The world leader in serving science. Jonathan L. Josephs and Aaron O. Bailey. Life Sciences Mass Spectrometry

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1 Characterization of Monoclonal Antibodies Using UHPLC-HRAM MS The power of the Q Exactive BioPharma for online intact, subunit analysis, middle down, and peptide mapping Jonathan L. Josephs and Aaron O. Bailey. Life Sciences Mass Spectrometry The world leader in serving science

2 Q Exactive BioPharma offers a complete characterization solution for BioPharma customers? 2

3 Two Types of Orbitrap Measurements for Intact Proteins Average mass resolution High Mass Range (HMR) mode Works for all proteins (mabs) Lowest resolving power Isotopic resolution Protein mode Small / medium-sized proteins (mab subunits) Highest resolving power Average mass Monoisotopic mass ReSpect deconcolution algorithm Xtract deconvolution algorithm 3

4 Intact Protein Analysis in BioPharma Finder Workflow software for intact protein mass determination Supports all Orbitrap mass spectrometers Includes 2 deconvolution algorithms: Xtract for isotopically resolved proteins ReSpect TM for isotopically unresolved proteins (e.g. IgG) Batch processing/automation 4

5 Sliding Window Deconvolution is the smart choice for chromatography [ [ [ [ [ t t t I t i t I t i t I t i t n s y e n v i e l a R e n s y e n v i e l a R e n s y e n v i e l a R e mab RT(min) RT(min) RT(min) mab + 2 drugs mab + 4 drugs Components integrated 5

6 Q Exactive BioPharma offers a complete characterization solution for BioPharma customers? 6

7 Subunit Analysis in Protein Mode on Q Exactive Plus LC-MS analysis of IdeS-digested, reduced Trastuzumab Fc LC Fd Time (min) Fc * 28+ * R=1, Sliding Window Xtract TM deconvolution LC.2 ppm LC * Fc A2GF 1.9 ppm mass * Fd.1 ppm Fc A2G1F 1.4 ppm Fd * *

8 Subunit Analysis in Protein Mode on Q Exactive HF LC-MS analysis of reduced Trastuzumab Light Chain R = 1, Full MS * 24+ * LC HC Time (min) Heavy Chain R = 15, Full MS R = 2, SIM MS l a t i v e I n t e n s i t y R e LC.8 ppm Mass Xtract TM deconvolution for monoisotopic masses ReSpect TM deconvolution for average masses l a t i v e I n t e n s i t y R e HC A2GF 2.8ppm 1.26 Mass HC A2G1F 1.2ppm

9 Denaturing LC-MS in HMR mode Sensitive intact analysis of Trastuzumab antibody * ng ( pg).5 ng 1. ng 1. ng. ng. ng Q Exactive Plus in HMR Mode with MAbPac RP 2.1 x mm, 2 µl/min.9 ppm ppm ppm pg injection Mass ReSpect TM deconvolution 1. ppm ppm ppm Mass ng injection 9

10 Navigating Intact Protein complexity with High Mass Range (HMR) mode * 49+ MAbPac RP 5 1 Time (min) 3 7 R=17, Denaturing MS is compatible with reverse phase HPLC separation. MAbPac SEC Time (min) * R=35, Native MS is best for co-eluting high complexity samples like ADCs HMR mode intact analysis of Trastuzumab shows same answer in denaturing or native conditions 1

11 Two reasons why Native MS intact analysis is a powerful addition to a comprehensive ADC characterization stategy Cysteine-linked Lysine-linked (1) Preservation of structural non-covalent interactions (2) Increased separation in high mass range denatured native

12 Native LC-MS of Brentuximab vedotin, cysteine-linked ADC Time (min) Size Exclusion LC-MS Intact Brentuximab vedotin 3 7 DAR2 DAR4 DAR6 Challenges: Optimizing desalting (Size Exclusion LC) Optimizing desolvation (MS) Can achieve balance of clean spectra and preservation of non-covalent forms Average Drug-to-Antibody Ratio (DAR) 4.7 Relative Intensity DAR 2 x GF x GF 17.6 ( Da) 2 x GF , 2 x GF DAR8 2 x GF Mass Previously reported DAR static nanospray Dabaene et al., Anal Chem. 14 Nov 4;86(21):

