Column-to-column packing variation of disposable pre-packed columns for protein chromatography

In the biopharmaceutical industry, pre-packed columns are the standard for process development, but they must be qualified before use in experimental studies to confirm the required performance of the packed bed. Column qualification is commonly done by pulse response experiments and depends highly on the experimental testing conditions.

j ou rn a l h om ep a ge :w w w e l s e v i e r c o m / l o c a t e / c h r o m a

Susanne Schweigera, Stephan Hinterbergera, Alois Jungbauera,b,∗

a Austrian Centre of Industrial Biotechnology, Vienna, Austria

b Department of Biotechnology, University of Natural Resources and Life Sciences Vienna, Austria

a r t i c l e i n f o

Article history:

Received 9 August 2017

Received in revised form 23 October 2017

Accepted 24 October 2017

Available online 26 October 2017

Keywords:

Preparative chromatography

Peak analysis

Column qualification

Measurement precision

Packing variation

Column geometry

Aspect ratio

Column performance

a b s t r a c t

Inthebiopharmaceuticalindustry,pre-packedcolumnsarethestandardforprocessdevelopment,but theymustbequalifiedbeforeuseinexperimentalstudiestoconfirmtherequiredperformanceofthe packedbed.Columnqualificationiscommonlydonebypulseresponseexperimentsanddependshighly

ontheexperimentaltestingconditions.Additionally,thepeakanalysismethod,thevariationinthe3D packingstructureofthebed,andthemeasurementprecisionoftheworkstationinfluencetheoutcomeof qualificationruns.WhileafullbodyofliteratureonthesefactorsisavailableforHPLCcolumns,no com-parablestudiesexistforpreparativecolumnsforproteinchromatography.Wequantifiedtheinfluence

oftheseparametersforcommerciallyavailablepre-packedandself-packedcolumnsofdisposableand non-disposabledesign.Pulseresponseexperimentswereperformedon105preparativechromatography columnswithvolumesof0.2–20ml.Theanalyteacetonewasstudiedatsixdifferentsuperficial veloci-ties(30,60,100,150,250and500cm/h).Thecolumn-to-columnpackingvariationbetweendisposable pre-packedcolumnsofdifferentdiameter-lengthcombinationsvariedby10–15%,whichwasacceptable fortheintendeduse.Thecolumn-to-columnvariationcannotbeexplainedbythepackingdensity,but

isinterpretedasadifferenceinparticlearrangementinthecolumn.Sinceitwaspossibletodetermine differencesinthecolumn-to-columnperformance,weconcludedthatthecolumnswerewell-packed Themeasurementprecisionofthechromatographyworkstationwasindependentofthecolumnvolume andwasinarangeof±0.01mlforthefirstpeakmomentand±0.007ml2forthesecondmoment.The measurementprecisionmustbeconsideredforsmallcolumnsintherangeof2mlorless.Theefficiency

ofdisposablepre-packedcolumnswasequalorbetterthanthatofself-packedcolumns

©2017TheAuthor(s).PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCC

BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/)

Small scale columns of up to 20ml are frequently used in

biomanufacturingforprocess development,scale-downstudies,

explorationofthedesignspace,andtroubleshooting.For

prepar-ativeseparations,columnscaneitherbeboughtasready-to-use

pre-packedcolumnsortheyarepackedby theuserhimself.In

thelattercases,onlythebulkresinandtheemptycolumn

hard-wareareboughtfromthemanufacturer.Pre-packedpreparative

columnshavebecomepopularbecausethelaboriouscolumn

pack-ingcanbe outsourced[1] Pre-packed columnsareavailable in

non-disposableanddisposabledesigns.Non-disposablecolumns

aremadeofhighqualitymaterialssuchasglasswallsandcould

∗ Corresponding author at: University of Natural Resources and Life Sciences,

Department of Biotechnology, Muthgasse 18, 1190 Wien, Austria.

E-mail address: alois.jungbauer@boku.ac.at (A Jungbauer).

bere-packedwithadifferentmedium bythecustomer,similar

toself-packedcolumns In comparison, disposablecolumnsare madeofcheapermaterialssuchaspolypropyleneandcannotbe re-packed.Ifthecolumnlifetimeisover,theyarediscared Dis-posablecolumnsmustbesimpleandeasytomanufactureinorder

toyieldaffordablecolumns.Self-packedchromatographycolumns arecommonlytestedbeforeusetocheckthepackingqualityand

toidentifydefectsinordertoensurethereproducibilityofruns Frequently,pre-packedcolumnsareusedbycustomerswithonly limitedadditionalqualificationsincethecolumnsareassumedto havethesamepackingquality.However,onlylimitedinformation

isavailabletoprovethisassumptionforpreparative chromatog-raphycolumnsontheprocessdevelopmentscale[2].Differences

inthecolumn-to-columnperformancewereinvestigatedonlyfor process-scalechromatographycolumnswithdiameterslargerthan

40cm [3,4] Ample of literature is also available for analytical

[5–10],semi-preparativeand preparativeHPLCcolumns[11,12] The column-to-column variation is more pronounced than the

https://doi.org/10.1016/j.chroma.2017.10.059

0021-9673/© 2017 The Author(s) Published by Elsevier B.V This is an open access article under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/

knowl-edge,acomparisonofthepackingqualityofpre-packedcolumns

tothatofself-packedcolumnshasnotbeenperformed

Thepackedbeditselfishighlyheterogeneousinboththeaxial

andradialdirections[13–15].Themorehomogeneousthepacking,

thelowerthepeakdispersion,measuredeitherasheightequivalent

toatheoreticalplate(H)orskewness[16].Itisknownthatthe

pack-ingmethod[17]andthepropertiesofthechromatographymedium

[2,18]influencethestructureofthepackedbed.Furthermore,the

material[19,20]andthesurfaceproperties[21]ofthecolumnwall

haveaninfluenceoncolumnperformancesincetheychangethe

packingbehavior.Thepackingdensityisanimportantfactorto

con-siderforevaluationofthecolumnperformance.Itinfluencespeak

retentionandwidth,sinceitisdirectlyrelatedtotheextraparticle

porosity.Apartfromthepackedbed,thecolumnperformancealso

dependsonthedesignofthecolumnheader[22]aswellasonfrits

andfilters[23]

