Rapidly Characterize Antibody- Drug Conjugates and Derive Drug-to-Antibody Ratios Using LC/MS

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1 Rapidly Characterize Antibody- Drug Conjugates and Derive Drug-to-Antibody Ratios Using LC/MS Ning Tang, Ph.D. Agilent Technologies Santa Clara, California, USA

2 Heterogeneity in ADCs Complexity T-DM1 (Genentech) ADC Disulfide shuffling Pyroglutamate mab Drug Deamidation Oxidation Fragmentation Glycosylation (G0, G1, G2) Truncation (Lys 0, 1, 2) Heterogeneity in ADC - More complex than mabs alone - Dependent on linker & payload stability (hydrolysis, degradation, etc.) - Dependent on the conjugation chemistry

3 Mass Spectrometry Always Separate Different Drug Conjugates MS also provides information on relative amounts of antibodies loaded with 1, 2, 3, 4 etc. drugs (without prior separation) MS also provides information on unconjugated antibody MS can detect other conjugation products, e.g. - Cross linked species - Species with linker attached but no drug

4 Agilent DAR Characterization Workflow LC/MS DAR Calculation Report ADC Sample Prep Reduction Deglycosylation LC/MS DAR Calculation Report ADC ADC purification from serum LC/MS DAR Calculation Report ADC Sample Prep Digestion Clean up LC/MS/MS Payload Mapping Report

5 LC/MS Setup LC: Agilent 1290 UHPLC MS: Agilent 6550 ifunnel Q-TOF Column: Agilent PLRP-S 1000A 8 µm 150 x 2.1 mm Column temp: 80 C Flow rate: 0.4 ml/min

6 Conjugation Adds Heterogeneity DAR1 DAR2 DAR3 DAR4 DAR5 DAR6 DAR7 DAR8

7 MassHunter ADC DAR Calculator Automatic calculation of DAR value Intuitive peak integration Built-in support for intact and reduced ADCs Easy analysis of different types of ADC molecules (Lyslinked, Cys-linked and site-specific ADCs) Flexible control to define peaks of interest Dedicated ADC DAR report in PDF and MS Word

8 Intact ADC DAR Calculation calculated DAR DAR peak list shows all components and % area for each species

9 Automated Workflow for Deglycosylation & Reduction in DAR Characterization

10 Reduced ADC DAR Calculation heavy chain light chain

11 Making these technologies accessible A front-end to LC and LC/MS systems that allows investigators to submit samples without being experts in the technology with few instrument specialists who are responsible for multiple systems 11

12 MassHunter Walkup: 3 Step Sample Submission 12

13 MassHunter Walkup: 3 Step Sample Submission 13

14 MassHunter Walkup: 3 Step Sample Submission 14

15 MassHunter Walkup: 3 Step Sample Submission 15

16 DAR Calculation Automation Workflow in Walkup ADC = Antibody Drug Conjugates DAR = Drug Antibody Ratio

17 ADC DAR Determination Workflow for Serum Samples SA- W SA- W Sample Preparation LC/MS DAR Calculation

18 Affinity Purified T-DM1 at 6 Different Loading Amounts AF-1000 ng; ~80ng on column. AF-125 ng; ~10ng on column DAR=3. DAR=3.5 5 DAR=3.5 AF-500 ng; ~40ng on column. AF-62.5 ng; ~5ng on column DAR=3.5 DAR=3.5 AF-250 ng; ~20ng on column. AF ng; ~2.5ng on column. DAR=3.5 DAR=3.4

19 Reproducibility of Affinity Purified T-DM1 at 6 Different Loading Amounts AF: Affinity purified AF-1000 ng; ~80ng on column. AF-500 ng; ~40ng on column. AF-250 ng; ~20ng on column. AF-125 ng; ~10ng on column AF-62.5 ng; ~5ng on column AF ng; ~2.5ng on column.

20 ADC Peptide Mapping Workflow Using AssayMAP Bravo and LC/MS/MS Sample Preparation LC/MS BioConfirm

21 Identification of Conjugated Peptides A T-DM1 20X Herceptin B EIC: T-DM1 Herceptin

22 Signature Elution Pattern and Fragment Ions A LTVDK[ ]SR B b 2 y 2 2+ y 5 y y y b 2 -H O 2 b 3 -H O 2 Drug Fragments C Precursor Drug Fragment GPSVFPLAPSSK[ ] STSGGTAALGCLVK y ion b ion D y 1 x b 4 -H O b 5 -H O Counts vs. Mass-to-Charge (m/z) Drug Fragments b 5 y 5 y 6 b 7 y 7 b 8 y 8 y 11

23 Sequence Coverage and Conjugation Sites Identification Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGS RSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTAS VVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC Heavy Chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADS VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKG PSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVV TVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPG K: Confirmed drug Identified conjugation sequence site (Light-9; Heavy 20) K: Potential drug conjugation site (Heavy 5) K: Unconjugated site identified (Light 4; Heavy 6) Total (Light-13; Heavy31)

24 Summary Determining ADC DAR is an important aspect of ADC characterization Workflow can be enabled in various ways New DAR calculator makes obtaining DAR value easy and straight forward Sample preparation can be streamlined and automated using the AssayMAP Bravo platform for reproducibility and scalability Localization of conjugation sites can be achieved by digesting the protein and analyzing by LC/MS/MS

25 Acknowledgments Application development Jing Chen Alex Zhu Ravindra Gudihal Steve Murphy Maryann Shen Software development Tanner Stevenson Robert Williams

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