Monoclonal Antibody CharacterizationAchieving Higher Throughput and Productivity

AnalyticsPurity

AggregatesCharge VariantsPeptide Mapping

Glycans

Clinical DevelopmentCell Culture and

Purification ProcessDevelopment

Pre-CommercializationFormulation

Lot Release TestingStability Studies

Post-CommercializationProduct Improvements,

Patent Extensions,Biobetters

Drug DiscoveryTarget Identification and Validation

MAb Generation

Preclinical DevelopmentCell Line Development

Clone Screening and Selection

2

Dionex Solutions to Accelerate Monoclonal Antibody R&D and Characterization

The throughput and productivity challenge•IncreasingnumberofMAbcandidatesenteringtheclinicalpipeline

•Advancesinautomationinupstreamprocesseslikecellcultureandpurificationprocessdevelopment,andformulationscreening

•StrictQuality-by-Design(QbD)guidelinerequiringenhancedantibodycharacterization

Allresultinalargeincreaseinsamplerequestsforcharacterization.

The analytical solutionsDionexprovidesbest-in-classanalyticalsolutionsforMAbtherapeutics,improvingthroughputandproductivity,thusreducingtimetomarket.

•Best-in-classcolumnsforhigh-resolutionandhigh-throughputcharacterizationusingion-exchangechromatography(IEC)andsize-exclusionchromatography(SEC)

•MAbcharacterizationplatformstoincreasesamplethroughputandstreamlinemultistepworkflows

•PowerfulandflexibleChromeleon®ChromatographyDataSystemsoftware

•InnovativeUltiMate®3000RSLCsystemforUHPLCsolutionsforfastMAbcharacterizationandpeptidemapping

•HighperformanceMAbglycananalysis

3

MAb Samples Separation by IEC or SEC Fraction Collection Desalting on RP

C18 Column MS Analysis

Samples from Cell Line Development

Titer AssayProtein A (Column 1)

Titer AssayProtein A (Column 2)

Selection of High-TiterProducing Clones

Samples from Process Purification or Formulation

Samples from Process Purification or Formulation

SEC or IEC Column 1

SEC or IEC Column 2

Data back to Process Purification or Formulation

Data back to Process Purification or Formulation

Assay 1Aggregates by SEC

Assay 2Charge Variants by IEC

MAb aggregate or charge variant characterization by LC/MS

High-throughput MAb titer assay using parallel or dual Protein A column setup

High-throughput MAb aggregate and charge variant characterization using parallel setup

Dual method MAb aggregate and charge variant characterization using parallel setup

Samples from Cell Line Development

MAb Captureby Protein A

Aggregate Analysisby SEC

Charge VariantAnalysis by IEC Clone Selection

MAb clone selection workflow

High-throughput MAb characterization workflows

Whatever Your Workflow, You Can Achieve Higher Throughput and Productivity

Multistep MAb characterization workflows

UltiMate 3000 MAb Analysis Platform with dual ternary gradient pumps and autosampler with integrated fraction collector.

The Dionex MAb Characterization Platform— For Your Throughput and Productivity Needs

Autosampler with fraction collection and re-injection•Dualvalvedesignallowsinjection,fractioncollection,andre-injection

•Optimizedforautomatedworkflowslike:

- Automatedmultistepworkflowautomation

- Proteinpurification

- SamplefractionationanddesaltingpriortoMSdetection

- Samplederivatizationlikedigestionorneutralizationbetweenmultistepseparations

Dual gradient pump design: 2-in-1 system•MultistepAutomation:allowsautomationoftwoormoremethodslikeProteinAcapture,SECaggregate,andIECchargevariantanalysis

•TandemAnalyses:shortensruntimesbyutilizingthepowerofoff-linecolumnregeneration

•ParallelLC:doublethroughputforproductivity,cost,andspacesaving

UltiMate 3000 MAb fully biocompatible platform•Titaniumpumps;PEEK™fluidics,valvesandinjectionneedle

