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©2017 Waters Corporation 1COMPANY CONFIDENTIAL
LC-MS/MS for Bioanalytical Protein Quantification: Life of a Biotherapeutic
Infliximab Case Study
©2017 Waters Corporation 2COMPANY CONFIDENTIAL
Identification of Unique Signature PeptidesMRM Optimization of Unique Peptides Internal Standard OptionsProtein Level Sample Preparation DigestionPeptide Level Sample PreparationMS Quantification
Outline
©2017 Waters Corporation 3COMPANY CONFIDENTIAL
Case Study Goal
How do we get there?
Let’s take a look at the overall LC/MS workflow for protein quantification via the surrogate peptide approach…
The goal of this study is to develop a discovery bioanalytical method for the quantification of infliximab in rat and mouse plasma, with the intent to extend to human at a later date
©2017 Waters Corporation 4COMPANY CONFIDENTIAL
Why the Surrogate Peptide Approach?
©2017 Waters Corporation 5COMPANY 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)
PurifiedPeptides
Purified Protein
DataProcessing
TargetLynx
©2017 Waters Corporation 6COMPANY CONFIDENTIAL
Identification of Unique Signature PeptidesMRM Optimization of Unique Peptides Internal Standard OptionsProtein Level Sample Preparation DigestionPeptide Level Sample PreparationMS Quantification
Outline
©2017 Waters Corporation 7COMPANY CONFIDENTIAL
Find similar sequences of light and heavy chains (LC and HC)– Protein BLAST against the NCBI database
Align the mAb sequence against closely related LC/HC sequences– SIM alignment tool (ExPASy Proteomics Server)
Generate proteotypic peptides in-silico while maintaining sequence alignment– Trypsin used in this study, cleaves at lysine and arginine– Text editor (ex. TextPad, Notepad)
Assess uniqueness of the un-matched tryptic peptides from therapeutic mAb– Peptide BLAST against the NCBI database
Identification of Unique Proteotypic Peptides
©2017 Waters Corporation 8COMPANY CONFIDENTIAL
Infliximab is a chimeric human-murine anti-human tumor necrosis factor (TNF) monoclonal antibody.
Infliximab (Remicade) Sequence
Remicade Light chain [2]: DILLTQSPAILSVSPGERVSFSCRASQFVGSSIHWYQQRTNGSPRLLIKYASESMSGIPSRFSGSGSGTDFTLSINTVESEDIADYYCQQSHSWPFTFGSGTNLEVKTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTEDTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
http://www.drugbank.ca/drugs/DB00065
Formula: C6428H9912N1694O1987S46Molecular Weight: ~ 149.1 kD
Light Chain
Heavy
Cha
in
©2017 Waters Corporation 9COMPANY CONFIDENTIAL
Step 1: BLAST Search Infliximab Light Chain to Identify Closest Related Protein in Database
Protein BLAST search screen
Infliximab LC sequence
https://blast.ncbi.nlm.nih.gov/Blast.cgi
©2017 Waters Corporation 10COMPANY CONFIDENTIAL
Closely related to LC of Human IgG
Results of BLAST search for Infliximab LC
BLAST Result for LC
Protein sequence differences and gaps
©2017 Waters Corporation 11COMPANY CONFIDENTIAL
Step 2: Align the mAb Sequence Against Closely Related Sequences in ExPASy SIM Alignment Tool
SIM Protein Alignment for Infliximab LC
Paste infliximab LC sequence
Paste the identified (see step 1), closely related human IgG LC sequence
http://web.expasy.org/sim/
©2017 Waters Corporation 12COMPANY CONFIDENTIAL
ExPASy SIM Alignment Results: Infliximab LC vs. Human IgG LC
ExPASy SIM Protein Alignment for Infliximab LC vs. Human IgG LC
Highlights protein sequence differences while maintaining sequence alignment
©2017 Waters Corporation 13COMPANY 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
Infliximab LC: Tryptic Peptides
Gaps = tryptic peptide differences, maintaining sequence alignment
“matched” non-unique peptides
“un-matched” potentially unique peptides
©2017 Waters Corporation 14COMPANY CONFIDENTIAL
Step 4: Assess uniqueness of the un-matched tryptic peptides using peptide BLAST search
https://blast.ncbi.nlm.nih.gov/Blast.cgi
ASQFVGSSIHWYQQR
Peptide Level BLAST
Infliximab LC
Long List of Possibilities
©2017 Waters Corporation 15COMPANY CONFIDENTIAL
Step 4: Detailed View of Closely Related Database Matches
Mouse
Monkey
Goal: BA method for rat and mouse with extension to human= ID peptides unique in rat, mouse, human
“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 speciesThe alignment and gaps show the differences
ASQFVGSSIHWYQQR
Infliximab
Human
©2017 Waters Corporation 17COMPANY 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, ratASQFVGSSIHWYQQR: 80% Mouse IgG peptide-Specificity in human, rat, and mouseYASESMSGIPSR:90% Mouse IgG peptide-Specificity in human, rat, and mouse
Infliximab LC Tryptic Peptides
©2017 Waters Corporation 18COMPANY CONFIDENTIAL
Follow same process as LC:
Infliximab HC Tryptic Peptide Identification
BLAST search full chain
SIM Alignment of Infliximab HC
BLAST Search Peptides from Infliximab HC
Generate Tryptic Peptides In-silico
Remicade Heavy chain [2]: EVKLEESGGGLVQPGGSMKLSCVASGFIFSNHWMNWVRQSPEKGLEWVAEIRSKSINSATHYAESVKGRFTISRDDSKSAVYLQMNSLRTEDTGVYYCSRNYYGSTYDYGQGTTLTVSXASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Unique Infliximab HC Tryptic Peptides
©2017 Waters Corporation 19COMPANY CONFIDENTIAL
List of Unique Infliximab Tryptic Peptides
Light Chain– DILLTQSPAILSVSPGER (human & rat)– ASQFVGSSIHWYQQR (human, rat &
mouse)– YASESMSGIPSR (human, rat & mouse)– LC conserved region human IgG1
peptides (no specificity in human plasma)o DSTYSLSSTLTLSK
Heavy Chain– LEESGGGLVQPGGSMK (rat & human)– GLEWVAEIR (rat & human)– SINSATHYAESVK (mouse, rat &
human)– HC conserved region human IgG1
peptides (no specificity in human plasma)o GPSVFPLAPSSKo STSGGTAALGCLVKo DTLMISRo VVSVLTVLHQDWLNGK
©2017 Waters Corporation 21COMPANY 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)Signature Peptide Sequences
– Ideally rule out: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
©2017 Waters Corporation 22COMPANY CONFIDENTIAL
Identification of Unique Signature PeptidesMRM Optimization of Unique Peptides Internal Standard OptionsProtein Level Sample Preparation DigestionPeptide Level Sample PreparationMS Quantification
Outline
©2017 Waters Corporation 23COMPANY CONFIDENTIAL
Skyline Software: Download to Acquisition PC
https://skyline.ms/project/home/software/Skyline/begin.view
©2017 Waters Corporation 24COMPANY CONFIDENTIAL
Developed by the MacCoss laboratory in the University of Washington
Best Practice:Our Approach = Skyline Workflow
Start Here!
