Introducing the Thermo Scientific Q Exactive HF-X and ZipChip Technology for BioPharma Applications

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1 Introducing the Thermo Scientific Q Exactive HF-X and ZipChip Technology for BioPharma Applications Jonathan L. Josephs Ph.D. Marketing Director Life Sciences BU The world leader in serving science

2 Thermo Scientific Q Exactive BioPharma MS 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 Thermo Scientific 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 t t Sliding Window Deconvolution is the Smart Choice for Chromatography [ [ t [ I t [ [ i t I t i t I t i t y s e n n v i e l a R e y s e n n v i e l a R e y s e n 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 Comparison of Sliding Window and Standard Deconvolution Mass Relative Intensity Sliding window detects the higher mass ADC components missed by standard deconvolution

7 Thermo Scientific Q Exactive BioPharma MS Offers a Complete Characterization Solution for BioPharma Customers? 7

8 Subunit Analysis in Protein Mode on Thermo Scientific Q Exactive Plus MS LC-MS Analysis of IdeS-digested, Reduced Trastuzumab Fc LC Fd Time (min) Fc * 28+ * R=14, Sliding Window Xtract TM deconvolution LC.2 ppm LC * Fc A2GF 1.9 ppm mass * Fd.1 ppm Fc A2G1F 1.4 ppm Fd * *

9 Subunit Analysis in Protein Mode on Thermo Scientific Q Exactive HF MS LC-MS Analysis of Reduced Trastuzumab Light Chain R = 12, Full MS * 24+ * LC HC Time (min) Heavy Chain R = 15, Full MS R = 24, SIM MS R e l a t i v e I n t e n s i t y 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 Mass HC A2G1F 1.2ppm

10 Denaturing LC-MS in HMR mode Sensitive Intact Analysis of Trastuzumab Antibody 1 * ng (1 pg).5 ng 1. ng 1. ng 1. ng 1. ng Q Exactive Plus in HMR Mode with MAbPac RP 2.1 x 5mm, 25 µl/min ppm ppm ppm pg injection Mass 5 ReSpect TM deconvolution 1. ppm ppm ppm Mass 1 ng injection 1

11 Navigating Intact Protein Complexity with High Mass Range (HMR) Mode * MAbPac RP 5 1 Time (min) 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 11

12 Two Reasons Why Native MS Intact Analysis is a Powerful Addition to a Comprehensive ADC Characterization Strategy Cysteine-linked Lysine-linked (1) Preservation of structural non-covalent interactions (2) Increased separation in high mass range denatured native

13 Intact Analysis of Trastuzumab Emtansine Lysine-linked ADC Denaturing vs. Native Conditions Denatured R=17,5 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 1 5 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 Understanding Micro-heterogeneity and Critical Quality Attributes (CQAs) Biologics are Always Complex Mixtures Oxidation Deamidation Disulfide bond scrambling Sequence variants Molecular Weight Drug-to- Antibody Ratio Chain Pairing Isoform pattern Carbohydrate structures Subunit analysis Comprehensive characterization of therapeutic protein CQAs requires analysis from multiple perspectives. 15

16 Thermo Scientific UHPLC + Orbitrap MS + Data Analysis Software A Powerful Solution to the Challenge of BioPharma Characterization Thermo Scientific Vanquish Horizon UHPLC system Thermo Scientific Q Exactive Plus/HF/HF-X BioPharma Orbitrap Mass Spectrometer Thermo Scientific BioPharma Finder Data Analysis Software Platform 16

17 Thermo Scientific Q Exactive HF-X MS Specifications Scan rate Up to 4 Hz Resolution 24, (FWHM) at 2 Mass range Mass Accuracy Dissociation Multiplexing Detectors Polarity Switching Scan Functions 5 to 6, Up to 8, in BioPharma option 3 ppm external, 1 ppm internal Source CID, HCD Up to 1 precursor ions Orbitrap device one full cycle in <1 sec (one full positive mode scan and one full negative mode scan at a resolution setting of 6,) FS: Full Scan AIF: All Ion Fragmentation, SIM: Selected Ion Monitoring, PRM: Parallel Reaction Monitoring, DIA: Data Independent Acquisition, ddhcd: data dependent HCD Options BioPharma option 17

18 Thermo Scientific Q Exactive HF-X MS: Improved Architecture, Improved Performance High Capacity Transfer Tube (HCTT) Ultra-High Field Orbitrap Mass Analyzer HCD Cell C-Trap Electrodynamic Ion Funnel Advanced Active Beam Guide (AABG) HyperQuad Mass Filter with Advanced Quadrupole Technology (AQT) 18

