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Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development
PLEASE STAND BY… the webinar
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Webinar Series Science
Sponsored by:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
Webinar Series Science
14 November, 2012
Joey Studts, Ph.D. Boehringer Ingelheim Pharma GmbH & Co. KG Biberach, Germany
Timothy Blanc ImClone Systems Branchburg, NJ
Bahram Fathollahi, Ph.D. PerkinElmer San Francisco, CA
Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development
© 2009 PerkinElmer © 2009 PerkinElmer © 2009 PerkinElmer © 2009 PerkinElmer
Bahram Fathollahi Microfluidics R&D November 14, 2012 Science/AAAS Microfluidics Webinar
Microfluidic Assays for High Throughput Screening of Protein Quality in Bioprocess Development
What is a Microfluidic Device?
An integrated device with controlled transport of sample and reagents in microchannels to carry out bio/chemical analysis Advantages Miniaturization Fast analysis/high throughput Small sample volume Integration Automation
4
Microfluidic Chip Format and Fabrication Planar Chip Single use Samples added to wells manually
Sipper Chip Reusable Samples introduced automatically
from microtiter plate
Chip Fabrication Chips manufactured using photolithography
and wet etching technology Quartz and glass substrate Highly reproducible and precise control of
channel geometry Typical depth - 5 µm to 25 µm Typical width - 10 µm to 100s µm
Sipper (autosampler)
5
Electrophoretic Separation in Microfluidic Chips
SAMPLE LOAD
AND INJECTION
SEPARATION DETECTION
Sample Reservoir
Waste Reservoir
Sample Load
Sample Separation
LIF detection
Sample Injection
Buffer Reservoirs
B C D
A
B
A
∆V ∆V ∆V ∆V
6
Commercial Microfluidic Electrophoresis Platforms
Bioanalyzer 2100
Experion
Higher Throughput
LabChip |DX/GX/GXII
Planar chip format
Sipper chip format
• RNA & DNA analysis
• Proteins
• N-Glycans (LabChip Only)
• Digital information
• Easy-to-use
• Time and cost savings
7
8
Protein Characterization Assays on LabChip GX II
Microchip
LabChip GX II
Multiple HT Screening Assays on a Single Microfluidic Platform
1) Microchip CE-SDS – 40 sec/sample Protein expression optimization Assess purity
Reduced and non-reduced Titer Quantify impurities
Product- and process-related Monitor fragmentation and Ab assembly
2) N-Glycan Profiling – 60 sec/sample Measure relative amounts of major N-linked glycans
3) Charge Variant Profiling – 68 to 90 sec/sample Identify and measure relative amounts of basic, main,
acidic variants
Microchip CE-SDS Assay Workflow
Crude or purified protein sample (2µL)
Add Sample Buffer Heat denature ~15min
Prepare Gel/Dye-
Solution and Ladder
Add Reagents to chip
Place Ladder and Buffer on GXII
Reusable chip Up to 400 samples
~ 40 sec per sample ~ 75 min for 96-well plate
Protein Analysis
E-gram
Plate View
Sizing, Conc., and
Purity
Identified expected protein
peaks
Gel View
9
10
Attributes Assay Instrument time Microchip CE-SDS
Concentration ProA HPLC RP HPLC
2-5 min ~20 min
< 1 min
Assembly, covalent aggregation nrCE-SDS 30-50 min < 1 min
Fragmentation, Degree of N-glycosylation rCE-SDS 30-50 min < 1 min
10
12
14
16
18
20
22
24
26
28
30
LC HC
NGHC LM
8
Fluorescence
Time (seconds)
0
250
500
750
1000
1250
13 18 23 28 33 38 43 48 53
HHL Aggregates (covalent)
Monomer
HL
In-process sample
Reducing Non-reducing
X. Chen et al .Electrophoresis 2008, 29, 4993-5002
Microchip CE-SDS
Conventional CE-SDS
Microchip CE-SDS vs. Conventional CE-SDS
30 seconds
HT N-Glycan Profiling Sample Prep Workflow
Denaturing plate
PNGase-F Plate
Glycan Labeling Plate
Denature and Reduce mAb in Denaturing Buffer
Add Denatured Reduced mAb to Enzyme Plate
Transfer to Dye Plate
Heat Assay Plate at 55 °C for ~2h
Heat (Denatured mAb + Enzyme) at 37 °C for ~1h
Dilute with separation Buffer
Read 96-well plate on LabChip GXII ~1.5h
G1F’ G1F G0F
G2F M5 G0