©2017 Waters Corporation 25COMPANY CONFIDENTIAL
SkyLine
In silco 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
©2017 Waters Corporation 27COMPANY CONFIDENTIAL
Skyline MRM Method Creation: Infliximab LC
With Skyline, importing a protein sequence elicits an in silico digestion algorithm and MRM transition prediction
Skyline Protein Import
©2017 Waters Corporation 28COMPANY CONFIDENTIAL
Skyline MRM Method Creation: Infliximab LC
Skyline> MassLynx Method Export
©2017 Waters Corporation 29COMPANY CONFIDENTIAL
Method Conditions
LC System: ACQUITY UPLCMS System: Waters Xevo TQ-S, ESI+
Column: ACQUITY UPLC Peptide BEH C18, 300Å, 1.7 µM, 2.1 mm x 150 mm
Column Temperature: 55 °CSample Temperature: 10 °CMobile Phase A: 0.1 % Formic Acid in waterMobile Phase B: 0.1 % Formic Acid in waterGradient: 1 min hold at 10% B,
then 10-55% B in 6 minutes
Skyline LC/MS Conditions
MS ConditionsCapillary (KV): 3.00Cone (V): 30Source Temperature (°C): 150Desolvation Temperature (°C): 1000Cone Gas Flow (L/Hr): 150Desolvation Gas Flow (L/Hr): 1000Collision Gas Flow (mL/min): 0.15Nebuliser Gas Flow (Bar): 7.00
©2017 Waters Corporation 30COMPANY 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
©2017 Waters Corporation 31COMPANY CONFIDENTIAL
Skyline MRM Method Creation: Narrowing Down MRMs
Skyline Protein Import
©2017 Waters Corporation 32COMPANY CONFIDENTIAL
Sample Analysis with MassLynx: Skyline imported methods
Narrowed down Skyline methods are imported into MassLynx for LC/MS acquisition
MassLynx imported Skyline Methods
©2017 Waters Corporation 33COMPANY CONFIDENTIAL
Skyline MRM Method Optimization: CE Optimization
Transitions are optimized by collision energy and the results are visualized
The optimized method can be exported automatically
Skyline MRM CE Optimization
©2017 Waters Corporation 34COMPANY CONFIDENTIAL
DILLASQ
DSTY
YASE
Final Optimized Skyline Method for Instrument Import: Peptides, Precursors, and Fragments
©2017 Waters Corporation 35COMPANY CONFIDENTIAL
Exporting a Final MRM Method to MassLynx
When you’ve narrowed down your best optimized transitions, it is important to check back in the transitions settings to make sure the “Use optimization values when present” is checked. This allows Skyline to choose the best CE per transition to export into your method instead of the original predicted CE.
Options include transition or precursor.
©2017 Waters Corporation 36COMPANY CONFIDENTIAL
Optimized Skyline MassLynx Method: Generic and signature peptides
Optimized MRM method for HC and LC Infliximab tryptic peptides
Individual MRMs for DILL peptide
©2017 Waters Corporation 37COMPANY CONFIDENTIAL
Infliximab Skyline Optimization vs. Manual: DILLTQSPAILSVSPGER (LC)
Infliximab Skyline
Time2.00 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)
3.94e6Area
29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER)
3.94e6Area
5.5926370
Skyline Optimization633.1>546.3More sensitive transition!
Manual Optimization633.1>731.8
5.59140139
Alternate MRM transition
©2017 Waters Corporation 38COMPANY CONFIDENTIAL
Infiximab Unique Signature PeptidesInfliximab Skyline
Time2.00 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
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
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 21: MRM of 3 Channels ES+ 948.526 > 545.268 (Infliximab_LC DILLTQSPAILSVSPGER.2)
6.51e5Area
29Dec2016_Infliximab_Optimized_003 11: MRM of 3 Channels ES+ 598.629 > 631.307 (Infliximab_LC ASQFVGSSIHWYQQR.3)
3.49e6Area
4.42;114896
29Dec2016_Infliximab_Optimized_003 15: MRM of 3 Channels ES+ 642.798 > 359.204 (Infliximab_LC YASESMSGIPSR.2)
6.19e6Area
3.64;246053
29Dec2016_Infliximab_Optimized_003 7: MRM of 2 Channels ES+ 515.924 > 576.281 (Infliximab_HC LEESGGGLVQPGGSMK.3)
4.93e6Area
3.88;170856
29Dec2016_Infliximab_Optimized_003 8: MRM of 2 Channels ES+ 536.793 > 171.113 (Infliximab_HC GLEWVAEIR.2)
9.39e6Area
5.21;329917
29Dec2016_Infliximab_Optimized_003 5: MRM of 3 Channels ES+ 469.569 > 603.791 (Infliximab_HC SINSATHYAESVK.3)
5.03e7Area
3.38;1735473
5.58;22653
DILLTQSPAILSVSPGER-LC
ASQFVGSSIHWYQQR-LC
YASESMSGIPSR-LC
LEESGGGLVQPGGSMK-HC
GLEWVAEIR-HC
SINSATYAESVK-HC
633.1>731.8
598.6>631.3
642.8>359.2
515.9>576.3
536.8>171.7
469.6>603.8
©2017 Waters Corporation 39COMPANY CONFIDENTIAL
Infliximab Generic, Human IgG1, Signature Peptides
Infliximab Skyline
Time2.00 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
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
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
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 2: MRM of 4 Channels ES+ 418.221 > 506.276 (Infliximab_HC DTLMISR.2)
1.73e7Area
3.90;549369
