LabChip GXII for High Throughput Analysis of Protein ... - PerkinElmer

LabChip GXII for High Throughput Analysis of Protein
Quality
Bahram Fathollahi
Microfluidics R&D
May 20, 2010
COGS Meeting 2010
1

LabChip Platforms
on-chip capillary electrophoresis
Bioanalyzer 2100
LabChip GXII
Protein analysis
N-Glycan analysis
DNA sizing & quantification
RNA quality assessment
Higher Throughput
Experion
Improved Data Quality
Labor Savings vs. Gels
Digital information
Easy-to-use
2
2

LabChip GXII Applications
MicroChip-CE SDS
• Crude lysate analysis
• Protein expression
• Antibody analysis
• Protein purification
• Fusion protein analysis
• Formulation stability
• Detection of covalent
aggregates (up to 500 kDa)
• Pre-labeled proteins for high
sensitivity
MicroChip-CE
• N-Glycan profiling
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HT Protein Assay: Microchip-CE-SDS
HT Protein Assay
• Integrates the entire SDS-PAGE process onto a
microfluidic chip
• Determine size, quantification, and purity
• Automatic sampling from 96- or 384-well plate
• Fast sample analysis - 41 seconds per sample
• Can run crude samples
• 96-well plate analyzed in < 1.25 h
• Can do >400 samples per day per instrument
Microchip CE
• 2 μl of sample
• Minimal sample preparation
• 21 CFR Part 11 support
4

Protein Assay Workflow
Crude or purified
protein sample
Add denaturing
sample buffer
Heat
Denature
2μl
96- or 384-well plate
Add reagents to chip
well
Product Quality -
Titer, % purity,
degree of
glycosylation, ….
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Automated Sample Analysis Window
Identified
expected protein
peaks
Plate View
Gel View
Electropherogram
Sizing, Conc.,
and
Purity results
Well table view,
Peak table view
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(1) (3) (2)
Resolution Ladder Mix
(1) Fermentas PAGE Ruler Unstained Protein Ladder
(2) Fermentas Unstained Molecular Weight Marker
(3) Mix
Lowe er Marker
10kDa
14.4 and 15kDa 45 and 50kDa
MIX
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Staccato Protein Workstation: Workflow Automation
Features
• Integrated sample
processing & analysis
• Walk away automation
• Easy-to-use software
• Enables complex DOE
experiment
Scalable design: Twister plate loading, liquid handling
sample prep, p and fully integrated system
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Protein Assay Specifications
Type
Sizing Range
Specification
Protein 100 Assay: 14 - 100 kDa
Protein 200 Assay: 14 - 200 kDa ( can detect higher Mw proteins
by increasing the separation and sample-load time)
Sizing Accuracy* ± 20%
Resolution*
Linear Dynamic Range*
Relative Concentration CV*
Sensitivity*
Analysis Time per Sample
± 10% difference in MW across the sizing range, 50% valley
Resolution is comparable to a 10 cm 4-20% SDS-PAGE gel
5 - 2000 μg/mL
30% up to 120 kDa relative to the ladder
5 μg/mL (10 ng) Carbonic Anhydrase
Sensitivity is equivalent to Coomassie stain
Protein 100 Assay: ~34 seconds/sample
Protein 200 Assay: ~41 seconds/sample
Chip Lifetime
Analysis Time Per Plate
Maximum Salt Concentration
4 96-well plates
96-well plate

Conventional CE-SDS vs LabChip: Reduced mAb
CE-SDS
Microchip CE-SDS
Chen X. et al. Microchip assays for screening monoclonal antibody protein quality. Electrophoresis, 29 (2008) 4993-5002
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CE-SDS vs. LabChip: Non-Reducing mAb
cence
Fluores
550
40 sec
Microchip-CE-SDS
