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Webinar:Webinar:'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical 'Tips and Tricks' for Biopharmaceutical
Characterization using SECCharacterization using SEC
Waters CorporationWaters Corporation
January 27, 2012January 27, 2012
©2012 Waters Corporation 1
Waters Commitment
To develop, commercialize and market columns that when used on Waters ACQUITY UPLC® systems, give the speed, sensitivity,
l ti d th d d ibilit th t h t b i l resolution, and method reproducibility that has not been previously achieved for the characterization of biological macromolecules with traditional HPLC.
©2012 Waters Corporation 2
Liquid ChromatographyProtein Separation Modesp
Protein Structure
Primary, Secondary, Tertiary Structure
Net Charge
Carbohydrate Groups
HydrophilicGroups
Hydrophobic Regions
Aromatic Groups
Regions
Disulfide H d
©2012 Waters Corporation 3
Disulfide Linkages
HydrogenBonding
Liquid ChromatographyProtein Separation Modesp
Protein Structure
Primary, Secondary, Tertiary Structure
Net Charge
Carbohydrate Groups
HydrophilicGroups
Hydrophobic Regions
Aromatic Groups
Regions
Disulfide H d
©2012 Waters Corporation 4
Disulfide Linkages
HydrogenBonding
Agendag
Size-Exclusion Chromatography– Theory and practiceTheory and practice
– ACQUITY UPLC Columns for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns
ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Application
o SEC-MS Applications
– Insulin and Small Protein Applications
– Factors Influencing Component Resolution
– Considerations to extending column life
©2012 Waters Corporation 5
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 6
Size Exclusion Chromatographyg p y
No adsorption to surface of particles
Large molecules elute Large molecules elute before small molecules
Large molecules cannot access poresaccess pores
Small molecules access pores within particle
dimer
monomer
©2012 Waters Corporation 7
Common SEC applications:Biotherapeutics Types
Monoclonal Antibodies
Biotherapeutics Types
Antibody Conjugates
Fc Fusion Proteins
Synthetic OligonucleotidesSynthetic Oligonucleotides
Protein Subunit Vaccines
Recombinant Proteins
©2012 Waters Corporation 8
Agendag
Size-Exclusion Chromatography– Theory and practiceTheory and practice
– ACQUITY UPLC Columns for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns
ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Application
o SEC-MS Applications
– Insulin and Small Protein Applications
– Factors Influencing Component Resolution
– Considerations to extending column life
©2012 Waters Corporation 9
UPLC Systems for Biopharmaceutical Analysisp y
Wide range of applicationsg pp Complete Solutions
– Instrumentationo UPLC System (s)
• ACQUITY UPLC System• ACQUITY UPLC System• ACQUITY UPLC H-Class System• ACQUITY UPLC H-Class Bio System
o UV, FLR, PDA and MS DetectionsA li ti S ifi Ch i t io Application Specific Chemistries• Developed and designed with applications• QC Tested with application• Optimizes for UPLC
o Software • Data Analysis• Information management
Focused on customer application requirements
©2012 Waters Corporation 10
pp q
Advantages of UPLC Technologyfor SEC Separations
Requires Columns and Instrumentation to Minimize Band Spreading
HPLC
p
Broad BandBroad PeakLess Sensitivity
HPLC
Narrow PeakIncreased Sensitivity
Waters UPLC®
Technology
Less SensitivityLess Resolving Power
Increased SensitivityIncreased Resolving Power
©2012 Waters Corporation 11
Effect of System Dispersion on ACQUITY UPLC BEH200 SEC 1.7 µm separationµ p
UP-SEC0.30
ACQUITY UPLC BEH200 SEC 1.7 µm 4.6 x 300mm
USP Res= 2.37
AU
0.10
0.20
0.00
ACQUITY UPLC BEH200 SEC 1 7 µm
HP-SEC
USP Res= 1.37
AU
0 10
0.15
0.20
0.25
BEH200 SEC 1.7 µm 4.6 x 300mm
0.00