Columnperformanceis typicallyqualified bypulseresponse

experimentsofasmallnon-interactingsolute.Forsmallmolecules,

themainfactorcontrollingcolumnperformanceishydrodynamic

dispersionand notmass transfer.Thisallows evaluation ofthe

column packing,which would beimpossible with a

biomacro-molecule.Itisassumedwhenthecolumnispackedwellenough

togivegood performance values for smallmolecules, it isalso

suitableforbiomacromolecules.Pulseresponseexperimentsare

highlydependentonthetypeofexperimentaltestingset-upused

andthemethodofpeakanalysis.Thetestingsolutehasanimpact

onthepeakshape [24]andthereforemustbekeptconstantfor

comparativestudies.Theamountoftheinjectedsample affects

thestatisticalmoments ofapeak [25]andpeakanalysisisalso

influenced by noise and baseline drift[26–29] Proper baseline

correctionandsettingoftheintegrationintervalsstillallowsthe

determinationofhighermomentswithagoodaccuracy[30].The

twomostcommonlyusedpeakanalysismethodsaredirect

numer-ical integration and peak fitting to a predefined function The

exponentially modified Gaussian (EMG)function [31,32] is the

mostpopularfunctionforpeakfittingandprovidesrobustresults

[33],especiallyforpeakswithhighexperimentalnoise.TheEMG

wasderivedbyconvolutionofaGaussianpeakwithanexponential

decayfunction.However,thereisnophysicalreason,whyapeak

shouldfollowtheshapeofanEMG[34].Therefore,itfailstofit

severecasesoftailingorfronting[34].Thepeakparameters

deter-minedbyEMGfittingcanonlybeasgoodasthefitandhencedonot

reflecttherealpeakpropertieswhenthefitisbad.Incomparison,

directnumericalintegrationprovidesthemostexactresults[33],

presumingthebaselinedriftismoderateandthedataaresmooth

andwithoutanynoise

Inthisstudy,weanalyzedtheperformanceof0.2–20.0ml

pre-packedandself-packedpreparativechromatographycolumnsof

differentlengthsanddiametersthathadbeenpackedwithdifferent

chromatographymediainordertoshedlightonthescale-downof

proteinchromatography.Thecolumnshavebeentestedby

injec-tionofanon-interactingsoluteatdifferentflowrates.Thepeak

wasevaluatedbynumericalintegrationandEMGfittingandthe

firstandsecondpeakmomentsandpeakskewnesswerecalculated

andstatisticallyevaluatedwithrespecttocolumn-to-column

vari-ation,measurementprecisionoftheworkstation,columntypes,

andcolumndimensions

2.1 Chemicals

TrisandsodiumchloridewerepurchasedfromMerckMillipore

and acetonewasobtainedfromVWR chemicals.Silicaparticles

(surfaceplain,size1␮m,50mg/mlsuspensioninwater)were pur-chasedfromKiskerBiotechGmbH&CoKG

2.2 Columns

Pre-packedMiniChromandValiChromcolumnsfromRepligen (previouslyAtoll)wereused.MiniChromcolumnsareofdisposable designwhileValiChromcolumnsarenon-disposablecolumns.The wallsoftheMiniChromcolumnsaremadeofpolypropylene,while theValiChromcolumnsaremadeofglass.Theadaptersofboth columntypesaredesigneddifferentlyandhavedifferentvolumes Thedisposablecolumnsareavailableat2–3pre-definedlengths

In contrast,thenon-disposablecolumnsarecustom-made with anyrequiredlength.Allpre-packedcolumnshavethesamefrit andfilteratthetopandatthebottomofthecolumn (polypropy-lene/polyethylenefibre,weight200g/m2,thickness0.42mm).The columnswerepackedwith4differentmedia:MabSelectSuRe(GE Healthcare,85␮mparticlediameter),ToyopearlGigacapS–650M (Tosoh, 75␮m particle diameter), Toyopearl SP–650M (Tosoh,

65␮m particle diameter) and Toyopearl Phenyl–650M (Tosoh,

65␮mparticlediameter).MabSelectSuReisacompressibleProtein

Amediumwithhighlycross-linkedagaroseasbackbone.Both, Toy-opearlGigacapS–650MandToyopearlSP–650Mmediaarestrong cationexchangemediawithamethacrylatebackbone.The Giga-capresinhasanadditionalpolymerlinkerbetweenthebackbone and the sulfopropyl functionalization Toyopearl Phenyl–650M hasthesamebackboneasSP–650Mbutisahydrophobic inter-action medium since it is functionalized with a phenyl ligand group.MiniChromcolumnsweresuppliedincompletesetsofall availablecolumnsizeswiththefollowingdiameter-length com-binations(inmm):5–10,5–25,5–50,8–20,8–50,8–100,11.3–50 and 11.3–100 Eachof those column dimensionswas delivered threetimespre-packedwitheitherMabSelectSuReorToyopearl GigacapS–650M.ThreeadditionalcolumnspackedwithMabSelect SuRewereavailableinthe11.3–50dimension.Eachcolumn dimen-sion was available once pre-packed with Toyopearl SP–650M and Toyopearl Phenyl–650M ValiChrom columns packed with MabSelect SuReand ToyopearlSP–650Mweredeliveredin the followingdiameter-lengthcombinations(inmm):5–100,5–150, 5–200,8–150,8–200,8–250,11.3–100, 11.3–150and11.3–200 ValiChromcolumns packedwithToyopearl Phenyl–650M were availableinthefollowingdiameter-lengthcombinations(inmm): 5–100,5–200,8–150,8–200,11.3–150and11.3–200

