tips and tricks' for biopharmaceutical characterization ... · ch. 2. ch. 2 o. si eto oet si o...
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©2012 Waters Corporation 1
'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical Characterization using SECCharacterization using SEC
Waters CorporationWaters Corporation
©2012 Waters Corporation 2
Waters Commitment
To develop, commercialize and market columns that, when used on Waters ACQUITY UPLC® systems, provide speed, sensitivity, resolution, and reproducibility not previously achieved for characterization of biological macromolecules by traditional HPLC.
©2012 Waters Corporation 3
Liquid Chromatography Protein Separation Modes
Protein Structure
Primary, Secondary, Tertiary Structure
Carbohydrate Groups
Hydrophobic Regions
Disulfide Linkages
HydrophilicGroups
Aromatic Groups
HydrogenBonding
Net Charge
©2012 Waters Corporation 4
Liquid Chromatography Protein Separation Modes
Protein Structure
Primary, Secondary, Tertiary Structure
Carbohydrate Groups
Hydrophobic Regions
Disulfide Linkages
HydrophilicGroups
Aromatic Groups
HydrogenBonding
Net Charge
©2012 Waters Corporation 5
Agenda
Size-Exclusion Chromatography– Theory and practice– ACQUITY UPLC Columns for SEC
o ACQUITY BEH450 SEC, 2.5 µm Columnso ACQUITY BEH200 SEC, 1.7 µm Columnso ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Applicationo SEC-MS Applications
– Insulin and Small Protein Applications– Factors Influencing Component Resolution– Considerations to extending column life
©2012 Waters Corporation 6
Principles of Size Exclusion Chromatography Principles of Size Exclusion Chromatography of Proteinsof 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
©2012 Waters Corporation 7
Size Exclusion Chromatography
No adsorption to surface of particles
Large molecules elute before small molecules
Large molecules cannot access pores
Small molecules access pores within particle
dimermonomer
©2012 Waters Corporation 8
Monoclonal Antibodies
Antibody Conjugates
Fc Fusion Proteins
Synthetic Oligonucleotides
Protein Subunit Vaccines
Recombinant Proteins
Common SEC applications: Biotherapeutics Types
©2012 Waters Corporation 9
Agenda
Size-Exclusion Chromatography– Theory and practice– ACQUITY UPLC Columns for SEC
o ACQUITY BEH450 SEC, 2.5 µm Columnso ACQUITY BEH200 SEC, 1.7 µm Columnso ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Applicationo SEC-MS Applications
– Insulin and Small Protein Applications– Factors Influencing Component Resolution– Considerations to extending column life
©2012 Waters Corporation 10
UPLC Systems for Biopharmaceutical Analysis
Wide range of applications
Complete Solutions– Instrumentation
o UPLC System (s)• ACQUITY UPLC System• ACQUITY UPLC H-Class System• ACQUITY UPLC H-Class Bio System
o UV, FLR, PDA and MS Detectionso Application Specific Chemistries
• Developed and designed with applications• QC Tested with application• Optimized for UPLC
o Software • Data Analysis• Information management
Focused on customer application requirements
©2012 Waters Corporation 11
Columns and Instruments both Minimize Band Spreading
Broad BandBroad PeakLess SensitivityLess Resolving Power
HPLC
Advantages of UPLC Technology for SEC Separations
Narrow PeakIncreased SensitivityIncreased Resolving Power
Waters UPLC®
Technology
©2012 Waters Corporation 12
Effect of System Dispersion on ACQUITY UPLC BEH200 SEC 1.7 µm separation
Large system dispersion decreases resolution
Sample: Human polycolonal IgG
ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm
UP-SEC
ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm
HP-SEC
USP Res= 1.37
USP Res= 2.37
AU
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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
©2012 Waters Corporation 13
