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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

6.9

23

7.7

48

8.2

30

9.0

98

AU

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00

12.3

51

14.3

25

15.3

75

17.2

64

AU

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

10.00 11.00 12.00 13.00 14.00 15.00 16.00 17.00 18.00 19.00

BA/B

A

A*

Compe tor FPP300 Å C4 1.8 µm

AB C

TEST CONDITIONS

Time, min Time, min

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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.

1

De-glycosylation of protein

2

Labeling ofreleased glycans

3

Separation by HILIC

4MS used to

identify glycans during

development

5

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.

5.0 5.5 6.0 6.5 7.0 7.5 8.0

Time, min

11.5 12.5 13.5 14.5 15.5 16.5 17.5

Time, min

HALO 1000 Å Diphenyl

HALO 400 Å Diphenyl

Figure 10. Intact Denosumab Separation Using HALO 1000 Å and HALO 400 Å Diphenyl Columns

TEST CONDITIONS