Understanding and Overcoming Separation Challenges in the … · 2020-05-20 · Separation...
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1 advanced-materials-tech.com Overview of the Drug Development Stages Biotherapeutics go through multiple development stages before they are ready to be sold commercially. The list below details the stages: 1. Selection of a new or matching protein sequence 2. Identification of lead clone(s) using the DNA sequence for proposed protein 3. Cell culture development 4. Process development and final process “lock” – no more changes after this stage 5. Clinical trials and pharmacological assessment 6. Scale-up to commercial quantities 7. Obtain data for regulatory filings 8. Obtain approval and prepare for product launch Within this process there are different primary analytical needs at each development stage. The identification of lead clones requires titer (protein concentration/run) and protein sequence verification. At this stage, there is also generation of a Master Cell Bank (MCB) and Working Cell Bank (WCB). For cell culture development, titer is still required along with ion exchange (IEX) chromatography to identify charge states, and size exclusion chromatography (SEC) to monitor monomer and aggregates. During process development multiple batches are made which require complete analysis to define the biotherapeutic drug. Included in this is an assessment of the diversity of the protein that is produced. For example, a monoclonal antibody (mAb) has a primary sequence with 150 kDa molecular weight and then there are a variety of post translational modifications (PTMs) and glycosylation modifications that are possible. See Figure 1 for a representative structure of a mAb. These variations create different forms of the same mAb --- theoretically millions of possibilities! Thankfully, nature does not support all of the variations. Separation techniques and mass spectrometry are used to identify these different forms of the mAb. Additionally, stability studies are conducted at this stage. Table 1 shows an overview of some properties Understanding and Overcoming Separation Challenges in the Biological Drug Development Process Figure 1. Representative Structure of a mAb CHARACTERISTIC PROPERTY MEASURED Protein Backbone Amino acid sequence Molecular Weight Amino acid composition Charge profile distribution PTMs Glycosylation Galactosylation Galactose-a-1,3 galatose Sialylation N-glycolylneurominic acid Core fucosylation High mannose structure Low abundance glycan species Aglycosylation Protein Backbone Modifications N-terminal variation C-terminal variation Deamidation Oxidation C-terminal amidation Glycation Higher Order Structure Protein Folding Disulfide connectivity Free cysteine Enthalpy of unfolding Tertiary structure Spectroscopic properties Aggregation Percent Monomer Aggregates Fragmentation Sub-visible particles Hydrodynamic radius Formulation and Drug Product Properties Protein extinction coefficient Protein concentration Solution properties Formulation Components Container Closure Components Process Impurities Leachables and Extractables Stability Profile Comparative stress stability Stress stability Long term stability studies Host Derived Impurities Host Cell Protein Host Cell DNA Table 1. Properties Measured for Physicochemical Characterization of Biological Drugs
Transcript of Understanding and Overcoming Separation Challenges in the … · 2020-05-20 · Separation...
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Overview of the Drug Development StagesBiotherapeutics go through multiple development stages before they are ready to be sold commercially. The list below details the stages:
1. Selection of a new or matching protein sequence 2.Identificationofleadclone(s)usingtheDNA
sequence for proposed protein 3. Cell culture development 4.Processdevelopmentandfinalprocess“lock”–no
more changes after this stage 5. Clinical trials and pharmacological assessment 6. Scale-up to commercial quantities 7.Obtaindataforregulatoryfilings 8. Obtain approval and prepare for product launch Within this process there are different primary analytical needsateachdevelopmentstage.Theidentificationofleadclonesrequirestiter(proteinconcentration/run)andproteinsequenceverification.Atthisstage,thereisalsogenerationofaMasterCellBank(MCB)andWorkingCellBank(WCB).Forcellculturedevelopment,titerisstillrequiredalongwithionexchange(IEX)chromatographytoidentifychargestates,andsizeexclusionchromatography(SEC)tomonitormonomerandaggregates.Duringprocessdevelopmentmultiplebatches are made which require complete analysis todefinethebiotherapeuticdrug.Includedinthisis an assessment of the diversity of the protein that isproduced.Forexample,amonoclonalantibody(mAb)hasaprimarysequencewith150kDamolecularweight and then there are a variety of post translational
modifications(PTMs)andglycosylationmodificationsthatarepossible.SeeFigure1forarepresentativestructureofamAb.ThesevariationscreatedifferentformsofthesamemAb---theoreticallymillionsofpossibilities!Thankfully,naturedoesnotsupportallof the variations. Separation techniques and mass spectrometry are used to identify these different formsofthemAb.Additionally,stabilitystudiesareconducted at this stage.
