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Improving characterization of monoclonal antibodiesMaking a better biotherapeuticNovember 19, 2014
Brought to you by the Science/AAAS Custom Publishing Office
Participating Experts
Niomi Peckham, M.Sc.Alexion PharmaceuticalsCheshire, Connecticut
Sponsored by:
Improving characterization of monoclonal antibodiesMaking a better biotherapeuticNovember 19, 2014
Webinar Series
I-Jane Chen, Ph.D.PerkinElmerHopkinton, MA
N‐LinkedOligosaccharideProfilingandExo‐glycosidaseSequencingUsingaLab‐on‐a‐ChipFormat
N‐LinkedOligosaccharideProfilingandExo‐glycosidaseSequencingUsingaLab‐on‐a‐ChipFormatNiomi Peckham, Scientist, Analytical Sciences
Alexion Pharmaceuticals, Cheshire CT
Niomi Peckham, Scientist, Analytical Sciences
Alexion Pharmaceuticals, Cheshire CT
4
GlycosylationofAntibodies
• (a) Introduction to Protein Structure: Second Edition, Branden & Tooze
N-LinkedOligosaccharide linked to
asparagine at Asn-X-Ser, Asn-X-Thr, or Asn-X-Cys
O-LinkedOligosaccharide linked to serine
or threonine
5
N‐LinkedOligosaccharides
Asn
Mannose
Fucose
N-acetylglucosamine
Galactose
Sialic Acid
Sources of heterogeneity Bisecting GlcNAc Galactosylation Sialylation/Fucosylation High Mannose
Bioactivity Pharmacokinetics Immunogenicity
Commercial importance
6
N‐LinkedOligosaccharideStructures
G2F
G1F
G0F
MAN‐5
Asn
Asn
Mannose
Fucose
N-acetylglucosamine
Galactose
Asn
Asn
Asn
G0F‐GN
(Biantennary Hybrid)
7
Caliper(PerkinElmer)LabChip® GXII
SystemComponents–
• LabChipGXIIInstrument
• LabChipGXIISoftware
• LabChipReagent/Chips:
• DNA(1K,5K,and12K)• RNA(100– 6000nucleotides)• HTProtein(14to350+kDa)• HTPicoProtein(10pg/μL– 100ng/μL)• N‐LinkedGlycanProfiling• ChargeVariant(New!pI7.0to9.5)
8
CaliperProfilerPro™GlycanProfilingKit• Analogous to HPLC or CE Glycan Profiling
• Complement to MALDI‐ToF‐MS for mass profiling
• Developed for neutral N‐linked glycans (e.g. Man5, G0F, G1F, G2F)
• Separation by molecular weight
• High Throughput – 96 well plate format, reagents pre‐aliquoted
• Measures relative amount of N‐Linked neutral glycans
• estimated LoQ of 1%
• Sample Requirements
• Minimum one step purified (e.g. Protein A), pH ~7.0
• Normalized to 5 mg/mL, as low as 2 mg/mL
9
N‐LinkedOligosaccharideProfiling:Method• Denaturation of protein with SDS‐βME solution at 70°C• PNGase digestion removes N‐Linked glycans
• specifically cleaves the N‐acetyl glucosamine – asparagine linkage
• Label glycans with fluorophore• Needed to visualize glycans• Reductive amination
Dye
PNGase
Asn
• Separate electrophoreticaly by size on chip• Detect by laser induced fluorescence
10
WorkflowoftheGlycanAssay
45 samples (duplicates) in < 6 hours
Reconstitute ladder and marker
Clean chip and add gel and marker
Place chip, ladder, wash buffer and samples in GXII
96 wells –separation in ~1.5 hour
Sample Preparation – < 4 hours
Chip Preparation ‐ < 15 minutes
11
SeparationofGlycanStandards
12
mAb#1Profile
13
ProfilesofOthermAbs
mAb #3
mAb #2
14
mAb#1SampleData
Average Man‐5 *G0F‐GN *G0 G0‐F *G'1‐F *G1‐F G2‐f
Reference 1.69 2.98 4.68 77.13 6.24 5.38 1.93Sample 2.49 3.37 3.97 76.70 6.53 5.26 1.70
*Peaks have been tentatively assigned.
