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![©2017 Waters Corporation 6COMPANY CONFIDENTIAL
Infliximab is a chimeric human-murine anti-human tumor necrosis
factor (TNF) monoclonal antibody.
Infliximab (Remicade) Sequence
Remicade Light chain [2]:
DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQS
HSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Remicade Heavy chain [2]:
EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTE
DTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
http://www.drugbank.ca/drugs/DB00065
Formula:
C6428H9912N1694O1987S46
Molecular Weight: ~ 149.1 kD](https://image.slidesharecdn.com/apa2017diegorodriguez3-170301135609/85/Apa-2017-diego-rodriguez-3-6-320.jpg)
![©2017 Waters Corporation 16COMPANY CONFIDENTIAL
Peptide Options for Quantification:
Infliximab (Remicade)
Van Dongen et al. 61st ASMS, MP525 Minneapolis Minnesota,
USA 9-13 June 2013.
Remicade Light chain [2]:
DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQS
HSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Remicade Heavy chain [2]:
EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTE
DTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Conserved region: blue (Human IgG1 Sequence)
Variable regions: red
CDR regions: green
Unique signature
Generic signature
http://www.drugbank.ca/drugs/DB00065
Formula:
C6428H9912N1694O1987S46
Molecular Weight: ~ 149.1 kD
Final Selection of Tryptic Peptide Options
Choices for quantification are based on sensitivity
and specificity](https://image.slidesharecdn.com/apa2017diegorodriguez3-170301135609/85/Apa-2017-diego-rodriguez-3-16-320.jpg)
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- 1. ©2017 Waters Corporation1COMPANY CONFIDENTIAL LC-MS/MS for Bioanalytical Protein Quantification: Life of a Biotherapeutic Infliximab Case Study
- 2. ©2017 Waters Corporation2COMPANY CONFIDENTIAL
- 3. ©2017 Waters Corporation3COMPANY CONFIDENTIAL Humira Linear Range: 25-500 mg/mL m/z 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 % 0 100 m/z 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 % 0 100 m/z 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 % 0 100 m/z 2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400 % 0 100 5132015 Blank Rat Plasma_250pt0 ugmL 151 (2.947) 1: TOF MS ES+ 2.00e52743.07842693.2227 2645.1912 2598.7388 2554.0410 2468.8767 2428.4585 2314.6531 2211.0151 2794.7969 2848.5042 2904.3540 2907.5510 2965.6665 3022.9172 3085.7578 3154.8474 3223.6106 3370.1011 5132015 Blank Rat Plasma_100pt0 ugmL 150 (2.930) 1: TOF MS ES+ 1.30e52794.82502743.0637 2693.2292 2645.1558 2598.7661 2554.0063 2510.7832 2428.46562314.6995 2244.6521 2848.5764 2904.3823 2962.4216 3022.9009 3026.2302 3089.1956 3154.9912 3291.5718 5132015 Blank Rat Plasma_50pt0 ugmL 150 (2.930) 1: TOF MS ES+ 8.57e42794.81862743.0356 2645.1282 2598.7595 2553.9998 2510.6736 2428.4788 2314.6135 2200.6035 2848.5479 2904.3762 2907.5068 2965.6443 3022.8723 3026.9216 3155.0083 5132015 Blank Rat Plasma_25pt0 ugmL 151 (2.947) 1: TOF MS ES+ 4.26e42743.04222693.2717 2645.1138 2598.8704 2554.0063 2468.8838 2351.42552314.69952204.6226 