Infliximab case study: LC-MS/MS for Bioanalytical Protein Quantification

1. ©2017 Waters Corporation 1COMPANY CONFIDENTIAL
LC-MS/MS for Bioanalytical Protein
Quantification: Life of a Biotherapeutic
Infliximab Case Study

2. ©2017 Waters Corporation 2COMPANY CONFIDENTIAL

3. ©2017 Waters Corporation 3COMPANY 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 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)
Purified
Peptides
Purified
Protein
Data
Processing
TargetLynx

5. ©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

6. ©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

7. ©2017 Waters Corporation 7COMPANY CONFIDENTIAL
Infliximab LC and HC Tryptic Peptide Identification

8. ©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

9. ©2017 Waters Corporation 9COMPANY CONFIDENTIAL
 Closely related to LC of Human IgG
Results of BLAST search for Infliximab LC
Protein sequence differences and gaps

10. ©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/

11. ©2017 Waters Corporation 11COMPANY CONFIDENTIAL
ExPASy SIM Alignment Results: Infliximab LC
vs. Human IgG LC
Highlights protein sequence
differences while maintaining
sequence alignment

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 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 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

14. ©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

15. ©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

16. ©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

17. ©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

18. ©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

19. ©2017 Waters Corporation 19COMPANY CONFIDENTIAL
Developed by the MacCoss laboratory in the University of Washington
Best Practice:
Our Approach = Skyline Workflow
Start Here!

20. ©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

21. ©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

22. ©2017 Waters Corporation 22COMPANY CONFIDENTIAL
Skyline MRM Method Creation: Infliximab LC

23. ©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

24. ©2017 Waters Corporation 24COMPANY CONFIDENTIAL
Sample Analysis with MassLynx: Skyline
imported methods
 Narrowed down Skyline methods
are imported into MassLynx for
LC/MS acquisition

25. ©2017 Waters Corporation 25COMPANY CONFIDENTIAL
Skyline MRM Method Optimization: CE
Optimization
 The optimized method can
be exported automatically

26. ©2017 Waters Corporation 26COMPANY 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 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

28. ©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

29. ©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

30. ©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

31. ©2017 Waters Corporation 31COMPANY 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 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

33. ©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

34. ©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

35. ©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)

36. ©2017 Waters Corporation 36COMPANY 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 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

38. ©2017 Waters Corporation 38COMPANY CONFIDENTIAL
2014
2014
2012
Xevo TQ-S
2010
2016
Xevo TQ Family
Data coming soon!
New data available!

39. ©2017 Waters Corporation 39COMPANY 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 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

41. ©2017 Waters Corporation 41COMPANY CONFIDENTIAL
ionKey Microfluidics
ESI Assembly Incoming FlowEEProm Memory Electrical Connections
Analytical ColumnColumn Heater Sensor

42. ©2017 Waters Corporation 42COMPANY 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 Corporation 43COMPANY 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 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

45. ©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

46. ©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