higher quality peptide separations using hybrid particle

Charged Surface Hybrid C18 for

High Resolution LC and LC/MS Peptide

Separations

©2013 Waters Corporation 1

Higher Quality Peptide Separations Using

Hybrid Particle-Based Reversed-Phase Columns and CSH Technology

Reversed Phase Peptide SeparationsReversed Phase Peptide Separations

� Peptide separations – critically important– peptide mapping, bottom-up proteomics …– reversed phase– challenges remain

Peptide

TFA

TFA

LC performance

MS performance

� Ionic analytes – peptides– Secondary interactions - Poor peak shape

– Overloading at very low loads (<< neutrals)


� Still needed:– high resolution, high sensitivity peptide

separations regardless of eluent additive

– i.e. formic acid (FA) separations for LC-MS

– Overloading at very low loads (<< neutrals)

– MS signal suppressing ion pairing agents needed

Charged Surface

Hybrid


AgendaAgenda

� The Peak Shape Problem – Column Chemistries– CSH Technology– Peak Capacity

� CSH130 C18 and separations without TFA– LC-MS of protein digests– Small protein separations


– Small protein separations

� Peptide Mapping a Therapeutic mAb– CSH130 C18 for LC-UV-MS – Disulfides and Deamidation

� Peptide Separation Technology (PST) Columns – Quality control - QC tested with digests

� Analytical Standards and Reagents (ASR)

The Peak Shape Problem


The Peak Shape Problem

Competitor’s “Industry Standard” Competitor’s “Industry Standard” 5 µm Porous Silica C18 5 µm Porous Silica C18


Competitor’s “Industry Standard” C18 2.1 x 250 mm, Porous 5 µm, 300Å ACQUITY UPLC® H-Class Bio

2% ACN for 1 min, then to 50% ACN over 60 min0.3 mL/min40°CUV @ 214 nm / Xevo® G2 QTOF5.6 µg MassPREP Peptide Mixture

Peptide Sequence

1 RASG-1 RGDSPASSKP

2 Angiotensin 1-7 DRVYIHP

3 Bradykinin RPPGFSPFR

4 Angiotensin II DRVYIHPF

5 Angiotensin I DRVYIHPFHL

6 Renin Substrate DRVYIHPFHLLVYS

7 Enolase T35 WLTGPQLADLYHSLMK

8 Enolase T37 YPIVSIEDPFAEDDWEAWSHFFK

9 Melittin GIGAVLKVLTTGLPALISWIKRKRQQ

MassPREP™ Peptide Mixture

Bridged Ethanes

In Silica Matrix

U.S. Patent No. 6,686,035 B2

and others patent pending

Organo Silica Hybrid Particles

Ethylene Bridged Hybrid - BEH Technology™

©2013 Waters Corporation 7Anal. Chem. 2003, 75, 6781-6788

Tetraethoxysilane Bis(triethoxysilyl)ethane

+4

Polyethoxysilane

Si

EtO

EtO OEtEtO

Si

EtO

EtOCH2

EtO

CH2Si

OEt

OEt

OEtSi

EtO

O

CH2 CH2

SiO

Si

EtO

OEt

Si O

O

OEt

O

Si

O

Si

OEt

O

O

OEt

Et

Et

n

Organo Silica Hybrid Particles– pH stability

– Reduced ionic interactions

– Basis of Peptide Separation Technology

Small Particle SizeSmall Particle Size

Porous

Particle

Peptides

Mobile Phase

1500 Da Peptide

23.5 µm


� Diffusion distances decrease– Reduced Eddy diffusion– Improved mass transfer kinetics

� Column efficiency� Narrower peaks

Adsorption Equilibria

Diffusion-related band broadening

0

1

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2

Velocity (mm/sec)

H (mm)

1.7 µm

2.1 mm ID40

µL/min400 µL/min

Waters BEH130 C18 1.7 µm Waters BEH130 C18 1.7 µm

3 4 5 6

3 4 5 6

Competitor’s “Industry Standard”