29Dec2016_Infliximab_Optimized_003 9: MRM of 4 Channels ES+ 559.939 > 708.849 (Infliximab_HC FNWYVDGVEVHNAK.3)
2.02e7Area
4.67;650255
29Dec2016_Infliximab_Optimized_003 12: MRM of 3 Channels ES+ 603.34 > 805.439 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
5.12e6Area
29Dec2016_Infliximab_Optimized_003 3: MRM of 2 Channels ES+ 419.755 > 654.382 (Infliximab_HC ALPAPIEK.2)
2.99e7Area
3.84;1004329
29Dec2016_Infliximab_Optimized_003 16: MRM of 3 Channels ES+ 643.841 > 359.204 (Infliximab_HC EPQVYTLPPSR.2)
9.18e5Area
3.64;31544
29Dec2016_Infliximab_Optimized_003 19: MRM of 2 Channels ES+ 848.715 > 764.357 (Infliximab_HC GFYPSDIAVEWESNGQPENNYK.3)
4.71e6Area
29Dec2016_Infliximab_Optimized_003 13: MRM of 2 Channels ES+ 625.312 > 234.145 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
1.99e6Area
29Dec2016_Infliximab_Optimized_003 1: MRM of 3 Channels ES+ 394.729 > 588.335 (Infliximab_HC SLSLSPGK.2)
6.02e6Area
3.63;215260
Time2.00 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
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
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
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 12: MRM of 3 Channels ES+ 603.34 > 805.439 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
5.12e6Area
29Dec2016_Infliximab_Optimized_003 3: MRM of 2 Channels ES+ 419.755 > 654.382 (Infliximab_HC ALPAPIEK.2)
2.99e7Area
3.84;1004329
29Dec2016_Infliximab_Optimized_003 16: MRM of 3 Channels ES+ 643.841 > 359.204 (Infliximab_HC EPQVYTLPPSR.2)
9.18e5Area
3.64;31544
29Dec2016_Infliximab_Optimized_003 19: MRM of 2 Channels ES+ 848.715 > 764.357 (Infliximab_HC GFYPSDIAVEWESNGQPENNYK.3)
4.71e6Area
29Dec2016_Infliximab_Optimized_003 13: MRM of 2 Channels ES+ 625.312 > 234.145 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
1.99e6Area
29Dec2016_Infliximab_Optimized_003 1: MRM of 3 Channels ES+ 394.729 > 588.335 (Infliximab_HC SLSLSPGK.2)
6.02e6Area
3.63;215260
29Dec2016_Infliximab_Optimized_003 17: MRM of 4 Channels ES+ 751.883 > 234.092 (Infliximab_LC DSTYSLSSTLTLSK.2)
1.32e6Area
4.81;50235
29Dec2016_Infliximab_Optimized_003 10: MRM of 3 Channels ES+ 593.827 > 418.23 (Infliximab_HC GPSVFPLAPSSK.2)
1.96e7Area
4.76;737428
5.30;162081
5.55;64156
5.86;166012
DSTYSLSSTLTLSK
GPSVFPLAPSSK
DTLMISR
FNWYVDGVEVHNAK
VVSVLTVLHQDWLNGK
ALPAPIEK
EPQVYTLPPSR
GFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSK
SLSLPGK
751.9>234.1
593.8>418.2
418.2>506.3
559.9>708.8
603.3>805.4
409.7>654.4
643.8>359.2
848.7>764.3
625.3>234.1
394.7>588.31
©2017 Waters Corporation 40COMPANY CONFIDENTIAL
Infliximab Specificity in Matrix: Generic Signature VVSVLTVLHQDWLNGK (HC)
remicade buffer 50
Time4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20
%
0
100
4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20
%
0
100
4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20
%
0
100
11Dec2014_digest_direct_1025_mabs MRM of 6 Channels ES+ 603 > 1110 (VVSVLTVLHQDWLNGK)
4.84e5Area
5.54;16050
03May2016_Infliximab_AutomationReagentStability_016 MRM of 10 Channels ES+ 603.3 > 1110.6 (Generic VVSVLTVLHQDWLNGK)
4.31e3Area
11Dec2014_digest_direct_1027_mabs MRM of 6 Channels ES+ 603 > 1110 (VVSVLTVLHQDWLNGK)
2.34e5Area
5.53;8028
remicade buffer 50
Time4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20
%
0
4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20%
0
4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20
%
0
100
11Dec2014_digest_direct_1025_mabs MRM of 6 Channels ES+ 603 > 1110 (VVSVLTVLHQDWLNGK)
4.84e5Area
5.54;16050
11Dec2014_digest_direct_1023_mabs MRM of 6 Channels ES+ 603 > 1110 (VVSVLTVLHQDWLNGK)
1.14e7Area
5.53;391409
11Dec2014_digest_direct_1029_mabs MRM of 6 Channels ES+ 603 > 1110 (VVSVLTVLHQDWLNGK)
1.26e7Area
5.52;430796
Infliximab 50 µg/mL Buffer Digest
Blank Rat Plasma
50 µg/mL Rat Plasma
Blank Human Plasma
50 µg/mL Human Plasma
Generic Human IgG PeptideVVSVLTVLHQDWLNGK
©2017 Waters Corporation 41COMPANY CONFIDENTIAL
Infliximab Specificity in Matrix: Unique Signature DILLTQSPAILSVSPGER(LC)
remicade buffer 50
Time4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20
%
0
4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20
%
0
4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20
%
0
100
11Dec2014_digest_direct_1025_mabs MRM of 6 Channels ES+ 633 > 731 (DILLTQSPAILSVSPGER)
2.67e6Area
5.32;71906
03May2016_Infliximab_AutomationReagentStability_016 MRM of 10 Channels ES+ 633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER)
1.53e6Area
11Dec2014_digest_direct_1027_mabs MRM of 6 Channels ES+ 633 > 731 (DILLTQSPAILSVSPGER)
6.75e5Area
5.32;19165
remicade buffer 50
Time4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20
%
0
