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1 Practical Considerations for LC/MS Bioanalysis of Proteins via the Surrogate Peptide Approach Thank you for joining us! Our Webinar will begin shortly. While you are waiting, please feel free to browse our library of Webinar content: Click below to learn more about our NEW GlycoWorks RapiFluor-MS N-Glycan Analysis Kit and our NEW ACQUITY UPLC Glycoprotein BEH Amide 3Å 1.7 µm Columns Waters Corporation 1

2 Friendly Reminders Please use text chat functionality to submit questions during the Webinar. Upon conclusion, follow up information will be available: Recorded version of today s presentation PDF Copies of today s slides Discount Offers on BioSeparations Products Product specific information Reference materials 215 Waters Corporation 2

3 Today s Presenter Erin E. Chambers, Ph.D. Director, Technology Advancement Pi Principal i lapplications Chemist it Waters Corporation, Milford, MA USA Erin Chambers is the Director of the Technology Advancement group and a Principal Applications Chemist, providing pharmaceutical applications development and support for Waters. Erin s primary scientific roles are to support regulated and discovery Bioanalysis, and develop bioanalytical methods for drug molecules, both large and small. She is responsible for sample preparation, mass spectrometry, t and LC method development, and also provides customer and in-house training on these topics. Her most recent focus has been on peptide and protein bioanalysis. Her primary research interests include diabetes and Alzheimer s disease. 215 Waters Corporation 3

4 Practical Considerations for LC/MS Bioanalysis of Proteins via the Surrogate Peptide Approach Erin E Chambers, PhD 215 Waters Corporation 4

5 Topics Challenges in Workflow Options and Typical Steps Antibodies and Choice of Representative Peptide Effect of Protein and Peptide-level l clean-up Addressing the Challenges with a Flexible Kit-based Approach ADC Quantification What about intact antibody analysis? 215 Waters Corporation 5

6 Challenges for an LC/MS Approach Many possible workflow options How do I choose? What steps should I include/omit for my protein? Method Development Many steps to optimize i Lack of standardization approaches reagent choice/quality different labs, different results Reproducibility Long term lot-to-lot reproducibility/traceability Sensitivity 215 Waters Corporation 6

7 Possible Protein Bioanalysis Workflows for Surrogate Peptide Approach 215 Waters Corporation 7

8 Sample Preparation Method Development Challenges: Digestion Optimization of protein denaturation Optimization of protein reduction/alkylation: time, temp, reagent; omit? Finding the appropriate enzyme, protein : enzyme ratio, temp Digestion time optimization Testing digestion reproducibility 215 Waters Corporation 8

9 Optimization Complexity: Enzyme native peptide 13 C 15 N peptide Digestion Efficiency 1 9 native peptide 13 C 15 N peptide TypeA ($473/mg) Increasing Digestion Time Type B ($9.2/mg) Type C ($.5/mg) 3 2 Type A ($.3/mg) Type B ($82/mg) Increasing ($13/mg) Protein:Trypsin Ratio Type A Protein to trypsin ratio Type C ($.7/mg) Type A ($15/mg) Type B ($.2/mg) Vendor #1 Vendor #2 Vendor #3 Vendor #4 215 Waters Corporation 9

10 Optimization Complexity: Reducing Agent Concentration There is a sweet spot for reducing agent concentration For a representative generic signature peptide (GPSV), area counts reached a peak in the middle of the concentration range For the unique signature peptide of Remicade, area counts increased with decreasing concentrations of reduction reagent Area Decreasing Concentration 215 Waters Corporation 1

11 Optimization Complexity: Reduction Temperature So many literature protocols Different reagents, different optimal temperature Reagent 1 prefers higher temperature Reagent 2 prefers lower temperature Reducing Agent 1 6 C Reducing Agent 2 6 C Area Normalized to 8C Area Normalized to 8C One must carefully choose the specific reducing agent, the concentration of each reagent, and the time and temperature 4 variables to optimize! 215 Waters Corporation 11

12 Optimization Complexity: Reduction/Alkylation Time Peptide area increases with decreased reduction time Peptide area decreases with decreased alkylation time Area Reduction Time Alkylation Time Area Decreasing time Decreasing time (normalized to longest time) (normalized to longest time) 215 Waters Corporation 12