15
mAb#1SampleData– ProcessChange
Relative Percent Man‐5 G0F‐GN G0 G0‐F(G1'F + G1F)
G2‐F
ReferencemAb #1 Reference 2.12 2.86 4.63 76.85 11.96 1.60
SampleClone xyz, New supplement 1.20 1.82 1.68 44.10 39.76 9.98
16
ProteinAssay;MonitoringDeglycosylation
• May be required in USP Chapter <212> Oligosaccharide Analysis
• Evaluated with Caliper Protein Assay
• May be performed during Labeling step (2 hours)
17
PeakIdentification
• Limitation of Lab‐on‐a‐Chip; peak identification
• Caliper data on human IgG glycans, standards
• Experimental Approach
– Compare to standards (G0F, G1F, G2F, Man‐5, 6, 7, 8, 9)
– Utilize MALDI‐ToF‐MS data (N‐Linked Oligosaccharide Mass Profiling) as a guide
– Exo‐glycosidase Sequencing as an orthogonal technique
18
N‐LinkedOligosaccharideSequencing• Traditionally done on single peak or excised band• Exo‐glycosidase Sequencing
– PNGase Digest mAb, ethanol precipitate– Digest with Exoglycosidases (Prozyme)– Label with ProfilerPro – Analyze with GXII
• For molecules with few species present (mAb) all species may be sequenced simultaneously
• Data interpretation can be challenging
Enzyme R1 R2 R3 R4 R5 R6Sialidase A - x x x x -β(1-4) Galactosidase - - x x x -β-N-Acetylhexosaminidase - - - x x -α-Mannosidase (Jack Bean) - - - - x xα-Mannosidase (X. manibotis)
- - - - x x
19
Exo‐glycosidase DigestionsExo‐glycosidase Sequencing of an N‐Linked Biantennary Oligosaccharide
Neuraminidase (Sialidase)
β‐Galactosidase
β‐N‐Acetylhexosaminadase
α‐Mannosidase (α1‐2,3,6)
α‐Mannosidase (α1‐6)
20
N‐LinkedOligosaccharideSequencing;G2F
• Sequencing of G2F standard as a control
• Use enzyme specificity and digest mobility to generate linkage
• Galactose = 1 GU• Mannose = 0.8 GU• Fucose = 0.5 GU• GlcNac = 1.1 GU
• α‐mannosidase digest of Man‐5 as a control
21
N‐LinkedOligosaccharideSequencing;Step1
Sialidase Digest
• No change in profile
• No significant sialyation of mAb#1
Digest Control
22
N‐LinkedOligosaccharideSequencing;Step2
Sialidase DigestG1'F + G1F
G2F
G0F
G1?
Sialidase + β‐Galactosidase
Man‐7?G0F‐GN?
G0?
G1 may not resolve from G1F and G0F
Man‐7 elutes with G1F
All forms with terminal Gal shift to G0F
Confirms G2F, G1F
23
N‐LinkedOligosaccharideSequencing;Step3
Sialidase + Β‐Galactosidase
Sialidase + β‐Galactosidase + β‐N‐Acetylhexosaminidase
G0F
G0F‐GN? G0?
G0? And G0F‐GN? shift with Hexosaminidase
Preliminary assignment based on abundance and size
G0F digested by Hexosaminidase only
Confirms G0F
24
N‐LinkedOligosaccharideSequencing;Step4
Sialidase + β‐Galactosidase + β‐N‐Acetylhexosaminidase
Sialidase + β‐Galactosidase + β‐N‐Acetylhexosaminidase + α‐Mannosidase
Man‐5
Man‐5 at 10.8 GU digested by
Mannosidase only
All digested by mannosidase
Some incomplete digests possible
25
SummaryandFutureDirection
• Method appears to be reproducible and offers a high‐
throughput option for process development samples
• Has the potential to be used for release and stability
• Many biopharmaceutical companies adopting
• USP Chapter <1084> Glycoprotein and Glycan Analysis –
Introduction and Choice of Analysis Methods; cites CE
separations
Participating Experts
Sponsored by:
Brought to you by the Science/AAAS Custom Publishing Office
Improving characterization of monoclonal antibodiesMaking a better biotherapeuticNovember 19, 2014
Webinar Series
Niomi Peckham, M.Sc.Alexion PharmaceuticalsCheshire, Connecticut
I-Jane Chen, Ph.D.PerkinElmerHopkinton, MA
2727 © 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer© 2009 PerkinElmer
I-Jane Chen, Ph. D.Sr. R&D Engineer, Microfluidics November 19, 2014
LabChipCapillary Electrophoresis: The Next Generation of High Throughput Protein Characterization
2828
What is lab-on-a-chipMicro fabricationSurface properties and ElectrokineticsSample injection and resolutionAutomated system and high throughput performance
Content
2929
What is lab-on-a-chipMicro fabricationSurface properties and ElectrokineticsSample injection and resolutionAutomated system and high throughput performance
3030
Lab-on-a-Chip Technology- a.k.a. micro total analysis system
Miniaturization
Integration
Automation
Detection
Analysis
For research use only. Not intended for diagnostic procedures.