Table 1 shows an overview of some properties
Understanding and Overcoming Separation Challenges in the Biological Drug Development Process
Figure 1. Representative Structure of a mAb
CHARACTERISTIC PROPERTY MEASURED
Protein Backbone
Amino acid sequenceMolecular WeightAmino acid compositionCharge profile distribution
PTMs Glycosylation
GalactosylationGalactose-a-1,3 galatoseSialylationN-glycolylneurominic acidCore fucosylationHigh mannose structureLow abundance glycan speciesAglycosylation
Protein Backbone Modifications
N-terminal variationC-terminal variationDeamidationOxidationC-terminal amidationGlycation
Higher Order Structure
Protein FoldingDisulfide connectivityFree cysteineEnthalpy of unfoldingTertiary structureSpectroscopic properties
Aggregation
Percent MonomerAggregatesFragmentationSub-visible particlesHydrodynamic radius
Formulation and Drug Product Properties
Protein extinction coefficientProtein concentrationSolution propertiesFormulation ComponentsContainer Closure ComponentsProcess ImpuritiesLeachables and Extractables
Stability ProfileComparative stress stability Stress stabilityLong term stability studies
Host Derived Impurities Host Cell ProteinHost Cell DNA
Table 1. Properties Measured for Physicochemical Characterization of Biological Drugs
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measuredforphysicochemicalcharacterization.Theyaregroupedbywhichaspectofcharacterizationisbeing addressed. The methods listed in redutilizeHPLCseparations.
HALO®BioClassofferscolumnsthatfittheseparation needs for several of the methods used for physicochemicalcharacterization.WhetherintactmAbanalysis,reducedandalkylatedheavyandlightchainfragmentanalysis,proteindigests,orglycananalysis,AdvancedMaterialsTechnologyofferstailoredparticlesolutions to meet the most challenging separation requirements.Figure2showsthevariousparticlesizesandbondedphasesthatcompriseHALO®BioClassproducts. There are two particle designs for protein analysis:a1000Å2.7µmparticleanda400Å3.4µm
particle.The1000ÅparticleisusedfortheultimateresolutionofmAbsandotherlargeproteins,whilethe400Åparticleisdesignedforfastanalysisofthesecompoundsatlowerbackpressures.Forpeptideanalysis,AdvancedMaterialsTechnologyoffersthreeparticlesizesfromwhichtochoose:2µmcolumnswhichofferthehighestefficiencyandaredesirableforUHPLCsystems,2.7µmcolumnsforagoodcompromisebetweenefficiencyandbackpressure,and5µmcolumnswherelongercolumnscanbeutilizedandalsoplaced in series for high resolution while maintaining moderatebackpressure.Forglycananalysis,anapplicationspecificcolumnisavailablewhichutilizesaproprietarybondedHILICphase.AlloftheHALO®BioClass products are designed for high temperature operation and long column lifetimes.
Figure 2. Particles available from AMT for Biomolecule Analysis
160 Å 2 micron particle 160 Å 2.7 micron particle 160 Å 5 micron particle
PEPTIDE
1000 Å 2.7 micron particle 400 Å 3.4 micron particle 90 Å 2.7 micron particle
PROTEIN GLYCAN
BONDED PHASESC4ES-C18DIPHENYLPHENYL-HEXYLES-CNPROPRIETARY POLY-HYDROXY
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Peptide MappingPeptide mapping is widely used and needed for both characterizationandreleaseofbiologicalmolecules.Typically,aproteinisdigestedusinganenzyme(Trypsin,Chymotrypsin,Lys-C,etc.)andthenthepeptides generated are separated using reversed phase chromatography. This process generates a sample with manypeaks(50-100ormore),whichrequiresahighefficiencyseparation.Figure4showsthecomparisonofatrastuzumabtrypticdigestrunonaHALO160ÅES-C18,2.7µmcolumncomparedtoa1.7µmFPPC18column.WhileslightlymorepeaksareobservedontheFPPcolumn,itisatacostoftwicethebackpressureoftheHALO160ÅES-C18column.