LabChip GXII
microchip
11
Man5 G0
G0F
G1F
G2F
G1’F
UNK
G2 UNK
UNK
G1
Digested and Labeled N-Glycan Profile
12
Methods of Characterizing Charge Heterogeneity Three methods of separation used in the biotherapeutics industry: Ion Exchange Chromatography (IEC)
Extension of HPLC systems Proteins interact with charged resin, based on pI and hydrophobicity
Capillary Zone Electrophoresis (CZE) Based on Capillary Electrophoresis (CE) systems Proteins migrate at different speeds, based on pI and hydrodynamic drag
Isoelectric Focusing (icIEF) Based on imaged CE systems Proteins migrate to a particular position within a pH gradient, based on pI
Limitation of current methods: Lack of speed
13
Analysis Time IEC icIEF CZE Microchip-CZE
Per Sample 30 - 90 min 15 - 20 min 8 - 30 min 1 - 2 min
Per 96-Well Plate 48 - 144 hrs 24 - 32 hrs 16 - 48 hrs 1.8 - 2.9 hrs
HT Protein Charge Variant Assay
Variant Detection Conventional methods use absorbance at 280 nm LabChip detects via LIF Dye must be conjugated to protein variants Dye must absorb & emit at wavelengths that are compatible with instrument optics Dye must conserve charge of protein variants
14
Rel
ativ
e A
mou
nt (%
)
Acidic Variants
Main Variant
Basic Variants
Before Labeling After Labeling
From icIEF analysis
Charge profile does not change
Direct Comparison With Alternative Methods
15
Minutes0 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 3.20 3.40
8.58
5
8.71
2M
ain
Isof
orm
- 8.
810
8.94
89.
044
8.00 8.20 8.40 8.60 8.80 9.00 9.20 9.40 9.60
HT PCV
Conventional CZE ~10min
icIEF 15 min
Acidic Basic
Acidic Basic Acidic Basic
Microchip-CZE 60 sec
Small-Scale High Throughput Process Development
Mother Plate
Purity Assessment
N-Glycan profiling
Charge Variant
BioTx workstation
Daughter Plate
Analytics for determination of critical quality attributes
16
Clone selection/cell
culture optimization
Screening high number of cell clones in small-scale bioreactors
µscale chromatographic purification
Sample prep/ µscale purification
Daughter Plate
Daughter Plate
Informatics
Higher demand of product quality requires multi-factorial DOE studies in upstream and downstream process development
Process parameter optimization
Optimization and
Scale-up
Process understanding and decisions
Microfluidics R&D Tobias Wheeler Lucy Sun Rajendra Singh Mai Ho Hui Xu Melissa Takahashi Roger Dettloff
Chip and Reagent Fabrication Joan McAuliffe Ken Summers Trang Le Khushroo Gandhi
Software Advit Bhatt Dan Camporese Renil Das
17
Acknowledgements
Thank you!
Marketing & App Support Rick Bunch Krystyna Hohenauer Nate Cosper Seth Cohen Linda Gary Marsha Paul
Process Science Germany, 2012
Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development
14 November 2012
Project Timelines Focus on Critical Bottlenecks in Development
19
Exploratory Research
Preclinical Development
Development Clinical Dev. Phase I,II
Phase IIb/ III Registration
Market Supply
Overall Project Timeline
• Critical bottleneck is time to the clinic
• Reduce time to clinic to shorten time to market Process
Development/ optimization
GMP Pilot Plant
Ph I - Scale up Cell line
non GMP scale up,
Tox material
• Fast track development by applying “platform technologies”
• Material supply: 1 g - 2 kg
Bottleneck to Clinic
• Often is data on the critical path in development
• Early process understanding critical to efficiency and safety
More data Better Decisions Efficient Development Better Products
BI-PurEx: Lean Development Strategy Understanding the product & the process
25
• Cell line development is on critical path • Rate limiting step is cell growth, very little material with which to
gain knowledge • Choosing the correct cell line for each product is most critical • Impacts product quality, functionality, expression levels,
comparability… • Early knowledge and correct decisions will have an significant
impact on efforts and timeline throughout the development process.