13 Intact analysis of Trastuzumab Emtansine lysine-linked ADC Denaturing vs. Native conditions Denatured R=17, Native R=35, Native MS allows greater separation of co-eluting species charge states 13

14 Native LC-MS in High Mass Range (HMR) mode Intact ADC Analysis of Trastuzumab Emtansine R=7, zoom Size Exclusion Chromatography ADC Buffer salts can be diverted to waste Time (min) Mass GF/G1F DAR Mass Accuracy (ppm) Relative Abundance D D D D D D D D D Trastuzumab Emtansine Lysine-linked ADC Average Drug-to-Antibody Ratio (DAR)

15 Q Exactive BioPharma offers a complete characterization solution for BioPharma customers 15

16 Workflow for a middle down experiment 1gG (1kDa) IdeS Reduction Sample Prep LC (25kDa) Fd (25kDa) Fc/2 (25kDa) LC-MS Fc/ LC LC Time (min) Fd Type of fragmentation available: Q Exactive Plus/HF: HCD Orbitrap Tribrid Fusion/Lumos: HCD, CID, ETD and EThcD (UVPD - not commercial) 16

17 Workflow for a middle down experiment 1gG (1kDa) IdeS Reduction Sample Prep LC (25kDa) Fd (25kDa) Fc/2 (25kDa) LC-MS Fc/ LC LC Time (min) Fd MS/MS Due to the complexity of the spectra in top-down analysis, high resolution is required 17

18 Sequence coverage with HCD fragmentation on an Q Exactive Plus (Trastuzumab) HCD MS/MS R=1, z=26 z=25 z=7 z=6 z=6 z=8 z=6 z=6 z=7 z=4 z=4 z= Xtract TM deconvolution Light Chain 49% residue cleavages Fd 38% residue cleavages mass Fc 39% residue cleavages 18

19 Q Exactive BioPharma offers a complete characterization solution for BioPharma customers 19

20 Trastuzumab peptide mapping Trastuzumab Accurate peptide IDs are based on high fidelity MS/MS spectra visualized in a color coded display by BioPharma Finder software.

21 Top 15 most abundant glycoforms measured by peptide mapping Protein Residue # Modification Category Sequence Confidence Automatic detection by BioPharma Finder Average % Abundance % Abundance Run 1 % Abundance Run 2 % Abundance Run 3 Heavy Chain 3 N3+A2GF Glycoform EEQYNSTYR Heavy Chain 3 N3+A2G1F Glycoform EEQYNSTYR Heavy Chain 3 N3+A1GF Glycoform EEQYNSTYR Heavy Chain 3 N3+A2G2F Glycoform EEQYNSTYR Heavy Chain 3 N3+A2G Glycoform EEQYNSTYR Heavy Chain 3 N3+M5 Glycoform EEQYNSTYR Heavy Chain 3 N3+A1G1F Glycoform EEQYNSTYR Heavy Chain 3 N3+A1G Glycoform EEQYNSTYR Heavy Chain 3 N3+A2G1 Glycoform EEQYNSTYR Heavy Chain 3 N3+Unglycosylated Glycoform EEQYNSTYR Heavy Chain 3 N3+A2S1G1F Glycoform EEQYNSTYR Heavy Chain 3 N3+A1G1 Glycoform EEQYNSTYR Heavy Chain 3 N3+M6 Glycoform EEQYNSTYR Heavy Chain 3 N3+A2S1GF Glycoform EEQYNSTYR Heavy Chain 3 N3+M4 Glycoform EEQYNSTYR

22 Top 15 most abundant glycoforms measured by peptide mapping Automatic detection by BioPharma Finder R e l a t i v e A b u n d a n c e Predicted MS/MS b (GnM) b b b Y1-F² M1² M2F² M3² Acquired MS/MS A1G² Y

23 Thank you! Eugen Damoc Kai Scheffler Stephane Houel Thomas Moehring Alexander Makarov Jessica Wang Jennifer Sutton Michael Blank Terry Zhang Kate Comstock Keeley Murphy David Brant Tina Settineri Ken Miller Paul Gazis Shanhua Lin Shane Bechler Seema Sharma David Horn Fred Zinnel Mark Sanders Keith Waddell Brenda Kesler 23