ACQUITY UPLC BEH200 and BEH125 SEC 1.7 µm Columns, and BEH450 SEC 2.5 µm Columns
Application Areas– Molecular weight ranges dependent on pore size:
o BEH450: 100,000 to 1,500,000 Daltonso BEH200: 10,000 to 450,000 Daltonso BEH125: 1,000 to 80,000 Daltons
– Determination of protein / peptide molecular weight
– Quantifying protein / peptide aggregates primarily in therapeutic monoclonal antibodies, EPO, and Insulin
– Determination of size heterogeneity in a sample
©2012 Waters Corporation 14
BEHBEH™™ Technology ParticlesTechnology Particles Bridged EthylSiloxane/Silica HybridBridged EthylSiloxane/Silica Hybrid
Bridged EthanesIn Silica Matrix
Polyethoxysilane(BPEOS)
Si
EtO
O
CH2 CH2
Si O
Si
EtO
OEt
Si O
O
OEtO
Si
O
Si
OEt
O
OOEt
Et
Et
n
+ Si
EtOEtO
CH2EtO
CH2Si
OEt
OEtOEt
Tetraethoxysilane(TEOS)
Bis(triethoxysilyl)ethane(BTEE)
4 Si
EtO
EtO OEtEtO
1
Anal. Chem. 2003, 75, 6781-6788
U.S. Patent No. 6,686,035 B2
©2012 Waters Corporation 15
HPLC to UPLC SEC Comparison
Murine monoclonal antibody - Scaled load Conditions: 0.4 mL/min; 25mM Sodium Phosphate, pH 6.8, 0.15 M NaCl
AU
0.000
0.005
0.010
0.015
0.020
0.025
0.030
0.035
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0.045
0.050
0.055
0.060
0.065
0.070
Minutes2.00 4.00 6.00 8.00 10.00
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
2.26 % Aggregate 2.24 %
Aggregate
HPLC 100% Silica-Diol
SEC 250Å 5µm7.8 x 300 mm
ACQUITY UPLC BEH200 SEC,1.7
µm4.6 x 300mm
8.00 30.008.00 30.00
©2012 Waters Corporation 16
Calibration Curves of ACQUITY UPLC BEH450, BEH200, and BEH125 SEC Columns
©2012 Waters Corporation 17
Protein Adsorption and SizeProtein Adsorption and Size-- Exclusion ChromatographyExclusion Chromatography
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 solution)
SEC particles frequently coated with a hydrophilic reagent to minimize 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 material
©2012 Waters Corporation 18
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 19
Lysozyme, pKi = 10.7
Suggestive of DIOL Bleed
AU
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0.70
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
HPLC 100% Silica-DiolSEC 250Å 4µm4.6 x 300 mm
Injection 19Injection 618
Comparative SEC Column Life
Suggestive of DIOL Bleed
Lysozyme, pI = 10.7
©2012 Waters Corporation 20
BEH200 shows minimal secondary interactions even after 600 injections
AU
0.00
0.02
0.04
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0.12
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0.20
0.22
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
ACQUITY BEH200 SEC, 1.7 µm4.6 x 150 mm
Injection 19Injection 618
Lysozyme, pKi = 10.7
Suggestive of DIOL BleedAU
0.00
0.10
0.20
0.30
0.40
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0.60
0.70
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
HPLC 100% Silica-DiolSEC 250Å 4µm4.6 x 300 mm
Injection 19Injection 618
Comparative SEC Column Life
Suggestive of DIOL Bleed
Lysozyme, pI = 10.7
©2012 Waters Corporation 21
Column StabilityColumn Stability
Calibration Curve
100
10000
1000000
0.5 1 1.5 2 2.5 3
Elution volume (mL)
Log
Mw
0 hours12 hours24 hours36 hours48 hours60 hours
Protein MWThyroglobulin 669000Ferritin 440000Aldolase 150000BSA 66000Ovalbumin 44000Carbonic Anhydrase 29000Ribonuclease A 13700Aprotinin 6500Uracil 112
Protein standard analyzed over 48 hours
Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm
Elution volume for all proteins within 0.2% RSD
©2012 Waters Corporation 22
AU
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0.04
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0.08
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
AU
0.00
0.02
0.04
0.06
0.08
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
AU
0.00
0.02
0.04
0.06
0.08
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
