Comprehensive Characterization of Trastuzumab usinga Single-Shot Peptide Mapping Approach with CESI-MS
Edna Betgovargez1, Bryan Fonslow1, Anna Lou1, Zhiqi Hao2, David Horn2, Alain Beck3, Jean-Marc Busnel4, and Hans Dewald1
1AB Sciex LLC, Brea, CA; 2Thermo Fisher Scientific Inc., San Jose, CA; 3Pierre Fabre, Saint-Julien-en-Genevois, France; 4Beckman Coulter Inc., Brea, CA

CESI 8000 Configuration

Relative Abundance

Table 1. Summary of quantified degradative PTMs from triplicate analyses.

Asn300 glycosylation hotspot analysis

RESULTS

130115_cems_tras-20kv5psi60sec60mi_03

1/15/2013 8:47:17 PM

130115_cems_tras-20kv5psi60sec60mi_03 #17155 RT: 38.24 AV: 1 NL: 1.70E6
F: FTMS + p NSI d Full ms2 1030.07@hcd27.00 [100.00-3175.00]
45- H2O

274.0921

40

100% Sequence Coverage of Trastuzumab

3530
366.1395
25
20
292.1025

15
105256.0815
215.0547

232.0812

0200

220245.0771

240

339.1666

296.7999
260

280

300

320

350.1456

340

439.2334

380.0956

360

380

400

420

440

452.2511
460

130115_cems_tras-20kv5psi60sec60mi_03 #17201 RT: 38.33 AV: 1 NL: 1.32E6
F: FTMS + p NSI d Full ms2 1035.40@hcd27.00 [100.00-3190.00]- H2O- H2O274.0921

3025

Figure 3. Triplicate sequence coverages of heavy (left) and light (right) chains
130115_CEMS_Tras-20kv5psi60sec60mi_01_OK

1/15/2013 6:06:11 PM

RT: 20.03 - 50.05
31.99
30.67
30.97
30.16

10080
25.56

60

25.70
28.62

26.50

40
20

35.65
36.00 36.73 38.14
38.50 40.17 41.40 42.07 43.23

21.08 22.01 23.10 24.22 25.24

0100

46.74 47.42 49.00

40220308.0973

256.0812
240260342.1301
389.1811 403.2826

280

300

320

340

360

380m/z400452.2505

423.2210420440460Figure 8. MS/MS spectra of NANA- and NGNA-containing glycopeptides(EEQYNSTYR) from the heavy chain.

CK60

293.1051
270.1452
215.0548 227.1030

NL: 1.52E9m/z=
307.1357-307.1511 F:
ms MS
130115_CEMS_Tras20kv5psi60sec60mi_01
_OK

27.19

80366.1394

290.0870

1546.31

44.57

2010
NL: 3.17E10
TIC F: ms MS
130115_CEMS_Tras- 5
20kv5psi60sec60mi_01
_OK
0200

33.92 34.35

33.42
32.39

30.00

2023.81 24.68 25.92 27.05

21.62 22.27

0100

27.54
27.78

29.57 30.38

31.93

33.39

35.68 36.47

38.02 38.87

40.65 41.24

42.58 43.79 44.66

45.92

47.15 47.83

49.54
NL: 5.50E8m/z=
174.0916-174.1004 F:
ms MS
130115_CEMS_Tras20kv5psi60sec60mi_01
_OK

80

AEK60
402020.65

0100

21.81

23.34 24.34

26.44 27.61

27.95 29.47 30.32
28.5480

33.29

34.56

36.77 37.32

38.83

40.78

42.77 43.36 43.96

46.57 47.20

48.95
NL: 2.80E9
m/z=
448.2658-448.2882 F:
ms MS
130115_CEMS_Tras20kv5psi60sec60mi_01_OKTISK60402027.05 28.13

0100

28.70 30.00

33.23
33.62

48.32

60
33.79
33.89
22.53

02226

35.40 36.49 36.75 38.00

31.36 31.72 32.37

27.84
24283032

NL: 1.76E8
m/z=
1343.6294-1343.6966
F: ms MS
130115_CEMS_Tras20kv5psi60sec60mi_01_OK

