June 9th, 2013 AB SCIEX – Beckman Coulter Partner workshop – ASMS Complete sequence coverage in one injection followed by posttranslational modifications

June 9th, 2013 AB SCIEX – Beckman Coulter Partner workshop – ASMS

Complete sequence coverage in one injection followed by posttranslational

modifications and major N-glycosylation characterization of monoclonal

antibodies by sheathless CESI-MS/MS

R. Gahoual1, J-M. Busnel2, J. Chicher3, L. Kuhn3,

P. Hammann3, A. Beck4, Y.N. François1, E. Leize-Wagner1

1Laboratory of Mass Spectrometry of Interactions and Systems, University of Strasbourg (CNRS-UDS UMR 7140)

2Beckman Coulter, Brea (CA, USA)

3IEsplanade Proteomic Facility, IBMC, University of Strasbourg (CNRS-UDS-UPR 9002)

4Centre d’immunologie Pierre Fabre (CIPF)

EUPA – Saint-MaloOctober 17th, 2013

2

Separation in capillary electrophoresis

electrophoretic mobility

electroosmotic mobility

• Analytes are separated depending on their charge and size

• CE provides fast separation

great efficiency

low sample consumption

3

Web of scienceSM search using term “capillary electrophoresis and mass spectrometry”

Num

ber

of p

ublic

atio

nsCE–MS Coupling

4

CE-ESI-MS Coupling

Advantages of CE-MS Great efficiency

Selectivity

Sensitivity

Structural information

Drawbacks of CE-MS Low sample volume (high concentration)

Compatibility of background electrolyte to MS

Difficulty to maintain electrical field

Ultra-low flow rate

5

“sheath liquid” interface is the most common

Addressing CE-MS Limitations

• Over 30 publications describing new interfaces• 3 different categories (sheath liquid, junction liquid, sheathless)

6

CE is a miniaturized technique performing ultra-low flow rates

Decreasing the flow allows for increased sensitivity in the ESI-MS1

“Sheathless” CE-ESI-MS

1Wilm, Mann International Journal of Mass Spectrometry 1994, 136, 167–180

Addressing CE-MS Limitations

7

CESI Interface

30 µm ID separation capillary with outlet portion etched by HF, provides electrical contact

Originally developed by M. Moini at U. of Texas and further developed by Beckman Coulter Inc.

8

CESI Interface

• No sheath liquid is necessary anymore to perform CE-ESI-MS

nano flow rates and increased sensitivity

What are the accessible flow rates?

9

10

CESI Interface Achievable Flow rates

• CESI-MS infusion of intact protein sample

Spray could be obtained using flow rate as low as 4 nL/min

Conditions : Myoglobin 1 μM (in 10% acetic acid), Flow rates 3, 7 - 170 nL/min, Capillary voltage: -1400V, Investigated m/z : 848,94

0 20 40 60 80 100 120 140 160 1800

2

4

6

8

10

12

14

16

18

20

Measured flow rate (nL/min)

CE

sys

tem

ap

pli

ed p

ress

ure

(p

si)

0 5 10 15 20 25

0

1

2

3

Gahoual et al, Analytical and Bioanalytical Chemistry 2013, online available

Conditions : Flowrate 4 - 700 nL/min Capillary voltage : - 1400V, Investigated m/z : 2196

0 300 600 9001E+017

1E+019

1E+021

Flow rate (nL/min)

Sen

siti

vity

(co

un

ts/m

ol)

Infusion of Myoglobin 250nM (in 20mM AceNH4 pH 6,7)

Influence of the flow rate on sensitivity

46 fold increase in sensitivity by decreasing the Flow rate

from 350 to 10 nL/min

Gahoual et al, Analytical and Bioanalytical Chemistry 2013, online available

12

1. Introduction

2. Rapid and multi-level characterization of monoclonal antibody through CESI-MS workflow

Content

13

Monoclonal Antibodies (mAbs)

• Highly specific to the targeted antigen

• Opening new pathways for treatments

• Over 40 mAbs currently approved by FDA (15 in oncology)

• Complex and heterogeneous protein

necessity of precise and high throughput characterization

challenge to analytical sciences

14

Trastuzumab (Herceptin)

Average mass: 148,057 Da (1,328 a.a.)