13 Sensitivity and Sample Prep: What is Enough and How do I Get There? Specific: Anti-human in Rat Generic: Protein A in Human None: Direct Human Plasma 1 ng/ml 5 ng/ml 5 ng/ml 1 ng/ml 1 ng/ml 5 ng/ml 5 ng/ml 5 ng/ml Blank Plasma Blank Plasma Blank Plasma 215 Waters Corporation 13

14 Topics Challenges in Workflow Options and Typical Steps Antibodies and Choice of Representative Peptide Unique or generic signature peptide o Impact on 3 vs 5-step protocol Effect of Protein and Peptide-level clean-up Addressing the challenges with a Flexible Kit-based Approach ADC Quantification What about intact antibody analysis? 215 Waters Corporation 14

15 Infliximab (Remicade) Remicade Light chain [2]: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQS HSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTE DTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Conserved region: blue variable regions: red CDR regions: green Unique signature Generic signature Van Dongen et al. 61st ASMS, MP525 Minneapolis Minnesota, USA 9-13 June 213. Formula: C 6428 H 9912 N 1694 O 1987 S 46 Molecular Weight: ~ kd Waters Corporation 15

16 Trastuzumab (Herceptin) Conserved region Surrogate Peptides Anti-HER2 Light chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPS RFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Anti-HER2 Heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRY Q Q ADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPPKSCDKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Unique Signature Generic Signature Formula: C 647 H 112 N 1726 O 213 S 42 Molecular Weight: ~ kda (claims are 148 package insert) Waters Corporation 16

17 Bevacuzimab (Avastin) Bevacizumab light chain DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTIS SLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDN ALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Bevacizumab heavy chain EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSK STAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE ALHNHYTQKSLSLSPGK Unique Signature Generic Signature Drug Bank Formula: C 6538 H 134 N 1716 O 233 S 44 Molecular Weight: ~ 149. kd Waters Corporation 17

18 Adalimumab (Humira) Light chain: DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQSGVPS RFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Heavy chain: EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITWNSGHIDY ADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSLDYWGQGTLVTVS Q SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSC DKTHTCPPCPAPELLG GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Unique Signature Generic Signature C 6428 H 9912 N 1694 O 1987 S 46 Protein average weight Drug Bank Waters Corporation 18

19 Peptide Choice: Impact on Protocol for Unique Peptides Eliminate reduction/alkylation? 3 step, normalized to 5 step protocol Area For unique signature peptides from 4 monoclonal antibody drugs, 3 step protocol works well 215 Waters Corporation 19

20 Peptide Choice: Impact on Protocol for Generic Peptides Eliminate reduction/alkylation? 3 step, normalized to 5 step protocol Area For generic signature peptides from 4 monoclonal antibody drugs, 5 step protocol is more efficient, more often than not 215 Waters Corporation 2

21 Labeled Antibody Internal Standard: SILu Mab SILuMab Heavy Chain EVQLVESGGGLVQPGGSLRLSCVASGFTLNNYDMHWVRQGIGKGLEWVSKI GTAGDRYYAGSVKGRFTISRENAKDSLYLQMNSLRVGDAAVYYCARGAGRW APLGAFDIWGQGTMVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ QPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SILuMab Light Chain QSALTQPRSVSGSPGQSVTISCTGTSSDIGGYNFVSWYQQHPGKAPKLMIY DATKRPSGVPDRFSGSKSGNTASLTISGLQAEDEADYYCCSYAGDYTPGV VFGGGTKLTVL GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTV AWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQ VTHEGSTVEKTVAPTECS Labeled Peptides: DTLMISR Heavy Chain (IgG1, IgG2, IgG3, IgG4) FNWYVDGVEVHNAK Heavy Chain (IgG1) VVSVLTVLHQDWLNGK Heavy Chain (IgG1, IgG3, IgG4) NQVSLTCLVK Heavy Chain (IgG1, IgG2, IgG3, IgG4) GFYPSDIAVEWESNGQPENNYK Heavy Chain (IgG1, IgG4) AGVETTTPSK Light Chain (lambda) YAASSYLSLTPEQWK Light Chain (lambda) 215 Waters Corporation 21

22 Intact Murine mab Mass Check Standard (Waters) Murine mab Light chain: DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSG TDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKD INVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Q Murine mab Heavy chain: QVQLKESGPGLVAPSQSLSITCTVSGFSLLGYGVNWVRQPPGQGLEWLMGIWGDGSTDYNSALKSRISITK DNSKSQVFLKMNSLQTDDTAKYYCTRAPYGKQYFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTDSMVTL GCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVP RDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAHTQPREEQFN STFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFF PEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG Conserved region: blue Variable regions: in red Signature peptides: BOXED Signature Generic 215 Waters Corporation 22

