Kappaselect

GEHealthcareDatafile28-9448-22AACustomDesignedMediaAntibodyfragments,especiallyFabs,aregettingincreasedattentionaspotentialbiopharmaceuticalsbecausetheyhavesomeadvantagesovermonoclonalantibodies(MAbs).Forexample,Fabsshowimprovedpharmacokineticsfortissuepenetrationandcanbindtotargetsinaccessibletoconventionalantigen-bindingsites.KappaSelectisanaffinitymediumdesignedforthepurificationofFab(kappa)fragments,enablinganefficientcapturestepwithhighpurityandyield.KappaSelectispartofGEHealthcare’sCustomDesignedMediaprogram.KappaSelectaffinitychromatographymediumprovides:• Efficient, industrial-scale capture of Fabs by affinity chromatography• High binding capacity for Fabs • Rigid agarose base matrix allows high flow rates and processingoflargesamplevolumesforincreasedthroughput• Non-mammalian derived product reduces regulatory concernsintheproductionofFabsforclinicalapplications• Low ligand leakage, which ensures increased Fab purity andproductivityCharacteristicsofthemediumKappaSelectisbasedonahighlyrigidagarosebasematrixthat allows high flow rates and low back pressure at large scale.KappaSelectisanaffinitymediumfeaturingaligandthatbindstotheconstantregionofthekappalightchain(i.e.,fragmentslackingtheconstantregionofthekappalightchainwillnotbind;Fig1).KappaSelectisthereforecapableofbindingothertargetmoleculescontainingtheconstantregionofthekappalightchain,forexample,IgAandIgM.Theligandisattachedtothematrixviaalonghydrophilicspacerarmtomakeiteasilyavailableforbindingtothetargetmolecule(Fig2).Theligandisbasedonasingle-chainantibodyfragmentthatisscreenedforhumanIgkappa.Theligandisproducedinayeastexpressionsystem,wherefermentationandsubsequentpurification/formulationisperformedintheabsenceofmammaliancomponents.ThecharacteristicsofKappaSelectaresummarizedinTable1.KappaSelectFig1.AntibodystructureandbindingsiteforKappaSelectligandtoFabfragment.Fig2.PartialstructureofKappaSelect.OOOHHNLigandHNVLCLVHCHVLCLVHCHVLCLVHCHVLCLVHCHVLCLVHCHFabFcHeavychainLightchain–S–S––S–S––S–S––S–S–IgGBindingsiteforKappaSelectligandFabF(ab)2—S—S—=Papaindigestion=Pepsindigestion=Disulfidebridge=Carbohydrate–S–S–Table1.MaincharacteristicsofKappaSelectMatrixHighlycross-linkedhigh-flowagaroseParticlesize175µm(d50v)Ligand Recombinant protein (Mr13000),producedinS.cerevisiae,thatbindstoconstantregionofFabkappalightchainLigand density Approx. 5 mg/ml of mediumBindingcapacity2Approx.11mgFab/mlofmediumFlowvelocityAtleast600cm/hina1mcolumnwith20cmbedheightat20°Cusingbufferswiththesameviscosityaswaterat3bar(0.3MPa)pHstabilitylongterm3–10shortterm2–12Workingtemperature34°Cto30°C1d50visthemeanparticlesizeofthecumulativevolumedistribution2DeterminedusingmultiwellplatesandpolyclonalFabasreagent3 Recommended long-term storage conditions: 4°C to 8°C, 20% ethanol209/200828-9448-22AAPrinciplesGeneralaffinitychromatographyprinciplesexploitanimmobilizedligandthatadsorbsaspecificmoleculeorgroupofmoleculesundersuitablebindingconditionsanddesorbsthemduringsuitableelutionconditions.Theseconditionsdependonthetargetmolecule,feedcomposition,andthechromatographymedium,andthesemustbestudiedtogetherwithotherchromatographicparameters(e.g., sample load, flow velocity, bed height, regeneration, cleaning-in-place,etc.)toestablishtheconditionsthatwillbindthelargestamountoftargetmoleculeintheshortesttimeandwiththehighestproductrecovery.AtypicalprotocolforusingKappaSelect,withrecommendedbuffers, is described below:Equilibration/washingbuffer:Phosphatebufferedsaline(PBS),pH7.4(0.01Mphosphatebuffer,0.0027MKCl,0.14M NaCl)Elutionbuffer:0.1Mglycinebuffer,pH31.PackthecolumnwithKappaSelect.2.Equilibratewith10columnvolumes(CV)ofequilibrationbuffer.3. Load the sample.4.Washwithwashingbuffer.5.Elutewith5to10CVofelutionbuffer.ImmediatelyadjustelutedfractionstophysiologicpHbyaddingneutralizationbuffer(e.g.1MTris,pH7.5–8.5).Regeneration should restore the original function of themedium.Dependingonthenatureofthesample,regenerationisnormallyperformedaftereachcycle,followedbyre-equilibrationinstartbuffer.Inordertopreventbuild-upofcontaminantsovertime,morerigorousprotocolsmayhavetobeapplied(seeCleaning-in-placeandsanitization).StabilityTheligandisimmobilizedtotheagarosebasematrixviastableamidebondsthatensurehighchemicalstabilityandlowleakage.Figure3showsthestabilityofKappaSelectafterstorageindifferentsolutionsofvariouspHat20°Cduringone week. Ligand leakage is low in the pH range 2 to 12, andtherewasonlyaminoreffectonFab-bindingcapacitywhenKappaSelectwasstoredinsolutionsofpH1,2and12(oneweekat20°C;Fig4).AtpHvalues12,bothcarbonandnitrogenarereleasedwhichindicateshydrolysisoftheligand.LeakageassayAnassayfordeterminationofligandleakageisavailablefrom BAC BV (Bio Affinity Company, Netherlands) through theirwebsite().Fig3.StabilityofKappaSelectatdifferentpH.02040608010012002468101214pHLeakage(ppm)carbonnitrogenCleaning-in-place(CIP)andsanitizationAstudywasperformedwhereKappaSelectwastreatedwithvariouscommonlyusedCIPsolutions.TheFabbindingcapacitywasdeterminedaftersettimeintervals(Fig5).KappaS