•Fullcompatibilitywithallbiologicalbuffers

•Preventsironpoisoningofcolumns

•Maintainsproteinmodifications

•Lesssystemcorrosionandmaintenance

4

The Dionex MAb Characterization Platform— For Your Throughput and Productivity Needs

Chromeleon Chromatography Data System (CDS) software FeaturesofChromeleonCDSsoftware:

•Automatedpeakintegration

•Automatedcontroloffractioncollectionintoautosampler

•Automatedmethodtemplateandsequencegenerationformulti-dimensionalworkflows

•Automaticrejectionofsamplesbasedonsetcriteriae.g.,automatedrejectionofcloneswithlowantibodytiter

•Dilutionsorvariableinjectionvolumesforthenextsteps

•One-clickreportgeneration

•Validationreporttemplatesandsequencesforincreasedautomationofmethodvalidation

Fully integrated solutions for R&D, analytical method development and QA/QC.

5

Boost Productivity with Automated Multistep MAb Capture and Characterization

Automate your multistep MAb capture and characterization— reduce hands-on time and increase productivity

Step 1: Protein A capture of IgG (MAb) and automated fraction collection.

Step 2: SEC aggregate analysis. Step 3: IEC charge variant analysis.

Dual Ternary Gradient Pump. 2-in-1 Pump Design.

Autosampler with Injection, Fraction Collection, and Re-Injection. Two sampling technologies in one.

The Multistep MAb Analysis Platform allows the purification and analysis of hundreds of MAb samples. Cell culture fluid samples containing antibodies are injected onto a Protein A column for antibody recovery, then the purified antibody samples are automatically injected, first onto the SEC column, and then onto the IEC column—fully automated.

0 1 2 4 53-500

4000

Minutes

mAU

Purified Antibody

0 10 20 40 50 60 70 80 9030-5

35

Minutes

Basic Variants

Lysine Variants

Acidic Variants

mAU KK

K

0 5 10 15 20 25

0

200

Minutes

mAU

Aggregates

MAb Monomer

Pump 1

Waste

TCC L R

UV OUT IN

Pump 2

SCX

SECProtein A

The diagram shows the flowpath configuration of the Protein A, SEC and IEC columns for fully automated MAb capture followed by size and charge characterization.

6

Accelerate Product Development by Increasing Sample Throughput

Analyze over a 1000 MAb samples a day by utilizing the power of off-line column regeneration

With the same samples perform multiple separation steps including SEC and IEC in a parallel configuration

0 5 10 15 20 25

0

200

Minutes

mAU

Aggregates

MAb Monomer

0 10 20 40 50 60 70 80 9030-5

35

Minutes

Basic Variants

Lysine Variants

Acidic Variants

mAU KK

K

Dual-Gradient PumpWaste

Column 2

DetectorColumn 1

Off-line Column Regeneration Flow

Autosampler

Analysis Flow

Dual-Gradient Pump

Detector

DetectorColumn 1

Column 2From Left Pump

Autosampler

From Right Pump

Analysis 1 Regen.WithoutAlternatingRegeneration

WithAlternatingRegeneration

Analysis 1 Regen.

Regen.

Regen.Analysis 2

Analysis 2

Analysis 3

Analysis 3

7

8

Monoclonal Antibody Characterization Using High-Resolution Columns

Charge Characterization with IEC•Glycosylation

•Sialylation

•C-TerminalLysine

•Deamidation

•Glutaminecyclization

•Maleuricacidadduct

•Oxidation

•Cysteinylation

•Disulfiderelated

•Succinimide

•Isomerization

ColumnProPac®WCX-10(4×250mm)ProPacWCX-10HT(4×50mm)

NEW!MAbPac™SCX-10(4×250mm)MAbPacSCX-10(4×150mm)MAbPacSCX-10HT(4×50mm)MAbPacSCX-10forLC/MS(2×250mm)

ColumnProPacHIC(4.6×250mm)ProPacHIC(4.6×100mm)ProPacHIC-10(2.1×100mm)ProPacHIC-10(7.8×75mm)

