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.

Abenefitofthisworkflowautomationisdeliveringthecellsinaformatwhereautomationcanbeusedtoexpressproteinandpurifyandrecovertheexpressedprotein.

PipetteTipColumnforAutomatedPlasmidPurification.Completeautomationofplasmidminiandmidiscalepurificationsisaccomplishedwithpipettetipcolumns.(A)Thecolumnisbaseduponapipettetip.Athinfritscreenatthebottomofthepipettetipcolumnholdstheresininplace.(B)Thecolumnsareconfiguredtobeusedwitharoboticliquidhandler,includingthoseequippedwitha96-channelhead.

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

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)

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)

PhyTipColumnDesignPhyTipcolumnsarebaseduponpipettetipsofeither200µLor1mLvolumesizes.Thinfritscreensretaintheresininsideadiscretearea.Achoiceofresinsandresinvolumesareavailable.

Top screen - retains resin

Bottom screen - retains resin

Resin maintained in discrete area

TherangeofbidirectionalcolumnsizesavailableExtractioncolumnsRaininTip5-320µLbedOtherrobots5and20µLbedRangeofstandardresinsProteinA,G,ProPlus,Ni-NTAIMAC,GST,ProPlus,Streptavidin.CustomresinsavailableOthercolumntypesNormalphaseReversephaseIonexchangeVirtuallyALLchromatographicbiomoleculeseparationachievableinthisformat96atatimeProtein

PhyTipcolumnsavailablefor96-sampleparallelprocessingPhyTipcolumnsarecompatiblefor96-channelliquidhandlingrobotsincludingAgilent,Beckman,Caliber,DynamicDevices,HamiltonandPerkinElmer.Proteinpurificationdeliverstheindustry’shighestconcentrationfromdeepwellplatesamples.

CaptureEfficiencyasaFunctionofResidenceTimePipettetipcolumnspackedwithaffinityresinareusedforparallelandautomatedmammalianproteinpurification.SeveraldifferentbedsizescolumnsofProteinAwereusedtocaptureanIgG.Within2to3minutesofbackandforthflowinteractionthecaptureiscompleteregardlessofthebedsize.

CaptureEfficiencyControlledbyBack-and-forthCyclesThedatafrompreviousslideisshownasafunctionofnumberofcapturecycles.Within5–7backandforthcycles)ofbackandforthflowinteractionthecaptureiscomplete.

ProteinACapacityIgGrecoveryfroma5,10,20,40,80,160,and320µLbedcolumnsisalinearplotofcapacityvs.columnbedvolume.Theseparationconditionsdevelopedwithsmallmicrocolumnswithbackandforthflowcanbeappliedtoallsizedcolumnsincludingmanufacturingcolumns.

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.