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IDENTIFICATIONANDQUANTIFICATION OFDRUGS,METABOLITES, DRUGMETABOLIZINGENZYMES, ANDTRANSPORTERS Concepts,Methods,andTranslationalSciences
IDENTIFICATIONAND QUANTIFICATION OFDRUGS, METABOLITES,DRUG METABOLIZING ENZYMES,AND TRANSPORTERS Concepts,Methods,and TranslationalSciences Editedby SHUGUANG MA SWAPAN K.CHOWDHURY
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Contributors FarahAlQaraghuli DepartmentofPharmaceuticalSciences,SchoolofPharmacyand PharmaceuticalSciences,TheStateUniversity ofNewYorkatBuffalo,Buffalo,NY,United States
RavindraVarmaAlluri ClinicalPharmacology andSafetySciences,R&DBioPharmaceuticals, AstraZeneca,Cambridge,UnitedKingdom
SophieM.A.Argon DepartmentofPharmaceutics,SchoolofPharmacy,Universityof Washington,Seattle,WA,UnitedStates
PiyushBajaj DrugSafetyResearchandEvaluation,TakedaPharmaceuticalInternationalCo., Cambridge,MA,UnitedStates
TashingaE.Bapiro DMPK,ResearchandEarly Development,OncologyR&D,AstraZeneca, Cambridge,UnitedKingdom
AbdulBasit DepartmentofPharmaceutics, UniversityofWashington,Seattle;DepartmentofPharmaceuticalSciences,Washington StateUniversity,Spokane,WA,UnitedStates
AndreasBrink RochePharmaResearch andEarlyDevelopment,RocheInnovation CenterBasel,F.Hoffmann-LaRocheLtd,Basel, Switzerland
TingtingCai LaboratoryTestingDivision, WuXiAppTec,Nanjing,China
JoseCastro-Perez AgiosPharmaceuticals,Inc., Cambridge,MA,UnitedStates
JaeH.Chang PreclinicalDevelopment,ORIC Pharmaceuticals,SouthSanFrancisco,CA, UnitedStates
EugeneChia-TeChen DepartmentofDrug MetabolismandPharmacokinetics,Genentech, SouthSanFrancisco,CA,UnitedStates
MarieCroft PharmaronABS,Germantown, MD,UnitedStates
LiamEvans HyphaDiscoveryLtd.,Oxfordshire,UnitedKingdom
RaymondEvers DepartmentofPharmacokinetics,PharmacodynamicsandDrugMetabolism, Merck&CoInc.,Kenilworth,NJ,UnitedStates
RobertS.Foti Pharmacokinetics,PharmacodynamicsandDrugMetabolism,Merck ResearchLaboratories,Boston,MA,United States
AdrianJ.Fretland DMPK,ResearchandEarly Development,OncologyR&D,AstraZeneca, Waltham,MA,UnitedStates
ChristopherGemski TranslationalResearch BioassayandImmunogenicityGroup,Drug MetabolismandPharmacokineticDepartment, TakedaPharmaceuticalsInternationalCo., Cambridge,MA,UnitedStates
AnimaGhosal IndependentConsultant, Piscataway,NJ,UnitedStates
JiaHao DrugMetabolism,GileadSciences Inc,FosterCity,CA,UnitedStates
SatyajeetHaridas TranslationalResearchBioassayandImmunogenicityGroup,Drug MetabolismandPharmacokineticDepartment, TakedaPharmaceuticalsInternationalCo., Cambridge,MA,UnitedStates
SimonHauri RochePharmaResearchandEarly Development,RocheInnovationCenterBasel, F.Hoffmann-LaRocheLtd,Basel,Switzerland
NinaIsoherranen DepartmentofPharmaceutics,UniversityofWashington,Seattle, WA,UnitedStates
WenyingJian DMPK,JanssenR&D,Spring House,PA,UnitedStates
KevinJohnson DrugMetabolismandPharmacokinetics,Genentech,SouthSanFrancisco, CA,UnitedStates
BarryJones DMPK,ResearchandEarlyDevelopment,OncologyR&D,AstraZeneca,Cambridge,UnitedKingdom
RobertS.Jones DrugMetabolismandPharmacokinetics,Genentech,SouthSanFrancisco, CA,UnitedStates
JanFelixJoseph FreieUniversitaetBerlin,InstituteofPharmacy—PharmaceuticalAnalysis; FreieUniversitaetBerlin,Departmentof Biology,Chemistry,Pharmacy,CoreFacility BioSupraMol,Berlin,Germany
S.CyrusKhojasteh DrugMetabolismand Pharmacokinetics,Genentech,SouthSan Francisco,CA,UnitedStates
YurongLai DrugMetabolism,GileadSciences Inc,FosterCity,CA,UnitedStates
HoaLe DrugMetabolism,GileadSciences, FosterCity,CA,UnitedStates
XiaominLiang DrugMetabolism,GileadSciencesInc,FosterCity,CA,UnitedStates
LimingLiu ProductDevelopment,Curon BiopharmaceuticalLtd,Shanghai,People’s RepublicofChina
FilipeLopes RochePharmaResearchand EarlyDevelopment,RocheInnovationCenter Basel,F.Hoffmann-LaRocheLtd,Basel, Switzerland
JustinQ.Ly DrugMetabolismandPharmacokinetics,Genentech,SouthSanFrancisco,CA, UnitedStates
ShuguangMa DrugMetabolismandPharmacokinetics,GenentechInc.,SouthSanFrancisco, CA,UnitedStates
RoshiniMarkandu DMPK,ResearchandEarly Development,OncologyR&D,AstraZeneca, Cambridge,UnitedKingdom
RosalindeMasereeuw DivisionofPharmacology,UtrechtInstituteforPharmaceutical Sciences,Utrecht,TheNetherlands
J.EricMcDuffie Investigative&Mechanistic Toxicology,JanssenResearch&Development, SanDiego,CA,UnitedStates
KaushikMitra DepartmentofDrugMetabolismandPharmacokinetics,JanssenResearch andDevelopment,Springhouse,PA,United States
DianaMontgomery DepartmentofPharmacokinetics,PharmacodynamicsandDrug Metabolism,Merck&CoInc.,Kenilworth,NJ, UnitedStates
AlexandraL.Orton DMPK,ResearchandEarly Development,OncologyR&D,AstraZeneca, Cambridge,UnitedKingdom
KatieH.Owens DepartmentofPharmaceutics, SchoolofPharmacy,UniversityofWashington,Seattle,WA,UnitedStates
AxelPahler RochePharmaResearchand EarlyDevelopment,RocheInnovationCenter Basel,F.Hoffmann-LaRocheLtd,Basel, Switzerland
MariaKristinaParr FreieUniversitaetBerlin, InstituteofPharmacy—PharmaceuticalAnalysis,Berlin,Germany
ShefaliPatel DMPK,JanssenR&D,Spring House,PA,UnitedStates
IchikoD.Petrie DepartmentofPharmaceutics,SchoolofPharmacy,Universityof Washington,Seattle,WA,UnitedStates
RichardPhipps HyphaDiscoveryLtd.,Oxfordshire,UnitedKingdom
ChandraPrakash AgiosPharmaceuticals,Inc., Cambridge,MA,UnitedStates
BhagwatPrasad DepartmentofPharmaceutics, UniversityofWashington,Seattle;Department ofPharmaceuticalSciences,WashingtonState University,Spokane,WA,UnitedStates
IsabelleRagueneau-Majlessi Departmentof Pharmaceutics,SchoolofPharmacy,UniversityofWashington,Seattle,WA,UnitedStates
VenkateshPillaReddy DMPK,Researchand EarlyDevelopment,OncologyR&D;DepartmentsofModelingandSimulation,Early OncologyDrugMetabolismandPharmacokinetics,R&DOncology,AstraZeneca, Cambridge,UnitedKingdom
EllenRiddle DrugMetabolism,GileadSciences Inc,FosterCity,CA,UnitedStates
QianRuan PharmaceuticalCandidateCharacterization,Bristol-MyersSquibb,Princeton, NJ,UnitedStates
SimoneSchadt RochePharmaResearchand EarlyDevelopment,RocheInnovationCenter Basel,F.Hoffmann-LaRocheLtd,Basel, Switzerland
DhavalK.Shah DepartmentofPharmaceutical Sciences,SchoolofPharmacyandPharmaceuticalSciences,TheStateUniversityofNew YorkatBuffalo,Buffalo,NY,UnitedStates
JuliaShanu-Wilson HyphaDiscoveryLtd., Oxfordshire,UnitedKingdom
KellyMacLennanStaiger DrugMetabolism, GileadSciencesInc,FosterCity,CA,UnitedStates
JonathanSteele HyphaDiscoveryLtd.,Oxfordshire,UnitedKingdom
ManthenaV.S.Varma MedicineDesign, WorldwideR&D,PfizerInc.,Groton,CT, UnitedStates
MatthewP.Wagoner DrugSafetyResearch andEvaluation,TakedaPharmaceuticalInternationalCo.,Cambridge,MA,UnitedStates
NaidongWeng DMPK,JanssenR&D,Spring House,PA,UnitedStates
StephenWrigley HyphaDiscoveryLtd., Oxfordshire,UnitedKingdom
CaishengWu SchoolofPharmaceuticalSciences,XiamenUniversity,Xiamen,China
