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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

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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

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

N‐LinkedOligosaccharides

Mannose

Fucose

N-acetylglucosamine

Galactose

Sialic Acid

Sources of heterogeneity Bisecting GlcNAc Galactosylation Sialylation/Fucosylation High Mannose

Bioactivity Pharmacokinetics Immunogenicity

Commercial importance

N‐LinkedOligosaccharideStructures

MAN‐5

Mannose

Fucose

N-acetylglucosamine

Galactose

G0F‐GN

(Biantennary Hybrid)

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)

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

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

PNGase

• Separate electrophoreticaly by size on chip• Detect by laser induced fluorescence

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

SeparationofGlycanStandards

mAb#1Profile

ProfilesofOthermAbs

mAb #3

mAb #2

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.

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

ProteinAssay;MonitoringDeglycosylation

• May be required in USP Chapter <212> Oligosaccharide Analysis

• Evaluated with Caliper Protein Assay

• May be performed during Labeling step (2 hours)

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

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

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)

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

N‐LinkedOligosaccharideSequencing;Step1

Sialidase Digest

• No change in profile

• No significant sialyation of mAb#1

Digest Control

Sialidase DigestG1'F + G1F

Sialidase + β‐Galactosidase

Man‐7?G0F‐GN?

G1 may not resolve from G1F and G0F

Man‐7 elutes with G1F

All forms with terminal Gal shift to G0F

Confirms G2F, G1F

Sialidase + Β‐Galactosidase

Sialidase + β‐Galactosidase + β‐N‐Acetylhexosaminidase

G0F‐GN? G0?

G0? And G0F‐GN? shift with Hexosaminidase

Preliminary assignment based on abundance and size

G0F digested by Hexosaminidase only

Confirms G0F

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

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

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I-Jane Chen, Ph. D.Sr. R&D Engineer, Microfluidics November 19, 2014

LabChipCapillary Electrophoresis: The Next Generation of High Throughput Protein Characterization

What is lab-on-a-chipMicro fabricationSurface properties and ElectrokineticsSample injection and resolutionAutomated system and high throughput performance

Content

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

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

Photolithography to make glass chips

Glass or Quartz

2. Develop

3. Etch

5. Bond1. Expose

Etched Channel Plate

Glass or Quartz Well Plate

Photo resist

4. Strip

6. Sipper

Microchannels- after etching & bonding

Bonded Channels

Substrate with channels etched in

“Grooves” on glass substrate

Substrate forms the ceiling2 substratestotally fused Into one

What is lab-on-a-chipMicro fabrication

Surface properties and ElectrokineticsSample injection and resolutionAutomated system and high throughput performance

Surface property matters

Hydrolysis

Siloxane Silanol

pKa of silanol = 5.3

At pH 7.5

Electrokinetic Fluid Actuation

velectrokinetic = velectroosmotic + velectrophoretic

- - - - - - - - - -+ ++

+ + + + + + +

- - - - - - - - - -+ + + + ++ + + + +

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

“Gated” Injection

column

sample

buffer

Sample loading using cross junctions-Electrokinetic or hydrodynamic

sample as small as 1 nL is loaded

“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!

Electrophoretic separations

Separate molecules based on their mobility under applied electric fieldMobility, µ, is velocity per unit applied electric field

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

221 wwtR

tt FWHMw 7.14

2/)44( 2,1, tt

121212

Resolution

Gaussian distribution:

Time separation:

222det

2otherdiffusionectorinjtot

Adding dispersion (band-broadening) contributions:

diffusion

Injector dispersion

Diffusion dispersion

Detector dispersion

Resolution: general expression

tottot

Rdetinjdetinj

221 wwtR

Sample plug size impact on resolution:

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

Automated sample loading and separation

What is lab-on-a-chipMicro fabricationSurface properties and ElectrokineticsSample injection and resolution

Automated system and high throughput performance

Work flow of labchip

5353 For research use only. Not intended for diagnostic procedures.

staining

destaining

Raw Profile of Reduced mAb

LCLowerMarker

System Peaks (SDS)

Raw Profile of Non-Reduced mAb

Intact mAb

Fragments

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

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

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

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

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

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

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

Closing remarks

Actuators

Sensors

Cell culture Drug screening PCR…

ElectrochemicalSPR..

Fluorescence labelAutomatedSimpleTime

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!

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

Flow rate profiles-Pressure driven and electrokenitic driven

Flow rate profile

(a) Pressure driven

(b) Electrokineticdriven

Sample plug profile

flow of bulk

flow of ions/bulk

“Reverse Pinch” Injections

column

wastesample

buffer

“Double T” Junction

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

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

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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