Advanced Characterization of Antibody Drug Conjugates (ADCs) by Liquid Chromatography and Mass Spectrometry (LC/MS) John Gebler, Ph.D.

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1 Advanced Characterization of Antibody Drug Conjugates (ADCs) by Liquid Chromatography and Mass Spectrometry (LC/MS) John Gebler, Ph.D. Director Waters Corporation 1

2 2015 Waters Corporation 2

3 2015 Waters Corporation 3

4 Intact ADC 2015 Waters Corporation 4

5 Cysteine Conjugation Location Possibilities Positional Isomers Waters Corporation 5

6 Intact ADC Characterization Waters Corporation 6

7 HIC LC/UV Characterization HIC 4.6mm x 100 mm, 2.5 µm A: 125 mm Phosphate buffer, ph 6.7 with 2.5 M (NH 4 ) 2 SO 4 B: 125 mm phosphate buffer, ph 6.7 C: Isopropyl alcohol D: Water Gradient table: Non-porous Time Flow (ml/min) % A % B %C % D Initial Waters Corporation 7

8 Cys ADC DAR via Intact Mass Analysis SEC-LC/Native MS (Deglycosylated) UV A280 QTof TIC Cys-Low 25 µg load Cys-Med 25 µg load Cys-High 25 µg load Cys-mAb 25 µg load Ammonium Acetate ph mm No acid - No organic ADC Salt 2015 Waters Corporation 8

9 SEC-LC/MS Combined Raw Spectra Sample Combined Raw Spectra Zoomed Charge State Cys-Low Cys-Med Cys-High Cys-mAb 2015 Waters Corporation 9

10 UNIFI Report of LC/MS (SEC) Deconvoluted Intact MS Spectra Mass accuracy Distribution % Total DAR Individual DAR UV TIC Waters Corporation 10

11 Lysine-Conjugated ADCs NH 2 H 2 N H 2 N H 2 N NH 2 H 2 N H 2 N NH 2 NH Waters Corporation 11

12 LC (RP)/MS Method G0F/G1F MaxEnt1 Deconvoluted Mass Spectrum G0F/G0F G1F/G1F G1F/G2F G2F/G2F 2015 Waters Corporation 12

13 DAR Comparison of MS Reversed-phase LC/MS by Waters Lys-Low DAR=1.96 Lys-Med DAR=3.40 Lys-High DAR= Waters Corporation 13

14 2D HIC/RP/MS Waters Corporation 14

15 Multiple Isomers Resolved by HIC Calculated DAR from HIC Analysis a 6c 6b Expected DAR Retention time (min) Area % Area Individual DAR Total DAR Waters Corporation 15

16 2D-LC/MS (HIC/RPLC) for Isoform Structure Elucidation HIC (1 st Dimension) 4 Heart Cut 2D LC-MS RPLC/MS (2 nd Dimension) peak 2 2 peak Possible sub-units using RPLC Retention Time (min) peak 1 peak 2 G0F 23,575.0 Da 74,439.0 Da 125,302.0 Da G1F 23,575.0 Da 101,727.0 Da 101,727.0 Da 125,302.0 Da G2F Mass [Da] Mass [Da] 2015 Waters Corporation 16

17 Positional Isomers Determination Using 2D-LC/MS 1 st Dimension B) DAR 6a/b 2 nd Dimension Possible Structures Deconvoluted Mass Spectrum peak 1 peak 2 G0F Heart-cut TIC [Counts] peak 1 peak 2 23,575.0 Da 102,484.0 Da 23,575.0 Da 102,484.0 Da G1F = 51,620.0 Da 74,439.0 Da G2F Retention time [min] Retention time [min] Mass [Da] Mass [Da] C) DAR 6c peak 3 Heart-cut peak 1 peak 2 peak 3 TIC [Counts] peak 2 peak 1 23,575.0 Da 102,484.0 Da G0F G0F = Retention time [min] Retention time [min] 51,620.0 Da 74,439.0 Da 23,575.0 Da 74,439.0 Da G1F 51,620.0 Da G1F Mass [Da] Mass [Da] Mass [Da] 2015 Waters Corporation 17

18 Kadcyla - Trastuzumab Emtansine Peptide Mapping Updates Waters Corporation 18

19 Kadcyla - Trastuzumab Emtansine MCC = C12H13O3N DM1 + MCC = C47H61N4O13SCl Waters Corporation 19

20 MS E Chromatograms (LC/MS): Trypsin Trypsin Peptide Mapping Tmab DM1 Kadcyla Tmab 2015 Waters Corporation 20

21 MS E Chromatograms (LC/MS): AspN AspN Peptide Mapping 2:D3 unmodified 2:D3 lys-conjugated Tmab DM1 Kadcyla Tmab 2015 Waters Corporation 21

22 UV 254 Chromatograms: Trypsin Digest Tmab DM1 Kadcyla Tmab 2015 Waters Corporation 22

23 Control mab & Lysine-Conjugated Sample UV 254 Chromatograms: AspN Digest Tmab DM1 Kadcyla Tmab 2015 Waters Corporation 23

