Key factors influencing ADME properties of therapeutic proteins

17 Pages·2016·0.49 MB·English

by  Jay Tibbitts

MABS
2016,VOL.8,NO.2,229–245
http://dx.doi.org/10.1080/19420862.2015.1115937
REVIEW
Key factors influencing ADME properties of therapeutic proteins: A need for ADME
characterization in drug discovery and development
JayTibbittsa,DavidCanterb,RyanGraffb,AlisonSmithb,andLeslieA.Khawlib
aUCBCelltech,Slough,SL13WE,UK;bDepartmentofPathology,KeckSchoolofMedicineoftheUniversityofSouthernCalifornia,LosAngeles,CA,USA
ABSTRACT ARTICLEHISTORY
Protein therapeutics represent a diverse array of biologics including antibodies, fusion proteins, and Received5August2015
therapeuticreplacementenzymes.Sincetheirinception,theyhaverevolutionizedthetreatmentofawide Revised13October2015
range of diseases including respiratory, vascular, autoimmune, inflammatory, infectious, and Accepted29October2015
neurodegenerative diseases, as well as cancer. While in vivo pharmacokinetic, pharmacodynamic, and KEYWORDS
efficacy studies are routinely carried out for protein therapeutics, studies that identify key factors Absorption;antibody-drug
governingtheirabsorption,distribution,metabolism,andexcretion(ADME)propertieshavenotbeenfully conjugate(ADC);biologics;
investigated.Thoroughcharacterizationandin-depthstudyoftheirADMEpropertiesarecriticalinorder biotherapeutics;distribution;
to support drug discovery and development processes for the production of safer and more effective excretion;imaging;labeling;
biotherapeutics. In this review, we discuss the main factors affecting the ADME characteristics of these metabolism;monoclonal
large macromolecular therapies. We also give an overview of the current tools, technologies, and antibody(mAb);neonatalFc
approaches available to investigate key factors that influence the ADME of recombinant biotherapeutic receptor(FcRn);
pharmacokinetics;
drugs,anddemonstratehowADMEstudieswillfacilitatetheirfuturedevelopment.
subcutaneousbioavailability
Introduction
either 1) measure effect site concentrations directly (often
Ithasbeennearly40ysincebiologistsfirstlearnedtoproduce impracticalinhumans),2)assumethatdrugattheeffectsiteis
human growth hormone and insulin. The discovery of recom- inequilibriumwiththebloodcompartment,3)usenonclinical
binant protein technology revealed the potential of proteins as absorption, distribution, metabolism, and excretion (ADME)
therapeuticagents;apotentialwhichhasbeenincreasinglyreal- data to derive an informed estimate, or 4) employ the mecha-
izedthroughouttheinterveningyears.1Whatstartedwithrela- nistic mathematical models to characterize and predict the
tively small, native proteins has gradually expanded to include time-courseofdrugeffectsintissues,ateffectsites,andincom-
monoclonal antibodies (mAbs), cytokines, replacement plexwiththepharmacologicalreceptor.
enzymesandmorerecently,adiversearrayofproteinproducts. Therapeuticproteinshavetraditionallybeenadministeredto
Theseproteinproductsmergetogetherbiologicandpharmaco- patients intravenously, which is both inconvenient and expen-
logic elements yielding engineered antibody derivatives (e.g., sive. As the popularity of these therapeutics has grown, their
nanobodies, Fabs, scFvs), antibody-drug conjugates (ADCs), route of administration has increasingly shifted toward non-
fusions of therapeutic proteins with native and non-native intravenous delivery methods. These delivery methods include
products, and bispecific antibodies. This burgeoning diversity inhalation and parenteral administration (subcutaneous (SC)
of protein therapeutics has resulted in a concomitant increase and intramuscular) along with depot formulations facilitating
in the number of biologics in clinical development, with more sustained-release and other formulations which are thought to
than400molculescurrentlyinclinicaltrialsaroundtheworld. improveSCdeliverybyincludinghelperenzymessuchashyal-
Thesemoleculesarebeingassessedfortheirpotentialtotreata uronidase.Inordertomaximizethedevelopmentandapplica-
variety of diseases, including cancer, immunological disorders, tionofsuchapproacheswemustgainabetterunderstandingof
andinfectiousdiseases.2Despite thispromise,thereisasober- the mechanisms and determinants underlying the absorption
ing attrition rate for biologics in the clinic, with only 12% of ofthesehighmolecularweighttherapeuticagents.Evenforthe
those molecules entering the clinic and reaching the market.3 seemingly well characterized therapeutic class of mAbs, our
The causes for this attrition may vary, but lack of efficacy is ability to accurately predict human bioavailability (F) and
often identified as a major contributor.4 Optimizing efficacy absorptionkineticsremainspoor.
requires, among other things, sufficient drug delivery to the The evolution from more native protein therapeutics (e.g.,
intended target site. Indeed, a key pillar proposed for improv- cytokines, antibodies) to biotherapeutics with more novel and
ing the clinical success rate is the confirmation of sufficient complexstructuresincludingpolyethyleneglycol(PEG)-conju-
drugexposureattheeffectsite.4Toachievethisgoal,onemust gated proteins or peptides, fusion proteins, and ADCs, has
CONTACT JayTibbitts [email protected];LeslieA.Khawli [email protected]
PublishedwithlicensebyTaylor&FrancisGroup,LLC©JayTibbitts,DavidCanter,RyanGraffb,AlisonSmith,andLeslieA.Khawli
ThisisanOpenAccessarticledistributedunderthetermsoftheCreativeCommonsAttribution-Non-CommercialLicense(http://creativecommons.org/licenses/by-nc/3.0/),whichpermitsunre-
strictednon-commercialuse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.Themoralrightsofthenamedauthor(s)havebeenasserted.
230 J. TIBBITTSETAL.
introduced new challenges related to the stability, catabolism, discuss these effects asthey pertain toSCabsorption,as thisis
and elimination of these products. These characteristics can themostcommonnon-intravenousrouteofadministration.
affect the observed pharmacology as well as the pharmacoki- While drug delivery technology platforms have advanced
netics(PK)oftheproteintherapeutic.Regulatoryagenciesrec- significantly, inhalation and oral administration of biothera-
ognize the challenges and potential value of determining the peuticagentsremainformidablechallengesforformulationsci-
ADME characteristics of therapeutic proteins, as evidenced by entists. As large, hydrophilic, and chemically labile molecules,
theinclusionofasectionondispositionintheEuropeanMedi- most biotherapeutics are virtually excluded from traditional
cines Agency’s Guideline on the Clinical Investigation of the tablet and capsular formulations.6 Consequently, most biolog-
Pharmacokinetics of Therapeutic Proteins.5 While the agency ics currently on the market are injected. Unfortunately, this
acknowledgesthatstudiesofthedispositionoftherapeuticpro- routeofdrugadministrationtendstoincurlowpatientcompli-
teins may not be necessary, it suggests that “specific studies of ance and a higher cost of therapy. Despite new advancements
therouteofeliminationandmetabolism…andidentificationof inmodernoralandpulmonarydeliverydevices,certainbarriers
metabolites in vitro should be considered and discussed on a still compromise the absorption of biologics through these
case-by-case basis” and that active metabolites should be mea- routes.Theoralrouteofdrugdeliveryposesachallengedueto
sured.Fortherapeuticproteinswithnon-nativeconformations, degradationofbiotherapeuticsinthegastrointestinal(GI)tract.