4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20
%0
4.60 4.80 5.00 5.20 5.40 5.60 5.80 6.00 6.20
%
0
100
11Dec2014_digest_direct_1025_mabs MRM of 6 Channels ES+ 633 > 731 (DILLTQSPAILSVSPGER)
2.67e6Area
5.32;72436
11Dec2014_digest_direct_1023_mabs MRM of 6 Channels ES+ 633 > 731 (DILLTQSPAILSVSPGER)
4.30e5Area
11Dec2014_digest_direct_1029_mabs MRM of 6 Channels ES+ 633 > 731 (DILLTQSPAILSVSPGER)
8.76e6Area
5.32;264330
Infliximab 50 µg/mL Buffer Digest
Blank Rat Plasma
50 µg/mL Rat Plasma
Blank Human Plasma
50 µg/mL Human Plasma
Unique Signature PeptideDILLTQSPAILSVSPGER
©2017 Waters Corporation 42COMPANY CONFIDENTIAL
Identification of Unique Signature PeptidesMRM Optimization of Unique Peptides Internal Standard OptionsProtein Level Sample Preparation DigestionPeptide Level Sample PreparationMS Quantification
Outline
©2017 Waters Corporation 43COMPANY 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
©2017 Waters Corporation 44COMPANY CONFIDENTIAL
Peptide Level IS: TrastuzumabC13N15 Labeled Trastuzumab (FTISADTSK) peptide with terminal
overhang– Added just prior to digestion
Time2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00
%
0
100
2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00
%
0
100
7pt5ugmL_2hr_bicarb_070214_002 MRM of 8 Channels ES+ 489.2 > 729.4 (FTISADTSK(13C15N) HC)
4.33e57.41
7pt5ugmL_2hr_bicarb_070214_002 MRM of 8 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
4.33e5
7.41
8.65
C13N15 FTISADTSK Trastuzumab PeptideInternal Standard
FTISADTSK Trastuzumab Peptide
©2017 Waters Corporation 45COMPANY CONFIDENTIAL
Labeled Antibody Internal Standard: SILu™Mab
SILuMab Heavy ChainEVQLVESGGGLVQPGGSLRLSCVAS GFTLNNYDMHWVRQGIGKGLEWVSK IGTAGDRYYAGSVKGRFTISRENAKD SLYLQMNSLRVGDAAVYYCARGAGR WAPLGAFDIWGQGTMVTVSS|ASTKG PSVFPLAPSSKSTSGGTAALGCLVK DYFPEPVTVSWNSGALTSGVHTFPAVLQ SSGLYSLSSVVTVPSSSLGTQTYIC NVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG
SILuMab Light ChainQSALTQPRSVSGSPGQSVTISCTGT SSDIGGYNFVSWYQQHPGKAPKLMI YDATKRPSGVPDRFSGSKSGNTASLT ISGLQAEDEADYYCCSYAGDYTPGV VFGGGTKLTVL|GQPKAAPSVTLFP PSSEELQANKATLVCLISDFYPGAV TVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS
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)
©2017 Waters Corporation 46COMPANY CONFIDENTIAL
direct 50ug/ml SPE*
Time-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
10018Dec2014B_spe_direct_1037 MRM of 10 Channels ES+
603.3 > 1110.6 (TNO Furlong Remicade signatureVVSVLTVLHQDWLNGK)3.07e5
prot a spe blk plasma silumab
Time-0.00 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
10015Jan2015_HerceptinProtA_spe_1009 MRM of 10 Channels ES+
423.225 > 516.284 (SILUMAB IS-DTLMIS[R].HEAVY-3)8.58e5
Chromatography: SILuMab IS Labeled Antibody Drug Signature Peptides
1 2 3
mAb PeptideRetention Time
(min)4 Unique Herceptin FTISADTSK HC 3.315 Generic STSGGTAALGC[+57.0]LVK HC 4.056 Generic DSTYSLSSTLTLSK LC 4.397 Generic GPSVFPLAPSSK HC 4.508 Unique Remicade DILLTQSPAILSVSPGER LC 5.179 Generic VVSVLTVLHQDWLNGK HC 5.41
4 5 6 7 8 9
SILUMAB IS PeptideRetention Time
(min)1 DTLMIS[R] HC 3.562 YAASSYLSLTPEQW[K] HC 4.773 VVSVLTVLHQDWLNG[K] HC 5.41
SILuMabInternal Standard
Antibody Drug Unique and Generic Signature Peptides
©2017 Waters Corporation 47COMPANY CONFIDENTIAL
Intact Murine mAb Mass Check Standard (Waters)
Murine mAb Light chain:DVLMTQTPLSLPVSLGDQASISCRSSQYIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPLTFGAGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC Murine mAb Heavy chain:QVQLKESGPGLVAPSQSLSITCTVSGFSLLGYGVNWVRQPPGQGLEWLMGIWGDGSTDYNSALKSRISITKDNSKSQVFLKMNSLQTDDTAKYYCTRAPYGKQYFAYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTDSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAHTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPG
Conserved region: blue Variable regions: red Signature peptides: BOXED
Unique SignatureGeneric Signature
©2017 Waters Corporation 48COMPANY CONFIDENTIAL
Generic Internal StandardWorkflow check Standard
prot A spe blk ISTD murine mab
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
10014Aug2015_RemicadeSPE_ProteinA_01004 MRM of 11 Channels ES+
983.95 > 397.21 (Murine 4)4.91e6
prot a spe 50 ug/ml
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00
%
0
10014Aug2015_RemicadeSPE_ProteinA_01038 MRM of 11 Channels ES+
633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER)8.35e6
1
2
3
4
5
6
7
8
9
Murine mAbInternal Standard
Infliximab Unique and Generic Signature Peptides
Retention Time(min)
1 MNSLQTDDTAK 4.062 VNSAAFPAPIEK 5.073 NTQPIMDTDGSYFVYSK 5.374 SVSELPIMHQDWLNGK 5.66
Murine mAb IS Peptides
Retention Time(min)
5 SINSATHYAESVK* 4.226 DSTYSLSSTLTLSK 5.437 GPSVFPLAPSSK 5.488 DILLTQSPAILSVSPGER* 6.029 VVSVLTVLHQDWLNGK 6.16