0.05
0.10
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 12
Large system dispersion decreases resolution Sample: Human polycolonal IgG
Minutes
ACQUITY UPLC BEH200 and BEH125 SEC1.7 µm Columnsµ
Application Areas
– Molecular weight ranges dependent on pore size:Molecular weight ranges dependent on pore size:
o BEH200: 10,000 to 450,000 Daltons
o BEH125: 1,000 to 80,000 Daltons
– Determination of protein / peptide molecular weight
– Quantitation of protein / peptide aggregates primarily in therapeutic monoclonal antibodies, EPO, and Insulin
– Determination of size heterogeneity in a sample
©2012 Waters Corporation 13
BEH™ Technology ParticlesBEH™ Technology ParticlesBridged EthylSiloxane/Silica HybridBridged EthylSiloxane/Silica Hybridg y yg y y
Si
EtO CH2 CH2
Si OOEt
SiOEt
O EtEtO OEtEtO
O
Si
EtO
Si O
O
OEtO
O
Si OOEt
Et
+ Si
EtOEtO
CH2EtO
CH2Si
OEt
OEtOEt
4 Si
EtO
EtO OEtEtO
1
Polyethoxysilane(BPEOS)
EtO OEt nTetraethoxysilane
(TEOS)Bis(triethoxysilyl)ethane
(BTEE)
Bridged EthanesIn Silica Matrix
©2012 Waters Corporation 14
Anal. Chem. 2003, 75, 6781-6788
U.S. Patent No. 6,686,035 B2
HPLC to UPLC SEC Comparisonp
0.065
0.070
0.065
0.070
HPLC 100% ACQUITY UPLC
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 UPLC BEH200 SEC,1.7
µm4.6 x 300mm
AU
0.030
0.035
0.040
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 15
Murine monoclonal antibody - Scaled load
Conditions: 0.4 mL/min; 25mM Sodium Phosphate, pH 6.8, 0.15 M NaCl
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
Calibration Curves for ACQUITY UPLC SEC Columns
BEH200, SEC, 1.7um
Thyroglobulin (~ 669,000 Da)
IgG (~ 150,000 Da)
Aprotinin (~ 6,500 Da)
BEH125, SEC, 1.7um Uracil (~ 112 Da)
©2012 Waters Corporation 16
Protein Adsorption and SizeProtein Adsorption and Size--Exclusion ChromatographyExclusion Chromatographyg 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 solution)p ( p p y )
SEC particles frequently coated with a hydrophilic reagent tominimize non-desired ionic interactions between proteins and minimize non-desired ionic interactions between proteins and packing material
M bil h dditi ( 150 M N Cl) d Mobile phase additives (e.g., 150mM NaCl) may decrease non-desired ionic interactions between proteins and packing material
©2012 Waters Corporation 17
BEH SEC Particle 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.
©2012 Waters Corporation 18
Comparative SEC Column Life
Lysozyme, pKi = 10.7
0 60
0.70
HPLC 100% Silica-Diol
p
Lysozyme,
Suggestive of DIOL Bleed
AU
0.30
0.40
0.50
0.60 HPLC 100% Silica DiolSEC 250Å 4µm4.6 x 300 mm
Injection 19Suggestive of DIOL Bleed
pI = 10.7
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
Injection 19Injection 618
©2012 Waters Corporation 19
Comparative SEC Column Life
Lysozyme, pKi = 10.70.60
0.70
HPLC 100% Silica-DiolLysozyme, pI 10 7
Suggestive of DIOL BleedAU
0 20
0.30
0.40
0.50 SEC 250Å 4µm4.6 x 300 mm
Injection 19Suggestive of DIOL Bleed
pI = 10.7
0.22
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
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
©2012 Waters Corporation 20
BEH200 shows minimal secondary interactions even after 600 injections
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
Influence of Ionic Strength on Peak Shape and Retention
0 06
0.08
p
Conventional 100% Silica-Diol CoatedSEC Column 4.6 x 300 mm 10 mMlysozyme
AU
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
Minutes
AU
0.04
0.06
0.08
25 mMlysozyme
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
100 mMlysozyme
©2012 Waters Corporation 21
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 Retentionp
AU
0 12
0.18
0.24
0 12
0.18
0.24
ACQUITY BEH200 SEC 1.7 µm column, 4 6 150
10 mMlysozyme
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
4.6 x 150mm
