ZipChip TM. Microfluidic CE-ESI Biotherapeutics CE-ESI-MS Applications. Please visit us at

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1 ZipChip TM Microfluidic CE-ESI Biotherapeutics CE-ESI-MS Applications Please visit us at // Actionable Intelligence HPMS & On-board Analytics / Slide 1 June 2016

2 // ZipChip CE-ESI Biotherapeutics Analysis Overview Fast, high-resolution ZipChip CE-ESI analysis of three minutes or less for intact glycoprotein, IgG, and ADC charge variants. Powerful back-end Orbitrap MS detection. Charge variants separated and identified through ZipChip CE-ESI MS. Associated glycoforms also detected and identified. Intact biotherapeutics charge variant detection and glycoform separation not achievable by LC/MS. Compatible with Exactive Plus EMR MS and NEW Q Exactive Plus EMR MS for native work For ADC ZipChip analysis, accurate DAR distributions obtained via fast ZipChip CE-ESI MS Minimal sample preparation required due to ZipChip on-line desalting. Low volume nl injections advantageous for precious in vivo biologics samples. Negligible carryover. Optimized CE method available on the ZipChip HR for fast intact biologics analysis. Data dependent top-down MS/MS can be run in same experiment. Powerful. Routine biotherapeutics peptide-mapping experiments can be achieved with ZipChip HS (10 cm) or ZipChip HR (22 cm) chips. ZipChip CE-ESI MS significantly faster than LC/MS peptide mapping. Excellent high throughput capability for this experiment via ZipChip. // Actionable Intelligence HPMS & On-board Analytics / Slide 2 June 2016

3 // ZipChip CE-ESI MS of Infliximab Monoclonal Antibody (Intact) 1 mg/ml Infliximab 10% 2-propanol 0.2% acetic acid ZipChip HR (22 cm separation channel) Decreasing electrophoretic mobility Unique, Fast Separation of Charge Variants (thru C-terminal lysine loss) by ZipChip CE followed by ESI-MS Analysis. These charge variants not separated by LC/MS <3 min analysis time Basic Acidic Redman, Batz, Mellors, Ramsey Anal. Chem, 2015, 87, 2264 // Actionable Intelligence HPMS & On-board Analytics / Slide 3 June 2016

4 // ZipChip CE-ESI MS of Intact Infliximab Monoclonal Antibody Near Native Conditions Basic Variants Acidic Variants Powerful MS information can also be gained for the additional Acidic and Basic Charge Variants and associated Glycoforms Top Down MS/MS Possibilities // Actionable Intelligence HPMS & On-board Analytics / Slide 4 June 2016

5 // ZipChip CE-ESI MS of Intact Infliximab mab Glycoform ID Glycoforms for each IgG charge variant can be identified from the deconvoluted mass spectrum ZipChip CE separation essential // Actionable Intelligence HPMS & On-board Analytics / Slide 5 June 2016

6 // Infliximab mab - Deconvoluted MS for Charge Variants + Glycoforms 128 Da 2-K Two C-terminal Lysines Highest charged, fastest CE elution 127 Da 1-K Partial Truncation of C-terminal Lysines 0-K Total Truncation of C- Terminal Lysines Least charged, slowest CE elution // Actionable Intelligence HPMS & On-board Analytics / Slide 6 June 2016

7 // Fast and Unique ZipChip CE Separations for an IgG-2 0-K 1 mg/ml IgG-2 10% 2-propanol 0.2% acetic acid 130 Da 2-K 131 Da 1-K Again C-terminal lysine truncation charge variants observed. Ratios much different relative to Infliximab 1-K 2-K PTM peak 0-K (major) // Actionable Intelligence HPMS & On-board Analytics / Slide 7 June 2016

8 // ZipChip CE-ESI Separations for Another IgG mg/ml IgG-2 10% 2-propanol 0.2% acetic acid Deconvolution of the above spectrum Different charge variants and associated glycoforms separated by ZipChip CE and identified. MS mining would likely reveal more IgG characterization information Fast separation just over 2 min Redman, E. A.; Mellors, J. S.; Starkey, J.; Ramsey, J. M. Anal. Chem. 2016, 88(4), // Actionable Intelligence HPMS & On-board Analytics / Slide 8 June 2016