mAb Peptides
Intact Murine mAb Mass Check Standard (Waters)
©2017 Waters Corporation 50COMPANY CONFIDENTIAL
Identification of Unique Signature PeptidesMRM Optimization of Unique Peptides Internal Standard OptionsProtein Level Sample Preparation DigestionPeptide Level Sample PreparationMS Quantification
Outline
©2017 Waters Corporation 51COMPANY CONFIDENTIAL
Sample Preparation Options
Protein Level Clean-up prior to digeston (Optional)– None
o Whole serum/plasma digestion– Affinity-based
o Generic: Protein A/G with binding affinity to various immunoglobulins in various species
o Anti-human: kappa with binding affinity to light chains on all human immunoglobulins
o Specific Receptor: anti-idiotypic binding– MWT cut-off filters– Depletion Plates– Gel Filtration
©2017 Waters Corporation 52COMPANY CONFIDENTIAL
spe 10 ug/ml
Time0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50
%
0
100
0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 9.50
%
0
100
16Oct2015_4mAb_HumanPL_3St_ProtA_1038 MRM of 15 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
1.28e7Area
4.12;486885
16Oct2015_4mAb_HumanPL_3St_direct_1035 MRM of 15 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
1.28e7Area
4.1195194
GE Multi-Trap Protein A Clean-Up of Infliximab from Plasma
Direct Digestion of Plasma
Protein A Clean-up Prior to Digestion
Infliximab SINSATHYAESVK Tryptic Peptide
©2017 Waters Corporation 53COMPANY CONFIDENTIAL
100uL SPE, Protein A, 1:5 Worthington, DTT, 17hr digestion
Time7.50 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)
6.43e4Area
100ngmL_plasma_051514_002 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
6.43e4Area
8.49722
50ngmL_plasma_051514_002 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
6.43e4Area
8.50345
blank_plasma_051514_002 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
6.43e4Area
8.49163
100uL SPE, Kappa
Time7.50 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)
1.30e5Area
100ngmL_plasma_051314_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
1.30e5Area
8.43810
50ngmL_plasma_051314_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
1.30e5Area
8.43401
blank_plasma_051314_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
1.30e5Area
50uL SPE, whole plasma, 1:12.5 Worthington, DTT
Time7.50 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)
2.60e4Area
8.38493
100ngmL_plasma_052114_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
2.60e4Area
8.4179
50ngmL_plasma_052114_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
2.60e4Area
blank_plasma_052114_003 MRM of 6 Channels ES+ 485.2 > 721.4 (FTISADTSK HC)
2.60e4Area
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
©2017 Waters Corporation 54COMPANY CONFIDENTIAL
Identification of Unique Signature PeptidesMRM Optimization of Unique Peptides Internal Standard OptionsProtein Level Sample Preparation DigestionPeptide Level Sample PreparationMS Quantification
Outline
©2017 Waters Corporation 55COMPANY CONFIDENTIAL
ProteinWorks™ eXpress Digest Kits
Key AttributesStandardized protocols
– Eliminates Method Development for Discovery Studies– Digestion and quantification of a diversity of proteins– 3 and 5-Step Digestion to accommodate variety of proteins and signature
peptide typesScalable, flexible formatReliable and reproducibleHigh SensitivityQuantification Total antibody analysis for ADCs
– Detection of conjugated peptidesNo capital investment required
©2017 Waters Corporation 56COMPANY CONFIDENTIAL
Peptide
Std. Curve Range (mg/mL) Weighting
Linear fit (r2)
Mean % Accuracy
of all points
DILLTQSPAILSVSPGER 0.25-500 1/x 0.998 100.01
QC Conc (ug/mL)Mean Cal. Conc
(ug/mL) Std. Dev. %CV Mean AccuracyDILLTQSPAILSVSPGER* 0.35 0.33 0.02 5.80 93.2SILUMAB-DTL(IS) 3.50 3.79 0.02 0.49 108.2
35.00 39.58 0.17 0.44 113.1350.00 350.02 3.09 0.88 100.0
QC Conc (ug/mL)DILLTQSPAILSVSPGER* 0.35 0.34 0.00 0.89 96.5SILUMAB-VVSV (IS) 3.50 3.65 0.05 1.47 104.2
35.00 36.51 0.73 2.01 104.3350.00 350.80 3.90 1.11 100.2
* Signature
Accurate and Precise Quantification using ProteinWorksInfliximab Direct Plasma Digestion
Linear, Precise and
Accurate
Single digit %CVs
©2017 Waters Corporation 57COMPANY CONFIDENTIAL
Identification of Unique Signature PeptidesMRM Optimization of Unique Peptides Internal Standard OptionsProtein Level Sample Preparation DigestionPeptide Level Sample PreparationMS Quantification
Outline
©2017 Waters Corporation 58COMPANY CONFIDENTIAL
Peptide Level Clean Up:
Peptides
Purified Peptides
Target AnalyteSensitivity
Interfering Digest Reagents, Buffer and Matrix Salts,
Phospholipids
Achieving Recovery of Target Peptides
High Levels of Unwanted
Endogenous Peptides
Should I clean up my
peptides?