GraemeC.Young GlaxoSmithKlineResearch andDevelopmentLtd.,DavidJackCentre, Ware,UnitedKingdom
JingjingYu DepartmentofPharmaceutics, SchoolofPharmacy,UniversityofWashington,Seattle,WA,UnitedStates
LushanYu InstituteofDrugMetabolismand PharmaceuticalAnalysis,ZhejiangUniversity, Hangzhou,People’sRepublicofChina
SuZeng InstituteofDrugMetabolismand PharmaceuticalAnalysis,ZhejiangUniversity, Hangzhou,People’sRepublicofChina
HaeyoungZhang DepartmentofPharmaceutics,UniversityofWashington,Seattle, WA,UnitedStates
WanyingZhang DepartmentofPharmaceuticalSciences,SchoolofPharmacyand PharmaceuticalSciences,TheStateUniversityofNewYorkatBuffalo,Buffalo,NY, UnitedStates
AndyZ.X.Zhu DepartmentofDrugMetabolismandPharmacokinetics,TakedaPharmaceuticalsInternationalCo.,Cambridge,MA, UnitedStates
MingsheZhu MassDefectTechnologies,Princeton,NJ,UnitedStates
Foreword Itismygreatpleasuretowritetheforewordforthisexcellentbook.Thestudyof drugmetabolismanddispositionisamature science,butstillessentialindrugdiscovery anddevelopment.Indeed,aquicksearchof PubMed,using“drugmetabolism”asthe searchterm,cameupwithmorethan 18,000hits.Oneoftheearlierarticlesisby BernardBrodie,consideredtobethefounder ofmodernpharmacologyandamajorcontributortothestudyofdrugmetabolism. Hisarticle—publishedintheJournalofPharmacyandPharmacologyin1956—istitled “PathwaysofDrugMetabolism”anditisbased onalectureattheUniversityofLondon [1] Itdescribeshisworkoverthedecadeand someofitisbasedoncollaborationswith otherpioneersinthefieldsuchasJulius Axelrod.Onesentencestillresonatesvery muchanditactuallycoversmuchofthematerialdescribedinthisbook:
Finally,itisthoughtthatadetailedknowledgeofenzymesinvolvedindrug“detoxification”mighthelpthemedicinalchemistto developcompoundsofeitherhighorlowstabilityinthebody,whicheverwouldbemoredesirableingainingadesiredtherapeuticresult.
Drugmetabolismscienceshaveadvanced tremendouslysincethepublicationofthisarticleandthisprogresswastoalargeextent enabledbyadvancesintheavailabilityof biochemicalreagentsandbioanalyticaltechniques,inparticularmassspectrometry. Bothacademicandindustrialscientistshave
identifiedmanybreakthroughsthathave ultimatelycontributedtobringingdrugsto patientswithanurgentneedforbettertreatments.Itisverygratifyingthatthenumber ofdrugsapprovedbytheFDAisincreasing steadilywith38NMEs(newmolecularentity)and10BLAs(biologicallicenseapplication)approvedbytheUSFoodandDrug Administration(FDA)in2019 [2].Thelevel ofinnovationisbestillustratedbyabouthalf oftheapproveddrugsin2019havinga breakthroughdesignation.Drugmetabolism andpharmacokinetics(DMPK)scientistsare fullyembeddedinprojectteamsindrugdiscoveryandworkhandinhandwithmedicinalchemistsontheidentificationofdrugs withsuperiorproperties.Thesescientists havebecomeverygoodatdialingoutthe “knownunknowns”suchasmetabolismbycytochromeP450enzymes.However,thishas actuallyresultedinhavingtodealwithmuch morecomplexdrugdispositionpathwaysinvolvinglesswell-studieddrugmetabolizing enzymesandalsomoreandmoredrug transporters.Thistrendisalsofueledbya shifttowardmoreandmoredrugshaving beyondthe“ruleoffive”molecularproperties [3].Indeed,theaveragemolecular weightofdrugsapprovedbytheFDAin 2016and2017wasmorethan500Daand themedianclogPofdrugsapprovedfrom 2008to2017isnow3.3,inpartdrivenbytrying,forexample,todisruptprotein-protein interactions.Anadditionalconsequenceof thisshiftinpropertiesisthatinvitroto invivoextrapolationofADMEproperties
topredictthehumanpharmacokineticshas becomemorecomplexandthesamecanbe saidofpredictingdrug-druginteraction— manyofwhichnowinvolvedrugtransporters.Ifanything,theinvolvementof DMPKscientistsisnowmoreimportantthan everbecauseofthepursuitofnovelmolecular modalitiesinacademiaandinthepharmaceuticalindustry;afewthatcometomind areantibody-drugconjugates,proteindegraders,andmacrocyclicpeptides.DMPK scientistscanhelpmakedrugsoutofthese hard-to-drugmodalities.Finally,theintegrationofthisdiversearrayofinvitroandinvivo dataisenabledbycomputationalmodeling andsimulation.Physiologicallybasedpharmacokinetic(PBPK)modelingisaverypowerfultooltopredicthumanpharmacokinetics andstudydrug-druginteractionaswellas thepharmacokineticsinspecialpopulations andinfants.Pharmacokinetic/pharmacodynamicmodelinganditsextension,quantitativesystemspharmacology,canhelp translatepreclinicalfindingstotheclinic.
Thisbookiscomprisedofmanyexcellent chapterswrittenbyexpertsinthefieldthat addresssomeofthechallengeshighlighted inthepreviousparagraph.Thefirstsection focuseson“Techniquesforidentifyingand quantifyingdrugsandmetabolites.” Chapters 1–3 introducethereaderstothelatestadvancesinbioanalysis,inparticularmass spectrometryfortheanalysisofdrugs,their metabolites,andendogenousbiomarkers.In contrastto20yearsago,high-resolution massspectrometryhasnowbecomeroutine andithashadagreatimpactonthestudy ofbiotransformation.Ofcourse,massspectrometryisfrequentlynotsufficienttoidentifythedefinitivestructureofametabolite, andhence Chapter4 focusesonstrategies forgeneratingmetabolitesandcharacterizationviaNMR.Supercriticalfluidchromatographyhasbecomeeasiertointerfaceandit cangreatlyfacilitatechiralseparation;the
lattertopiciscoveredin Chapter5.Thelast chapterinthefirstsection(Chapter6) focusesonacceleratormassspectrometry, apowerfultooltostudyADMEandthe absolutebioavailabilityinhumans.
Thesecondsectionaddresses“Drugmetabolizingenzymes,transportersanddrugdrug interactions.”CytochromeP450-mediated andnon-cytochromeP450-mediatedmetabolismisdiscussedin Chapters7and8. Invitrotoinvivopredictionofdrug-druginteractionisdescribedin Chapter9 while Chapter10 specificallyfocusesontherole oftransportersindrugdispositionand drug-druginteraction,and Chapter11 on theclinicalrelevanceofthesedrug-drug interactions.MakingpredictionsusingPBPK modelingreliesonaccurateknowledgeof physiologicconstantsand,mostrecently, massspectrometryhasbeenusedtodeterminetheabundanceofdrugmetabolizing enzymesandtransportersinvarioustissues. Thisisaddressedin Chapter12.Finally, Chapter13 focusesondisease-druginteractionsmediatedbytherapeuticproteinssuch asinterleukins.
Thethirdsectionfocuseson“Strategyrelatedtodrugmetabolismandsafety.”Metabolitesinsafetytestingcontinuestobea highlyrelevantareaofresearchindrugdiscoveryanddevelopmentanditisaddressed in Chapter14.Acloselookatthe(patent)literatureindicatesthatfindingdrugsthatbalancepotencyandmetabolicstabilitycanstill beproblematic,andthereforemanycompaniesincorporatedeuteriumtoenhance metabolitestabilityandlowerthedose— see Chapter15 fordetails.Thefocusonnovel chemicalspaceandmoremoleculardiversity hasintroducedmorechiralityinmolecules andtheimpactofthatonpharmacology,toxicology,anddrugmetabolismisdescribedin Chapter16.Drug-inducedliverinjuryand newpredictivemodelsforrenalinjuryaredescribedin Chapters17and18,respectively.
Finally, Chapter19 focusesonimmunogenicityasakeycomponentofantibody development.