23 Murine Monoclonal Antibody prot A spe blk ISTD murine mab 14Aug215_RemicadeSPE_ProteinA_ MRM of 11 Channels ES > (Murine 4) 4.91e6 Murine mab Internal Standard Murine mab IS Peptides Retention Time (min) 1MNSLQTDDTAK VNSAAFPAPIEK NTQPIMDTDGSYFVYSK SVSELPIMHQDWLNGK prot a spe 5 ug/ml 14Aug215_RemicadeSPE_ProteinA_138 1 Antibody Drug Unique and Generic Signature Peptides Time MRM of 11 Channels ES > ( Remicade DILLTQSPAILSVSPGER) 8.35e6 mab Peptides Retention Time (min) 5 SINSATHYAESVK* DSTYSLSSTLTLSK GPSVFPLAPSSK DILLTQSPAILSVSPGER* VVSVLTVLHQDWLNGK Time Generic Internal Standard Workflow check Standard d 215 Waters Corporation 23

24 Topics Challenges in Workflow Options and Typical Steps Antibodies and Choice of Representative Peptide Effect of Protein and Peptide-level clean-up Precipitation pre-treatment Generic and Specific Affinity Capture of Protein Peptide purification from digest Addressing the challenges with a Flexible Kit-based Approach ADC Quantification What about intact antibody analysis? 215 Waters Corporation 24

25 Albumin Interferences: HSA peptides and Trastuzumab peptide p MEOH 1:1 25Jan215_Herceptin_dilution_MEOH_12 1 Trastuzumab (1ug/mL) 5.18 MRM of 24 Channels ES > 68.3 (Herceptin-FTISADTSK HC) 7.3e Jan215_Herceptin_dilution_ACN_114 1 Albumin Peptides MRM of 24 Channels ES+ TIC 2.22e Jan215_Herceptin_dilution_MEOH_12 A.2 Range: Aqueous Time Waters Corporation 25

26 PPT Pretreatment: Albumin depletion and Herceptin Recovery Normalized to Direct Digestion Incubation (1 ul Plasma) Reduced recovery of trastuzumab (Herceptin) seen with organic solvents with greatest albumin removal Solvent 4 in 1:1 ratio to plasma yields greatest balance of albumin removal and increased area counts for trastuzumab 215 Waters Corporation 26

27 Off-line Sample Preparation Options for Purification at the Protein Level Off line Sample Preparation Technique General Proteins Monoclonal Antibodies Discovery Development/ Clinical Sensitivity None x x x low MW cutoff filters x x x x low Depletion Plates x x x x low Protein A/G x x x medium Kappa (anti human) x x high Specific Capture Reagent x x x high 215 Waters Corporation 27

28 Protein A/G Cross Reactivity Can be used as generic purification in discovery for certain pre-clinical species 215 Waters Corporation 28

29 Removal of Endogenous Background: Effect of Protein-Level Purification Specific: Anti-human in Rat Generic: Protein A in Human None: Direct Human Plasma 215 Waters Corporation 29

30 Effect of Protein-Level Purification : Detection Limit Comparison Specific: Anti-human in Rat Generic: Protein A in Human None: Direct Human Plasma 1 ng/ml 1 ng/ml LLOQ 5 ng/ml LOD ~5 ng/ml 1 ng/ml LOD >1 ng/ml 215 Waters Corporation 3

31 Conclusions: Comparison of Pre-fractionation Techniques Absolute LLOQ/LOD for Kappa anti-human and Protein A similar Kappa S:N much higher and integration much cleaner Protein A LLOQ may be 2-3X higher/worse due to interferences and ease of integration LLOQ/LOD for whole digest 2-1X worse than Kappa or Protein A NSB may occur after very specific clean-up at protein level! Too clean? All techniques yield linear curves Anti-human 3 orders magnitude o Due to capacity When more beads are used, the linear range could be extended o Will NSB impact be even greater using more beads? Protein A 4 orders magnitude Whole plasma digestion 3 4 orders magnitude 215 Waters Corporation 31

32 Matrix Effects at the Signature Peptide Level 1 nm trastuzumab u ab in ~15 area counts Solvent A human serum digest 1 nm trastuzumab in serum digest ~5-7 area counts = 2-3X lower! Addressing the problem with sample prep 215 Waters Corporation 32

