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©2012 Waters Corporation 1
UPLC Peptide Mapping and NUPLC Peptide Mapping and N--GlycanGlycanFingerprintingFingerprinting
Dr. Stephan Koza, Principal Applications Chemist Waters CorporationMilford, Massachusetts
©2012 Waters Corporation 2
Resolution ManagementResolution ManagementCurrent Status in UPLCCurrent Status in UPLC
11
4 kkNRs
Maximized in UPLC with
• Small Particle Packing Material• Low Dispersion System
• Optimized Detectors
Maximized in UPLC with
• Wide Range of Column Chemistries• Complete Application Solutions• Specialized Column Chemistry
N L/dp
©2012 Waters Corporation 3
Waters Column Manufacturing and Waters Column Manufacturing and Quality Control TestingQuality Control Testing
Over 30 tests per batch of media totaling over 300 QC response factors
©2012 Waters Corporation 4
ApplicationApplication--specific Columnsspecific ColumnsACQUITY UPLC BEH Glycan ColumnACQUITY UPLC BEH Glycan Column
Chromatographic Testwith
Biomolecule Standards
Chemical Tests
Individual Column Tests
©2012 Waters Corporation 5
Interrogating Protein StructureInterrogating Protein Structure
Analyze the intact protein Measure aggregation Map the protein Identify modifications Determine glycan structure Determine amino acid
composition Measure nutrients for cell
growth
National University of Ireland, Galway, University Road, Galway, Ireland
Wikemedia
©2012 Waters Corporation 6
UPLCUPLC®® TechnologyTechnologyfor Peptide for Peptide MappingMapping
©2012 Waters Corporation 7
UPLC Peptide MappingUPLC Peptide MappingApplication SolutionApplication Solution
Improved Peptide Mapping – Higher resolution– Faster run times– Focused maps– Improved sensitivity and dynamic range
o Enhanced resolution reveals trace componentso Reduced peak volume increases detector response
Unique Peptide Separations Technology Columns– Good peak shape with formic acid and TFA– Separation of glycopeptides– 130Å and 300Å– Column QC test with peptide map
Complete System Solution with UV or MS detection with BiopharmaLynx
Auto•Blend Technology
©2012 Waters Corporation 8
Time20.00 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 52.00 54.00 56.00
AU
2.0e-2
3.0e-2
4.0e-2
5.0e-2
6.0e-2
7.0e-2
8.0e-2
9.0e-2
1.0e-1
Time30.00 35.00 40.00 45.00 50.00 55.00 60.00 65.00 70.00 75.00 80.00 85.00 90.00
AU
1.0e-2
2.0e-2
3.0e-2
4.0e-2
5.0e-2
6.0e-2
7.0e-2
90 min
55 min
HPLC 2.1 x 250 mm, 3.5 mm
UPLC 2.1 x 150 mm, 1.7 mm
Reduce Run TimeReduce Run TimeComparable ResolutionComparable Resolution
©2012 Waters Corporation 9
Time8.00 10.00 12.00 14.00 16.00 18.00
AU
2.5e-2
5.0e-2
7.5e-2
1.0e-1
1.25e-1
1.5e-1
1.75e-1
2.0e-1
2.25e-1
2.5e-1
2.75e-1
0.25
1.00.5
105.02.0
5020
100
pmol
Peptide QuantitationPeptide QuantitationLinearityLinearity
©2012 Waters Corporation 10
Time28.30 28.35 28.40 28.45 28.50 28.55 28.60 28.65 28.70 28.75 28.80 28.85 28.90 28.95 29.00 29.05
AU
2.0e-2
2.5e-2
3.0e-2
3.5e-2
4.0e-2
4.5e-2
5.0e-2
5.5e-2
6.0e-2
6.5e-2
7.0e-2
7.5e-2
8.0e-2
8.5e-2
9.0e-2
9.5e-2
1.0e-1
1.05e-1
1.1e-1
1.15e-1
1.2e-1
1.25e-1
1.3e-1
1.35e-1
1.4e-1
0.2%
0.5%
1%
2%**
Peptide MapPeptide MapTrace ContaminantTrace Contaminant
©2012 Waters Corporation 11
Reduce Development Time:Reduce Development Time:UPLC can Provide Higher Throughput UPLC can Provide Higher Throughput AnalyticsAnalytics
Customer Challenge:– Increased sample load from stability studies– Analysis of oxidation/deamidation critical for choosing correct
formulation– Peptide map method required hours of run time
Business Impact– Bottleneck in the development process– Wrong formulation could lead to clinical trial failure
©2012 Waters Corporation 12
Focused Peptide Maps using UPLCFocused Peptide Maps using UPLC®®
33--6X Improvement in Productivity6X Improvement in Productivity
“We were able to get much faster modification analysis with no significant loss in resolution, allowing us to move the samples through the process much faster”
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0 10 20 30 40 50 60