Largest tryptic peptide of themixture: Mw=6713.3144 Da

80

2034

36

38

40.41 41.23 42.05

44.06 45.24 46.66 47.15

4044424648.63
4850

Time (min)Figure 4. Separation and identification of very small and large peptides

Degradative PTM hotspot analysis130115_CEMS_Tras-20kv5psi60sec60mi_01_OK

1/15/2013 6:06:11 PM

RT: 29.00 - 39.48
NL: 2.81E9m/z= 628.00-628.01
MS130115_CEM S_Tras20kv5psi60sec60mi_01_OKNL: 2.81E9m/z= 932.50-932.51
MS130115_CEM S_Tras20kv5psi60sec60mi_01_OK

24
2220

Relative Abundance

1816
14
12
108

N-terminal PyroGlu

6

N-terminal Glu

420
29.0

29.5

30.0

30.5

31.0

31.5

32.0

32.5

33.0

33.5

34.0
34.5
Time (min)

35.0

35.5

36.0

36.5

37.0

37.5

38.0

38.5

39.0

RT: 28.00 - 35.01
NL: 5.87E9m/z= 418.22-418.23
MS130115_CEM S_Tras20kv5psi60sec60mi_01_OKNL: 5.87E9m/z= 426.21-426.22
MS130115_CEM S_Tras20kv5psi60sec60mi_01_OK

24
2220

Relative Abundance

18Figure 1. Trastuzumab schematic representing the major PTMs
identifiable by mass spectrometry (disulfide bridge pairings, mainglycoforms, heavy chain N-terminal pyroglutamic acid, C-terminal
lysine clipping, deamidation hot spot in CDRs) and theirassociated mass differences.21614

10

CONCLUSIONS

86

MetOx255

Met25542028.0

28.5

29.0

29.5

30.0

30.5

31.0

31.5
Time (min)

32.0

32.5

33.0

33.5

34.0

34.5

•

35.0

Figure 5. Separation, identification, and relative quantification of N-terminal
pyroglutamate and methionine oxidation on the heavy chain.
1/15/2013 6:06:11 PM•

Asn51 deamidation

RT: 28.00 - 35.00
25

NL: 3.79E9

•

15
10

NL: 3.79E9

50
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

m/z=
543.2665-543.2670
MS130115_CEMS_Tras20kv5psi60sec60mi_01
35.0_OK•Time (min)RT: 26.00 - 34.00
10

NL: 2.67E98

Trp280dioxidation

Trp280

6420
26.0

26.5

27.0

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

Time (min)Lys329 glycation

NL: 7.88E7

REFERENCES

m/z=570.5985-570.6035
MS
130115_CEMS_Tras20kv5psi60sec60mi_01
34.0_OK

Lys3292

NL: 2.00E7

Busnel et al., Anal. Chem. 2010, 82, 9476–9483.
Beck et al., Anal. Chem. 2012, 84, 4637−4646.

m/z=422.9170-422.9195
MS
130115_CEMS_Tras20kv5psi60sec60mi_01
_OK

2520

TRADEMARKS/LICENSING

15

NL: 2.00E710
5026.0m/z=559.9380-559.9400
MS130115_CEMS_Tras20kv5psi60sec60mi_01_OK1

RT: 26.00 - 34.00
3530

CESI-MS was capable of analyzing very small (< 300 Da)
and very large peptides (>8000 Da) under the same
experimental conditions
100% sequence coverage was reproducibly achieved foreach chain with a single run and single enzymatic digestion
from 100 fmol of a monoclonal antibody
Multiple degradative PTM hotspots were reproducibly
identified and quantified with relative abundances over threeorders of magnitude
Reproducible, in-depth characterization and quantification ofa glycosylation hot spot with relative abundances over threeorders of magnitude was achieved