Thefourthandlastsectionisdedicatedto “Translationalsciences.”Theuseofgeneticallymodifiedrodentsisexploredfurther in Chapter20,while Chapter21 speaksto theuseofCRISPRtoadvanceinvitroADME models.Invitrotoinvivoextrapolationof hepaticandrenalclearanceisdiscussedin Chapter22.Thebreadthofourscienceis nicelyillustratedby Chapter23 whichdescribestheroleofmathematicalmodeling intranslationalsciences.Last,butbyno meansleast, Chapter24 elegantlydefines waystopredictthehumanefficaciousdose usingPK/PDmodeling.
Asisthecasewithmanycompilations,a lotofefforthasgoneintoassemblingan
excellentsetofchaptersthatdescribethe stateoftheartinADMEsciencesasit relatestodrugdiscoveryanddevelopment. Theeffortsbyallauthors,andinparticular theeditorsShuguangMaandSwapan Chowdhury,aregreatlyappreciated.This bookwillbeusedasaresourceformany yearstocome.
CornelisE.C.A.Hop
References [1] B.B.Brodie,Pathwaysofdrugmetabolism,J.Pharm. Pharmacol.8(1)(1956)1–17.
[2] A.Mullard,2019FDAdrugapprovals,Nat.Rev. DrugDiscov.19(2)(2020)79–84.
[3] M.D.Shultz,Twodecadesundertheinfluenceof theruleoffiveandthechangingpropertiesofapprovedoraldrugs,J.Med.Chem.62(4)(2019) 1701–1714.
Preface Some15yearsago,oneofus(SKC)compiledthefirsteditionofthisbookthat coveredthemostup-to-dateinformation previouslyavailableonthestrategies, methods,applications,andimplicationsof scientificdataontheroleofenzymesand transportersinthedispositionofpharmaceuticals.Thebookwasagreatsuccessand waswidelyusedasavaluableresourceby scientistsinbothindustryandacademia. Sincethen,alothaschangedinthefieldof drugmetabolism,includinghowrecent scientificadvancesarebeingutilizedtodiscoveranddevelopsafermedicines.Therefore,ithasbecomeapparentthatanew editionofthebookisrequiredtofullycapturetheseprofoundchanges,whichinvolves theimplementationofnewertechnologiesin thediscoveryanddevelopmentofmedicines totreatawiderangeofmaladies.Withmuch enthusiasmfromthepublisher,wecollaboratedtoassembleacomprehensivetreatise thatwouldcapturehowthelatestscientific findingsarehavingafundamentalimpact ontheutilizationofthesenoveladvances indrugresearch.Thissecondeditionis completelyupdatedandprovidesanoverviewofthelastdecade’snumerousimprovementsinanalyticaltechnologiesforthe detectionandquantificationofdrugs,metabolites,andbiomarkers.Thisneweditiongoes beyondconventionalLC-MSandfeatures all-newchapters,including:howtoevaluate drugabsorption,distribution,metabolism,
andexcretion(ADME),thepotentialforhepaticandrenaltoxicity,immunogenicityof biotherapeutics,andtranslationaltoolsfor predictinghumandosage,safety,andefficacyofsmallmoleculesandbiologics.
Thisbookisorganizedintofoursections: (1)techniquesforidentifyingandquantifyingdrugsandmetabolites,(2)drugmetabolismenzymes,transporters,anddrug-drug interactions,(3)strategiesrelatedtodrug metabolismandsafety,and(4)translationalsciences.Thebookcontains24chapters coveringthemostrecent,novelscientific breakthroughsandhowtheyareutilizedto developmedicinesinthemodernera.Itis oursincerehopethatthismaterialwillserve asanimportanttoolanddeskreferencefor pharmacologists,toxicologists,clinicalscientists,andstudentsinterestedinthefields ofpharmacology,biochemistry,anddrug metabolism.
Finally,wewishtoacknowledgethecontributionsofthemanyscholarswhoparticipatedinandcontributedtothisbookfrom conceptionandpassionintoprint.Wealso wanttoextendourgratitudetothecontributionsoftheeditorialstaffandproduction manageratElsevier,andlastbutnotleast, thefamiliesoftheeditorsfortheirencouragement,love,andsupport.
ShuguangMa SwapanK.Chowdhury
1 Bioanalysisofsmallandlarge moleculedrugs,metabolites,and biomarkersbyLC-MS NaidongWeng,ShefaliPatel,WenyingJian
DMPK,JanssenR&D,SpringHouse,PA,UnitedStates
1Introduction
Bioanalysis,oftenshortenedtoBA,isasubdisciplinewithinpharmaceuticalresearchand development(R&D).Contemporarybioanalysisquantitativelyanalyzesverylowquantity buthighlyvariablelevels(pg/mL-μg/mL)ofdrugcandidates,theirmetabolites,endogenous biomarkers,etc.inextremelycomplicatedbiologicalmatricessuchasplasma,blood,urine, andtissueswhichareharvestedfromdifferenttypesofanimalspecies(rodents,dogs, nonhumanprimates,etc.)andhumans [1].Bioanalysissupportsdiscovery,nonclinical (tox),andclinicalstudies(Fig.1). Fig.1 showstypicalstudiesanintegratedbioanalyticalfunctionwouldsupport.
Bioanalyticaldataareusedforcalculatingpharmacokineticparameterssuchasbioavailability,bioequivalence,drugandmetabolitesexposure,clearance,theirdistributioninto variousbodyorgans,correlationofpharmacokinetics(PK)effectsandpharmacodynamics (PD)changes,etc.Thus,bioanalysisplaysapivotalroleinmovingdrugcandidatesfromearly discoveryallthewaytoregulatoryfilingandpostmarketsurveillanceintheentiredrugdiscoveryanddevelopmentprocess.Intoday’sdynamicdrugdiscoveryanddevelopment environment,bioanalyticalscientistsnotonlyprovidepivotaldatabutalsoactivelyengage inproject/programgo/no-godiscussions,alongwithcolleaguesfromotherfunctionalareas. Whilethemostessentialelementofbioanalysisistouseanalyticalchemistryknowledgeand state-of-the-artinstrumentstoprovidereliableandaccuratemeasurement,knowledgefrom relevantdisciplinessuchasbiotransformation,pharmacokinetics,biology,pharmacology, etc.isinvaluableforensuringappropriateconductofbioanalysis.
1Bioanalysisofsmallandlargemoleculedrugs,metabolites,andbiomarkers
Discovery In vitro
Plasma protein binding
Transporter
Inhibition-induction
Metabolic stability
Plasma-blood distribution
In vivo
Salt form selection
Formulation
Dose range finding
Preclinical Clinical Short term (2wk, 4 wk) TOX
Long term (3mts +) TOX
Reproductive TOX
Carcinoma studies
Micronucleus studies
Animal ADME
Bioavailability (IV/ORAL )
SAD, MAD
Metabolite assessment (MIST)
Food effect
Drug-drug interaction
Comparator study
Human ADME
Population PK
Special population study (renal impaired, pediatric, etc.)
Adaptive design clinical trial
Bioequivalence
Tissue distribution Drug-like?Known liabilities?Superior efficacy?
FIG.1 Exemplarybioanalyticalsupportindrugdiscoveryanddevelopment.TOX,toxicology;SAD,singleascendingdosestudy;MAD,multipleascendingdosestudy;ADME,absorption,distribution,metabolism,andelimination; IV,intravenous.
Inthisbookchapter,wetrytoprovideabriefoverviewofcontemporarybioanalysisusing theLC-MSplatform.Itisanimpossibletasktoprovideadetailedandcomprehensivereview ofLC-MSbioanalysisinabookchapter.Thecasestudiesandliteraturearenodoubtincompleteandbiasedtowardourownexperiencesandpublications.Interestedreadersare referredtotheexcellentbioanalysishandbookbyLietal.foramorecomprehensiveoverview ofthisdiscipline [1].Nevertheless,wehopethereaderscanappreciatethecomplexityand dynamicsofmodernLC-MS-basedbioanalysisforsmallandlargemoleculedrugs,metabolites,andbiomarkers.