Cell Line Development Process Development USD & DSD
Scale-up USD & DSD
Pilot Scale GMP
Product & Process are critical
Product Understanding Glyco Patterns
26
• Initial challenges in Glycosylation • Mainly controlled by cell line decision • Purification normally has very little impact on glycans • Impacts comparability and functionality of molecule • Critical to get early knowledge and impact cell line development decisions!
20 30 40 50 60 70
8 7
6A 4A
S:/B
PQC/
QC/Q
CAS/
LG7/
FOB/
Calip
er D
aten
abgl
eich/
T1-H
EX-1
10-0
9.op
j/13.
01.2
011
Zug
Retention Time [min]
Ex 330
nm
/ Em
420 n
m
6 5 4
3
2
1
HPLC: 20 min/run MS: >> 20 min/run
Product Understanding High Throughput Glyco Analysis
27
T9 Std. 5 mg/mL
T9-HEX2-101-01
T9-HEX2-101-02
T9-HEX2-101-03
T9-HEX2-101-04
T9-HEX2-101-05
T9-HEX2-101-06
T9-HEX2-101-07
T9-HEX2-101-08
T9-HEX2-101-10
Man5 7,48 4 6,3 6,15 8,39 3,24 3,1 4,28 4,83 3,07
NGA2 3,09 1,94 1,23 0,92 6,65 1,73 1,16 1,19 2,54 2,31
NGA2F 16,95 14,71 17,45 0,37 0,73 12,03 9,15 12,17 14,2 11,52
NA2G1F 3,66 1,79 1,89 0 0,73 1,54 0,73 1,21 1,55 1,61
NA2 0,06 0,1 0 0 0 0 0,07 0,08 0,04 0,15
NA2F 0,24 0,26 0,14 0,37 0,22 0,17 0,1 0,17 0,17 0,26
0
2
4
6
8
10
12
14
16
18
20%
CGU
(Cali
per G
luco
se U
nits
)
Standard Clone 1 Clone 2 Clone 3 Clone 4 Clone 5 Clone 6 Clone 7 Clone 8 Clone 9
• Using Microfluidic Assay – all pools are analyzed for glycan map prior to cell line decision.
Product Understanding High Throughput Glyco Analysis
28
DS01 [5mg/mL] Inj. 1 Inj. 2 Inj. 3 Mean SD VK Std.
Method Man5 4,05 4,27 4,24 4,19 0,12 2,85 4,52
NGA2 2,93 2,89 2,83 2,88 0,05 1,75 2,73
NGA2F 59,61 57,5 59,1 58,79 1,10 1,87 60,48
NA2G1F 14,2 13,8 13,82 13,94 0,23 1,62 20,6
NA2F 1,7 1,83 1,43 1,65 12,34 12,3 1,9
HT - Microfluidic Assay • High Throughput • Very reproducible • Comparable to standard
Methods.
Product Understanding Product Based Impurities
29
Standard 97.1 % Purity Clone 1 94.1 % Purity Clone 2 94.6 % Purity Clone 3 95.1 % Purity Clone 4 94.3 % Purity Clone 5 94.2% Purity
• Product purity influenced by purification process development • Microfluidic Assay helps point out critical impurities to focus later
development efforts and to track in cell line decisions.
Electorpherogram from reduced mAb sample
Product Understanding Product Purity – Charge Heterogeneity
30
7.03
0 7.17
8
7.30
4
7.41
7
7.55
5 7.65
5
7.76
3
7.84
2
Minutes6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90
iCE
16.1
36
16.5
55
17.7
66 18.2
79
19.1
20
19.7
60
20.9
13
Minutes13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00 2
WCX
BPG
BPG BPG
APG
APG APG
MP
MP
MP
Qualitative analysis no longer enough • Impact on safety and efficacy Critical to Comparability • Impacted by cell line • Impacted by fermentation process • Impacted by purification process
High throughput method required to look
at all samples
acidi
c Pe
akgr
oup
- 18.