AU
0.12
0.18
0.24
0.12
0.18
0.24
25 mM
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
100 mM
©2012 Waters Corporation 22Flow 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
BEHBEH200 SEC, 1.7um BatchBatch--toto--Batch Batch ReproducibilityReproducibilityp yp y
©2012 Waters Corporation 23
BEHBEH125 SEC, 1.7um BatchBatch--toto--Batch Batch ReproducibilityReproducibilityp yp y
AU
0.06
0.09
1
3
24
Batch 1 Analyte pl MW
1. Thyroglobulin, 0.1 mg/mL 4.6 669,000
2. Ovalbumin, 0.3 mg/mL 4.5 44,200
A
0.00
0.03
Minutes 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
3. Ribonuclease A, 0.3 mg/mL 9.6 13,700
4. Uracil, 0.05 mg/mL N/A 112
AU
0.03
0.06
0.09
Batch 2
0.00
Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
0.09
AU
0.03
0.06
Batch 3
©2012 Waters Corporation 24
0.00
Minutes 0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Conditions: 100mM Sodium Phosphate pH 6.8; 0.3 mL/min; 30°C; 4.6x150mm
Column ReproducibilityColumn Reproducibilityp yp yAU
0 00
0.05 Batch 1, Column 1
0.00
AU
0.00
0.05 Batch 1, Column 2
AU
0.00
0.05Batch 1, Column 3
AU
0.00
0.05Batch 2, Column 1
AU
0.00
0.05
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
Batch 2, Column 2
©2012 Waters Corporation 25
Minutes
Humanized monoclonal antibody Conditions: 25mM Sodium Phosphate, pH 6.8, 0.15 M Sodium Chloride, 0.4 mL/min Retention times within 0.2min
Agendag
Size-Exclusion Chromatography– Theory and practiceTheory and practice
– ACQUITY UPLC Columns for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns
ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Application
o SEC-MS Applications
– Insulin and Small Protein Applications
– Factors Influencing Component Resolution
– Considerations to extending column life
©2012 Waters Corporation 26
Monoclonal Antibodyy
©2012 Waters Corporation 27
Adapted from Alain BeckCenter of Immunology
Protein Structure
NNative
UUnfolded
SolubleAggregates Insoluble
AggregatesNative UnfoldedorI
Intermediate
Aggregates
Addressing particulate and aggregation issues of therapeutic protein products, Shi, L, PEGS, May 2011
Soluble aggregates and insoluble particles may affect
©2012 Waters Corporation 28
Soluble aggregates and insoluble particles may affect immunogenicity and efficacy of biotherapeutic
Orthogonal Techniques for Characterization
AUC
Dynamic Light Scattering Counter principle
Static Light Scattering
FFF-MALS
AUCFlow Imaging Microscopy
Light Microscopy
SEC MicroscopeVisual Inspection
nm µm mm cm10 10 10100 100 100
monomersoligomers
subvisible particles Visible particles
Aggregates Particles
©2012 Waters Corporation 29
E. Freud, PDA Visual Inspection Forum, Oct- 2009
Aggregates Particles
Column LifetimeColumn Lifetime
0.015 Dimer = 0.46%USP Res = 2.35
AU
0.000
0.005
0.010 Injection 2
-0.005
0.010
0.015
I j ti 497
mAb
Dimer = 0.49%USP Res = 2.27
AU
0.000
0.005
0 0 0Injection 497
mAb
-0.005
Minutes3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50
©2012 Waters Corporation 30
Humanized monoclonal antibody Conditions: 25mM Sodium Phosphate buffer, 0.15 M Sodium Chloride, pH 6.8; 280 nm Column: 4.6 x 300 mm
Effect of Flow RateEffect of Flow Rate
AU 0.010
0.020
0.030
0.4 mL/minFlow 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
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
0.000
0.010
0.020
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
0.35 mL/min
AU 0 010
0.020
0.030
Minutes
0 2 mL/min0.000
0.010
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
0.2 mL/min
©2012 Waters Corporation 31
Triplicate injections overlaid
No observable trend in aggregation with flow rate
Murine monoclonal antibody (63 µg load)
Effect of Particle Size:Effect of Particle Size:Analysis of LMW SpeciesAnalysis of LMW Speciesy py p
0.010BEH200 SEC 1.7µm
