Comprehensive Characterization of Monoclonal Antibody and Antibody Drug Conjugate on a Hybrid Quadrupole-Orbitrap Mass Spectrometer

Figure 2. (A) Single scan (10 microscans) FTMSbroadband mass spectrum of intact monoclonalantibody trastuzumab (IgG1) under nativeconditions, acquired at 30,000 resolving power (atm/z 200). (B) Expanded view of most abundantcharge state (z=26) with baseline resolvedglycoform pattern. (C) Deconvolution spectrumobtained from BioPharma Finder 4.0 using SlidingWindow ReSpect algorithm providing average massaccuracy below 7 ppm for all trastuzumab variants.

RESULTSIntact Mass Analysis of Herceptin Under Native Conditions

Full MS spectra of intact Herceptin monoclonal antibody were acquired using SEC LC-MS see Figure 2). OrbitrapExploris 480 provides accurate intact mAb mass analysis under native conditions using ReSpect deconvolution.

ABSTRACTPurpose: Evaluate the performance of the new Thermo Scientific™ Orbitrap Exploris™ 480 massspectrometer equipped with BioPharma option for complete characterization of monoclonal antibodies andantibody-drug conjugates

Methods: (1) Intact mass analysis under native and denaturing conditions using SEC LC-MS and RP LC-MS, (2) Top-down and middle-down analysis using RP LC-MS/MS, and (3) Peptide mapping using RP LC-MS/MS

Results: Demonstrated excellent performance of the new Orbitrap Exploris 480 mass spectrometer forcomplete characterization of antibodies

INTRODUCTIONThe complexity of modern therapeutic proteins, such as monoclonal antibodies (mAbs) and antibody-drug conjugates(ADCs) presents a great analytical challenge. MS approaches such as peptide mapping, top-down/middle-down andintact mass analysis are critical during development and production as those can provide complementary informationneeded for in-depth insight into the structure and composition of biopharmaceuticals. Native MS analysis allows probingmolecules while preserving critical structural features, such as cysteine-linked ADCs and produces decreased chargestates thus improves spatial resolution. Here we demonstrate the capabilities of the new mass spectrometer to fullycharacterize therapeutic proteins across a wide range of analysis, from peptide mapping to native intact mass analysis ona single platform.

MATERIALS AND METHODSSample PreparationHerceptin® (trastuzumab) IgG mAb (Genentech), Sigma SILuTM mAb universal standard and SigmaMAb Antibody DrugConjugate Mimic (Lot # SLCC0520) were used as standard samples. For native intact mass analysis using SEC LC-MS,the antibodies and ADC samples were injected without any further dilution. For subunit analysis, samples were eitherreduced in 4M GgHCl/50mM TCEP or first digested with FabRICATOR (Genovis) enzyme according to themanufacturer’s protocol and then reduced. For peptide analysis, antibodies were proteolytically digested using theSMART Digest kit following reduction with 10mM DTT.

Test Method(s)Intact denatured and native mass measurements of the intact antibodies and ADC were performed using either SEC LC-MS or RP LC-MS. The top-down, middle-down, and peptide mapping experiments were performed using RP LC-MS/MS.All measurements were carried out on an Orbitrap Exploris 480 mass spectrometer with BioPharma option.

Data AnalysisData analysis was performed using Thermo Scientific™ BioPharma Finder™ 3.2 software.

Figure 1. TNG templates and method editor. The Thermo Scientific Orbitrap Exploris 480 MS BioPharma methodeditor is intuitive and features a drag-and-drop user-friendly interface with a library of pre-defined templates for standardmethods and easy instrument setup, enabling the analyst to focus on the science rather than the instrument setup.

CONCLUSIONS In this work we demonstrated outstanding performance of the Orbitrap Exploris 480 mass spectrometer for complete

antibody characterization from intact and subunit to peptide level.

TRADEMARKS/LICENSING© 2020 Thermo Fisher Scientific Inc. All rights reserved. ReSpect is a trademark of Positive Probability Ltd. Herceptin isa trademark of Genentech, Inc.. Silu is a trademark of Sigma. All other trademarks are the property of Thermo FisherScientific and its subsidiaries. This information is not intended to encourage use of these products in any manner thatmight infringe the intellectual property rights of others.

PO65796-EN 0422S

IYPTNGYTR N55 HC Deamidation Full MS and MS2

Unmodified

Deamidated

Mass = 1083.5349

Mass = 1084.5189

Full MS MS2

0

50

100

y1175.1192

a2249.1603

y2276.1679

b2277.1544

y7++404.7023

y4496.2494

y5610.2964

y6711.3439

y7808.3961

y8971.4697

200 300 400 500 600 700 800 900m/z

0

50

100

Rel

ativ

e Ab

unda

nce

y6++356.6679

y7++405.1977

y8++486.7252

y4496.2519

y5

611.2842

y6

712.3348

y8

973.4506

200 300 400 500 600 700 800 900 1000m/z

Rel

ativ

e Ab

unda

nce

IYPTNGYTR Peptide

IYPTDGYTR (+ deamidation)+ 0.98402 (Da)

+ 0.98402 (Da)

+ 0.98402 (Da)

Intact native & denatured MS

Subunit analysis

Peptide Mapping

Top-down Analysis of Reduced Herceptin

Residue cleavages: Light Chain: 62% Heavy Chain: 36.7%

Data processed using BPF: minimum required fitter score: 80, S/N cutoff: 7, fragment mass

tolerance: 10 ppm

R = 7,5K

R = 240K(A)

