uplc peptide mapping and n-glycan fingerprinting · 3 bradykinin rppgfspfr 4 angiotensin ii...

©2012 Waters Corporation 1

UPLC Peptide Mapping and NUPLC Peptide Mapping and N--GlycanGlycanFingerprintingFingerprinting

Dr. Stephan Koza, Principal Applications Chemist Waters CorporationMilford, Massachusetts


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


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


ApplicationApplication--specific Columnsspecific ColumnsACQUITY UPLC BEH Glycan ColumnACQUITY UPLC BEH Glycan Column

Chromatographic Testwith

Biomolecule Standards

Chemical Tests

Individual Column Tests


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


UPLCUPLC®® TechnologyTechnologyfor Peptide for Peptide MappingMapping


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


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


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


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


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


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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Linear, segmented, focused


Separation of PENNY Peptide and its Separation of PENNY Peptide and its Deamidated PeptidesDeamidated Peptides


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


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


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


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


UPLCUPLC®® TechnologyTechnologyfor Glycans Analysisfor Glycans Analysis


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


22--AB Labeled Dextran LadderAB Labeled Dextran LadderUPLC Separation UPLC Separation


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


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


22--AB Labeled High Mannose GlycansAB Labeled High Mannose Glycans(RNase b) UPLC Separation (RNase b) UPLC Separation

EU

-0.50

0.00

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1.00

1.50

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2.50

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4.50

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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


2AB glycans from Fetuin (10 pmol)2AB glycans from Fetuin (10 pmol)


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


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


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


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


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


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


Pc,4σ = 348


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


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

uplc peptide mapping and n-glycan fingerprinting · 3 bradykinin rppgfspfr 4 angiotensin ii...

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