LMW peakmAb mAb d
LMW species
AU
0.000
0 010250Å, 4µmHPLC 100% Sili Di l
dimer
AU
0.000
0.010 HPLC 100% Silica-Diol
Å
AU
0.000
0.010 300Å, 5µmHPLC 100% Silica-Diol
AU
0.000
0.010
2 00 4 00 6 00 8 00 10 00 12 00 14 00
290Å, 5µm HPLC 100% Silica-Diol
©2012 Waters Corporation 32
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00
Humanized monoclonal antibody biotherapeutic Conditions: 25 mM Sodium Phosphate, 0.15 M Sodium Chloride Flow rates and Injection volumes scaled for column dimensions
Column StabilityColumn Stabilityyy
Calibration Curve
Protein MWThyroglobulin 669000Ferritin 440000Aldolase 150000BSA 66000
1000000
0 hours
Ovalbumin 44000Carbonic Anhydrase 29000Ribonuclease A 13700Aprotinin 6500Uracil 112
10000Log
Mw
12 hours24 hours36 hours48 hours60 hours
1000.5 1 1.5 2 2.5 3
Elution volume (mL)
©2012 Waters Corporation 33
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
Precautions in SECPrecautions in SEC--MSMS
Protein structure in solution depends on
– pHpH
– Ionic strength
– Buffer and salt
– Additives
Good ionization conditions are different from conditions for biological activitybiological activity
Validation required when buffer is changed
Special uses are valuable
– Fast desalting
– Clips
©2012 Waters Corporation 34
LC/MS Compatible Mobile Phaseon ACQUITY UPLC BEH200, SEC, 1.7um Q , ,
AU
0.10
0.20PBSPBS
0.00
0.10
0.30
100 M A i F t100 M A i F t
AU
0.10
0.20
100mM Ammonium Formate100mM Ammonium Formate
0.00
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
©2012 Waters Corporation 35
Similar retention time/ peak shape observed with MS compatible mobile phases
Humanized Monoclonal Antibody:MS Compatible/Native Mobile Phase
0 010
0.015
0.020
0.025
U 1 0 2
1.25e-2
1.5e-2
1.75e-2 26.00100mM Ammonium Formate
AU
-0.005
0.000
0.005
0.010
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
17.05
20.02
AU 0.010
0.015
0.020
14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
PBS
-0.005
0.000
0.005
Minutes
4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50 10.00
©2012 Waters Corporation 36
Flow Rates: 100mM Ammonium Formate - 0.15mL/min, PBS- 0.4 mL/min Lower flow rate for MS compatibility
SEC-MSHumanized Monoclonal Antibodyy
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 37
MS: Xevo G2 Q Tof Conditions: 100mM Ammonium Formate, Flow rate: 0.15 mL/min Post UV detection additive: ACN, 0.8% Formic acid
Extracted 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
p
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 38
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
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
HC Detect Clips• No Sample Concentration required
LCUV
280
HC HC
HC-HC
TIC
Mass Spectrum LC
Mass Spectrum
LCHC HCHC-HC
©2012 Waters Corporation 39
Conditions: System, ACQUITY UPLCTM with TUV optical detector and Synapt G2 QTof MS
Flow Rate: 0.2 ml/min 0.1%TFA and 0.1%FA in 30% ACN
Agendag
Size-Exclusion Chromatography– Theory and practiceTheory and practice
– ACQUITY UPLC Columns for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns
ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Application
o SEC-MS Applications
– Insulin and Small Protein Applications
– Factors Influencing Component Resolution
– Considerations to extending column life
©2012 Waters Corporation 40
Resolution of Proteins and Peptides (Aqueous)
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 41
-
/ BEH125 column provides increased resolution throughout the lower end of the
peptide mass range (132 29,000).
ACQUITY UPLC BEH125 SEC 1.7µm Column Reproducibility
1
% RSD Retention Time Range
p y
0.6
0.7
0.8
0.9
n Ti
me
Ran
ge
0.2
0.3
0.4
0.5
%R
SD/R
eten
tion
0
0.1
Peptide/Protein
©2012 Waters Corporation 42
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.
