Automated High Throughput Purification of Mammalian ... · Automated High Throughput Purification...
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AutomatedHighThroughputPurificationofMammalianProteinsbyTransientTransfectionAbstract1ofthepaper:“ImplementationofanAutomatedHigh-ThroughputPlasmidDNAProductionPipeline”KarenBilleci1,ChristopherSuh2,TinaDiIoia1,LovejitSingh1,RyanAbraham1,AnneBaldwin1,andStephenMonteclaro1JLabAutom.2016Feb8.pii:2211068216630547.[Epub]GenentechBiologicsResourceManagement,1DNAWay,SouthSanFrancisco,CA94080PhyNexus,Inc,3670CharterParkDr. SuiteA,SanJose,CA95136CorrespondingAuthor:Billeci.karen@gene.comHighthroughputproductionandcharacterizationofmammalianproteinsandtherapeuticproteincandidatesisbecomingthestandardrequirementforlifescienceresearch.ThisisenabledbycompletelyautomatedhighthroughputDNAproduction,transienttransfectionandproteinpurification.Thedriverforimplementingthisprocessistheneedforgeneratingrecombinantmammalianproteinswithproperposttranslationalmodificationsthatareproperlyfolded.Althoughthetransfectedmammaliancellsformedthroughtransienttransfectionareonlyfleeting,thesecellsareabletoproduceuseableamountsofresearchproteinsfromclonedgenesandgenevariantsofinterest.Thispaperhighlightstechnologythatintegratesplasmidproductionpurificationforcompletelyautomatedtransienttransfectionofmammaliancellsandhighthroughputmammalianproteinproduction
AutomatedTransientTransfectionandProteinProduction
STEP1 Automatedtransformation,platingandcolonygrowth
STEP2 Automatedcolonypickingandgrowthin96or24wellformat
STEP3 Automatedplasmidpurificationfromcellpellet
STEP4 Automatednormalizationofconcentrationofpureplasmids
STEP5 Automatedtransienttransfectionofmammaliancells
STEP6 AutomatedPurificationofMammalianProteins
Theautomationoftransienttransfectionofmammaliancellsrequiresthatsampleformattingandsampleprocessingareconducivetoautomatedworkflow.Configurationforautomationmustbeginwithtransformationandcontinuewithcolonypicking,plasmidpurificationfromcellpelletandnormalizationofpurifiedplasmid.
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Abenefitofthisworkflowautomationisdeliveringthecellsinaformatwhereautomationcanbeusedtoexpressproteinandpurifyandrecovertheexpressedprotein.
PipetteTipColumnforAutomatedPlasmidPurification.Completeautomationofplasmidminiandmidiscalepurificationsisaccomplishedwithpipettetipcolumns.(A)Thecolumnisbaseduponapipettetip.Athinfritscreenatthebottomofthepipettetipcolumnholdstheresininplace.(B)Thecolumnsareconfiguredtobeusedwitharoboticliquidhandler,includingthoseequippedwitha96-channelhead.
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AutomatedMammalianProteinProductionviaTransientTransfectionFlowPathAFrozenglycerolstocksofE.colitransformedwithplasmidsstocksareusedtoinoculate1.4mLmediumin96-wellformat.Afterculturegrowth,plasmidDNAispurifiedusingLysateDirectPhyTipcolumns.Forhighthroughputplasmidminipreppurifications,96sampleplatesaremanagedusingahotelandthesystemiscapableofprocessingmorethan24,000samplesamonth.Afterpurification,softwareandautomatedliquidhandlingareusedtonormalizetheDNAconcentrations.TheplasmidDNAisusedfortransienttransfectionof4mLmammaliancellcultures.Four24-wellculturesareincubatedtoexpresssecretedprotein.Supernatantisreformattedinto96-wellformatwherePhyTipcolumnspackedwithproteinaffinityresinisusedtopurifyrecombinantmammalianproteins.