24 2:T19-20 Conjugated Peptides Elute in Pairs Unassigned Ions -OH Loss of Drug C30H41NO6Cl +H Waters Corporation 24

25 2:D7 Unconjugated vs. Conjugated 2:D7 unconjugated 2:D7 conjugated Two pairs 2015 Waters Corporation 25

26 2:D7 Lys site DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK -Drug y32& y33& y27& y29& y35& y36& 2015 Waters Corporation 26

27 2:D7 Lys site DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK -Drug y18& y19& y26& y34& y41& 2015 Waters Corporation 27

28 Peptide Mapping Results Light Chain Heavy Chain Constant Fab Variable Fab Constant Fab Variable Fab Hinge Constant Fc Trypsin Digest AspN Digest Lys Position Trypsin MSE DDA MSE DDA AspN Peptides Drug y/b Drug y/b Drug y/b Drug y/b Peptides 39 1:T3+ n/a n/a 42 1:T3-4+ yes >5 yes >5 yes >5 n/a 1:D3 45 1:T4-5+ yes >5 n/a 103 1:T7-8+ n/a n/a 107 1:T8-9+ yes 3 yes 1 n/a n/a 1:D :T yes >5 yes > :T yes 3 yes 3 yes >5 yes >5 1:D :T yes 3 yes > :T yes >5 yes >5 yes 0 yes 1 1:D :T yes >5 yes >5 yes >5 yes 3 1:D :T yes >5 yes > :T yes 3 yes >5 yes >5 yes >5 1:D :T yes >5 yes >5 30 2:T2-3+ yes >5 yes >5 yes >5 yes >5 2:D1 43 2:T4-5+ yes >5 yes >5 yes >5 yes >5 2:D2 65 2:T7-8+ yes 2 yes 3 yes >5 yes 3 2:D3 76 2:T9-10+ yes >5 yes >5 yes >5 yes >5 2:D :T yes >5 yes > :T yes >5 yes >5 yes 1 yes >5 2:D7 150, 208 2:T n/a n/a 208 2:T15+ yes >5 yes > :T yes >5 yes >5 yes >5 yes >5 2:D :T yes 0 yes :T yes 3 yes 0 yes >5 yes 4 2:D :T n/a n/a 225, 249 2:T yes >5 yes :T20+ yes >5 yes >5 yes >5 yes >5 2:D10 249, 251 2:T yes 5 yes > :T yes >5 yes >5 yes 4 yes 2 2:D13 291, 293 2:T yes >5 yes > :T24+ yes 1 yes 1 n/a n/a 2:D :T yes >5 yes > :T yes 1 yes :T yes 3 yes :T yes >5 yes > :T yes >5 yes >5 yes 3 yes >5 2:D :T yes 0 n/a 343 2:T yes 2 yes :T yes >5 yes > :T n/a n/a 395 2:T yes >5 yes >5 yes >5 yes >5 2:D :T n/a n/a n/a n/a 2:D :T yes 3 yes :T yes 2 yes >5 yes >5 yes 2 2:D Waters Corporation 28

29 Variable FAB Constant FAB Relative intensity Light Heavy Light Lys Site Lys Site Constant Fc Relative intensity Heavy Lys Site 2015 Waters Corporation 29

30 Key Technologies for ADCs LC-UV ACQUITY UPLC H-Class System with TUV Detector (or, ACQUITY UPLC H-Class Bio System) Empower 3 Software LC-MS Waters Biopharmaceutical Platform Solution with UNIFI: ACQUITY UPLC H-Class System with TUV Detector Xevo G2-XS QTof Mass Spectrometer UNIFI Scientific Information System Columns Hydrophobic interaction (HIC): Protein-Pak HiRes HIC Intact & subunit analysis: ACQUITY UPLC Protein BEH C4 Peptide mapping: ACQUITY UPLC Peptide CSH C18 Released glycan analysis: ACQUITY UPLC Glycan BEH Size exclusion chromatography: ACQUITY UPLC Protein BEH SEC Bioseparations Columns Advisor 2D LC-MS ACQUITY UPLC H-Class Bio System with 2D Technology Xevo G2-XS QTof Mass Spectrometer UNIFI Scientific Information System Waters Corporation 30

31 Contributors & More Details Acknowledgements Weibin Chen Henry Shion Robert Birdsall Ying Qing Yu Liuxi Chen ADC Resources Waters.com/ADC Molecular insights video Presentations by collaborative industry thought leaders on ADCs (Slideshare) Case studies & applications work Key application notes Automating the Determination of Drug-to-Antibody Ratio (DAR) of Antibody Drug Conjugates (ADCs) Based on Separation by Hydrophobic Interaction Chromatography (HIC) Development of Integrated Informatics Workflows for the Automated Assessment of Comparability for Antibody Drug Conjugates (ADCs) Using LC-UV and LC-UV/MS 2015 Waters Corporation 31

32 Thank you 2015 Waters Corporation 32