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©2018 W
aters Corporation
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Autom
ated Protein D
igestion: Does it A
dd Value?
D
r Ian Edw
ards P
harmaceutical B
usiness Developm
ent Manager
23rd N
ov, 2018
©2018 W
aters Corporation
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Goals of P
resentation
Large Molecule B
ioanalysis: Why A
utomation?
Hybrid LC
-MS
/MS
using the Surrogate P
eptide Approach: The W
orkflow
Autom
ation Method D
evelopment: P
rotein Digestion &
Affinity P
urification
©2018 W
aters Corporation
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Large Molecule B
ioanalysis �
Ligand binding assay (LBA
) is the preferred method for large m
olecule bioanalysis (e.g. ELIS
A)
–E
asy to use, high sensitivity, and high throughput –
Can have specificity issues w
ith protein isoforms &
biotransformations
�LC
-MS
/MS
using the surrogate peptide approach is an alternative or complem
entary approach –
Direct, highly selective, m
ass measurem
ent –
Good sensitivity and dynam
ic range
�H
ybrid LC-M
S/M
S has em
erged combining best of both w
orlds –
High selectivity and sensitivity
–C
omplex m
ulti-step workflow
�
More and m
ore groups moving tow
ards automation
–R
educe complexity of the w
orkflow
–Focus on high value tasks
©2018 W
aters Corporation
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�Large m
olecule bioanalysts rely on high quality, reproducible data to support pre-clinical and clinical studies for drug discovery
Bioanalysts know
what they w
ant and need
I want to increase
the pro
du
ctivity of m
y lab!
I want to
minim
ize hum
an error!
I need reliable
sample preparation
I need rep
rod
ucib
ility betw
een assays
I don’t want to
need an autom
ation specialist.
Walk-aw
ay tim
e!
Ensured com
parab
ility betw
een manual and
automated preparations
Easy d
eplo
yability
©2018 W
aters Corporation
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Autom
ation - What’s Stopping You?
What is stopping
you from
achieving your goals?
Red
uction
of
Error
Co
nsisten
cy
Scien
tists mo
re ab
le to fo
cus
on
alternative
tasks
Cost
Increase
Prod
uctivity
Integ
ration o
f W
orkflow
s
Techn
ical D
ifficulties
©2018 W
aters Corporation
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Protein B
ioanalysis: Surrogate Peptide Approach
WO
RKFLO
W
Identify unique peptides and
transitions
Protein clean-up (optional)
Peptide clean-up (optional)
Data
processing
Protein digestion
LC-M
S
Results
Optim
ize / fine-tune
MS conditions
©2018 W
aters Corporation
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Surrogate P
eptide Approach: The options are overw
helming!
WO
RKFLO
W
Protein digestion
Peptide clean-up (optional)
Protein clean-up (optional)
Non-S
pecific Capture
Protein P
recipitation P
rotein A
Protein G
S
pecific Capture
Anti D
rug A
ntigen Capture
Anti H
uman
Device
Magnetic B
eads 96-W
ell Plate
Tips
Denaturation
Organic S
olvent H
igh Heat
Digestion E
nzyme
Trypsin LysC
A
spN
Reduction/A
lkylation Iodoacetam
ide TC
EP
D
ithiothreitol
Solid P
hase Extraction
Reverse P
hase Ion E
xchange W
eak/Strong C
ation W
eak/Strong A
nion P
hospholipid Rem
oval M
acro Elution
Evaporation µE
lution D
ilution
©2018 W
aters Corporation
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�E
xperiment 1: A
ssessing manual preparation against the initial m
ethod �
Experim
ent 2: Assessing the pipetting steps
�E
xperiment 3: A
ssessing the sample handling (m
anually adding samples, autom
ating each other step) �
Experim
ent 4: Heating assessm
ent �
Experim
ent 5: Increased temperature control
