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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.
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