2848.5107 2904.3823 2962.5107 3022.8560 3026.2751 3089.1726 m/z 2720 2740 2760 % 0 100 m/z 2720 2740 2760 %0 100 m/z 2720 2740 2760 % 0 100 m/z 2720 2740 2760 % 0 100 5132015 Blank Rat Plasma_250pt0 ugmL 151 (2.947) 2.00e52743.0784 2739.3311 2716.3662 2746.1213 2748.4795 5132015 Blank Rat Plasma_100pt0 ugmL 150 (2.930) 1.25e52743.0637 2738.9309 2737.7749 2746.0850 2749.1294 2750.7810 5132015 Blank Rat Plasma_50pt0 ugmL 150 (2.930) 8.44e42743.0356 2740.7654 2737.8540 2746.0999 2748.3723 2749.6160 2751.4180 5132015 Blank Rat Plasma_25pt0 ugmL 151 (2.947) 4.26e42743.0422 2742.1853 2716.8633 2746.1492 2748.8291 2749.8157 250 mg/mL 100 mg/mL 50 mg/mL 25 mg/mL Generic Affinity Capture of Humira from Rat Plasma mass 148000 148250 148500 148750 % 0 100 5132015 Humira 50 ugmL_3 147 (2.879) 1: TOF MS ES+ 5.49e5148078.0000 148221.0000 148363.0000 148510.0000 mass 148000 148250 148500 148750 % 0 100 5132015 Humira 100 ugmL_4 151 (2.947) 1: TOF MS ES+ 7.53e5148078.0000 148222.0000 148370.0000 148529.0000 Deconvoluted Spectra
- 4. ©2017 Waters Corporation4COMPANY CONFIDENTIAL LC-MS/MS Protein Quantification Workflow Protein-level clean-up (optional) LC-MS Results Protein in Plasma/Serum Peptides Digestion ID unique peptides & transitions Optimize/Fine-tune MRM transitions Peptide-level clean-up (optional) Purified Peptides Purified Protein Data Processing TargetLynx
- 5. ©2017 Waters Corporation5COMPANY CONFIDENTIAL Identification of Unique Signature Peptides MRM Optimization of Unique Peptides Internal Standard Options Protein Level Sample Preparation Digestion Peptide Level Sample Preparation MS Quantification Outline
- 6. ©2017 Waters Corporation6COMPANY CONFIDENTIAL Infliximab is a chimeric human-murine anti-human tumor necrosis factor (TNF) monoclonal antibody. Infliximab (Remicade) Sequence Remicade Light chain [2]: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQS HSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTE DTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK http://www.drugbank.ca/drugs/DB00065 Formula: C6428H9912N1694O1987S46 Molecular Weight: ~ 149.1 kD
- 7. ©2017 Waters Corporation7COMPANY CONFIDENTIAL Infliximab LC and HC Tryptic Peptide Identification
- 8. ©2017 Waters Corporation8COMPANY CONFIDENTIAL Step 1: BLAST Search Infliximab Light Chain to Identify Closest Related Protein in Database Infliximab LC sequence https://blast.ncbi.nlm.nih.gov/Blast.cgi
- 9. ©2017 Waters Corporation9COMPANY CONFIDENTIAL Closely related to LC of Human IgG Results of BLAST search for Infliximab LC Protein sequence differences and gaps
- 10. ©2017 Waters Corporation10COMPANY CONFIDENTIAL Step 2: Align the mAb Sequence Against Closely Related Sequences in ExPASy SIM Alignment Tool Paste infliximab LC sequence Paste the identified (see step 1), closely related human IgG LC sequence http://web.expasy.org/sim/
- 11. ©2017 Waters Corporation11COMPANY CONFIDENTIAL ExPASy SIM Alignment Results: Infliximab LC vs. Human IgG LC Highlights protein sequence differences while maintaining sequence alignment