�E
xperiment 6, 7, 8, 9: S
ample sealing assessm
ent �
Experim
ent 10: Specific heating assessm
ent �
Experim
ent 11, 12: Room
temperature incubation m
anipulation �
Experim
ent 13, 14: Assessing variability of piercing sam
ples �
Experim
ent 15, 16: Format change
�E
xperiment 17, 18, 19 : N
ew accessory assessm
ent (plate format change)
�E
xperiment 20, 21, 22: D
eck reconfiguration �
Experim
ent 23, 24, 25: Method C
onfirmation
Optim
izing a Com
plex, Autom
ated Method:
Experiments ad nauseum
©2018 W
aters Corporation
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Protein B
ioanalysis: Surrogate Peptide Approach
WO
RKFLO
W
Identify unique peptides and
transitions
Protein clean-up (optional)
Peptide clean-up (optional)
Data
processing
Protein digestion
LC-M
S
Results
Optim
ize / fine-tune
MS conditions
©2018 W
aters Corporation
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0.00
20.0
0
40.0
0
60.0
0
80.0
0
100
.00
120
.00
AFV
ESD G
YS SIN
S LEES
ASQ
D
ILL YA
SE SLSL
DTL
ALP
A G
PSV
VV
SV D
STY
CR
P In
fliximab
Surro
gate Pe
ptid
es (10 µ
g/mL in
plasm
a)
Au
tom
ated D
igestion
Co
mp
ared to
Man
ual D
igestion
Au
tom
ated raw
area co
un
ts of e
ach p
ep
tide are
no
rmalize
d to
the
raw area
cou
nts o
f their re
spe
ctive man
ually p
rep
ared
samp
les
Man
ual %
RSD
Au
tom
ated A
rea N
orm
alized
Au
tom
ated %
RSD
�M
anually and automated protein digestion perform
ed using the Ham
ilton STA
R
–C
-Reactive P
rotein and infliximab
–35 µL of plasm
a –
Tryptic digestion with reduction and alkylation
Fully Autom
ated Digestion P
roof of Concept
Automated vs. M
anual
Com
parable manual vs. autom
ated digestion
Single digit %
RS
Ds
%
©2018 W
aters Corporation
11 C
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Protein B
ioanalysis: Surrogate Peptide Approach
WO
RKFLO
W
Identify unique peptides and
transitions
Protein clean-up (optional)
Peptide clean-up (optional)
Data
processing
Protein digestion
LC-M
S
Results
Optim
ize / fine-tune
MS conditions
©2018 W
aters Corporation
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Peptide C
urve Range
(µg/mL)
r 2 W
eighting M
ean A
ccuracy (%
)
SQV
FFK
0.10-250 0.9989
1/x 99.62
YA
SESISGIPSR
0.50-250
0.9957 1/x
98.90 D
ILLTQSPV
ILSVSPG
ER
0.10-250 0.9979
1/x 100.04
DTLM
ISR
0.10-250 0.9982
1/x 100.56
ALPA
PIEK
0.10-250 0.9951
1/x2
99.56 G
PSVFPLA
PSSK
0.10-250 0.9942
1/x2
99.91 TTPPV
LDSD
GSFFLY
SK
0.50-250 0.9953
1/x 99.90
Peptide Q
C C
onc. (µg/m
L)
Calc.
Conc.
(µg/mL)
%C
Vs
Mean
Accuracy (%
) R
eplicates
SQV
FFK
0.25 0.23
7.06 92.35
2 of 3 4.0
4.14 3.27
103.47 3 of 3
40.0 40.13
4.37 100.30
3 of 3 200.0
192.96 6.07
96.47 3 of 3
YA
SESISGIPSR
4.0
3.75 0.82
93.80 3 of 3
40.0 40.78
1.34 101.97
3 of 3 200.0
184.99 4.90
92.50 3 of 3
DILLTQ
SPVILSV
SPGER
0.25 0.24
13.13 89.80
3 of 3 4.0
4.03 10.51
100.65 2 of 3
40.0 40.12
2.40 100.30
3 of 3 200.0
186.89 4.15
93.43 3 of 3
DTLM
ISR
0.25 0.22
0.64 88.40
2 of 3 4.0
4.15 2.96
103.67 3 of 3
40.0 42.34
2.79 105.83
3 of 3 200.0
189.57 1.78
94.77 3 of 3
ALPA
PIEK
0.25 0.24
0.64 88.40
3 of 3 4.0
4.31 2.16
103.67 3 of 3
40.0 39.43
0.00 105.83
3 of 3 200.0
180.35 1.78
94.77 3 of 3
GPSV
FPLAPSSK
0.25 0.25
5.90 100.67
3 of 3 4.0
4.32 4.74
107.93 3 of 3
40.0 38.57
2.18 96.43
3 of 3 200.0
187.51 0.97
93.77 3 of 3
TTPPVLD
SDG
SFFLYSK
4.0
4.14 3.27
103.47 3 of 3
40.0 40.13
4.37 100.30
3 of 3 200.0
192.96 6.07
96.47 3 of 3
�C
etuximab spiked into rat plasm
a at various concentrations (0.1-250 µg/m
L) –
Standard curve (N
=2) and QC
samples (N
=3)
�D
igested from 40 µL of plasm
a on the H
amilton M
icrolab STAR w
ithout any need for m
anual intervention.