chemical rxn/labeling
mixingSample separation
sample purification
heating
detection
sample loading
3131
Capillary electrophoresis in lab-on-a-chip
Detection Window
Separation Channel
Sampleloading
Automatic sampling from microplates
On-chip non-covalent FL staining and destaining
Integrates the entire SDS-PAGE process onto a microfluidic chip
Fast sample analysis - 40 seconds per sample
For research use only. Not intended for diagnostic procedures.
3232
What is lab-on-a-chipMicro fabricationSurface properties and ElectrokineticsSample injection and resolutionAutomated system and high throughput performance
3333
Photolithography to make glass chips
Light
Glass or Quartz
Mask
2. Develop
3. Etch
5. Bond1. Expose
Etched Channel Plate
Glass or Quartz Well Plate
For research use only. Not intended for diagnostic procedures.
Photo resist
4. Strip
6. Sipper
3434
Microchannels- after etching & bonding
For research use only. Not intended for diagnostic procedures.
Bonded Channels
Substrate with channels etched in
“Grooves” on glass substrate
Substrate forms the ceiling2 substratestotally fused Into one
3535
What is lab-on-a-chipMicro fabrication
Surface properties and ElectrokineticsSample injection and resolutionAutomated system and high throughput performance
3636
Surface property matters
Si
Si
Si
Si
Si
O
O
O
O
Si
Si
Si
Si
Si
OH
O
OH
OH
OHOH
OH
OH
Hydrolysis
Heat
Siloxane Silanol
pKa of silanol = 5.3
Si
Si
Si
Si
Si
O-
O
O-
O-
O-O-
O-
O-
At pH 7.5
For research use only. Not intended for diagnostic procedures.
3737
Electrokinetic Fluid Actuation
velectrokinetic = velectroosmotic + velectrophoretic
+ -
- - - - - - - - - -+ ++
+ + + + + + +
- - - - - - - - - -+ + + + ++ + + + +
FLOW
+-
+
++
For research use only. Not intended for diagnostic procedures.
3838
What is lab-on-a-chipMicro fabricationSurface properties and ElectrokineticsSample injection and resolutionAutomated system and high throughput performance
3939
Sample loading using 6-way valves
Position A: Sample loop is filled Position B: Contents of the loop are injected onto the column
At least 25 nL sample needs to be loaded
http://www.vici.com/support/app/app11j.php
4040
“Gated” Injection
column
waste
sample
buffer
Sample loading using cross junctions-Electrokinetic or hydrodynamic
For research use only. Not intended for diagnostic procedures.
sample as small as 1 nL is loaded
4141
“Pinched” injection
Sample loading using cross junctions
column
wastesample
buffer
Plug size is determined by1. Channel width2. Currents applied
For a 10 µm wide channel, the plug size is << than CE!
For research use only. Not intended for diagnostic procedures.
4242
Electrophoretic separations
Separate molecules based on their mobility under applied electric fieldMobility, µ, is velocity per unit applied electric field
E
sign
al
x
For research use only. Not intended for diagnostic procedures.
4343
E
sign
al
x
For research use only. Not intended for diagnostic procedures.
Electrophoretic separations
Separate molecules based on their mobility under applied electric fieldMobility, µ, is velocity per unit applied electric field
4444
DL
w1
E
w2
x
sign
al
Ds
For research use only. Not intended for diagnostic procedures.
