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Accelerate mAb

Characterization Using

Automated Sample Prep

David Knorr, Ph.D.

Automation Solutions

Ning Tang, Ph.D.

LC/MS

15 February 2012

Method Specific

Protein Sample Processing Workflows

Manufacturing

process

Biological

system

Clinical

patient

Crude Sample Prep

Glycan Profiling

Peptide Mapping

Oxidation Characterization

Sample Titer

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Agilent AssayMAP Bravo®

Liquid Handling Platform

Agilent Bravo® Liquid Handler

•Best-in-class liquid handling

•Accessorize deck for any

application

•VWorks control software

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AssayMAP Cartridges Chromatography columns on a micro scale

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AssayMAP Bravo Head Different pipetting modes

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AssayMAP Bravo vs. Pipette Tips Quantitative recovery in fewer steps / lower volume

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Single pass

Accurate flow control

No air bubbles

Multi pass

Variable flow control

Air bubbles

Current AssayMAP BioPharma Portfolio

General Ab Capture

Protein A

Protein Digestion

Trypsin

Sample Clean Up

Reverse Phase DIY Captures

Streptavidin

• In development

• Contaminant ELISA

• N-glycan sample prep

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Form-Driven Protocols

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Protein A for Ab Purification

Sample Analysis

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Protein A Performance Characteristics

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Quantitative Binding & Elution to 100 μg

No load volume effect over linear range

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 20 40 60 80 100

A28

0

µg hIgG loaded

10 µL

30 µL

90 µL

250 µL

0

40

80

120

160

200

0 50 100 150 200 250

µg

Elu

ted

µg hIgG Loaded

Protein A Performance Characteristics

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0.0

0.2

0.4

0.6

0.8

0 20 40 60 80 100

A2

80

Sample Number

%CV = 1.26

µg hIgG loaded 11 15 19 25 33 44 58 76 100 132 173 228

%CV 1.6 1.3 1.4 1.0 1.4 1.2 1.4 1.4 1.9 1.9 1.5 1.0

Purification is consistent across a rack…

…and across cartridges.

User- Defined Affinity Purification with

Streptavidin (SA-W)

Anything you can biotinylate can be a capture ligand!

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Streptavidin (SA)

Custom affinity purification examples

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Elution of Hu IgG from biotinylated protein A

Elution of FLAG-BAP from biotinylated αFLAG SA

0

20

40

60

80

100

0 20 40 60 80

μg h

IgG

Elu

ted

μg of Biotin-Protein A Immobilized

On-Cartridge Trypsinization

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Fra

ctio

na

te, C

lea

n u

p, A

na

lyze

Sam

ple

On Bravo Deck or Offline

Trypsin Cartridge (TR)

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TR-W comparisons

Minutes

4 6 8 10 12 14 16 18 20 22

mA

U

20

40

60

80

100

120

140

160

180

200

mA

U

20

40

60

80

100

120

140

160

180

200DAD: Signal A, 215 nm/Bw:4 nm

Urea 1.11 pre-digest

urea 1.11 pre-digest 9-29-2011 1-12-00 pm.dat

DAD: Signal A, 215 nm/Bw:4 nm

Urea 1.11 digest

urea 1.11 digest 9-29-2011 5-20-09 pm.dat

Analyte: BSA

Denaturant: 6M Urea (1.2M final)

Flow Rate: 2 μL/min.

Load: 67 μg

RP-HPLC

TR-W comparisons

Minutes

4 6 8 10 12 14 16 18 20 22

mA

U

0

20

40

60

80

100

120

140

160

180

200

mA

U

0

20

40

60

80

100

120

140

160

180

200DAD: Signal A, 215 nm/Bw:4 nm

TFE 18.1 pre-digest

tfe 18.1 pre-digest 9-29-2011 4-18-06 pm.dat

DAD: Signal A, 215 nm/Bw:4 nm

TFE 4.50 digest

tfe 4.50 digest 9-29-2011 6-53-15 pm.dat

Analyte: BSA

Denaturant: 50% TFE (5% final)

Flow Rate: 2 μL/min.

Load: 67 μg

RP-HPLC

Urea

TFE

Elution and coverage comparable to in-solution digestion.

In Progress: antibody digestion with LC/MS characterization.