19 Key Technologies of Thermo Scientific Q Exactive HF-X MS High Capacity Transfer Tube Electrodynamic Ion Funnel 28 Hz 32 ms maxit 32 ms 4 Hz 16 ms 16 ms Ultra-High Field Orbitrap Optimized Scan Matrix and new Transient lengths 19

20 Optimized Scan Matrix Q Exactive HF Hz 32 ms maxi T Q Exactive HF-X 28 Hz 4 Hz 32 ms maxit 16 ms 16 ms 32 ms MS/MS 32 ms Longer maxit Reduced scan overhead ahcd Similar maxit Reduced scan overhead ahdc Brighter ion beam, reduced scan overhead, and accelerated HCD (ahcd) is boosting acquisition speed Advantage for both MS and MS/MS mode Fast and high quality MS/MS acquisition up the 4 Hz with new 16 msec transient (7,5 resolution setting) Orbitrap detection Maximum fill time with precursor ions Inter and intra scan overheads 2

21 Intact Antibody Analysis with High Mass Range Mode Thermo Scientific Q Exactive Plus MS with BioPharma Option Orbitrap scanning up to 8 Flexibility for denaturing conditions or Native MS Column: MAbPac RP (reversed phase LC) Denatured Column: MAbPac SEC-1 (native size exclusion LC) Native

22 Analysis of Intact Trastuzumab under Native Conditions in HMR Mode Improved S/N ratio on the Q Exactive HF-X by a factor of ~ z= z= z= z= z=26 z= z=27 z= NL: 1.16e5 z= NL: 4.1e6 z= z= N=152.3 z= N= N= N= N= N= N= N= S/N=76.65 (1.16e5/152.5) S/N=828 (4.1e6/499.5) Q Exactive HF Q Exactive HF-X TIC, size exclusion chromatography using Acclaim SEC column Time (min) salt peak salt peak SEC LC-MS analysis of intact Trastuzumab monoclonal antibody using Acclaim SEC column, 4.6 x 3 mm, 3 µl/min flow rate, 5 mm ammonium acetate. Full MS, HMR mode, 25 8, resolution setting 3k, 1 µscans. Spectra show an average of 3 scans (1 µscans each). 22

23 Intact Analysis of Trastuzumab Emtansine Lysine-linked ADC Denaturing vs. Native Conditions ADC construction can create layers of sample heterogeneity Native MS allows greater inter-charge state separation Native MS allows True Intact analysis of heterogeneous samples No deglycosylation step Denatured Native

24 Choosing the Right Strategy for Intact Protein Analysis Denaturing MS Native MS Most sensitive detection of simple mabs and subunits, low abundance protein isoforms High resolution separations Reverse Phase (RP) Most confident analysis of heterogeneous mixtures, ADCs, glycoproteins, PEGylation On-line desalting Size Exclusion (SEC) 24

25 Intact What is native? Protein What MS: is What denatured?? is Native? What is Denatured? Native conditions 1% aqueous Moderate (physiological) ph Organic content ph = Very high or very low Denaturing conditions Static nanospray MS Very weak non-covalent interactions best analyzed with static nanospray infusion Size Exclusion Chromatography MS Easy to use. Enables online desalting. Signifcant acetonitrile concentration and highly acidic ph cause protein unfolding Reverse Phase Chromatography MS 25

26 Is What Ion is native? Exchange What Chromatography is denatured?? Compatible with LC-MS? Native conditions 1% aqueous Moderate (physiological) ph Organic content ph = Very high or very low Denaturing conditions Static nanospray MS Size Exclusion Chromatography MS Ion Exchange Chromatography MS??? Reverse Phase Chromatography MS 26

27 Highly Cited Article on Trastuzumab Charge Variant Analysis (Salt-based Elution) Acidi c Main Basic Harris et al., 21 1 x deamidation on light chain residue D3 LC-UV - 2 mm NaCl gradient in 2 mm NaPO 4 Unmodified mab 27

28 Asparagine Deamidation Results in Very Small Mass Increase, Very Small pi Decrease Isoaspartic Acid Calculated isoelectric point (pi) change upon Trastuzumab deamidation Asparagine (N) (pk a = 8.8) Succinimide Aspartic Acid (D) (pk a = 3.9) Unmodified Δ pi = -.7 units 1 x Deamidation pi = pi = N D Δ mass = Monoisotopic Average Da.9848 Da Data source for pka values: ExPasy 28

29 Elution by ph Gradient, Most Sensitive ESI in Native Cation Exchange LC-MS R R - COO - COO H 2 O + NH 3 H 2 O H 2 O H 2 O H 2 O H 2 O Binding conditions ph = ~ neutral Protein pi = basic R R COO - - COO - OH - OH - OH NH 2 - OH - OH - OH Eluting conditions ph = basic constant 5 mm ammonium acetate buffer 29