Electrophoretic separations
Separate molecules based on their mobility under applied electric fieldMobility, µ, is velocity per unit applied electric field
4545
Electrophoretic separations: performance
Resolution: the ability to differentiate
between multiple species often quoted as the smallest
mobility (or size) difference that can be measured
Sensitivity the ability to detect the
presence of a species often described as the
concentration or mass that yields a peak of some minimum signal-to-noise ratio
DL
w1
E
w2
x
sign
al
Ds
For research use only. Not intended for diagnostic procedures.
4646
221 wwtR
tt FWHMw 7.14
2/)44( 2,1, tt
tR
Dt
w1 w2
sign
al
121212
11E
LvL
vLttt
Resolution
Gaussian distribution:
Time separation:
For research use only. Not intended for diagnostic procedures.
4747
222det
2otherdiffusionectorinjtot
Adding dispersion (band-broadening) contributions:
22
2
12
Ewinj
inj
32 2
EDL
diffusion
22
2
12
Ewdet
det
Injector dispersion
Diffusion dispersion
Detector dispersion
For research use only. Not intended for diagnostic procedures.
Resolution: general expression
4848
2,1,
12
2
11
tottot
EL
R
32
22
2
22,
22
22,
31
12
1
21,
21
21,
12
21212
21212
2
11
ELD
Ew
Ew
ELD
Ew
Ew
EL
Rdetinjdetinj
221 wwtR
Sample plug size impact on resolution:
For research use only. Not intended for diagnostic procedures.
shrink , reduce required L (mm vs. cm)
e.g. loading 2 nL sample into a 50 µm diameter capillary, the plug is 100 µm long!
injection plug size
5050
Automated sample loading and separation
5151
What is lab-on-a-chipMicro fabricationSurface properties and ElectrokineticsSample injection and resolution
Automated system and high throughput performance
5252
Work flow of labchip
For research use only. Not intended for diagnostic procedures.
5353 For research use only. Not intended for diagnostic procedures.
Work flow of labchip
5454
staining
For research use only. Not intended for diagnostic procedures.
Work flow of labchip
5555 For research use only. Not intended for diagnostic procedures.
Work flow of labchip
5656 For research use only. Not intended for diagnostic procedures.
Work flow of labchip
5757 For research use only. Not intended for diagnostic procedures.
Work flow of labchip
5858
destaining
For research use only. Not intended for diagnostic procedures.
Work flow of labchip
5959 For research use only. Not intended for diagnostic procedures.
Work flow of labchip
6060
Raw Profile of Reduced mAb
LCLowerMarker
System Peaks (SDS)
HC
NGHC
For research use only. Not intended for diagnostic procedures.
6161
Raw Profile of Non-Reduced mAb
Intact mAb
Fragments
For research use only. Not intended for diagnostic procedures.
6262
Chen, X. et al, Electrophoresis. 29, 2008, 4993-5002
LabChip Produces Comparable Data Faster
“Microchip CE-SDS… provides sufficient resolution and sensitivity for this purpose but on a time scale approximately 70 times faster (41 s vs 50 min per sample) than conventional CE separation”
LabChip
PA800
Comparison of microchip and conventional CE-SDS.Antibody samples were denatured, reduced andanalyzed with LabChip or ProteomeLab PA800.
Antibody Percent Glycosylation: LabChip vs. CE-SDS
Analytical & Formulation,Thousand Oaks, Amgen Inc. CA, USA
For research use only. Not intended for diagnostic procedures.
6363
Low Level Impurity Detection
Impurities of as low as 0.5% are quantified reproducibly Lysozyme was spiked into the sample at 1% of total protein and was readily
identified, with a S:N of 9:1 In the Amgen publication in Electrophoresis the authors reported the LOD and
LOQ of the method to be 1 and 3.3. mg/ml
For research use only. Not intended for diagnostic procedures.
6464
Chen, X. et al, Electrophoresis. 29, 2008, 4993-5002
Protein Fragmentation Analysis
• LabChip and conventional CE-SDS show comparable profiles
• LabChip profile was generated in 40 seconds vs. ~15 minutes by conventional CE-SDS
Antibody Fragmentation: LabChip vs. CE-SDS
Analytical & Formulation,Thousand Oaks, Amgen Inc. CA, USA
CE-SDS LabChip
For research use only. Not intended for diagnostic procedures.
6565
How simple is it?
Add 2 uL sample to 7 uL sample buffer.Heat to 70 oC for 10 min. Heat 12 uL ladder in
parallel.