2Complexityofcontemporarybioanalysis Bioanalyticalsupportisrequiredforimportantdecision-makingforalltypesofstudies, fromdiscovery(non-GLP),todevelopment(GLP),andtoclinical(GCP)studies.Yetthere aremanyuniqueandcomplicatedattributesofcost,quality,andtimelydeliveryateachof theabovementionedstages(orsubstagewithineachstage).Forbioanalysis,thequality andintegrityofthebioanalyticaldataareultimatelythemostimportantattributes.Theright scientificandcompliancevigormustbeappliedtoeachstudy.Thecurrentregulatorylandscapeforregulatedbioanalysisishighlycomplicated,withguidancefrommultipleregional healthauthorities [2–6].Sinceguidancefromdifferentregionsarenottotallyharmonized,and compoundedbyindividualinterpretationofdifferentinspectors,itisstillquiteachallengeto fullyunderstandandexecutetherightlevelofcompliancethatcanbeacceptableintheglobal filing.Effortsarecurrentlybeingmadetoharmonizetheguidelinesintoonesingleglobal guidelineICH-M10 [7]
Whiletimelydeliveryofbioanalyticaldatatosupportprojectdecisionsisamust-doitemto meettheever-tighteningdrugdiscoveryanddevelopmenttimelines,costisanotherattribute thatshouldnotbeoverlooked.Thecostofbioanalysisactivitiesshouldbecarefullymanaged toensurethatitstayswithinthepredeterminedbudget.Ontheotherside,theapplicationofa
I.Techniquesforidentifyingandquantifyingdrugsandmetabolites
tieredapproach,whichtypicallyconsistsofthreetiersofassayqualification—screeningassay,qualifiedassay,andvalidatedassaywiththeincreasedlevelsofvalidationparameters— canbeusedwithabalanceofscientificvigorandcost [8].TheEuropeanBioanalyticalForum (EBF)recommendsexercisingthisapproachfor“nonregulated”nonclinicalbioanalysisin drugdevelopmentandusing“fit-for-purpose”elementsinmetabolitequantitationfor establishingsafetycoverage(MIST);urinebioanalysis;andtissuebioanalysis [9,10].Of course,theactualimplementationofwhichtiertousedependsoneachindividualstudy. Forexample,forurinebioanalysis,whileaqualifiedassaycouldbeusedformostclinical studiesforunderstandingtheurinaryexcretionofadrugcandidate,validatedassaysshould beappliedifrenalclearanceisthemainrouteofelimination(PKendpoint)and/orthedrug targetactionisatthekidney.
Thereisanexpansionofbioanalysisscopeoverthepastdecades.Intheearlydays, bioanalysisfocusedonsupportingsmallmoleculePKandbioequivalence(BE)fromstandard formulationssuchastabletsandcapsules.Analysisofmetabolitesandbiomarkersrarely occurred.Currently,PKandBEforbothsmallandlargemolecules,aswellasmanyhybrid formsoflargeandsmallmoleculessuchasantibodydrugconjugate(ADC),aresupported bybioanalysis [11,12].Evenforsmallmolecules,advancementinformulationpresentsnew challengesforbioanalysis.Forexample,liposomesarewidelyappliedinthepharmaceutical industryduetotheiruniquecapabilitiessuchasencapsulatingandprotectingthetherapeutic analytesfromdegradation,controllingthereleaserate,facilitatingon-targetdelivery,and reducingtoxicityfordrugs [13,14].Forliposomaldrugproductdevelopment,validated bioanalyticalmethodstodeterminetheconcentrationoftheencapsulatedandnonencapsulated forms(boththeprotein-freeandprotein-bound)oftheactivesubstanceinbiologicalsamples shouldbeemployed.However,establishingsuchbioanalyticalassayscanbequitechallenging duetothepotentialraptureoftheliposomeduringsamplecollection,shipping,storage,and analysis,whichcanartificiallyelevatethenonencapsulatedconcentrations [15,16].
Peptideandproteindrugshaveevolvedinrecentyearsintomainstreamtherapeutics, representingasignificantportionofthepharmaceuticalmarket [17].Peptidesandproteins exhibithighlydiversestructuresandbroadbiologicalactivitiesashormones,neurotransmitters,structuralproteins,andmetabolicmodulatorsandthereforehaveasignificantroleas boththerapeuticsandbiomarkers.Unspecificproteolysisisamajoreliminationpathway forpeptidesandproteinsinsteadoftheoxidativehepaticmetabolismthatistypicalformost smallmoleculedrugs [18].InadditiontothetypicalPKsupport,bioanalysishasbeenactively engagedinnewlydevelopedareassuchasPKPDcorrelation,biomarkerresearchintarget engagement/tissuedistribution,simultaneousquantitationandmetaboliteidentification forsmallmoleculesandbiologics,andantidrugantibody(ADA),etc.,formanyofwhichboth scienceandregulatoryrequirementsarestillevolving.
Historically,bioanalysissupportcanbecategorizedintotwodistinctareas—liquidchromatographyforsmallmolecules,usuallychemicallysynthesized,andligandbindingassay forlargemolecules,suchasthosewhichmonoclonalantibodiestypicallyproducebycell culture.Withthedevelopmentofmoderninstrumentation,especiallymassspectrometers, measurementoflargemoleculesbyliquidchromatographyinconjunctionwithamassspectrometer(LC-MS)hasbecomeareality.ThereisalsoaneedtouseLC-MStoinvestigatethe invivofateofsomeofthenewmodalitiessuchasahalf-lifeextendedpeptideconstructed byconjugatingorfusinganotherwiseshort-livedpeptidewithanantibodyorFctoextensivelyextenditsinvivohalf-lifethroughdecreasedclearanceandincreasedstability [19].
LC-MShasalsobeenincreasinglyusedfordiscoveringandmeasuringendogenousproteinor peptidebiomarkers [20,21].
3Bioanalyticalrequirementsforsupportingdiscovery,nonclinical,andclinical studies
Letustakeafurtherlookatbioanalyticalrequirementsforsupportingdiscovery, nonclinicaldevelopment,andclinicalstudiesusingLC-MSmethods.Thesethreestages shouldnotbeviewedasthreeseparateandconsecutiveonessinceeachcanextendwellinto thenextstage,inparticularbetweennonclinicaldevelopmentandclinicalphases.Manyofthe longer-termtoxicologystudiesarerunninginparallelwiththeclinicalstudies.Discovery bioanalyticalsupportisperformedundernon-GLPconditions.GenericLC-MSmethodsusing gradientmobilephaseelutionandproteinprecipitationsampleextractionaretypicallyusedto analyzethedrugcandidatesinplasmaandtissues.Theresultsareusedtorankthedrugcandidatesaswellastoobtainotherimportantinformationsuchastissuedistributionandbasic pharmacokineticparameters,forexample,Cmax,Tmax,AUC,T1/2,andclearance.Usuallyan internalbioanalysis/PKguidelineisusedfortheconductofthistypeofstudy.Somelevelof methodverificationsuchasaccuracy,precision,andcriticalstabilityisperformedbeforethe sampleanalysis.RunacceptancecriteriaareusuallyalittlebitwiderthanthoseusedinGLP studies.EventhoughdiscoverybioanalysisdoesnotfollowstrictGLPrules,thedatagenerated inthediscoverystagearestillpivotalforcompoundselection,andthereforemanycompanies institutesomelevelsofcompliancetoensuredataintegrity.Nonclinicalbioanalyticalsupport indevelopmentisunderGLPregulationswherethedatageneratedshouldwithstandrigorous regulatoryscrutiny [2,3].Foreachmethod,vigorousmethoddevelopmentandvalidationis performedasperregulatoryguidanceandinternalStandardOperationProcedures(SOPs), eventhoughmethodvalidationitselfisnon-GLP.Anydeviationduringthesampleanalysis, whichfollowstheGLPprinciples,shouldbethoroughlyinvestigated,documented,andjustified.Oncethecandidatemovesintotheclinicalstage,automationandothermeansofimprovingthethroughputofsampleanalysisbecomemorerelevant.Thesameclinical bioanalyticalmethodcanbeusedbymultiplebioanalyticalchemistswithinorevenamong differentorganizationstomeetthedemandforanalyzingalargenumberofsamples.Atthe clinicalstage,especiallyforstudiesinfirstinhuman(FIH),abioanalyticalmethodwithbetter sensitivitythanthoseinnonclinicalstudiesmayberequired.Extensivemethodoptimization forbothsamplepreparationandLC-MSconditionstomaximizerecoveryandminimizematrix effectsisoftenpursued.
