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The world leader in serving science Global BioPharma Summit Advanced Fragmentation Techniques for BioPharma Characterization

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The world leader in serving science

Global BioPharma Summit

Advanced Fragmentation Techniques for BioPharma Characterization

2

Different modes of fragmentation to answer different questions or for different assays

(Figure from Coon et al., Analytical Chemistry, 2009)

Peptide Mapping

Site occupancy of PTM

for mAb or site of

conjugation for ADC

Top-Middle down

3

MS Tools for Major Biopharma Characterization Workflows

Mode of fragmentation: HCD

CID

ETD

EThcD

ETciD

(UVPD - not commercial)

Mode of fragmentation: HCD

For all the routine

characterization workflows.

From routine to the

most challenging tasks.

4

Etanercept: O-glycosylation

5

O-glycosylation of Etanercept

Etanercept

(trade name Enbrel)LPAQVAFTPYAPEPGSTC18RLREYYDQTAQMC31C32SKC35SPGQHAKVFC45TKTSDTVC53DS

C56EDSTYTQLWNWVPEC71LSC74GSRC78SSDQVETQAC88TREQNRIC96TC98RPGWYC104ALS

KQEGC112RLC115APLRKC121RPGFGVARPGTETSDVVC139KPC142APGTFSNTTSSTDIC157R

PHQIC163NVVAIPGNASMDAVC178TSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPST

SFLLPMGPSPPAEGSTGDEPKSC240DKTHTC246PPC249PAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTC281VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN

GKEYKC341KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC387LVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC445SVMHEALHNH

YTQKSLSLSPGK

1) 3 N-glycan sites

2) 13 reported O-glycosylation sites

Sialic acids from the O-glycan were removed prior trypsin digestion

Peptide mapping was performed on the tryptic digest

6

Results for peptide SMAPGAVHLPQPVSTR after processing HCD data

RT: 27.13 - 35.10 SM: 7G

27.5 28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0 32.5 33.0 33.5 34.0 34.5 35.0

Time (min)

0

50

100

0

50

100

0

50

100

Rela

tive A

bundance 0

50

10032.43

34.11

31.57

30.40

29.17 32.24 32.39 32.6030.96 31.12 33.2732.9829.83 33.9733.8129.72

29.07

27.82 29.9929.60 32.7130.26 30.53 31.0430.69

27.75

35.0533.7430.26 32.43 33.4429.81 33.9530.73

NL: 5.21E5

m/z= 549.9566-549.9620 F: ms MS 20170406_Etanercept_Ocore_HCD30_test2

NL: 3.95E7

m/z= 671.6666-671.6734 F: ms MS 20170406_Etanercept_Ocore_HCD30_test2

NL: 6.19E7

m/z= 1189.5621-1189.5739 F: ms MS 20170406_Etanercept_Ocore_HCD30_test2

NL: 2.68E5

m/z= 686.5336-686.5941 F: ms MS 20170406_Etanercept_Ocore_HCD30_test2SMAPGAVHLPQPVSTR

SMAPGAVHLPQPVSTR +

SMAPGAVHLPQPVSTR +

S186MAPGAVHLPQPVS199T200R

HCD

7

8

Data acquisition method on a Fusion Lumos: ETD methods

Flexibility and ease of use to design acquisition method

Charge 3ETD reaction time = 60 ms

Charge 4ETD reaction time = 40 ms

Charge 5 - 7ETD reaction time = 20 ms

Scan priority

DDA method used to collect data to identify the sites of O-glycosylation.

9

Data acquisition method on a Fusion Lumos: ETD methods

Flexibility and ease of use to design acquisition method

10

Data acquisition method on a Fusion Lumos: ETD methods

Other useful methods:

* HCD followed by ETD for every precursors.

* HCD followed by ETD when specific product ions are present in the HCD MS2 spectrum

11

12

13

14

15

Results for peptide SMAPGAVHLPQPVSTR after processing ETD data

RT: 27.13 - 35.10 SM: 7G

27.5 28.0 28.5 29.0 29.5 30.0 30.5 31.0 31.5 32.0 32.5 33.0 33.5 34.0 34.5 35.0

Time (min)

