Analysis of Antibodies and Antibody Drug Conjugates using Xevo G2-XS QTof MS

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1 Analysis of Antibodies and Antibody Drug Conjugates using Xevo G2-XS QTof MS Guillaume Béchade, PhD Life Sciences Business Development Manager, Central Europe MS Tech Days München, 06-XI Waters Corporation 1

2 Introduction LC/MS for the characterization of biotherapeutics monoclonal antibodies (mabs) antibody-drug-conjugates (ADCs) Increased MS sensitivity and resolution improve the data quality provide better assessment to the structure of biomolecules 2014 Waters Corporation 2

3 The Xevo G2-XS QTof provides increased sensitivity with no loss in resolution 2014 Waters Corporation 3

4 Performance Characteristics (Resolution vs. Sensitivity) The graph of resolution versus sensitivity Sensitivity (relative) G2-S Sens Mode G2-XS Sens Mode G2-S Res Mode G2-XS Res Mode Resolution/ Waters Corporation 4

5 Intact Mass Analysis 2014 Waters Corporation 5

6 LC/MS Intact Mass Analysis of Antibody on Xevo G2-XS A fast, simple, and highly sensitive method for intact mass assessment Mass Spectrum 1 µg on column +50 Charge state Improved feature/glycoform definition 2014 Waters Corporation 6

7 LC/MS Intact Mass Analysis of Antibody on Xevo G2-XS A fast, simple, and highly sensitive method for intact mass assessment Mass Spectrum 1 µg on column +50 Charge state Improved feature/glycoform definition G0F/G1F Glycoform Mass Error (ppm) G0F/G0F 0.7 MaxEnt1 Deconvoluted Mass Spectrum G0F/G0F G1F/G1F G0F/G1F 0.0 G1F/G1F 4.0 G1F/G2F 8.0 G2F/G2F 20.0 G1F/G2F G2F/G2F 2014 Waters Corporation 7

8 LC/MS Intact Antibody Mass Analysis at different on-column loading by Xevo G2-XS 2014 Waters Corporation 8

9 LC/MS Intact IgG1 Antibody showing increased sensitivity and improved feature definition on Xevo G2-XS Xevo G2-XS QTof 1 µg on column Xevo G2-XS QTof 1 µg on column Xevo G2-S QTof 1 µg on column Xevo G2-S QTof 1 µg on column 1 µg >10-fold improvement in sensitivity +50 Charge state More information from Improved feature definition With XS Collision Cell Without XS Collision Cell 2014 Waters Corporation 9

10 LC/MS analysis of deglycosylated intact ADC showing drug distribution profile Mass Spectra - Xevo G2-XS QTof D2 D3 Zoomed Mass Spectrum +53 Charge state D1 D4 D5 MaxEnt1 Deconvoluted Mass Spectrum D6 D7 D Waters Corporation 10

11 Subunit Mass Analysis 2014 Waters Corporation 11

12 LC/MS Subunit Mass Analysis of IgG1 Antibody Reduction Alkylation LC + HC Light Chain +13 LC HC Resolution Mode (40K Resolution) Near baseline isotopic resolution of LC (~25kDa) achieved by Xevo G2-XS 0.5 µg on column 2014 Waters Corporation 12

13 LC/MS Subunit Mass Analysis of IgG1 Antibody Reduction Alkylation LC + HC Light Chain +13 LC HC Resolution Mode (40K Resolution) Near baseline isotopic resolution of LC (~25kDa) achieved by Xevo G2-XS 0.5 µg on column Mass Spectrum +18 Charge state Sensitivity Mode (30K Resolution) +36 Charge state Mass Spectrum 2014 Waters Corporation 13

14 LC/MS Subunit Mass Analysis of IgG1 G2-XS vs. G2-S at Equivalent Resolution LC HC 0.5 µg on column Light Chain LC Xevo G2-XS QTof (Sensitivity mode) Heavy Chain Xevo G2-XS QTof (Sensitivity mode, 30K Res) Xevo G2-XS QTof Xevo G2-S QTof (Resolution mode) Xevo G2-S QTof (Resolution mode, 30K Res) 20-fold improvement in sensitivity for LC with no reduction in resolution 20-fold improvement in sensitivity for HC with no reduction in resolution 2014 Waters Corporation 14

15 Middle-down Analysis 2014 Waters Corporation 15

16 Middle-down (Targeted MS/MS) Analysis of Antibody Light Chain 1. Reduction LC HC + 2. Reduction & Alkylation +19 LC alkylated Mixture of LC with/without alkylation +19 LC m/z 2014 Waters Corporation 16

17 Middle-down (Targeted MS/MS) Analysis of Antibody Light Chain 1. Reduction LC HC + 2. Reduction & Alkylation +19 LC alkylated Mixture of LC with/without alkylation +19 LC m/z Partially reduced LC without alkylation Partially reduced LC with alkylation Targeted MS/MS (m/z ) Targeted MS/MS (m/z ) The XS collision cell delivers increased resolution and allows efficient fragmentation for middle-down analysis of LC 2014 Waters Corporation 17