5 µm C18


Waters ACQUITY UPLC BEH130 C182.1 x 150 mm, Porous 1.7 µm, 130Å

Peptide Sequence

1 RASG-1 RGDSPASSKP









Si

EtO

O

CH2 CH2

SiO

Si

EtO

OEt

Si O

O

OEt

O

Si

O

Si

OEt

O

O

OEt

BEH130 C18

Bridged Ethyl Hybrid

PoreSize(Å)

Ligand

30 40

Time (min)

3 4 5 6

A New Column Chemistry A New Column Chemistry –– CSH130 C18 CSH130 C18


Waters ACQUITY UPLC CSH130 C182.1 x 150 mm, Porous 1.7 µm, 130Å

Peptide Sequence

1 RASG-1 RGDSPASSKP









A New Column A New Column Chemistry Chemistry –– CSH130 C18 CSH130 C18


Charged Surface Hybrid (CSH) TechnologyCharged Surface Hybrid (CSH) Technology

Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography.

P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. WalterWaters White Paper 720003929EN 2011

� Expands upon the robust BEH particle technology

patent pending


Peptide

� Expands upon the robust BEH particle technology

� CSH130 C18 = BEH130 base particle + low level of basic moieties+ trifunctional C18/end cap

� Acidic pH

Positive Surface Charge

100%

Peak CapacityPeak Capacity

� Peak Capacity =– The number of peaks that can be separated within a retention window

Neue, U. D., J Chromatogr A 2005, 1079 (1-2), 153-61.