- 12. ©2017 Waters Corporation12COMPANY CONFIDENTIAL Trypsin cleaves peptides on the C-terminal side of lysine (K) and arginine (R) amino acid – Tryptic peptides can be found using freeware, such as Skyline and Protein Prospector , but these tools will not maintain sequence alignment Text editor (ex. TextPad, Notepad) Step 3: Generate Tryptic Peptides, Maintaining Sequence Alignment Gaps = tryptic peptide differences, maintaining sequence alignment “matched” non-unique peptides “un-matched” potentially unique peptides
- 13. ©2017 Waters Corporation13COMPANY CONFIDENTIAL Infliximab, a chimeric murine mAb should have close sequence homology to mouse Tryptic Peptide Specificity: 3 Possibilities for Infliximab LC vs. IgG1kappa 1 too long (45 AA) DILLTQSPAILSVSPGER: Mouse IgG peptide-Specificity in human, rat ASQFVGSSIHWYQQR: 80% Mouse IgG peptide-Specificity in human, rat, and mouse YASESMSGIPSR:90% Mouse IgG peptide-Specificity in human, rat, and mouse
- 14. ©2017 Waters Corporation14COMPANY CONFIDENTIAL Step 4: Assess uniqueness of the un-matched tryptic peptides using peptide BLAST search https://blast.ncbi.nlm.nih.gov/Blast.cgi ASQFVGSSIHWYQQR Long List of Possibilities
- 15. ©2017 Waters Corporation15COMPANY CONFIDENTIAL Step 4: Detailed View of Closely Related Database Matches Mouse Monkey “Unique” < 100% sequence homology It found infliximab (100% homology) Unique for mouse, monkey and human (homology of 93, 85 and 78%) , thus it could be used to quantify in those species ASQFVGSSIHWYQQR Infliximab Human
- 16. ©2017 Waters Corporation16COMPANY CONFIDENTIAL Peptide Options for Quantification: Infliximab (Remicade) Van Dongen et al. 61st ASMS, MP525 Minneapolis Minnesota, USA 9-13 June 2013. Remicade Light chain [2]: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQS HSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTE DTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Conserved region: blue (Human IgG1 Sequence) Variable regions: red CDR regions: green Unique signature Generic signature http://www.drugbank.ca/drugs/DB00065 Formula: C6428H9912N1694O1987S46 Molecular Weight: ~ 149.1 kD Final Selection of Tryptic Peptide Options Choices for quantification are based on sensitivity and specificity
- 17. ©2017 Waters Corporation17COMPANY CONFIDENTIAL Best Practice for Signature Peptide Choice Once you have a selection of Unique Peptides: Size – Ideally 8-24 amino acids (6-7 sometimes okay) Avoid o Peptides with modifications (i.e. oxidation or deamidated) o Those containing Cys, Met, N-terminus Gln, Trp, or Asn-Gly LC/MS characteristics – Strong MS ionization (increased sensitivity) – Chromatographic retention, peak shape/width – Specificity in Matrix
- 18. ©2017 Waters Corporation18COMPANY CONFIDENTIAL Identification of Unique Signature Peptides MRM Optimization of Unique Peptides Internal Standard Options Protein Level Sample Preparation Digestion Peptide Level Sample Preparation MS Quantification Outline
- 19. ©2017 Waters Corporation19COMPANY CONFIDENTIAL Developed by the MacCoss laboratory in the University of Washington Best Practice: Our Approach = Skyline Workflow Start Here!