whererelianceonwelldescribedADMEpropertiesmaynotbe Alternatively, the nasal cavity and lung can be effectively tar-
possible, the need for dedicated ADME investigations may geted for drug absorption, thereby avoiding the proteolytic
becomeparamount. enzymes and first-pass metabolism of the GI tract. Although
As the realm of protein therapeutics grows, both in interest similar threats to protein therapeutics (such as metabolic
and diversity, it becomes clear that a greater understanding of enzymesandmacrophages)arefoundinthelungs,thegeneral
theADMEpropertiesofthesemoleculeswillbecriticaltotheir metabolicactivitiesandpathwaysoftherespiratoryepithelium
design, development, and use. In this review, we discuss the differfromthoseobservedintheGItract.7Furthermore,while
relationships between protein therapeutic diversity, current thealveolarepitheliumandcapillaryendotheliumremainfairly
knowledge, and the available tools to assess ADME properties. impermeable to many hydrophilic substances of large molecu-
We hope to illustrate the benefit of utilizing these tools as a larsize,theendothelialjunctionsallowforthepassageoflarger
means to gain a better mechanistic understanding of the PK, moleculesofabout4–6nm–thesizeofmanybiotherapeutics.8
pharmacodynamics(PD),andmetabolismofproteintherapeu- Additionally, the mucosal lining of the pulmonary epithelium
ticsandtoemphasizetheimportanceofunderstandingbiother- andthesurfactantcoatingthealveolihavehighconcentrations
apeutic ADME as a way to drive forward the selection of of protease inhibitors, and presumably protect peptides and
successfuldrugcandidates. proteins from degradation. Nonetheless, this protection
appears tobe anexceptionratherthanthe rule,as membrane-
associated (on epithelium and endothelium) and intracellular
ProteintherapeuticdiversityandADME (inmacrophages,lymphocytes,neutrophilsandmastcells)pro-
teases and peptidases can readily degrade administered pepti-
The current chemical space defining protein therapeutics is
des and proteins.9 Although oral administration remains a
vast. These molecules come in many unique forms, with vari-
challenge in the delivery of therapeutic biologics, inhalation
ous structural components that each affect the molecule’s
poses a promising potential route of administration if current
behavior.Inthissection,wetouchonafewcomponentsofbio-
challenges can be overcome. Until then, the focus will remain
therapeuticdiversitywithafocusonhoweachofthesecompo-
onSCdeliveryforthesemolecules.
nents can affect the ADME properties of the therapeutic by
Relatively little is known about the mechanism of SC
introducingstructuraldiversity.
absorption of proteins in different species, and conflicting
results have been reported on the effects of the delivery route
andsite ofadministrationonproteinPK.10Upon SCadminis-
InfluenceofmolecularmassonADME
tration,therapeuticproteinsaredeliveredtothehypodermis,a
Perhapsthemoststrikingcharacteristicoftherapeuticproteins space consisting of adipose tissue separated by a fibrovascular
istherangeinmolecularweightobservedinthesetherapeutics. network.11-13Withinthehypodermisliesanetworkofextracel-
While there is some uncertainty about the mass distinction lular matrix (ECM) consisting of a number of matrix proteins
between peptides and proteins, it is often stated that peptidic including collagen, proteoglycans, and glycosaminoglycans,
molecules above 3–5 kgdaltons (kDa) are defined as proteins. which together confer a net negative charge to the ECM that
Usingthisdefinition,therangeinmassoftherapeuticproteins mayhaveaneffectontheinteractionsoftheECMwithpoten-
spansfrommoleculesassmallascalcitonin(3.5kDa)tomAbs tial therapeutic proteins. Therapeutic proteins move through
(150 kDa). It is important to distinguish between molecular the ECM via diffusion and convection thereafter entering sys-
weightandphysicalsize,whichcanberepresentedbymeasure- temic circulation through blood or lymphatic capillaries. The
mentssuchasthehydrodynamicradius.Thesetwoparameters fraction undergoing lymphatic absorption increases with an
tendtocorrelate,particularlyforglobularproteins,butnotnec- increaseinmolecularsize.15,16Thediffusionvelocityofsmaller
essarilyforproteinstetheredtolessstructuredmoietiessuchas proteins (<15 kDa) is greater than their convection velocity
PEGylatedproteinsandothermorenovelconstructs.Neverthe- allowingthemtodiffuserapidlythroughtheECMandprimar-
less, the mass of a therapeutic protein, regardless of its overall ilyintothebloodcapillaries.13,18Thediffusionvelocityoflarger
shape, can have dramatic effects on its absorption. We will moleculesisdramaticallyhinderedbythemesh-likeframework
MABS 231
of the ECM limiting their local migration.15,16 Therefore con- due to the presence of a non-membranous diaphragm across
vection is believed to be the primary mechanism responsible the opening.27,28 Discontinuous endothelium (liver, spleen,
for antibody transport away from the SC injection site. Bulk bonemarrow)hasthegreatestpermeability.26,29Itischaracter-
flow of interstitial fluid (IF) drives these larger molecules ized by large fenestrations lacking diaphragms, pores within
toward blind-ended lymphatic capillaries which, unlike the individualcells,andapoorlyformedbasementmembrane.27,28
endothelium of continuous blood capillaries, expand in Passive movement via convection or diffusion between the
response to increases in IF pressure allowing fluid to flow in cells is thought to be the more dominant process for protein
but not out. Once biotherapeutics enter the lymphatic system, therapeutic distribution.31 Diffusion into and out of tissues is
they eventually drain into the venous system. This passage dependent on the concentration gradient between the plasma
through the lymphatic system is considerably faster than their and interstitial fluid and the surface area of the exchange area.