0

50

100

0

50

100

0

50

100

Rela

tive A

bundance 0

50

10032.43

34.11

31.57

30.40

29.17 32.24 32.39 32.6030.96 31.12 33.2732.9829.83 33.9733.8129.72

29.07

27.82 29.9929.60 32.7130.26 30.53 31.0430.69

27.75

35.0533.7430.26 32.43 33.4429.81 33.9530.73

NL: 5.21E5

m/z= 549.9566-549.9620 F: ms MS 20170406_Etanercept_Ocore_HCD30_test2

NL: 3.95E7

m/z= 671.6666-671.6734 F: ms MS 20170406_Etanercept_Ocore_HCD30_test2

NL: 6.19E7

m/z= 1189.5621-1189.5739 F: ms MS 20170406_Etanercept_Ocore_HCD30_test2

NL: 2.68E5

m/z= 686.5336-686.5941 F: ms MS 20170406_Etanercept_Ocore_HCD30_test2

S186MAPGAVHLPQPVS199T200R

SMAPGAVHLPQPVSTR

SMAPGAVHLPQPVSTR

SMAPGAVHLPQPVSTR

SMAPGAVHLPQPVSTR

ETD

All of the O-glycosylation sites of peptide SMAPGAVHLPQPVSTR were successfully identified

16

Etanercept: leveraging ETD and MSn

for disulfide bond mapping

17

Disulfide bond mapping: Etanercept

Etanercept

(trade name Enbrel)

LPAQVAFTPYAPEPGSTC18RLREYYDQTAQMC31C32SKC35SPGQHAK

VFC45TKTSDTVC53DSC56EDSTYTQLWNWVPEC71LSC74GSRC78SSD

QVETQAC88TREQNRIC96TC98RPGWYC104ALSKQEGC112RLC115APL

RKC121RPGFGVARPGTETSDVVC139KPC142APGTFSNTTSSTDIC157RPHQIC163NVVAIPGNASMDAVC178TSTSPTRSMAPGAVHLPQPVST

RSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSC240DKTHT

C246PPC249PAPELLGGPSVFLFPPKPKDTLMISRTPEVTC281VVVDV

SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW

LNGKEYKC341KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM

TKNQVSLTC387LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

FFLYSKLTVDKSRWQQGNVFSC445SVMHEALHNHYTQKSLSLSPGK

18

Disulfide bond mapping: Etanercept

Etanercept

(trade name Enbrel)

LPAQVAFTPYAPEPGSTC18RLREYYDQTAQMC31C32SKC35SPGQHAK

VFC45TKTSDTVC53DSC56EDSTYTQLWNWVPEC71LSC74GSRC78SSD

QVETQAC88TREQNRIC96TC98RPGWYC104ALSKQEGC112RLC115APL

RKC121RPGFGVARPGTETSDVVC139KPC142APGTFSNTTSSTDIC157RPHQIC163NVVAIPGNASMDAVC178TSTSPTRSMAPGAVHLPQPVST

RSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSC240DKTHT

C246PPC249PAPELLGGPSVFLFPPKPKDTLMISRTPEVTC281VVVDV

SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW

LNGKEYKC341KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM

TKNQVSLTC387LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS

FFLYSKLTVDKSRWQQGNVFSC445SVMHEALHNHYTQKSLSLSPGK

The N-terminal side of this sample is not as expected. The first 2 amino acids are missing.

19

Disulfide Bond Mapping: Etanercept

Etanercept

(trade name Enbrel)

LPAQVAFTPYAPEPGSTC18RLREYYDQTAQMC31C32SKC35SPGQHAKVFC45TKTSDTVC53DS

C56EDSTYTQLWNWVPEC71LSC74GSRC78SSDQVETQAC88TREQNRIC96TC98RPGWYC104ALS

KQEGC112RLC115APLRKC121RPGFGVARPGTETSDVVC139KPC142APGTFSNTTSSTDIC157R

PHQIC163NVVAIPGNASMDAVC178TSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPST

SFLLPMGPSPPAEGSTGDEPKSC240DKTHTC246PPC249PAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTC281VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN

GKEYKC341KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC387LVKGFYP

SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC445SVMHEALHNH

YTQKSLSLSPGK

The N-terminal side peptide contains one cysteine.

The HCD search result of the non-reduced sample suggests that the N-terminal peptide is

linked to two other peptides through disulfide bonds.