Resolution of Small Protein
BioSuite 125 4µm UHR 4.6 x 300 mm column
ACQUITY UPLC BEH125 SEC 1.7µm 4.6 x 300 mm column
0.60
0.70
0.80
0.60
0.70
0.80
0.60
0.70
0.80
0.60
0.70
0.80
4.6 x 300 mm column 4.6 x 300 mm column
USP Rs= 1.94
AU
0.20
0.30
0.40
0.50
AU
0.20
0.30
0.40
0.50
AU
0.20
0.30
0.40
0.50
AU
0.20
0.30
0.40
0.50
USP Rs= 1.94
USP Rs= 3.29
0.00
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
0.00
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
0.00
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
0.00
0.10
Minutes0.00 2.00 4.00 6.00 8.00 10.00 12.00
-
- Conditions: 25mM Sodium Phosphate, 150mM Sodium Chloride, pH 6.8, 0.4 mL/min, sample 2 mg/mL
©2012 Waters Corporation 43
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.
HPLC/UPLC Column Comparison:Insulin
0.007
0.008
0.009
0.010
AU
0.05
0.10
0.15
0.20
0.25
0.30
ACQUITY UPLC BEH125 1.7µm(4.6 x 300 mm)
AU
0.002
0.003
0.004
0.005
0.006 0.00
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 = 15K
0.000
0.001
Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00
0.018
0.020
0.30
0.40
0.50
USP Plate Count 15KFlow Rate = 0.4 mL/min
HMWP 10µm
AU
0004
0.006
0.008
0.010
0.012
0.014
0.016 AU
0.00
0.10
0.20
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 = 3K
HMWP 10µm(7.8 x 300 mm)
.
0.000
0.002
0.004
Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00
USP Plate Count = 3KFlow Rate = 0.5 mL/min
Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v), Wavelength : 276 nm, Injection volume:
©2012 Waters Corporation 44
(Waters HMWP) tested to perform in the European Pharmacopoeial method. Increase in HMW resolution observed in shorter run-times
BEHBEH125 SEC, 1.7um Column LifeInsulin Analysis
0.004
0.005
Injection 26
y
ACQUITY UPLC BEH125, SEC 1.7µm4 6 x 300 mm
AU
0.000
0.001
0.002
0.003
j4.6 x 300 mm
-0.002
-0.001
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
0.005
AU
0.001
0.002
0.003
0.004
Injection 853
-0.002
-0.001
0.000
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
©2012 Waters Corporation 45
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
Minutes50 3 00 3 50 00 50 5 00 5 50 6 00 6 50 00 50 8 00 8 50 9 00
Column Stability for Insulin Analysis
Retention Time USP Resolution
y 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 46
and the resolution between insulin monomer and dimer peaks are maintained.
Effect of Pore Size:Insulin
ACQUITY UPLC BEH125 SEC 1.7 µm
ACQUITY UPLC BEH200SEC 1. 7µm
AU
-0.026
-0.024
-0.022 Control Control
AU
0 000
0.002
0.004
0.006Sample 1 Sample 1Fragment
0.000
AU
-0.042
-0.040 Sample 2 Sample 2
-0.044
Minutes4.00 5.00 6.00
Minutes6.00 7.00 8.00 9.00
Conditions: Mobile Phase: L-arginine (1.0 g/L) /acetic acid (99%)/acetonitrile; 65/15/20 (v/v/v) Wavelength : 276 nm
©2012 Waters Corporation 47
(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
Effect of Particle Size:Insulin
-0.022
ACQUITY UPLC BEH125, 1.7µm4.6 x 300mm
BioSuite125 UHR, 4µm4.6 x 300mm
Insulin HMWP, 10µm7.8 x 300mm
Control Control Control
AU
-0.026
-0.024 Rs= 2.21 Rs= 2.08Rs= 3.37HMW
AU
0 000
0.002
0.004
0.006Sample 1 Sample 1 Sample 1
Rs= 1.93Rs= 1.95
Rs= 2.63
Fragment
0.000
AU
-0.042
-0.040 Sample 2 Sample 2Sample 2
Rs= 1.92Rs= 1.88
Rs= 2.63
-0.044
Minutes4.00 5.00 6.00
Minutes6.00 7.00 8.00 9.00 10.00
Minutes14.00 16.00 18.00 20.00
©2012 Waters Corporation 48
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
Agendag
Size-Exclusion Chromatography– Theory and practiceTheory and practice
– ACQUITY UPLC Columns for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns
ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Application
o SEC-MS Applications
