Agilent LC/MS Solutions for

Characterizing Therapeutic Proteins

Ning Tang, Ph. D

Agilent Technologies

Intact protein

Peptide mapping

Glycan analysis

Synthetic peptide

Oligo-nucleotide

BioConfirm

BioConfirm

Intact protein

analysis

Peptide mapping

Glycan analysis

Synthetic peptide

Oligo-nucleotide

Acquire data Integrate and extract MS Deconvolute

Match protein sequences and predict PTMs

Report generation

Intact Protein Workflow

A New Deconvolution Algorithm -- pModResolves Overlapped Peaks

IgG 1

IgG2

IgG1 and IgG2mix

These two peaks are 39 Da apart

Not resolved by Maximum Entropy

Resolved by pModIgG1 and IgG2mix

pMod

Acquire data Extract compounds

Match protein sequences and identify PTMs

Sequence coverage

Report generation

Peptide Mapping Workflow

Bravo Liquid Handler• Best-in-class precision• Parallel processing

Introduction – AssayMAP Technology Components

Miniature Chromatography Cartridges• Single pass quantitative

binding & elution

• Reversed-phase• Strong-cation exchange

Positive Displacement Pipetting• Precise, controlled flow rate• No air bubbles

Simple User Interface• Push button control• Easy method transfer

Features

Automated in-solution digestion and cleanup of BSA

Cleanup cartridges: capacity and recovery dynamic range

***Quantitative elution in 10 µL for many applications***

Peptide Mapping Fast gradient with AdvanceBio Peptide Map Column

15

20

30

60

Deamidation

6x10

0

0.25

0.50.75

1

1.25

1.5

1.75

22.25

2.5

2.75

3

3.25

3.53.75

4

4.25

4.5

4.755

5.25

5.5

5.75

A(4

8-63

)

A(4

8-63

)

A(4

8-63

)

Counts vs. Acquisition Time (min)5.8 6 6.2 6.4 6.6 6.8 7 7.2 7.4 7.6 7.8 8 8.2 8.4 8.6

2x10

0

0.2

0.4

0.6

0.8

1* 586.6531

587.3201

587.6537

588.3230

1x10

0

0.5

1

1.5

2

* 586.9787

587.6476

587.9820586.6519 588.6514585.7555585.2533

1x10

0

0.5

1

1.5

586.9778

587.6471

587.9813588.6503

Counts (%) vs. Mass-to-Charge (m/z)585 585.5 586 586.5 587 587.5 588 588.5 589

Deamidation

3x10

0

0.5

1

1.5

2

2.5

3 227.1755b

2

709.8431y

132+

766.3856y

142+

628.3125y

12,b

12-NH32+

586.6502340.2608b

3 511.2581y

143+

822.9272y

152+

277.1549542.7799387.1989 473.5691

3x10

0

0.25

0.5

0.75

1

1.25

1.5 227.1741 766.8799710.3377628.8048

340.2605

249.1592 587.3139512.2570

447.2619 823.4211390.2353

3x10

0

0.5

1

1.5

2

710.3353227.1761 766.8816628.8038

586.9786511.5849340.2612

542.7781249.1630

824.4254475.3254390.2355

Counts vs. Mass-to-Charge (m/z)200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540 560 580 600 620 640 660 680 700 720 740 760 780 800 820 840 860 880

599.8006y11

2+

600.2981y11

2+

600.2898y11

2+

511.5849y14

3+

511.5826y14

3+

586.9768542.7818

y102+

542.7799y10

2+

542.7781y10

2+

340.2605b3

340.2612b3

227.1761b2

227.1741b2

628.8038y12

2+

628.8048y12

2+

710.3377y13

2+

710.3353y13

2+

766.8799y14

2+

766.8816y14

2+

823.4211y15

2+

823.4254y15

2+

N G Y I L

D G Y I L

D G Y I L

Acquire data Extract compounds

Database search and identify

glycans

Calculate percentage and

report generation

mAb Glycan Workflow

6x10

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

1.1

1.2

1.3

1.4

1.5

1.6+ESI TIC Scan Frag=160.0V Antibody_Standard_Reference.d

1 1

Counts vs. Acquisition Time (min)1 2 3 4 5 6 7 8 9 10 11

5x10

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

5.5

6

6.5

7

7.5

8

2100 0A 0G N: +ESI ECC Scan Frag=160.0V Antibody_Standard_Reference.d

Counts vs. Acquisition Time (min)8.9 9 9.1 9.2 9.3 9.4 9.5 9.6 9.7 9.8 9.9 10

mAb-Glyco Chip Kit – A Comprehensive Workflow Solution

mAb-Glyco chipReagent Pack

Structure Viewer

Total workflow solution

Glycan Database

Reporting Templates

Glycan Profiling to Support In-Process Development

Agilent mAb-Glyco Chip Analysis

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

G2F G1F G1

G0F G0

Man5

G0F-G

lcNAc

G0-GlcN

AcG1F

-GlcN

AcG1-G

lcNAc A1F

A1-G

al

Man9

Man8

Man7

Man6

Man3

Man4

Man2

Man3F

162

163

164

165

166

Intact PSA Analysis

0102030405060708090

100

Hex

NA

c1H

ex4H

exN

ac2

Hex

5Hex

NA

c2H

ex6H

exN

Ac2

Hex

4Hex

NAc

3Neu

Ac1

Hex

3Hex

NA

c5(S

O3)