migration through the ECM,13 marking interstitial convection Convection is dependent on both the surface area of exchange
astherate-limitingstepofabsorption. and the pressure gradient across the vessel. Which process is
An accurate description of the quantitative relationship dominantforagiventherapeuticproteindependsprimarilyon
between molecular size and the fraction absorbed by lym- size and charge, but for molecules the size of antibodies the
phatics remains elusive, particularly across species.13 Devia- movement is likely governed by convection.32 For antibodies,
tionsfromourcurrentmodelsarisefromstudiesreportingthat whichlieatthehighendoftheproteintherapeuticmassscale,
somemid-sizedproteins,rangingfrom5.6–60kDa,showlittle the extent of tissue distribution has been thoroughly studied,
lymphatic absorption.18 The cause of this discrepancy is not and there appears to be little species or molecule dependency
known,butmayberelatedtospeciesandinjectionsitedifferen- in the non-specific tissue (i.e., not target-mediated) distribu-
ces. What is even less understood is the effect of molecular tion. The estimated interstitial fluid-to-plasma ratio for anti-
weightonbioavailabilityasthereappearstobelittlecorrelation bodies and other large therapeutic proteins at steady state
between these parameters.11,21 This lack of correlation, while ranges from »0.3-1 (derived from physiologically based PK
notintuitive,maybeattributedtoamultitudeoffactorsaffect- modeling).33,34 As described above, the ratio is thought to be
ing bioavailability such as target binding, catabolism, and the tissuedependent,withtissuespossessingnon-fenestratedendo-
administration site. As such, the major parameters affecting thelium at the lower end of the scale while those tissues with
these processes are thought to include the roles of lymph and fenestrated or discontinuous endothelium at the higher end of
bloodcapillariesinsystemicabsorption,cross-speciesdifferen- the scale. Direct measurements of the interstitial fluid concen-
ces in hypodermis morphology and physiology, drug formula- trations of therapeutic proteins have not been reported; how-
tion,stabilityofthemolecule,thesiteofinjection,thedepthof ever, studies of interstitial fluid concentrations of albumin and
injection, as well as the molecular properties of the proteins IgG in skin have reported an interstitial fluid to plasma ratio
themselves.5,14,22-24 More work needs to be done in order to (0.5–0.6) generally consistent with these predictions.35,36 Little
more precisely characterize the contributions of these compli- informationhasbeenpublisheddescribingtheinterstitialfluid-
catingfactorstotheSCabsorptionofbiologics. to-plasma ratios for other proteins, but it is reasonable to
Onceabiotherapeuticreachessystemiccirculation,mass(or expect that the same processes govern those proteins and that
size)caninfluenceitsdistribution. Tissue distribution isdeter- smaller proteins would have higher ratios. Once within the
minedbythereversiblemovementofmoleculesintoandoutof interstitial space, many of the same dynamics and influences
tissuesfromthecirculation.Movementacrossthevascularbar- previously described for absorption still apply (e.g.,, interac-
rier into the interstitial fluid of tissues can occur either by tions with ECM) and the movement of protein therapeutics
movementacrosstheendothelialcellsthatmakeupthisbarrier, and other macromolecules (including PEGylated molecules)
orbetweenthosecells.Thesizeandcharge oftherapeuticpro- into and through the tissues is governed primarily by convec-
teinsgenerallylimitsmovementacrossthelipidbilayersconsti- tion, though still dependent on mass and volume.37,38 As
tuting the cell membranes unless facilitated by an active described above, larger molecules have more limited distribu-
transport process, such as the neonatal Fc receptor (FcRn) of tiontotheextracellularspace,anddistributeintoandoutoftis-
IgG. The contribution of active transport to tissue distribution sues more slowly.33,39,40 This can have an effect on molecule
isanactiveareaofstudythatwediscussinthecontextofFcRn choice for different disease states, as has been nicely demon-
below. Movement between cells (paracellular) is generally stratedfortumorsbyseveralgroups.41
dependentonthenatureoftheendotheliumliningthecapillar- Disease state can also influence vascular permeability and
ies in that tissue. Three types of capillary endothelium have transport, often leading to increased permeability.27,28 In the
beendescribed thatexhibitdistincttransportmechanisms.25-29 caseoftumors,alargebodyofresearchexists onsolutemove-
Continuous non-fenestrated endothelium, found in muscle, ment into and through tumors, which are known to have per-
CNS, skin, and lung, is thought to be the most resistant to turbed vascular function and physiologic fluid flow.31-34,42-44
transport, restricting movement to water and solutes less than Forotherdiseasestates,muchlessisknownabouttheeffectof
3 nm in radius. In the brain, a special case of non-fenestrated changes in vascular permeability and other disease processes
endothelium exists where tight junctions between endothelial on the movement of protein therapeutics into and out of
cells act as a stringent barrier to paracellular transport.29,30 affectedtissues.Therapeuticproteinsareeliminatedviaseveral
Continuousfenestratedendothelium(renalglomeruli,synovial mechanisms, including proteolytic degradation in the plasma
tissue, intestinal mucosa) is more permissive due to the pres- or extracellular fluid, renal filtration, and cellular uptake fol-
enceoffenestrationsbetweencells,butstillretainsahighresis- lowed by proteolytic degradation (e.g., receptor-mediated
tance to the passage of large macromolecules (e.g., albumin) uptakebycellsurfacereceptortargetsorFcreceptors,andfluid
232 J. TIBBITTSETAL.
phasepinocytosis).Ofthese,renalfiltrationfollowedbytubular describedabove,theinterstitialspacehasanetnegativecharge,
uptake and degradation is the most dependent on the mass of and proteins with a net positive charge may have delayed
theproteintherapeutic.Ithasbeenlongknownthattherateof absorption following SC dosing, likely due to charge-charge
renaleliminationofproteinsisinverselyrelatedtosize.45Com- interactions in the SC space.11,13 Changes in charge can also
monly quoted dogma states that proteins above »50 kDa are dramaticallyaffectdistribution,asdescribedbyHongetal.42In
notsubjecttorenaleliminationduetotheinabilityofsuchpro- this study antibodies and antibody fragments with greater net
teinstopassthroughtherenalglomerularbarrier.Whileuseful, positive charge were found to have increased renal clearance
this maxim is not entirely true because size is only one factor and greater tissue distribution, likely due to increased interac-
governing renal excretion of proteins, with charge and tubular tion with negative charges on the cell surface. Other examples
reabsorptionplayingimportantrolesinrenalclearanceaswell. are described in a review of the effects of antibody charge by
Given the high perfusion rate of the kidney, even solutes that Boswell et al.50 Changes in pI of more than one pI unit can
are inefficiently filtered can be subject to substantial excretion havephysiologicconsequences,includingtheincreasedplasma
in the glomerular filtrate. For example, considerable evidence clearance and tissue retention described above. However, it is
existsindicatingthatrelativelylargequantitiesofalbuminpass important to keep in mind that for most therapeutic proteins,
intotheglomerularfiltratewiththevastmajorityreabsorbedin the range of charge heterogeneity within the product is small,
the renal tubules.46 Renal excretion can also play a significant andseveralstudieshavedemonstratedthatsuchminorchanges
role in the elimination of large therapeutic proteins, as evi- inchargehavelittleoverallimpactonPKanddistribution.50,51
denced by a 30% decreased clearance of a 63 kDa peptibody The effects of charge on protein therapeutics can also be
fusion protein in subtotal nephrectomised rats.47 Similar evi- observed in relation to their altered elimination. Charge is
denceexistsforPEGylatedpeptideswheretherenalelimination known to affect renal filtration at the glomerulus due to nega-
of parent molecule (»45 kDa) or the 40 kDa PEG component tive charges on the glomerular basement membrane.50 Thus,
was greater than 70%.49 Readers interested in the mechanisms negatively charged molecules undergo less renal filtration than
of renal elimination of macromolecules are directed to reviews more positively charged molecules of a similar size.48 In addi-
by Harraldsson et al 48 and Maack.45 The effect of therapeutic tion, the renal tubular epithelium, a site for reabsorption of
protein size on other routes of elimination is less well under- some protein therapeutic molecules from the glomerular fil-
stood,butsizeisnotlikelytoaffectprocessessuchasreceptor- trate, has a net negative charge, which can facilitate improved
mediated endocytosis, vesicular transport, or cellular reabsorption of more positively charged proteins.50 Other
pinocytosis. recent studies from the last 2 y have tried to correlate changes
As mentioned previously, size and shape are often but not in charge with the PK of antibody therapeutics. These studies
alwayscorrelated.Biotherapeuticsaretypicallythoughttohave also emphasize the point that balancing charge in the comple-
a generallyglobular shape. A notable exception may bePEGy- mentarity-determining regions (CDR), variable domain (Fv),
lated proteins, where PEG molecules (which often range from orframeworkofthetherapeuticantibodycanleadtoimproved
1-50 kDa) are conjugated to a protein or peptide, usually to PK,themagnitude of whichislikely dependentontherelative
enhanceitsPKorstability.49TheconjugationofPEGincreases influence of charge imbalance and other factors affecting the
the mass of a protein or peptide, resulting in similar ADME molecule’sdisposition.52-54Lietal.provideddirectevidenceof
effectstothosedescribedabove.However,PEGhasverydiffer- improved antibody PK following the incorporation of a more
ent physiochemical characteristics than proteins, including negativechargeonthevariablelight(VL)frameworks.53Avar-
greaterflexibility,lowerdensity,anddistinctaqueousbehavior. iantwithapIof8.61exhibitedrapidclearancewhereasamole-
As such, the ADME characteristics of native therapeutic pro- cule with pI of 6.10 exhibited relatively slow clearance. Both
teins and PEGylated proteins of a similar size may differ. This variants exhibitedcomparable binding to ratFcRn,butbiodis-
topicisdiscussedingreaterdetailbelow. tribution studies showed that the high pI variant was catabo-
lized in the liver and spleen. These results suggest antibody
chargecanhaveaneffectonPKthroughalterationsinantibody
InfluenceofchargeonADME
catabolismindependentofFcRn-mediatedrecycling.However,
Other physicochemical characteristics of therapeutic proteins in another study, Schoch et al. demonstrated that the charge
contribute to their diversity and affect their ADME properties. distribution on the variable fragment (Fv) domain is involved
Thesurfacechargeofatherapeuticproteinisapropertyofthe in excessive FcRn binding.54 This excessive binding prevents
aminoacidsequenceoftheproteinandthepHofitssurround- efficient FcRn–IgG dissociation at physiological pH, thereby
ings. This physicochemical property is complex and heteroge- reducingFcRn-dependentterminalhalf-lives.