- 20. ©2017 Waters Corporation20COMPANY CONFIDENTIAL SkyLine In silico generation of MRM transitions for peptides Output scouting MRM method MassLynx/Xevo TQ-XS Acquisition of MRM traces for all proposed transitions SkyLine Comparison of overlaid MRM traces, to pick optimal transitions & collision energies Output final MRM method MassLynx/Xevo TQ-XS Sample analysis Overview of Skyline Workflow
- 21. ©2017 Waters Corporation21COMPANY CONFIDENTIAL Skyline MRM Method Creation: Infliximab LC With Skyline, importing a protein sequence elicits an in silico digestion algorithm and MRM transition prediction
- 22. ©2017 Waters Corporation22COMPANY CONFIDENTIAL Skyline MRM Method Creation: Infliximab LC
- 23. ©2017 Waters Corporation23COMPANY CONFIDENTIAL Green light implies peak detection with great sensitivity, yellow light is peak detection with moderate sensitivity, and red light is minimal to no peak detection. HOWEVER, the lights are not always right. First Skyline Import: Filtering Potential Infliximab peptides
- 24. ©2017 Waters Corporation24COMPANY CONFIDENTIAL Sample Analysis with MassLynx: Skyline imported methods Narrowed down Skyline methods are imported into MassLynx for LC/MS acquisition
- 25. ©2017 Waters Corporation25COMPANY CONFIDENTIAL Skyline MRM Method Optimization: CE Optimization The optimized method can be exported automatically
- 26. ©2017 Waters Corporation26COMPANY CONFIDENTIAL Infliximab Skyline Optimization vs. Manual: DILLTQSPAILSVSPGER (LC) Infliximab Skyline Time 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 % 0 100 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 4.00 4.25 4.50 4.75 5.00 5.25 5.50 5.75 6.00 6.25 6.50 6.75 7.00 % 0 100 29Dec2016_Infliximab_Optimized_003 14: MRM of 3 Channels ES+ 632.686 > 545.268 (Infliximab_LC DILLTQSPAILSVSPGER.3) 3.94e6 Area 29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER) 3.94e6 Area 5.59 26370 Skyline Optimization 633.1>546.3 More sensitive transition! Manual Optimization 633.1>731.8 5.59 140139 Alternate MRM transition
- 27. ©2017 Waters Corporation27COMPANY CONFIDENTIAL Identification of Unique Signature Peptides MRM Optimization of Unique Peptides Internal Standard Options Protein Level Sample Preparation Digestion Peptide Level Sample Preparation MS Quantification Outline
- 28. ©2017 Waters Corporation28COMPANY CONFIDENTIAL Internal Standard Options Peptide Level (added just prior to digestion) – Labeled Peptide – Extended Tag Labeled peptide Protein Level (added prior to denaturation, reduction and alkylation) – Generic Labeled protein – Generic Unlabeled protein – Unique Labeled protein
- 29. ©2017 Waters Corporation29COMPANY CONFIDENTIAL Identification of Unique Signature Peptides MRM Optimization of Unique Peptides Internal Standard Options Protein Level Sample Preparation Digestion Peptide Level Sample Preparation MS Quantification Outline
- 30. ©2017 Waters Corporation30COMPANY CONFIDENTIAL Sample Preparation Options Protein Level Clean-up prior to digestion – None o Whole serum/plasma digestion – Affinity-based o Generic: Protein A/G o Anti-human o Specific Receptor – MWT cut-off filters – Depletion Plates – Gel Filtration
- 31. ©2017 Waters Corporation31COMPANY CONFIDENTIAL 100uL SPE, Protein A, 1:5 Worthington, DTT, 17hr digestion Time 7.50 8.00 8.50 9.00 % 0 100 7.50 8.00 8.50 9.00 % 0 100 7.50 8.00 8.50 9.00 % 0 100 7.50 8.00 8.50 9.00 % 0 100 500ngmL_plasma_051514_002 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 6.43e4 Area 100ngmL_plasma_051514_002 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 6.43e4 Area 8.49 722 50ngmL_plasma_051514_002 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 6.43e4 Area 8.50 345 blank_plasma_051514_002 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 6.43e4 