Mapping the Drug Conjugation Sites of an Antibody Drug Conjugate Using Automated

Sample Preparation and LC/MS AnalysisAlex Zhu1, Jing Chen2

1Agilent Technologies, Inc. Wilmington, DE2Agilent Technologies, Inc. Madison, WI

WCBP 2016

P123T

Introduction

Conclusions

In recent years, antibody drug conjugates (ADCs) that combine thespecificity of monoclonal antibodies (mAbs) with the potency ofcytotoxic molecules have gained significant interest as a promisingapproach to achieve more selective treatment for cancers. ADCs aremore complex and heterogeneous compared to the unmodifiedbiotherapeutic antibodies due to multitude of potential drugconjugation sites and the variation of site occupancy among thesepotential conjugation sites. In order to obtain full characterization ofthe ADC molecule, peptide mapping experiments can be carried out toprovide in-depth site specific information about the ADC conjugationsites.

In this work, an integrated peptide mapping workflow was developedfor in-depth characterization of antibody drug conjugates. Robust andreproducible automated in-solution trypsin digestion wasdemonstrated using an automated sample preparation platform.Excellent mass accuracy and sensitivity were achieved by highresolution accurate mass Q-TOF LC/MS system to ensurecomprehensive sequence coverage and accurate peptideidentification. Automated data extraction, sequence matching anddrug conjugation site identification were also demonstrated. For theADC investigated, 98.7% sequence coverage was achieved. Morethan 29 out of 44 lysine sites were identified with drug conjugation.

Experimental

Results and Discussions

Automated trypsin digestion of T-DM1:

Antibody digestions were performed on the Agilent AssayMAP Bravo (Santa

Clara, CA) using the In-Solution Digestion Protocol. T-DM1 and Herceptin

were diluted to 1.5 mg/ml. 10 µl of T-DM1 was dispensed into well A1-H1 of

a 96-well Eppendorf PCR plate and 10 µl of Herceptin was dispensed into

well A2-H2 of the same plate. Proteins in each vial were reduced by DTT,

alkylated by iodoacetamide and followed by trypsin digestion.

LC system and Columns:

Agilent 1290 Infinity LC. Agilent ZORBAX Eclipse Plus C18, RRHD,

2.1×100mm, 1.8µm

MS System:

Agilent 6550 iFunnel Q-TOF LC/MS System with Agilent JetStream ion

source

Data Analysis

Compounds were extracted by molecular extraction (MFE), then matched

against the ADC sequence with the following modifications using Agilent

MassHunter BioConfirm Software. All identified peptides were confirmed by

at least one MS/MS spectrum.

• An integrated peptide mapping workflow was developed for in-depth

characterization of antibody drug conjugates.

• Robust and reproducible automated in-solution trypsin digestion was

demonstrated for ADCs using the Agilent AssayMAP Bravo system.

• Excellent mass accuracy and sensitivity were achieved using the

Agilent 1290 UHPLC/6550 Q-TOF LC/MS system allowing

comprehensive sequence coverage and accurate peptide identification.

• Automated data extraction, sequence matching, and drug conjugation

site identification were provided using Agilent MassHunter Bioconfirm

Software.

• For T-DM1, 98.7% sequence coverage was achieved. At least 29 out of

44 lysine sites were confirmed to be drug conjugation sites.

LC Parameters Agilent 1290 Infinity LC System

Mobile phase A 0.1% formic acid in Water

Mobile phase B 0.1% formic acid in Acetonitrile

Gradient 0 min → 5%B

16 min → 50%B

18 min → 90%B

19 min → 90%B

Post time 3 min

Column Temperature 55°C

Flow rate 0.5 mL/min

MS parameters Agilent 6550 Q-TOF LC/MS System

Ion mode Positive ion mode

Drying gas temperature 150 °C

Drying gas flow 13 L/min

Nebulizer 35 psi

Sheath gas temperature 250 °C

Sheath gas flow 12 L/min

Capillary voltage 3500 V

Nozzle voltage 500 V

Acquisition parameters Extended dynamic range, 2 GHz; Auto

MS/MS mode for drug conjugation site

identification; MS only mode for

reproducibility assessment

MS range

MS/MS range

300-2000 m/z

100-2000 m/z

Sample Preparation LC/MS BioConfirm

A

B

T-DM1

Herceptin

20X

T-DM1

Herceptin

EIC: 547.22

Bb

2

Drug Fragments

y

-546.215

2+y 5

y

-546.216

y

-546.214

y 2

b

-H O3

2

b

-H O2

2

ALTVDK[+956.36]SR

GPSVFPLAPSSK[+956.36]

STSGGTAALGCLVK

C

PrecursorDrug Fragment

y ionb ion

3x10

0

0.25

0.5

0.75

1

1.25

1.5

1.75

2

2.25

2.5

2.75

3

3.25

3.5547.2185

769.4205

485.2178

689.4037893.7515

323.1689 1067.0313618.3130242.1126

Counts vs. Mass-to-Charge (m/z)150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 1050 1100 1150 1200

Drug Fragments

Dy 1

b

-H O4

2

b

-H O5

2

b 5

y 5

y 6

b 7

y 7

b 8

y 8

y 11

GPSVFPLAPSSK[+956.36]

STSGGTAALGCLVK

A B

VEIK[+956.36]R C

CV= 6.84%

CV= 8.30%

CV= 8.72%

CV= 7.65%

<10%

10% - 20%

>20%

229

5

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS

RSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV

VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV

THQGLSSPVTKSFNRGEC

Identified sequence

K: Confirmed drug conjugation site --- 29 (Light-9; Heavy 20)

K: Potential drug conjugation site --- 5 (Heavy 5)

K: Unconjugated site identified --- 10 (Light—4; Heavy 6)

Total --- 44 (Light-13; Heavy31)

Light Chain

Heavy Chain

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSV

KGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPS

VFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP

SSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS

RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK

EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES

NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Figure 1. ADC Peptide mapping workflow uses Agilent AssayMAP Bravo for automated protein digestion, Agilent 1290 UHPLC/6550 QTOF system for

LC/MS peptide mapping analysis, and BioConfirm Software for data analysis

98.7% sequence coverage was achieved for T-DM1. All 44 lysine sites were

covered by at least one unique peptide, with 29 confirmed drug conjugated sites

(Green), 5 potential drug conjugation sites (Yellow; unique drug conjugated

peptides were identified based on both MS and MS/MS spectra, but the exact

conjugation site could not be unambiguously identified among the multiple lysine

sites on the peptide), and 10 unconjugated sites (Blue; no evidence of conjugation

found).

Figure 2. Base peak chromatograms (A) and extracted ion

chromatograms of 547.22 (Known fragment ion for DM1) from

MS/MS spectra (B) for digested T-DM1 and Herceptin

Figure 3. Representative precursor EICs and MS/MS spectra of two drug conjugated

peptides LTVDKSR and GPSVFPLAPSSKSTSGGTAALGCLVK. The drug conjugated peptides

formed diastereomers and eluted in pairs.

Figure 4. Reproducibility of the peptide mapping workflow. A) overlay of

eight precursor EICs for two identified drug conjugated peptides. B)

Scatter plot of integrated EIC peak area for four representative drug

conjugated peptides. C) Pie chart of the number of peptides with

different % CV.