1. The use of Monitoring Peptides from Proteolytic Digests for LCMS MRM-based Investigations of in vivo Protein Degradation Tao Niu, Lin-Zhi Chen, Jeffrey X. Duggan*, Jesse Jones Boehringer Ingelheim Pharmaceuticals, Inc, Ridgefield, CT USA * presenting author Current Regulatory Developments and Challenges in Biotherapeutics Bioanalysis by Hybrid LBA/LCMS AAPS Annual Meetingand Exposition, (#216), October 27, 2015, Orlando, FL

2. Refer to a Previously Published White Paper that Discusses the Use of Monitoring Peptides in LCMS Protein Quantification Recommendations for Validation of LC-MS/MS Bioanalytical Methods for Protein Biotherapeutics Rand Jenkins 1, Jeffrey X. Duggan 2, Anne-Françoise Aubry 3, Jianing Zeng 3, Jean W. Lee 4, Laura Cojocaru 5, Dawn Dufield 6, Fabio Garofolo 7, Surinder Kaur 8, Gary A. Schultz 9, Keyang Xu 8, Ziping Yang 10, John Yu 2, Yan J. Zhang 3, Faye Vazvaei 11,12 AAPSJ 17(1),1-16 (2015) “Because LC-MS/MS technology readily supports multi-analyte testing, it is possible to obtain qualitative structure-related information simultaneously with quantification of the target protein by measuring multiple peptides in the assay. This information is potentially valuable for gaining insights into possible biotransformation of the protein in vivo, and it may also be useful for assay trouble-shooting.” “In addition to the surrogate peptide for quantification, one or more secondary peptides may be chosen from the list of potential peptide candidates for use as “monitoring” peptide(s)” “Any notable change in the ratio(s) that is observed to trend over a PK or TK profile may be indicative of biotransformation (e.g., protein subunits have been cleaved), or that the integrity of the molecule has been otherwise altered in vivo.” …The following example will demonstrate the use of monitoring peptides to assess the molecular integrity of a muiti-subunit protein therapeutic in vivo 2

3. Monitoring peptide: a specific peptide within the protein molecule or subunit that is unique, free from significant matrix interference, and distinct from the surrogate peptide that is used for quantitation. Each monitoring peptide must have the following basic properties: Unique to the analyte protein Sufficiently free from matrix interference Sufficient sensitivity and stability Located in subunit or other part of interest in the biotherapeutic Not used for quantification Monitoring Peptides for Use in Assessing the integrity of Biotherapeutics in vivo 3

4. LC/MS/MS Binary Biotherapeutic Assay with Immunopurification 4 LC/MS/M S lys-C/trypsin digestion all peptides: biotinylated capture RGT Drug capture RGT biotinylaton Streptavidin magnetic beads drug- capture RGT complex drug-RGT complex on beads 1 2 2 1 √

5. Animal SC and IV PK study results, peptide 2 Quantitation IV Dosing, BI XYZAB, Peptide 2 5

6. TK Study Concentration Ratios Based Upon Quantitation with peptide 2 Vs Peptide 1, SC Dose 6 SubjectTimePep 1aPep 2Ratio 10 75.7 76.20.99 16 73.5 740.99 124 39.2 52.40.75 148 32 42.80.75 196 27.1 28.40.95 1144 22.3 19.91.12 1192 0 00.00 1288 2.55 00.00 30 77.9 85.40.91 36 70.8 67.11.06 324 60.4 71.10.85 348 50.3 52.60.96 396 37.2 46.50.80 3144 33.1 460.72 3192 2.45 00.00 3288 0 00.00 50 153 1401.09 56 74.4 74.71.00 524 40.4 40.70.99 548 35 33.71.04 596 33 34.20.96 5144 22.6 24.70.91 5192 7.11 00.00 5288 2.33 00.00 Lines diverge slightly with concentration; should compare back-calculated concentrations or response-corrected areas P2 P1 Y=.0357x 2 +1.7x - 0.0005 Y= 0.007x 2 +1.43 -0.0003 P2 P1

7. Examine Intactness In Vivo of a Complex Multi Subunit Construct IgG-based construct with 4 additional subunits Subunits covalently bound, necessary for activity The drug was administered to Cyno monkeys on Day 1 and Day 11 TK samples were taken for 72 then 96 hours post dose Was the molecule intact or was there cleavage / degradation Used both Monitoring Peptide and Intact HRLCMS approaches 7

8. IgG Construct Components and Monitoring Peptide Locations Is this construct stable in vivo? 8 L- subunits Peptide linker A B B A