33 Peptide Clean-up: Mixed-mode Cation Exchange SPE Oasis MCX Single SPE method recovers unique and generic signature peptides with high efficiency Strong cation exchange mixedmode (Oasis MCX) best overall µelution format eliminates dry down and concentrates up to 15X Oasis WCX 215 Waters Corporation 33

34 Example 1: Incremental Improvement in Signal Combining Clean-up Options 1 1: MRM of 2 Channels ES > (FTISADTSK HC) 6.23e5 Area Direct plasma digestion: no clean-up : MRM of 2 Channels ES > (FTISADTSK HC) 6.23e5 Area Protein A plasma clean-up : MRM of 2 Channels ES > (FTISADTSK HC) e5 Area Protein A plasma clean-up, Oasis MCX digest clean-up Time Waters Corporation 34

35 Example 2: Mixed-mode Cation Exchange Clean-up of Unique Herceptin Peptide Before SPE Before SPE After SPE Summary Strong cation exchange most universal >9 recovery for unique and generic signature peptides tested Eliminates digest reagents Increases sensitivity Eliminates phospholipids Digest concentration without evaporation Increased system robustness 215 Waters Corporation 35

36 Topics Challenges in Workflow Options and Typical Steps Antibodies and Choice of Representative Peptide Effect of Protein and Peptide-level clean-up Addressing the challenges with a Flexible Kit-based Approach Reproducibility and Standardization di ti Broadly applicable protocol Sensitivity ADC Quantification What about intact antibody analysis? 215 Waters Corporation 36

37 Solution to the Complexity: Waters Kits Prototype Kits: A Few Key Attributes Processing time for 3-step: ~2 hours for antibodies in plasma Processing time for 5-step: ~3 hours for antibodies in plasma Standardized protocols Scalable, flexible format Reduced variability High Sensitivity Eliminates Method Development for Discovery Studies No capital investment required 215 Waters Corporation 37

38 Large Molecule Drugs 29 sales ($Billion) Anti-infective Oncology Pegasys PEGIntron Herceptin Remicade Rituxan Avastin Erbitux 5.7 Humira Rheumatology Endocrine CNS Norditropin SimpleXx.8 Avonex Lantus 4.3 Tysabri Levemir NovoMix 1.1 Rebif Humalog Enbrel NovoRapid /NovoLog 6.1 Neulasta Blood NovoSeven Procrit/Eprex Kogenate Monoclonal Ab Other protein Modified slide from McKinsey and Company US Patent Expiration Date Data Source: Evaluate Pharma 215 Waters Corporation 38

39 Reproducibility:Waters Prototype Kits Mean Area CV s Intra-kit Inter-kit 215 Waters Corporation 39

40 Standardized Protocol: Remicade (infliximab) Unique Signature Peptide prot a no spe 35 ug/ml 22Dec214_ProtA_nospe_142a Sm (Mn, 5x5) g/ml MRM of 1 Channels ES > (TNO Remicade signaturedilltqspailsvspger) 1.37e7 Area Dec214_ProtA_nospe_129 Sm (Mn, 5x5) MRM of 1 Channels ES > (TNO Remicade signaturedilltqspailsvspger) 1.58e6 Area 35. g/ml Dec214_ProtA_nospe_121 Sm (Mn, 3x3) MRM of 1 Channels ES > (TNO Remicade signaturedilltqspailsvspger) 2.91e5 Area 3.5 g/ml Dec214_ProtA_nospe_112 Sm (Mn, 3x3) MRM of 1 Channels ES > (TNO Remicade signaturedilltqspailsvspger) 3.7e4 Area.35 g/ml Dec214_ProtA_nospe_15 Sm (Mn, 5x5) MRM of 1 Channels ES > (TNO Remicade signaturedilltqspailsvspger) 1.23e4 Blank plasma Area Time 215 Waters Corporation 4