Influence of Ionic Strength on Peak Shape and Retention
Conventional 100% Silica-Diol Coated SEC Column 4.6 x 300 mm
Flow rate: 0.5 mL/min; Mobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8
10 mM
25 mM
100 mM
lysozyme
lysozyme
lysozyme
©2012 Waters Corporation 23
Influence of Ionic Strength on Peak Shape and Retention
AU
0.00
0.06
0.12
0.18
0.24
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
0.18
0.24
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
AU
0.00
0.06
0.12
0.18
0.24
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
0.18
0.24
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
ACQUITY BEH200 SEC 1.7 µm column, 4.6 x 150mm
Flow rate: 0.5 mL/min; Mobile phase: 10, 25 or 100 mM sodium phosphate, pH 6.8
10 mM
25 mM
AU
0.00
0.06
0.12
0.18
0.24
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
0.18
0.24
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
100 mM
lysozyme
©2012 Waters Corporation 24
BEHBEH125 SEC, 1.7um BatchBatch--toto--Batch Batch ReproducibilityReproducibility
AU
0.00
0.03
0.06
0.09
Minutes 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
1
3
24
AU
0.00
0.03
0.06
0.09
Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
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0.00
0.03
0.06
0.09
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Batch 1
Batch 2
Batch 3
Conditions: 100mM Sodium Phosphate pH 6.8; 0.3 mL/min; 30°C; 4.6x150mm
Analyte pl MW
1. Thyroglobulin, 0.1 mg/mL 4.6 669,000
2. Ovalbumin, 0.3 mg/mL 4.5 44,200
3. Ribonuclease A, 0.3 mg/mL 9.6 13,700
4. Uracil, 0.05 mg/mL N/A 112
BEH125 SEC Protein StandardPart No. 186006519
©2012 Waters Corporation 25
BEHBEH200 SEC, 1.7um BatchBatch--toto--Batch Batch ReproducibilityReproducibility
BEH200 SEC Protein StandardPart No. 186006518
©2012 Waters Corporation 26
AU
0.00
0.05
0.10
AU
0.00
0.05
0.10
AU
0.00
0.02
0.04
0.06
0.08
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00
21
3 4 5
6
ACQUITY BEH200 SEC(300mm)
ACQUITY BEH200 SEC and BEH450 SEC(150mm + 150mm)
ACQUITY BEH450 SEC(300mm)
Compounds: 1. Thyroglobulin Dimer (1,340 KDa), 2. Thyroglobulin (667 KDa), 3. IgG (150 KDa), 4. BSA (66 KDa), 5. Myoglobin (17 KDa), 6. Uracil (112 Da)
2
1
3 45
6
2
1
3 4 5
6
Combining Pore Sizes for Added Method Development Flexibility
BEH450 SEC Protein StandardPart No. 186006842
©2012 Waters Corporation 27
Column ReproducibilityColumn ReproducibilityAU
0.00
0.05
AU
0.00
0.05
AU
0.00
0.05
AU
0.00
0.05
AU
0.00
0.05
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Batch 1, Column 1
Batch 1, Column 2
Batch 1, Column 3
Batch 2, Column 1
Batch 2, Column 2
Humanized monoclonal antibody
Conditions: 25mM Sodium Phosphate, pH 6.8, 0.15 M Sodium Chloride, 0.4 mL/min
Retention times within 0.2min
©2012 Waters Corporation 28
Agenda
Size-Exclusion Chromatography– Theory and practice– ACQUITY UPLC Columns for SEC
o ACQUITY BEH450 SEC, 2.5 µm Columnso ACQUITY BEH200 SEC, 1.7 µm Columnso ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Applicationo SEC-MS Applications
– Insulin and Small Protein Applications– Factors Influencing Component Resolution– Considerations to extending column life
©2012 Waters Corporation 29
Monoclonal Antibody
Adapted from Alain BeckCenter of Immunology
©2012 Waters Corporation 30
Protein Structure
Addressing particulate and aggregation issues of therapeutic protein products, Shi, L, PEGS, May 2011
NNative
UUnfolded
orI
Intermediate
SolubleAggregates Insoluble
Aggregates
Soluble aggregates and insoluble particles may affect immunogenicity and efficacy of biotherapeutic
©2012 Waters Corporation 31
Orthogonal Techniques for Characterization
E. Freud, PDA Visual Inspection Forum, Oct- 2009
nm µm mm cm10 10 10100 100 100
monomersoligomers
subvisible particles Visible particles
Aggregates Particles
SEC