– The best metric for determining the quality of gradient separations


0%

50%

Peak Height

tgradient

wh

2.35σ

4σw4σ13.4%

9 peakscould resolve ~300-400

Peak Capacity Peak Capacity -- FA FA vsvs TFATFA

220

270

320

370

c,4σ


20

70

120

170

220

0,00 0,05 0,10

P c,4

Percent TFA0.050.05

0.100.00

0.000.10

% TFA% FA

Competitor’s “Industry Standard” C18 5 µm2.1 x 250 mm


220

270

320

370

c,4σ

BEH130 C18 1.7 µm2.1 x 150 mm


20

70

120

170

220

0,00 0,05 0,10

P c,4

Percent TFA


0.050.05

0.100.00

0.000.10

% TFA% FA

220

270

320

370

c,4σ


BEH130 C18 1.7 µm2.1 x 150 mm

Competitor’s SPP “Peptide” C18 1.7 µm2.1 x 150 mm


20

70

120

170

220

0,00 0,05 0,10

P c,4

Percent TFA


0.050.05

0.100.00

0.000.10

% TFA% FA


220

270

320

370

c,4σ

Competitor’s SPP “Peptide” C18 1.7 µm2.1 x 150 mm

BEH130 C18 1.7 µm2.1 x 150 mm

CSH130 C18 1.7 µm2.1 x 150 mm

20%

90%


20

70

120

170

220

0,00 0,05 0,10

P c,4

Percent TFA0.050.05

0.100.00

0.000.10

% TFA% FA


270

320

370

MS Signal MS Signal -- FA FA vsvs TFATFA

CSH130 C18 1.7 µm

Competitor’s SPP “Peptide” C18 1.7µm

BEH130 C18 1.7 µm

4

2

Fold Decrease in MS Peak Area

Peak Capacity MS Signal


20

70

120

170

220

0,00 0,05 0,10

P c,4σ

Percent TFA0.050.05

0.100.00

0.000.10

% TFA% FA

Competitor’s “Industry Standard” C18 5 µm

12

10

8

6

0,00 0,05 0,10

Fold Decrease in MS Peak

Percent TFA0.050.05

0.100.00

0.000.10

% TFA% FA

350

400

450High Mass Load

CSH C18

BEH C18

A 6 µg of mixture

LoadabilityLoadability

Attribute – how much analyte can be loaded before peak shape deteriorates

CSH130 C18 1.7 µm

Typical Mass Load

350

400

450Low Mass Load

CSH C18

BEH C18

BCSH C18

BEH C18

0.6 µg of mixture

CSH130 C18 1.7 µm

BEH130 C18 1.7 µm

Low Mass Load*Previously shown


Pc,4σ

150

200

250

300

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10

Pc,4σ

Percent TFA0.05

0.05

0.00

0.10

0.10

0.00

% TFA

% FA

BEH130 C18 1.7 µm

150

200

250

300

0.00 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.10

Pc,4σ

Percent TFA

0.100.10

0.000.05

0.05

0.00

0.10

0.10

0.00

BEH130 C18 1.7 µm

CSH130 C18 and Separations without TFA


CSH130 C18 and Separations without TFA

LCLC--MSMS


0.1% TFA

10x drop in sensitivityWaters ACQUITY UPLC CSH130 C18

2.1 x 150 mm, 1.7 µm, 130Å 2% ACN for 1 min,then to 50% ACN over 60 min0.3 mL/min40°C

ACQUITY UPLC H-Class BioXevo G2 QTof500 pmol MassPREP Enolase Digestp/n 186002337

CSH130 C18 CSH130 C18 vsvs Other ChemistriesOther Chemistries

0.1% FA


Improvement for both optical and MS detection

LCLC--MS MS Retention and SelectivityRetention and Selectivity


Large Peptides/Small ProteinsLarge Peptides/Small Proteins0.1% FA0.1% FA

130 Å

Peptide/Protein kDa

1 Bradykinin 1.1

2 Renin Substrate 1.8

3 Ubiquitin 8.6

4 Cytochrome C (Equine) 12.4

5 Insulin(Bovine) 5.7

6 Melittin 2.8


300 ÅACQUITY UPLC H-Class BioUV @ 214 nm / Xevo G2 QTOF 1 µg each component

2.1 x 150 mm columns2% ACN for 1 min,then to 50% ACN over 60 min0.3 mL/min40°C

Peptide Mapping of a Therapeutic mAb


Peptide Mapping of a Therapeutic mAb

Peptide Mapping a Therapeutic Peptide Mapping a Therapeutic mAbmAb

� Trastuzumab (Herceptin; Genentech) – Breast Cancer, Anti-HER2– One of the highest grossing therapeutic mAbs (~5 billion $/yr)

– Biosimilars

– Basis for a new ADC (Trastuzumab emantansine)

o Phase III clinical trials completed

� High peak capacity at mass loads to detect trace modifications and


� High peak capacity at mass loads to detect trace modifications and thoroughly characterize

o Disulfide linkages

o Deamidation

o Oxidation

o Glycosylation

o Conjugation in ADCs

JCO 2010;28:2698-2704

NonNon--Reduced LysReduced Lys--C Peptide MappingC Peptide Mapping

Anal Biochem 2011, 411 (2), 284-91.

A recent Amgen protocol:

� Non-Reduced Lys-C Digests– Minimal complexity + disulfides preserved

– 27 different linear peptides

– 8 different disulfide linked peptides

– 150 to 11,000 Da


Heavy Chain

Light Chain

1-30 31-43 44-65 66-76 77-124 125-136 137-150 151-213 214-216 217 218-221 222-225 226-251 252-277 278-291 292-320 321-323 324-325 326-329 330-337 338-341 342-343 344-363 364-373 374-395 396-412 413-417 418-442 443-449

1-42 43-45 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-214

1-30 31-43 44-65 66-76 77-124 125-136 137-150 151-213 214-216 217 218-221 222-225 226-251 252-277 278-291 292-320 321-323 324-325 326-329 330-337 338-341 342-343 344-363 364-373 374-395 396-412 413-417 418-442 443-449

1-42 43-45 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-214

S-S S-S

S-SS-S S-S

S-S S-S

S-S S-S

S-S S-S

S-SS-S

S-S

S-S

S-S

L:

H:

H:

L:

1-42 43-45 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-214

1-30 31-43 44-65 66-76 77-124 125-136 137-150 151-213 214-216 217 218-221 222-225 226-251 252-277 278-291 292-320 321-323 324-325 326-329 330-337 338-341 342-343 344-363 364-373 374-395 396-412 413-417 418-442 443-449

1-30 31-43 44-65 66-76 77-124 125-136 137-150 151-213 214-216 217 218-221 222-225 226-251 252-277 278-291 292-320 321-323 324-325 326-329 330-337 338-341 342-343 344-363 364-373 374-395 396-412 413-417 418-442 443-449