41 Standardized Protocol: Remicade (infliximab) Generic Signature Peptide prot a no spe 35 ug/ml 22Dec214_ProtA_nospe_142a Sm (Mn, 5x5) g/ml MRM of 1 Channels ES > (TNO Furlong Remicade signaturevvsvltvlhqdwlngk) 1.9e7 Area Dec214_ProtA_nospe_129 Sm (Mn, 3x3) MRM of 1 Channels ES > (TNO Furlong Remicade signaturevvsvltvlhqdwlngk) 2.16e6 Area 35. g/ml Dec214_ProtA_nospe_121 Sm (Mn, 2x3) MRM of 1 Channels ES > (TNO Furlong Remicade signaturevvsvltvlhqdwlngk) 2.52e5 Area 3.5 g/ml Dec214_ProtA_nospe_112 Sm (Mn, 1x1) MRM of 1 Channels ES > (TNO Furlong Remicade signaturevvsvltvlhqdwlngk) 3.78e4 Area.35 g/ml Dec214_ProtA_nospe_15 Sm (Mn, 1x1) MRM of 1 Channels ES > (TNO Furlong Remicade signaturevvsvltvlhqdwlngk) 2.34e3 Blank plasma Area Time 215 Waters Corporation 41

42 Standardized Protocol: Remicade (infliximab) Unique Signature Peptide Peptide DILLTQSPAILSVSPGER DILLTQSPAILSVSPGER* Std. Curve Range Linear fit Mean Accuracy of all (ug/ml) Weighting (r 2 ) points /x /x * Quantified using SILUMAB VVSV (IS) Otherwise quant using DTL Silumab peptide as IS Compound name: Remicade DILLTQSPAILSVSPGER Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 1. Residual. -1. Conc 75. Response Conc Waters Corporation 42

43 Standardized Protocol: Remicade (infliximab) Unique Signature Peptide Mean Cal. Conc QC Conc (ug/ml) (ug/ml) Std. Dev. CV Mean Accuracy DILLTQSPAILSVSPGER* SILUMAB DTL(IS) Mean Cal. Conc QC Conc (ug/ml) (ug/ml) Std. Dev. CV Mean Accuracy DILLTQSPAILSVSPGER* SILUMAB VVSV (IS) * Signature 215 Waters Corporation 43

44 Standardized Protocol: Remicade (infliximab) Generic Signature Peptide Peptide Std. Curve Range (ug/ml) Weighting Linear fit (r 2 ) Mean Accuracy of all points GPSVFPLAPSSK /x STSGGTAALGC[+57]LVK /x DSTYSLSSTLTLSK /x DSTYSLSSTLTLSK* /x VVSVLTVLHQDWLNGK /x VVSVLTVLHQDWLNGK* /x *Quantified using SILUMAB VVSV (IS) Compound name: Furlong Remicade VVSVLTVLHQDWLNGK Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None 1. All others are quant using DTL Silumab pepitde as IS Compound name: Merck DSTYSLSSTLTLSK Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None Residual. -1. Conc Residual Conc Response Conc ponse Resp Conc Waters Corporation 44

45 Standardized Protocol: Remicade (infliximab)generic Signature Peptide Peptide QC Conc (ug/ml) Mean Cal. Conc (ug/ml) Std. Dev. CV Mean Accuracy GPSVFPLAPSSK SILUMAB DTL(IS) Mean Cal. Conc QC Conc (ug/ml) (ug/ml) Std. Dev. CV Mean Accuracy STSGGTAALGC[+57]LVK SILUMAB DTL(IS) Mean Cal. Conc QC Conc (ug/ml) (ug/ml) Std. Dev. CV Mean Accuracy DSTYSLSSTLTLSK SILUMAB DTL(IS) Mean Cal. Conc QC Conc (ug/ml) (ug/ml) Std. Dev. CV Mean Accuracy DSTYSLSSTLTLSK SILUMAB VVSV (IS) Mean Cal. Conc Peptide QC Conc (ug/ml) (ug/ml) Std. Dev. CV Mean Accuracy VVSVLTVLHQDWLNGK SILUMAB DTL(IS) Mean Cal. Conc QC Conc (ug/ml) (ug/ml) Std. Dev. CV Mean Accuracy VVSVLTVLHQDWLNGK SILUMAB VVSV (IS) Waters Corporation

46 Standardized Protocol: Herceptin (trastuzumab) Unique Signature Peptide Compound name: FT peptide Correlation coefficient: r = , r^2 = Calibration curve: * x Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Oi Origin: i Exclude, Weighting: 1/x, Ai Axis trans: None Trastuzumab (Herceptin) Whole Plasma Digest: Linearity 1 ng/ml 5 µg/ml Respons se 2 1 Std. Conc ug/ml Area IS Area Conc. Dev Conc Waters Corporation 46

47 Standardized Protocol: Herceptin (trastuzumab) Unique Signature Peptide Compound name: FT peptide Correlation coefficient: r =.99968, r^2 = Calibration curve: * x Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: i Exclude, Weighting: 1/x, Axis trans: None Trastuzumab (Herceptin) Kappa affinity: Linearity 5 ng/ml 5 µg/ml Response Std. Conc Area IS Area Conc. Dev Conc Waters Corporation 47