9 // Peptide Mapping - ZipChip CE-ESI MS For IgG Tryptic Digest IgG2 Monoclonal Antibody Tryptic Digest, 0.1 mg.ml BGE: 50% methanol, 2% formic acid (ph2.2) ZipChip HS (10 cm) Full scan MS ( m/z) No Additional Sample Preparation Increased coverage of light and heavy peptides relative to LC Analysis time < 3 min run time can be extended for greater sequence coverage if required (see next) IgG peptide maps typically 60 mins or more by LC/MS large time savings via ZipChip CE-ESI 9 // Actionable Intelligence HPMS & On-board Analytics / Slide 9 June 2016

10 // Peptide Mapping Beckman/Sciex CESI 8000 MS For a IgG Tryptic Digest C-ESI 8000 (BPC) Considerably Slower IgG Peptide Maps by Conventional CE-MS (> 40 min) LC/MS IgG Peptide Maps typically min B,C Exacted Ion Traces for Glycopeptides CE Migration Time (min) // Actionable Intelligence HPMS & On-board Analytics / Slide 10 June 2016

11 // Peptide Mapping, ZipChip CE-ESI, Different IgG Tryptic Digest Q Exactive HF, DDA MS and MS2 Many scans across peptide peaks IgG Tryptic Digest, ~1.25 ng injected ZipChip HR (22 cm) 50% Acetonitrile 5% Acetic Acid Longer ~10 min run time achieved via HR Chip and BGE selection. Peak Capacity ~145 in <10 minutes Full MS MS/MS // Actionable Intelligence HPMS & On-board Analytics / Slide 11 June 2016

12 // ZipChip HR Peptide Mapping, Glycoprotein, Exactive EMR MS Beta Galactosidase Digest 0.1 mg/ml (860 nm) ZipChip HR with 22 cm separation channel longer digest run time of ~6 min improves coverage Exactive Plus EMR Orbitrap MS, Full scan MS BGE: 50% acetonitrile, 1% formic acid (ph 2.2) Sequence Coverage >90% Significantly faster analysis versus LC/MS // Actionable Intelligence HPMS & On-board Analytics / Slide 12 June 2016

13 // β-galactosidase Tryptic Digest - Q Exactive HF MS - DDA MS and MS/MS ZipChip HR (22 cm) Q Exactive HF DDA MS and MS/MS BGE: 50% Methanol, 1% Formic Acid <5 minutes Run Sequence Coverage >90% Full MS MS/MS 13 // Actionable Intelligence HPMS & On-board Analytics / Slide 13 June 2016

14 Relative Abundance // BSA (66,463 Da) Peptide Map on High Resolution ZipChip (22 cm) c:\xcalibur\...\bg_2016x03x23xxhr002xx02 03/23/16 10:08:06 BSA digest 0.5 um, 50% MeOH 2% FA, 60 s injection RT: Background subtracted BPI electropherogram NL: 3.99E7 Base Peak MS bg_2016x03x23 xxhr002xx02 Exactive Plus EMR Orbi MS ZipChip HR channel (22 cm) Run time ~8 min Width at base: 1.5 sec Extracted ion electropherogram Width at base: 2.1 seconds NL: 1.08E7 Base Peak m/z= MS bg_2016x03x23 xxhr002xx02 Can collect many MS scans across peaks excellent coverage Time (min) // Actionable Intelligence HPMS & On-board Analytics / Slide 14 June 2016

15 2016x05x05xx01_ /05/16 09:53:39 ADH digest 100 mm AmAcet 50% 2-propanol 5% HAc, 60s inj // Modulating Analysis Time via BGE Viscosity RT: Total run time <10 min Avg. peak width: ~2.4s Avg. scans/peak: ~ Total run time <13 min Avg. peak width: ~3.1s Avg. scans/peak: ~ Time (min) PRELIMINARY WORK IN PROGRESS // Actionable Intelligence HPMS & On-board Analytics / Slide 15 June Total run time <23 min Avg. peak width: ~4.6s Avg. scans/peak: ~ NL: 1.27E8 Base Peak MS bg_2016x0 5x04xx04 50/50 ACN/Water 5% Acetic Acid η = 0.77 cp NL: 1.06E8 Base Peak MS bg_2016x0 5x04xx09 50/50 MeOH/Water 5% Acetic Acid η = 1.62 cp NL: 9.05E7 Base Peak MS 2016x05x0 5xx01_ /50 2-propanol/Water 5% Acetic Acid η = 3.04 cp