Static Light Scattering
FFF-MALS
AUC
Dynamic Light Scattering
MicroscopeVisual Inspection
Flow Imaging Microscopy
Counter principle
Light Microscopy
©2012 Waters Corporation 32
AU
-0.005
0.000
0.005
0.010
0.015
AU
-0.005
0.000
0.005
0.010
0.015
Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50
Column LifetimeColumn Lifetime
Humanized monoclonal antibody
Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm
Column: 4.6 x 300 mm
Injection 2
Injection 497
Dimer = 0.46%USP Res = 2.35
mAb
mAb
Dimer = 0.49%USP Res = 2.27
©2012 Waters Corporation 33
AU
0.000
0.010
0.020
0.030
Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00
AU
0.000
0.010
0.020
0.030
Minutes3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
AU
0.000
0.010
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
Effect of Flow RateEffect of Flow Rate
Triplicate injections overlaidNo observable trend in aggregation with flow rate Murine monoclonal antibody (63 µg load)
0.4 mL/min
0.35 mL/min
0.2 mL/min
Flow Rate (mL/min) 0.2 0.35 0.4Average 2.87 2.83 2.79Std Dev 0.04 0.02 0.02% RSD 1.45 0.70 0.57
% Aggregate
©2012 Waters Corporation 34
Effect of Particle Size:Effect of Particle Size: Analysis of LMW SpeciesAnalysis of LMW Species
AU
0.000
0.010
AU
0.000
0.010
AU
0.000
0.010
AU
0.000
0.010
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00
250Å, 4µmHPLC 100% Silica-Diol
BEH200 SEC 1.7µm
300Å, 5µmHPLC 100% Silica-Diol
290Å, 5µm HPLC 100% Silica-Diol
LMW peakmAb mAb dimer
LMW species
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 35
Agenda
Size-Exclusion Chromatography– Theory and practice– ACQUITY UPLC Columns for SEC
o ACQUITY BEH450 SEC, 2.5 µm Columnso ACQUITY BEH200 SEC, 1.7 µm Columnso ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Applicationo SEC-MS Applications
– Insulin and Small Protein Applications– Factors Influencing Component Resolution– Considerations to extending column life
©2012 Waters Corporation 36
Precautions in SECPrecautions in SEC--MSMS
Protein structure in solution depends on– pH– Ionic strength– Buffer and salt– Additives
Good ionization conditions are different from conditions for biological activity
Validation required when buffer is changed
Special uses are valuable– Fast desalting– Clips
©2012 Waters Corporation 37
AU
0.00
0.10
0.20
AU
0.00
0.10
0.20
0.30
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
LC/MS Compatible Mobile Phase on ACQUITY UPLC BEH200, SEC, 1.7um
Similar retention time/ peak shape observed with MS compatible mobile phases
100mM Ammonium Formate
PBS
100mM Ammonium Formate
PBS
©2012 Waters Corporation 38
AU
-0.005
0.000
0.005
0.010
0.015
0.020
0.025
AU
-0.005
0.000
0.005
0.010
0.015
0.020
Minutes4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
Time14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
AU
0.0
2.5e-3
5.0e-3
7.5e-3
1.0e-2
1.25e-2
1.5e-2
1.75e-2 26.00
17.05
20.02
Humanized Monoclonal Antibody: MS Compatible/Native Mobile Phase
Flow Rates: 100mM Ammonium Formate - 0.15mL/min, PBS- 0.4 mL/min
Lower flow rate for MS compatibility
100mM Ammonium Formate
PBS
©2012 Waters Corporation 39
SEC-MS Humanized Monoclonal Antibody
MS: Xevo G2 Q Tof
Conditions: 100mM Ammonium Formate, Flow rate: 0.15 mL/min
Post UV detection additive: ACN, 0.8% Formic acid
UV @ 280
TIC
Scan500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
%
4
500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700
AU
0.0
2.0e-3
4.0e-3
6.0e-3
8.0e-3
1.0e-2
1.2e-2
1.4e-2
1.6e-2
1.8e-2
2: Diode Array 280 0.0500Da
Range: 6.757e-1
13.22
25.27
16.58
19.50
1: TOF MS ES+ TIC
7.58e619.49
15.35
16.62
23.7425.06
1 2
3
1
2
3
©2012 Waters Corporation 40
Herceptin 50%ACN, .4% FA_100mm Amm Form_0.15 mL/min_40CV_AutoQua
m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800
%
0
100
m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800