30 Method for Native Weak Cation Exchange (WCX) LC-MS Thermo Scientific ProPac WCX-1 Weak cation exchange column Buffer A = 5 mm AmAce, ph 7 Buffer B = 5 mm AmAce, ph 1 Flow rate =.3 ml/min Time (min) % Buffer B Thermo Scientific Vanquish Horizon UHPLC system Buffer salts Trastuzumab Time (min) Thermo Scientific Q Exactive HF-X Orbitrap MS with BioPharma Option 6, resolution setting 1 microscans % Buffer B On-column sample concentration and online desalting are key features of native cation exchange LC-MS 3

31 Native WCX-MS Method is Comparable to Separation in Literature Acidi c Main Unmodified Basic 1 Acidic Main Unmodified Basic 9 8 Deamidated LC D Deamidate d LC D Time (min) Harris et al., 21 LC-UV - 2 mm NaCl gradient in 2 mm NaPO 4 Salt elution Thermo Scientific Q Exactive HF-X BioPharma LC-MS ph 7-1 gradient (1-8%) in 5 mm NH 4 CH 3 CO 2 ph elution 31

32 Native WCX-MS: 1 ul Injection of Formulation Trastuzumab (21 ug/ul) Buffer salts Time (min) Herceptin Time (min)

33 Raw Data Reflect Mass Accuracy of Unmodified and Deamidated Trastuzumab Simulated Spectrum Experimental Data Unmodified Deamidated Unmodified ppm Deamidated Base Peak XIC 3 ppm R= R= Deamidated Unmodified Time (min) 33

34 Sliding Window Deconvolution is the Smart Choice for Chromatography Automatically performs Xtract or ReSpect deconvolution along separations timescale Removes user bias from deconvolution analysis Improves quality of batch analyses Sensitive and confident identification + relative quan All biologics benefitted - simple mabs, complex ADCs [ [ [ [ [ R e l a t i v e I n t e n s i t y R e l a t i v e I n t e n s i t y R e l a t i v e I n t e n s i t y RT(min) RT(min) mab mab + 2 drugs mab + 4 drugs RT(min) LC peak area automatically integrated 34

35 ReSpect Sliding Window Results for Native WCX-MS with Automatic Annotation Deconvolution XIC TIC RT (min) Modification Average Mass (Da) Theoretical Mass (Da) Matched Mass Error (ppm) Relative Abundance RT Range 2xA2G1F,1xDeamidation xA2GF,1xA2G1F,1xDeamidatio n xA2GF,1xDeamidation xA2G1F xA2GF,1xA2G1F xA2GF

36 NIST mab Lys-variants are Readily Separated and Detected by Native WCX-MS Δ =.5 Δ =.6 pi = 8.47 pi = 8.53 Native WCX-MS method baseline resolves pi differences of <.5 units 1 Lys pi = Lys 2 Lys Time (min) Lys 1 Lys 2 Lys Without separation true impurities will cause mass interferences Data source for pka values: ExPasy Calculator/ProteinToo l 36

37 Intact What is Protein native? MS What Separation is denatured?? Solutions also Include Capillary Electrophoresis (CE) Native conditions 1% aqueous Moderate (physiological) ph Organic content ph = Very high or very low Denaturing conditions Static nanospray MS Size Exclusion Chromatography MS Ion Exchange Chromatography MS Capillary Electrophoresis MS Reverse Phase Chromatography MS 37

38 98 Devices ZipChip System The 98 Devices ZipChip system interface directly mounts onto the front end of a compatible mass spectrometer The ZipChip system uses integrated microfluidic technology to prepare, separate samples by capillary electrophoresis (CE), and then electrospray (ESI) analytes directly into mass spectrometers (MS) Each analysis only consumes a few nanoliters of sample containing pico grams to nano grams of analytes Most analyses can be completed in a few minutes Zip Chip HS Zip Chip HR Separation channel length (cm) 1 22 On Chip De-salting capability Yes Yes Integrated ESI Emitter Yes Yes EEPROMS Yes Yes Max chip life (# of injections) Recommended use Small molecules or Big molecules or simple sample mixture complex sample mixture Typical anlaysis time Up to 3 min Up to 12 min 38

