Mapping the Drug Conjugation Sites of an Antibody Drug … · 2016-09-04 · Mapping the Drug...
Transcript of Mapping the Drug Conjugation Sites of an Antibody Drug … · 2016-09-04 · Mapping the Drug...
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.