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0 10 20 30 40 50 60
Linear, segmented, focused
©2012 Waters Corporation 13
Separation of PENNY Peptide and its Separation of PENNY Peptide and its Deamidated PeptidesDeamidated Peptides
©2012 Waters Corporation 14
Compatible with widest range of bioseparation applications Compatible with all modes of chromatography
– RP, IEX, SEC, HILIC Suitable for widest range of samples Biocompatible system for analysis in high salt mobile phases Good recovery of biological macromolecules Incorporates Auto•Blend™ Plus TechnologyTrue UPLC PerformancePhenomenal HPLC performance (up to 2 mL/min)
ACQUITY UPLC HACQUITY UPLC H--Class Bio SystemClass Bio System
©2012 Waters Corporation 15
Benefits of Auto•Blend and Auto•Blend Benefits of Auto•Blend and Auto•Blend Plus Technology with Quaternary SystemPlus Technology with Quaternary System
Efficient method development– Explore more separation alternatives– Test intermediate conditions– Minimize errors in mobile phase preparation
Routine analyses– Minimize errors
in mobile phase preparation– Conserve costly solvent– Switching among
multiple methods
Patent pending
©2012 Waters Corporation 16
AutoAuto●●Blend Blend
Auto•Blend Technology allows the automatic blending of up to four solvents in accurate proportions and to run any sequence of isocratic, binary, ternary, quaternary gradients for routine method operation, automatic method development, convenient method transfer, or system flushing
Routine assays become more rugged (less human error)
Water
1%TFAIsopropanol
Acetonitrile Water
1%TFAIsopropanol
Acetonitrile
95%
0%
0%
5%
45%
0%
50%
5%
100% Water0% Acetonitrile0.05% TFA
50% Water50% Acetonitrile0.05% TFA
©2012 Waters Corporation 17
Peptide MappingPeptide MappingReversedReversed--phase chromatographyphase chromatography
The peptide mapping sample list started on Friday and ran automatically through the weekend. The data was ready to review upon return to work on Monday. Every third sample is shown, demonstrating excellent reproducibility in both resolution and retention
©2012 Waters Corporation 18
UPLCUPLC®® TechnologyTechnologyfor Glycans Analysisfor Glycans Analysis
©2012 Waters Corporation 19
UPLC Glycan Analysis SolutionUPLC Glycan Analysis Solution
UPLC Solution– UPLC– Glycan Separation Technology columns provide high resolution, selectivity,
sensitivity, reproducibility and throughputo BEH Chemistry – HILIC columno Application QC-Tested
– FLR detection provides a wide dynamic range Improved resolution and quantitative assay of all glycans of monoclonal
antibodies and glycoproteins– Separation of Man5 and Man6 from other glycans – Separation of acidic sugars including poly-sialylated glycans
Analyses Time Mass Spectra can complement the UPLC/FLR separation and biological data
in glycan analysis in that it can be useful for confirmation of expected structures
Central part of our Comprehensive Glycoprotein Analysis with complementary Waters techniques– Intact Mass Analysis – UPLC Peptide Mapping
©2012 Waters Corporation 20
22--AB Labeled Dextran LadderAB Labeled Dextran LadderUPLC Separation UPLC Separation
©2012 Waters Corporation 21
UPLCUPLC®® SeparationSeparationHuman IgG Human IgG NN--linked Glycanslinked Glycans
1 G02 G0F3 Man54 G0FGN5 G16 G1Fa7 G1Fb8 G1FGN9 Man610 G211 G2F12 G1F+SA13 G2F+SA
1
4
5
6
78
9
10
11
2
3
12
13
Peak identification was done in LC-MS under same gradient conditions
©2012 Waters Corporation 22
Murine IgG OligosaccharidesMurine IgG OligosaccharidesHPLC vs. UPLC BEH Glycan ColumnHPLC vs. UPLC BEH Glycan Column
T im e30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00
EU
x 1
0e4
0 .000
5000.000
10000.000
15000.000 G0F
Man6
G2F
Man5
G1F
Time
2 x 150 mm - 3 µm
100.0
T im e5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0
0 .0 0 0
2 5 0 0 .0 0 0
5 0 0 0 .0 0 0
7 5 0 0 .0 0 0
1 0 0 0 0 .0 0 0
1 2 5 0 0 .0 0 0
1 5 0 0 0 .0 0 0
35.0
Man5
Man6
G1F
G2F
G0F
2.1 x 150 mm - 1.7 µm
EUEU
©2012 Waters Corporation 23