16 // ZipChip CE-ESI MS - Reduced/Alkylated IgG mg/ml 50% Acetonitrile 1% Formic Acid Thermo Exactive Plus EMR MS More powerful IgG characterization information through fast ZipChip CE-ESI mabs reduced and alkylated with DTT/IAM using standard protocols // Actionable Intelligence HPMS & On-board Analytics / Slide 16 June 2016

17 // ZipChip CE-ESI MS of Intact Antibody Drug Conjugate Antibody Drug Conjugate, 1 mg/ml ZipChip HR (22 cm separation channel) BGE: 10% IPA, 0.2% acetic acid (ph 3.5) Excellent CE Separations seen for ADCs Very challenging LC analysis Redman, Mellor, Starkey, Ramsey, Anal. Chem., 2016, 88(4), 2220 // Actionable Intelligence HPMS & On-board Analytics / Slide 17 June 2016

18 // ZipChip CE-ESI MS of an Intact Antibody Drug Conjugate 1 mg/ml ADC 10% 2-propanol 0.2% Acetic Acid A wealth of ADC species, glycoforms and charge variants revealed by ZipChip CE-ESI MS Less than 2.7 min analysis. Redman, E. A.; Mellors, J. S.; Starkey, J.; Ramsey, J. M. Anal. Chem. 2016, 88(4), // Actionable Intelligence HPMS & On-board Analytics / Slide 18 June 2016

19 // Deconvolution of DAR Spectra Overlaid spectra from each DAR peak in the separation Avg. mass shift due to conjugation: 3145 Da Redman, E. A.; Mellors, J. S.; Starkey, J.; Ramsey, J. M. Anal. Chem. 2016, 88(4), // Actionable Intelligence HPMS & On-board Analytics / Slide 19 June 2016

20 // DAR Distribution Species CE-MS Area % Infusion Area % ice Area % DAR-0 7.7% 8.6% 5.8% DAR % 33.2% 29.1% DAR % 39.0% 44.8% DAR % 16.5% 16.7% DAR-4 2.1% 2.7% 3.6% Avg. DAR Close agreement between ZipChip CE-MS and established ice DAR Distributions ice data was provided by collaborators Redman, E. A.; Mellors, J. S.; Starkey, J.; Ramsey, J. M. Anal. Chem. 2016, 88(4), // Actionable Intelligence HPMS & On-board Analytics / Slide 20 June 2016

21 // ZipChip CE-ESI MS - Reduced/Alkylated Lysine-Linked ADC Drug Conjugation Occurs Primarily on the Light Chain 0.25 mg/ml ADC 50% Acetonitrile 1% Formic Acid Thermo Exactive Plus EMR // Actionable Intelligence HPMS & On-board Analytics / Slide 21 June 2016

22 // ZipChip CE-ESI MS - Analysis of Whole Blood Lysate Biomarkers for diabetes management Blood glucose Hours Serum albumin Weeks Red blood cell Months Lifespan in vivo 1000x Dilution of Whole Blood Lysate 50% Acetonitrile 3% Acetic Acid // Actionable Intelligence HPMS & On-board Analytics / Slide 22 June 2016

23 // Summary Combining ZipChip with Conventional MS Analytical Capabilities Fast unique analysis (~3 min per sample) Low sample volume injection, nl scale Key for early stage, high value samples Low impact on MS minimize downtime/analytical drift Little to no sample preparation, on-chip de-salting Unique measurement capabilities: Antibodies and ADCs Glycoproteins, Antibodies Antibody Drug Conjugates Amino Acids, Metabolites Peptide Mapping, Proteomics Small Molecules Zip Chip fully-integrated Interface for commercial Mass Spectrometer Introductory Price $25K Zip Chips (rated for ~100 samples) - $300/$400 Current interfacing with Thermo Q Exactive (QE, QE Plus, QE HF) and Exactive EMR Orbitrap MS platforms. Also available on the Thermo LTQ-Orbitrap, Ion Trap and QQQ MS systems. // Actionable Intelligence HPMS & On-board Analytics / Slide 23 June 2016

24 // Thank You 908 Devices is headquartered in Boston s Innovation District. // Actionable Intelligence HPMS & On-board Analytics / Slide 24 June 2016