Why Fused-Core® columns are especially useful for biomolecule separationsWhile many separations can be done with multiple typesofmoderncolumns,oneofthemajoradvantagesofFused-Core® columns is their ability to generate high efficiencyseparationswithreasonablebackpressure.Furthermore,multiplecolumnsmayberuninseriessincethebackpressureissufficientlylow.Incontrastwithsub-2µmfullyporousparticle(FPP)columns,highpressures would prevent one from coupling several columnstogether.Figure3showsseveraldifferent
particletypesandtheirbackpressuresrelativetoa5µmFPP.Therelativebackpressureishighestforthe1.7µmFPPcolumnwithavalueof8.7.However,the2µmFused-Core®particlehasarelativebackpressureof6.2,the2.7µmFused-Core®particlehasarelativebackpressureof3.4,andthe5µmFused-Core® particle has arelativebackpressureof1.2.LowbackpressurenotonlyenablesFused-Core®columnstoberuninseries,but it also means longer lifetimes of pump seals since they are not running at such high pressures.
Figure 3. Back Pressure of HALO® and FFP Particles of Various Sizes Relative to 5 µm FPP Back Pressure
Figure 4. Comparison of HALO 160 Å ES-C18 to FPP 130 Å C18 for a Tryptic Digest
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TEST CONDITIONS
SincethebackpressureislowwiththeHALO160ÅES-C18column,a150mmlengthcolumncanbeusedtoincreasetheefficiencyoftheseparation.SeeFigure5.ThebackpressureisnowsimilartotheFPPcolumn,andmorepeaksareobserved(156vs.140).Ultimateperformanceisachievedwhenthegradienttimeisextendedto90minutes,yielding189peaks.Thisis35%morepeaksthanobservedwiththeFPPcolumn!
Figure 5. Comparison of 30 minute Gradient to 90 minute Gradient using HALO 160 Å ES-C18
ToutilizethehigherperformanceatlowerpressurebenefitofSPPcolumns,anexampleofcolumnsruninseriesisshowninFigure6.Figure6Bdemonstratesthegainsinresolutionthatcanbeattainedbycouplingtwo2.1x150mmHALO160ÅES-C18columnsfora70%increaseinresolutioncomparedtousingasingle2.1x100mmcolumnFigure6Ctakestheexampleonestepfurtherandcouplesthree2.1x150mmHALO160ÅES-C18columnsfora110%gaininresolutioncomparedtowhenone2.1x100mmHALO160ÅES-C18columnisused!
TEST CONDITIONS
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Figure 6. Effect of Column Length on Resolution with HALO 160 Å ES-C18 Columns
Figure 7. Comparison of HALO 1000 Å C4 to Different Pore Size and Particle Size Columns for the Separation of Intact IgG2
AnotheradvantageofFused-Core® columns for biomolecule separations is their particle morphology. The thin shell with large pores around a solid silica core provides proteins unrestricted access to the bonded phase and shorteroveralldiffusionpathscomparedtosub-2µmFPPparticlecolumns.InFigure7,aseparationofanIgG2iscomparedacrossseveraldifferentproteincolumns.Figure7AshowslimitedresolutionofthemultipleisoformsoftheIgG2usinga1.8µm300ÅFPPcolumn.Figure7Bshowsa5µm300ÅSPPcolumnbeginningtoresolvesomeoftheIgG2isoforms.Incontrast,maximumresolutionisobtainedbyusingtheHALO1000ÅC4columnasshowninFigure7C.Forintactproteinanalysis,itisimperativetousecolumnswithporesthatarewideenoughtoaccommodate large biomolecules so the bonded phase accessibility to the protein will lead to better resolution andultimatelythebestcharacterizationdetails.