1-42 43-45 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-214Disulfide Bond

Lys-C Cleavage(C-terminal Side of Lys)

400

Method ConsiderationsMethod Considerations

ACQUITY UPLC BEH130 C18 1.7µm

ACQUITY UPLC CSH130 C18 1.7µm 2.1 x 150 mm

1.2

0.02% TFA0.08% FA

CSH130 C18

Elevated Temperature (60°C)� Optimize peak capacity

� Small compromise to MS Sensitivity

� Improved peak shape and recovery

� 5-11 kDa species


12

10

8

6

4

2

150

200

250

300

350

0.00 0.02 0.04 0.06 0.08 0.10

Peak Capacity

Percent TFA

0.0

0.2

0.4

0.6

0.8

1.0

92.5 95 97.5 100 102.5 105 107.5 110 112.5

A210

Time (min)

40°C

60°C

5-11 kDa Species BEH130 C18

Anal Chem 2011, 83 (15), 5912-9.Anal Biochem 2011, 411 (2), 284-91.MAbs 2010, 2 (4)J Biol Chem 2009, 284 (51), 35390-402.

CSH130 C18

BEH130 C18

MS Signal

NonNon--Reduced LysReduced Lys--C Peptide MapsC Peptide MapsTrastuzumabTrastuzumab


LCLC--UVUV--MS with an MS with an MSMS--Compatible Mobile PhaseCompatible Mobile Phase

CSH130 C18

Low TFA mobile phase

Optimized Peak Capacity +MS signal ≥ UV signal

ACQUITY H-Class Bio


m/z200 400 600 800 1000 1200 1400 1600 1800

ACQUITY H-Class BioCSH130 C18 1.7 µm

UV Detector

ESI-MSXevo G2 QTof

3+

2+

0.3

0.5

0.7

0.9

1.1

A210

L:208-214 x H

:222-225

H364-373 x H:418-442

H:252-277 x H:324-325

L:127-145 x L:191-207

H:1-30 x H:77-124

H:226-251 x H:226-251

L:1-42 x L:46-103

H:137-150 x H:151-213

Disulfide CharacterizationDisulfide Characterization

CSH130 C18 - Optimized Gradient

Non-Reduced


-1.1

-0.9

-0.7

-0.5

-0.3

-0.1

0 10 20 30 40 50 60 70 80 90 100 110 120 130

A210

Time (min)

0.1

L:208-214

L:191-207

H:137-150

H364-373

H:418-442

H:252-277

H:1-30

L:127-145

H:226-251

L:46-103

H:151-213

H:77-124

L:1-42

Reduced

Blank

1-42 43-45 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-214

S-S S-S

S-SS-S S-S S-SS-S

S-S

S-S

1-42 43-45 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-2141-42 43-45 46-103 104-107 108-126 127-145 146-149 150-169 170-183 184-188 189-190 191-207 208-214

222-225 226-251 252-277

1-30 31-43 44-65 66-76 77-124 125-136 137-150 151-213 214-216 217 218-221 222-225 226-251 252-277 278-291 292-320 321-323 324-325 326-329 330-337 338-341 342-343 344-363 364-373 374-395 396-412 413-417 418-442 443-449

Assaying Assaying DeamidationDeamidation

44GLEWVARIYPTNGYTRYADSVK65

CSH130 C18 - Optimized Gradient

� Binding region from the heavy chain

� Prone to deamidation


0

0.1

0.2

0.3

0.4

0.5

59 60 61 62 63 64 65

A210

Time (min)

0E+0

3E+5

853 854 855 856

H:44-65

**

92 % Asn

7 % isoAsp1 % Asp

Intensity

0E+0

2E+4

853 854 855 856

0E+0

5E+3

853 854 855 856m/z

Mass shift+0.98 Da

*

*

Deamidated

Unmodified

ConclusionsConclusions

� CSH130 C18 Peptide Separations– Improved loadability and greater peak capacity vs. other C18 columns

– Excellent peak shape with both TFA and FA mobile phases (highly compatible with MS)