6
500
Migration Corrected Migration Corrected
450
Time, Relative Time, Relative
400
350
Peak Number seconds Area minutes Area Delta
300
1 18.6 0.4% 15.5 0.7% 0.3%
250
200
150
100
50
0
-50
5
1 2 3 4
6
7
16 18 20 22 24 26 28 30 32
Time (seconds)
8
5
9
0
1
LC-90
CE-SDS
2 20.5 0.1% 18.7 0.2% 0.1%
3 23.9 0.3% 22.2 0.3% 0.0%
4 26.4 0.5% 25.2 0.5% 0.0%
5 27.8 2.0% 26.7 2.4% 0.4%
6 29.0 96.7% 27.8 95.9% 9% 08% 0.8%
Total Impurities 3.3% 4.1% 0.8%
35 minute separation
Baseline
variability
Data is quite consistent, despite
differences in:
• Instrument
• Sample preparation buffer
• Detection method
• Some impurities are known to
be sensitive to sample
preparation conditions
Siemiatkoski et al. Use of LabChip 90 in Product Development of a
Recombinant Fusion Protein. Protein Summit 2008, London UK
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Crude Lysate Analysis
Microchip-CE SDS vs SDS-PAGE
Expressed protein 47.7 kDa
Expressed protein 48kDa
* Lysate sample and SDS-PAGE data courtesy of Structural GenomiX - San Diego, CA
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Analysis of Subunit Vaccine Expression in
Pseudomonas
High throughput
expression
monitoring with
analysis on the
LabChip GXII led
to a 10-fold
increase in yield
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Protein Expression Optimization using DOE
Optimal expression condition
is easily observed
Swalley, S.E. et al., Quantitative Analysis of Protein Expression, Anal. Biochem, 351 (2006) 122-127
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LabChip GX Dependent Process Development for mAb Purification
Analysis of
mAb yield
and purity
with varying
purification
parameters
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Purification Process Development
Affinity resin capacity determination
Protein are introduced continuously into the column and unbound fractions are
monitored o by UV and Labchip90.
20.0 the
15.0
10.0
Proteins are detected in
the unbound the
dynamic binding
capacity of the column
has been reached
5.0
Slide courtesy of X.Le Saoût – COGS 2008 meeting
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High Sensitivity: Derivatization of Proteins
with FL dye
• Replaces on-chip staining with off-chip labeling in order to increase sensitivity by
10X or more
• NHS esters reaction with amine groups of lysine residues
O O
N O
Dye
NH 3
O NH
Dye
MAb
Dye Succinimidyl Ester
O
Intact Antibody
Reduction
2 + 2
O
N
O
O
O
Dye
NH
NH
O
Dye
Dye
MAb
Heavy Light
Chain Chain
Dye Succinimidyl Ester
O
HC and LC
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Linearity of intact mAb at constant dye
concentration
ti
• linearity of 4 orders of magnitude for high sensitivity Pico protein assay
100000
10000
HT Protein Express 200
HT Pico Protein Express
R 2 = 0.999
R 2 = 0.9997
Time Corrected
Area
1000
100
10
1
0.01 0.1 1 10 100 1000 10000
MAb Concentration (μg/mL)
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Low Level Impurity Detection: Non-Covalent
On-Chip Staining
• Impurities of as low as 1% are clearly identified
• Lysozyme was spiked into the sample at 1% of total protein and was
readily identified, with a S:N of 9:1
HC
NGHC
LC
Ly
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Low Level Impurity Detection: Covalent
Off-Chip Staining
i
• Carbonic anhydrase was spiked into mAb sample at 0.01%, 0.05% and 0.5% of the
total protein at 1 mg/ml.
• ~ 100X improvement in sensitivity!