48 Standardized Protocol: Avastin (bevacizumab), QC Statistics Peptide QC Conc (ug/ml) Mean Cal. Conc (ug/ml) Std. Dev. CV Mean Accuracy GPSVFPLAPSSK SILUMAB DTL(IS) * Mean Cal. Conc QC Conc (ug/ml) (ug/ml) Std. Dev. CV Mean Accuracy STSGGTAALGC[+57]LVK SILUMAB DTL(IS) Mean Cal. Conc QC Conc (ug/ml) (ug/ml) Std. Dev. CV Mean Accuracy DSTYSLSSTLTLSK * * * * SILUMAB DTL(IS) * Outside of curve dynamic range 215 Waters Corporation 48

49 Standardized Protocol: Avastin (bevacizumab) prot a no spe.5 ug/ml 9Jan215_ProtA_nospe_137 Sm (Mn, 2x2) MRM of 1 Channels ES > (Merck DSTYSLSSTLTLSK) 2.13e6 1. g/ml Area Jan215_ProtA_nospe_135 Sm (Mn, 2x2) MRM of 1 Channels ES > (Merck DSTYSLSSTLTLSK) 5. g/ml 1.15e6 Area Jan215_ProtA_nospe_131 Sm (Mn, 2x2) MRM of 1 Channels ES > (Merck DSTYSLSSTLTLSK) 2.5 g/ml 6.4e5 Area Jan215_ProtA_nospe_128 Sm (Mn, 2x2) MRM of 1 Channels ES > (Merck DSTYSLSSTLTLSK) 5.91e g/ml Area Jan215_ProtA_nospe_126 Sm (Mn, 2x2) MRM of 1 Channels ES > (Merck DSTYSLSSTLTLSK).5 g/ml 5.3e Area Jan215_ProtA_nospe_121 Sm (Mn, 2x2) MRM of 1 Channels ES > (Merck DSTYSLSSTLTLSK).25 g/ml 5.46e5 259 Area Jan215_ProtA_nospe_12 Sm (Mn, 2x2) MRM of 1 Channels ES > (Merck DSTYSLSSTLTLSK).1 g/ml 4.71e Area Jan215_ProtA_nospe_16 Sm (Mn, 2x2) MRM of 1 Channels ES > (Merck DSTYSLSSTLTLSK) Blank Plasma 4.4e5 Area 548 Time Waters Corporation 49

50 Standardized Protocol: Humira (adalimumab) Unique Signature Peptide Peptide: APYTFGQGTK 1-5 ug/ml Compound name: Humira Correlation coefficient: r = , r^2 = Calibration curve:.388 * x Response type: Internal Std ( Ref 2 ), Area * ( IS Conc. / IS Area ) Curve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None ug/ml 14Aug215_Dir_5step_SPE_mabs_HumPlsm_25p_41 Sm (Mn, 2x3) ; g/ml MRM of 12 Channels ES > (Humira APYTFGQGTK LC) 4.93e4 Area Residual Aug215_Dir_5step_SPE_mabs_HumPlsm_5p_38 Sm (Mn, 2x3) MRM of 12 Channels ES+ 5.29; > (Humira APYTFGQGTK LC) 1.33e4 5. g/ml Area -2. Conc Response Aug215_Dir_5step_SPE_mabs_HumPlsm_1p_33 Sm (Mn, 2x3) MRM of 12 Channels ES ; > (Humira APYTFGQGTK LC) 1.88e3 Area 1. g/ml Aug215_Dir_5step_SPE_mabs_HumPlsm_5p_3 Sm (Mn, 2x3) MRM of 12 Channels ES ;53 1 Conc > (Humira APYTFGQGTK LC) Area 5. g/ml Aug215_Dir_5step_SPE_mabs_HumPlsm_Blk_2 Sm (Mn, 2x3) MRM of 12 Channels ES > (Humira APYTFGQGTK LC) 1.65e3 Area Time Waters Corporation Blank plasma