– 130Å pore size optimal for species up to 8-10 kDa


– Unique selectivity (basic residues)

o Less retentive

� Exceptional chemistry for peptide separations– Peptide mapping

... proteomics and peptide isolation

Peptide Separation Technology Columns


Peptide Separation Technology Columns

New Addition to the Suite of New Addition to the Suite of Waters Peptide Separation Technology Waters Peptide Separation Technology

� Peptide Separation Technology– Peptide C18 Columns– QC Tested with Digests

� BEH Technology– BEH130 C18 and BEH300 C18– Industry Leading Performance for Most Applications


– Industry Leading Performance for Most Applications– Two Pore Sizes– Particle Sizes: 1.7 µm, 3.5 µm, 5 µm– Analytical, Nano and Prep Columns

� Now even more tools in the toolbox … CSH Technology– CSH130 C18– Best columns for formic acid separations– Unique selectivity

UPLC UPLC andand HPLCHPLC

0.2

0.4

0.6

0.8

1.0A214

0.2

0.4

0.6

0.8

1.0

A214

0.1 % FA

1.7 µm

CSH130 C182.1 x150 mm

0.1 % TFA

~8000 psi

High peak capacity separations not limited to UPLC

CSH130 C18 Peptide Separation Technology Columns

Available Now: Upcoming:


0.0

10 20 30 40 50

Time (min)

0.0

0.2

0.4

0.6

0.8

1.0

14.5 24.5 34.5 44.5 54.5 64.5 74.5

A214

Time (min)

0.0

10 20 30 40 50

Time (min)

0.0

0.2

0.4

0.6

0.8

1.0

13.5 23.5 33.5 43.5 53.5 63.5 73.5

A214

Time (min)

2.5 µm XP~3000 psi

Longer Run Time

LowerPressure

Method Transfer

Available Now: Upcoming:Analytical Columns Nano (75, 150, 300 µm ID)1.7 µm Prep Columns (5 µm) 2.5 µm XP3.5 µm

Quality ControlQuality Control

CSH130 C180.1 % Formic Acid


Quality ControlQuality Control

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Minutes2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

AU

0.40

0.60

0.80

1.00

1.20Batch 108

T1

T13-T14

T14

T4

T9-T10

T10

T8

T15

T19C

T19

Batch 110

T12-T13 T12

� CSH130 C18

� Batch-to-Batch Reproducibility

� Cytochrome C Digest, 0.1% Formic Acid


0.00

0.20

0.40

Minutes2.00 4.00 6.00 8.00

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Minutes2.00 4.00 6.00 8.00

Batch 116

AU

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Minutes2.00 4.00 6.00 8.00

Batch 102

*Data provided by S. McCall/P. Iraneta

Formic Acid

� Each new column will perform comparably toone previously used

Analytical Standards and Reagents (ASR)Analytical Standards and Reagents (ASR)

Digestion Standards

MassPREP Peptide Mixture

Cytochrome C Digestion StandardPart Number: 186006371


Rapigest SF

Amgen Digestion ProtocolAnal Chem. 2009 81(4):1686-92

Useful LiteratureUseful Literature

� Charged Surface Hybrid (CSH) Technology and Its Use in Liquid Chromatography– P.C. Iraneta, K.D. Wyndham, D.R. McCabe, and T.H. Walter

– Waters White Paper 720003929EN 2011

� Increasing Peak Capacity in Reversed Phase Peptide Separations with Charged Surface Hybrid (CSH) C18 Columns– M.A. Lauber, S.M. Koza, K.J. Fountain

– Waters Application Note 720004568EN 2013


� Peptide Mapping and Small Protein Separations with Charged Surface Hybrid (CSH) C18 and TFA-Free Mobile Phases– M.A. Lauber, S.M. Koza, K.J. Fountain

– Waters Application Note 720004571EN 2013

� High Resolution Peptide Separations with a Charged Surface C18 Stationary Phase– M.A. Lauber, S.M. Koza, S.A. McCall, B.A. Alden, P.C. Iraneta, and K.J. Fountain

– Manuscript in Preparation 2013

higher quality peptide separations using hybrid particle

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