Add 35 uL water to sample, mix, and spin. Add 120 uL water to ladder, mix, transfer to
ladder tube.
Add DTT or IAM to sample buffer to create reducing or non-reducing buffers
Load wells on chip with gel/dye, destaingel, and marker solution.
Place chip on instrument for priming.
Add dye to gel matrix, mix, pass through spin filter.
Chip Prep Sample Prep
40 seconds/sample analysis, that is 400 sample analyses/5 hour!
No dye crosslinking stepsAutomated staining and destaining steps
For research use only. Not intended for diagnostic procedures.
6666
Thorough Process Development leads to Success
Target Discovery
Process Development
Clinical Manufacturing
Commercial Manufacturing
Clone Selection
Cell Line Development
Protein Purification
Bioprocess Scale Up Formulation
For research use only. Not intended for diagnostic procedures.
6767
The potential design space is vast, the relationships between process parameters and critical quality attributes must be determined experimentally
High Throughput Process Development
Parameter Choices Process Outcome Cell line Media components Feed strategy Control settings for pH, T, O2,
CO2 Chromatography resin Binding and Elution Buffers Residence Time
Growth Titer Purity Potency Glycosylation Charge heterogeneity Stability
High throughput analytics are needed to address multi-factorial DOE studies in Upstream, Downstream and Formulations Development
For research use only. Not intended for diagnostic procedures.
6868
Closing remarks
Actuators
Sensors
Cell culture Drug screening PCR…
ElectrochemicalSPR..
Fluorescence labelAutomatedSimpleTime
6969
PerkinElmer, Inc. All rights reserved . Some of the information contained in this presentation was obtained from third party sources, as cited. PerkinElmer, LabChip, and the LabChip logo are registered trademarks of PerkinElmer, Inc. and/or its parent, affiliates, and/or subsidiary companies (collectively “PerkinElmer”). The PerkinElmer logo is a registered trademark of PerkinElmer, Inc. All references to other company names, products, trademarks and/or registered trademarks are the property of their respective holders
Thank You for Your Attention!
7070
Miniaturization: How Small Can We Go?
Volume1 L
(10-6 L)1 nL
(10-9 L)1 pL
(10-12 L)1 fL
(10-15 L)
Cube dimension 1 mm 100 m 10 m 1 m
Diffusion time across the cube*
1000 s 10 s 100 ms 1 ms
Number of molecules
in 1 nM6x108 6x105 600 0.6
(*Assume a molecular diffusivity of 5x10-6 cm2/sec typical for small molecules)L = (2Dt)1/2
For research use only. Not intended for diagnostic procedures.
7171
Flow rate profiles-Pressure driven and electrokenitic driven
Flow rate profile
(a) Pressure driven
(b) Electrokineticdriven
Sample plug profile
Ti Td
Ti Td
flow of bulk
flow of ions/bulk
7272
“Reverse Pinch” Injections
For research use only. Not intended for diagnostic procedures.
column
wastesample
buffer
7373
“Double T” Junction
7474
Electrokinetic Dispersion Around a 90 Bend
Electric field gradient around the corner induces an electrokineticvelocity gradient, causing sample band dispersion.
(Simulation by Microcosm’s NetFlow CFD package)
J. Molho, PhD thesis, Stanford University, 2001
7575
Fluorescence Detection
Fluorescence• Molecules absorb light over a given wavelength
range and emit light at a higher wavelength• At low concentration the intensity of the emitted
light is proportional to dye concentration
Molecule Excited Molecule
Ground state Molecule
Fluorescence -hn
Energy LossAbsorption
7676
Double Layer and Electroosmotic Flow
Ion distribution near a charged surface: Electric double layer
Electroosmotic flow in an electric field
http://alcheme.tamu.edu/?page_id=6823
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Participating Experts
Sponsored by:
Brought to you by the Science/AAAS Custom Publishing Office
Improving characterization of monoclonal antibodiesMaking a better biotherapeuticNovember 19, 2014
Webinar Series
Niomi Peckham, M.Sc.Alexion PharmaceuticalsCheshire, Connecticut
I-Jane Chen, Ph.D.PerkinElmerHopkinton, MA
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Improving characterization of monoclonal antibodiesMaking a better biotherapeuticNovember 19, 2014
Brought to you by the Science/AAAS Custom Publishing Office
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