ColumnMAbPacSEC-1,5µm,300Å(4.0×300mm)MAbPacSEC-1,5µm,300Å(4.0×150mm)

Hydrophobicity Characterization with HIC•Isomerization

•Succinimide

•Oxidation

•Amidation

•Aggregation

•Clipping

Size Characterization with SEC•Monomers,aggregates,andfragments

•Undernon-denaturingconditionsusingbothhigh-andlow-saltmobilephasesandvolatileeluentsforLC/MS

9

Monoclonal Antibody Characterization Using High-Resolution Columns

0

0

80

5 10 15 3020 25Minutes

Main Peak

Methionine OxidationmAU

28041

27562

mAU

Minutes0 10 20 30 40 50 58

0

10

11

12 13 14 15 16 17

9

8

7

6

5 4

1 2 3

A B

Basic Variants

Lysine Variants

Acidic VariantsKK

K

KK

K

0 3 6 9 12 15

0

200

Minutes

mAU

27618

MAbPac SEC-1

Competitor T

MAb

MAb

B

5 6 7 8 9

0.00

2.001.43%

2

mAU

0 3 6 9 12 15

0

150

Minutes

mAU

2

1

27619

A

A. MAbPac SCX-10 column is the next generation IEC column for MAb charge characterization from Dionex.B. ProPac WCX-10 column (inset) is the industry gold standard for charge variant characterization.

ProPac HIC-10 column - Separation of populations of MAb variants using hydrophobic interaction chromatography (e.g., methionine oxidation monitoring).

B. MAb analysis in volatile buffer for LC/MS—MAb Pac SEC-1 vs the competitor’s column.

A. MAb analysis using the MAbPac SEC-1 column demonstrating excellent ruggedness.

10

Fast MAb Characterization and Peptide Mapping

Method acceleration using the new MAbPac SCX-10 column, 4 × 150 mm

Fast MAb peptide mapping—application of UHPLC and reduced particle size

0-2

22

5 4510 15 20 25 30 35 40Minutes

mAU

28042

-2

18

-1

10

46.020.0

46.020.0

46.024.0

Gradient time: 30 minTotal analysis time: 45 min

Gradient time: 15 minTotal analysis time: 30 min

Gradient time: 7 minTotal analysis time: 15 min

0-10

120

10 12020 4030 50 70 80 90 110100Minutes

60

mAU

28043

-10

130-10

175

5 µm

3 µm

2 µm

Performing peptide mapping with smaller particle columns allows the user to achieve identical resolution in less time. Here a method transfer was performed using 2.1 × 100 mm columns packed with 5, 3, and 2 µm particles respectively on the UltiMate 3000 Rapid Separation LC (RSLC).

Elution profiles of a MAb sample from a 4 × 150 mm MAbPac SCX-10 column with different gradient elution times. The gradient time and total analysis time is indicated in the chromatogram.

11

The Preferred and Proven Platform for MAb Glycan Characterization

Monitoring release of sialic acids from human lgG N-linked oligosaccharides by HPAE-PAD.

Profiling MAb hydrolysate on the CarboPac® PA20 column, with and without an AminoTrap™ precolumn.

MAb glycosylation—why measure it?•Increasingrelevance–biosimilars

•Glycosylationcanaffect:

- Biologicalactivity- Pharmacokineticsandclearancein vivo- Stability- Immunogenicity

•Analysisofglycosylationisimportantto:

- Meetregulatoryrequirement- Ensureproductconsistency- Developnewgenerationdrugswithmodifiedglycosylation

Dionex solution•HighPerformanceAnion-ExchangewithPulsedAmperometricDetection(HPAE-PAD)

- Theworkhorseincarbohydrateanalysis- HighSensitivity—0.1to1pmoledetectionlimits- Label-Free—Noderivatizationnecessary

ICS-5000—New capillary system for carbohydrate analysis.