4Currentregulatorylandscapeforbioanalysis BothnonclinicaldevelopmentandclinicalbioanalyticalsupportarerequiredtofollowinternalSOPsandregulatoryrecommendations.DozensofSOPsareusuallyusedtoaddress manyaspectsofbioanalyticalactivitiesrangingfrominstrumentqualification,maintenance, andcalibration,tobioanalyticalmethodvalidation,samplestorage,analysisanddestruction, todatamanagementandarchiving,etc.ThegoaloftheseSOPsistoensurethehighestdata
TABLE1
Bioanalyticalfullvalidation,partialvalidationandcrossvalidation [2] Fullvalidation
Usedformeasuringanalyte concentrationsinbiologicalsamples. Afullvalidationofabioanalytical methodshouldbeperformedwhen establishingabioanalyticalmethod forthequantificationofananalytein clinicalandinpivotalnonclinical studies
Developingandvalidatinganassay forpivotalregulatorysubmission (INDorNDA)
Implementingananalyticalmethod thatisreportedintheliterature
Partialvalidation
Modificationstoafullyvalidated analyticalmethodmaybeevaluated bypartialvalidation.Partial validationcanrangefromaslittleas oneaccuracyandprecision determinationtoanearlyfull validation.Theitemsinapartial validationaredeterminedaccording totheextentandnatureofthe changesmadetothemethod
Analyticalsitechangeusingsame method(i.e.,bioanalyticalmethod transfersbetween laboratories)
Achangeinsampleprocessing procedures
Crossvalidation
Wheredataareobtainedfrom differentmethodswithinoracross studies,orwhendataareobtained withinastudyfromdifferent laboratoriesapplyingthesame method,comparisonofthosedatais needed,andacrossvalidationofthe appliedanalyticalmethodsshould becarriedout
Dataareobtainedfromdifferent fullyvalidatedmethodswithina study
Dataareobtainedwithinastudy fromdifferentlaboratorieswiththe samebioanalyticalmethod
integritywhichcanpasstheregulatoryscrutiny.TheseSOPsareusuallydraftedbasedonspecificregulationsandtheregulatoryguidancefromtheFoodandDrugAdministration(FDA) andotherinternationalregulatoryagencies,suchastheEuropeanMedicinesAgency(EMA), andotherfederal/staterequirements,aswellaspoliciesateachinstitute.OneofthemostimportantregulatoryguidelinesistheUSFDAguidanceonbioanalyticalmethodvalidation [2], whichprovidesageneralguidelineonestablishingimportantparameters,someofwhichare specificity/selectivity,sensitivity,linearityrangesfromalowerlimitofquantitation(LLOQ)to anupperlimitofquantitation(ULOQ),accuracy,precision,stability,matrixeffects,carryover, recovery,dilutionintegrity,incurredsamplereanalysis(ISR),etc.Themethodshouldalso demonstratereproducibilityandaccuracyforincurredsamplesanditssuitabilityforits intendeduse.However,itisuptoeachinstitutetoformulateitsownSOPsbasedonthisgeneral guidance.Typically,methodvalidationscanbecategorizedintofull,partial,andcrossvalidations(Table1).Mostofthevalidationsareconductedintheformoffullvalidation,whereasthe partialorcrossvalidationcanbeusedwhenafullvalidationisnotrequired,asshowninthe examplesin Table1.
5Generalconsiderationsforbioanalysisforsamplecollection Duringtheprojectdiscussion,itisimportanttounderstandtheobjectiveofthestudyso thatimportantparameterssuchasmatrixtype,samplevolume,waysofsampling(regular samplingormicrosampling),analytestobemeasured(parentorparentplusmetabolites), anticoagulantselection,conditionofcentrifugationforharvestingplasma,samplestorage container,shippingconditionandinstruction,samplestoragecondition( 20°Cvs. 70°C),etc.canbeprovidedtotheprojectteam.Whileduringtheearlystageofdrug
discovery/developmentitisnotalwayspossibletoconductthefullspectrumofastability test,itis,however,alwaysgoodpracticetoevaluatewhetherthereispotentialinstability ofametabolitethatcanimpacttheaccuratequantitationoftheparentanalyteand/orthemetabolites.DetailedPKorTK(toxicokinetic)samplecollectioninformation,includinglabeling, willbeenteredintothelabmanualorstudydesign.Thefollowingisjustonetypicalexample inalabmanualsupportingaPhase1clinicalstudy.Itincludesthreepartsoftheinstruction (materialandlabeling;preparationofPKsamples;andshipmentofPKsamples).Asonecan seefromthisexample,anextremelydetailedinstructionisneededtoensuresuccessfulconductofbioanalysis.
Materialsandlabeling:
•BloodmustbecollectedinaglassorplasticK2EDTAcontainingbloodcollectiontubes (e.g.,Vacutainer).
•Notubeswithseparationgelshouldbeused.Theresultingplasmasamplesmustbestored inpolypropylenestoragetubeswithpolypropyleneorpolyethylenecaps.
•Alltubesandcontainerswillbelabeledwithpreprintedlabels.Thepreprintedinformation willincludethestudynumber,participantidentificationnumber,treatmentortreatment period,scheduledsamplingdayandtimeasstipulatedintheflowchart,andtheanalyte nameifapplicable.Nootherinformationwillbewrittenonthelabels.Labelsshouldbe attachedtothestoragetubesatleast12hbeforebeingfrozentoensureproperadherence.
•Labelsshouldbeappliedtothesampletubesasfollows:
• Applylabelstothesampletubessothattheydonotoverlapandobscureany information.Ifpossible,exposeanareabetweenthetwoendsofthelabeltoallow viewingofthecontentsofthetube.
• Donotaltertheorientationofthelabelonthesampletube.
• Applylabelstoalltubesinthesamemanner.
Preparationofpharmacokineticsamples:
•Collect4mLofbloodintotheappropriateK2EDTA-containingcollectiontube(e.g., Vacutainer)ateachtimepoint.
•Recordtheexactdateandtimeofsampling.
•Beforeprocessing,gentlyinvertthetubes8–10timestoaffordmixing;bloodsamplesmust bekeptonmeltingiceatalltimesduringprocessing.
•Centrifugebloodsampleswithin1hofcollectioninacentrifugeat1300g for10minat4°C, toyieldapproximately1.5–2mLofplasmafromeach4-mLwholebloodsample.Plasma samplesmustbekeptonmeltingiceatalltimesduringprocessing.
•Transferplasmaimmediatelytoaprelabeledpolypropylenetube.Plasmasamplesmustbe keptonmeltingiceatalltimesuntilstorageinafreezer.
•Storeplasmasamplesinanuprightpositioninafreezer,atasettemperatureat 20°Cuntil transfertothebioanalyticalfacility.
•Thetimebetweenbloodcollectionandfreezingtheplasmawillnotexceed2h.
•Shipsamplesaccordingtotheinstructionsprovided.Shipspecimenstothebioanalytical facility,sortedbyparticipant,samplecollectiondate,andtime.
•Questionsregardinghandlingtheplasmapharmacokineticspecimensshouldbe addressedtothecontactpersonforthesponsor.
Shipmentofpharmacokineticsamples:
•Allpharmacokineticsampleswillbesenttothebioanalyticalfacilityinasingleshipment attheendofthestudyorinmultipleshipmentsasagreeduponwiththebioanalytical scientist.
•Aninventorylistmustbeincludedwitheachshipment.Thesponsor-providedlogscanbe usedasaninventorylist.Theinventorylistmustnoteeachspecimendrawnforeach participantandnoteanymissingspecimens.
•Forallinternationalshipments,WorldCourierwillbeused.Fordomesticshipments,a reliabledomesticcourier,suchasFederalExpress,willbeused.
•Assoonastheshipmentdayandairbillnumber(s)areavailable,thesitewillsendan e-mailtotheprincipalinvestigator,samplemanagementteam,andsitemanagerofthe bioanalyticalfacility.Thee-mailmustspecifythestudynumber,thenumberof pharmacokineticsamples,thetimeofshipmentpickupandthetrackingnumberand includeanelectronicsampleinventory.
•Notifythebioanalyticalscientistandthecourier,atleast24hinadvanceoftheplanned shipment.Providethecourierwiththeappropriateaccountnumbertobeused,if applicable.
•Unlessagreementsweremadewiththeprincipalinvestigator,sampleswillbeshippedvia overnightdeliveryonlyonMondaythroughWednesday,excludingholidays.
•Preferablythefrozensampleswillbeshippedinboxes,sortedbyparticipantandsampling time.Boxeswillbepackedinbagsthatcanwithstanddryiceconditions(e.g.,cryogenic bags).
•Packthefrozensamplesinasufficientquantityofdryiceinappropriatecontainers,to maintainafrozenstateforatleast3days.
•Forallbiologicalsamples,followtheInternationalAirTransportAssociation(IATA) regulationsforshipment.
•Ensurethatthetotalpackageweightdoesnotexceed27.2kg(60pounds).
•Labelthepackagewiththesponsornameandstudynumber.
•Includeareturnaddress(whichincludestheinvestigator’sname)ontheoutsideofeach shippingcontainer.
•Complywithallcourierregulationsfortheshipmentofbiologicalspecimens(includeall paperwork).
•Retainalldocumentsindicatingthedate,time,andsignature(s)ofperson(s)makingthe shipment,inthestudyfiles.