(B)800 1200 1600 2000 2400 2800

m/z

50601.26

50763.64

50925.60

G0F

G1F

G2F

+1.8 ppm

+2.8 ppm

+0.8 ppm

SIM (480K)

1582 1586 1590m/z

HC-G0F32+

HC-G1F32+

1582.0 1582.5m/z

32+

800 1200 1600 2000m/z

ReSpect

Light Chain

Heavy Chain

938.5 939.0m/z

25+

Top-Down fragmentation

Figure 3: Intact mass (A) and (B) Top-Down analysis of reduced Herceptin

Heavy ChainLight Chain

(B)

(C)(A)

Xtract

Middle-down Analysis of Ides-digested Reduced Herceptin

IdeS-digested & reduced Herceptin MD

Fc: 54% residue cleavages

Fd’: 52% residue cleavages

FcLC

Fd'

(A)

(D)

934 936 938 940 942m/z

FcFc-G0F Fc-G1F

R=240K

600

1016.0 1016.5m/z

Fd'

25+

R=240K

(B)

(C)

27+

27+

Herceptin monoclonal antibody was analyzed after FabRICATOR® digestion followed by reduction. The resulted 23-25kDasubunits were analyzed to produce baseline resolved MS spectra with a resolution setting of 240,000 (at m/z 200). Forsubunit fragmentation experiments the 100 Th isolation window was centered at m/z 900.

Herceptin subunits

M exp. (monoisot.)

∆M (ppm)

Fc - G0F 25,220.50382 +1.6

Fc - G1F 25,382.53286 +0.7

LC 23,428.56981 +2.0

Fd' 25,367.54613 +1.1

600 800 1000 1200 1400 1600 1800 2000m/z

600 800 1000 1200 1400 1600 1800 2000m/z

4600 5000 5400 5800 6200 6600m/z

R = 60K0 5 10

Time (min)

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce 4.24

146500 147500Mass

G0F,G1F

146822.40

G1F,G1F

146984.37G0F,G0F

146660.72

G1F,G2F

147145.74

7.4 ppm

10.6 ppm

6.3 ppm

1 ppm

ReSpectTM

G0,G0F

146513.624.1 ppm

Average Drug-to-Antibody Ratio (DAR)

4.56

Figure 10. DAR calculation of Sigma ADC based on most

abundant glycoform (G0F/G1F).

Herceptin peptide mixture obtained after performing a Thermo Scientific™ SMART Digest™ was analyzed using RPLC-MS/MS and resulted in 100% sequence coverage as shown in Figure 5.

2 4 6 8

(DAR8)28+

(DAR0)26+

0

RT 4.3 min

147000 148000 149000 150000 151000 152000 153000Mass*

* * **

zoom

R=60K

27+

(A2)

(B2)

0 5 10Time (min)

0

20

40

60

80

100

Rel

ativ

e Ab

unda

nce

4.58

(C)

(D2)

(A1)

(D1)

(B1)

G0F/G1FDAR

Mass Accuracy

(ppm)

Relative Abundance

(%)DAR0 6.5 9.19DAR2 4.4 85.74DAR4 2.1 100DAR6 7.8 88.66DAR8 11.4 49.25

mAb ADC

mAb ADC

Figure 4: RP-LC separation (A) Intact mass analysis (B-C) and Middle-Down analysis of Herceptin SubunitsLC separation (A) and Full MS spectra (B, C) at 240k resolution for the Herceptin Fc, and Fd’ subunits. Very accurateintact masses for the Fc, LC and Fd’ subunits are obtained (see table) and middle-down (MD) analysis thereof providesvery good sequence coverage for all subunits (MD cleavage map for Fc and Fd’ (D)).

Figure 7: Analysis of Silu mAb and ADC under native conditions allowing DAR determinationNative size exclusion chromatography (SEC) coupled to mass spectrometry for the analysis of the SigmaMAbAntibody (1) and its Drug Conjugate Mimic (2). (A1-A2) Corresponding base peak chromatograms. (B1-B2) Orbitrapfull MS spectra acquired on Orbitrap Exploris 480 with BioPharma Option at R=60,000 (at m/z 200). (C) A detailedview of the 27+ charge state for ADC mimic. (D1-D2) ReSpect deconvolution results for mAb (D1), and more complexresults for ADC (D2) showing a distribution of 0-8 dansyl-cadaverine SMCC linker-drug attachments.

Herceptin Characterization at peptide level

Native Intact Mass Analysis of SIGMA Silu mAb And SIGMA ADC Mimic

Light Chain Heavy Chain

05

101520253035404550556065707580859095

100

Relat

i veInt

ensit

y

4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36RT (min)

Figure 6 : MS and MS/MS results for IYPTNGYTR N55 Heavy chainpeptide of Trastuzumab with and without deamidation. MS andzoomed main mass peak (A) and fragmentation for the IYPTNGYTRpeptide in the heavy chain with and without deamidation (B).Deamidation assessment in Biopharma Finder modification plot (C)showing the % abundance for asparagine (N) and glutamine (Q)deamidation observed in the light and heavy chains of trastuzumab.

Figure 5: Herceptin peptide mapping results showing base peak chromatograms and sequence coverage

Kristina Srzentić1, Eugen Damoc2, Krisztina Radi3, Tom Buchanan3 , Angela Criscuolo2, Jennifer Sutton1 1Thermo Fisher Scientific, Massachusetts, US, 2Thermo Fisher Scientific, Germany, and 3Thermo Fisher Scientific, UK

(A) (B) (C)