m/z=542.7730-542.7770
MS130115_CEMS_Tras20kv5psi60sec60mi_01
_OK

Asn51

20

Samples were prepared following a classical but short (2hour digestion) protocol using DTT, iodoacetamide, RapiGest,and trypsin. CESI experiments were carried out with a CESI 8000
prototype system equipped with a temperature controlled
autosampler and a power supply with the ability to deliver up to30 kV. Prototype fused-silica capillaries with porous tip wereused. Solutions of 10% acetic acid and 200 mM ammonium
acetate (pH 4) were employed as background electrolyte (BGE)
and leading electrolyte, respectively. CESI separations were
performed at 20 kV, and about 100 fmol (45 nL of a 2 mMsolution) of each tryptic digest were injected per analysis. Eluted
peptides were analyzed using a bench-top quadrupole Orbitrap
mass spectrometer with a data-dependent top ten HCD method.
High resolution HCD spectra were analyzed using Protein Pilot TM(AB SCIEX software) and Byonic (ProteinMetrics).

Table 2. Summary of quantified glycoforms from N300 glycosylation on theheavy chain from triplicate analyses.

12130115_CEMS_Tras-20kv5psi60sec60mi_01_OK

METHODS

480m/z40

Representative mAb (Trastazumab)

Figure 7. Separation by glycan structure and relative quantification ofglycopeptide forms (EEQYNSTYR) from the heavy chain.

Figure 2. Illustrations of CESI 8000 sprayer and cartridge.

Relative Abundance

In the biopharmaceutical industry, comprehensive
characterization of therapeutics-based monoclonal antibodies(mAbs) is crucial, as impurities and heterogeneity can impact
therapeutic safety and/or efficacy. mAbs are large glycosylated
proteins (≈ 150 kDa) containing several post-translational
modifications (PTMs). Peptide mapping of mAbs can provide
critical qualitative and quantitative information about impurities
and heterogeneity.
Capillary electrophoresis (CE) has exceptional separation
efficiency and capabilities for peptide mapping of mAbs by mass
spectrometry. We have previously reported on the developmentof an electrospray ionization (ESI) emitter integrated with capillaryelectrophoresis (CE), combining CE separation and ESI into asingle dynamic process (CESI).1 We present data for the bottomup proteomic peptide mapping of a leading, representative
monoclonal antibody, Trastuzumab (Herceptin) using a single
protease, trypsin. A Beckman Coulter CESI 8000 system soldthrough SCIEX Separations, a part of AB SCIEX, coupled to aThermo Q-Exactive was used for the analysis.
The results illustrate the benefits of the integration of CE
and ESI in a single dynamic process with high resolution mass
spectrometry. That is, the CESI separation provides both theseparation efficiency of CE and low-nanoflow electrospray
conditions to provide the sensitivity to achieve 100% sequence
coverage of the heavy and light chains of this mAb. At flow rates
below 20 nL/min, ionization efficiency is maximized and ion
suppression is dramatically reduced, allowing strong ionization ofmodified peptides and glycopeptides. As all analytes migrate
through the capillary, all peptides, both large and small (3 – 65
amino acids) were identified, separated, and quantified. With thecomprehensive (100%) sequence coverage, identification andquantification of mAb purity, stability, and glycoform heterogeneity
is possible from a single CESI-MS analysis. In particular, we canidentify degradative hotspots like aspargine deamidation,
methionine oxidation, and glutamic acid cyclization from a mere
100 fmol. Exploiting the reduced ion suppression effects from thelow-nanoflow ESI regime, low abundance PTMs were identified
and quantified in the presence of peptides with higher responses
and abundances. A glycosylation hotspot is highlighted bycomprehensive characterization at N300. Collectively theseresults indicate the capabilities for CESI-MS coupled with highresolution mass spectrometry to quickly and comprehensively
characterize a therapeutic mAb using peptide mapping from asmall amount of sample with a single protease.

Relative Abundance

INTRODUCTION

26.5

27.0

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

Time (min)Figure 6. Separation, identification, and relative quantification of asparagine
deamidation, tryptophan dioxidation, and lysine glycation on the heavy chain.

m/z=476.9340-476.9375
MS
130115_CEMS_Tras20kv5psi60sec60mi_01
34.0_OKFor Research Use Only. Not for use in diagnostic procedures.
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490.197480