22--AB Labeled High Mannose GlycansAB Labeled High Mannose Glycans(RNase b) UPLC Separation (RNase b) UPLC Separation
EU
-0.50
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
6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00
6.00 minutes 24.00
2AB-
2AB-
2AB-
2AB-
2AB-
3 isomers
ACQUITY UPLC BEH Glycan, 1.7µm, 2.1 x 150 mm
©2012 Waters Corporation 24
2AB glycans from Fetuin (10 pmol)2AB glycans from Fetuin (10 pmol)
©2012 Waters Corporation 25
BatchBatch--toto--Batch Reproducibility of ACQUITY Batch Reproducibility of ACQUITY UPLCUPLC®® BEH BEH GlycanGlycan Material Using Material Using 22--AB Labeled Human AB Labeled Human IgGIgG NN--Linked Linked GlycansGlycans
Batch 1
Batch 2
Batch 3
Batch 4
©2012 Waters Corporation 26
Sneak Peek: Peptide Mapping Sneak Peek: Peptide Mapping Column DevelopmentColumn Development
An Ideal Peptide Mapping Column
1.High resolution2.Ideal peak symmetry (TFA and Formic Acid)3. Recovery of all peptides (hydrophobic,
hydrophilic, acidic, basic, small, and large4.Rugged and reproducible
©2012 Waters Corporation 27
Peak Capacity and MS SensitivityPeak Capacity and MS SensitivityFA FA vsvs TFATFA
12
10
8
6
4
2
20
70
120
170
220
270
320
370
0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10
Fold
Dec
reas
e in
MS
Peak
Are
a (R
eala
tive
to 0
.1%
FA)
Peak
Cap
acity
Percent TFA
Prototype C18
BEH C18 ~100%
~20%
0.010.09
0.020.08
0.050.05
% TFA% FA
0.000.10
0.100.00
©2012 Waters Corporation 28
MassPREPMassPREP Peptide MixturePeptide Mixture0.1% FA0.1% FA
10 20 30 40 50 60Time (min)
0.0
1.0
10 20 30 40 50 60
A21
4
Time (min)
1 2
Prototype C182.1 x 150 mm, 1.7 µm
Pc,4σ = 3052 3
5 67
9
841Vo
0.0
1.0
A21
4
BEH C182.1 x 150 mm, 1.7 µm
Pc,4σ = 1561 2 3 4 5 67
8
9
BEH C182.1 x 150 mm, 1.7 µm
Pc,4σ = 156
Prototype C182.1 x 150 mm, 1.7 µm
Pc,4σ = 305
* Pc,4σ calculated based on FWHM for peptides 2-7 (2 replicates)
1 RASG-1 RGDSPASSKP2 Angiotensin 1-7 DRVYIHP3 Bradykinin RPPGFSPFR4 Angiotensin II DRVYIHPF5 Angiotensin I DRVYIHPFHL6 Renin Substrate DRVYIHPFHLLVYS7 Enolase T35 WLTGPQLADLYHSLMK8 Enolase T37 YPIVSIEDPFAEDDWEAWSHFFK9 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ
Acquity H-Class BioA - Water with 0.1% FAB - ACN with 0.1% FA2% ACN for 1 min, then to 50% ACN over 60 min0.3 mL/min40°CUV @ 214 nm 5.6 µg MassPREP Peptide Mixture10 µL injection
©2012 Waters Corporation 29
MassPREPMassPREP Peptide MixturePeptide Mixture0.1% TFA0.1% TFA
* Pc,4σ calculated based on FWHM for peptides 2-7 (2 replicates)
Acquity H-Class BioA - Water with 0.1% TFAB - ACN with 0.1% TFA2% ACN for 1 min, then to 50% ACN over 60 min0.3 mL/min40°CUV @ 214 nm 5.6 µg MassPREP Peptide Mixture10 µL injection
1 RASG-1 RGDSPASSKP2 Angiotensin 1-7 DRVYIHP3 Bradykinin RPPGFSPFR4 Angiotensin II DRVYIHPF5 Angiotensin I DRVYIHPFHL6 Renin Substrate DRVYIHPFHLLVYS7 Enolase T35 WLTGPQLADLYHSLMK8 Enolase T37 YPIVSIEDPFAEDDWEAWSHFFK9 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ
0.0
1.0
A21
4
BEH C182.1 x 150 mm, 1.7 µm
Pc,4σ = 2941 23 4 5 6
78
9
10 20 30 40 50 60Time (min)
0.0
1.0
10 20 30 40 50 60
A21
4
Time (min)
CSH C182.1 x 150 mm, 1.7 µm
Pc,4σ = 3481 23
4 56 7
8
9
BEH C182.1 x 150 mm, 1.7 µm
Pc,4σ = 294
Prototype C182.1 x 150 mm, 1.7 µm
Pc,4σ = 348
©2012 Waters Corporation 30
Prototype C18Prototype C18LCLC--UVUV--MSMS
Peak AreaMS-100555UV-51185
Peak AreaMS-98701UV-96527
0E+0
2E+5
4E+5
6E+5
8E+5
0
0.2
0.4
0.6
0.8
52 54 56 58 60 62
MS
Inte
nsity
A21
0
Time (min)
Low TFA mobile phase
Optimized Peak Capacity +MS signal ≥ UV signal
Acquity H-Class BioWater with 0.02% TFA/0.08% FAACN with 0.02% TFA/0.08% FA0.5% ACN for 5 min, then to 40% ACN over 120 min0.3 mL/min60°CUV @ 210 nm / Xevo G2 QTOF (Res Mode, 2 Hz)36 µg humanized mAb Lys-C Digest
©2012 Waters Corporation 31
Benefits of UPLCBenefits of UPLC®®Technology for Technology for BiomoleculesBiomolecules
UPLC® Technology brings improved resolution, sensitivity, and speed to Peptide Mapping and Glycan Analyses
Batch-to-Batch Reproducibility and QC testing to ensure the performance of every ACQUITY UPLC® PST and BEH GlycanColumn
©2012 Waters Corporation 32