Area 8.49 163 100uL SPE, Kappa Time 7.50 8.00 8.50 9.00 % 0 100 7.50 8.00 8.50 9.00 % 0 100 7.50 8.00 8.50 9.00 % 0 100 7.50 8.00 8.50 9.00 % 0 100 500ngmL_plasma_051314_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 1.30e5 Area 100ngmL_plasma_051314_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 1.30e5 Area 8.43 810 50ngmL_plasma_051314_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 1.30e5 Area 8.43 401 blank_plasma_051314_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 1.30e5 Area 50uL SPE, whole plasma, 1:12.5 Worthington, DTT Time 7.50 8.00 8.50 9.00 %0 100 7.50 8.00 8.50 9.00 % 0 100 7.50 8.00 8.50 9.00 % 0 100 7.50 8.00 8.50 9.00 % 0 100 500ngmL_plasma_052114_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 2.60e4 Area 8.38 493 100ngmL_plasma_052114_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 2.60e4 Area 8.41 79 50ngmL_plasma_052114_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 2.60e4 Area blank_plasma_052114_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC) 2.60e4 Area Effect of Protein Level Clean-Up: Trastuzumab Blank Plasma Blank Plasma Blank Plasma 50 ng/mL 50 ng/mL 50 ng/mL 100 ng/mL100 ng/mL100 ng/mL 500 ng/mL 500 ng/mL Specific: Anti-human in Rat Generic: Protein A in Human None: Direct Human Plasma The level of sample prep needed will be based on the sensitivity and specificity required for the assay? 500 ng/mL8.43 3412 8.50 1707
- 32. ©2017 Waters Corporation32COMPANY CONFIDENTIAL Identification of Unique Signature Peptides MRM Optimization of Unique Peptides Internal Standard Options Protein Level Sample Preparation Digestion Peptide Level Sample Preparation MS Quantification Outline
- 33. ©2017 Waters Corporation33COMPANY CONFIDENTIAL ProteinWorks™ eXpress Digest Kits Standardized protocols – No Method Development – Diverse proteins Reliable and reproducible High Sensitivity Quantification Total antibody analysis for ADCs – Detection of conjugated peptides
- 34. ©2017 Waters Corporation34COMPANY CONFIDENTIAL Identification of Unique Signature Peptides MRM Optimization of Unique Peptides Internal Standard Options Protein Level Sample Preparation Digestion Peptide Level Sample Preparation MS Quantification Outline
- 35. ©2017 Waters Corporation35COMPANY CONFIDENTIAL ProteinWorks µElution SPE Kit: Mixed-mode Specificity Maximizing Instrument Up-Time, Recovery & Sensitivity Orthogonal to RP LC method! Remove interfering buffer salts and digest reagents Recover unique and generic signature peptides with high efficiency using a single SPE method Minimize sample loss with µElution format Concentrate the sample up to 15x 0 20 40 60 80 100 120 Oasis MCX One generic protocol achieves high recovery for a diversity of tryptic peptides Peptides Purified Peptides Peptide-level clean-up (optional)
- 36. ©2017 Waters Corporation36COMPANY CONFIDENTIAL Infliximab Peptide Level Clean-Up: Mixed-Mode SPE with Oasis MCX direct 100ug/ml Time 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 % 0 100 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 % 0 100 18Dec2014B_nospe_dir_1033 MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK) 3.65e6 Area 4.40;149708 18Dec2014B_spe_direct_1040 MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK) 3.65e6 Area 4.41 73225 Direct Plasma Digestion No SPE Direct Plasma Digestion SPE DSTYSLSSTLTLSK Tryptic Peptide
- 37. ©2017 Waters Corporation37COMPANY CONFIDENTIAL Identification of Unique Signature Peptides MRM Optimization of Unique Peptides Internal Standard Options Protein Level Sample Preparation Digestion Peptide Level Sample Preparation MS Quantification Outline
- 38. ©2017 Waters Corporation38COMPANY CONFIDENTIAL 2014 2014 2012 Xevo TQ-S 2010 2016 Xevo TQ Family Data coming soon! New data available!
