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Page 1
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
Page 2
Page 3
Agilent AssayMAP Bravo®
Liquid Handling Platform
Agilent Bravo® Liquid Handler
•Best-in-class liquid handling
•Accessorize deck for any
application
•VWorks control software
Page 4
AssayMAP Cartridges Chromatography columns on a micro scale
Page 5
AssayMAP Bravo Head Different pipetting modes
Page 6
AssayMAP Bravo vs. Pipette Tips Quantitative recovery in fewer steps / lower volume
Page 7
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
8
Form-Driven Protocols
Page 9
Protein A for Ab Purification
Sample Analysis
10
Protein A Performance Characteristics
Page 11
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
Page 12
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!
13
Streptavidin (SA)
Custom affinity purification examples
Page 14
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
Page 15
Fra
ctio
na
te, C
lea
n u
p, A
na
lyze
Sam
ple
On Bravo Deck or Offline
Trypsin Cartridge (TR)
Page 16
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
Page 17
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)
Page 18
Complete elution in 2 column
volumes
Quantitative binding
and elution to ~400 μg
Cleanup with a leading pipette tip
Page 19
Reverse Phase Cleanup of BSA
Example Workflow – from Sample to Analysis (in progress)
Page 20
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
21
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
22
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 %
23
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)
Page 24
AssayMAP Cartridge Performance Different loads and sample backgrounds
Page 25
mAb Characterization Using LC/MS
February 17, 2012
Agilent Confidential
26
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.
Page 36
Thank You !
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