2H

ex5H

exN

Ac4

Hex

5Hex

NA

c2dH

ex1N

euA

c1H

ex4H

exN

Ac3

dHex

1Neu

Ac1

Hex

5Hex

NAc

3Neu

Ac1

Hex

6Hex

NA

c3dH

ex1

Hex

3Hex

NA

c4dH

ex1N

euA

c1H

ex4H

exN

Ac4N

euA

c1H

ex5H

exN

Ac4

dHex

1H

ex6H

exN

Ac4

Hex

3Hex

NAc

5Neu

Ac1

Hex

5Hex

NA

c5H

ex6H

exN

Ac3N

euA

c1H

ex4H

exN

Ac4

dHex

1Neu

Ac1

Hex

5Hex

NAc

4Neu

Ac1

Hex

6Hex

NA

c5dH

ex1

Hex

5Hex

NA

c5dH

ex1N

euA

c1H

ex4H

exN

Ac5N

euA

c1H

ex4H

exN

Ac3

dHex

1Neu

Ac2

Hex

3Hex

NAc

6Neu

Ac1

Hex

6Hex

NA

c3dH

ex1N

euA

c1H

ex4H

exN

Ac5N

euA

c1SO

3H

ex5H

exN

Ac4

dHex

1Neu

Ac1

Hex

6Hex

NAc

4Neu

Ac1

Hex

4Hex

NA

c5dH

ex1N

euA

c1H

ex3H

exN

Ac6

dHex

1Neu

Ac1

Hex

4Hex

NA

c5dH

ex1N

euA

c1S

O3

Hex

5Hex

NAc

6Neu

Ac2

Hex

3Hex

NA

c6dH

ex1N

euA

c1S

O3

Hex

4Hex

NAc

5Neu

Ac2

Hex

4Hex

NA

c5dH

ex2N

euA

c1H

ex5H

exN

Ac5

dHex

1Neu

Ac1

Hex

5Hex

NAc

4Neu

Ac2

SO3

Hex

6Hex

NA

c3dH

ex1N

euA

c2H

ex5H

exN

Ac4

dHex

1Neu

Ac2

Hex

6Hex

NAc

4Neu

Ac2

Hex

4Hex

NA

c5dH

ex1N

euA

c2H

ex5H

exN

Ac5N

euA

c2H

ex3H

exN

Ac6

dHex

1Neu

Ac2

Hex

6Hex

NA

c3dH

ex1N

euA

c2H

ex6H

exN

Ac5

dHex

1Neu

Ac2

Hex

6Hex

NA

c5dH

ex1N

euA

c3

It is possible to determine PSA glycosylation due to its single glycosylation site and moderateprotein MW. The glycan profiles of two PSA samples are compared in this experiment. StandardPSA (top Left) is more highly sialyated than the High Isoform PSA (top Right). The bar chart(bottom) shows the glycosylation differences between the two samples (green = standard PSA).

High pIIsoform PSA PSA

PSA

M. Ly, ASMS 2013 WP430

Glycopeptides Analysis

01020304050

Hex5He

xNac2

Hex4He

xNAc3d

Hex1

Hex4He

xNAc3N

euAc1

Hex3He

xNAc5(SO

3)2

Hex6He

xNAc3

Hex5He

xNAc2d

Hex1Neu

Ac1

Hex4He

xNAc4N

euAc1

Hex5He

xNAc4d

Hex1

Hex3He

xNAc5N

euAc1

Hex5He

xNAc2d

Hex2Neu

Ac1

Hex5He

xNAc5

Hex6He

xNAc3N

euAc1

Hex4He

xNAc4d

Hex1Neu

Ac1

Hex5He

xNAc4N

euAc1

Hex4He

xNAc5N

euAc1

Hex6He

xNAc5

Hex5He

xNAc4N

euAc1(SO

3)He

x6He

xNAc3d

Hex1Neu

Ac1

Hex4He

xNAc5(SO

3)Neu

Ac1

Hex5He

xNAc4d

HexN

euAc1

Hex6He

xNAc4N

euAc1

Hex4He

xNAc5d

Hex1Neu

Ac1

Hex5He

xNAc5N

euAc1

Hex3He

xNAc6d

Hex1Neu

Ac1

Hex4He

xNAc5d

Hex1Neu

Ac1(SO

3)He

x5He

xNAc4N

euAc2

Hex5He

xNAc4d

Hex2Neu

Ac1

Hex4He

xNAc5d

Hex2Neu

Ac1

Hex6He

xNAc3d

Hex1Neu

Ac2

Hex5He

xNAc4d

Hex1Neu

Ac2

Hex4He

xNAc5d

Hex1Neu

Ac2

Hex5He

xNAc5N

euAc2

For PSA, glycopeptide analysis showed heterogeneity of the peptide inaddition to complex glycosylation. Glycoforms from different peptideswere summed.

M. Ly, ASMS 2013 WP430

Released Glycan AnalysisHPLC Chip (HILIC)

0

10

20

30

40

50

60

70

80

90

100

2-AB reductively aminated glycans separated on HILIC HPLC-Chip/MS weredetected on a QTOF and their relative quantitation is plotted above. Isoformswere summed and normalized relative abundance (percents} are plotted versusglycan type, organized by increasing MW.

Intact Protein

Peptide Mapping

Analysis Comparison

AGILENT MASSHUNTEREASY-ACCESS SOFTWARE

Walk-up solution for the biochemist with no LC/MS experience

Walkup LC/MS for BiologistsThree steps to get the results

1. Identify User2. Enter Protein Information3. Place Samples Where Directed

Receive confirmation report by e-mail

Behind the Scene - Administration

All system setup, user group set up, method setup, queue management and error clearing is done from here

Automatic calibration of TOF on Wakeup or through Events Scheduler

E-mail Notification of System Problems