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Transcript of Apa 2017 diego rodriguez 3
©2017 Waters Corporation 1COMPANY CONFIDENTIAL
LC-MS/MS for Bioanalytical Protein
Quantification: Life of a Biotherapeutic
Infliximab Case Study
©2017 Waters Corporation 2COMPANY CONFIDENTIAL
©2017 Waters Corporation 3COMPANY CONFIDENTIAL
Humira Linear
Range:
25-500 mg/mL
m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400
%
0
100
m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400
%
0
100
m/z2100 2200 2300 2400 2500 2600 2700 2800 2900 3000 3100 3200 3300 3400
%
0
100
m/z2100 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
2428.45852314.6531
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
2510.67362428.4788
2314.6135
2200.6035
2848.5479
2904.3762
2907.5068
2965.64433022.8723
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/z2720 2740 2760
%
0
100
m/z2720 2740 2760
%0
100
m/z2720 2740 2760
%
0
100
m/z2720 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
mass148000 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
mass148000 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
©2017 Waters Corporation 4COMPANY 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 5COMPANY 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
©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
©2017 Waters Corporation 7COMPANY CONFIDENTIAL
Infliximab LC and HC Tryptic Peptide Identification
©2017 Waters Corporation 8COMPANY 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
©2017 Waters Corporation 9COMPANY CONFIDENTIAL
Closely related to LC of Human IgG
Results of BLAST search for Infliximab LC
Protein sequence differences and gaps
©2017 Waters Corporation 10COMPANY 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/
©2017 Waters Corporation 11COMPANY CONFIDENTIAL
ExPASy SIM Alignment Results: Infliximab LC
vs. Human IgG LC
Highlights protein sequence differences while maintaining sequence alignment
©2017 Waters Corporation 12COMPANY 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
©2017 Waters Corporation 13COMPANY 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
©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
Long List of Possibilities
©2017 Waters Corporation 15COMPANY 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
©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
©2017 Waters Corporation 17COMPANY 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
©2017 Waters Corporation 18COMPANY 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
©2017 Waters Corporation 19COMPANY CONFIDENTIAL
Developed by the MacCoss laboratory in the University of Washington
Best Practice:
Our Approach = Skyline Workflow
Start Here!
©2017 Waters Corporation 20COMPANY 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
©2017 Waters Corporation 21COMPANY CONFIDENTIAL
Skyline MRM Method Creation: Infliximab LC
With Skyline, importing a
protein sequence elicits an
in silico digestion algorithm
and MRM transition
prediction
©2017 Waters Corporation 22COMPANY CONFIDENTIAL
Skyline MRM Method Creation: Infliximab LC
©2017 Waters Corporation 23COMPANY 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 24COMPANY CONFIDENTIAL
Sample Analysis with MassLynx: Skyline
imported methods Narrowed down Skyline methods
are imported into MassLynx for
LC/MS acquisition
©2017 Waters Corporation 25COMPANY CONFIDENTIAL
Skyline MRM Method Optimization: CE
Optimization
The optimized method can
be exported automatically
©2017 Waters Corporation 26COMPANY 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>546.3
More sensitive transition!
Manual Optimization633.1>731.8
5.59140139
Alternate MRM transition
©2017 Waters Corporation 27COMPANY 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
©2017 Waters Corporation 28COMPANY 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 29COMPANY 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
©2017 Waters Corporation 30COMPANY 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
©2017 Waters Corporation 31COMPANY 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 PlasmaBlank Plasma Blank Plasma
50 ng/mL 50 ng/mL 50 ng/mL
100 ng/mL100 ng/mL100 ng/mL
500 ng/mL500 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 32COMPANY 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
©2017 Waters Corporation 33COMPANY 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
©2017 Waters Corporation 34COMPANY 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
©2017 Waters Corporation 35COMPANY 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)
©2017 Waters Corporation 36COMPANY 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 37COMPANY 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
©2017 Waters Corporation 38COMPANY CONFIDENTIAL
20142014
2012
Xevo TQ-S
2010
2016
Xevo TQ Family
Data coming soon!
New data available!
©2017 Waters Corporation 39COMPANY 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
Linear, Precise and Accurate Single digit
%CVs
LLOQ of 10ng/mL35 µL sample, Protein A, ProteinWorks digestion and MCX peptide clean-up
©2017 Waters Corporation 40COMPANY 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
©2017 Waters Corporation 41COMPANY CONFIDENTIAL
ionKey Microfluidics
ESI Assembly Incoming FlowEEProm Memory Electrical Connections
Analytical ColumnColumn Heater Sensor
©2017 Waters Corporation 42COMPANY CONFIDENTIAL
Infliximab High Analytical Sensitivity Using
Integrated Microflow
Time5.50 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
©2017 Waters Corporation 43COMPANY CONFIDENTIAL
m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300
%
0
100
m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300
%
0
100
m/z2400 2500 2600 2700 2800 2900 3000 3100 3200 3300
%
0
100
m/z2400 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
2792.83572689.2893
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
2924.1621250 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
©2017 Waters Corporation 44COMPANY 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
©2017 Waters Corporation 45COMPANY 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
©2017 Waters Corporation 46COMPANY 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