- 39. ©2017 Waters Corporation39COMPANY CONFIDENTIAL High Sensitivity Infliximab Quantification: Xevo TQ-S Triple Quadrupole Time 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 % 0 100 2.20 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 4.20 4.40 4.60 4.80 5.00 5.20 5.40 % 0 100 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK ) 2.20e4 Area 4.15 361 MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK ) 2.20e4 Area 10 ng/mL infliximab Blank plasma SINSATHYAESVK Unique Signature Peptide Linear, Precise and Accurate Single digit %CVs LLOQ of 10ng/mL 35 µL sample, Protein A, ProteinWorks digestion and MCX peptide clean-up
- 40. ©2017 Waters Corporation40COMPANY CONFIDENTIAL Segmented quadrupole second stage – focused ion beam – enhanced ion transmission to detector Constant RF voltage at 1st stage across horizontal plates -> less energetic collisions/fragmentation – better for labile compounds Wider profile first stage, more space for ion cloud – less collision w/ plates
- 41. ©2017 Waters Corporation41COMPANY CONFIDENTIAL ionKey Microfluidics ESI Assembly Incoming FlowEEProm Memory Electrical Connections Analytical ColumnColumn Heater Sensor
- 42. ©2017 Waters Corporation42COMPANY CONFIDENTIAL Infliximab High Analytical Sensitivity Using Integrated Microflow Time 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 % 0 100 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 % 0 100 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 % 0 100 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 % 0 100 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 % 0 100 5.50 5.75 6.00 6.25 6.50 6.75 7.00 7.25 7.50 % 0 100 Std_21Sep_49 MRM of 6 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK) 4.07e6 Std_21Sep_39 MRM of 6 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK) 4.70e5 Std_21Sep_29 MRM of 6 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK) 4.66e4 Std_21Sep_1 MRM of 6 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK) 1.33e4 Std_21Sep_60 MRM of 6 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK) 1.14e4 blank_21Sep_8 MRM of 6 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK) 9.15e3 5.94 7.377.19 blank 10,000 ng/mL 1000 ng/mL 100 ng/mL 10 ng/mL 1 ng/mL For research use only. Not for use in diagnostic procedures
- 43. ©2017 Waters Corporation43COMPANY CONFIDENTIAL m/z 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 % 0 100 m/z 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 % 0 100 m/z 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 % 0 100 m/z 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 % 0 100 3252015 C4 Herceptin ADC 10000X Dilution_2 156 (3.051) Cm (144:169) 1: TOF MS ES+ 6.08e32904.37552847.4702 2740.01492689.4590 2593.2161 2904.7944 2963.8701 3089.8511 3090.2375 3252015 C4 Herceptin ADC 1000X Dilution_2 151 (2.947) Cm (143:161) 1: TOF MS ES+ 1.01e42847.47022792.9436 2792.8357 2689.2893 2904.2434 2904.7063 3025.3850 3252015 C4 Herceptin ADC 100X Dilution_2 151 (2.947) Cm (145:165) 1: TOF MS ES+ 4.98e42848.01612794.3059 2724.7688 2644.5042 2884.9858 2904.7285 3252015 C4 Herceptin ADC 10X Dilution_2 150 (2.930) Cm (144:165) 1: TOF MS ES+ 2.68e52794.32762742.5422 2644.6094 2598.2388 2847.9941 2885.3372 2924.1621 250 ng 25.0 ng 2.5 ng 0.25 ng Trastuzumab ADC (De-Glycosylated) High Sensitivity Spectra Trastuzumab MW: 145165.1575 g/mol Linker and Drug: 958.5322 g/mol 1 Drug + 1 Antibody: 146123.6 g/mol
- 44. ©2017 Waters Corporation44COMPANY CONFIDENTIAL Intact Mass Analysis on T-DM1 +Drug 0 +Drug 1 +Drug 2 +Drug 3 +Drug 4 +Drug 5 +Drug 6 +Drug 7+Linker +Linker +Linker +Linker +Linker Antibody with 0-7 drugs observed Linker-only peaks observed
- 45. ©2017 Waters Corporation45COMPANY CONFIDENTIAL Conclusions Waters offers a Total Solution – Software tools – Sample preparation – LC/MS – Service and Application support Innovative tools for the latest performance – Xevo TQ-XS improved Step Wave and Detector – ionKey microfluidics – Xevo G2-XS
- 46. ©2017 Waters Corporation46COMPANY CONFIDENTIAL Acknowledgements Mary Lame Nikunj Tanna Paula Orens Caitlin Dunning Erin Chambers Catalin Doneanu Steven Calciano Ian Edwards Logan Umberger Mark Wrona Yun Alelyunas Hua Yang