%
0
100
m/z1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000 4200 4400 4600 4800
%
0
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
2353.5356
3530.13483706.4951
3801.68433901.6064
3905.8140
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+ 1.97e3
2652.85842648.5669
2460.41241251.3574
2801.4185
2968.89673154.9690
3370.08893616.4006
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
1985.9363
2056.27692166.3523 2382.8904
2647.5525
Extracted Spectrum
Deconvoluted molecular weight determined using MaxEnt1
Intact IgG MW 148,221Peak 1
ClipMW 100,764Peak 2
Low MW SpeciesPeak 3
©2012 Waters Corporation 41
SECSEC--UVUV--MSMS: : A generic methodology for A generic methodology for screening intact and reduced antibodiesscreening intact and reduced antibodies
Desalting LC/HC Resolution Detect Clips• No Sample Concentration required
HCMass Spectrum LC
Mass Spectrum
LC
HC
HC-HC
LC
HC-HC
HC
UV280
TIC
Conditions: System, ACQUITY UPLCTM with TUV optical detector and Synapt G2 QTof MSFlow Rate: 0.2 ml/min 0.1%TFA and 0.1%FA in 30% ACN
©2012 Waters Corporation 42
Agenda
Size-Exclusion Chromatography– Theory and practice– ACQUITY UPLC Columns for SEC
o ACQUITY BEH450 SEC, 2.5 µm Columnso ACQUITY BEH200 SEC, 1.7 µm Columnso ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Applicationo SEC-MS Applications
– Insulin and Small Protein Applications– Factors Influencing Component Resolution– Considerations to extending column life
©2012 Waters Corporation 43
- -
-
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
BSA
(66,
000)
Ova
lbum
in(4
4,00
0)Ca
rb. An
hyd .
(29
,000
)
Myo
glob
in(1
6,90
0)
Ubi
quiti
n(8
,565
)RA
SG (
898)
G-G
-G
(189
)G
-G (
132)
Ura
cil (
112)
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
ACQUITY UPLC BEH125 SEC 1.7um4.6 x 300mm
BioSuite125 UHR SEC 4.6 x 300mm
BSA
(66,
000)
Ova
lbum
in(4
4,00
0)Ca
rb. An
hyd .
(29
,000
)
Myo
glob
in(1
6,90
0)
Ubi
quiti
n(8
,565
)RA
SG (
898)
G-G
-G
(189
)G
-G (
132)
Ura
cil (
112)
A2
14
Resolution of Proteins and Peptides (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 44
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
G-GG-G
-GRASG
Aprotin
inUbiq
uitin
Cytoch
rome C
Myoglo
binCarb
. Anh
yd.
Ovalbu
min
BSABSA D
imer
Insuli
n Mon
omer
Insuli
n Dim
er
Peptide/Protein
%R
SD/R
eten
tion
Tim
e R
ange
% RSD Retention Time Range
ACQUITY UPLC BEH125 SEC 1.7µm Column Reproducibility
Table 1. Retention time reproducibility for 5 ACQUITY UPLC BEH125 SEC 1.7 µm columns (4.6 mm x 30cm) using aqueous and organic (insulin separation method only) mobile phases.
©2012 Waters Corporation 45
-
-
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
BioSuite 125 4µm UHR 4.6 x 300 mm column
ACQUITY UPLC BEH125 SEC 1.7µm 4.6 x 300 mm column
USP Rs= 1.94
USP Rs= 3.29
Resolution of Small Protein
Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min, sample 2 mg/mL
USP monomer/aggregate resolution was 1.7 times greater on the BEH125 1.7µm SEC column as compared to 4 µm pore diol-coated silica column.
©2012 Waters Corporation 46
.
AU
0.000
0.001
0.002
0.003
0.004
0.005
0.006
0.007
0.008
0.009
0.010
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
AU
0.000
0.002
0.004
0.006
0.008
0.010
0.012
0.014
0.016
0.018
0.020
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
AU
0.00
0.10
0.20
0.30
0.40
0.50
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
Rs = 2.1USP Plate Count = 3KFlow Rate = 0.5 mL/min
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Rs = 3.7USP Plate Count = 15KFlow Rate = 0.4 mL/min
ACQUITY UPLC BEH125 1.7µm(4.6 x 300 mm)
HMWP 10µm(7.8 x 300 mm)
HPLC/UPLC Column Comparison
Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Injection volume:
(Waters HMWP) tested to perform in the European Pharmacopoeial method.
