 Waters Corporation 27
Auto•Blend Plus™ Technology
Enter Buffer
Enter Buffer System
Concentration
Enter pH andSalt Gradient
©2011 Waters Corporation 28
Salt Gradient
Effect of pH on Selectivity pH 6
0.020
1 2
p y
pH 6 0
Ribonuclease A 1
cytochrome c 2
3
AU
0.000
0.010
1 3
20.020
pH 6.0Aprotinin 3
2
1 32
AU
0.000
0.010 pH 6.2
2
1 3
AU
0.000
0.010
0.020
pH 6.4
1 3
2
AU
0.000
0.010
0.020
pH 7.2
©2011 Waters Corporation 29
Minutes10.00 15.00 20.00 25.00 30.00 35.00
pH can be used to alter selectivity of proteinsColumn: Protein-Pak Hi Res CM 4.6 x 100 mm column
AutoBlend Plus Buffer Management forProtein Ion Exchange Method Developmentg p
Fluid Path of ACQUITY UPLC H-Class Bio System configured for multi-buffer use
— The optional solvent select valve provides 6 selectable choices that are associated with particular method names.
— The proportions of the four selected reservoirs are calculated with Auto•Blend Plus™ Technology to meet the requirements of the method using the defined buffer system specified with the method
©2011 Waters Corporation 30
meet the requirements of the method, using the defined buffer system specified with the method.
— The four physical reservoirs, labeled A, B, C, and D1-D6 can be assigned as needed as the Acid, Base, Salt , and Aqueous components.
Enhanced Method Development viaAutoBlend Plus and Automated Column ManagementManagement
d l f h
©2011 Waters Corporation 31
Automated Column Management for Protein Ion Exchange.
The method specifies the column to be used and the bypass (B) permits matching the buffer to the column.
Use of AutoBlend Plus and Automated Column Switching for mAb Separation Method Scouting
0.055
0.060
i k i
AU
0.020
0.025
0.030
0.035
0.040
0.045
0.050 Protein-Pak™ HiRes Q
0.000
0.005
0.010
0.015
0.050
0.055
0.060
AU
0.015
0.020
0.025
0.030
0.035
0.040
0.045
Protein-Pak™ HiRes SP
0.000
0.005
0.010
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 25.00 26.00 27.00 28.00
©2011 Waters Corporation 32
IEX Summaryy
Total system solution with combination of ACQUITY UPLC H Class Bio System and column
— Auto•Blend Plus™ Technology allows for change of pH and ionic strengths without preparation of different buffers
— Simplifies methods developmentp p
Protein-Pak Hi Res IEX column benefits
— Consistent batch-to-batch performance (tested with protein — Consistent batch-to-batch performance (tested with protein standards)
— Minimal column related carryover
Stable over a wide pH range— Stable over a wide pH range
©2011 Waters Corporation 33
Agendag
Ion-Exchange Chromatography— Theory and practice y p
— Protein-Pak Hi Res IEX Columns
— Method Development Strategies
o Auto•Blend Plus Technology and ACQUITY UPLC H-Class Bio gy QSystem
Size-Exclusion Chromatography— Theory and practice
— ACQUITY UPLC for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns — Factors Influencing Component Resolution— Ways to Maximize SEC Column Life
©2011 Waters Corporation 34
Principles of Size ExclusionChromatography of Proteins
Separates proteins by their size in solution (Stokes radius)
Separations are Isocratic
Tends to be used as a “Polishing” isolation step or as an analytical technique to determine presence of protein aggregates
Generally a “lower resolving” technique compared to other methods such as ion-exchange or reversed-phase methods
©2011 Waters Corporation 35
Size Exclusion Separation of Proteinsp
©2011 Waters Corporation 36
Common 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 pressure—Increased pressure
—Changes in resolution
Tailing of specific proteins
Resolution
Throughput
©2011 Waters Corporation 37
Agendag
Ion-Exchange Chromatography— Theory and practice y p
— Protein-Pak Hi Res IEX Columns
— Method Development Strategies
o Auto•Blend Plus Technology and ACQUITY UPLC H-Class Bio gy QSystem
Size-Exclusion Chromatography— Theory and practice
— ACQUITY UPLC for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns— Factors Influencing Component Resolution— Ways to Maximize SEC Column Life
©2011 Waters Corporation 38
Advantages of UPLC Technologyfor SEC Separations
Requires Columns and Instrumentation to Minimize Band Spreading
Waters UPLC®HPLC
p
Narrow PeakIncreased Sensitivity
Waters UPLC®
TechnologyBroad BandBroad PeakLess Sensitivity
HPLC
Increased Resolving Power
Less SensitivityLess Resolving Power
©2011 Waters Corporation 39
Effect of LC System Dispersion onBEH200 SEC mAb Separationp
Larger system dispersion decreases component resolution
0.20
0.30 BEH200 SEC 1.7um Column (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)
©2011 Waters Corporation 40
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
UPLC-SEC vs HPLC-SECof mAb monomer and aggregatesgg 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
©2011 Waters Corporation 41
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
ACQUITY BEH200 SEC 1.7 µm ColumnsQ µ
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.