20

Leveraging ETD and MSn for disulfide bond mapping

Workflow of the MS2 ETD – MS3 HCD for disulfide bond peptide identification

Ultra-High Field Orbitrap

Analyzer

Dual-Pressure

Linear Ion Trap

Ion Routing

Multipole

3 fragment channels after ETD of disulfide bond peptide

21

Leveraging ETD and MSn for disulfide bond mapping

Workflow of the MS2 ETD – MS3 HCD for disulfide bond peptide identification

Ultra-High Field Orbitrap

Analyzer

Dual-Pressure

Linear Ion Trap

Ion Routing

Multipole

3 fragment channels after ETD of disulfide bond

peptide

22

Peptide: AQVAFTPYAPEPGSTC18R-EYYDQTAQMC31C32SK-VFC45TK

MS

2 E

TD

S

pe

ctr

um

de

co

nv

olu

ted

sp

ectr

um

23

Peptide: AQVAFTPYAPEPGSTC18R-EYYDQTAQMC31C32SK-VFC45TK

MS3 HCD spectrum of AQVAFTPYAPEPGSTC18R.

24

Peptide: AQVAFTPYAPEPGSTC18R-EYYDQTAQMC31C32SK-VFC45TK

MS3 HCD spectrum of EYYDQTAQMC31C32SK using a) the Orbitrap

or b) the ion trap mass analyzers.

25

Peptide: AQVAFTPYAPEPGSTC18R-EYYDQTAQMC31C32SK-VFC45TK

MS3 HCD spectrum of VFC45TK.

26

Antibody Drug Conjugates

27

ADCs are heterogeneous mixture

Lysine-linkedCysteine-linked

FDA-approved ADCs

Brentuximab vedotin

Adcetris®

Trastuzumab Emtansine

Kadcyla® Light Chain: 12 Lysines

Heavy Chain: 32 Lysines

Peptide mapping

Goal: Identifying conjugated peptides and sites of conjugation

28

29

Trastuzumab emtansine: conjugated peptides

Subset of identified conjugated peptides

TKPRASQDVNTAVAWYQQKPGKALPAPIEKTISKAKGQPRADYEKHK

# K

2

1

2

2

3

2

1

30

31

32

Trastuzumab emtansine site occupation: ETD spectrum

20160525_fsn10361_TDM1_smart_mNBA_targetETD_0P3_20ms_04pmNBA_2AfteCali #5880 RT: 46.41 AV: 1 NL: 3.55E4F: FTMS + p ESI d Full ms2 [email protected] [150.0000-2000.0000]

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

900.1154z=3

649.9147z=5

812.3932z=4

548.2264z=1 1083.1908

z=3

304.1607z=1

1350.1721z=2

982.1383z=3 1160.5668

z=1

696.0023z=3

763.3984z=3

1255.1453z=2 1624.7864

z=2

1451.6891z=?

1376.7234z=1

1540.7869z=1

419.1882z=?

1726.8646z=1

176.1024z=?

518.2562z=?

1861.9128z=1

260.1471z=?

359.6571z=?

Z3

299.1840 (+1)

( -3.0 ppm)

Z4

692.3253 (+2)

( -1.9 ppm)

Dmass = 1084.4594 DaK (128.0949) + MCC-DM1 (956.3644)

z3z4

ASQDVNTAVAWYQQK39PGK42APK

45.99 min; m/z= 649.916 (+5); ETD

ETD spectrum allows unambiguous

identification of the conjugation site

33

Trastuzumab emtansine site occupation

20160525_fsn10361_TDM1_smart_mNBA_targetETD_0P3_20ms_04pmNBA_2AfteCali #5880 RT: 46.41 AV: 1 NL: 3.55E4F: FTMS + p ESI d Full ms2 [email protected] [150.0000-2000.0000]

200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e A

bu

nd

an

ce

900.1154z=3

649.9147z=5

812.3932z=4

548.2264z=1 1083.1908

z=3

304.1607z=1

1350.1721z=2

982.1383z=3 1160.5668

z=1

696.0023z=3

763.3984z=3

1255.1453z=2 1624.7864

z=2

1451.6891z=?

1376.7234z=1

1540.7869z=1

419.1882z=?

1726.8646z=1

176.1024z=?

518.2562z=?

1861.9128z=1

260.1471z=?

359.6571z=?

c14

790.3785 (+2)

( -3.3 ppm)

c17

931.4632 (+2)

( -2.1 ppm)

Dmass = 282.1694 DaK (128.0949) + P (97.0528) +G (57.0215)

c17c14

ASQDVNTAVAWYQQKPGKAPK

45.99 min; m/z= 649.916 (+5); ETD

34

Trastuzumab emtansine: conjugated peptides

Subset of identified conjugated peptides

# K

2

1

2

2

3

2

1

35

36

Middle-down: ETD

37

Main parameters that can be controlled for ETD fragmentation

on an Orbitrap™ Fusion™ mass spectrometer.