9. Process Workflow  In Silico analysis –Tryptic fragments –Exclusion rules  Confirm Peptides after proteolysis in buffer  Determine peptides’ sensitivity and dynamic range  Confirm peptides in matrix: using immunocapture analytical method a)- Anti human Fc for capture of and IgG (monkey plasma matrix) b)-Captures the main subunit to which others are covalently bound  Determine subunit peptide/Fc peak area ratio across required concentrations  Perform retrospective analysis on study samples. 9 LC/MS/M S Trypsin Digestion All Peptides

10. In Silico Digestion To identify potential tryptic peptides for quantitation # AA >= 6# AA <= 18 Ignore if MetIgnore if Cys Ignore Multiple Modification Sites2 or 3 Charge States Y-ions onlym/z product > m/z parent Peptide selection criteria:

11. 11 LC/MS/MS 0.5 mg/mL in 0.1% FA Trypsin Digestion All Peptides To identify the best tryptic peptides for quantitation Neat Solution Digestion RapiGest TCEP Iodoacetamide

12. Post Neat Solution Digestion 12 17 peptide candidates remain after neat digestion of the protein standard …Next spike protein drug into matrix and subject to immunopurification as intended for the assay

13. 13 Monkey Plasma Assay Workflow with Immuno Capture and Off Bead Digestion LC/MS/MS Samples * Trypsin Digestion 7 Peptides 13 Transitions Spike Biotinylated Capture Ab Goat Anti-Human IgG (Fc) Antibody (Monkey Absorbed) Biotinylation Drug-Ab Complex Drug-Ab Complex on Beads KingFischer Flex Off-Bead Digestion Immuno - purified Drug Streptavidin Coated Magnetic Beads Elute Wash

14. Monkey Plasma Results Post Immunopurification 14 Confirmed Peptides:

15. LLOQ Chromatography & Specificity 15 Peptide LLOQ * (ng/mL) (14aa)K.3/y8 50 (14aa)K.2/y7 200 (10aa)R.2/y7 500 (10aa)R.2/y8 500 (11aa)K.2/y7 50 (11aa)K.2/y8 50 (15aa)K.3/y9 50 * S/N >= 10, peak area> 100

16. LLOQ & Specificity 16 Peptide LLOQ * (ng/mL) (6aa)R.2/y5 10 (6aa)R.2/y6 200 (5aa)K.2/y5 10 (5aa)K.2/y4 50 (17aa)K.3/y8 50 (17aa)K.3/y7 200 * S/N >= 10, peak area> 100

17. Dynamic Range 17 *Weighting: 1/concentration ^2 (14aa)K.3/y8 (10aa)R.2/y7 Dynamic Range (ng/mL) Nominal Conc. (ng/mL) Calculated Conc. (ng/mL) Accuracy (%) 50-5000 5047.695.2 200244122 50047595 10001020102 2000176087.9 5000490098 Dynamic Range (ng/mL) Nominal Conc. (ng/mL) Calculated Conc. (ng/mL)Accuracy (%) 500-10000 500547109 100081181.1 2000196097.8 50005020100 1000011100111

18. Potential Monitoring Peptide MRMs: Dynamic Range 18 *Weighting: 1/concentration ^2 (11aa)K.2/y7 (15aa)K.3/y9 Dynamic Range (ng/mL) Nominal Conc. (ng/mL) Calculated Conc. (ng/mL) Accuracy (%) 50-10000 5050.1100 200209104 50045891.6 100088388.3 2000194096.9 50005360107 1000011100111 Dynamic Range (ng/mL) Nominal Conc. (ng/mL) Calculated Conc. (ng/mL) Accuracy (%) 50-10000 5048.296.3 200252126 50040981.9 100078578.5 2000185092.3 50005870117 1000010800108

19. Potential Monitoring Peptide MRMs: Dynamic Range 19 *Weighting: 1/concentration ^2 Dynamic Range (ng/mL) Nominal Conc. (ng/mL) Calculated Conc. (ng/mL) Accuracy (%) 50-5000 5048.596.9 200239120 50044589 100085785.7 2000189094.6 50005710114 (6aa)R.2/y5 Dynamic Range (ng/mL) Nominal Conc. (ng/mL) Calculated Conc. (ng/mL) Accuracy (%) 50-5000 5050.8102 200202101 50041683.1 100087087 20002310115 50005610112 (5aa)K.2/y5 Dynamic Range (ng/mL) Nominal Conc. (ng/mL) Calculated Conc. (ng/mL) Accuracy (%) 50-5000 5050.2100 200206103 50047194.3 100085085 2000195097.5 50005990120 (17aa)K.3/y8