neous, resulting in a mixture of charge variant species within In the development of therapeutic monoclonal antibodies,
the population of therapeutic protein product. Charge hetero- research and development has focused largely on the IgG1,
geneity is typically caused by deamination, isomerization, or IgG2, and IgG4 isotypes, with the IgG1 isotype remaining the
post-translational modification, which can cause a change in mostpopulartodate.55AlthoughallIgGvariants,eachofthese
thenetchargeofaprotein,therebyleadingtotheformationof isotypes contain slight differences in their hinge and constant
acidic and basic variants.51 Most therapeutic proteins have an regions that affect their functionality and half-life in vivo. In
isoelectric point (pI) in the range of 5-9, with most antibodies order to identify possible ADME differences between different
beingslightlypositivelycharged,withapIof7–9.10,51Thissur- isotypes,Datta-Mannanetal.showedthattheeffectofbalanc-
face charge can lead to interactions with other molecules and ing the CDR net positive charge on nonspecific binding was
tissue constituents in the body, thereby affecting ADME. As more significant for theIgG4versusthe IgG1 molecule.52 This
MABS 233
differential effect was connected to the degree of influence on proteinwouldaffectnotonlyitseliminationbutalsoitsdistri-
cellulardegradationinvitroandinvivoclearance,distribution bution. As the protein size increases, the proportional increase
andmetabolism.InthemoreextremecaseoftheIgG4isotype, in size conferred by glycosylation is lower, which would likely
balancing the charge yielded greater than fold7- improvement resultinmoremodesteffectsondistribution.Forexample,the
in peripheral exposure, as well as significantly reduced tissue glycosylation of a mAb adds only 10–15% to the mass of the
catabolism and subsequent excretion of proteolyzed products protein.65 The importance of glycosylation on the distribution
inurine.Consequently,balancingCDRchargeontheIgG1iso- oftherapeuticproteinsisnicelyexemplifiedbyenzymereplace-
type had a more subtle influence on non-specific binding and menttherapiesforthetreatmentoflysosomal storagediseases.
yielded only a modest alteration in clearance, distribution and Thedistributionoftheseexogenouslyadministeredenzymesis
elimination. oftendeterminedbytheinteractionofmannoseandmannose-
6-phosphate molecules on the enzyme with the respective
receptors on tissues.66 Thus, tissue distribution is determined
InfluenceofglycosylationonADME
by the relative density of these receptors on cells.67 Unfortu-
Glycosylationisapost-translationalmodificationofproteinsby nately, this limits distribution of the enzyme to cells with high
which carbohydrates (glycans) are added to specific amino receptor expression (e.g., reticuloendothelial cells) and limits
acids. Such modification is a natural phenomenon associated distribution to other affected organs that may be less highly
with the production of therapeutic proteins in eukaryotic pro- expressing(e.g.,,kidney,lung).
ductionsystems.Theglycosylationofatherapeuticproteincan
bedependentonanumberoffactors,includingcellproduction
InfluenceofdiverseproteinmodificationsonADME:
system and cell culture conditions. This interesting, but highly
Mechanismofactionenhancementandhalf-lifeextension
complex,topicisoutsidethescopeofthisreviewandinterested
readers are directed to some excellent reviews on the sub- Researchershaveexploredanumberofstrategiestoenhanceor
ject.56,57 The amount and nature of glycosylation can dramati- optimize the efficacy of protein therapeutics, often by modify-
cally affect the behavior of the proteins. The most commonly ing the protein structure, fusing a smaller protein to another
describedrolesforglycosylationarerelatedtoreceptorbinding protein fragment, or chemically conjugating an active peptide
andFceffectorfunctioninantibodies,withrecombinanteryth- orchemicaltoaprotein.Thesemodificationstypicallyresultin
ropoietin and afucosylated rituximab as illustrative exam- either an enhancement in the pharmacologic activity or a pro-
ples.58,59 However, the glycosylation profile of a therapeutic longationofthedrugexposureinthecirculation.Twotypesof
protein can substantially affect its PK and distribution. Intro- modifications can have a major impact on the pharmacologic
ducing N-linked glycosylation to proteins as a strategy for and ADME characteristics of these molecules. First, conjuga-
improvingsystemicresidencetime(generallybyincreasingsize tion of moieties that enhance or modify the pharmacology
or modifying binding to glycoprotein receptors) has been (e.g., ADCs, radioimmunoconjugates, immunotoxins) has the
exemplified by darbepoetin alfa (a hyperglycosylated form of potentialtochangenotonlytheefficacyandtoxicityassociated
erythropoietin),follicle-stimulatinghormone,andwithasingle with the therapeutic protein to which they are conjugated, but
chain diabody.58,60 For antibodies and fusion proteins possess- also the ADME characteristics. For example, the catabolism
inganantibodyFc,theeffectofglycosylationonPKanddistri- andcatabolicproductsofanunconjugatedantibodymaybeof
bution is less clear. Human antibodies contain N-linked littleinterestduetotheclosesimilaritytoendogenousantibod-
glycansatAsn297oftheFc,andsomeantibodiesalsopossess ies. In contrast, the potentially potent catabolic products of an
N-glycosylationsitesintheFvregion.61Theseglycansaretypi- ADC can have pharmacologic effects that may differ from the
callyinaccessibletocellularreceptors(e.g.,,mannoseorasialo- parent ADC due to dissimilar distribution and elimination
glycoprotein receptors) that could facilitate binding and properties.Whilethedistributionofanunconjugatedantibody
increased clearance. Work by Huang et al.61 has shown that to tissues not expressing target may be quite benign, this may
changes in the N-glycosylation of neither the Fc nor the Fv notbetrueforanADCwherenon-specificuptakeandcatabo-
regionsofanantibodyaffectclearanceinmice.However,other lism can have serious consequences. Estimates of tissue cyto-
studies have demonstrated that Fc glycosylation can indeed toxin levels based on the established understanding of mAb
have effects on PK. By following the glycan patterns over time distribution may be helpful, but work by Alley et al.68 shows
followingmAbadministrationinhumans,itwasobservedthat that the proportion of free (and presumably active) to conju-
high mannose-5 glycan forms were more rapidly eliminated gated cytotoxin varied widely between tissues. This suggests
compared to other glycoforms.62 Wright et al. found that an thatsometissuesmaybemoreeffectiveinreleasingoraccumu-
IgG with a high mannose glycan also exhibited more rapid lating active cytotoxin. In most cases, conjugation of a small
elimination inmice.63PK anddistributionstudiesinmiceofa molecule cytotoxin to a mAb has been shown to have little
series of Fc fusion proteins differingin their levels of O-linked impactonmAbclearanceanddistribution.69,70Thereare,how-
sialylationshowedthatvariantsthatweredesialylatedorwitha ever, some examples where conjugation has been shown to
lowlevelofsialylationhadmorerapidclearanceandgreatertis- affectdistributionresultinginadverseoutcomes.Ananti-Lewis
sue uptake than those with higher sialylation.64 However, this YmAbwasfoundtohavetypicalbiodistributioninpatientsby
was not thought to be mediated by the asialoglycoprotein scintigraphy,butuponconjugationwithcalicheamicindemon-
receptor.Theinfluenceofglycosylationontissuedistributionis strated increased clearance, decreased tumor uptake, and
not well described. One would rationally assume that the increased liver uptake that was associated with liver toxic-
increased size provided by extensive glycosylation of a small ity.71,72Theeffectofdrugloadingonclearanceandtoxicitywas