• Isolation window

• AGC target for precursor ions and reagent

• Reaction time

• Supplemental energy

38

Light Chain of Trastuzumab

ETD settings: 300Da isolation window, 3E5 precursor, 7E5 reagent, 10 ms reaction time

Due to the complexity of the spectra in top-down analysis, high resolution is required

MS/MS

39

Effect of parent ion AGC and ETD reaction time on sequence coverage (Trastuzumab)

% residue cleavages for AGC target value of 3E5 or 1E6 at different ETD reaction times

Ion Routing Multipole

C-Trap

Ion Trap

HPC

LPC

MP0Q1

1st parent ion isolation in Q1

Orbitrap

1st parent: ETD dissociation

1st parent trapped in the ion trap

Injection of ETD reagent

ETD

LC

40

Effect of parent ion AGC and ETD reaction time on sequence coverage (Trastuzumab)

% residue cleavages for AGC target value of 3E5 or 1E6 at different ETD reaction times

Fc FdLC

41

Effect of parent ion AGC and ETD reaction time on sequence coverage (Trastuzumab)

% residue cleavages for AGC target value of 3E5 or 1E6 at different ETD reaction times

LC

42

Middle Down: Orbitrap Tribrid Fusion Lumos

High sequence coverage for the light chain, Fc and Fd

were obtained from the combined ETD and EThcD

experiments.

LC Fd

Fc

43

NIST mAb: middle down experiment using ETD and UVPD F

c (

52

%)

Fd

(43

%)

LC

(6

0%

)

One acquisition with ETD 10 ms One acquisition with UVPD 12 ms

Fc

(3

2%

)F

d(2

6%

)L

C (

40

%)

ETD 10 ms + UVPD 12 ms

Fc

(6

7%

)F

d(6

1%

)L

C (

78

%)

44

NIST mAb: middle down experiment using ETD and UVPD F

c (

52

%)

Fd

(43

%)

LC

(6

0%

)

One acquisition with ETD 10 ms One acquisition with UVPD 12 ms

Fc

(3

2%

)F

d(2

6%

)L

C (

40

%)

ETD 10 ms + UVPD 12 ms

Fc

(6

7%

)F

d(6

1%

)L

C (

78

%)

45

Hydrogen Deuterium Exchange

46

HDX-MS workflow

MS1

47

Moving from MS only to MS/MS HDX experiment using ETD

•Minimal deuterium scrambling ETD measurement with different instruments and source

conditions(No deuterium is retained on Histidine due to fast exchange with solvent)

HHHHHHIIKIIK

0.0

0.5

1.0

1.5

2.0

2.5

3.0

C2 C3 C4 C5 C6 C7 C8 C9 C10

De

ute

riu

m C

on

tent

Orbitrap Fusion

Literature

(Martin Zehl, Kasper D. Rand, Ole N. Jensen, and Thomas J. D. Jørgensen

J. AM. CHEM. SOC. 2008, 130, 17453–17459 9 17453)

Under normal operation condition, minimal scrambling is observed on the ETD Spectrum

48

Moving from MS only to MS/MS HDX experiment using ETD

Significant change was observed around Z8-Z10 is

consistent with the C fragments plot. Combine

the two plot results, the potential binding site was

predicted.

Deuterium labeled protein and protein+ligand

biding sample’s C and Z fragments mass

differences vs. sequence position. From C3 to C14

mass differences were from 0.03 to 0.2. Start from

C17, the Δ mass increased to around 1 and stay at

the same level for the rest of the C fragments. C14

was identified as significant change area.

Pinpoint the Protein Ligand Binding Site with ETD

Deuterium labeled protein and protein+ligand

biding sample’s C and Z fragments mass

differences vs. sequence position. From C3 to C14

mass differences were from 0.03 to 0.2. Start from

C17, the Δ mass increased to around 1 and stay at

the same level for the rest of the C fragments. C14

was identified as significant change area.

49

Summary

• ETD is a must have type of

fragmentation for in-depth

characterization of biotherapeutic

proteins.

• High sequence coverage for middle-

down experiments allows quick

characterization assays.

• Orbitrap Fusion Lumos offers multiple

types of fragmentation and ease of use

to tackle the most challenging task.

50

Thank you !

Acknowledgements

• Kelly Broster

• Rowan Moore

• Terry Zhang

• Aaron Bailey

• Kyle D’Silva

• Michael Blank

• Simon Cubbon

• Stephane Houel

• Jonathan Josephs

• John Rontree

• Jennifer Sutton

• Mark Sanders

• Helene Cardasis

• Seema Sharma

• Romain Huguet

• Vlad Zabrouskov