IgG1

A. Beck et al., Anal. Chem. 2012, 84, 4637-4646

-N300GlcSTY-

15

Bottom-up Approach

• Peptide separation and detection by NanoLC-MS/MS Eksigent nanoLC™ 2D plus system with cHiPLC® System AB SCIEX TripleTOF® 5600 System

• Peptide identification using search algorithm

Mascot algorithm

• Protein enzymatic digestion

Tryptic digestion (conventional in solution protocol)

16

• Trastuzumab peptide mapping using nanoLC-MS/MS

74.6 % 95.4 %

EVQLVESGGG LVQPGGSLRL SCAASGFNIK DTYIHWVRQA PGKGLEWVAR

IYPTNGYTRY ADSVKGRFTI SADTSKNTAY LQMNSLRAED TAVYYCSRWG

GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT

YICNVNHKPS NTKVDKKVEP KSCDKTHTCP PCPAPELLGG PSVFLFPPKP

KDTLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN

STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ

VYTLPPSREE MTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV

LDSDGSFFLY SKLTVDKSRW QQGNVFSCSV MHEALHNHYT QKSLSLSPG

DIQMTQSPSS LSASVGDRVT ITCRASQDVN TAVAWYQQKP GKAPKLLIYS

ASFLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQQ HYTTPPTFGQ

GTKVEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV

DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG

LSSPVTKSFN RGEC

Necessity to perform a different proteolytic digestion and to compile different injections to obtain full sequence coverage

mAb sequence coverage

17

CESI Workflow

18A. Beck et al., Anal. Chem. 2012, 84, 4637-4646



LC : -N30T – (D/isoD, +1 Da)HC : -N55T – (D/isoD, +1 Da)HC : -N387T – (D/isoD, +1 Da)

19

Bottom-up Approach

• Peptide separation and detection by CESI-MS/MS

AB SCIEX TripleTOF 5600 System

• Sequence characterization by MS/MS peptide mapping

• Research of glycosylation and posttranslational modifications

• Protein enzymatic digestion

Same Sample of trastruzumab (same protocol)

20

Trastuzumab MS/MS peptide mapping

Amino acid sequence characterization (trastuzumab)



GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK

DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT










LSSPVTKSFN RGEC

100% sequence coverage could be achieved

in 1 injection through only purely tryptic unmodified peptides

21

mAb MS/MS Peptide Mapping

MS/MS spectrum of digested peptides HT33

LTVDK(288.182 ; 2+)

MS/MS spectrum of digested peptides HT21

DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK(1198.290 ; 6+)

CE allows separation and detection of a wide variety of peptides

22

Variable domain retraced on 98/120 AAs for the HC

Variable domain retraced on 99/107 AAs for the LC

MS/MS spectra obtained through the CESI interface allowed characterization of almost the entire variable domain

mAb MS/MS Peptide Mapping

23A. Beck et al., Anal. Chem. 2012, 84, 4637-4646



LC : -N30T – (D/isoD, +1 Da)HC : -N55T – (D/isoD, +1 Da)HC : -N387T – (D/isoD, +1 Da)

24

PTMs Hot-spot Characterization

6 PTMs hot spots characterized on the same CESI-MS/MS analysis

• Hot-spots detected by CESI-MS/MS :

HC E1 cyclization

HC N55 and N387 deamidation

HC M255 and M431 oxidation

LC N30 deamidation

• Peptides detected intact and modified

N*

N

25

E1/pE1 Characterization

EVQLVESGGGLVQPGGSLR

y(16)y(15)

y(13)y(14)

y(11)

b(3)

b(4)

b(2)

