Full automation and validation of a flexible ELISA platform

JournalofPharmaceuticalandBiomedicalAnalysis70 (2012) 580–586

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JournalofPharmaceuticalandBiomedicalAnalysis

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a

Shortcommunication

Fullautomationandvalidationofa exibleELISAplatformforhostcellproteinandproteinAimpuritydetectioninbiopharmaceuticals

GuillaumeRey,MarkusW.Wendeler

NovartisPharmaAG,TRDBiologics–AnalyticalR&D,CH-4002Basel,Switzerland

article

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abstract

Articlehistory:

Received20March2012Accepted22May2012

Available online 3 June 2012

Keywords:ELISA

ImmunoassayRobot

Bioanalytics

DownstreamprocessdevelopmentImpurity

Monitoringhostcellprotein(HCP)andproteinAimpuritiesisimportanttoensuresuccessfuldevel-opmentofrecombinantantibodydrugs.Here,wereportthefullautomationandvalidationofanELISAplatformonaroboticsystemthatallowsthedetectionofChinesehamsterovary(CHO)HCPsandresidualproteinAofin-processcontrolsamplesand naldrugsubstance.TheELISAsetupisdesignedtoservethreemaingoals:highsamplethroughput,highqualityofresults,andsamplehandling exibility.Theprocessingofanalysisrequests,determinationofoptimalsampledilutions,andcalculationofimpuritycontentisperformedautomaticallybyaspreadsheet.Upto48samplesinthreeunspikedandspikeddilu-tionseachareprocessedwithin24h.Thedilutionofeachsampleisinpiduallypreparedbasedonthedrugconcentrationandtheexpectedimpuritycontent.Adaptabledilutionprotocolsallowtheanalysisofsampledilutionsrangingfrom1:2to1:2×107.Thevalidityofresultsisassessedbyautomatictestingfordilutionallinearityandspikerecoveryforeachsample.ThisautomatedimpurityELISAfacilitatesmulti-projectprocessdevelopment,iseasilyadaptabletootherimpurityELISAformats,andincreasesanalyticalcapacitybycombining exiblesamplehandlingwithhighdataquality.

© 2012 Elsevier B.V. All rights reserved.

1.Introduction

Anincreasingnumberofbiopharmaceuticalsindevelopmentandtheintroductionofqualitybydesign(QbD)forthemanufactureofbiologicsgenerateagrowingnumberofsamplesforanalytics[1,2].Consequently,analyticalsupportofprocesscharacterizationcaneasilybecomeabottleneck.Automationofgenericmethodsisonestrategytoincreaseanalyticalcapacityandthusallowparallelsupportofmultipledevelopmentprojects[3–5].

Duringthedownstreamprocessingofbiopharmaceuticalssev-eralprocess-derivedimpuritiessuchashostcellproteins(HCPs),residualDNA,and–dependingonthepuri cationprocedure–pro-teinAmustbeseparatedfromtherecombinantproduct[6,7].HCPsandproteinAmayhaveimmunogeniceffectsandthereforetheirremovalmustbecarefullymonitored[8].Inearlydevelopment,proteinimpuritiesarecommonlymonitoredusinggenericenzyme-linkedimmunosorbentassays(ELISAs)toassessproductpurityand

Abbreviations:BSA,bovineserumalbumine;CHO,Chinesehamsterovary;ELISA,enzyme-linkedimmunosorbentassay;HCP,hostcellproteins;LOD,limitofdetec-tion;LOQ,limitofquanti cation;PBS,phosphatebufferedsaline;QbD,qualitybydesign.

Correspondingauthor.Tel.:+41616967065;fax:+41616966808.

E-mailaddress:markus(M.W.Wendeler).

processconsistency[9–11].Althoughgenericmethodslendthem-selvestoautomation,theheterogeneityofsamplesderivedfromdifferentprojectsandpuri cationprocessstepsposesachallengeforautomationintermsofparallelsamplepreparation.Forexam-ple,theHCPcontentofsamplesfromdifferentpuri cationstepsrangesfromseveralmillionppminthebulkharvesttolevelsinthelowppmrangeorevenbelowthelimitofdetection(LOD)inthepuri eddrugsubstance,andrequiresinpidualsampledilutionstoavoidmultipleroundsofreanalysis[12,13].Toallowinpid-ualsampledilutionsandparallelprocessingofdifferenttypesofsampleinthesameautomatedassay,a exiblesampledilutionprocedureisneeded.

Here,wereportthefullautomationofagenericELISAplat-formsupportingHCPandproteinAimpuritydetectionforthedevelopmentoftherapeuticproteins.Thefollowingstepswereautomated:(i)samplepreparationwithdilutionfactorscover-ingsevenordersofmagnitude,(ii)thecompleteELISAprocedure,and(iii)rawdatacaptureincludingdatareductionandimpu-ritycontentdetermination.Calculationofsampledilutions,resultanalysis,andthevaliditycheckofresultsarealsoautomatedusingspreadsheetprocessing.TheautomatedELISAplatformwasvalidated,comparedtotherespectivemanualprocedures,andiscurrentlyusedtosupportproteinimpuritydeterminationforearlyprocessdevelopmentofbiopharmaceuticalsproducedinCHOcells.

0731-7085/$–seefrontmatter© 2012 Elsevier B.V. All rights reserved.http://www.77cn.com.cn/10.1016/j.jpba.2012.05.027

G.Rey,M.W.Wendeler/JournalofPharmaceuticalandBiomedicalAnalysis70 (2012) 580–586

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2.Materialandmethods

2.1.Antibodies,standards,andtestsamples

Polyclonalrabbitanti-CHOHCPantibodyandbiotinylatedrab-bitanti-CHOHCPF(ab )2werecustommade(Biogenes,Berlin,Germany).ConcentratedsupernatantofmocktransfectedCHOcellswasusedforimmunizationandforCHOstandard.Rabbitanti-proteinAantibody(Sigma–Aldrich,Buchs,Switzerland)andbiotinylatedchickenanti-proteinAantibody(OEMconcepts,TomsRiver,USA)wereusedfortheproteinAELISA.MabSelectSuReproteinAwasusedasastandard(GEHealthcare,Glattbrugg,Switzerland).TestsamplesanalyzedintheHCPandproteinAELISAwerein-processsamplesfrombiopharmaceuticalprojectscurrentlyunderdevelopment.

2.2.Roboticsystem,peripheralinstruments,anddatahandlingsoftware

ASTARletroboticsystem(HamiltonRobotics,Bonaduz,Switzerland)wascustom-designedtopreparesampledilutionsandacompletesandwichELISAontwo96wellmicrotiterplates.Itisequippedwith8inpidualpipettingchannels,ahorizon-talshaker(VariomagTeleshake,H+PLabortechnikAG,Germany),twotransportdevices(CO-REGripperandiSWAPRoboticHand;HamiltonRobotics,Bonaduz,Switzerland),anintegratedplate-washer(Powerwasher384,Tecan,Maennedorf,Switzerland),andanintegratedmultidetectionmicroplatereader(SpectraMaxM4,MolecularDevices,Sunnyvale,USA).TheautomatedELISAmethodwasprogrammedusingthesoftwareMICROLAB®STARVectorSoft-ware4.1(HamiltonRobotics,Bonaduz,Switzerland).Automatedd …… 此处隐藏：25301字，全部文档内容请下载后查看。喜欢就下载吧 ……