Increase in HMW resolution observed in shorter run-times
©2012 Waters Corporation 47
AU
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
Minutes2.50 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
Comparable absolute retention time change observed for both columns
Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Sample: Human Insulin ( 4mg/mL), Injection volume: 5 µL
AU
-0.002
-0.001
0.000
0.001
0.002
0.003
0.004
0.005
Minutes2.50 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
Injection 853
Injection 26
BEHBEH125 SEC, 1.7um Column Life Insulin Analysis
ACQUITY UPLC BEH125, SEC 1.7µm4.6 x 300 mm
©2012 Waters Corporation 48
0
1
2
3
4
5
6
0 100 200 300 400 500 600 700 800 900
Injection Number
Ret
entio
n Ti
me
(min
)
0.0
1.0
2.0
3.0
4.0
5.0
USP
Res
olut
ion
Retention Time USP Resolution
Column Stability for Insulin Analysis
Over 800 injections the retention time of the insulin monomer peak and the resolution between insulin monomer and dimer peaks are maintained.
©2012 Waters Corporation 49
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
ACQUITY UPLC BEH125 SEC 1.7 µm
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
ACQUITY UPLC BEH200SEC 1. 7µm
©2012 Waters Corporation 50
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
ACQUITY UPLC BEH125, 1.7µm4.6 x 300mm
Minutes6.00 7.00 8.00 9.00 10.00
BioSuite125 UHR, 4µm4.6 x 300mm
Minutes14.00 16.00 18.00 20.00
Insulin HMWP, 10µm7.8 x 300mm
Control
Sample 1
Sample 2
Control
Sample 1
Sample 2
Control
Sample 1
Sample 2
Rs= 2.21
Rs= 1.93
Rs= 1.92
Rs= 2.08
Rs= 1.95
Rs= 1.88
Rs= 3.37
Rs= 2.63
Rs= 2.63
HMW
Fragment
Effect of Particle Size: Insulin
Improved resolution of HMW and Fragment peaks observed with BEH125 1.7 um column
Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Column dimensions: 4.6 x 300mm BEH125 and BioSuite125, 7.8 x 300mm Insulin HMWP, Flow rate: 0.4 mL/min (HMWP: 0.5 mL/min), Injection volumes: scaled
©2012 Waters Corporation 51
Agenda
Size-Exclusion Chromatography– Theory and practice– ACQUITY UPLC Columns for SEC
o ACQUITY BEH450 SEC, 2.5 µm Columnso ACQUITY BEH200 SEC, 1.7 µm Columnso ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Applicationo SEC-MS Applications
– Insulin and Small Protein Applications– Factors Influencing Component Resolution– Considerations to extending column life
©2012 Waters Corporation 52
Factors Influencing ResolutionFactors Influencing Resolution
Resolution increases with lower injection volumes
Resolution increases with lower flow rate– Ideal flow rate is lower than typically running, however will sacrifice
speed
Resolution increases with column length
Baseline resolution typically achieved at 50%-100% molecular weight difference
©2012 Waters Corporation 53
AU
0.00
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
Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50
Loading Capacity:Loading Capacity: Undiluted Monoclonal Antibody Undiluted Monoclonal Antibody BiopharmaceuticalBiopharmaceutical
Humanized IgG (20 mg/mL), 4.6 x 150 mm column
Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm
5 µL, 100µg
10 µL, 200µg
15 µL, 300µg
21.0%
21.2%
Aggregates
21.8%
Injection Volume, Total Load
15 µL, 300µg
Aggregates
21.8%
Injection Volume, Total Load
©2012 Waters Corporation 54
AU
-0.020
-0.010
0.000
0.010
0.020
0.030
0.040
Minutes3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00
AU
-0.020
-0.010
0.000
0.010
0.020
0.030
0.040
Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
Effect of Column Length:Monoclonal Antibody
Murine monoclonal antibody (load: 6.4 µg - 150mm; 12.7 µg -300mm)
Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8;214 nm
300 mm
150 mm98.88%
98.76%USP Res= 2.811.22%
USP Res=2.071.12%
mAbaggregates
mAbaggregates
©2012 Waters Corporation 55
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