324
Mai
npea
k - 1
9.21
2
basic
Pea
kgro
up -
19.8
21
Minutes13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 31
Aci
dic
Pea
k 1
- 7.0
36
Aci
dic
Pea
k 2
- 7.1
91
Aci
dic
Pea
k 3
- 7.3
08 Mai
n P
eak
- 7.4
09
Bas
ic P
eak
1 - 7
.543
Bas
ic P
eak
2 - 7
.633
Minutes.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 7.60 7.70 7.80 7.90 8.00
acid
ic P
eakg
roup
- 18
.332
Mai
npea
k - 1
9.23
5
basi
c P
eakg
roup
- 19
.772
Minutes13.00 14.00 15.00 16.00 17.00 18.00 19.00 20.00 21.00 22.00
iCE
WCX
Clone Z Clone X
Clone Z Clone X
Clone Z Clone X
R&D 1 Clone Z
Aci
dic
Pea
k 1
- 7.0
35
Aci
dic
Pea
k 2
- 7.1
80
Aci
dic
Pea
k 3
- 7.3
04
Mai
n P
eak
- 7.4
17
Bas
ic P
eak
1 - 7
.554
Bas
ic P
eak
2 - 7
.635
Bas
ic P
eak
3 - 7
.759
Bas
ic P
eak
4 - 7
.841
Minutes6.85 6.90 6.95 7.00 7.05 7.10 7.15 7.20 7.25 7.30 7.35 7.40 7.45 7.50 7.55 7.60 7.65 7.70 7.75 7.80 7.85 7.90 7.95
R&D 1 Clone Z
R&D 1 Clone Z
iCE
WCX
Sensitivity very high Ability to detect differences between clones
Ability to detect
differences to reference material.
Product Understanding Product Purity – Charge Heterogeneity
Process Understanding RAPPTor®
32
Fully automated purification screening platform From resin and plate preparation to step elution Screen 96 variables and generate of up to 500 sample per day
Fully automated sample preparation and analytical assays Automated homogeneous assays to screen critical output parameters
Tecan 150 – HT Screening Tool Tecan 200 - HT Analytical Tool
Separate but Seamless
Rapid Automated Protein Purification Technology
Process Understanding High-Throughput Parallel Chromatography
33
gaining process knowledge earlier in a shorter timeframe with less material demand
288
72
4
33
9
4
0 100 200 300
Serial Column runs
RoboColumn runs
Filterplate run
time (h)
robustness study (11 runs) initial screening (96 runs)
• Microfluidic Assay allows analysis of impurities profile for each of the samples generated.
Process Understanding Critical Impurities
34
Sample Name % Purity Type
Standard 0,22 Unknown
AT Prod Pool 0,88 Unknown
DF Prod Pool 0,87 Unknown
AEX Prod Pool 0,88 Unknown
CEX Prod Pool 0,76 Unknown
Bulk Prod Pool 0,79 Unknown
• Assay set-up not complex, carried out within development lab • Data delivered within hours, not days • Can take next step to test fractionation of individual process steps • IMPACT on real time decsions!
Process Understanding Determination of aggregates
35
Size exclusion chromatography Molecular weight standard
HPLC TSK G3000 SWXL, 7.8 x 300 mm, 5 µm, 20 µL
ACQUITY UPLC BEH200 SEC, 4.6 x 150 mm, 1.7 μm,4 µL ~ 8 min
~ 20 min
Acquity UPLC H-Class: • Shorten analysis time by
factor of 2.5 • Increase resolution through
smaller particle diameter • Lower injection volumes • Low carry over • Applicable to WCX, RP….
Process Understanding Analytical Throughput: Method portfolio
36
UPLC- SEC 96-well UV detection
Analytical techniques with increased throughput. Understand impact of each step on product quality and contaminant removal.