Peptide-level clean-up (optional)
System Robustness
©2017 Waters Corporation 59COMPANY 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
020406080
100120
Oasis MCX
One generic protocol achieves high recovery for a diversity of tryptic peptides
©2017 Waters Corporation 60COMPANY CONFIDENTIAL
Infliximab Peptide Level Clean-Up: Mixed-Mode SPE with Oasis MCX
direct 100ug/ml
Time1.00 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)
3.65e6Area
4.40;149708
18Dec2014B_spe_direct_1040 MRM of 10 Channels ES+ 751.88 > 836.47 (Merck DSTYSLSSTLTLSK)
3.65e6Area
4.4173225
Direct Plasma Digestion No SPE
Direct Plasma Digestion SPE
DSTYSLSSTLTLSK Tryptic Peptide
©2017 Waters Corporation 61COMPANY CONFIDENTIAL
Identification of Unique Signature PeptidesMRM Optimization of Unique Peptides Internal Standard OptionsProtein Level Sample Preparation DigestionPeptide Level Sample PreparationMS Quantification
Outline
©2017 Waters Corporation 62COMPANY CONFIDENTIAL
Place Holder for MS Introduction
Options for TQ or Accurate MassPros and ConsData for TQ, G2-XS
©2017 Waters Corporation 63COMPANY CONFIDENTIAL
High Sensitivity Infliximab Quantification:Xevo TQ-S Triple QuadrupoleLinear, Precise and Accurate
Single digit %CVs
LLOQ of 10ng/mL35 µL sample, Protein A, ProteinWorks digestion and MCX peptide clean-up
©2017 Waters Corporation 64COMPANY CONFIDENTIAL
High Sensitivity Infliximab Quantification: Xevo TQ-S Triple Quadrupole
Time2.20 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 )
2.20e4Area
4.15361
MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
2.20e4Area
10 ng/mL infliximab
Blank plasma
SINSATHYAESVK Unique Signature Peptide
©2017 Waters Corporation 65COMPANY CONFIDENTIAL
Coming soon
G2-XS Data
©2017 Waters Corporation 66COMPANY CONFIDENTIAL
Conclusions
©2017 Waters Corporation 67COMPANY CONFIDENTIAL
Acknowledgements
Mary LameNikunj TannaPaula OrensCaitlin DunningErin ChambersCatalin DoneanuSteven Calciano Ian EdwardsLogan UmbergerMark WronaYun AlelyunasHua Yang
©2017 Waters Corporation 68COMPANY CONFIDENTIAL
Supplemental Information
©2017 Waters Corporation 69COMPANY CONFIDENTIAL
Skyline Optimization vs. Manual Optimization - DILL
Infliximab Skyline
Time2.00 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)
3.94e6Area
29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 633.1 > 731.8 ( Remicade DILLTQSPAILSVSPGER)
3.94e6Area
5.5926370
Skyline Optimization
Manual Optimization
5.59140139
©2017 Waters Corporation 70COMPANY CONFIDENTIAL
High Sensitivity Antibody Quantification: Remicade (infliximab)
Linear, Precise and Accurate
Single digit %CVs
©2017 Waters Corporation 71COMPANY CONFIDENTIAL
High Sensitivity Antibody Quantification: Remicade (infliximab)
Time2.20 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 )
2.20e4Area
4.15361
MRM of 11 Channels ES+ 469.6 > 603.8 (Remicade SINSATHYAESVK )
2.20e4Area
LLOQ of 10ng/mL35 µL sample, Protein A affinity sample clean-up and
digestion
10 ng/mL infliximab
Blank plasma
SINSATHYAESVK Unique Signature Peptide
©2017 Waters Corporation 72COMPANY CONFIDENTIAL
Protein BLAST: Infliximab HC
Protein BLAST search screen
Infliximab HC sequence
©2017 Waters Corporation 73COMPANY CONFIDENTIAL
Protein BLAST: Infliximab HCBLAST
Result for HCProtein sequence differences and gaps
©2017 Waters Corporation 74COMPANY CONFIDENTIAL
Align the mAb sequence against closely related sequences: ExPASy SIM Alignment
SIM Protein Alignment for Infliximab HC
©2017 Waters Corporation 75COMPANY CONFIDENTIAL
ExPASy SIM Alignment Results: Infliximab HC
ExPASy SIM Protein Alignment for
Infliximab HC vs. Human IgG HC
Protein sequence differences maintaining sequence alignment
©2017 Waters Corporation 76COMPANY CONFIDENTIAL
Generate Tryptic Peptides Maintaining Sequence Alignment: Infliximab HC
Infliximab HC: Tryptic Peptides
Trypsin cleaves peptides on the C-terminal side of lysine (K) and arginine (R) amino acid
©2017 Waters Corporation 77COMPANY CONFIDENTIAL
Assess uniqueness of the un-matched tryptic peptides from therapeutic mAb: Infliximab HC
GLEWVAEIR: Variable and Complimentary Determining Region (CDR)
LEESGGGLVQPGGSMK: Variable Region
SINSATHYAESVK:Variable and Complimentary Determining Region (CDR)
This a chimeric murine mAb and variable region should have very close sequence homology to mouse
Peptide-Level BLAST
Infliximab HC100% sequence homology mouse
~85% sequence homology mouse & human
100% sequence homology to mouse
©2017 Waters Corporation 78COMPANY CONFIDENTIAL
Tryptic Peptide Specificity: Trastuzumab HC vs. IgG1kappa 1
Heavy Chain
too long (25 AA)
LEESGGGLVQPGGSMK and GLEWVAEIR : Mouse IgG peptides-Specificity in humanSINSATHYAESVK:~85% Mouse IgG peptide-Specificity in mouse, rat and humna
This a chimeric murine mAb and variable region should have very close sequence homology to mouse
©2017 Waters Corporation 79COMPANY CONFIDENTIAL
Skyline Optimization vs. Manual Optimization - GLEW
Infliximab Skyline
Time2.00 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 8: MRM of 2 Channels ES+ 536.793 > 488.283 (Infliximab_HC GLEWVAEIR.2)
8.47e6Area
5.21;292371
29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 536.793 > 773.43 (GLEWVAEIR)
8.47e6Area5.21
250364
Skyline Optimization
Manual Optimization
©2017 Waters Corporation 80COMPANY CONFIDENTIAL
Skyline Optimization vs. Manual Optimization - GPSV
Infliximab Skyline
Time2.00 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 10: MRM of 3 Channels ES+ 593.827 > 418.23 (Infliximab_HC GPSVFPLAPSSK.2)
1.96e7Area
4.76;737428
29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 593.83 > 699.4 (Generic GPSVFPLAPSSK)
1.96e7Area4.77
606398
Skyline Optimization
Manual Optimization