HALO 1000 Å C4 2.7 µmCompetitor SPP 300 Å C8 5 µm
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TEST CONDITIONS
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Figure 8. HILIC Analysis using HALO Glycan for a Highly Sialylated Protein
Proposed strategies for Developing versatile platform methods that can be used or easily modified for all stages and needs in drug development Withinbiotherapeuticsdrugdevelopmentthethreemainareasforseparationsareprocesssupport,characterization,andfinalrelease/QC.Processsupportcanberapidandlowresolution,butmoreefficientseparationscanbedevelopedandthen“detuned”usingthesamecolumnandmobilephase,thusnotrequiringacompletenewqualificationorvalidation.Characterizationmethodscanbedesignedtobeveryhighlyefficient,focusingonthehighestresolution,usuallywithlongertimes.Thisisoftenacceptablesincethesefullcharacterizationsareonlydoneonalimitedbasis.Similartotheprocesswork,highefficiencymethodscanbe“detuned”forfinalrelease/QCandstabilitytobefasterwhenthehighestresolutionissecondarytotime.
Forinstance,onecoulddevelophighresolutionmethodsusinglongSPPcolumns(150mmorlonger)witheither2.7µmor5µmparticlesandwidepore,1000Å.Thesemethodswillbeusedforthecharacterizationofthebiotherapeuticdrug.Themethodscanthenbemodifiedbyshorteningthecolumnlengthand/orincreasingflowrateforprocesssupport,QCworkandreleaseassays.Theadvantageofthisapproachisthatthesamecolumnisused for multiple purposes and translation from one method to another is easy.
Toillustratetheaboveconcept,thedevelopmentofacharacterizationmethodandQCmethodforglycananalysiswillnowbediscussed.Glycananalysisconsistsofseveralsteps:
First,theproteinisdeglycosylated.ThenthereleasedglycansarelabeledwithaUVorfluorescentsmallmoleculetag.ThelabeledglycansareseparatedusingtheHALO®GlycaninHILICmode.Massspectrometryisusedtoidentifytheglycansduringdevelopmentwhilefinalquantitationisperformedusingfluorescence.Figure8showsa90minuteHILICseparationofthereleasedandlabeledglycansfromahighlysialylatedprotein.
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De-glycosylation of protein
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Labeling ofreleased glycans
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Separation by HILIC
4MS used to
identify glycans during
development
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Final quanitification done by
fluorescence
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TheHILICmethodusingaHALO®Glycancolumnwasabletoseparate>70glycanspecieswithapeakcapacityof~200.Alimitationofthemethodwasthatonly1µLofsamplecouldbeinjectedduetosamplesolventinteractionswhilerunninginHILIC.Additionally,thek*valuewas178,whichismuchhigherthantherecommendedrangeof5-10.SeeEquation1forthek*formula:
Equation1
(wheretGisthetimeofthegradient,Fistheflowrate,Vmisthecolumnvolumeand is the difference in starting and endingmobilephasecomposition).
Inordertoreducek*,thetimeofthegradientwasreduced(shortertime)andtheflowratewasalsoreduced.Figure9showsthecomparisonofthe90minutegradienttothe45minutegradient.
Figure 9. Comparison of 90 min and 45 min gradients for Glycan Analysis
Notonlyisthemethod(twice as fast),butalsothepeakheightsarehigherwhich(increased the sensitivity) of themethod.Inadditionthek*hasbeenreducedto44from178.Thiscanbecounterintuitive,butisanexampleofhow proper use of theory can lead to better separations.
AnotherstrategyimplementingmethodsforbothcharacterizationofintactmAbsandaquickreleaseassayisshowninFigure10.Awidepore,2.7µmHALO1000ÅDiphenylcolumncouldbeusedforthedetailedcharacterizationworkup.Thenforthereleaseassay,the3.4µm400ÅDiphenylcolumncouldbeusedforahigherspeedanalysisduetoitsthinshell.Inthiscase,completeresolutionmaynotberequiredrather,onlythecriticalqualityattributesneedtobeidentifiedsincethefullcharacterizationwascompletedatanearlierstageofthebiotherapeutic drug development process.
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ConclusionBiological drug development requires testing at many stages during and after the process. Separations,especiallyHPLC,areveryimportantinthistesting.Process,Characterization,andFinalRelease/QCallhavespecificrequirements.UseoftheHALO®Fused-Core®columnscanbebeneficialinalldevelopmentalstagestomaximizeanalyticalefficiency.
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Figure 10. Intact Denosumab Separation Using HALO 1000 Å and HALO 400 Å Diphenyl Columns
TEST CONDITIONS
