Advanced Fragmentation Techniques for BioPharma Characterization

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1 Advanced Fragmentation Techniques for BioPharma Characterization Global BioPharma Summit The world leader in serving science

2 Different modes of fragmentation to answer different questions or for different assays Peptide Mapping Site occupancy of PTM for mab or site of conjugation for ADC Top-Middle down (Figure from Coon et al., Analytical Chemistry, 2009) 2

3 MS Tools for Major Biopharma Characterization Workflows For all the routine characterization workflows. Mode of fragmentation: HCD From routine to the most challenging tasks. Mode of fragmentation: HCD CID ETD EThcD ETciD (UVPD - not commercial) 3

4 Etanercept: O-glycosylation 4

5 O-glycosylation of Etanercept Etanercept (trade name Enbrel) LPAQVAFTPYAPEPGSTC 18 RLREYYDQTAQMC 31 C 32 SKC 35 SPGQHAKVFC 45 TKTSDTVC 53 DS C 56 EDSTYTQLWNWVPEC 71 LSC 74 GSRC 78 SSDQVETQAC 88 TREQNRIC 96 TC 98 RPGWYC 104 ALS KQEGC 112 RLC 115 APLRKC 121 RPGFGVARPGTETSDVVC 139 KPC 142 APGTFSNTTSSTDIC 157 R PHQIC 163 NVVAIPGNASMDAVC 178 TSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPST SFLLPMGPSPPAEGSTGDEPKSC 240 DKTHTC 246 PPC 249 PAPELLGGPSVFLFPPKPKDTLMIS RTPEVTC 281 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKC 341 KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC 387 LVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC 445 SVMHEALHNH YTQKSLSLSPGK 1) 3 N-glycan sites 2) 13 reported O-glycosylation sites Sialic acids from the O-glycan were removed prior trypsin digestion Peptide mapping was performed on the tryptic digest 5

6 Results for peptide SMAPGAVHLPQPVSTR after processing HCD data HCD RT: SM: 7G Time (min) SMAPGAVHLPQPVSTR S 186 MAPGAVHLPQPVS 199 T 200 R SMAPGAVHLPQPVSTR + SMAPGAVHLPQPVSTR + NL: 5.21E5 m/z= ms MS _E HCD30_tes NL: 3.95E7 m/z= ms MS _E HCD30_tes NL: 6.19E7 m/z= ms MS _E HCD30_tes NL: 2.68E5 m/z= ms MS _E HCD30_tes 6

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8 Data acquisition method on a Fusion Lumos: ETD methods Flexibility and ease of use to design acquisition method Scan priority Charge 3 ETD reaction time = 60 ms Charge 4 ETD reaction time = 40 ms Charge 5-7 ETD reaction time = 20 ms DDA method used to collect data to identify the sites of O-glycosylation. 8

9 Data acquisition method on a Fusion Lumos: ETD methods Flexibility and ease of use to design acquisition method 9

10 Data acquisition method on a Fusion Lumos: ETD methods Other useful methods: * HCD followed by ETD for every precursors. * HCD followed by ETD when specific product ions are present in the HCD MS2 spectrum 10

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15 Results for peptide SMAPGAVHLPQPVSTR after processing ETD data ETD All of the O-glycosylation sites of peptide SMAPGAVHLPQPVSTR were successfully identified RT: SM: 7G SMAPGAVHLPQPVSTR Time (min) SMAPGAVHLPQPVSTR S 186 MAPGAVHLPQPVS 199 T 200 R SMAPGAVHLPQPVSTR SMAPGAVHLPQPVSTR NL: 5.21E5 m/z= ms MS _E HCD30_test NL: 3.95E7 m/z= ms MS _E HCD30_test NL: 6.19E7 m/z= ms MS _E HCD30_test NL: 2.68E5 m/z= ms MS _E HCD30_test 15

16 Etanercept: leveraging ETD and MSn for disulfide bond mapping 16

17 Disulfide bond mapping: Etanercept Etanercept (trade name Enbrel) LPAQVAFTPYAPEPGSTC 18 RLREYYDQTAQMC 31 C 32 SKC 35 SPGQHAK VFC 45 TKTSDTVC 53 DSC 56 EDSTYTQLWNWVPEC 71 LSC 74 GSRC 78 SSD QVETQAC 88 TREQNRIC 96 TC 98 RPGWYC 104 ALSKQEGC 112 RLC 115 APL RKC 121 RPGFGVARPGTETSDVVC 139 KPC 142 APGTFSNTTSSTDIC 157 RPHQIC 163 NVVAIPGNASMDAVC 178 TSTSPTRSMAPGAVHLPQPVST RSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSC 240 DKTHT C 246 PPC 249 PAPELLGGPSVFLFPPKPKDTLMISRTPEVTC 281 VVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKC 341 KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTC 387 LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSC 445 SVMHEALHNHYTQKSLSLSPGK 17