©2017 Waters Corporation 81COMPANY CONFIDENTIAL
Infliximab Mary
Time2.00 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 12: MRM of 3 Channels ES+ 603.34 > 805.439 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
5.12e6Area
29Dec2016_Infliximab_Optimized_004 MRM of 13 Channels ES+ 603.3 > 1110.6 (Generic VVSVLTVLHQDWLNGK)
5.12e6Area
Skyline Optimization vs. Manual Optimization - VVSV
Skyline Optimization
Manual Optimization
5.86175812
5.866451
©2017 Waters Corporation 82COMPANY CONFIDENTIAL
Internal Standard Comparison
Best curve fit from generic mab IS using peptide with RT closest to analyte– Curve fit from extended tag peptide IS very close
Average accuracy of standard curve points comparable between extended peptide IS (9%) and mab IS (12%)
Worst fit and accuracy from mab IS using peptide with very different RT
Curve FitAvg Acc of Std Curve Points RSD of IS
Extended Tag Pep IS 0.988 9% 17%
SILuMab IS, close RT 0.998 12% *8%
SILuMab IS, diff RT 0.979 33% 19%
*mab IS 8% RSD through entire workflow, includes protein A and digestion
©2017 Waters Corporation 83COMPANY CONFIDENTIAL
©2017 Waters Corporation 84COMPANY CONFIDENTIAL
LC-MS/MS Protein Quantification Challenges Identification and LC/MS optimization of unique surrogate peptides is complex
and laborious, – Workflow can be challenging, particularly for small molecule bioanalytical scientists
Currently no end-to-end informatics platform exists to helps a scientist navigate and method develop for the surrogate peptide approach
Simplification and LC/MS platforms with improved informatics platforms are need– Current approaches require multiple informatics free-ware
o BLAST protein search (NCBI database)o ExPASY SIM Alignmento Protein Prospectoro Skylineo MassLynx BioLynx Application/UNIFI…
Many possible sample preparation workflow options– Lengthy method development times– Many steps to optimize– Lack of standardization– Sensitivity and reproducability issues
©2017 Waters Corporation 85COMPANY CONFIDENTIAL
Manual Optimization Process
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Protein Prospector (UCSF)– In silico tryptic digestion DILLTQSPAILSVSPGER (LC)– Prediction of precursors and MRM fragments
Possible Approach: Manual Optimization
Possible Precursors Potential fragment ions
http://prospector.ucsf.edu/prospector/cgi-bin/msform.cgi?form=msproduct
DILLTQSPAILSVSPGER (LC)
©2017 Waters Corporation 87COMPANY CONFIDENTIAL
Confirmation of Precursor Presence and Retention Time
DILL 2nd Charge State
Time3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00
%
1
3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00
%
1
26Jan2017_DILLMRM_005 1: MRM of 1 Channel ES+ 632.69 > 632.69 (DILL 3rd Charge State)
1.81e7Area
5.92;710710
26Jan2017_DILLMRM_006 1: MRM of 1 Channel ES+ TIC (DILL 3rd Charge State)
1.81e7Area
5.92;710710
Infliximab digested in buffer
Blank buffer digest
DILL Peptide 632.73+
©2017 Waters Corporation 88COMPANY CONFIDENTIAL
MassLynx Manual Optimization: Infliximab DILLTQSPAILSVSPGER (LC)
MS/MS Spectra of MH+3Precursor
50 µg/mL Infliximab DILL
m/z200 225 250 275 300 325 350 375 400 425 450 475 500 525 550 575 600 625 650 675 700 725 750 775 800 825 850 875 900 925 950 975 1000 1025 1050 1075
%
0
100
20Jan2017_Infliximab_ManualOpScans_013 237 (5.937) Cm (237) Daughters of 633ES+ 9.28e5610.8
575.8
474.6230.5
215.2
458.3
342.5
308.9
261.5
388.9
423.1
526.4
509.2
495.5
541.3
563.0
732.0
645.3
658.8
706.9676.2
844.2
798.7
783.2752.9
1029.7
925.9
907.5
889.1
959.0
939.7 1007.0
b2 +1H
b3 +1H MH+3
y7 +1H
633.5
b17 +3H
y8 +1H
b11 +3Hy9 +1H
y4 +1H
y6 +1H
y10 +1H545.3y5 +1H
DILL 2nd Charge State
Time3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00
%
1
3.10 3.20 3.30 3.40 3.50 3.60 3.70 3.80 3.90 4.00 4.10 4.20 4.30 4.40 4.50 4.60 4.70 4.80 4.90 5.00 5.10 5.20 5.30 5.40 5.50 5.60 5.70 5.80 5.90 6.00 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00
%
1
26Jan2017_DILLMRM_005 1: MRM of 1 Channel ES+ 632.69 > 632.69 (DILL 3rd Charge State)
1.81e7Area
5.92;710710
26Jan2017_DILLMRM_006 1: MRM of 1 Channel ES+ TIC (DILL 3rd Charge State)
1.81e7Area
5.920
©2017 Waters Corporation 89COMPANY CONFIDENTIAL
MassLynx Manual Optimization: Infliximab DILLTQSPAILSVSPGER (LC)
0
2000
4000
6000
8000
10000
12000
14000
16000
18000
5 10 15 20 25 30 35
Peak
Are
a
CE Ramp of DILL
632.69>458.24 Area
632.69>545.27 Area
632.69>731.37 Area
MRM CE Optimization of MH+3Precursor
©2017 Waters Corporation 90COMPANY CONFIDENTIAL
Optional ExPasy Peptide Level BLAST searching
©2017 Waters Corporation 91COMPANY CONFIDENTIAL
This can be done in NCBI data base too. See notes section
Infliximab Peptide level BLAST:ExPASY example ASQFVGSSIHWYQQR
Mouse
Rat
Monkey
Human
http://web.expasy.org/blast/
©2017 Waters Corporation 92COMPANY CONFIDENTIAL
Optional Visualization
©2017 Waters Corporation 93COMPANY CONFIDENTIAL
Confirming Species Specificity Multiple Sequence Alignment Tool: Example below is mAb, mouse, human and monkey
Infliximab Peptide level BLAST:NCBI example ASQFVGSSIHWYQQR
Mouse
Rat
Monkey
Human
http://web.expasy.org/blast/
InfliximabMouseHumanMonkey
https://blast.ncbi.nlm.nih.gov/Blast.cgi
©2017 Waters Corporation 94COMPANY CONFIDENTIAL
Additional Scrren Shot Detail from Skyline
©2017 Waters Corporation 95COMPANY CONFIDENTIAL
Reasons why you want BLAST searching and filtering to narrow down peptide options
Text in box is why you import multiple methods– Each method is one peptide, one precursor, with its predicted fragments
Skyline Exporting MRM Method: Fragment Determination
Too many transitions for 11 minute method. Doesn’t allow enough points (≈1.0) across the peak for useful data. So we must narrow it down even further.