100%

50%

0%

E*VQLVESGGGLVQPGGSLR

b(4)

y(6)

y(8)

y(12)

y(13) y(14)

y(16)

y(17)

y(15)

y(11)

y(7)

y(1)

100%

50%

0%

26

N55 Deamidation Characterization

IYPTNGYTR

N

b(8)

y(8)971.476

y(7)808.419

y(6)711.363y(5)

610.312

y(4)496.266

y(3)439.243

y(2)276.175

y(1)175.126

b(7)b(3)

b(2)

100%

50%

0%

IYPTN*GYTR

N*

y(8)972.472

y(7)808.972

y(6)712.367y(5)

611.317

y(4)496.266

y(3)439.243

y(2)276.175

y(1)175.126 b(3)

b(2)

100%

50%

0%

27

M431 Oxydation Characterization

WQQGNVFSCSVM*HEALHNHYTQK

y(16)

M*

y(12)y(5)y(4)

y(3) y(6)y(7)

y(11)

100%

50%

0%

WQQGNVFSCSVMHEALHNHYTQK

y(23)

y(17)

y(18)

y(16)

y(14)y(15)

y(13)y(12)

y(11)

y(9)y(7)

M100%

50%

0%

28

Trastuzumab glycosylation characterization

Structural characterization

204.0854

366.1242

1216.4796 1318.0205

+MS2(1318.0016), 29.5458-29.5458eV, 46.7min #4186

0

20

40

60

80

100

Intens.[%]

200 400 600 800 1000 1200 1400 1600 m/z

2+2+

2+

2+

2+

2+

HT24

Relative abundance 47.5%

MS/MS spectrum of HT24 – G0F (1217.510, 2+)

+MS2(1581.4166), 45-45eV, 47.1min #4212

0

20

40

60

80

100

Intens.[%]

200 400 600 800 1000 1200 1400 1600 m/z

2+2+

Relative abundance 0.71%

HT24

MS/MS spectrum of HT24 – H5N4F1 (1581.190, 2+)

• CESI-MS/MS method in data dependent analysis acquisition allowed to detect 13 different

glycosylations including fragmentation spectra for 9 of them in a single analysis

Trastuzumab

29

Glycosylation profiling

Glycosylation distribution

H4N4F1 (G

1F)

H3N4F1 (G

0F)

H5N4F1 (G

2F)

H3N4 (G0)

H4N4 (G1)

H4N3F1 (G

1F-GlcN

ac)

H5N4F1SiA

1

H3N3F1 (G

0F-GlcN

ac)

H5N2 (Man

5)

H4N4F1SiA

1

H4N3 (G1-G

lcNac)

H4N3F1SiA

1

H5N4 (G0)

H3N3 (G0-G

lcNac)

H5N4F1SiA

2

H4N3SiA1

H4N5F1

H5N3SiA1

H5N3

H5N3SiA1

H6N4SiA1

H6N4F1

H5N5F1

H5N4F1SiA

1NSiA1

0

10

20

30

40

50

60

70

80

28.26

47.46

5.35 5.431.57 3.04 3.88

1.62 0.48 0.78 0.30 0.81 0.31 0.71

Trastuzumab

30



H4N4F1 (G

1F)

H3N4F1 (G

0F)

H5N4F1 (G

2F)

H3N4 (G0)

H4N4 (G1)

H4N3F1 (G

1F-GlcN

ac)

H5N4F1SiA

1

H3N3F1 (G

0F-GlcN

ac)

H5N2 (Man

5)

H4N4F1SiA

1

H4N3 (G1-G

lcNac)

H4N3F1SiA

1

H5N4 (G0)

H3N3 (G0-G

lcNac)