Fully Autom
ated Digestion and S
PE P
rotein Quantification:
Cetuxim
ab with SILuM
Ab Internal Standard A
ccurate & R
obust Quantification
Fully automated, standardized protein digestions for the quantification of
surrogate peptides
©2018 W
aters Corporation
13 C
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Protein B
ioanalysis: Surrogate Peptide Approach
WO
RKFLO
W
Identify unique peptides and
transitions
Protein clean-up (optional)
Peptide clean-up (optional)
Data
processing
Protein digestion
LC-M
S
Results
Optim
ize / fine-tune
MS conditions
©2018 W
aters Corporation
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�M
anual vs. Autom
ated (Ham
ilton STA
R ) A
ffinity Capture B
enchmarking
–A
nti Hum
an IgG capture using m
agnetic beads –
Etanercept, inflixim
ab, and trastuzumab purified from
50 µL rat plasma
–D
igested manually using P
roteinWorks A
uto-eXpress Low
5 Digest K
it and provided protocol
Fully Autom
ated Affinity C
apture Proof of C
oncept: Autom
ated vs. Manual
0.00
20.0
0
40.0
0
60.0
0
80.0
0
100.00
120
.00
Surro
gate Pe
ptid
es from
Inte
rnal Stan
dard
Solu
tion
(10 µ
g/mL in
TBS)
Au
tom
ated
Pre
paratio
n C
om
pare
d to
Man
ual P
reparatio
n A
uto
mate
d raw
area co
un
ts of e
ach p
ep
tide
are n
orm
alized
to th
e raw
area co
un
ts of th
eir
resp
ective
man
ually p
rep
ared sam
ple
s
Man
ual %
RSD
Au
tom
ated A
rea N
orm
alized
Au
tom
ated %
RSD
Acceptance criteria for
reproducibility +/-15%
Acceptable area intensity com
parison +/-25%
On average, less than +/-6%
difference in area w
hen com
pared to manually
prepared samples!
Single digit %
RS
Ds %
©2018 W
aters Corporation
15 C
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Protein B
ioanalysis: Surrogate Peptide Approach
WO
RKFLO
W
Identify unique peptides and
transitions
Protein clean-up (optional)
Peptide clean-up (optional)
Data
processing
Protein digestion
LC-M
S
Results
Optim
ize / fine-tune
MS conditions
©2018 W
aters Corporation
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3000.0 ng/mL
Time
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
01Nov2018_ENBhercAntiHuman_043
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
7.79e6Area
5.90;449329
01Nov2018_ENBhercAntiHuman_034
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
7.79e6Area
5.9051684
01Nov2018_ENBhercAntiHuman_025
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
7.79e6Area
01Nov2018_ENBhercAntiHuman_016
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
7.79e6Area
01Nov2018_ENBhercAntiHuman_050
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
7.79e6Area
300.0 ng/mL
Time
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
01Nov2018_ENBhercAntiHuman_043
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.23e6Area
01Nov2018_ENBhercAntiHuman_034
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.23e6Area
5.9046911
01Nov2018_ENBhercAntiHuman_025
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.23e6Area
5.904685
01Nov2018_ENBhercAntiHuman_016
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.23e6Area
01Nov2018_ENBhercAntiHuman_050
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.23e6Area
30.0 ng/mL
Time
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
01Nov2018_ENBhercAntiHuman_043
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
2.71e5Area
01Nov2018_ENBhercAntiHuman_034
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
2.71e5Area
01Nov2018_ENBhercAntiHuman_025
