Purification Strategies to Process.docx
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Purification Strategies to Process.docx
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PurificationStrategiestoProcess
PurificationStrategiestoProcess5g/LTitersofMonoclonalAntibodies
Alteringtheorderofoperations,usingnewresins,andincreasingdynamicbindingcapacitycanobviatetheneedformajorfaciltychanges.
Mar2,2009
By:
MelodyTrexler-Schmidt,StefanieSze-Khoo,AmberR.Cothran,BinhQ.Thai,SandySargis,BenedicteLebreton,BrianKelley,GregoryS.Blank
BioPharmInternationalSupplements
ABSTRACT
Monoclonalantibodypurificationprocesseshavebeenchallengedrecentlytorecover5g/Lcellculturetitersinexistingmanufacturingfacilities.ThisrequiresthemodificationofaplatformpurificationprocesscomprisingProteinAchromatographyastherobustcapturestepfollowedbytwoionexchangechromatographysteps.Becausebufferandpooltankvolumesaretheprimaryfacilitybottlenecks,processdevelopmenteffortshavefocusedprimarilyontheorderofunitoperations,thedynamicbindingcapacityofcurrentandnewresins,reducingpoolmanipulationsandbufferconsumption,andelutionbufferselection.Thisarticlepresentsexamplesinwhichnewoperatingconditionsorpurificationtechnologiescanhelptoaccommodateincreasesintiterwithoutextensivechangestothemanufacturingfacilityorequipmentrequirements.
Lonza,Ltd.,Basel,Switzerland
Overthepastfiveyears,therehasbeenadramaticincreaseinthetitersofrecombinantproteins,particularlyintheproductionofIgGmonoclonalantibodies(MAbs)usingChinesehamsterovary(CHO)cells.Improvementsincelllines,mediacomposition,andcellcultureoperatingconditionshaveallcontributedtohigherexpressionlevels.Inmostcases,thesechangeshavehadlittleornoimpactonmanufacturingequipmentandfacilities.However,astitershaveincreasedbyanorderofmagnitudeormoreusingexistingcellculturebioreactors,therehasbeenaconcomitantincreaseinthestartingmassofproteinenteringdownstreampurification.Thisdevelopmenthasresultedinashiftinemphasisfromincreasingvolumetricproductivityforlow-titerprocessestohandlingsignificantincreasesintheamountofproteinduringdownstreamprocessing,particularlyinexistingfacilities.Althoughincrementalincreasesintiterscanbehandledbyincreasingthescaleofpurificationunitoperations(e.g.,usinglargerchromatographycolumnsandfilters),atsomepointlinearscalingexceedsthephysicallimitsofexistingfacilities.
Toaddressthepurificationchallengesofhighantibodytiters,itisimperativetodeterminethespecificlimitationsofacompany'smanufacturingfacilityorfacilities.Thiscanbestbeaccomplishedbyusingamodelthatperformsafacilityfitusinganexistingprocess.Suchamodelcanidentifythebottlenecksforeachunitoperationataspecificmanufacturingfacilityasafunctionofantibodytiter.Insomecases,itmaybenecessarytousemultiplemodeliterationstofindabottleneck,modelachangetotheprocessorfacility,andthendeterminewherethenextbottleneckmaybe.Forthepurposesofthisarticle,weassumethatonecellculturebatchispurifiedtoproduceonebulk.Alternativeprocessoptionssuchassplitbatchprocessesmaybeconsidered,andthesecouldpotentiallyallowhigherrecoverabletitersfromonecellculturebatch.
PlatformProcessBottlenecks
Figure1
AlmostallcurrentantibodypurificationplatformprocessesuseProteinAaffinitychromatographyasthecapturestepbecauseitisrobust,widelyapplicable,andfacilitatestheimplementationofasuccessfulstandardpurificationprocess.1,2Figure1showstwoplatformpurificationprocessesthatwehaveimplemented.Aftercellandproductseparationduringharvestoperations,ProteinAaffinitychromatographyisusedastheinitialcapturestepwithlow-pHviralinactivationintheProteinApool.Dependingontheplatform,thisisfollowedbyeitherthecationexchangechromatographystepinbind-and-elutemode3ortheanionexchangechromatographystepinflow-throughmode.Thevirusfiltrationoperationisplacedbetweenthetwoionexchangechromatographysteps.Afterthethirdchromatographystep,ultrafiltration–diafiltrationisperformedusingtangentialflowfiltration(TFF)forfinalproductformulation.
PotentialvolumebottleneckscausedbyincreasedtitersareincludedforeachunitoperationofeachprocessshowninFigure1.Underneatheachpotentialbottleneckareprocessoptionsthatshouldbeconsideredforoptimizationtoavoidfacilitybottlenecks,andthosehavingthegreatestimpactareshowninred.Wehavefoundthattheprimarybottlenecksinexistingfacilitiesarebufferandpooltankvolumes,whereassecondarybottlenecksincludepumpcapacitiesorpipediametersthatlimitflowrates.Forthesereasons,theuseoflargerchromatographycolumnsorfiltersurfaceareasisnotsufficienttohandlehighertiters.