234 J. TIBBITTSETAL.
demonstrated by the observation that highly loaded ADCs component of these molecules is subject to different metabolic
exhibited more rapid clearance and greater toxicity,73 possibly processes, and cellular or organ elimination may also differ.
due to perturbations in the antibody structure, resulting in The metabolism of PEGs is dependent on molecular weight,
scavenging by Fc-gamma receptors or inefficient neonatal Fc withminimalmetabolismbeingobservedforPEGmoleculesof
receptor(FcRn)recycling.Forimmunotoxins,i.e.,proteintox- the size typically used for therapeutic conjugation (>5 kDa).79
insconjugatedtoantibodiesorantibodyfragments,thereisless What catabolism does occur can be of the peptide or protein
known. It was reported that conjugation of gelonin to an anti- conjugated to the PEG.16,80,81 Elimination of PEGylated pro-
body against a tumor antigen (gp240) resulted in a nearly teins can occur via elimination of the parent molecule or via
fold2- increase in plasma clearance of the conjugate as com- catabolism of the protein or peptide component with subse-
pared to the unconjugated antibody, in addition to uptake of quent elimination of the PEG component. In general, renal
the conjugate into spleen and kidney.74 Such examples suggest eliminationappearstobethepredominantrouteofelimination
thatcarefulinvestigationoftheeffectsofconjugationondistri- for PEG and PEGylated proteins, with a rate that is inversely
butionandclearanceareimportantaspectsinthedevelopment proportional to PEG or PEGylated protein size.41,49 Interest-
ofADCs. ingly, PEGylated protein clearance appears to be somewhat
The second type of modificationis theconjugation orengi- slower than that of a similarly sized protein. While a direct
neeringofproteinstoimprovetheirresidencetimeinthebody. comparisonwasnotfound,theclearanceofaF(ab’)2oradiFab
Asdescribedabove,smallproteinsarerapidlyclearedfromthe with amassof»100kDawas10–fold15-faster thanthatof an
body and this can limit their pharmacologic efficacy. Many IgG,36,82 whereas Fab-PEGs can have clearances with only a
strategieshavebeenusedtoaddressthisproblemforsmallpro- fold2- difference from comparable IgGs.49,83 The reasons for
teins, and these typically fall into 2 general categories: those thismaybeduetodifferencesinhydrodynamicradiusbetween
that increase the hydrodynamic volume (i.e., increase in size), globularproteinsandlinearorbranchedhydrophilicpolymers
and those that utilize FcRn-mediated recycling in addition to resultinginslowerrenalclearanceforpolymerconjugatedpro-
volumeincrease.75 teins.41 A full discussion of this is beyond the scope of this
review,butotherreviewsofthistopicareavailable.41,49,83,84
Increaseinsize
IncreaseinsizeinconjunctionwithFcRn-mediated
The first category involves increasing the size of a small thera-
recycling
peuticproteintolimiteliminationbythekidney.Thisincludes
such approaches as conjugation with hydrophilic polymers (e. Thesecondcategoryforreducingtherapeuticproteinclearance
g.,, polyethylene glycol, polydextrans, starch), increasing the leveragesbothanincreaseinsizeandtheFcRn-mediatedphys-
amountofglycosylation,orrecombinantextensionofthether- iologic recycling process of IgG and albumin. This can be
apeutic protein amino acid sequence to increase its size (e.g., achieved by fusing or conjugating the therapeutic protein or
XTEN).76 This discussion will focus on proteins and peptides peptide to an immunoglobulin Fc (e.g.,, etanercept, abatacept,
conjugatedtoPEGasmostoftheliteraturedescribessuchmol- romiplostim) or albumin, or by engineering the therapeutic
ecules. Molecular size has a substantial effect on therapeutic proteinwithabindingsiteforalargercirculatingnativeprotein
proteinADMEproperties, asdescribedpreviously. However, a (e.g.,albumin-bindingFabs,albumin-bindingdomainantibod-
small protein conjugated with a hydrophilic polymer may not ies),75,76 thereby enabling binding of the Fc- or albumin-bind-
possessthesameADMEpropertiesasanunconjugatedprotein ing construct to FcRn. The FcRn is a major histocompatibility
ofasimilarmass. complex class I-related receptor composed of an a chain and
Differences in absorption and distribution between protein b(2) microglobulin expressed by cells of many tissues of the
therapeutics and polymer-conjugated proteins of similar mass body.ItfunctionsinthereducedpHoftheendosomebymeans
mightbeexpectedbasedonthepotentialdifferencesinhydro- ofpH-dependentbinding(highaffinityatpH6,lowaffinityat
dynamic radius. Per unit of mass, PEG is thought to possess a pH 7.4) to albumin or IgG (via the Fc), followed by recycling
muchgreatermolecularvolumelikelyduetotheextendedcon- and release of bound protein at the cell surface where FcRn
formation of the PEG polymer and the ability to coordinate affinity is considerably lower.85 The addition of an FcRn bind-
water molecules.41 In addition, the molecular charge of PEG ing site to a therapeutic protein will thus reduce plasma clear-
and other polymers is considerably different than that of pro- ance of the biotherapeutic by facilitating its recycling in the
teins,whichwillinfluenceinteractions withendogenous mole- endosome andpreventing degradation inthe lysosome.ForFc
culesintissues.However,SCabsorptionofPEGylatedproteins fusionsorconjugates,whileonemightexpectthattheclearance
appearstooccuratasimilarrateandmagnitudeastherapeutic shouldbeidenticaltothatofanativeIgG,itiscommonlynoted
proteinsofasimilarmass.11,49,77Anumberofstudiesofthetis- that the plasma clearance of these constructs is substantially
suedistributionofpolymerconjugatedproteinshavebeencon- greater, possibly due to differences in binding to FcRn or
ducted and those related to PEG have been summarized in a increased renal clearance due to their smaller size.75,86 By
recent review byBaumann etal.49 PEG sizeplayed a clear role changing the size of the construct and including binding to
indistribution,butthefewstudiesthathaveevaluatedthisrela- FcRn,onemightalsoexpect differencesinabsorptionanddis-
tionship have not identified a consistent trend.78 The catabo- tribution from that seen with the unaltered protein or peptide,
lism/metabolism and elimination of PEGylated and other andthishasbeencommonlyobserved.37,87,88
polymer-conjugatedproteinsmaydifferfromthatoftherapeu- TheadditionofFcRnbindingcouldhaveeffectsonabsorp-
tic proteins consisting solely of amino acids. The polymer tion and distribution independent of the increase in molecular
MABS 235
size,andtherelativeroleofeachofthesecontributorsremains lung,gut,liver,kidney,heart,andspleen.93Usinganalternative
unclear.TheroleofFcRnintheADMEofproteintherapeutics approach, studies in normal mice using human IgG1 FcRn
is still an active area of research, with most of this work being binding variants with fold6- greater binding affinity (pH
conducted with antibodies. The effect of FcRn binding on dependency not noted) or no binding to FcRn found little or
absorption has proved to be controversial due to potentially nodifferenceintissue/plasmaAUCratiosbetweenthevariants
conflicting data. Studies have been conducted with wild-type andwild-typeIgGs.94Thereasonsforthediscrepancybetween
andFcRn knockout(KO)mice,aswellas primates using anti- studies is not known, but could be attributed to the different
bodies with varying FcRn binding characteristics. A fold3- durations of the studies, or physiologic differences between
improvement in SC bioavailability of an IgG1 in wild-type wild-type and FcRn KO mice that affect the balance between
mice vs. FcRn KO mice was described, suggesting thatFcRn is uptake and return of antibody from tissue. At this time, gaps
importantinSCabsorption.77Usingaseriesofanti-Abmouse remaininourmechanisticunderstandingoftheeffectsofbind-
IgG2a FcRn binding variants, Deng et al.89 found similar bio- ingtoFcRnonabsorptionanddistributionoftherapeuticpro-
availability (F D 76–86%) for the wild type and a variant anti- teins, but, as illustrated in the studies described above, some
body with enhanced FcRn binding at pH 6 and pH 7.4; a insightsarebeginningtoemerge.