Resolution increases with lower flow rates
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 56
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
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
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
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
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
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
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: ACQUITY UPLC 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 57
Effect of Salt Anion on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shape
Different anions of sodium salt additive
Buffer: 10mM sodium phosphate, pH 6.8 and 200mM of additive ( unless otherwise noted)
Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil
©2012 Waters Corporation 58
Effect of Salt Cation on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shape
Different cations of chloride salt additive
Buffer: 10mM sodium phosphate, pH 6.8 and 200mM of additive
Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil
©2012 Waters Corporation 59
Agenda
Size-Exclusion Chromatography– Theory and practice– ACQUITY UPLC Columns for SEC
o ACQUITY BEH450 SEC, 2.5 µm Columnso ACQUITY BEH200 SEC, 1.7 µm Columnso ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Applicationo SEC-MS Applications
– Insulin and Small Protein Applications– Factors Influencing Component Resolution– Considerations to extending column life
©2012 Waters Corporation 60
UP-SEC Recommendations
H Class Bio system– Biocompatible system for the analysis of biological molecules
o Eliminate system corrosiono Best sample recovery
• Limit sample adsorption• Limit damage to molecules, especially oxidation
o Eliminate adduct formation in MS detection– Based on ACQUITY UPLC H-Class System (Quaternary) – True UPLC performance– Compatible for all modes of chromatography– Incorporates Auto●Blend Plus™ Technology
©2012 Waters Corporation 61
Excipients
Added to increase protein stability, minimize protein-protein interactions
Inhibit adsorption of proteins to vials
Can affect protein aggregation
Can affect biotherapeutic efficacy and immunogenicity
Common excipients– Carbohydrates (Sucrose, Treahlose)– Surfactants (Triton X-100, Polysorbate (Tween) 80, Polysorbate
(Tween) 20, Brij 35, Puronic F-68)– Human serum albumin
©2012 Waters Corporation 62
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
0 100 200 300 400 500 600 700 800 900 1000
Injection Number
USP
Pla
te C
ount
Column Lifetime
Figure 1: Effect of using a 30 mm guard column on column efficiency for a monoclonal antibody. The arrows indicate where the guard column was changed.
Column and guard Column without guard
©2012 Waters Corporation 63
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50
Effect of Column Guard on Lifetime : Monoclonal Antibody
mAb formulation with excipients (Tween 80)
Improved mAb peak tailing with use of guard column
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
0.28
Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Injection 2Injection 902
Injection 6Injection 488
No Guards Guards Replaced every 200 injections
©2012 Waters Corporation 64
BEH200 SEC, 1.7um 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 65
Injection 10
Injection 627
AU
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
Bacterial growth
Injection 10
Injection 627
System pressure increases slightly over lifetime (~50 psi) Analysis of frit indicated bacterial growth
©2012 Waters Corporation 66
AU
0.000
0.005
0.010
0.015
AU
0.000
0.010
0.020
0.030
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 16.00
Interaction with Flow Cell under Native Conditions
SEC-PDA chromatogram of bovine serum album (BSA) (5 mg/mL in H20) shows the effect of flow cell material on peak shape. BSA monomer exhibits extensive peak tailing.