Lab Chip GXII
Automated ELISA Anti-CHO HCP conjugated
acceptor bead
Streptavidin-coated
Donor bead
Excitation 680 nm Emission 615 nm 1O2
Host cell proteins
CHO HCP AlphaScreenTM (768 data points overnight)
iCE280
Boehringer Ingelheim Biopharma Process Science
37
Thanks for your attention!!!
Thanks to many colleagues at Boehringer Ingelheim especially: to H. Kaufmann, D. Ambrosious to, J. Stolzenberger, S. Combe, O. Haas, S. Müller, D. Dieterle
Boehringer Ingelheim, Bibearch, Germany
Company Confidential Copyright © 2010 ImClone LLC
The Use Of Microfluidics Technology in the Pharmaceutical Industry: MicroChip Electrophoresis as a High Throughput Process Analytical Platform to support the Development of Therapeutic Monoclonal Antibodies
Tim Blanc and Qinwei Zhou ImClone Systems. Branchburg, NJ 08876
39 Company Confidential Copyright © 2010 ImClone LLC
Introduction
Rapid analytical separations is an important application stemming from the development of Microfluidics. One such separation technique is MicroChip Electrophoresis (MCE). MCE can be performed in several modes to provide valuable insights to the relationship between bioprocess parameters and the quality attributes of biopharmaceutical products. Recombinant Monoclonal antibodies commonly exist as heterogeneous mixtures of product variants. Several factors contribute to product variants, one such factor is glycosylation. Using Glycosylation as the example, It is important to understand what the relationship is between process parameters and glycan distribution expressed on the antibody. An effective Quality by Design (QbD) approach uses design of experiments (DOE) to determine the most important process parameters, termed Critical Process Parameters (CPPs). However the number of samples required for the experimental design can be high. Testing this number of sample to gain the most thorough process understanding is time and cost prohibitive by standard methods. Discussed will the regulatory and biopharmaceutical issues that have accelerated the need for rapid analytical separation. Then, will be discussed application on MCE that have been developed to address those needs.
40 Company Confidential Copyright © 2010 ImClone LLC
The New Quality Paradigm • ICH Q8 (R2) Pharmaceutical Development
• ICH Q9 Quality Risk Management
• ICH Q10 Pharmaceutical Quality Systems
• ICH Q11 Development and Manufacture of
Drug Substance ICH Q11: “Risk management, and scientific knowledge are used more
extensively to identify and understand process parameters and unit operations that impact critical quality attributes (CQA’s) and develop appropriate control strategies applicable over the lifecycle of the drug substance
41 Company Confidential Copyright © 2010 ImClone LLC
Quality Risk
Management
Product Knowledge
Quality Systems
Process Knowledge
Critical Process
Parameters
Critical Quality
Attributes
Control Strategy
Quality Risk Management: Linking Process Knowledge, Product Knowledge and Quality Systems
Ref.- Quality Attributes of Recombinant Thereapeutic Proteins: An Assessment of Impact on Safety and Efficacy as part of a Quality by Design Development Approach Eon-Duval, A. et.al BIOTECHNOL. PROG., 2012, Vol.00, No. 00
42 Company Confidential Copyright © 2010 ImClone LLC
Good Business too
IND Phase I Phase II Phase III BLA
Process Development and Scale-up (Changes)
Tox & PK Studies
Comparability Studies- Linking Commerical scale product back to original material use in Tox. & PK Studies
Assure QCA’s are maintained within acceptable ranges (QTPP), Quality Target Product Profile, throughout Process Development. Maintain link to Tox. & PK studies.
43 Company Confidential Copyright © 2010 ImClone LLC
Heterogeneity of Monoclonal Antibodies
Therapeutic Mab Product Contents
Product Variants
Process Related Impurities Trace amounts (ppb-ppm) • Residual Host Cell DNA, • Residual Host Cell Protein, • Residual Protein-A.
ProductRelated Impurities 1-5% • Antibody Fragments, • Aggregates
Product Variants Post-Translational Modifications:
• Glycan Variants, • C-Terminal Truncation, • Deamidation, etc.