0.5%
0.05%
0.01%
Background mAb trace
Spiked CA
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Antibody N-linked Glycans
Complex
Mannose
Hybrid
GlcNAc
Fucose
Galactose
High Mannose
• Glycosylation patterns of therapeutic proteins can influence
- Efficacy and safety of therapeutic antibodies
- Protein-protein interactions
- Solubility
- Intracellular trafficking
• Important to characterize and monitor the glycosylation pattern during cell
culture development
Antibody image courtesy of Dr. Greg Flynn
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Neutral N-Glycan Types
High
Mannose
M5 M6* M7* M8 M9
Hybrid
M5G1F M5G0F M5G0 M5G1
Complex
G2F G1F G0F G2 G1 G0
G0F‐GlcNac
slide courtesy of Dr. Greg Flynn
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IgG Charged N-Glycan Types
Sialylated
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Traditional N-Glycan Mapping (3-5 Days)
Sample
Digestion (PNGase-F)
Removal of Protein
Carbohydrate labeling (2-AB, APTS)
Removal of labeling
Reagent
CE-LIF
Chromatography/MS
• NP-HPLC
• RP-HPLC
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Goals for HT N-Glycan Screening Assay in
Early Development
• Identification and determination of relative amounts of
major N-Glycans (Man 5, G0, G0F, G1F isomers, and
G2F)
– Sialylated glycans?
– Low level glycans? gy
• Ease-of-use
• High throughput and fast turnaround
– Analysis of 96-well plate sample in < 8 hours
– Complete from digestion to results
• Robust and Reproducible
• Easy to transfer and establish in QC environment
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HT Glycan Profiling on LabChip System
Denature and Reduce mAb in Denaturing Buffer
LabChip GXII
Add Denatured Reduced mAb to Enzyme Plate
Denaturing plate
Heat (Denatured mAb + Enzyme) at 37 °C for ~1h
microchip
Transfer to Dye Plate
PNGase‐F Plate
Heat Assay Plate at 55 °C for ~2h
G0F
G1F
G1F’
G2F
Dilute with separation Buffer
Read 96-well plate on LabChip GXII ~1.5h
Glycan Labeling Plate
G0
M5
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HT Glycan Assay Performance Specifications
8ul with concentration range of 1.25mg/ml to
Amount of sample required:
7.5mg/ml (10ug to 60ug of MAb total).
Reproducibility of %area:
CV < 10% for a peak >= 2.5% of total glycan.
1ng of G0F standard. (Smallest amount of labeled
Limit of detection:
G0f standard that can be detected.)
>95% of all N-Linked glycans will be released from
Deglycosylation:
mAb.
Appropriate for neutral glycans found on MAbs.
Usable size range: Some charged glycans may run outside of our
usable range.
Sizing ing reproducibility: CV < 2.5%
< 0.5% of previous sample can be detected in
Carryover:
following sample.
Sample prep, chip prep, and < 8 hours for a 96-well plate.
analysis time:
Number of samples per chip Up to 192 samples before chip needs to be
prep: reloaded with fresh gel.
Number of samples per chip Up to 384 samples before chip needs to be
lifetime: replaced.
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PNGase-F Digestion
Observed complete deglycosylation
• Digested IgG (red trace) shows shift towards NGHC
when compared to undigested IgG (blue trace)
HC
NGHC
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Use of Internal Standards for Peak Identifications
• Individual neutral glycan standards show good resolution of the main glycans
• The two G1F isomers, differing only in the branch position of terminal galactose, are resolved
Mixture of Glycan Standards
G1
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Sialylated Glycan Standards
•Mono- and di-sialylated glycan standards, with and without internal fucose (A1, A1F, A2, and
A2F) were purchased from ProZyme (Hayward CA)
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Use of Glucose Ladder For Peak Identification
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Identification of Glycan Peaks
Expected Peaks Function in Analysis Settings
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Intermediate-Assay Reproducibility
•Labeled N-glycans released from three different mAbs at starting concentrations of 1.25 mg/mL, 2.5mg/mL, and 7.5
mg/mL were analyzed.
•The %RSD was calculated for 35 reactions (complete from deglycosylation to labeling and separation) per mAb, 2
repeat injections, 4 different chips and instruments, and 2 users.