Conditions: 25mM sodium phosphate, 150mM sodium chloride, pH 6.8, 0.4 mL/min, Injection volume: 4 µL, Wavelength: 280 nm; Column: ACQUITY UPLC BEH200 SEC 1.7 µm column, 4.6 x 300mm
Standard Teflon AF 10mm flow cell
Titanium 5 mm flow cell
©2012 Waters Corporation 67
Auto•Blend Plus™ Technology AQUITY H-Class Bio
Program methods directly in units of pH and Molarity
Calculation of required proportions from physical constants– pH is calculated using Henderson-Hasselbalch equation with
pKa provided– Typically use pKa corrected for salt concentrationOR:– Empirical calibration table covering operating range of buffer
and salts selected
Proportions calculated at each pump stroke for best fidelity
Independent gradients for pH and salt concentration
Can produce a near true linear pH gradients as required
©2012 Waters Corporation 68
Instrument Control Method Auto•Blend™ to Auto•Blend Plus™
Click to Convertto Auto•Blend PlusTM
©2012 Waters Corporation 69
Typical Gradient Table Auto•Blend™ Plus
©2012 Waters Corporation 70
Auto•Blend Plus™ PH and Salt Gradients
©2012 Waters Corporation 71
Calibration Options Auto•Blend™ Plus
Select to Use Empirical rather than Henderson- Hasselbalch
©2012 Waters Corporation 72
Auto•Blend™ Plus Calibration Options - Delivered pH
6.35
6.45
6.55
6.65
6.75
6.85
6.95
7.05
7.15
1 2 3 4 5 6 7 8 9
MeasuredpH
Fraction Number
Programmed
pK
Corrected pK
Empirical
©2012 Waters Corporation 73
Auto●Blend Plus™ Method Development
AU
0.00
0.05
0.10
AU
0.00
0.06
0.12
AU
0.00
0.05
0.10
AU
0.00
0.05
0.10
AU
0.00
0.05
0.10
Minutes0.00 5.00 10.00 15.00
pH 6.1
pH 7.6
pH 7.1
pH 7.0
pH 6.9
A mixture of proteins was separated using cation exchange chromatography- alpha- Chymotrypsinogen A (peak A), Ribonuclease A (peak B), and Cytochrome C (peak C) –
A
A
A+B
A
A
B
B
B
B
C
C
C
C
C
©2012 Waters Corporation 74
SEC of Humanized Monoclonal AbSEC of Humanized Monoclonal Ab Effect of AutoBlend Plus pH AdjustmentEffect of AutoBlend Plus pH Adjustment
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
AU
0.00
0.10
0.20
0.30
0.40
0.50
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
A: 17.26%B: 2.74%C: 20.00%D: 60.00%
A: 2.74%B: 17.26%C: 20.00%D: 60.00%
pH6.0
pH7.6
125mM NaH2 PO4
125mM Na2H2 PO4
1000mM NaCLH2 O
©2012 Waters Corporation 75
0.000
0.005
0.010
0.015
0.000
0.005
0.010
0.015
0.000
0.005
0.010
0.015
0.000
0.005
0.010
0.015
Minutes4.00 5.00 6.00 7.00 8.00
Minutes4.00 5.00 6.00 7.00 8.00
Minutes4.00 5.00 6.00 7.00 8.00
UV
Ab
sorb
ance
@ 2
80
nm
150 mM 250 mM 350 mMpH
6.0
6.5
7.0
7.5
[NaCl]
HMW
Monomer
LMW 1 LMW2
Developing a Robust SEC Method using AutoBlend Plus
©2012 Waters Corporation 76
Reference Material
Care and Use– Size Exclusion and Ion-Exchange Chromatography of Proteins using
the ACQUITY UPLC™ System,” 715002147, REV. A– “Size Exclusion and Ion-Exchange Chromatography of Proteins using
the ACQUITY UPLC H-Class System, ” 715002909, Rev A– “Controlling contamination in LC/MS and HPLC/MS Systems,”
715001307– “Improving the Lifetime of UPLC Size-Exclusion Chromatography
Columns Using Short Guard Columns,” Waters Technical Brief, 720004034en
– “Guidelines for Routine Use and Maintenance of Ultra-Performance Size-Exclusion and Ion-Exchange Chromatography Systems”, Waters Technical Brief, 720004182en
©2012 Waters Corporation 77
Summary: Waters ACQUITY UPLC SEC System Solution
New SEC column chemistries in 125Å, 200Å and 450Å pore sizes based on BEH particles– Reduced secondary interaction– Improved physical and chemical column lifetime– Improved column-to-column reproducibility– Improved resolution– Improved throughput
UPLC-SEC provides improved resolution, sensitivity, and higher throughput as compared to tradition HPLC– Improved resolution of monoclonal antibody aggregates and clipped forms
Complete system solution includes column chemistry and system– UPLC columns specifically designed for bioseparations– ACQUITY H-Class Bio System designed for the bioapplications– Auto●Blend Plus™ Technology provides convenience and efficiency
©2012 Waters Corporation 78
Acknowledgements
Paula Hong
Kenneth Fountain
Ed Bouvier
Sue Serpa
Bill Warren