©2017 Waters Corporation 96COMPANY CONFIDENTIAL
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 402.718 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
1.54e55.19
December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 510.292 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
2.65e55.18
December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 557.947 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
4.31e35.203.64
3.56 3.895.15
5.31 6.18 6.39
December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 657.361 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
3.14e55.18
December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 891.461 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
2.79e45.19
December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 1063.51 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
2.38e45.18
3.55 5.21
December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 1265.568 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
5.92e35.18
5.23
December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 1477.721 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
1.65e35.18
December2016_InfliximabMD_039 MRM of 32 Channels ES+ 625.312 > 74.06 (Infliximab_HC TTPPVLDSDGSFFLYSK.3)
3.76e45.193.91
3.56 4.02 5.05Time
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 147.113 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
4.93e43.13 5.44
3.77 4.66
December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 437.567 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
5.75e36.073.783.71
2.715.534.22
4.66 5.31 6.46 6.636.95
December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 655.846 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
2.00e55.44
5.47
December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 1110.569 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
1.53e45.42
5.48
December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 216.134 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
5.89e36.50
5.443.88 6.04 6.55
December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 854.973 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
1.83e55.43
December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 204.134 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
1.19e55.44
5.49
December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 1423.769 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
4.37e35.43
5.46
December2016_InfliximabMD_035 MRM of 32 Channels ES+ 603.34 > 74.06 (Infliximab_HC VVSVLTVLHQDWLNGK.3)
3.40e33.140.04 4.774.644.273.92 5.455.76
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 246.181 (Infliximab_HC SINSATHYAESVK.3)
2.25e53.13
December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 147.113 (Infliximab_HC SINSATHYAESVK.3)
1.47e63.13
December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 312.159 (Infliximab_HC SINSATHYAESVK.3)
2.32e43.12
6.695.695.535.21 6.566.16
December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 312.159 (Infliximab_HC SINSATHYAESVK.3)
2.32e43.12
6.695.695.535.21 6.566.16
December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 467.735 (Infliximab_HC SINSATHYAESVK.3)
7.64e53.13
December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 1092.532 (Infliximab_HC SINSATHYAESVK.3)
1.33e43.13
3.17
December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 1319.659 (Infliximab_HC SINSATHYAESVK.3)
226.88
December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 364.849 (Infliximab_HC SINSATHYAESVK.3)
1.61e43.13
2.58 6.794.113.773.22 5.664.71 5.57 6.695.75 7.01
December2016_InfliximabMD_028 MRM of 32 Channels ES+ 469.569 > 74.06 (Infliximab_HC SINSATHYAESVK.3)
8.73e43.13
If not for Skyline…
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
December2016_InfliximabMD_001 MRM of 32 Channels ES+ 948.526 > 1441.765 (Infliximab_LC DILLTQSPAILSVSPGER.2)
3.17e45.25
December2016_InfliximabMD_001 MRM of 32 Channels ES+ 948.526 > 563.317 (Infliximab_LC DILLTQSPAILSVSPGER.2)
9.31e35.26
5.30 6.326.08 6.95
December2016_InfliximabMD_001 MRM of 32 Channels ES+ 948.526 > 721.386 (Infliximab_LC DILLTQSPAILSVSPGER.2)
9.86e35.24
5.223.74 3.89 6.375.89
December2016_InfliximabMD_001 MRM of 32 Channels ES+ 948.526 > 563.317 (Infliximab_LC DILLTQSPAILSVSPGER.2)
9.31e35.26
5.30 6.326.08 6.95
December2016_InfliximabMD_001 MRM of 32 Channels ES+ 948.526 > 88.063 (Infliximab_LC DILLTQSPAILSVSPGER.2)
2.96e36.535.275.114.84 6.035.83 6.84
December2016_InfliximabMD_001 MRM of 32 Channels ES+ 948.526 > 957.536 (Infliximab_LC DILLTQSPAILSVSPGER.2)
1.87e45.26
5.28
December2016_InfliximabMD_001 MRM of 32 Channels ES+ 948.526 > 1125.626 (Infliximab_LC DILLTQSPAILSVSPGER.2)
1.32e55.25
December2016_InfliximabMD_001 MRM of 32 Channels ES+ 948.526 > 1028.573 (Infliximab_LC DILLTQSPAILSVSPGER.2)
5.28e35.265.10
5.30
December2016_InfliximabMD_001 MRM of 32 Channels ES+ TIC (Infliximab_LC DILLTQSPAILSVSPGER.2)
8.47e55.25
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 472.288 (Infliximab_LC YASESMSGIPSR.2)
6.79e43.40
December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 1121.526 (Infliximab_LC YASESMSGIPSR.2)
4.20e43.41
3.44
December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 88.063 (Infliximab_LC YASESMSGIPSR.2)
4.06e36.274.25
3.39 4.68 4.955.17 5.59
6.70
December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 756.287 (Infliximab_LC YASESMSGIPSR.2)
1.11e43.42
3.92 4.854.25 6.02
December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 813.308 (Infliximab_LC YASESMSGIPSR.2)
1.66e43.40
December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 747.382 (Infliximab_LC YASESMSGIPSR.2)
2.02e53.40
December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 322.14 (Infliximab_LC YASESMSGIPSR.2)
2.96e53.40
December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 1050.488 (Infliximab_LC YASESMSGIPSR.2)
3.78e53.41
December2016_InfliximabMD_004 MRM of 32 Channels ES+ 642.798 > 529.309 (Infliximab_LC YASESMSGIPSR.2)
3.70e53.41
Time1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00
%
0
100
December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 418.74 (Infliximab_LC DSTYSLSSTLTLSK.2)
2.43e44.49
4.54 5.24
December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 174.118 (Infliximab_LC DSTYSLSSTLTLSK.2)
1.05e44.49
4.56 6.37
December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 949.557 (Infliximab_LC DSTYSLSSTLTLSK.2)
1.74e54.49
December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 518.798 (Infliximab_LC DSTYSLSSTLTLSK.2)
3.91e44.49
4.47 4.52
December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 304.114 (Infliximab_LC DSTYSLSSTLTLSK.2)
2.24e54.49
December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 174.118 (Infliximab_LC DSTYSLSSTLTLSK.2)
1.05e44.49
4.56 6.37
December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 475.282 (Infliximab_LC DSTYSLSSTLTLSK.2)
3.09e34.51
4.39 5.224.71 6.77
December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 1300.699 (Infliximab_LC DSTYSLSSTLTLSK.2)
8.49e34.50
4.53
December2016_InfliximabMD_005 MRM of 32 Channels ES+ 751.883 > 74.06 (Infliximab_LC DSTYSLSSTLTLSK.2)
6.16e54.49
You’d have to individually pick through 2000+ chromatograms (only 4 peptides) for the most sensitive fragments.
©2017 Waters Corporation 97COMPANY CONFIDENTIAL
These transitions will be optimized further for collision energy. Repeat for each peptide and each charge state.
2nd Charge State: from 19 > 6 transitions
Importing Data: Filtering data/Exclusion