– Insulin and Small Protein Applications
– Factors Influencing Component Resolution
– Considerations to extending column life
©2012 Waters Corporation 49
Factors Influencing ResolutionFactors Influencing Resolutiongg
Resolution increases with lower injection volumes
Resolution decreases with increasing flow rate Resolution decreases with increasing 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 differenceg
©2012 Waters Corporation 50
Loading Capacity:Loading Capacity:Undiluted Monoclonal Antibody Undiluted Monoclonal Antibody BiopharmaceuticalBiopharmaceutical
2 40
2.60
pp
Injection Volume, Total LoadInjection Volume, Total Load
1.80
2.00
2.20
2.40
15 µL, 300µg15 µL, 300µg
AU1.20
1.40
1.6010 µL, 200µg
Aggregates
21.8%
Aggregates
21.8%
0 0
0.60
0.80
1.00
5 µL, 100µg
21.0%
21.2%
0.00
0.20
0.40
1 50 2 00 2 50 3 00 3 50 4 00 4 50
©2012 Waters Corporation 51
Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50
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
Effect of Column Length:Monoclonal Antibody
0.040
y
150 mm98.88%USP Res=2 07
AU
0.000
0.010
0.020
0.030 USP Res=2.071.12%
-0.020
-0.010
Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50
300
mAbaggregates
0.010
0.020
0.030
0.040 300 mm
98.76%USP Res= 2.811.22%
AU
-0.020
-0.010
0.000
mAbaggregates
©2012 Waters Corporation 52
Minutes3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00
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
Effect of Flow Rate on RsEffect of Flow Rate on Rs
0.30 0.2 mL/minRs= 2.4 IgG dimer
AU
0.00
0.10
0.20 ~1500 psigdimer
AU
0.10
0.15
0.20
0.25
0.4 mL/minRs= 1.8
~3000 psi
0.00
0.05
0.15 0.8 mL/min
AU
0.05
0.10Rs= 1.3
~6000 psi
©2012 Waters Corporation 53
0.00
Minutes0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00
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
Effect of Sample Load :Myoglobin
1 40
1.50
1 40
1.50
USPInjection USPInjection 1 70
1.80
1.90
1 70
1.80
1.90
1 70
1.80
1.90
USP Concentration USP Concentration
y g
Effect of Volume LoadEffect of Concentration
0.90
1.00
1.10
1.20
1.30
1.40
0.90
1.00
1.10
1.20
1.30
1.40
3.0235
2.6550
USPInjection Volume
3.0235
2.6550
USP Res
Injection Volume
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.10
1.20
1.30
1.40
1.50
1.60
1.70
1.10
1.20
1.30
1.40
1.50
1.60
1.70
3.262.5
3.2610
Res(mg/ mL)
3.262.5
3.2610
Res(mg/ mL)
AU
0.40
0.50
0.60
0.70
0.80
AU
0.40
0.50
0.60
0.70
0.80 3.7815 3.7815
AU
0.40
0.50
0.60
0.70
0.80
0.90
1.00
AU
0.40
0.50
0.60
0.70
0.80
0.90
1.00
AU
0.40
0.50
0.60
0.70
0.80
0.90
1.00 3.231.25
3.150.625
3.231.25
3.150.625
0.00
0.10
0.20
0.30
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
0.00
0.10
0.20
0.30
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
0.00
0.10
0.20
0.30
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
0.00
0.10
0.20
0.30
Minutes3.50 4.0
04.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50
0.00
0.10
0.20
0.30
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
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)
©2012 Waters Corporation 54
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 Salt Anion on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shapep
Different anions of sodium salt additive
©2012 Waters Corporation 55
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
Effect of Salt Cation on BEH200 SEC, 1.7um 4.6 x 150mm Peak Shapep
Different cations of chloride salt additive
©2012 Waters Corporation 56
Buffer: 10mM sodium phosphate, pH 6.8 and 200mM of additive Sample: Thyroglobulin, IgG, BSA, Myoglobin, Uracil
Agendag
Size-Exclusion Chromatography– Theory and practiceTheory and practice
– ACQUITY UPLC Columns for SEC
o ACQUITY BEH200 SEC, 1.7 µm Columns