39 98 Devices ZipChip System Hardware and Consumables ZipChip Interface Autosampler Zip Chips ZipChip Assay Kits Compatible with all Thermo Scientific Exactive and Q Exactive Hybrid Quadrupole- Orbitrap MS, as well as the Thermo Scientific LTQ- Orbitrap XL Hybrid Ion Trap- Orbitrap series MS instruments Also compatible with Thermo Scientific ion trap MS and select triple-stage quadrupole MS instruments Data collection, processing and reporting through Thermo Scientific Xcalibur software Fully automated and integrated into the ZipChip software Compatible with both 48-vial plate and 96-well plate Disposable chips Two types: HR chip and HS chip Each chip is a single piece of glass, about the same size and shape as a microscope slide Microfluidic channel and electrospray is integrated into both types of chips 5 chips per pack Three types of pre-packed assay kits are designed for intact protein, peptides, and metabolites analyses, respectively Individual bottles can be directly loaded into the 98 autosampler Packaged in individual bottles to match to 96 well-plate solvent usage Good for 5 injections per box of assay kit 39

40 ZipChip - Thermo Scientific Q Exactive HF-X MS Analysis of Intact Infliximab Antibody Lys Lys 3.5 Lys Time (min) 4.5 CZE-MS electropherogram of intact infliximab: separation of lysine-variants 3 ng of sample injected Relative Intensity Deconvoluted MS spectrum [GF/GF] 2 Lys Lys [GF/G1F] 2 Lys Lys [(GF/G2F)/(G1F) 2 ] 2 Lys Time (min) Sliding window deconvolution with Respect TM algorithm Mass z=26 z= z=25 z= Full MS spectra zoom RT 2.6 min RT 3.5 min RT 4.5 min z= Lys 1 Lys Lys Theoretical and experimental masses of infliximab glycoforms of lysine variants Lys-Variant Glycoform Theor. average mass Exp. average mass Mass (ppm) 2 Lys GF/G1F Lys GF/GF Lys (G1F)2 or (GF/G2F) Lys GF/GF Lys GF/G1F Lys (G1F)2 or (GF/G2F) Lys GF/GF Lys GF/G1F Lys (G1F)2 or (GF/G2F)

41 WCX and CE Show Complementarity, Reverse Elution Order of NIST mab Lys-variants lys 1 lys 2 lys Time (min) Basic Acidic ZipChip (Electrophoresis) Acidic Lys Lys Basic 2 Lys Time (min) Weak Cation Exchange (Chromatography) Time (min) % Buffer B 41

42 Conclusions Native LC-MS is an easy to use solution for analyzing micro-heterogeneous samples Thermo Scientific Q Exactive HF-X MS with BioPharma Option offers increased sensitivity for native mab analysis Native WCX-MS is a powerful method which expands offerings for native LC-MS intact protein analysis Online desalting On-column sample concentration Baseline resolves pi differences of <.5 units Without separation true micro-heterogeneous impurities can cause interferences and mass errors 42

43 Thank you! Genentech Wendy Sandoval Guanghui Han Wilson Phung Chengfeng Ren BioPharma Finder Team Paul Gazis Jennifer Sutton Pharma/BioPharma Team Michael Blank David Brant Kelly Broster Kate Comstock Simon Cubbon Stephane Houel Rowan Moore Keeley Murphy Kyle D Silva Terry Zhang John Rontree 43

44 Thermo Scientific Q Exactive BioPharma MS Offers a Complete Characterization Solution for BioPharma Customers 44

45 Peptide Mapping of Trastuzumab With Digestion Using SMART Digest T Trypsin Kit Vanquish UHPLC Q Exactive HF-X MS Digestion SMART Digest Kit LC-MS Data Analysis Sequence coverage Sequence variants ID of unexpected mods Modifications ID +quan TIC Modification Sumary Time (min) Thermo Scientific Acclaim TM VANQUISH TM C18 UHPLC column 2 min gradient ddtop5 Method 6k/15k resolution settings (MS/MS 2 ) Sequence coverage map with color coded peptides Identified peptides highlighted by color coded peak shading in chromatogram 1% sequence coverage based on MS/MS spectra 45

46 Workflow for a Middle Down Experiment 1gG (15kDa) IdeS Reduction Sample Prep LC (25kDa) Fd (25kDa) Fc/2 (25kDa) LC-MS Fc/ LC LC Time (min) Fd MS/MS Type of fragmentation available: Q Exactive Plus/HF: HCD Orbitrap Tribrid Fusion/Lumos: HCD, CID, ETD and EThcD (UVPD - not commercial) Due to the complexity of the spectra in top-down analysis, high resolution is required 46

47 Sequence Coverage with HCD Fragmentation on a Thermo Scientific Q Exactive Plus MS (Trastuzumab) HCD MS/MS R=14, 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 47

48 Thank you! Aaron Bailey Eugen Damoc Kai Scheffler Stephane Houel Alexander Harder 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 48