©2011 Waters Corporation 42
BEH Overview
The packing material is based on our patented Bridged Ethyl H b id b ti l d ff ti di l b di hi h Hybrid base particle and effective diol bonding, which provide a stable chemistry with minimal secondary interactions.
©2011 Waters Corporation 43
BEH200 SEC, 1.7um Column Batch Test
0 20
0.22
Analyte pI MW
1. Thyroglobulin, 3 mg/mL 4.6 669,000
2. IgG, 2 mg/mL (Vicam) 6.7 150,000
0.16
0.18
0.20
2
4
2. IgG, 2 mg/mL (Vicam) 6.7 150,000
3. BSA, 5 mg/mL 4.6 66,400
4. Myoglobin, 2 mg/mL 6.8, 7.2 17,000
5. Uracil, 0.1 mg/mL N/A 112
AU
0.12
0.14 35
0.06
0.08
0.10
1
0.02
0.04
©2011 Waters Corporation 44
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 Calibration BEH200 SEC, 1.7um Calibration CurveCurve
10000000
Analyte MWBlue Dextran 2,000,000Thyroglobulin 669,000β Amylase 200 000
100000
1000000IgG (~150,000 Da)
β-Amylase 200,000IgG, 150,000Amyloglucosidase 97000Conalbumin 75000BSA 66,400Ovalbumin 44 000
10000
100000
MW
Ovalbumin 44,000Carbonic Anhydrase 29,000Myoglobin 17,000Lysozyme 14,400Ribonuclease A 13,700Aprotinin 6 500
100
1000
Aprotinin 6,500Uracil 112
10
100
1 1.5 2 2.5 3 3.5 4 4.5 5
©2011 Waters Corporation 45
Elution Volume
Protein Adsorption and SizeProtein Adsorption and Size--Exclusion Exclusion ChromatographyChromatographyg p yg p y
Proteins can interact or adsorb onto the SEC packing material
These interactions create undesired and unpredictable retention of proteins (i.e. proteins not separated by size in p ( p p ysolution)
SEC particles frequently coated with a hydrophilic reagent toSEC particles frequently coated with a hydrophilic reagent tominimize non-desired ionic interactions between proteins and packing material
Mobile phase additives (e.g., 150mM NaCl) may decrease non-desired ionic interactions between proteins and packing
l
©2011 Waters Corporation 46
material
DIOL coating used to minimizenon-desired ionic interactionsbetween proteins and packing material
Ionic interactions between available negatively charged silanols on SEC particles and positive charges on proteins
p p g
SEC
-
SEC Particle
©2011 Waters Corporation 47
DIOL Loss increases non-desired ionic interactions between proteins and packing material packing material
SEC Particle
-
Particle
©2011 Waters Corporation 48
Comparative SEC Column Life
0.60
0.70
HPLC 100% Silica-DiolLysozyme, pKi = 10.7
AU
0 20
0.30
0.40
0.50 SEC 250Å 4µm4.6 x 300 mm
Injection 19
Suggestive of DIOL Bleed
0.00
0.10
0.20
Minutes5.50 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 14.50 15.00
0.22
jInjection 618
AU
0.10
0.12
0.14
0.16
0.18
0.20
ACQUITY BEH200 SEC, 1.7 µm4.6 x 150 mm
I j ti 19
0.00
0.02
0.04
0.06
0.08
0 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
Injection 19Injection 618
©2011 Waters Corporation 49
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 shows minimal secondary interactions even after 600 injections
NaCl will decrease ionic interactions between proteins and packing material
Ionic interactions between available negatively charged silanols on SEC particles and positive charges on proteins
p p g
Na +
Cl -
Na + Cl -
Na +
Na +
Cl - Cl -
SEC Na +
Na +
Na +
-
ParticleNa + Cl -
Na +
Na +
Na +
Cl
Cl -Cl -
Na +
©2011 Waters Corporation 50
Cl -
Na + Cl -Cl -
Influence of Ionic Strength on Peak Shape and Retention on Silica-based SEC
0 06
0.08
Conventional 100% Silica-Diol CoatedSEC Column 4.6 x 300 mm 10mM
N Cl
and Retention on Silica based SECA