ACQUITY BEH125 SEC 1 7um Columns o ACQUITY BEH125 SEC, 1.7um Columns
– Monoclonal Antibody Application
o SEC-MS Applications
– Insulin and Small Protein Applications
– Factors Influencing Component Resolution
– Considerations to extending column life
©2012 Waters Corporation 57
UP-SEC Recommendations
H Class Bio system– Biocompatible system for the analysis of biological moleculesBiocompatible system for the analysis of biological molecules
o Eliminate system corrosion
o Best sample recovery
Limit sample adsorption• 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 58
Excipientsp
Added to increase protein stability, minimize protein-protein interactionsinteractions
Inhibit adsorption of proteins to vials
Can affect protein aggregation
Can affect biotherapeutic efficacy and immunogenicity
Common excipients– Carbohydrates (Sucrose, Treahlose)y ( , )
– Surfactants (Triton X-100, Polysorbate (Tween) 80, Polysorbate (Tween) 20, Brij 35, Puronic F-68)
– Human serum albuminHuman serum albumin
©2012 Waters Corporation 59
Column Lifetime
9000
6000
7000
8000
9000
nt
3000
4000
5000
6000
USP
Pla
te C
oun
0
1000
2000
3000
Column and guard Column without guard
00 100 200 300 400 500 600 700 800 900 1000
Injection Number
Figure 1: Effect of using a 30 mm guard column on column efficiency for
©2012 Waters Corporation 60
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.
Effect of Column Guard on Lifetime :Monoclonal Antibody
0 24
y
0 28
No Guards Guards Replaced every 200 injections
0.18
0.20
0.22
0.24
0 20
0.22
0.24
0.26
0.28
Injection 2Injection 902
Injection 6Injection 488
AU0.12
0.14
0.16
AU
0 12
0.14
0.16
0.18
0.20
0.04
0.06
0.08
0.10
0 04
0.06
0.08
0.10
0.12
0.00
0.02
Minutes1.50 2.00 2.50 3.00 3.50 4.00 4.50
0.00
0.02
0.04
Minutes1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
©2012 Waters Corporation 61
mAb formulation with excipients (Tween 80)
Improved mAb peak tailing with use of guard column
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 um filter (i.e, Don’t inject particulates)
Use high purity water– 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 contaminationInlet 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 62
Extended: Store in the HPLC grade water with 10% methanol
Bacterial growth
0 12
0.14
g
Injection 6270.10
0.12
Injection 627
AU
0.06
0.08
Injection 100.02
0.04
Injection 10
0.00
Minutes0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
©2012 Waters Corporation 63
System pressure increases slightly over lifetime (~50 psi) Analysis of frit indicated bacterial growth
Interaction with Flow Cell under Native Conditions
0.015
Tit i 5 fl ll
AU
0.005
0.010 Titanium 5 mm flow cell
0.000
0.030 Standard Teflon AF 10mm flow cell
AU
0.010
0.02010mm flow cell
0.000
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
SEC-PDA chromatogram of bovine serum album (BSA) (5 mg/mL in H20) shows the effect of flow cell
©2012 Waters Corporation 64
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
Reference Material
Care and Use– Size Exclusion and Ion-Exchange Chromatography of Proteins using 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 Athe 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 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 Guidelines for Routine Use and Maintenance of Ultra Performance Size-Exclusion and Ion-Exchange Chromatography Systems”,Waters Technical Brief, 720004182en
©2012 Waters Corporation 65
Summary:Waters ACQUITY UPLC SEC System Solution New SEC column chemistries in 125 and 200Å pore size based on
BEH particles
Reduced secondary interaction– 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 66