6Diagnosisandmitigationofnonspecificadsorptionloss forurinebioanalysis Forurinesamples,itisalwaysadvisabletoconductanonspecificadsorptionlossexperimentbeforeprotocolfinalizationsothat,ifnecessary,themeansofpreventingnonspecific adsorptioncanbeimplemented.Nonspecificadsorptionhappensfrequentlyinurineassays becauseurinelacksproteinsandlipidsthatcanbindtotheanalytesorsolubilizelipophilic analytes [22].Acommonapproachtourinemethoddevelopmentwithafocusonovercoming
adsorptionissueswaspublished [23].Asimpleandquickwaytoidentifyanadsorptionproblemisthroughaseriesoftransfersandincubations.Inthistest,adrugsolutionincontrol urineispreparedatalowqualitycontrol(QC)levelinatestcontainer.Dryandcleantest containersthataremadefromthesamematerialandareofthesamesizeasthoseforfuture urinesamplesshouldbeused.Aportionoftheurinesolutionispouredintoanextcontainer, andthenthetransferprocessisrepeatedforafewmorecontainers.Betweentransfers,each solutioninthecontainershouldbeleftatroomtemperatureforabout5–20mintoallowadsorptiontotakeplacebeforecontinuingtothenexttransferstep.Finally,theurinesample fromeachtestcontainerisassayedtodeterminethecompoundresponse.Asequentialloss ofanalyteresponseaftereachtransferindicatestheexistenceofnonspecificadsorptionloss. Toovercometheissue,antiadsorptiveagentscanbeutilized,suchasbovineserumalbumin (BSA),zwitterionicdetergentssuchas3-[3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate(CHAPS),sodiumdodecylbenzenesulfonate(SDBS),beta-cyclodextrin,Tween80, andTween20.
7Tissuebioanalysis Knowledgeofdistributionofdrugs,metabolites,biomarkers,etc.atspecificlocationsin thebodyofanimalspeciesandhumansubjectsbecomesmoreandmoreimportantfordrug discoveryanddevelopment [24].Thisknowledgeisoftenobtainedthroughtheanalysisof tissuesamplesobtainedfromnonclinicaland,toalesserextent,clinicalstudies.Manyfactors canaffectthetissuedistributionofdrugsandmetabolites.Passivediffusionacrossthecell membraneistheprimarypathwayforthedrugtodistributetothetissue.However,transporterproteinscanalsoassistorminimizetheuptakeofthedrugsandmetabolitesinto thetissues.Othercontributingfactorssuchasdrugmetabolism,clearancerate,proteinbinding,permeability,molecularweight,log P,andpKa andotherphysicochemicalpropertiescan alsohaveasignificantimpactontissuedistribution.LC-MShasbecomethestandardtoolbox fortissuesampleanalysis [25].Differentfromplasma,whichisintheliquidform,tissuesamplesareinasolidorsemisolidformat.Therefore,fortypicalLC-MSassays,theyarehomogenizedpriortosampleextraction.Softtissuessuchasthebrain,liver,lung,andkidneycanbe easilyhomogenized.Toughtissuessuchasthemuscle,heart,stomach,intestine,andcolon aremorefibrousandneedamorevigoroushomogenizationprocedure.Ahighershearing forceandlongerdurationofprocessmayberequiredwhenarotorbladehomogenizeror beadsbeaterisused [26].Theheatgeneratedduringhomogenizationoftissuesmaycause degradationofthermolabileanalyte(s),andthereforecautionmustbeexercised.Hardtissues suchasskeletalbones,skin,andhairaremostlynonvascularizedtissuesandneedspecial treatment.Insomeextremecases,enzymaticdigestionorchemicaltreatmentmayberequired forsomehardtissues [27].Oncethetissuesampleishomogenized,thehomogenateisready forextractionandthesampleextractionapproachesusedforplasmasamplesshouldstillapply.However,differentfromplasmasamples,extractionrecoveryfromsolidtissuesamples cannotbeassumedtobe100%andfortifiedQCsamples,preparedintissuehomogenate,cannotmimictheincurredsamples [25].Similarly,theinternalstandard,whichwasroutinely usedtocompensateforincompleteorvariableextractionrecoveryfortheplasmasamples,
doesnottracktheanalytesinthetissues.Recoveryevaluationusingaradiolabeledincurred sample [28] andrecoveryevaluationwithorthogonalapproaches [29] arejusttwoexamples ofaddressingtheissueofrecoveryevaluation.
8Managingunstablemetabolitessuchasacylglucuronide Whenmovingintothedevelopmentphase,informationaboutbiotransformationofthe drugcandidateanditsmetabolitesbecomesmoreavailable.Whiletheparentdrugmay notbesubjectedtoglucuronidation,itsphase1metabolism,forexample,oxidationofalcohol tothecarboxylicacidgroup,maygeneratemetabolitesthatcanformacylglucuronide.Ifthe plasmasampleswerecollectedwithoutacidificationtostabilizetheacylglucuronide,the phase1carboxylicacidmetabolitemaybeoverestimatedduetothebreakdownofacylglucuronideduringsamplecollection,storage,andanalysis.Itshouldalsobenotedthatacidificationoftheplasmasampleswillaffecttheproteinbindingbetweenthedrugcandidateand theendogenousproteinssuchasalbumin [30].Iftheprotein-bindingmeasurementisneeded withtheincurredsamples,aseparatepoolofunbufferedplasmasamplesshouldbecollected. Acidificationofthesamplescouldalsoleadtotheinstabilityofothermetabolites.Inoneexampleofquantitationofdiclofenacandmetabolitesinmouseplasma,acidificationtostabilize diclofenacacylglucuronideresultedinincreasedoxidativedegradationof5-OHdiclofenac andascorbicacidwasaddedtothesamplestopreventthedegradation [31]
Aswecantellfromthissimpleacylglucuronideexample,abioanalyticalsamplecollection strategymayevolve,dependingonthestageofthedrugdiscoveryanddevelopment.Forexample,attheearlystageofdrugdevelopment,informationonparentexposuremightbe enoughforago-no-godecision.There,asimplestabilizationofacylglucuronidebyadjusting thepHisused.Withtheprojectmovingforwardandgainingofinformationregardingacyl glucuronidemigration,whichmayraisetoxicityconcern [32],themeasurementofboththe parentandacylglucuronidemetaboliteisneeded.Plasmaproteinbindingforboththeparent andacylglucuronidemetabolitemaybeneededtobetterpredicttheinitialhumandose.One wouldthenbecarefulaboutthepotentialfreefraction(Fu)changewithpHadjustment (nonphysiologicalpH).
9Generalconsiderationsforbioanalysisforextraction,chromatography, andMSdetection
Bioanalyticalmethodstypicallyconsistofanalyteextractionfrombiologicalsamples,liquidchromatographytoseparateanalytesofinterestfromendogenouscomponentsandmetabolitesthatmaycauseamatrixeffectorselectivityissue,andMSdetection,ofteninthe formatoftandemmassspectrometers,toenhanceassayselectivityandsensitivity.WhendevelopingaquantitativebioanalyticalLC-MSmethod,oneneedstoholisticallyconsiderall threepartsasoneintegratedsystemandsometimestrade-offsneedtobecarefullybalanced [33].Onewillalwaysneedtokeepinmindtheintegrityoftheanalyteduringthesampleextraction,postextraction,andchromatography.Itshouldalsobenotedthatsomelabile
metabolites,eventhoughnotbeingmeasured,canconverttotheanalyteduringsampleextraction,chromatography,andMSdetectionandcausequantitationbiasfortheanalytesof interest.
BiologicalsamplesareseldomamenabletodirectinjectionontotheLC-MSsystem. AnalytesofinterestneedtobeextractedfromthebiologicalmatricespriortoLC-MSanalysis. Thepurposeofextractionistoeliminateorreduceinterferenceswhichcanco-elutewiththe analytesandcausematrixeffectsorquantitationerrors,andtoconcentratetheanalytesand improvetheirdetection.Whenoptimizingtheextraction,analyteratherthanthematrixisthe focus.Thismeansthatanextractionmethodwiththehighestrecoveryfortheanalytemay alsosufferfromaseverematrixeffect.Thecommonlyusedsamplepreparationmethods aredirectinjection,diluteandshoot,proteinprecipitation,liquid-liquidextraction(LLE) (solid-phasesupportedliquid-liquidextraction),andsolid-phaseextraction(SPE),thelast threebeingthemostfrequentlyused.Forexample,proteinprecipitationiswidelyutilized andisalsofrequentlythepreferredextractionmethodduringdrugdiscoveryandpreclinical development.Analytesofinterestarereleasedfromproteinwhenaproteinprecipitation reagentsuchasorganicsolvents(e.g.,acetonitrile,methanol),acids,orsalt(e.g.,ammonium sulfate)isaddedtothebiologicalsamplestodenaturetheprotein.Theanalytestaysinthe supernatantafterthecentrifugationandcanbeanalyzeddirectlyorcangothroughevaporation/reconstitutionstepstomaketheinjectionsolutioncompatiblewiththechromatographiccondition.Thisprocedureisoflowcost,easytoperform,andcanbeperformedin the96-wellformat.Theextractionisalsoverymildandthusavoidspotentialanalytedegradationorconversionfrommetabolitetoparent.Withtheadvancementofthemodernmass spectrometer,assaysensitivityatlowng/mL,whichinmostcasesisadequatefordiscovery andnonclinicalstudies,iseasilyachievable.However,ifasimplesampleextractionproceduresuchasproteinprecipitationisused,onemayneedtocompensateforthepotential ion-suppressionorin-sourceconversionsuchasthatfromtheglucuronidemetaboliteto theparentcompoundbyusingamoreextensivechromatographicelution.Themechanism forsampleextractionandchromatographyideallyshouldbeorthogonalsothatabetter methodselectivityisprovided.Ontheotherside,SPEisverypowerfulforsamplecleanup asitprovidessomelevelofchromatographicseparationbetweentheanalytesandother matrixcomponents.SPEisalsoeasilyautomatableandprovideshighcapacityforanalyte enrichment.However,someoftheSPEconditionscanbeharsh,especiallyinthecaseof strongcationorstronganionSPEwhereextremepHconditionsareusedtoelutetheanalytes. Thismayleadtounwantedinstabilityofanalytesorthebreakdownofconjugatedmetabolites totheanalyteofinterest.Therefore,theseSPEconditionsshouldonlybeusedwhenstability andbiotransformationoftheanalytearewellestablished.
