Mapping the Drug Conjugation Sites of an Antibody Drug Conjugate Using Automated Sample Preparation and LC/MS Analysis Alex Zhu 1 , Jing Chen 2 1 Agilent Technologies, Inc. Wilmington, DE 2 Agilent Technologies, Inc. Madison, WI WCBP 2016 P123T Introduction Conclusions In recent years, antibody drug conjugates (ADCs) that combine the specificity of monoclonal antibodies (mAbs) with the potency of cytotoxic molecules have gained significant interest as a promising approach to achieve more selective treatment for cancers. ADCs are more complex and heterogeneous compared to the unmodified biotherapeutic antibodies due to multitude of potential drug conjugation sites and the variation of site occupancy among these potential conjugation sites. In order to obtain full characterization of the ADC molecule, peptide mapping experiments can be carried out to provide in-depth site specific information about the ADC conjugation sites. In this work, 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 using an automated sample preparation platform. Excellent mass accuracy and sensitivity were achieved by high resolution accurate mass Q-TOF LC/MS system to ensure comprehensive sequence coverage and accurate peptide identification. Automated data extraction, sequence matching and drug conjugation site identification were also demonstrated. For the ADC investigated, 98.7% sequence coverage was achieved. More than 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 μ l of T-DM1 was dispensed into well A1-H1 of a 96-well Eppendorf PCR plate and 10 μ l 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 B b 2 Drug Fragments y -546.21 5 2+ y 5 y -546.21 6 y -546.21 4 y 2 b -H O 3 2 b -H O 2 2 A LTVDK[+956.36]SR GPSVFPLAPSSK[+956.36] STSGGTAALGCLVK C Precursor Drug Fragment y ion b ion 3 x10 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 2.5 2.75 3 3.25 3.5 547.2185 769.4205 485.2178 689.4037 893.7515 323.1689 1067.0313 618.3130 242.1126 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 D y 1 b -H O 4 2 b -H O 5 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% 22 9 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.