modest increase in bioavailability (95%) for a variant with Our understanding of the role of FcRn in catabolism and
enhanced pH 6 binding (which would confer improved FcRn- elimination is still evolving. It is generally believed that the
mediatedrecyclingandtranscytosis);andsubstantiallylowerF binding to FcRn by an Fc moiety or albumin in the endosome
(41%) for a variant with no FcRn binding, also supporting a allowsrecyclingortranscytosis,thusprovidingprotectionfrom
role for FcRn in SC absorption.90 This is consistent with a endocytic degradation.85 The importance of this interaction is
mousestudyofhumananti-TNFIgG1FcRnvariantsthatdem- evidentfromstudiesshowingincreasedantibodyorfusionpro-
onstrated that an antibody variant with substantially increased tein clearance in animals lacking FcRn or increased clearance
FcRnaffinityatpH7.4(»25fold)andonlymodestlyincreased innormalanimalsofantibodieswithreducedFcRnbinding.90-
affinityatpH6(»1.75fold)reducedthehalf-lifeofthisvariant 95Whatislesswellunderstoodarethedetailsoftheunderlying
toapproximatelyhalfofthevalueofthenormalantibody.This mechanisms for this effect, specifically the major tissues and
decreased half-life is presumably due to less efficient recycling cells involved, the efficiency of the process, and the overall
by FcRn as a result of poor release of the antibody at the cell impactonclearanceanddistributiontovarioustissues.Studies
surface.89 Poor release of the antibody may lead to increased determining the key tissues involved in mAb catabolism are
intracellular catabolism of the protein, increasing systemic emerging and other aspects of FcRn-mediated tissue dynamics
elimination of the antibody and decreasing SC bioavailability. arecurrentlyunderstudy.93,94Alesswell-studiedroleforFcRn
In support of this hypothesis, in a rat study of rituximab, SC in therapeutic protein elimination is related to renal elimina-
co-administration of nonspecific IgG to saturate local FcRn tion. FcRn is expressed in the podocytes and brush border of
resultedindecreasedFofrituximab.18Incontrasttotheabove the proximal tubular epithelium of the kidney.96 In the podo-
findings, experiments in primates using several human IgG4 cytes,FcRnisthoughttoacttocleartheglomerularslitsoffil-
antibodies and corresponding variants with 15–60 fold tered IgG by transporting it into the urine where it is
increased FcRn binding at pH 6 found no difference in F presumably reabsorbed by FcRn in the proximal tubules.97
between the native and FcRn variant antibodies.91 From this However, this presumption may not hold as there is evidence
work, it appears that decreases in FcRn recycling (via FcRn that FcRn may act by transporting IgG from the interstitium
blocking or increased FcRn affinity at pH 7.4) can have sub- into the urine.96 It is clear from this discussion that the ability
stantialeffectsonF,whileincreasesinFcRnaffinityatpH6(as ofatherapeuticproteintobindtoFcRncansignificantlyaffect
ameansofattemptingtoincreaseFcRnrecycling)provideslit- its ADME characteristics, but that many of the dynamics and
tle improvement in F beyond that already achieved with the mechanismsofthisinteractionremaintobeelucidated.
nativeFc.
FcRn has the potential to play several roles in therapeutic
InfluenceoftargetbindingonADME
proteindistribution.Attheendothelialbarrierofthecapillaries,
FcRn could recycle Fc or albumin-containing therapeutic pro- Thedesiredpropertyofalltherapeuticproteinsisbindingtoits
teins back into circulation, thereby limiting their penetration therapeutic target. The specificity of this interaction is a key
into the interstitial space. Alternatively, FcRn could facilitate advantageforthisclassofmoleculesovermanyothertherapeu-
transcytosis of the same molecules into the interstitial space, ticagents.TheeffectoftargetbindingontheADMEproperties
thereby improving distribution relative to other similarly sized of the therapeutic protein depends on a number of factors
proteins. The effect of FcRn binding on distribution has been related to both the target and the therapeutic, including the
studied, again with IgGs, with disparate outcomes. Using tis- amount,turnoverrate,andlocationofthetargetandthebind-
sue/blood area under the curve (AUC) ratio (T/B ratio) as a ingaffinityofthedrugtothetarget.Littleinformationhasbeen
measure oftissuedistribution,theT/B ratioforahuman IgG1 reportedon thepotential effect of thetarget on the absorption
was higher in spleen, liver, kidney, and lung in FcRn a chain of protein therapeutics. For many therapeutic proteins, move-
KO micecompared to wild-type mice, while the T/B ratio was mentthroughtheSCspaceandtransitthroughthelymphsys-
higher in wild-type mice for fat, lymph node, skin and mus- tem will expose the drug to soluble or cell-surface target (e.g.,,
cle.92 Ina similar study using an FcRn b(2)-microglobulin KO TNF,IGF1R,VEGFR,CD11b).Thisinteractionwiththetarget
mouse model, tissue to plasma ratios were higher in muscle could result in receptor-mediated uptake and degradation of
andskinintheWTmicerelativetoKOwithlittledifferencein thetherapeuticproteinortheformationofimmunecomplexes
236 J. TIBBITTSETAL.
that are subsequently cleared. Both of these would result in a andlaborintensivewhenappliedtoalargenumberofproteins
decrease in absorption and bioavailability. Searches for studies andsamples.111Thus,higherthroughputanalyticalapproaches
specificallyevaluatingtheeffectoftargetbindingonFwerenot andinstrumentplatformsarecontinuouslybeingpursued.One
successful; however, there are examples whereby increasing F exampleisthe“genericELISA”whichusesareagentrecogniz-
withincreasingSCdosemayindicatesaturationofdegradation inghumanFc,allowingforthedetectionofanti-drugantibod-
or target binding at the site of administration or during the iesandPKstudiesofanyhumanFc-containingbiologics.