18 Disulfide bond mapping: Etanercept Etanercept (trade name Enbrel) LPAQVAFTPYAPEPGSTC 18 RLREYYDQTAQMC 31 C 32 SKC 35 SPGQHAK VFC 45 TKTSDTVC 53 DSC 56 EDSTYTQLWNWVPEC 71 LSC 74 GSRC 78 SSD QVETQAC 88 TREQNRIC 96 TC 98 RPGWYC 104 ALSKQEGC 112 RLC 115 APL RKC 121 RPGFGVARPGTETSDVVC 139 KPC 142 APGTFSNTTSSTDIC 157 RPHQIC 163 NVVAIPGNASMDAVC 178 TSTSPTRSMAPGAVHLPQPVST RSQHTQPTPEPSTAPSTSFLLPMGPSPPAEGSTGDEPKSC 240 DKTHT C 246 PPC 249 PAPELLGGPSVFLFPPKPKDTLMISRTPEVTC 281 VVVDV SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKC 341 KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM TKNQVSLTC 387 LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS FFLYSKLTVDKSRWQQGNVFSC 445 SVMHEALHNHYTQKSLSLSPGK The N-terminal side of this sample is not as expected. The first 2 amino acids are missing. 18

19 Disulfide Bond Mapping: Etanercept Etanercept (trade name Enbrel) LPAQVAFTPYAPEPGSTC 18 RLREYYDQTAQMC 31 C 32 SKC 35 SPGQHAKVFC 45 TKTSDTVC 53 DS C 56 EDSTYTQLWNWVPEC 71 LSC 74 GSRC 78 SSDQVETQAC 88 TREQNRIC 96 TC 98 RPGWYC 104 ALS KQEGC 112 RLC 115 APLRKC 121 RPGFGVARPGTETSDVVC 139 KPC 142 APGTFSNTTSSTDIC 157 R PHQIC 163 NVVAIPGNASMDAVC 178 TSTSPTRSMAPGAVHLPQPVSTRSQHTQPTPEPSTAPST SFLLPMGPSPPAEGSTGDEPKSC 240 DKTHTC 246 PPC 249 PAPELLGGPSVFLFPPKPKDTLMIS RTPEVTC 281 VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLN GKEYKC 341 KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC 387 LVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC 445 SVMHEALHNH YTQKSLSLSPGK The N-terminal side peptide contains one cysteine. The HCD search result of the non-reduced sample suggests that the N-terminal peptide is linked to two other peptides through disulfide bonds. 19

20 Leveraging ETD and MSn for disulfide bond mapping Workflow of the MS2 ETD MS3 HCD for disulfide bond peptide identification Dual-Pressure Linear Ion Trap Ultra-High Field Orbitrap Analyzer Ion Routing Multipole 3 fragment channels after ETD of disulfide bond peptide 20

21 Leveraging ETD and MSn for disulfide bond mapping Workflow of the MS2 ETD MS3 HCD for disulfide bond peptide identification Dual-Pressure Linear Ion Trap Ultra-High Field Orbitrap Analyzer Ion Routing Multipole 3 fragment channels after ETD of disulfide bond peptide 21

22 deconvoluted spectrum MS2 ETD Spectrum Peptide: AQVAFTPYAPEPGSTC 18 R-EYYDQTAQMC 31 C 32 SK-VFC 45 TK 22

23 Peptide: AQVAFTPYAPEPGSTC 18 R-EYYDQTAQMC 31 C 32 SK-VFC 45 TK MS3 HCD spectrum of AQVAFTPYAPEPGSTC 18 R. 23

24 Peptide: AQVAFTPYAPEPGSTC 18 R-EYYDQTAQMC 31 C 32 SK-VFC 45 TK MS3 HCD spectrum of EYYDQTAQMC 31 C 32 SK using a) the Orbitrap or b) the ion trap mass analyzers. 24

25 Peptide: AQVAFTPYAPEPGSTC 18 R-EYYDQTAQMC 31 C 32 SK-VFC 45 TK MS3 HCD spectrum of VFC 45 TK. 25

26 Antibody Drug Conjugates 26

27 ADCs are heterogeneous mixture Brentuximab vedotin Adcetris Trastuzumab Emtansine Kadcyla Light Chain: 12 Lysines Heavy Chain: 32 Lysines Cysteine-linked Lysine-linked FDA-approved ADCs Peptide mapping Goal: Identifying conjugated peptides and sites of conjugation 27

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29 Trastuzumab emtansine: conjugated peptides Subset of identified conjugated peptides # K ADYEKHK AKGQPR ALPAPIEKTISK ASQDVNTAVAWYQQKPGK TKPR 29

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32 Trastuzumab emtansine site occupation: ETD spectrum z 4 z 3 Z (+1) ( -3.0 ppm) Dmass = Da K ( ) + MCC-DM1 ( ) Z (+2) ( -1.9 ppm) ETD spectrum allows unambiguous identification of the conjugation site ASQDVNTAVAWYQQK 39 PGK 42 APK min; m/z= (+5); ETD Relative Abundance _fsn10361_TDM1_smart_mNBA_targetETD_0P3_20ms_04pmNBA_2AfteCali #5880 RT: AV: 1 NL: 3.55E4 F: FTMS + p ESI d Full ms @etd20.00 [ ] z=3 z= z=? z=? z= z=? z=? z=? z= z= z= z= z= z= z= z= z= z= z=1 z= z=? z= z= m/z 32