20. IgG Construct Components and Monitoring Peptide Locations Unique monitoring peptides for every unit of concern in the biotherapeutic 20 …6 peptides were chosen as monitoring peptides: one from each chain, one from each of the L-subunits L- subunits Peptide linker A B B A

21. Experimental stategy for determining in vivo peptide ratios reflecting relative drug intactness  All study samples previously run by ELISA  Drug concentration > 60 ug/mL plasma in all study samples  Capture reagent on beads begins to saturate at 3-4 ug/mL drug in plasma  Dilute all samples to ~2 ug/mL a)No bead saturation b)Good S/N even for peptides with lower sensitivity c)Small dynamic range of measurement 5.Purify all Cyno study samples by Anti-human Fc immunocapture 6.Determine peptide ratios for each subunit of interest (subunit/FC) 7.Declining peptide ratio across sampling period may indicate instability/subunit loss 21

22. Cyno TOX study: Very high exposure, all > 60ug/mL by ELISA Dilute all samples to ~ 2 uM, remove any response variation across range 22 Animal No.Time pointDilution (Fold) ELISA Conc. ( µ g/mL) Dilution (Fold) * Target LC/MS/MS Conc. ( µ g/mL) ** AD1: 0 hr4BLLOQ1 NA D1: 1 hr300002595.8911300 2.00 D1: 8 hr80001750.437900 1.94 D1: 24 hr4000531.442250 2.13 D1: 48 hr150096.48250 1.93 D1: 72 hr40069.58930 2.32 D11: 0 hr1500150.80175 2.01 D11: 1 hr300002723.5981400 1.95 D11: 8 hr200001997.0931000 2.00 D11: 24 hr4000980.692500 1.96 D11: 48 hr4000275.89150 1.84 D11: 72 hr4000179.585100 1.80 D11: 96 hr1500127.40360 2.12 D15: 24 hr4000951.904500 1.90 BD1: 0 hr4BLLOQ1 NA D1: 1 hr80001427.315700 2.04 D1: 8 hr80001164.326600 1.94 D1: 24 hr4000441.533200 2.21 D1: 48 hr4000149.8275 2.00 D1: 72 hr40079.41940 1.99 D11: 0 hr1500134.05770 1.92 D11: 1 hr300001652.835800 2.07 D11: 8 hr200001430.277700 2.04 D11: 24 hr4000647.16300 2.16 D11: 48 hr4000204.605100 2.05 D11: 72 hr4000143.42970 2.05 D11: 96 hr1500117.89660 1.96 D15: 24 hr4000644.789300 2.15

23. Cyno TOX study 23 n=4 L-subunit A / Fc L-subunit B / Fc

24. Cyno TOX study 24 n=4

25. Results of In Vivo Monitoring peptide analysis Ratios of all monitoring peptides to Fc peptide Constant across all dosing profiles Essentially constant for all 4 four subjects This indicates that the biotherapeutic subunits (L-subunits) are stable in vivo Confirmation experiments that could provide additional data Determine all monitoring peptides quantitatively (calibration curves) [GLP?] SL-IS for each peptide [more stable response] Peak area ratios corrected by IS and across calibration range … Another approach: Determine intact protein and possible fragments directly by HRLCMS (ESI-TOF) 25

26. Chromatograms, Extracted Spectra from the Drug Standard 26

27. In vivo stability, Intact molecule HRMS, Deconvoluted Spectra 27 Vial #Animal #Time point C21400417D1: 8 hr C31400417D1: 24 hr C51400417D11: 1 hr C61400417D11: 8 hr

28. Conclusions A PrD-LCMS method with 6 monitoring peptides for six regions was developed Unique peptides for each subunit (or region) were characterized:  Sufficient sensitivity and minimal matrix interference with existing method An immunopurification method with Fc capture was developed Reagent captures human IgG structure at Fc region All IgG subunits still attached remain captured, free components removed Study samples were diluted to ~ 2 ug/mL to avoid capture reagent saturation Monitoring peptide ratios were constant across the determined concentration range In vivo stability of the construct indicated by constant ratios during the Cyno study Additional confirmatory work by immunopurification and HRLCMS was performed Both approaches indicate that the protein construct was intact over the dosing profile This work demonstrates that monitoring peptide and intact HRLCMS methods can by used to evaluate in vivo stability / molecular intactness of multi subunit protein drugs 28

29. Acknowledgements 29 Tao Niu*: PhD candidate, BI DMPK summer intern, performed all lab work Dr. Lin-Zhi Chen, BI, Bioanalysis and metabolite profiling: Directly supervised all work Dr. Steve Norris:, Biotherapeutic bioanalysis: Project advice and planning Jessi Jones and Lee Frego, BI Research, : HRMS Work