Additionally,wepackedcolumnsinourlaboratorywith MabSe-lectSuReandToyopearlGigacapS–650MusingTricorn5columns (GE Healthcare) They are designed as non-disposable columns witha diameterof 5mm Tricorn 5 filters (ethylene propylene diene/polyethylene,porosity7␮m,thickness1.35mm)wereused

atthetopandatthebottomofthecolumnswithoutanyfrits.The columnswerepackedaccordingtooptimizedpackingprotocols withbedheightsintherangeof12–162mm

The described columns will hereafter be referred to as pre-packeddisposable(MiniChrom), pre-packed non-disposable (ValiChrom),andself-packed(Tricorn)columns

2.3 Workstation

AnÄKTATMpure25M2chromatography system(GE Health-care)wasused,whichwascontrolledwithUnicornsoftware6.4 Theextracolumntubingbetweenthepumps,valves,and detec-torswasusedasprovidedbythemanufacturer.Thesampleswere injectedviaaninjectionloop.Theinjectionvalvehasatotal vol-umeof44␮landthecolumnvalveof110␮l.Thedetectioncell

of theUVdetector hasavolume of15␮l.The tubingfrom the column valvetothecolumnand backwasvaried basedonthe columntypeused.TubingswithanIDof0.25mmandalengthof

thecolumnoutlettothecolumnvalvewereusedforpre-packed

disposablecolumns,forpre-packednon-disposablecolumnswith

diameter-lengthcombinationsof5–100and5–150andfor

self-packed columns For pre-packed non-disposable columns with

thediameter-lengthcombinationsof5–200,8–150,8–200,8–250,

11.3–100,11.3–150and11.3–200,thetubingfromthecolumnto

thecolumnvalvewas331mminlength

Theextracolumnvolumeandbandbroadeningwasdetermined

byinjections of acetonethrough theworkstation includingthe

tubingtoandfromthecolumn,whichwasconnectedbyaPEEK

con-nector,0.010”thru-holeand0.07␮lvolume.Theinfluenceofextra

columnvolumeandbandbroadeningisshowninthe

Supplemen-taryMaterial.Forverysmallcolumnstheextracolumnvolumewas

evenlargerthanthecolumnvolumeandalsoextracolumnband

broadeningwasveryhigh

2.4 Pulseresponseexperiments

Column performance was evaluated in triplicate by pulse

responseexperimentsatdifferentsuperficialvelocities(30,60,100,

150,250and500cm/h).Acetone(1%,v/v)wasusedforthepulse

Theinjectedpulsevolumeswere10␮lforallpre-packeddisposable

columns,pre-packednon-disposablecolumnswith5mmID,and

self-packedcolumns,50␮lforpre-packednon-disposablecolumns

with8mmID,and500␮lforpre-packednon-disposablecolumns

with11.3mmID.Forpulsesthroughtheextracolumnvolumeonly,

10␮lwereinjectedatalltheusedflowrates.Therunningbuffer

was50mMTris,0.9%(w/v)sodiumchloridepH8.0(pHadjusted

withhydrogenchloride)

2.5 Determinationofextraparticleporosity

The extra particle porosity of the pre-packed disposable

columnswasdeterminedbyinjectionofsilicananoparticles

(sur-faceplain)withadiameterof1000nm.Silicananoparticles(50␮l)

wereinjectedto theMabSelectSuRe columnsand 10␮l tothe

GigacapS–650Mcolumnswithaconcentrationof50mg/ml

Puri-fiedwaterwasusedasarunningbufferatasuperficialvelocityof

250cm/h.Theextracolumnvolumewasdeterminedbyinjections

throughtheworkstationandthetubingrangingtoandfromthe

columnattherespectiveflowrates.Theretentionvolumeatpeak

maximumwasusedforcalculationoftheextraparticleporosity

2.6 Peakanalysis

Thepeakswereautomaticallyanalyzedwithascriptwritten

inthestatisticalsoftwareR.Thescriptwasoptimizedforpeaks

obtainedbypulseresponseexperimentsandrunsstablyfordata

withonly one main peak and few smallerpeaks, which were

baselineseparated.Thepeakanalysisprocessstartedwithadata

reductionsteptoabout1000datapoints,thenseveralpeakswere

detectedandwerefittedtoalinearbaselinethroughnon-peakdata

points.Anotherpeakdetectionstepwasperformedwiththe

base-linecorrecteddata.Forpeakdetection,thedataweresmoothed

andthefirstderivativeofthedata,theslope,wascalculatedover

awindowsizeof30datapoints.Twothresholdlevelswereset:

level±1at±0.5%ofthemaximumpeakheightandlevel±2at±5%

ofthemaximumpeakheight.Assoonasthederivativeofthesignal

increasedabovelevel2andreturnedtolevel−2fromthe

nega-tive,apeakwasdetected.Thedetectedpeakthenstartedatlevel

1andendedatlevel−1.Apeakwasdefinedtohaveawidthof

atleast30datapointstomakethescriptmorerobust.For

cal-culationofthepeakmaximum,thedataweresmoothedinorder

tocorrectforsmallfluctuationsoftheoutputsignal,whichmight

biasthepeakmaximum.Thepeakwasintegratedfromthe

base-Fig 1.Schematic distinction between measurement precision (lower left panel, blue) and the packing variation (lower right panel, violet) Every column was tested

in triplicates The variation in the triplicate measurements (blue arrows) gives infor-mation on the measurement precision The difference between three individually packed columns gives information on the packing variation (violet arrows) Each cir-cle represents one measurement point The measurement precision was evaluated

in terms of mean (blue dashed lines) and standard deviation (blue error bars) for each column separately The packing variation was calculated based on the means

of the triplicate measurements of the individual columns (blue circles) Again, the mean (violet dashed line) and the standard deviation (violet error bar) were consid-ered (For interpretation of the references to colour in this figure legend, the reader

is referred to the web version of this article.)

linebetweenitsdetectedstarttoitsendpoints.Thecalculation

ofthepeakmomentsisdescribedinSection3,Theory.Fordirect numericalintegration,thestatisticalmomentsofthepeakswere calculatedasstatedin[38].Themomentswerecorrectedforthe extracolumncontributionstopeakretentionandbroadening con-sideringthedifferenttubinglengthsforeachcolumndimension Finally,thesecondmomentwascorrectedforthedifferent injec-tionvolumesbysubstractingthecontributionoftherectangular injectionpulse

2.7 Determinationofmeasurementandcolumn-to-column packingvariation

Threeindependently packed pre-packeddisposablecolumns wereavailableatdifferentdimensionspackedwithMabSelectSuRe andGigacapS–650M.Allcolumnswerepackedinthesamecolumn typewiththesameoptimizedmethod,withtheonlydifference beingthestructureofthepackedbed.Everycolumnwastestedin triplicate.Consideringeachcolumnseparately,themeanandthe standarddeviationofthetriplicatemeasurementsdescribedthe measurementprecisionofthesystem(seeFig.1).Columnswere excludedfordeterminationofthemeasurementprecisionifonly duplicateorsinglemeasurementswereavailable.Thepacking vari-ationcanbecalculatedbycomparingtheresultsofthedifferent columns.Forwell-packedcolumns,thecolumn-tocolumn varia-tionishigherthanthesame-columnrepeatability[11].Thepacking variationwascalculatedbycomparingthemeanofthetriplicate measurementsforeachcolumn.Ifthemeanswereclosetogether, thepackingwasrathersimilar,sotheresultswereeasily repro-ducible.Asameasureofthevarianceofthecolumnmeans,and

soforthedifferentpackings,thetotalmeanwiththerespective standarddeviationwasdetermined(Fig.1).Alowstandard

procedurewasdoneseparatelyforeachcolumndiameter-length

combinationforbothmedia.Onlycolumnsavailablethreetimes

withtriplicatemeasurementswereconsideredfortheanalysis(9

datapointsperdiameter-lengthcombination).Thecalculationof

themeasurementprecisionandthecolumn-to-columnvariation

wasmadeforthepeaksanalyzedbydirectnumericalintegration

2.8 Statisticaltesting

StatisticaltestsweredoneinthestatisticalsoftwareR.Thedata

weretestedfor statisticalsignificanceusinganalysisofvariance

(ANOVA),Student’st-tests,orlinearregression.Theassumption

ofnormaldistributionoftheresidualswasconfirmedusingthe

Kolmogorov-Smirnovtest.P-valuessmallerthan0.05were

con-sideredsignificant.Thep-valuesof pairedt-tests wereadjusted

withtheBonferroni method.Principalcomponent analysiswas

usedtodescribethelargestvariationsinthedata(Supplementary

Material).Thevariableswerecenteredandscaledfortheprincipal

componentanalysis

Thestatisticalmomentsofthepeaksweredeterminedbydirect

numericalintegration.Thefirstmoment(M1)isthemeanretention

volumeofa peak.Thesecondmoment(M2)isthevarianceofa

peakandisameasureofpeakwidtharounditscenterofgravity

Itisusedasameasureforcolumnefficiency.Thedeterminedfirst

andsecondmomentswerecorrectedbythecontributionsofthe

extracolumnvolumetothefirstandsecondmoments.Besides,the

secondmomentwascorrectedforcontributionsofthedifferent

injectionvolumesusingthefollowingequation

2inj= V

2

inj

12

whereinj2 isthepeak variance arisingfromtheinjectionof a

rectangularsampleplugandVinjistheinjectionvolume

Thethirdmoment(M3)isameasureforpeakasymmetry.The

degreeofasymmetryisdescribedbythepeakskewness,whichwas

calculatedby

skew= M3

Thepeakskewnessisnegativeforfrontingpeaks,zerofor

sym-metricalpeaks,andpositivefortailingpeaks.Theskewnesswas

notcorrectedforcontributionsoftheextracolumnvolume

Theheightequivalenttotheoreticalplate(H)wascalculatedby

H=M2∗L

whereListhecolumnlength.Columnefficiencywasevaluatedin

termsofreducedHETP(h),whichwasbycalculatedby

h= H

wheredpistheparticlediameterofthemedium.Thenominal

par-ticlediameterswereusedforthecalculationsasprovidedbythe

mediummanufacturers.Thereducedvelocityu’wascalculatedby

u’= u∗dp

D0

(8)

whereuisthesuperficialvelocityandD0isthemoleculardiffusivity

ofacetonewith1.16*10−5cm2/s

Fig 2. Number of runs for each of the chromatography media and column types.