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
2.71e5Area
5.904685
01Nov2018_ENBhercAntiHuman_016
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
2.71e5Area
5.89553
01Nov2018_ENBhercAntiHuman_050
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
2.71e5Area
3.0 ng/mL
Time
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
4.404.60
4.805.00
5.205.40
5.605.80
6.006.20
6.406.60
6.807.00
7.207.40
7.60
%0
100
01Nov2018_ENBhercAntiHuman_043
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.05e5Area
01Nov2018_ENBhercAntiHuman_034
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.05e5Area
01Nov2018_ENBhercAntiHuman_025
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.05e5Area
5.90;4685
01Nov2018_ENBhercAntiHuman_016
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.05e5Area
5.89553
01Nov2018_ENBhercAntiHuman_050
8: MRM
of 4 Channels ES+
TIC (Enbrel IC
[+57.0]TC[+57.0]R
PGW
YC[+57.0]ALSK)
1.05e5Area
ICTC
PGW
YC
ALSK
Blank
3 ng/mL
30 ng/mL
300 ng/mL
3,000 ng/mL
Fully Autom
ated Hybrid LC
-MS
/MS
Protein Q
uantification: Etanercept w
ith Trastuzumab Internal Standard
Peptide Linear
Dynam
ic Range
(ng/mL)
Weighting
Linear Fit (r 2)
Mean
Accuracy (%
) IC
TCPG
WYC
ALSK
1.0-10,000
1/x 0.9976
99.7 C
SSDQ
VETQ
AC
TR
5.0-10,000 0.9991
101.6 B
olded cysteine residue denotes the addition for 57 amu due to C
AM
modification from
alkylation.
Peptide Q
C C
onc. (ng/m
L)
Mean C
alculated C
onc. (ng/m
L)
Mean
Accuracy (%
) %
RSD
ICTC
PGW
YC
ALSK
3.00 3.03
102.03 10.07
30.00 28.90
96.33 4.20
300.00 300.43
100.13 2.13
3000.00 2944.30
98.13 3.16
CSSD
QV
ETQAC
TR
30.00 31.93
106.47 6.64
300.00 321.77
107.23 1.75
3000.00 3043.53
101.43 1.28
Bolded cysteine residue denotes the addition for 57 am
u due to CA
M m
odification from alkylation.
�A
ffinity and digestion performed using H
amilton STAR
–
Anti H
uman IgG
capture from 50 µL rat plasm
a using magnetic beads
–D
igestion using ProteinWorks A
uto-eXpress Low
Digest K
its –
Fully automated w
orkflow
©2018 W
aters Corporation
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PA
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Fully Autom
ated Hybrid LC
-MS
/MS
Protein Q
uantification: Etanercept w
ith Trastuzumab Internal Standard
50.00
100.00
150.00
05
1015
2025
3035
40
% Accuracy
Injection
Nu
mb
er
Intern
al Stan
dard
Perform
ance
FTIS %
RSD
=8.25%
IYP %RSD
=7.54%
Robustness of autom
ated sample preparation and LC
-MS
analysis workflow
% R
SD
s < 10% across the analytical run
©2018 W
aters Corporation
18 C
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PA
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�Large m
olecule bioanalysts rely on high quality, reproducible data to support pre-clinical and clinical studies for drug discovery
Autom
ated Protein D
igestion: D
oes it add value?
I want to increase
the pro
du
ctivity of m
y lab!
I don’t want to
need an autom
ation specialist.
Walk-aw
ay tim
e!
Easy d
eplo
yability
I need rep
rod
ucib
ility betw
een assays
Ensured com
parab
ility betw
een manual and
automated preparations
I need reliable
sample preparation
I want to
minim
ize hum
an error!
©2018 W
aters Corporation
19 C
OM
PA
NY C
ON
FIDE
NTIA
L
Deploym
ent of Autom
ated Protein D
igestion: D
oes it add value?
©2018 W
aters Corporation
20 C
OM
PA
NY C
ON
FIDE
NTIA
L
Acknow
ledgements
Paula O
rens, Applications S
cientist
Steven C
alciano, Senior P
roduct Marketing M
anager
Line Rørbæk O
lsen, Senior R
esearch Scientist, Lundbeck