Step1 PlasmidMidiPrep
Normalize[DNA]
1.4mLE.ColiCultures
PlasmidDNA50-150µg/µL
4mLMammalian CellCultures
CulturedMedia CellSupernatants
TransientTransfection
Protein Purification
FrozenGlycerolStocks
Inoculate Step2 Step3
Step4 Step5 Step6
Expression &
Re-array
Normalized PlasmidDNA
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AutomatedMammalianProteinProductionViaTransientTransfectionFlowPathBPlasmidsstocksareusedtransformcompetentE.colicells.Aftertransformation,thecellsareplatedontoLBagarplates.Acolonypickerisusedtoinoculate1.4mLmediumin96-wellformat.Afterculturegrowth,plasmidDNAispurifiedusingLysateDirectPhyTipcolumns.Forhighthroughputplasmidminipreppurifications,96sampleplatesaremanagedusingahotelandthesystemiscapableofprocessingmorethan24,000samplesamonth.Afterpurification,softwareandautomatedliquidhandlingareusedtonormalizetheDNAconcentrations.TheplasmidDNAisusedfortransienttransfectionof4mLmammaliancellcultures.Four24-wellculturesareincubatedtoexpresssecretedprotein.Supernatantisreformattedinto96-wellformatwherePhyTipcolumnspackedwithproteinaffinityresinisusedtopurifyrecombinantmammalianproteins.Mammalianproteinproductioncanbefullyautomatedusingliquidhandlingautomationandstandardlabware.Forapplicationsrequiring100µgofprotein,4mLtransfectionsaresufficient.Mini-scaleplasmidDNApurificationofExpressionconstructsfrom1.4mLE.coliculturesyieldsataconcentrationof50ng/µL.ThisDNAisnormalizedto10ng/µLpriortotransfection.
PlasmidStocks E.ColiAgarPlates
PickColonies
PlasmidMidiPrep
Normalize[DNA]
1.4mLE.coliCultures PlasmidDNA50-150ng/µL
4mLMammalian CellCultures
CulturedMedia CellSupernatants
TransientTransfection
Protein Purification
Transform &
Plate
Step1 Step2 Step3 Step4
Step5 Step6 Step7
Expression &
Re-array
Normalized PlasmidDNA 15-50ng/µL
(x12)
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AutomatedMammalianProteinProductionviaTransientTransfectionFlowPathCApplicationsrequiringalargermassofproteinrequireamidi-scaleplasmidDNApurificationofexpressionconstructs.PlasmidsstocksareusedtransformcompetentE.colicells.Aftertransformation,thecellsareplatedontoLBagarplates.Acolonypickerisusedtoinoculate4mLmediumin24-wellformat.Afterculturegrowth,plasmidDNAispurifiedusingLysateDirectPhyTipcolumns.Afterpurification,softwareandautomatedliquidhandlingareusedtonormalizetheDNAconcentrations.TheplasmidDNAisusedfortransienttransfectionof15mLmammaliancellculturesin50mLtubes.Culturesareincubatedtoexpresssecretedproteinandsupernatantisreformattedinto96-wellformat.PhyTipcolumnspackedwithproteinaffinityresinareusedtopurifyrecombinantmammalianproteins.Mammalianproteinproductioncanbefullyautomatedusingliquidhandlingautomationandstandardlabware.
E.ColiAgarPlates
PickColonies
PlasmidMidiPrep
Normalize[DNA]
4mLE.coliCultures
PlasmidDNA50-150ng/µL
15mLMammalian CellCultures
CulturedMedia CellSupernatants
TransientTransfection
Protein Purification
Transform &
Plate
Step1 Step2 Step3 Step4
Step5 Step6 Step7
Expression &
Re-array
Normalized PlasmidDNA 15-50ng/µL
(x12)
PlasmidStocks
(x96)
(x4)
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PhyTipColumnDesignPhyTipcolumnsarebaseduponpipettetipsofeither200µLor1mLvolumesizes.Thinfritscreensretaintheresininsideadiscretearea.Achoiceofresinsandresinvolumesareavailable.