Forlargemoleculebioanalysis,twoapproaches,namelythebottom-upandtop-down,are typicallyused [34].Thebottom-upapproachinvolvesanenzymaticdigestionandselectionof asurrogatepeptidethatcanrepresentthelargemolecule,whilethetop-downLC-MSmeasurestheintactlargemoleculedirectly.Bothapproachesstillrequireagoodsamplecleanto removetheabundantendogenousproteinsandotherinterferences.Inadditiontotheclassic samplecleanupproceduressuchasSPE,othernovelapproachesarealsodevelopedforproteinbioanalysis,notablyproteinprecipitation/pelletdigestion,abundantproteindepletion, andaffinityenrichment.Whilethebottom-upapproach,usingatriplequadrupolemassspectrometerforthedetection,typicallyhasasuperiorsensitivitytothetop-downapproachby
thehigh-resolutionmassspectrometersuchasTOF,thelatterhasseveraladvantagestoo [35] Forthetop-downintactproteinLC-MSanalysis,agenericMSmethodistypicallyused,and littlemethoddevelopmenttimeisrequired.Themethodisindependentoffragmentationefficiency,whichisoftennotthecaseforsurrogatepeptides.Theabilitytoacquirethecomplete informationofposttranslationalmodifications(PTM)andbiotransformationallows postacquisitiondatamining [19].Someofthecommonlyobservedchallengesforsample preparationoflargemoleculesincludelossofprotein/peptideduetononspecificadsorption; analyteinstabilityduetoproteases;poorreproducibilityofenzymaticdigestionandhighmatrixeffectsduetohighconcentrationofendogenousproteins;incompleterecoverydueto bindingofproteinorpeptidestohigh-abundanceproteinsandantibodies.Effortshavebeen madetoovercomethesechallenges.Possiblesolutionsfornonspecificadsorptionincludeuse oflow-adsorptionpolypropyleneandpolyethylenecontainers;useofsilanizedglasscontainers;avoidingpreparationoflowconcentrationsolutionsinamatrix-freeenvironment; addingacarriersuchasBSAtoanymatrix-freesolutions;andspikinghighconcentration stocksolutionsdirectlytoplasmaandpreparinglowconcentrationsampleswithserialdilution.Useofproteaseinhibitorssuchasdiisopropylflurophosphate(DFP),sodiumfluoride/ potassiumoxalate/trichloroaceticacid,4-(2-aminoethyl)benzenesulfonylfluoride,hydrochloride(AEBSF)—Pefabloc,orphenylmethylsulfonylfluoride(PMSF),storageofsamples inanultralowfreezer(< 60°C),andhandlingthesamplesinanicebatharerecommended aspossiblesolutionsforproteinandpeptideinstability.Thereareseveralpossiblesolutions toremoveabundantproteins [36].Thefirststepistouseurea,guanidine,orstrongacidto eliminateproteinbindingbetweentargetpeptide/proteinandendogenousabundantproteinssuchasalbumin.Ifthetargetprotein/peptideishighlyhydrophilic,TFAorTCAcan beusedtoprecipitateabundantproteinswhilekeepingtheanalytesintheaqueoussupernatant [37].SPE(anionorcationexchange)canbeusedaloneorinconjunctionwithprotein precipitation—forextractinghydrophilicproteins/peptides.Immuno-affinityextractionmay beusedforachievingadditionalselectivity [38].Thelargestchallengecurrentlyisstilltherelativelypoorsensitivity(comparedtoELISA),butithasbeensignificantlyimprovedwithnanoLC-MS/MSandabettersamplecleanupproceduresuchasimmune-affinityextraction.
ForLC-MSbioanalysis,chromatographyplaysapivotalroletoensureassayselectivityand robustness.Phase-IIconjugatedmetabolitessuchasglucuronideandglutathioneconjugates needtoberesolvedfromtheanalytesincetheycanbreakdownbacktotheanalyteintheion sourceandcauseassaybias [39].Isomersandendogenousinterferenceneedtoberesolved fromtheanalyte.CompoundssuchasphospholipidsanddosingvehicleslikePEGneedtobe resolvedfromanalytessincetheycancauseionsuppression [40].Reversed-phaseLChas beentraditionallyusedforthequantitativeLC-MS.Withanincreaseoforganicsolventconcentrationinthemobilephase,retentionoftheanalytedecreases.However,oneshouldbe awareofthepotentialbimodalretentiononthereversed-phasecolumn,leadingtoa U-shapedretentionprofilewheretheinitialretentiondecreasesuponincreasingtheorganic contentinthemobilephasebutafurtherincreaseintheorganiccontentresultsinincreased retentiontimeforcertaincompounds,especiallypolarbasicanalytes,duetotheirinteraction withtheresidualsilanolgroups.Thisbi-modelretentionmaycausearetentionshiftduring therunorirreproducibilityofthemethodwhenahighorganiccontentmobilephaseisused onareversed-phasecolumn [40].Thiscanalsoleadtothemismatchofinjectionsolventand chromatographywhichmayalsoleadtodistortedchromatographicpeaks [41].Inadditionto
reversed-phasechromatography,separationbasedonotherretentionmechanismscanalsobe usedcomplementarilysuchasHILICforpolaranalytes [42,43] andnormalphasechromatographyforchiralseparations [44].LargemoleculeLC-MSusuallyusesashorterchainstationaryphasesuchasC4,widerpoorsize(atleast300A ˚ ),andelevatedcolumntemperature.
TheprincipleofMSistheproductionofionsfromanalyzedcompoundsthatareseparated orfilteredbasedontheirmasstochargeratio(m/z).Mostoftheapplicationsforquantitative bioanalysisusetandemmassspectrometers(MS/MS)thatemploytwomassanalyzers—one fortheprecursorioninthefirstquadrupoleandtheotherfortheproductioninthethird quadrupoleafterthecollision—activateddissociationoftheprecursorioninacollisioncell (secondquadrupole).Betweenthehigh-pressureLCandtheMSoperatedunderahighvacuumenvironment,interfaceconnectionsthatoperateatatmosphericpressure,suchas electrosprayionization(ESI),atmospheric-pressurechemicalionization(APCI),andlessfrequentlyatmospheric-pressurephotoionization(APPI),havematuredintohighlyreliable techniquesnecessaryforquantitativeLC-MS/MSbioanalysis.Morerecently,application high-resolutionmassspectrometer(HRMS)inbioanalysishasdrawnalotofattention [45].Duetoitsenhancedspecificityusingthehigh-resolutionpoweranditscapabilityofsimultaneousquantitationandstructuralelucidation,HRMScouldleadtoapotentiallyrapid andreliablemethoddevelopmentforbioanalysisaswellassampleanalysis,thusgenerating bothcostandresourcesavings [46]
10SelectedapplicationsforLC-MSbioanalysis Itisnottheintentionofthisbookchaptertocovereveryaspectofquantitativebioanalysis. Rather,afewrelativelynewlydevelopedareasarefocusedontofurtherillustratetheimpact ofbioanalysisondrugdiscoveryanddevelopmentbyusingcasestudiesfromourownlab. Inthefirstexample,wewilldiscussthestatusofusingLC-MS,asacomplementarytoolto thetraditionalELISAmethods,toanalyzelargemolecules,particularlysomeofthenovel platformofbiotherapeuticssuchasantibody-drugconjugate(ADC)andhalf-lifeextended peptides.ThevalueofmultipleLC-MSmethodssuchasthebottom-upandtop-downaswell assimultaneousquantitationandcataboliteidentificationishighlighted.Thesecondexample isthedevelopmentofmicrosamplingtechnologywhichhasbeenwidelyusedtosupportboth preclinicalandclinicalstudies.Incomparisonwiththetraditionalplasmasamplingtechnology,microsamplingpresentssomeadditionalchallengesformethodestablishment,validation,andsampleanalysis,fromboththescientificandcompliancepointsofview. Biomarkershavebecomemoreandmoreimportantindrugdiscoveryanddevelopment, fromconfirmationoftargetengagementtopatientstratification.Asbiomarkersareendogenousmolecules,theirmeasurementisparticularlychallengingforbioanalyticalscientistsas thereisno“true”blankmatrixtopreparestandardsandQCsamples.Itisalsoverydifficultto confirmtheselectivityofabiomarkerassaydueitsendogenousnature.Variousstrategiesin thethirdexamplehavebeenproposedtomitigatethesechallenges.Lastly,theaccuratereadingofimportantmetabolitesinthebodyhasdrawnalotofattentionrecentlysincethepublicationoftheFDAMISTguidance [47].Toestablishsafetycoveragetoensureasafeclinical trialispivotal.Itisnotalwaysstraightforwardtoestablishtherightbioanalyticalstrategyfor metabolitemeasurement.Inthelastcasestudy,thestrategyformetabolitebioanalysissuchas
measuringpolarmetabolites,assessmentofchiralconversion,andselectionofinternalstandardwillbediscussed.