Thecolumnaspectratiowascalculatedby Columnaspectratio= L

dc

(9) wheredcisthecolumndiameter.Thebedaspectratiowas calcu-latedby

Bedaspectratio= dc

dp

(10)

4.1 Datadescription

Columnperformancewasevaluatedbypulseresponse exper-imentsin termsoffirstandsecondmomentaswellasreduced HETPandpeakskewness.Theimpactofthesuperficialflow veloc-ity,chromatographymedium,columntype,columndiameter,and columnlengthoncolumnperformancewasassessed.Intotal,105 columnswereanalyzedin1884runswithonerunrepresenting one pulse response experiment (Fig 2) 1169 runs were per-formedwithpre-packeddisposablecolumns,428withpre-packed non-disposablecolumnsand287withself-packedcolumns.Three differentmediumtypes(cationexchange,hydrophobicinteraction andProteinA)wereanalyzedtoobtainmorerepresentativeresults overvariouschromatography mediaandtoevaluatedifferences betweenthedifferentmediatypes.Thedatastructureand variabil-itywasevaluatedinmoredetailbyprinciplecomponentanalysis (SupplementaryMaterial).Acomparisonofnumericalintegration and EMGfittingof thepeakshowedthat numericalintegration

ismoresuitableforfrontingandnon-exponentiallytailingpeaks (SupplementaryMaterial).Consequently,alltheshowndatawere analyzedbydirectnumericalintegration

TheVanDeemtercurveshowsthat masstransferistherate limitingmechanism,sincethereducedHETPincreasedwiththe reduced velocity (Fig.3A) Consequently,especially therunsat higherreducedvelocitieswillpartlybecontrolledbydiffusional limitations ofacetoneinside thebeads andnot onlyreflect the differences in thedifferentpackings The reduced HETP widely variedwithinonereducedvelocity,becausecolumnsofdifferent typesanddimensionswereevaluated.Afewreducedplateheights arenegativebecauseforsomeofthecolumnstheextracolumn bandbroadeningwashigherthanthetotalbandbroadening.This

isattributedtothestatisticalvariationoftheresults.Thepacked mediumalsoinfluencedthecolumnperformance.However,the datamightbebiasedsincesomemediawerealsoavailablein pre-packednon-disposableandself-packedformat,whichhadlonger columnlengthsandmighthavebeenmoredifficulttopack There-fore,onlythepre-packeddisposablecolumnswereconsideredfor analyzingtheimpactof thepackedmedium oncolumn

perfor-Fig 3.Columnn performance for all columns (A) Van Deemter plot of all runs (B) Column performance of pre-packed disposable columns packed with the different media Data for a superficial velocity of 150 cm/h are shown (C) Variation of reduced HETP with column aspect ratio for all runs at all velocities (D) Variation of reduced HETP with bed aspect ratio for all runs at all velocities.

mance(Fig.3B).Asexpected,notallmediawereequallyeasyto

pack,asreflectedbysignificantlydifferentreducedHETPvalues

MabSelectSuRehadthelowestreducedHETPofabout4.5.Columns

packedwithMabSelectSuRearesoldveryfrequentlyandtherefore

representthemostoftenpackedcolumnsofthemanufacturer.It

isthereforereasonablethatahighlyoptimizedpackingprocedure

hasbeendevelopedovertime.Despitethereishardlyanytrend

visisblebetweenthereducedHETPandthecolumnaspectratio,a

linearmodelgaveasignificantslopeof0.1(Fig.3C).Consequently,

thereducedHETPinreasesslightlywiththeaspectratio.Thebed

aspectratiodoesnotchangewiththereducedHETP(Fig.3D), a

linearmodelfittedtothedatagaveanonsignificantslope

AsalreadyshownintheVanDeemterplot,thereducedvelocity

affectedthemeasuredperformanceparameters.Formoredetailed

analysis,thevariationofthemomentswithreducedvelocitywas

visualizedfordifferentcolumnvolumes.Asexpectedfromtheory,

thesecondpeakmomentincreasedwithcolumnvolumeandwith

thereducedvelocity(Fig.4A).Thereducedvelocitygreatly

influ-encedpeakwidth.Thisconfirmsthatpulseresponseexperiments

shouldalwaysberunatthesamereducedvelocityinorderthat

experimentsarecomparable

Thelargerthecolumn,themoresymmetricalarethepeaks(Fig

4B).Peaksofcolumnslargerthan5mlarerathersymmetrical,while

columnswithavolumesmallerthan1mldisplayedtailingdueto

thedominating extracolumneffects Thereducedvelocityused

fortesting hasa largeimpact onthemeasuredpeak skewness

forsmallcolumns.Consequently,theoutcomeofcolumn

perfor-mancetestscaneasilybechangedbychoosingadifferentreduced

velocity.Thelowertheflowrate,themoretailingoccurs.Thesame

effectwasobservedforpeaksthroughtheworkstationwithno col-umnconnected(datanotshown).Duetothelargeinfluenceofthe workstationinsmallcolumns,thepeakshapewassimilartopeaks measuredintheextracolumnvolume

4.2 MeasurementprecisionoftheÄKTApure25workstation

Theworkstationwillinfluenceeverypulseresponseexperiment sincethepulsewillnotonlybroadeninthecolumnitselfbutalso

intheextracolumnvolume.However,apartfromtheadditional bandbroadeningintroducedbytheworkstation,itwillalsoadda certainvariationtotheresults.Apulseresponseexperimentdone severaltimeswiththesamecolumnonthesameworkstationwill yieldslightlydifferentresultseachtime.Knowingthe measure-mentprecisionoftheworkstationallowstheevaluationofwhether

adifferenceinpeak parametersissignificantorjust withinthe typicaldatavariationrange

