19Nov IanEdwards Automated Protein Digestion Does it add ... · 2018 n 10 IAL 00 00 00 00 00 00 00...

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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

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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

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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

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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

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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

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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

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aters Corporation

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Surrogate P

eptide Approach: The options are overw

helming!

WO

RKFLO

W

Protein digestion



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

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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

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Protein B


WO

RKFLO

W


transitions



Data

processing

Protein digestion

LC-M

S

Results

Optim

ize / fine-tune

MS conditions

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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

%

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Protein B


WO

RKFLO

W


transitions



Data

processing

Protein digestion

LC-M

S

Results

Optim

ize / fine-tune

MS conditions

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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

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Protein B


WO

RKFLO

W


transitions



Data

processing

Protein digestion

LC-M

S

Results

Optim

ize / fine-tune

MS conditions

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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 %

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aters Corporation

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Protein B


WO

RKFLO

W


transitions



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


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

7.79e6Area

5.9051684


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

7.79e6Area


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

7.79e6Area


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


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

1.23e6Area


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

1.23e6Area

5.9046911


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

1.23e6Area

5.904685


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

1.23e6Area


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


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

2.71e5Area


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

2.71e5Area


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

2.71e5Area

5.904685


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

2.71e5Area

5.89553


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


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

1.05e5Area


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

1.05e5Area


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


8: MRM

of 4 Channels ES+

TIC (Enbrel IC

[+57.0]TC[+57.0]R

PGW

YC[+57.0]ALSK)

1.05e5Area

5.89553


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

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aters Corporation

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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

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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

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

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PA

NY C

ON

FIDE

NTIA

L

Deploym

ent of Autom

ated Protein D

igestion: D

oes it add value?

©2018 W

aters Corporation

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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

19Nov IanEdwards Automated Protein Digestion Does it add ... · 2018 n 10 IAL 00 00 00 00 00 00 00...

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