U
0.00
0.02
0.04
0.06
Minutes10.00 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 38.00 40.00 42.00 44.00 46.00 48.00 50.00
NaClLysozyme, pKi = 10.7
Minutes
AU
0.04
0.06
0.08 25mMNaCl
lysozyme
0.00
0.02
Minutes10.00 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 38.00 40.00 42.00 44.00 46.00 48.00 50.00
0 08
lysozyme
AU
0 00
0.02
0.04
0.06
0.08
100mMNaCllysozyme
©2011 Waters Corporation 51
0.00
Minutes10.00 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 38.00 40.00 42.00 44.00 46.00 48.00 50.00
Flow rate: 0.5 mL/min; Mobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8
Influence of Ionic Strength on Peak Shape and Retention on BEH200 SEC
AU
0 12
0.18
0.24
0 12
0.18
0.24 ACQUITY BEH200 SEC 1.7 µm Column, 4.6 x 150mm
10mMN Cl
and Retention on BEH200 SEC A
0.00
0.06
0.12
Minutes5.00 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 14.50 15.00
0.00
0.06
0.12
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 14.50 15.00
NaClLysozyme, pKi = 10.7
AU
0.12
0.18
0.24
0.12
0.18
0.24
25mMNaCl
0.00
0.06
Minutes5.00 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 14.50 15.00
0.00
0.06
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 14.50 15.00
0.240.24
AU
0.06
0.12
0.18
0.06
0.12
0.18 100mMNaCl
©2011 Waters Corporation 52Flow rate: 0.5 mL/min; Mobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8
0.00
Minutes5.00 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 14.50 15.00
0.005.00 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 14.50 15.00
Agendag
Ion-Exchange Chromatography— Theory and practice y p
— Protein-Pak Hi Res IEX Columns
— Method Development Strategies
o Auto•Blend Plus Technology and ACQUITY UPLC H-Class Bio gy QSystem
Size-Exclusion Chromatography— Theory and practice
— ACQUITY UPLC for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns — Factors Influencing Component Resolution— Ways to Maximize SEC Column Life
©2011 Waters Corporation 53
Factors Influencing Resolutiong
Resolution decreases with increasing volume and mass l dloads
Resolution decreases with increasing flow rateId l fl i l h i ll i h —Ideal flow rate is lower than typically running, however run time will be longer
l h l l hResolution increases with column length
Baseline resolution typically achieved at 50%-100% molecular weight difference—Proteins with less than 100% molecular weight difference
may not be acceptably resolved
©2011 Waters Corporation 54
Mass Loading Capacity onBEH200 SEC, 1.7um 4.6 x 150mm,
3.125 µg Total LoadRs = 1.7
400 µg Total LoadRs = 1.5
0 010
0.012
0 60
0.70
BSA
BSA
0.012 0.70
AU 0.006
0.008
0.010
AU
0.40
0.50
0.60
BSA dimer BSA dimer
0.002
0.004
0.10
0.20
0.30BSA dimer
0.000
Minutes1.00 2.00 3.00 4.00 5.00 6.00
0.00
Minutes1.00 2.00 3.00 4.00 5.00 6.00
©2011 Waters Corporation 55
Constant injection volume, 20 µL Resolution decreases for mass loads of 3- 400 µg
Volume Load Capacity onBEH200 SEC, 1.7um 4.6 x 150mm
1.60
,
Injection Volume
USP Resolution (Monomer-Dimer)
1.00
1.20
1.40 5 1.70
10 1.54
15 1.43
20 µL
AU
0.60
0.8020 1.27
5 µL
0 00
0.20
0.405 µL
0.00
Minutes1.80 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
Constant BSA concentration, 20 mg/mL
©2011 Waters Corporation 56
Resolution decreases for higher injection volumePoor peak shape observed at higher volumes
Effect of Column Length on mAb Rsg
0.015 BEH200, SEC, 1.7um4 6 x 150 mm
20.1% Aggregate
AU
0.005
0.0104.6 x 150 mm