Column: CarboPac PA200 and guardEluent: 0–5 min: 100 mM NaOH/5 mM NaOAc 60 min: 100 mM NaOH/180 mM NaOAcTemperature: 30 °CFlow Rate: 0.5 mL/minInj. Volume: 9 µL from 10 µL loopDetection: PAD (Au) Waveform A (TN 21)Samples: A) PNGase F digest of human lgG B) Sialic acids standard C) Neuraminidase digest of Sample A

A

B

C

Neu5Ac

302520151050Minutes

15

145

Neu5Gc

25520

nC

18778

1 23 4 5

6Mix of 6 standard

Protein hydrolysate

With AminoTrap

No AminoTrap

Column: CarboPac PA20 ( 3 × 150 mm) +/- AminoTrap (3 × 30 mm)Eluent: 12 mM NaOHFlow Rate: 0.5 mL/minDetection: Pulsed electrochemical, Au electrodeWaveform: Quadruple potential

Peaks: 1. Fucose 2. Galactosamine 3. Glucosamine 4. Galactose 5. Glucose 6. Mannose

Mix of 6 standard

Protein hydrolysate

80

nC

-500 5 10 15 20 25 30

Minutes

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Dionex products are designed, developed, and manufactured under an ISO 9001 Quality System.

Passion. Power. Productivity.

© 2011 Dionex CorporationCarboPac, Chromeleon, ProPac, and UltiMate are registered trademarks, and Amino Trap and MAbPac are trademarks of Dionex Corporation.PEEK is a trademark of Victrex PLC.LPN 2701 5M 01/11 Printed in U.S.A.

Selected Peer Reviewed Publications

1.Lyubarskaya,Y.;Houde,D.;Woodard,J.Murphy,D.;Mhatre,R.AnalysisofRecombinantMonoclonalAntibodyIsoformsbyElectrosprayIonizationMassSpectrometryasaStrategyforStreamliningCharacterizationofRecombinantMonoclonalAntibodyChargeHeterogeneity.Anal. Biochem.2006,348,24–39.

2.Vlasak,J.;Ionescu,R.HeterogeneityofMonoclonalAntibodiesRevealedbyCharge-SensitiveMethods.Curr. Pharm. Biotechnol.2008,9,468–481.

3.Valliere-Douglass,J.;Wallace,A.;Balland,A.SeparationofPopulationsofAntibodyVariantsbyFineTuningofHydrophobicInteractionChromatographyOperatingConditions. J. Chromatogr., A2008,1214,81–89.

4.Decrop,W.;Gendeh,G.;Swart,R.DevelopmentofanAutomatedMethodforMonoclonalAntibodiesPurificationandAnalysis.Chromatography Today,Jun2009,8–10.

5.Farnan,D.;Moreno,G.T.MultiproductHigh-ResolutionMonoclonalAntibodyChargeVariantSeparationsbypHGradientIon-ExchangeChromatography.Anal. Chem.2009,81(21),8846–8857.

6.Farnan,D.;Moreno,D.T.;Stults,J.;Becker,A.;Tremintin,G.;vanGils,M.InterlacedSizeExclusionLiquidChromatographyofMonoclonalAntibodies.J. Chromatogr., A2009,1216(51),8904–9.

7.Rea,J.C.;Moreno,G.T.;Lou,Y.;Parikh,R.;Farnan,D.High-ThroughputMulti-ProductLiquidChromatographyforCharacterizationofMonoclonalAntibodies.BioPharm International2010,23,44–51.

8.Grey,C.;Edebrink,P.;Krook,M.;Jacobsson,S.P.DevelopmentofaHighPerformanceAnionExchangeChromatographyAnalysisforMappingofOligosaccharides.J. Chromatogr., B Anal. Technol. Biomed. Life Sci.2009,877,1827–1832.

Downloadyourfree copyoftheDionex Protein Therapeutics Application Notebookatwww.dionex.com/proteintherapeutics.

TheDionexMAb Solutions eNewsletterisyourresourcetodiscovernewmethods,applications,strategiesandproductstoimproveMAbanalyticsquality,throughput,andproductivity.SubscribetothefreeMAb Solutions eNewsletteratwww.dionex.com/mab.

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