absorptionprocess.98 In recent years, advances in technology have made auto-
The effects of drug target on distribution can be evident mated unattended screening broadly accessible, and have fur-
immediatelyafterdosingwhere,forexample,ahighlyabundant therfacilitated the implementation of newrobotic systems and
orrapidlygeneratedtargetinplasmaorbloodcaninteractwith approachestoincreaseproductivityandefficiencyforscreening
the administered therapeutic protein, thereby limiting the during the development of biotherapeutics. Some of the major
apparent tissuedistributionofthefreedrug.99Cellsurfacetar- platformsincludetheGyrolabimmunoassayworkstation,which
getsintissuecansubstantiallyenhanceuptakeandretentionof iscurrentlybeingusedfordruganalyte(nanoliter)samplemea-
protein therapeutics at sites of target abundance. This is most surement and for PK studies commonly used in early efficacy
clearlyillustratedbythelargebodyofdata describingthehigh evaluations.Withareducedbloodsamplesizeof10–20mLfor
concentrationsofantibodythatcanbeachievedintumorswith each time point, serial bleeding can be conducted in the same
high levels of target.43,68,70,72,100-103 This phenomenon is not mouse over the time course for a PK study, minimizing the
limitedtotumors,asevidencedbyrecombinantIL-10distribu- inter-subject variability associated with non-serial sampling
tion to tissues with receptor overexpression and PK/PD evi- needed for a mouse PK study that relies on a conventional
denceforothertherapeuticproteins.98,104-107 ELISAforbioanalysis.111
Anothertechnologyparticularlyattractiveforbioanalysisof
proteins is MS. The advantages of MS over ELISA include the
ADMEenabling-technologiesandmethodsindrug
improved selectivity between structurally similar peptides and
discoveryanddevelopment
proteins, reduced requirements for specific reagents, improved
Incontrasttosmallmoleculedrugs,thedevelopmentofinvitro precision and accuracy, and a potentially higher throughput
and in vivo correlation tools for protein therapeutics lags rate.111,112 Quantitative liquid chromatography–mass spec-
behindduetothemorecomplexnatureofbiotherapeutics.The trometry (LC-MS) has also been applied to the quantitation of
lack of appropriate tools to study therapeutic exposure, meta- anti-drugantibodiesinhumanandcynomolgusmonkeyserum
bolic or metabolic-like biotransformation, and target engage- inthepresenceofhighcirculatingconcentrationsoftheprotein
ment in the vascular and tissue spaces makes ADME therapeutic.113 The development of quantitative mass spectro-
characterization of biologics more difficult as compared to metric assays has been evolving over the past decade and will
smallmoleculetherapeutics.108HighthroughputADMEmeth- continue to be the most powerful tool for ADME studies of
odsthatcurrentlyexistforsmallmoleculedrugshavenotbeen therapeuticbiologics.
asstronglypursuedforantibodyproteintherapeutics,reflecting
toacertaindegreethesubstantiallylowerattritionrateandthe
LabelingtechniquesforADMEandimagingstudies
associatedlowernumbersofpotentialclinicalproteintherapeu-
ticcandidates.109Thissectionrepresentsanattemptatsumma- Thelabelingoftherapeuticbiologicsisaninvaluabletechnique
rizingtheavailabletoolsandapproachesforgeneratingADME that has been used extensively to analyze PK and metabolism,
data. measure absolute tissue concentrations, and facilitate imaging
studies. Proteins possess unique sequences of amino acid resi-
dues, making them perfect targets for labeling. Generally, the
ADMEtoolsandtechnologies
typeoflabelandtheproteinconjugationstrategymustbecare-
CommonconsiderationsfortheADME-relatedissuesforthera- fullytailoredtoeachapplication.Radionuclidesareusedexten-
peutic biologics include target mediated clearance, FcRn recy- sively, but they have a short shelf-life and require special
cling for Fc-containing proteins, immunogenicity, isoform handlinganddisposal.Today,the2mainmethodsofradiolab-
heterogeneity, and metabolic stability, especially for relatively elingproteinsarehalogenationandthecomplexationofmetal-
low molecular weight proteins.110 Therapeutic biologics gener- lic radioisotopes. Radiohalogens (such as radioiodines) can be
ally have limited distribution in tissues. Therefore, most of the directly conjugated to proteins in one step, mainly via forma-
relevantADMEstudiesforproteintherapeuticsrelyon in vivo tion of a stable covalent bond. An alternative approach to the
evaluations,whicharebasedonavarietyoftechniquesaimedat direct method is the indirect incorporation of radiohalogens
following unlabeled/labeled drug/metabolite using either anti- into small organic molecules. These molecules contain activat-
body-basedormassspectrometry(MS)-basedtechniques.111 ing prosthetic groups allowing for iodination and conjugation
withproteinsundermildconditions.Thereaderisreferredtoa
reviewbyWilburforathoroughoverviewofthecurrentradio-
Quantitativeandanalyticaltools
iodinationmethodsandreagentsforconjugatelabeling.114
Immunoassays, such as the Enzyme-linked immunosorbent Some radiometallic nuclides (e.g., Tc-99m) can bind pro-
assay(ELISA),arethemostcommonlyusedtechniquesforthe teins directly by forming coordinate bonds with various func-
quantificationofproteinsinserumduetotheirhighsensitivity tional groups. However, the stability of such conjugates is
and specificity. However, such methods can become expensive questionable since most proteins, including mAbs, do not
MABS 237
possess functional groups capable of forming stable metal has been a major driver in the selection among labels.120
bonds.One solution tothis problem istoattachabifunctional Thus, appropriate choice of label allows tailoring of the
chelate that can be covalently conjugated to proteins and will properties of the labeled protein to the application required.
chelate a radiometal, thereby forming a stable radionuclide- Today,severallabels,includingradiohalogensandradiome-
chelate-protein label.115 Due to the diversity of the metallic tals, have fundamentally extended the possibilities of detection
radionuclides, many different variations of bifunctional chela- and imaging techniques and in turn caused the need for the
torsareuseddependingonthechoiceofradionuclide.Thesize, development of chemical methods for their conjugation. In
charge, and electron configuration of the metallic radionuclide fact, the advance of available imaging instrumentation com-
(e.g.,,indium-111,copper-64,zirconium-89)willdeterminethe binedwith wellcharacterizedlabelingchemistryfor antibodies
coordination number required of a bifunctional chelator in and other protein therapeutics has enabled detailed in vivo,
order to accommodate the radiometal ion. A recent review by dynamic and quantitative measurements of radiolabeled and
PriceandOrvigprovides aconvenientandaccessibleoverview fluorescent probes with quantitative whole-body autoradiogra-
ofthefieldofradiometalchelatingagents.116 phy(QWBA),andmolecularimaging,includingsingle-photon
The coupling of fluorescent moieties to proteins to create emission computed tomography (SPECT), positron emission
labeledproteinreagents has alsobecomearoutineand impor- tomography(PET),andopticalimaging.