10.1LC-MSoflargemolecules LBAistraditionallytheprimaryplatformforbioanalysisoflargemoleculesandhasthe advantagesofprovidingsuperiorsensitivityandhighthroughput.However,itmayalsosufferfromlimitationssuchascross-reactivity,requirementofhighlyspecificreagents,andnot beingabletodirectlyelucidatestructureinformation.Inthepastdecade,LC-MShasincreasinglybeenappliedforbioanalysisoflargemoleculesasacomplementarytechniqueofLBA. LC-MShastheuniqueadvantagesofbeinghighlyspecific,moreresistanttomatrixinterference,andlessstringentonreagentrequirement.Moreimportantly,LC-MScanprovidevaluablestructureinformationwhichmaybecriticalforunderstandingtheADMEpropertiesof proteindrugcandidates.
Asdiscussedearlier,therearegenerallytwoapproachesofanalyzingproteinsusing LC-MS:bottom-upandtop-down.Inthebottom-upworkflow,proteolytic(usuallytryptic) peptidesgeneratedbydigestionaremonitoredassurrogatesoftheprotein,typicallyonatriplequadruplemassspectrometer,whichaffordshighlyspecificandsensitivedetection. However,therecouldbesituationswhenthesurrogatepeptidedoesnotadequatelyrepresent thewholeproteinintermsofitsfunctionality,stability,orcoexistenceofmultipleisoforms. High-levelstructuralinformationsuchasintegrityofamultiplesubunitproteinorproteolytic catabolismislostduringdigestion.Thisiswhenthetop-downintactanalysisoftheprotein canplayanimportantrole.Thetop-downintactbioanalysisoflargeproteins(e.g.,mAb)has gainedpopularityinrecentyearsthankstoapplicationsoftheadvancedsamplepreparation technique,suchasimmune-affinitycapture,whichenableseffectivesamplecleanup [35]. IntactmassspectraobtainedfromHRMSmaycontainthepeaksofnotonlytheunchanged intactmoleculebutalsothatofcatabolites,makingsimultaneousquantitationandcatabolite identificationpossible [19].
Inboththebottom-upandtop-downapproaches,theworkflowinvolvesanup-frontsamplepreparation.Dependingonthenatureoftheanalyte,requiredsensitivity,andthebiologicalmatrix,methodologiesrangingfromproteinprecipitation,solidphaseextraction, abundantproteindepletion,andaffinitycapturecanbechosenorcombined,withincreased effectivenessinthesamplecleanup.Inthebottom-upapproach,thesurrogate(orsignature) peptideisdetectedandanalyzedinthesamewayassmallmolecules,onatriplequadruple instrumentbymultiplereactionmonitoring(MRM)analysis,orinsomelabs,onHRMSbya fullscanorproductionanalysis,aprocessalsoknownasparallelreactionmonitoring(PRM). Incomparison,intactproteinanalysishasmorediversifiedchoiceswhenitcomestoMSdetection [48].Peptidesorsmallproteinscanbemonitoredinthesamewayassmallmolecules orsurrogatepeptidesontriplequadrupoleorHRMS.LargeproteinssuchasmAbareheavily chargedwhenanalyzedunderESI,resultinginclustersofmultiplechargedionswhichrequireresolutiononHRMS.Peaksforaselectedchargedstate(e.g.,thethreemostabundant peaks)canbeextractedwithacertainextractionwindowtoreconstructanextractedionchromatogram(EIC)whichcanbeusedforquantitation [49].Thisapproachismorewidely adaptedbecauseitdoesnotrequireadditionalsoftwarefordataprocessingandthuscan
beuniversallyappliedtodataacquiredoninstrumentsfromdifferentvendors.Alternatively, multiplechargedionsaredeconvolutedtogeneratenon-orzero-chargedpeakswhichcanbe inturnquantifiedbasedontheintactmolecule,aprocessrequiringaspecificsoftwarefunction.Inthecasewhenmultiplecomponents,suchasthoseofcatabolitesordifferentglycosylationisoforms,arepresentinthesample,deconvolutionisamoredirectandeffective waytovisualizethedata.
10.1.1LC-MSbioanalysisofmAb Monoclonalantibodiesandrelatedproductsrepresentoneofthemostpromisingandfastgrowingclassesoftherapeutics.EventhoughthebioanalyticalplatformformAbhasbeen predominantlyLBA,therehavebeenincreasednumbersofapplicationsonquantitationof mAbusingLC-MS.Thisisparticularlytruefordiscoverystudysupportwhenaspecificreagentmaynotbereadilyavailableatanearlystagebeforetheidentificationofthefinalcandidatefordevelopment.ThegeneralworkflowfornonclinicalLC-MSbioanalysisofmAb involvedtrypsindigestionandLC-MRManalysisofsurrogatepeptides,whicharetypically theconservedsequencesofthehumanFcregiononthemAb.Thechoiceofsamplepreparationdependsontherequiredsensitivityandtypeofbiologicalmatrix.Proteinprecipitation followedbydigestionofthepellet,aprocessknownaspelletdigestion,hasbeenwidelyused becauseitiseasytooperateanddoesnotrequirespecificreagents [50].Forbettersensitivity, immune-affinitycaptureusingantihumanFccanprovideaneffectiveandselectivecleanup ofhumanmAbfromnonclinicalsamples.Forclinicalsamples,morespecificcapturingreagentssuchastheantiidiotypeantibodyagainstepitopeinCDRofthemAbcanbeused toavoidpullingdownendogenoushumanIgGs.Alternatively,thetargetproteinofthe mAbcanalsobeusedasanaffinitycapturereagent.Eitherway,cautionhastobeexercised toevaluateinterferencefromendogenoustargetsonthecapturingprocessandtounderstand whetherthemeasuredconcentrationsarethoseoffree(notoccupiedbytarget)ortotalmAb. DuetothehighlyspecificdetectionbyLC-MS,itispossibletomonitorcertainmodifications onthemolecules.Oneexampleisdeamidation,acommonandspontaneousbiotransformationofmAb.IthasbeenshownthatdeamidationatacertainregionofmAbcouldimpactits bindingaffinity,potency,safety,andpharmacokinetics [51].Thesurrogatepeptide containingAsn,thedeamidationproductAsp,andthesuccinimideintermediatecanbeseparatedonLCandmonitoredbyMRMtoevaluatetherelativelevelsofeachspecies,which helpsunderstandthedynamicsofthisinvivobiotransformation [52,53].
TheemergingtechniqueofintactproteinbioanalysishasalsobeenexemplifiedonmAbin afewpublications.Ourlabpublishedaworkflowofquantifyinglargeproteinsinbiological samplesbyusingimmune-affinitycapturecoupledtoLC-HRMSanalysis [35].The deconvolutedintactmassspectrawereprocessedforquantitationusingaresearchversion ofthesoftware.ThisdeconvolutedapproachwasalsocomparedwithEICanddemonstrated similarbioanalyticalperformanceintermsofsensitivity,linearity,accuracy,andprecision. Moreimportantly,theintactconcentrationdatageneratedforaninvivomonkeyPKstudy showedresultsconsistentwiththosebythemorecommonlyusedbottom-upapproach, confirmingthesuitabilityofquantifyingmAbusingtheintactquantitationmethod [49] Forbettersensitivityanddetectionresolution,mAbcanbebrokendownintorelatively smaller“intact”piecesbyusingreducingreagentstodissociatethelightchainsandheavy chains.Furthermore,mAbcanbespecificallycleavedaroundthehingeregionbytheIgG