•Total %RSD’s for the five main glycan peaks were all below 10% for peaks with relative amount greater than 2.5%.
IgG_A AVG % Area CV_10ug CV_20ug CV_60ug Total CV
G0 6.82 3.6% 3.4% 3.8% 6.3%
G0F 53.39 3.4% 1.6% 2.5% 3.2%
G1F 9.05 4.1% 1.9% 2.8% 4.1%
G1F' 9.28 4.6% 1.9% 2.1% 4.9%
G2F 2.05 12.2% 6.3% 2.2% 13.4%
Man5 4.61 4.9% 3.6% 6.3% 8.7%
IgG_B G0 10.55 5.8% 4.9% 4.2% 5.4%
G0F 47.78 2.4% 1.6% 1.2% 2.1%
G1F 5.70 5.1% 5.2% 4.3% 6.5%
G1F' 17.92 2.9% 2.0% 1.4% 3.2%
G2F 258 2.58 82% 8.2% 75% 7.5% 33% 3.3% 98% 9.8%
Man5 1.79 7.2% 8.8% 6.8% 12.1%
IgG_C G0 5.01 4.5% 3.2% 3.4% 6.8%
G0F 57.33 3.1% 2.1% 3.3% 3.1%
G1F 457 4.57 49% 4.9% 36% 3.6% 41% 4.1% 63% 6.3%
G1F' 13.92 3.5% 2.3% 1.8% 3.1%
G2F 1.25 14.4% 13.4% 6.7% 15.5%
Man5 5.58 4.9% 3.5% 3.3% 6.8%
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NPLC (2-AB labeled)
G0F Man5
Comparison to NPLC and CE-LIF
180 min
G1FA G1FB G2F
CE-LIF (APTS labeled)
Int Std
Man5
G0F
6min
G1FA G1FB
G2F
Microchip-CE (FL labeled)
G0F
45 sec
Int Std
Man5
G0
G1FB
G1FA
G2F
NPLC and CE profiles courtesy of Dr. Nate Lacher
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Relative Amounts Comparison
Glycan NP‐HPLC Microchip‐CE
% CPA %CPA
G0 06 0.6
Man5 2.00 3.4
G0‐GlcNAc 3.00 4.8
G0F 44.60 43.6
G1F 14.80 16.3
G1F' 19.40 20.3
G2F 6.90 6.9
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HT N-Glycan Assay: Summary of Key Features
• Integrates the CE-LIF process onto a microfluidic chip
• Fast separation time – 45 sec per sample; 96-well plate in ~90 min
• Reagents provided in 96-well plate format for convenience and ease of automation
• Rapid digestion ~ 1hr
• Labeling in presence of released antibody without requiring NaBH 3 CN
• Able to identify major N-glycan peaks, including mono- and di-sialylated glycans
• Software determines the relative amounts of major N-glycans
• Up to 200 samples per chip with single chip preparation
• Chip lifetime of 400 samples
• Capable of interfacing with plate robotics for complete automation of sample prep and
analysis
• 21CFR Part 11 software support
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Acknowledgements
Collaborators
– Greg Flynn - Amgen
– Joe Semiatkoski – Biogen Idec
– Xiaoyu Chen - Novartis
– Nate Lacher – Pfizer
Caliper Life Sciences
• Assay Development
– Mai Ho
– Raj Singh
• Microfluidics R&D
– Roger Dettloff
– Hui Xu
– Melissa Takahashi
• Chip Manufacturing
• SW Engineering
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Select Publications
38

LabChip System
Sampling of LabChip Users Quantitative Screening Assays
on One Platform
labchip@caliperls.com
• Antibody analysis
– Titer
– Purity for R and NR
• Crude lysate analysis
• Protein expression
• Fusion protein analysis
• Formulation stability
• Detection of covalent aggregates
• Pre-labeled proteins for high
sensitivity
• Purification process development
– Purity and Yield
• N-Glycan Profiling
39