Reverse Phase for Peptides Digest clean up and fractionation

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Analy

ze

Peptide D

igest

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

5 10 15 20 25 30 35 40 45 50

A2

80

Cumulative Elution Volume (µL)

2µL/min

5µL/min

10µL/min

20µL/min

Reverse Phase (RP)

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Complete elution in 2 column

volumes

Quantitative binding

and elution to ~400 μg

Cleanup with a leading pipette tip

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Reverse Phase Cleanup of BSA

Example Workflow – from Sample to Analysis (in progress)

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Protein A or

Streptavidin

Trypsin

Reverse Phase

Agilent HPLC

or LCMS

Automated Sample preparation with AssayMAP Bravo

On or Off Bravo

GlykoPrep™ N-Glycan Kit

Collaboration with

Digest with

N-Glycanase®

(PNGaseF)

Fluorescent

label

Purify

with Protein A

Crude Ab

sample

Denature &

immobilize

Purified

glycoprotein

Cleanup &

elute

Analysis by

HPLC, CE, LC/MS

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N-Glycan Profile Analysis

Conventional method

• Manual only

• 3 days

• 10 - 30 samples/run

AssayMAP method

• Manual or automated

• No drying steps

• 3 hours

• Up to 192 samples/run

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GlykoPrep System Results- (3 hr manual mode)

12 consecutive runs α-1 Acid Glycoprotein

Comparison to Standard Methods

Peak

Average

% Area

% CV

(n = 96)

1 9.8% 3.2%

2 20.0% 1.2%

3 20.3% 2.5%

4 11.1% 3.4%

5 13.1% 1.6%

6 9.5% 2.3%

0

5

10

15

20

25

0 20 40 60 80 100

% o

f To

tal P

eak

Are

a

Sample

Reproducibility

CV’s = 2 - 5 %

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Antibody Characterization Example

Monoclonal antibody production in CHO cells

• Hu IgG / mAb purification from CHO culture supernatants (CSS)

• Effect of high amounts of FBS

• Different initial antibody concentrations

– 10, 50, 100, 500 µg/mL (triplicate samples)

– Mimic screening to scale-up

MW determination

N-Glycan profiling (mAb-Glyco Chip)

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AssayMAP Cartridge Performance Different loads and sample backgrounds

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mAb Characterization Using LC/MS

February 17, 2012

Agilent Confidential

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Intact molecular

weight Glycan Analysis

Agilent’s LC/MS Systems for Biopharma Applications

•Intact protein analysis

•Peptide mapping

•PTM analysis

•Glycan analysis

HPLC-Chip QTOF/TOF

Applications

Intact Protein Analysis Zorbax SB300-C8 HPLC-Chip

February 17, 2012

Agilent Confidential

28

52+

Intact mAb Analysis

Control mAb

Sample 1

+CCS

Sample 2

-CCS

Sample 3

PBS

HPLC-Chip/MS Solution for Glycan Analysis mAb-Glyco Chip Workflow

February 17, 2012

Agilent Confidential

30

4 minutes

1 minute

TOF MS Detector

mAb sample

Expe

rime

ntal Tim

e

LC- or CE- Fluorescence

10-30 minutes

3-8 hours

2-4 days

MALDI MS

//

//

mAb-Glyco Chip MS

2 minutes

PNGase F, enzymatic N-glycan release

N-Glycan concentration

N-Glycan separation

mAb-Glyco Chip-LC/MS Solution

Total analysis time of 12 min with great chromatographic resolution

mAb-Glyco Chip-LC/MS Solution PCDL – Personal Compound Database and Library

Comparison of mAb-Glyco Chip with CE and MALDI

MS Results

0

10

20

30

40

50

60

70

Man-5 G0-GlcNAc G0-F G0 G1 G2

Re

lati

ve

mo

lar

dis

trib

uti

on

[m

ol%

]

MALDI-MS (N=34)

CE-LIF (N=43)

Agilent Glycan Workflow Chip (N=50)

Agilent Glycan Workflow Chip (N=50)

Agilent Glycan Workflow Chip (N=50)

mAb-Glyco Chip-LC/MS Solution Chip Reproducibility

Glycan Analysis

control

sample 1

sample 2

sample 3

G0F

G1F

G2F

Conclusions

• The Agilent AssayMap Bravo platform enables

automated sample preparation for biologic

characterization.

• Agilent HPLC-Chip LC/MS system provides fast

analysis of therapeutic proteins.

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Thank You !