USP Res= 1.44
0.000
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
0.020
BEH200 SEC 1 719 9%
AU
0 005
0.010
0.015BEH200, SEC, 1.7um4.6 x 300 mm
19.9% Aggregate
USP Res= 1.72
0.000
0.005
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
©2011 Waters Corporation 57
Comparable aggregate quantitation Human IgG (Sigma), (load: 10 µg - 150mm; 20 µg -300mm): TUV: 280 nm
Effect of Flow Rate on mAb Rs
AU 0.010
0.020
0.030
0.4 mL/min BEH200, SEC, 1.7um4.6 x 150 mm
0.000
Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
0.030
AU 0.010
0.020
0.030
AU 0.010
0.020
0.030
AU
0.000
0.010
0.0200.35 mL/min
0.000
Minutes2.00 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
0.000
Minutes2.00 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
0 020
0.030
Minutes2.00 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
AU
0.000
0.010
0.020
Mi t3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
0.2 mL/min
©2011 Waters Corporation 58
Triplicate injectionsNo observable trend in aggregation with flow rate
Minutes
Effect of Temperature onmAb SEC Peak Shapep
45 °CBEH200, SEC, 1.7um4 6 x 150 mm
0.63
0.70
0.7740 °C
4.6 x 150 mm
AU
0.35
0.42
0.49
0.56
30 °C
0.14
0.21
0.28
0.00
0.07
2.10 2.45 2.80 3.15 3.50
©2011 Waters Corporation 59
Chimeric IgG 0.4 mL/min, 25 mM Sodium Phosphate, 0.15m NaCl, pH 6.8
Effect of Salt Cation on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shapep
Different cations of chloride salt additive
©2011 Waters Corporation 60
Buffer: 10mM sodium phosphate, pH 6.8 and 200mM of additive Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil
Agendag
Ion-Exchange Chromatography— Theory and practice y p
— Protein-Pak Hi Res IEX Columns
— Method Development Strategies
o Auto•Blend Plus Technology and ACQUITY UPLC H-Class Bio gy QSystem
Size-Exclusion Chromatography— Theory and practice
— ACQUITY UPLC for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns — Factors Influencing Component Resolution— Ways to Maximize SEC Column Life
©2011 Waters Corporation 61
BEH200 SEC, 1.7um Care and Use:(Ways to extend column life)( y )
Preparation of SEC Mobile Phase and Needle Wash— Pre filter through 0 2 or 0 45 um filter (i e Don’t inject particulates)— Pre filter through 0.2 or 0.45 um filter (i.e, Don t inject particulates)— Use high purity water— Replace mobile phases weekly and do not “top off”
Use of BEH200 SEC, 1.7um Guard Column
Attention to SEC Eluent SinkersAttention to SEC Eluent Sinkers— Use titanium sinkers NOT stainless steel— Sinkers 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 - 20% of the
©2011 Waters Corporation 62
g y pmaximum recommended flow rate- Extended: Store in the HPLC grade water with 20% methanol
BEH200 SEC, 1.7um Guard E(4.6 x 30mm) Extends UPLC SEC Column Life
©2011 Waters Corporation 63
QC Protein Standards Mix onBacterial Contaminated, BEH200 SEC Column
BEH200, SEC, 1.7um4 6 x 150 mm4.6 x 150 mm
©2011 Waters Corporation 64
Summary:Waters ACQUITY UPLC SEC System SolutionQ y
New SEC column chemistry based on BEH particles
T UPLC tiTrue UPLC separation
Application benefits
— Reduced secondary interaction
— Improved physical and chemical column lifetime
— Improved column-to-column reproducibility
— Improved resolutionp
— Improved throughput
Synergistic combination of UPLC system and column
Higher throughput compared to traditional HPLC Higher throughput compared to traditional HPLC
©2011 Waters Corporation 65