33 Trastuzumab emtansine site occupation (+2) ( -3.3 ppm) (+2) ( -2.1 ppm) c 14 c 17 c 14 Dmass = Da K ( ) + P ( ) +G ( ) c 17 ASQDVNTAVAWYQQKPGKAPK min; m/z= (+5); ETD Relative Abundance _fsn10361_TDM1_smart_mNBA_targetETD_0P3_20ms_04pmNBA_2AfteCali #5880 RT: AV: 1 NL: 3.55E4 F: FTMS + p ESI d Full ms @etd20.00 [ ] z=3 z= z=? z=? z= z=? z=? z=? z= z= z= z= z= z= z= z= z= z= z=1 z= z=? z= z= m/z 33

34 Trastuzumab emtansine: conjugated peptides Subset of identified conjugated peptides # K

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36 Middle-down: ETD 36

37 Main parameters that can be controlled for ETD fragmentation on an Orbitrap Fusion mass spectrometer. Isolation window AGC target for precursor ions and reagent Reaction time Supplemental energy 37

38 Light Chain of Trastuzumab ETD settings: 300Da isolation window, 3E5 precursor, 7E5 reagent, 10 ms reaction time MS/MS Due to the complexity of the spectra in top-down analysis, high resolution is required 38

39 Effect of parent ion AGC and ETD reaction time on sequence coverage (Trastuzumab) % residue cleavages for AGC target value of 3E5 or 1E6 at different ETD reaction times LC Orbitrap LPC HPC Ion Trap MP0 Q1 Ion Routing Multipole ETD C-Trap 1 st parent ion isolation in Q1 1 st parent trapped in the ion trap Injection of ETD reagent 1 st parent: ETD dissociation 39

40 Effect of parent ion AGC and ETD reaction time on sequence coverage (Trastuzumab) % residue cleavages for AGC target value of 3E5 or 1E6 at different ETD reaction times LC Fc Fd 40

41 Effect of parent ion AGC and ETD reaction time on sequence coverage (Trastuzumab) % residue cleavages for AGC target value of 3E5 or 1E6 at different ETD reaction times LC 41

42 Middle Down: Orbitrap Tribrid Fusion Lumos LC Fd Fc High sequence coverage for the light chain, Fc and Fd were obtained from the combined ETD and EThcD experiments. 42

43 Fd (43%) Fc (52%) LC (60%) Fd (26%) Fc (32%) LC (40%) Fd (61%) Fc (67%) LC (78%) NIST mab: middle down experiment using ETD and UVPD One acquisition with ETD 10 ms One acquisition with UVPD 12 ms ETD 10 ms + UVPD 12 ms 43

44 Fd (43%) Fc (52%) LC (60%) Fd (26%) Fc (32%) LC (40%) Fd (61%) Fc (67%) LC (78%) NIST mab: middle down experiment using ETD and UVPD One acquisition with ETD 10 ms One acquisition with UVPD 12 ms ETD 10 ms + UVPD 12 ms 44

45 Hydrogen Deuterium Exchange 45

46 HDX-MS workflow MS1 46

47 Moving from MS only to MS/MS HDX experiment using ETD Deuterium Content Under normal operation condition, minimal scrambling is observed on the ETD Spectrum Orbitrap Fusion HHHHHHIIKIIK 2.0 Literature C2 C3 C4 C5 C6 C7 C8 C9 C10 (Martin Zehl, Kasper D. Rand, Ole N. Jensen, and Thomas J. D. Jørgensen J. AM. CHEM. SOC. 2008, 130, ) Minimal deuterium scrambling ETD measurement with different instruments and source conditions (No deuterium is retained on Histidine due to fast exchange with solvent) 47

48 Moving from MS only to MS/MS HDX experiment using ETD Pinpoint the Protein Ligand Binding Site with ETD Deuterium Deuterium labeled labeled protein protein and protein+ligand biding biding sample s sample s C and C Z and fragments Z fragments mass mass differences vs. sequence vs. sequence position. position. From From C3 to C3 C14 to C14 mass mass differences were were from from to 0.2. to 0.2. Start Start from from C17, C17, the Δthe mass Δ mass increased increased to around to around 1 and 1 stay and stay at at the same the same level level for the for rest the rest of the of C the fragments. C fragments. C14 C14 was was identified identified as significant as significant change change area. area. Significant change was observed around Z 8 -Z 10 is consistent with the C fragments plot. Combine the two plot results, the potential binding site was predicted. 48

49 Summary ETD is a must have type of fragmentation for in-depth characterization of biotherapeutic proteins. High sequence coverage for middledown experiments allows quick characterization assays. Orbitrap Fusion Lumos offers multiple types of fragmentation and ease of use to tackle the most challenging task. 49

50 Thank you! Acknowledgements Kelly Broster Rowan Moore Terry Zhang Aaron Bailey Kyle D Silva Michael Blank Simon Cubbon Stephane Houel Jonathan Josephs John Rontree Helene Cardasis Seema Sharma Romain Huguet Vlad Zabrouskov Jennifer Sutton Mark Sanders 50