Based on the triplicate measurements of all pulse response experiments,wewereabletocalculatethemeasurementprecision

oftheusedworkstation.Themeanandthecorrespondingstandard deviationofthetriplicatemeasurementsforthefirstandthesecond momentwerecalculatedandplottedagainsteachother.Novisual trendbetweentheabsolutemagnitudeofthemeananditsstandard deviationcouldbeobservedforthefirstmoment(Fig.5A).Alinear modelfittedtothedataconfirmedanon-significantslope,meaning thatthestandarddeviationofthefirstmomentwasindependent

ofthesizeofthefirstmomentandthereforecouldbeconsidered constant.Consequently,evencolumnslargerthantheonesusedin thisstudywouldhavethesamestandarddeviation.Thisisa

rea-Fig 4.Variation of column performance parameters with column volume and reduced velocity (A) Second Moment (B) Skewness.

Fig 5.Measurement precision of the ÄKTA pure 25 M2 workstation Variation of the standard deviation (SD) with the mean of the first (A) and second (B) moment of all runs at all velocities available in triplicates (565 data points) 95% of the data points are below the black dashed line.

sonableproposalsincethemeasurementprecisionoriginatesfrom

theworkstationitselfandnotfromthecolumnandthereforestays

constantirrespectiveofthecolumnvolume

Thestandarddeviationofthesecondmomentdependsonthe

sizeofthesecondmoment,sincetheslopeofalinearmodel

fit-tedtothedataofthesecondmomentwassignificant(Fig.5B)

However,thepredictedslopeissmall(0.014)andthereforeonly

aslightdependenceofthestandarddeviationwiththepeakwidth

wasobserved.Thelargerthecolumndiameter,thehigherthe

stan-darddeviationofthesecondmoment.Consequently,thestated

measurementprecisionshouldnotbeusedforextrapolationsto

columnswithanevenlargerdiameter

ThemeasurementprecisionoftheÄKTApure25workstation

issmallerthan±0.01mlforthefirstmomentand±0.007ml2for

thesecondmomentfor95%ofthedatapoints,whereasthelatter

parametermightbehigherifcolumnvolumeslargerthan20ml

areused.Theerrorrangesweregivenfor95%ofthedatapointsin

ordertogivemorereliableestimatesrepresentingthewholedata

rangeandnotthemean.TheRSDofthefirstmomentdependedon

thecolumnvolumeandwassmallerthan0.75%forcolumnslarger

than2ml.TheRSDofthesecondmomentwassmallerthan7.5%

forcolumnslargerthan2ml.However,theRSDmaybeupto25%

forcolumnssmallerthan1ml.Thepackedmediumhadnoeffect

onthemeasurementprecisionofthefirstandsecondmoment

4.3 Column-to-columnpackingvariation

Itiscommonlyassumedthatpre-packedcolumnshavethesame packingquality,sincetheyarepackedbyexpertswitha standard-izedpackingmethod.Thisisespeciallytrueforcolumnsofthesame batch,whichwerepackedsimultaneously.Weexaminedwhether thisassumptionwasvalidforpre-packeddisposablecolumns.We focusedonthevariationinthefirstandsecondmomentcaused

bythepackingofcolumnsof thesame size.Toverify,whether thecolumn-to-columnpackingvariationissignificantcompared

tothemeasurementprecision,wemadeanANOVAanalysison everycolumnlength-diametercombinationatacertainvelocity The column-to-column packingvariation wassignificantfor 59 outof70testedlength-diameterandvelocitycombinations.This meansthatthemajorityofthecolumnsandvelocities,thevariation betweenthedifferentpackingswaslargeenoughtobeidentified

assuchontopof themeasurementprecision.For theother11 columnsandvelocities,themeasurementprecisionmighteitherbe toolowtoidentifydifferencesinthepackingbetweenthedifferent columnsorthecolumnpackingswerethesame.Thecalculationof thecolumn-to-columnpackingvariationisdescribedinsection2.7

inmoredetail

Theabsolutestandarddeviationofthefirstandsecondmoment causedbythepackingdifferencesbetweenthecolumnsincreased withthe meanfirst and thesecond moment,respectively

(sig-Fig 6.Packing variation of pre-packed disposable columns Variation of the relative SD (RSD) expressed as the % of the mean for the first (A) and the second (B) moment of all runs available for three columns of the same size (70 data points) The data are shown for all superficial velocities.