Top screen - retains resin
Bottom screen - retains resin
Resin maintained in discrete area
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TherangeofbidirectionalcolumnsizesavailableExtractioncolumnsRaininTip5-320µLbedOtherrobots5and20µLbedRangeofstandardresinsProteinA,G,ProPlus,Ni-NTAIMAC,GST,ProPlus,Streptavidin.CustomresinsavailableOthercolumntypesNormalphaseReversephaseIonexchangeVirtuallyALLchromatographicbiomoleculeseparationachievableinthisformat96atatimeProtein
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PhyTipcolumnsavailablefor96-sampleparallelprocessingPhyTipcolumnsarecompatiblefor96-channelliquidhandlingrobotsincludingAgilent,Beckman,Caliber,DynamicDevices,HamiltonandPerkinElmer.Proteinpurificationdeliverstheindustry’shighestconcentrationfromdeepwellplatesamples.
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CaptureEfficiencyasaFunctionofResidenceTimePipettetipcolumnspackedwithaffinityresinareusedforparallelandautomatedmammalianproteinpurification.SeveraldifferentbedsizescolumnsofProteinAwereusedtocaptureanIgG.Within2to3minutesofbackandforthflowinteractionthecaptureiscompleteregardlessofthebedsize.
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CaptureEfficiencyControlledbyBack-and-forthCyclesThedatafrompreviousslideisshownasafunctionofnumberofcapturecycles.Within5–7backandforthcycles)ofbackandforthflowinteractionthecaptureiscomplete.
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ProteinACapacityIgGrecoveryfroma5,10,20,40,80,160,and320µLbedcolumnsisalinearplotofcapacityvs.columnbedvolume.Theseparationconditionsdevelopedwithsmallmicrocolumnswithbackandforthflowcanbeappliedtoallsizedcolumnsincludingmanufacturingcolumns.
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ProcedureforhighthroughputmammalianproteinproductionA. AutomatedPlasmidProduction
1)SetupE.colicultures.Aliquot1.4mLTBtoa2mLdeep-wellplatewithsquarewellsandroundbottom
2)Innoculatemediumwithsinglecolonyofinterestusingacolonypicker3)SealtheplatewithaThompsonseal4)Incubateat37°Cwithshakingat300rpmfor16hours5)HarvestcellswhenOD600reaches186)Spinplatefor15minutesat1,000rpm7)Decantspentmedia8)PlaceplateofcellpelletsonliquidhandlingrobotdeckandsetupforLysateDirectPhyTipColumn
purification9)MeasurePlasmidDNAconcentrationandnormalizeto50ng/µL
B.AutomatedTransientTransfection
1)Dispense850µLExpi293Fcellsat2.0e6cells/mLtoeachwellofa2mLdeepwellplate2)Incubateat37°C,8%CO2withshakingat1,000rpmwith3mmorbitaldiameterfor2hours3)Dilute20µLplasmidDNA,correspondingto1µg,with80µLofasolutionconsistingofDMEM
mediumsupplementedwith25kDaPEIat0.23mM.4)Usingautomationmixavolumeof85µLthreetimes.Incubateat4°Cfor10minutes.Thenaddto
thecells.5)Incubateat37°C,8%CO2,80%humiditywithshaking.6)Addfeedreagents24hoursposttransfectionaspermanufacturer’srecommendations.
C.AutomatedMammalianProteinPurification1)Spincellculturesat5kRPMfor15minutes2)Transfersupernatanttofresh2mLDeepWellPlate3)Use1mLPhyTipcolumnspackedwith20µLofProteinAresintopurifyproteins4)LoadPhyTipcolumnsandequilibratein1mLwith1cycleofprocessing.Whilemaintainingthetipof
thePhyTipcolumn1mmabovethebottomofthewell,onecycleconsistsofaspiratingactualvolumeless50µLat0.5mL/minute,pause20seconds,dispenseactualvolumeless50µLat0.5mL/minute,andpause20seconds.
5)Capturesampleusing4cyclesat0.25mL/minute6)Washin1mLWash1bufferusing1cycle7)Washin1mLWash2bufferusing1cycle8)Eluteusing60µLElutionbuffer.Foranyvolumelessthan250µL,aspirateanddispenseanadditional
230µL.Use4cycles.9)Neutralizebyadding15µLofneutralizationbufferandmixwith1cycleof75µL.