51 Standardized Protocol: High Sensitivity Remicade (infliximab) SINSATHYAESVK Unique Signature Peptide QC.35 ug/ml 14Aug215 _ RemicadeSPE_ ProteinA_ 15 1 Blank plasma MRM of 11 Channels ES > 63.8 (Remicade SINSATHYAESVK ) 2.19e4 Area Aug215_RemicadeSPE_ProteinA_11 MRM of 11 Channels ES > 63.8 (Remicade SINSATHYAESVK ) 2.76e4 Area.3535 g/ml 35 ng/ml Aug215_RemicadeSPE_ProteinA_119 MRM of 11 Channels ES > 63.8 (Remicade SINSATHYAESVK ) 2.4e5 Area.35 g/ml Aug215_RemicadeSPE_ProteinA_128 MRM of 11 Channels ES > 63.8 (Remicade SINSATHYAESVK ) 2.28e6 Area 3.5 g/ml Aug215_RemicadeSPE_ProteinA_135 ProteinA 135 MRM of 11 Channels ES > 63.8 (Remicade SINSATHYAESVK ) 2.5e7 Area 35. g/ml Aug215_RemicadeSPE_ProteinA_145 MRM of 11 Channels ES > 63.8 (Remicade SINSATHYAESVK ) 1.32e8 Area 35. g/ml Time 215 Waters Corporation 51

52 Competitor vs. Waters: Digestion, No Reduction/Alkylation Area for Unique Signature Peptides of 4 Monoclonal Antibodies, Normalized to Waters Prototype Kit Commerically Available Competitor Kit Waters Prototype Kit 2 Both protocols were completed in comparable time 215 Waters Corporation 52

53 Competitor vs. Waters Area for Generic Signature Peptides of 4 Monoclonal Antibodies, Normalized to Waters Prototype Kit Commerically Available Competitor Kit Waters Prototype Kit 215 Waters Corporation 53

54 Topics Challenges in Workflow Options and Typical Steps Antibodies and Choice of Representative Peptide Effect of Protein and Peptide-level clean-up Addressing the challenges with a Flexible Kit-based Approach ADC Quantification What about intact antibody analysis? 215 Waters Corporation 54

55 Trastuzumab Emtansine (T-DM1) Trastuzumab based 9 lysines MCC (Linker) Da SMCC linker Two-step conjugation Drug Maytansinoid Hydrophobic DM1 + MCC (Drug + Linker) Da ADCs from 3 sources Data courtesy of Liuxi Chen Bioconjugate Chemistry 214, 25, Waters Corporation 55

56 Why Does Drug Location Matter? The potential effect of drug loading, location and distribution: Efficacy and Toxicity CDR Binding Stability Pharmacokinetics ADC manufacturing process control PK E2 E4 E4 E8 Clin. Cancer Res. 1, Chemistry & Biology 2, , Waters Corporation 56

57 ADC Conjugated Peptide Quantification Strategy Control mab Lys-conjugated ADC Trypsin/Lys C Asp N Identification Quantification Peptide Mapping (MS E & DDA) Identifies conjugated peptides Confirms conjugation sites Generates accurate mass list of identified conjugated peptides Accurate Mass Screening (MS E ) Quantifies conjugated peptides across samples Automatically combines different charge states and adducts ions Automatically combines intensity ty from the same peptide at different RT Data courtesy of Liuxi Chen 215 Waters Corporation 57

58 MultiEnzymes for Lys ADC Quantification Trypsin Asp N Unconjugated peptide K K Conjugated peptide K K miscleavage Quantitation Relative abundance of conjugated peptides Relative site occupancy ratio Data courtesy of Liuxi Chen 215 Waters Corporation 58

59 Control mab vs T-DM1 - LC/MS E Chromatograms T-DM1 Data courtesy of Liuxi Chen Trypsin Tmab T-DM1 Conjugated peptide Elution window Asp N Tmab 215 Waters Corporation 59

60 Control mab vs T-DM1 - LC/MS E Chromatograms T-DM1 Data courtesy of Liuxi Chen 1x Trypsin Tmab T-DM1 1x Asp N Tmab 215 Waters Corporation 6

61 Representative Data: Relative Site Occupancy, AspN Results 3 Light Chain Heavy Chain Fab Heavy Chain Fc ve Site Occu upancy Relati 5 LC42 LC183 HC HC3 HC43 HC65 HC76 HC225 HC251 HC277 HC395 N term Lys Site Number Data courtesy of Liuxi Chen 215 Waters Corporation 61