nificantslopeofalinearmodel)(datanotshown).Therefore,we

consideredtheRSD(givenin%ofthemean).TheRSDofthefirst

momentwassmallerthan1%ofthemeanforallcolumnslarger

than1ml(Fig.6A),sowecanconcludethatthevariationin

pack-inghadnoimpactonthefirstmoment.TheRSDofthefirstmoment

decreasedwiththemean,whichcorrelateswiththecolumn

vol-ume.Consequently,thisparametershouldnotbehigherthan1%for

columnvolumesgreaterthanthosetested.TheRSDofthesecond

momentdidnotincreasewiththemeansincealinearmodelonly

gaveasignificantinterceptandnosignificantslope,andcan

there-forebeconsideredconstant(Fig.6B).Themajorityofthecolumns

showedaRSDofaround10–15%.Comparedtothefirstmoment,the

relativestandarddeviationofthesecondmomentwashigh.This

wasanexpectedoutcomesincethepackingqualitymainlyaffects

peakshapeandwidthandnotthepositionofthepeakmaximum

Theverysmallcolumnsshowthehighestpackingvariationofupto

20–30%buttheyalsoshowthelowestpackingvariationoflessthan

5%.Toourknowledge,itismoreeasytoreproduciblypackwider

columns,whichisthereasonwhytheverythincolumnsshowa

higherpackingvariation

WhenthevariationintheHETPwascalculated,wefoundamean

RSDof15%foralldatapoints.Thisvaluewascomparabletothe14%

RSDfoundforsemi-preparativeHPLCcolumnsand30%for

prepar-ative HPLC columns[11] Therefore, the disposablepre-packed

columnscanbeconsideredtobepackedreproduciblywithinthe

expectedrange,despitetheRSDis1%forthefirstand10–15%forthe

secondmoment.Ifhigherstandardswererequiredbycustomers,

forexampleapackingvariationoflessthan10%morethanhalf

ofthecolumnswouldneedtobediscarded,whichinturnwould

dramaticallyincreasethecostsofpre-packedcolumns.Considering

thatonlythemeasurementprecisioncanleadtoavariationofthe

secondmomentof7.5%,theobservedcolumn-to-columnpacking

variationcanbeconsideredacceptable

4.4 Influenceofcolumngeometryonpackingvariation

Wecomparedthecolumn-to-columnpackingvariationof

pre-packeddisposablecolumnswithdifferentdimensions

(diameter-lengthinmm:5–10,5–25,5–50,8–20,8–50,8–100,11.3–50and

11.3–100)toelucidatetheinfluencesofcolumnvolumeandaspect

ratioonthepackingvariation.Wealsoevaluatedwhetherone

col-umntype canbepackedtothesame standardsofqualitywith

variousmedia.Thisfactormightbeimportantformedium

screen-ingstudies,wheretheimpactofthemediumshallbeevaluated

insteadofthepackingquality.Wefocusedonevaluatingthe

vari-ationinthesecondpeakmomentsincethisparameterishighly affectedbypackingdifferencesasshowninsection4.5

Thediameter-lengthcombinationofthecolumnssignificantly influenced the column-to-column packing variation, while the medium type didnot (Fig.7A).Consequently, the columnsare equallypackedregardlessofthemedium.TheRSDofthesecond momentvariesbetween7and20%formostdiameter-length com-binations.Thehighvariationisattributedtothedifferentvelocities evaluated,since especially at highvelocities alsomass transfer contributestobandbroadening.Notrendwasseenbetweenthe packing variation and the column volume or the aspect ratio However,somediameter-lengthcombinationswereeasiertopack reproduciblyasillustratedbycolumnswith8mmIDand20and

50mm height This observation may be attributed todifferent packingproceduresusedfordifferentcolumnsizes.Especiallythe columnswith5mmIDshowahighpackingvariation

Theextraparticleporositywasdetermined(Fig.7B)toevaluate whethercolumn-to-columnpackingvariationwasduetodifferent packingdensity.However,thepackingdensitywasnotthecause forthevariationofthesecondmomentofthedifferentcolumns Whentheextraparticleporosityvariedwidely,thepackingcanstill

berepeatable.Forexample,thevariationinextraparticle poros-ity washigh for thesmall columns,even though theyshowed thesamepackingvariationasthelargeones.Hence,thereason forlargecolumn-to-columnvariationisexplainedbytheparticle arrangementinside thecolumn, sinceallotherfactorscouldbe excluded.Differencesinthepackingofprocessscale chromatogra-phycolumnswerealsoobservedby[4]andtheauthorsclaimedthat thesedifferencesdonothaveanimpactontheactualseparationof proteins

Whenwecomparethepackingvariationwiththecolumn per-formance measured as reduced HETP (Fig 7C), no correlation betweenpackingvariationandpackingqualitycanbeseen.Ahigh extraparticleporositydoesnotresultinlowcolumnperformance, whichwasalsoshownbyStanleyetal.[11]forHPLCcolumns.They alsoclaimedthatitisonlypossibletodeterminethe column-to-columnefficiencyforwell-packedcolumns.Sinceitwaspossible

todeterminedifferencesinthecolumn-to-columnefficiencywe canconcludethatthecolumnsarewell-packed

4.5 Influenceofcolumntypeoncolumnefficiency

Three different column types (pre-packed disposable, pre-packed non-disposableand self-packedcolumns) were investi-gated.Thepre-packeddisposablecolumnsaremadeof polypropy-lene,whereasthepre-packednon-disposablecolumnsareofhigher

Fig 7.Influence of column diameter and length on the column-to-column packing variation and packing quality of pre-packed disposable columns (A) Relative standard deviation (RSD) of the second moment caused by the packing variation for the differently packed columns of various column diameter-length combinations packed with Gigacap S–650 M and MabSelect SuRe at all evaluated superficial velocities (B) Extra particle porosity (␧) of the pre-packed disposable columns packed with MabSelect SuRe The error bars show the standard deviation between three equally packed columns * Standard deviation is exceptionally high because one of the three columns was treated under harsh conditions before the extra particle porosity was determined (C) Absolute variation in reduced HETP caused by the packing variation for columns of various diameter-length combinations packed with Gigacap S–650 M and MabSelect SuRe at all evaluated superficial velocities.