Critical Quality Attributes (CQA’s)
44 Company Confidential Copyright © 2010 ImClone LLC
Heterogeneity of Monoclonal Antibodies
Therapeutic Mab Product Contents
Product Variants
Multiple Sources give rise to Product Variants
45 Company Confidential Copyright © 2010 ImClone LLC
Critical Process Paramenters (CPP)
CPPs are independent process parameters most likely to affect the quality attributes of a product or intermediate
CPPs are determined by analytical testing, scientific judgment and on research, scale‐up or manufacturing experience
CPPs are controlled and monitored to confirm that the impurity profile is comparable to or better than historical data from development and manufacturing.
Quality attributes related to CPPs include:
• Product Variants Distribution (Glycosylation is a major contributor) • Product-Related Impurities • Process-Related Impurities
46 Company Confidential Copyright © 2010 ImClone LLC
Glycosylation
47 Company Confidential Copyright © 2010 ImClone LLC
Implications of Glycosylation Variants
• The greatest source of Mab Product Variants is Glycosylation
• Glycosylation is very closely tied to process parameters
• Aspects of glycosylation will frequently be CQA’s
Glycan Structural Characteristic Implication Fucose: Effector Function (e.g. ADCC) Sialic Acids (NANA, NGNA) PK modulation (up or down) NGNA Immunogenicity α-Galactose Immunogenicity
48 Company Confidential Copyright © 2010 ImClone LLC
MCE and Product Variants
Sample prep designed in microtiter plate format with immobilized enzyme to reduce sample prep from 2 days to ½ day (Glycans).
Rapid electrophoretic separation on Microchip with sensitive laser-induced fluorescence detection. (<60 seconds per sample)
Analytical separations in under 60 seconds for determinations of:
• Size Heterogeneity • Charge Heterogeneity • Glycosylation Heterogeneity
Throughput and Accuracy for thorough QbD testing, CPP determination
and Successful Control Strategy efforts
49 Company Confidential Copyright © 2010 ImClone LLC
Data is all numerical for statistical comparison
High Throughput Platform for PAT
MCE N-Linked Glycan Analysis
% G0F % G1F % G2F % α-Gal % Fucosylated % Sialylated (NGNA)
Glycosylation MCE-CZE
Charge-Based Evaluation
Relative Distribution Of Product Variants
% APG % NPG % BPD
Charge MCE-SDS
Size-Based Evaluation
% Purity (NR) % Aggregate % Fragment % Purity (Red.) % Non-Reducible % Aglyc HC
Size
50 Company Confidential Copyright © 2010 ImClone LLC
MCE Platform: Size Heterogeneity
MCE-SDS < 60 SECONDS per Samples
CE-SDS ≈ 60 Minutes Samples
51 Company Confidential Copyright © 2010 ImClone LLC
MCE Platform For QbD: Charge Heterogeneity
MCE < 60 SECONDS per Samples
IEC >60 MINUTES per Sample
52 Company Confidential Copyright © 2010 ImClone LLC
MCE Platform: Glycan Heterogeneity
MCE < 60 SECONDS per Samples
HPLC ≈ 60 Minutes Samples
53 Company Confidential Copyright © 2010 ImClone LLC
Conclusions
• Rapid Analysis time and easily automated.
• Allows more samples tested and better insight to Impact of process parameters.
• CPP’s determined and detailed process understanding.
• Effective Control Strategy.
• Relevant Drug Substance Specification (JOS).
Sponsored by:
Participating Experts:
Brought to you by the Science/AAAS Custom Publishing Office
Webinar Series Science
17 October, 2012
ADVANCING EPIGENETICS: NOVEL DRUG DISCOVERY STRATEGIES FOR EPIGENETIC TARGETS
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Webinar Series Science
14 November, 2012
Joey Studts, Ph.D. Boehringer Ingelheim Pharma GmbH & Co. KG Biberach, Germany
Timothy Blanc ImClone Systems Branchburg, NJ
Bahram Fathollahi, Ph.D. PerkinElmer San Francisco, CA
Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development
Brought to you by the Science/AAAS Custom Publishing Office Brought to you by the Science/AAAS Custom Publishing Office
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Webinar Series Science
14 November, 2012
Microfluidic Electrophoresis Assays for Rapid Characterization of Protein in Research and Development