62 Relative abundance of conjugated peptides from different sources (T-DM1) Relative Int tensity Trypsin results AspN results 12 N term L42 L17 N term H3 H43 H65 H76 H124 H3 H43 H65 H76 1 Relative e Intensity Company A Batch 1 Company A Batch 2 Company B 2 Company C Process 1 2 L126 L145 L149 L169 L183 L188 L19 L27 H136 H216 H217 Company C Process 2 Relat tive Intensity Data courtesy of Liuxi Chen H225 H249 H251 H277 H291 H293 H323 H325 H329 H337 H343 H363 H395 H417 H442 Lys Residue Number 215 Waters Corporation 62

63 Topics Challenges in Workflow Options and Typical Steps Antibodies and Choice of Representative Peptide Effect of Protein and Peptide-level clean-up Addressing the challenges with a Flexible Kit-based Approach ADC Quantification What about intact antibody analysis? 215 Waters Corporation 63

64 Improved Sensitivity Capability of ionkey for Deglycosylated gy y mab Analysis PWHM: 8.5s ng ng ng ng ng ng pg LOD 1 pg LOQ 4.51 Time m/z Waters Corporation 64 1 m/z m/z m/z m/z m/z man f Greg Ro Data courtesy o

65 Improved Sensitivity Capability of ionkey for Glycosylated y mab Analysis.4 ng LOD 1 ng LOQ No Glycoform Carryover f Greg Roman Data courtesy o 215 Waters Corporation 65

66 Generic Affinity Capture of Humira from Rat Plasma Blank Rat Plasma_25pt ugml 151 ( Blank Rat Plasma_25pt ugml 151 (2.947) 1: TOF MS ES e5 1 2.e g/ml m/z m/z Blank Rat Plasma_1pt ugml 15 ( Blank Rat Plasma_1pt ugml 15 (2.93) 1: TOF MS ES e e g/ml m/z m/z Blank Rat Plasma_5pt ugml 15 (2.93) 1: TOF MS ES Blank Rat Plasma_5pt ugml 15 ( e e g/ml Humira Linear Range: 25-5 g/ml Deconvoluted Spectra Humira 1 ugml_4 151 (2.947) 1: TOF MS ES e mass Humira 5 ugml_3 147 (2.879) 1: TOF MS ES e5 m/z Blank Rat Plasma_25pt ugml 151 (2.947) 1: TOF MS ES e g/ml m/z Blank Rat Plasma_25pt ugml 151 ( e m/z m/z mass Waters Corporation Data courtesy of Greg Roman 66

67 Trastuzumab ADC (De-Glycosylated): IonKey/MS Trastuzumab MW: g/mol C4 Herceptin ADC 1X Dilution_2 156 (3.51) Cm (144:169) 1: TOF MS ES e ng Linker and Drug: g/mol 1 Drug + 1 Antibody: g/mol m/z C4 Herceptin ADC 1X Dilution_2 151 (2.947) Cm (143:161) 1: TOF MS ES e4 2.5 ng m/z C4 Herceptin ADC 1X Dilution_2 151 (2.947) Cm (145:165) 165) 1: TOF MS ES e ng m/z C4 Herceptin ADC 1X Dilution_2 15 (2.93) Cm (144:165) 1: TOF MS ES e ng m/z Waters Corporation 67

68 Intact Mass Analysis on T-DM1 +Drug 2 +Drug 3 +Drug 4 +Drug 1 +Drug 5 +Drug +Linker +Linker +Linker +Linker +Linker +Drug 6 +Drug 7 Antibody with -7 drugs observed Linker-only peaks observed 215 Waters Corporation 68

69 Conclusions Protein BA workflows are complex Optimization of multiple aspects of multiple steps is required The number and nature of steps to include depends on many variables Waters prototype kits provide standardized, reproducible, and fully flexible solutions to accommodate multiple workflows with fast, optimized protocols For ADC analysis, conjugated peptides can be identified d and quantified using a 2-stage workflow, HRMS, and specifically designed software Intact analysis is closer than you might think 215 Waters Corporation 69

70 Acknowledgements Mary Lame Hua Yang Liuxi Chen Henry Shion Gregory Roman James Murphy Weibin Chen John Gebler Sherri Naughton Catalin Doneanu 215 Waters Corporation 7

71 Thank You for Attending! Post-Event Home Page: 3 Introductory Offer on Protein, Peptide and Glycan Separation Columns and Kits Full Webinar Recording of Today s Session w/pdf Slide Deck Compilation of TODAY S KEY Literature, Brochures etc For Questions and to Submit your Ideas for our Next Topic Please - mychemrep@waters.com 215 Waters Corporation 71