qualitywitha column wallmade ofglass Alsotheself-packed

columnshadaglasswallandweredesignedforre-use

ColumnswithanIDof11.3mmandlengthof100mmwere

com-paredtoelucidatethedifferencesbetweenpre-packeddisposable

and non-disposablecolumnssincethis wastheonly size

avail-ableinbothcolumntypes.Wefoundasignificantdifferenceinthe

reducedHETPbetweenthedisposableandnon-disposablecolumns

packedwithSP–650M,butnodifferencefor thosepackedwith

MabSelectSuRe(Fig.8A).Forcomparisonofthepre-packedwith

theself-packedcolumns,onlycolumnswithanIDof5mmanda

maximumlengthof60mmwereconsidered.Thisselectionallowed

ustocomparecolumnsofthesamedimensionsandtherebyavoids

biasesofeasierorhardertopackdimensions.Self-packedcolumns

packedwithMabSelectSuResignificantlydifferedfrompre-packed

disposablecolumnsbutshowedthesameefficiencywhenpacked

withGigacapS–650M(Fig.8B).However,itisworthnotingthatthe

packingprocedurewasoptimizedandthecolumnefficiencymight

beworseforcolumnswhicharenotwellpacked

Thediverseeffectsweobservedfordifferentmediamayoccur

becauseofchangesinthepackingbehaviorofthemediabetween

thedisposableandnon-disposablecolumnsandmayberelatedto

variationsintheirsurfacechargesandroughness

However, for HPLC columns it wasshown that the column

wallmaterialdoesnotinfluencecolumnefficiency[35]

Alterna-tively,thepackingoperatormighthaveaninfluenceonthecolumn

efficiencysincethedifferentcolumntypeswerepackedby

differ-entoperators.Besides,differentpackingsolutionsandprocedures

mighthavebeenused.Despitethedifferencesweobservedinpeak

shapebetweenthedifferentcolumntypes,thesedifferenceswere

alsopresentforthosemedia,wheretheefficiencyofbothcolumn

typeswasthesame.Thesameistrueconsideringthespecificdesign

ofthetopandbottomadapterandofthefilterandfritsinthe

col-umnresultingindifferentextracolumnvolumes.Furthermore,the volumeoftheadaptersofthepre-packednon-disposablecolumns waslargerthanthoseofthepre-packeddisposablecolumnsand stilltheyshowedbetterefficiency

Ingeneral,noclearevidenceofthesuperiorityofonecolumn typewasfound.Thespecificcombinationofacertainmediumand columntypeprobablyhasaninfluenceoncolumnefficiency.Forthe evaluatedmediaandcolumns,pre-packednon-disposablecolumns arebetterorequallypackedthanpre-packeddisposablecolumns Pre-packeddisposablecolumnswerebetterthanorequalin effi-ciencytothe self-packedcolumns.However,these resultsmay notbeapplicabletocolumnsofdifferentdimensionsorcolumns packedwithdifferentmedia

Statistical analysis of peaks made on independently packed columnsshowedasignificantinfluenceofthedifferentpackings comparedtothestandardfluctuationintroducedbythe measure-ment precisionof theworkstation The measurement precision

of theÄKTA pure25 workstation wasdetermined bytriplicate measurements for each column and was quantified as smaller than±0.01mlforthefirstmomentand±0.007ml2 forthe sec-ondmomentfor95%ofalldatapointsmeasured.Theimpactofthe workstationontheexperimentsdependsonthecolumnvolume evaluated.Whilethemeasurementprecisionisnegligibleforlarge columns,itshoulddefinitelynotbeneglectedforsmallcolumns, since thevariation is highcompared totheperformanceof the packedbed

The column-to-column variation of disposable pre-packed columnsdependsoncolumnvolumeandconsequentlyis consid-eredintermsofrelativestandarddeviation(RSD).TheRSDbetween

Fig 8.Influence of column type on column efficiency (A) Reduced HETP for pre-packed disposable and non-disposable columns with 11.3 mm ID and 10 mm bed height tested at all superficial velocities (B) Reduced HETP for pre-packed disposable and self-packed columns with a bed height lower than 60 mm tested at all superficial velocities.

columnsofthesamedimensionswaslowerthan1%forthefirst

momentandabout10–15%forthesecondmoment.Theonly

dif-ferencebetweentheevaluatedcolumnsisthepacking.Wefound

thatthevariationcannotbeexplainedbythepackingdensity,but

isratherattributedtotheheterogeneityinparticlestructure in

thecolumn.Thecolumm-to-columnpackingvariationofthe

sec-ondmomentissmall,consideringthatthemeasurementprecision

oftheworkstationaloneisaround7.5%forcolumnslargerthan

2mlandupto25%forcolumnssmallerthan2ml.Thevariation

ofthefirstandsecondmomentsleadstoaresultingvariationin

HETPof about15%.Thisisthevariation for anunretained

ace-tonepulseauserof pre-packedcolumnscanexpectif hebuys

twocolumnsofthesamedimensions.Consideringthatcolumns

aretypicallyusedwithretainedsolutes,which aremainlymass

transferlimited,hardlyanychangeinperformanceisexpected.For

theevaluatedcolumndimensionsandmedia,pre-packed

dispos-ablecolumnshadahigherorequalcolumnefficiencycomparedto

self-packedcolumns

Acknowledgements

ThisworkhasbeensupportedbytheFederalMinistryof

Sci-ence,ResearchandEconomy(BMWFW),theFederalMinistryof

Traffic,InnovationandTechnology(bmvit),theStyrianBusiness

PromotionAgencySFG,theStandortagenturTirol,theGovernment

ofLowerAustriaandZIT−TechnologyAgencyoftheCityofVienna

throughtheCOMET-FundingProgrammanagedbytheAustrian

ResearchPromotionAgencyFFG

Supplementarydataassociatedwiththisarticlecanbefound,

intheonlineversion,athttps://doi.org/10.1016/j.chroma.2017.10

059

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