H5N4F1SiA

2

H4N3SiA1

H4N5F1

H5N3SiA1

H5N3

H5N3SiA1

H6N4SiA1

H6N4F1

H5N5F1

H5N4F1SiA

1NSiA1

0

10

20

30

40

50

60

70

80

28.26

47.46

5.35 5.431.57 3.04 3.88

1.62 0.48 0.78 0.30 0.81 0.31 0.71

Trastuzumab

Possibility to detect very low abundant glycosylation

31



H4N4F1 (G

1F)

H3N4F1 (G

0F)

H5N4F1 (G

2F)

H3N4 (G0)

H4N4 (G1)

H4N3F1 (G

1F-GlcN

ac)

H5N4F1SiA

1

H3N3F1 (G

0F-GlcN

ac)

H5N2 (Man

5)

H4N4F1SiA

1

H4N3 (G1-G

lcNac)

H4N3F1SiA

1

H5N4 (G0)

H3N3 (G0-G

lcNac)

H5N4F1SiA

2

H4N3SiA1

H4N5F1

H5N3SiA1

H5N3

H5N3SiA1

H6N4SiA1

H6N4F1

H5N5F1

H5N4F1SiA

1NSiA1

0

10

20

30

40

50

60

70

80

28.26

47.46

5.35 5.431.57 3.04 3.88

1.62 0.48 0.78 0.30 0.81 0.31 0.71

Trastuzumab

Possibility to detect very low abundant glycosylation

Potential syalylated form detected (confirmation)

32

Trastu biosimilar MS/MS peptide mapping



GDGFYAMDYW GQGTLVTVSS ASTKGPSVFP LAPSSKSTSG GTAALGCLVK

DYFPEPVTVS WNSGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT










LSSPVTKSFN RGEC

Amino acid sequence characterization (Hz5D4)

Each peptide is correctly identified except K217 on the HC

Again complete sequence coverage obtained through tryptic unmodified peptides identification

33

Trastu biosimilar amino acid substitution characterization

175.1235

303.2118

+MS2(517.3055), 32.4557-32.4557eV, 23.4min #2347

0

20

40

60

80

100

Intens.[%]

150 200 250 300 350 400 450 500 m/z

R K D V

VD K R

157.1125

169.1372

211.1573

228.1825

244.1650

256.1768

357.2207

+MS2(314.6916), 20.2997-20.2997eV, 23.5min #2355

0

20

40

60

80

100

Intens.[%]

150 200 250 300 350 400m/z

K V

R V E

y

b

Trastu biosimilar MS/MS peptide mapping

MS/MS spectrum of ion 517.302 (1+) MS/MS spectrum of ion 314.692 (2+)

MS/MS spectra allowed to determine unambiguously Trastu biosimilar

amino acid substitution compared to trastuzumab

V D K R217 V E P K

34

mAb Characterization-Conclusion

• Single analysis of trastuzumab tryptic digest by CESI-MS/MS

Complete sequence coverage on both HC and LC

Characterization of 6 PTMs hot-spots

Structural characterization of 5 major N-glycosylations

100 fmol digested peptides injected

• Use of CE separation mechanism for mAb characterization

Possibility characterize modified and unmodified peptides

In some cases, separation of modified peptide (PTMs)

• This methodology is applicable to various mAbs

Acknowledgments

Emmanuelle Leize-WagnerRabah GahoualMichael Biacchi

Philippe HammannPhilippe Wolf

Lauriane KuhnJohanna Chicher

Esplanade Proteomic Facility(Strasbourg)

Laboratory of Mass Spectrometry of Interactions and System (LSMIS)

Jean-Marc BusnelHans DewaldJeff Chapman

Edna BetgovargezMichel Anselme

Centre d’Immunologie Pierre Fabre

Alain BeckElsa Wagner-Rousset

Marie-Claire Janin-BussatDaniel AyoubOlivier Colas

Acknowledgments

Gary ImpeyJean-Batiste Vincendet

Sujet de thèse déposée à l’EDSC Strasbourg

Documents

June 9th, 2013 AB SCIEX – Beckman Coulter Partner workshop – ASMS Complete sequence coverage in one injection followed by posttranslational modifications