18 Middle down Analysis of LC showing Increased Resolution gain with XS Collision Cell Partially reduced LC without alkylation Targeted MS/MS (m/z ) +8 Targeted MS/MS (m/z ) MS/MS peak ~11K Partially reduced LC with alkylation The increased resolution gain by XS collision cell allows efficient interpretation of higher m/z fragmentation ions for middle-down analysis 2014 Waters Corporation 18

19 Peptide Mapping 2014 Waters Corporation 19

20 LC/MS Peptide Mapping of NIST mab LC/MS E Peptide Map Trypsin Digest Denature & Alkylate NIST mab 0.9 pmol on column Xevo G2-XS QTof Coverage Map Time (min) The increased sensitivity and XS collision cell provide better fragment ions for peptide sequence conformation. Fragment Spectrum High abundant Peptide T1-2 Fragment Spectrum Low abundant Oxidized Peptide T1-2 (0.9%) 2014 Waters Corporation 20

21 LC/MSE Peptide Mapping of NIST mab Xevo G2-XS vs. Xevo G2-S 10-fold improvement in sensitivity Improved MS Spectrum Resolution 0.9 pmol on column Xevo G2-XS QTof The MS sensitivity gained by the Xevo G2-XS system provides better fragment ions for peptide sequence conformation. 4.5 pmol on column Xevo G2-XS QTof Xevo G2-S QTof Fragment Spectra Peptide T1-2, Oxidized Xevo G2-S QTof 2014 Waters Corporation 21

22 Released Glycan Analysis 2014 Waters Corporation 22

23 Sensitivity Increased (>10x) with XS in Released N-linked Glycan Analysis 2AB-labeled Human IgG Glycan Xevo G2-XS QTof 2 pmol FLR Xevo G2-S QTof 20 pmol FLR Sensitivity mode (30 K Res) BPI MS BPI MS Sensitivity mode (20K Res) Time Time Waters Corporation 23

24 Released N-linked Glycan Analysis at equivalent loadings with and without XS Collision Cell The extra MS sensitivity gained by the Xevo G2-XS system allows the detection/id of very low abundance of glycans. TIC 20 pmol 2AB-labeled Human IgG Glycans Xevo G2-S QTof TIC 20 pmol 2AB-labeled Human IgG Glycans Xevo G2-XS QTof CID of G2S2 Glycan (0.03% relative quantitation level) 2014 Waters Corporation 24

25 Conclusions Xevo G2-XS QTof Sensitivity gain with no reduction in resolution enhances feature definition enables detection of minor components/glycoforms of proteins >10-fold increased in sensitivity for a wide range of analytes intact mabs, ADCs and their subunits peptide sequence confirmation/ptm identification : more complete fragment ion series detection of very low level glycans in routine LC-FLR-MS analysis Near isotopic resolution of proteins with a MW of ~ 30kDa, low ppm mass accuracy results under routine LC/MS conditions. greater confidence on the mass measurement of large peptides/small proteins effective middle-down fragmentation analysis Waters continued developments in analytical technologies for biological molecules have permitted more accurate characterization of mab and ADCs Waters Corporation 25

26 There is one more thing 2014 Waters Corporation 26

27 IonKey/MS Enhanced MS by the turn of a key 2-40 x MORE SENSITIVITY UPLC PERFORMANCE PLUG n PLAY ROBUST RELIABLE SOLVENT SAVINGS UP TO 90% THE FUTURE OF MS 2014 Waters Corporation 27

28 IonKey/MS Enhanced MS by the turn of a key 2014 Waters Corporation 28

29 Intact protein analysis Reduced monoclonal antibody Beta ikey BEH 300 C4 Light Chain Heavy Chain BPI On column load : 0.02 µg Linear gradient from 20% to 55% B in 5 min. Flow rate= 3 µl/min Temp = 65 o C 2014 Waters Corporation 29

30 Intact protein analysis ESI-MS Spectrum of Trastuzumab Beta ikey BEH 300 C4 +54 m/z= ng/ul ng load Average of 10 MS scans Waters Corporation 30

31 Intact protein analysis Carryover Evaluation Beta ikey BEH 300 C4 54+ m/z = µg/ml, 2µL Peak Area (counts) R² = Conc (ng/ul) mab conc. (ug/ml) mab amount (ng) Peak area RSD (%) Waters Corporation 31

32 Intact protein analysis Carryover Evaluation Beta ikey BEH 300 C4 50 ng/ul mab 0.1% FA blank Carry-over 0.56 % 2014 Waters Corporation 32

33 Thank you Vielen Dank Merci! Asish Chakraborty Henry Shion Barbara Sullivan Ying Qing Yu Scott Berger Weibin Chen Alan Millar 2014 Waters Corporation 33

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