BenefitsofIncreasedResinCapacity
Oneapproachthatallowsgreateramountsofantibodytobepurifiedwithoutincreasingcolumnvolumeistoincreasethebindingcapacityofthechromatographystep.Increasingtheloadcapacitymeanstheoverallamountofprocessedantibodycanbeincreasedwithoutincreasingcolumnsizeorvolume.Sincebuffervolumesareusuallybasedoncolumnvolume,increasingtheloadmasswithoutincreasingthecolumnvolumerequiresnoincreaseinbuffervolumes.Thesameappliesforelutionconditionswherethepoolvolumewillonlybeaffectedslightlybyoverallproteinmasswhenusingafinalopticaldensity(OD)cutoff,ornotaffectedatallwhenusingconstantvolumepoolingcriteria.
Table1.MaximumexpressionlevelofaMAbthatcanbepurifiedasafunctionofProteinAdynamicbindingcapacity.Thisassumesnoothermodificationsweremadetothefacility,equipment,oroperatingconditions.ForProteinAchromatography,Table1showstheincreaseintitersthatcanbetoleratedinanexistingfacilitywithoutincreasingthesizeofthechromatographycolumn.Atadynamicbindingcapacity(DBC)of15g/L,amaximumtiterof1g/Lcanbeaccommodatedinthefacility.IftheDBCcanbedoubledto30g/L,thenthemaximumrecoverabletiterincreasesto3g/LandaDBCof45g/Laccommodatestitersofupto5g/L.AnincreaseinDBCcanbeachievedinseveralways:
byloweringtheflowrateatconstantbedheight,increasingthebedheightatconstantflowratetoincreaseresidencetime,and/orbychangingtoadifferentProteinAresinwithhighercapacity.4Inaddition,increasingthemaximummanufacturingloaddensityfromlowbreakthroughlevels(0.5–2%DBC)toslightlyhigherlevels(3–5%DBC)alsoenablesthepurificationof>5g/Ltitersandmostlikelyhasaninsignificantimpactonstepyield.DifferencesinDBCresultingfrompackingorresinlot-to-lotvariabilityareconsiderednegligible.
Figure2
ProteinAresinsalldemonstratehigherDBCswithdecreasingflowrate,althoughtheslopeofthecurvemayvaryfromresintoresin.AsshownwiththeDBCcurveforresinAinFigure2,theDBCcanbeincreasedfrom17g/Lat40columnvolumes(CV)/hto38g/Lbydecreasingtheflowrateto10CV/h,thereforedoublingtheamountofantibodyrecoveredwithnootherprocesschanges.Processingtimeandfacilitythroughputneedtobeassessedtodeterminewhetherreducingtheflowratealsoreducesoverallcapacity.5ThismaybeaddressedinpartbyreducingtheflowrateonlyfortheloadingphaseoftheProteinAstepandthenincreasingtheflowratefortheotherphasessuchasequilibration,wash,elution,andregeneration.Unpublisheddatafromourlaboratoriesindicatesthatstepyieldisnotaffectedbyoperatingtheseotherphasesathigherflowrates.Anotherpotentialstrategyistoconducttheloadphaseattwoflowrates,anddecreasetheflowrateduringthefinalstagesoftheloadphase.6
Table2showshowincreasingtheDBCaffectsacationexchangechromatographystepforaparticularantibodyandhighlightsthebottlenecksinanexistingfacility.Inthisexample,aDBCof50g/Lcanaccommodatea1g/Ltiterwithoutconstraints.Atiterof2g/Lisconstrainedbythebuffertankvolume(theequilibrationbufferinthiscase),andifthislimitationisremoved(perhapsbyin-linedilution),theconstraintbecomesthepooltankvolumeatatiterof3g/L.Increasingthebindingcapacityofthecationexchangeresinto75g/Lor100g/Lallowstitersof3g/Land4g/Ltobeaccommodated,respectively.Inbothcases,thebuffertanklimitationoccursatlowertitersthanthepooltankvolumelimitation.Thisassumesthatallotherprocessparametersremainunchangedwhenthecapacityincreases.However,onekeyconsiderationduringprocessdevelopmentistheseparationoftheantibodyandimpuritiesatthesehigherloaddensitiesorwithdifferent(highercapacity)resinsthatmayhavedifferentselectivities.
Table2.MaximumexpressionlevelofaMAbthatcanbepurifiedasafunctionofthedynamicbindingcapacityofcationexchangechromatography.Thisassumesnoothermodificationsweremadetothefacility,equipment,oroperatingconditions.
DecreasingBufferConsumptionandUsingIn-LineDilution
Table3.MaximumexpressionlevelofaMAbthatcanbepurifiedbasedondifferentprocessorfacilitymodifications
Asdiscussedabove,itisimportanttotakeaholisticviewoftheprocessandcarryoutmultiplemodeliterationsthatallowbottleneckstobeidentifiedandremoved,andsubsequentbottleneckstobeidentified.Inexaminingtheentirepurificationprocess,Table3showsthemaximumtiterthatcanbepurifieddownstreambyeachunitoperation.ThebaselineProteinAstepcanaccommodateatiterof1.3g/L,butmodificationoftheequilibrationandvariouswashphasestominimizetheProteinAbuffervolumesincreasesthisto1.6
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