tant procedure in biologics.Molecules thatabsorbin the near-
infrared(NIR)region(700–1000nm)canbeefficientlyusedto
DifferentADMEmethodsandapproaches
visualizeandinvestigateinvivomoleculartargetsbecausemost
tissues generate little NIR fluorescence. The most common The investigation of the ADME properties of therapeutic pro-
organicNIRfluorophoresaresmallmoleculeorganicdyessuch teins requires tools and experimental approaches that are spe-
aspolymethines.Theirphysical properties,biodistribution,PK cifictothis classofagents.Beforeembarkingoninvestigations
and applications for in vivo fluorescence imaging have been oftherapeuticproteinADME,itisworthwhiletoconsiderwhat
summarized in a recent review.117 Often, a succinimidyl-ester information is most critical for answering the questions at
functional group is attached to a fluorophore core and this hand. For instance, absorption studies may be interested only
functionality confers reaction specificity with primary amines intheamountofparenttherapeuticproteininserumorplasma
toformfluorophore-antibodyconjugates. after SC administration, thus requiring a simple parent drug
Ideally, the labeling procedures should minimally alter assay.Alternatively,astudymayalsobedesignedtoinvestigate
the properties of the molecule of interest, and the physical the mechanisms of the route of absorption (e.g., lymphatic or
half-life of a radionuclide should match the biological half- vascular)orthedegreeofcatabolismoccurringduringthispro-
life of the molecule to be labeled. However, a label may cess, requiring analysis in lymph tissue of both parent thera-
directly perturb the function of a protein, and the reaction peutic and perhaps catabolites. Distribution studies most
conditions used to introduce the label may inadvertently commonlyevaluateconcentrationsoftheparentdrugintissues
promote undesirable changes such as oxidation, deamida- ofinterest,butcanalsobedesignedtomeasureconcentrations
tion, side-chain isomerization, or aggregation.118 The of catabolic products and associated pharmacologically active
absence of gross changes in PK or molecular weight is not products(e.g.,cytotoxicdrugfromanADC).Inmostinstances,
always sufficient characterization of labeled proteins, and aquantitativemethodisrequiredasthisprovidesthemostuse-
binding or other functional assays are needed to assess the ful data for informative comparisons of molecules and strate-
integrity (e.g., immunoreactivity) of a labeled probe.119 gies,andisalsoidealforPK/PDandothermodelinganalyses.
Methods for labeling of proteins, in general, should also be Whenconsideringastrategyforinvestigatingproteinthera-
rapid and give high yields. The labeled protein that is peutic ADME, it is worthwhile to take into consideration the
obtained should be of high specific activity and should be followingcriteria:
labeled in a manner that results in a stable attachment of
the label. Optimization of reaction parameters is almost
Analyteofinterest
always a requisite of radiolabeling because radionuclides are
generally dilute and contain many minor impurities from AgoodplacetostartwhenplanningADMEstudiesistoiden-
processing of the target material as well as from the chemi- tify the critical analytes necessary to understand the ADME
cals used, which are present in more abundance than the characteristics of the biotherapeutic. There are a number of
radionuclides themselves.114 Any label should remain cou- considerations critical to study design that one should assess
pled to its conjugate protein for the duration of the experi- whendeterminingtheanalyte(s)ofinterest.Isoneinterestedin
ment and, ideally, make no difference to its behavior.120 parentorcatabolite?Inmanysituationstheanalyte(s)ofinter-
These are fundamental criterion in choosing an appropriate est is/are determined by the format of the therapeutic protein
label for a particular study. The label itself will remain andislikely tobetheparentmolecule,unlessoneisinterested
optically active or radioactive, and will be detected in any in identifying or measuring the catabolic products. If there is
biodistribution images, but it no longer reveals the presence evidenceor concern that the parent drug is not the only phar-
of the therapeutic protein per se. The distribution of macologically active species, the identification and ADME
excreted or catabolized labels can confound the biodistribu- assessment of active catabolites may be of interest to inform
tion and imaging of certain tissues, especially in and around PK/PD or drug design. For some situations, it is critical to
the hepatobiliary system, gut, kidneys, and urinary bladder. understandtheADMEofthepharmacologicallyactivemoieties
When imaging abdominal sites this can be a limitation and of the therapeutic protein, and perhaps any other components
238 J. TIBBITTSETAL.
that might contribute to its behavior. For example, for an Fc- feces) will require specialized techniques and equipment such
fusionitmightbeappropriatetoensure thatthesamplingand asmetabolismcagesandbiliarycannulation,particularlyifone
analytical method are capable of measuring both the presence is interested in understanding the mass balance of parent and
of the antigen-binding domain, confirming pharmacologic catabolite elimination. Such methodologies have been in use
activity, and the Fc domain, confirming the stability of the for small molecule drugs for many years, with the details
molecule. described in recent reviews.125,126 Measurement of analytes in
Fortherapeuticproteinswithmorethanonepharmacologi- tissue presents the researcher with several choices related to
callyrelevantcomponent(e.g.,,ADCs,bispecificantibodies),it analyticalmethodandstudydesign.Themostcommonlyused
may be necessary to ensure that the methods used are capable strategyisadestructiveapproachwherethesacrificeofindivid-
of confirming that all of the pharmacologically active compo- ualsorgroupsofanimalsatspecifiedtimepointsisfollowedby
nents of the molecule are intact. It may also be of interest to quantitation of the analytes of interest in specific tissues. The
measure the identities and amounts of the individual active investigator then has several options for analyte determination
components,forexample,toassessdifferencesinADMEofthe and quantification. The two most common are assaying indi-
active components upon ADC catabolism. A particularly ele- vidualtissuesseparatelyusingeitherradiometricmethods(i.e.,
gantexample isthe work of Alleyetal. investigating thetissue “cutandcount”)ornon-radiometricmethods(e.g.,immunoas-
distributionoftheantibodyandcytotoxininnormalandtumor say or MS). Alternatively, using radiolabeled material, one can
tissues following dosing with an ADC.68 This was achieved by employQWBAtogeneratea2-dimensionalwholebodyimage
radiolabelingeachcomponentwithaseparateradioisotopeand consistingofcrosssectionsoftheorganswhich,byexposingto
using tissue sampling to obtain highly quantitative tissue con- x-ray film, can provide a measure of analyte in each tis-
centrations. This work provided valuable insights into the dif- sue.101,127,128 While these methodscan provide excellentquan-
ferences in tissue accumulation and processing of this potent titative information, depending on theanalytical methodused,
therapeutic. In some situations, the identity of the catabolic they have the disadvantage of preventing serial assessment of
products may not be known at the time of the design of the tissue distribution within individual animals and can require
ADMEstudies,andthuscarefulconsiderationoftheanalytical relativelylargenumbersofanimals.Theselimitationscanoften
methods and study design required to ensure that all relevant be overcome by the use of non-destructive methods such as
catabolic products can be identified and quantitated is needed. imaging,includingPET, SPECT, andoptical imaging.Imaging
Thisisnicelyillustratedbythecatabolismandexcretionassess- allowsreal-time,serialassessmentoftissueconcentrations,but
ment conducted for ado-trastuzumab emtansine (T-DM1).121 insomesituationscanlackthenecessaryspatialresolutionand
BeyondADCs,therehasbeenmoreinterestinthebiotransfor- sensitivity.129,130 Ultimately, the choice of strategy and analyti-
mation (defined as the physical alteration of a biotherapeutic cal method is determined by balancing availability, sensitivity,
due to peripheral intermediate catabolism or truncation) of importanceofserialsampling,andneedforspatialresolution.
therapeuticproteinscontainingonlynativeaminoacid,partic-
ularlyasitrelatestounderstandingmoleculestabilityandcan-
Spatialresolution
didate selection.122 This strategy was used to evaluate the in
vivostabilityof3peptibodyanalogs;suchdatacouldbeusedto Fordistribution,andperhapsabsorption,spatialresolutioncan
guidedecisionsaboutmoleculedesignandprogression. be important in understanding the mechanistic aspects of
ADMEandmayalsoinformtherelationshipbetweendistribu-
tion and pharmacologic effect. Traditionally, these have been
Matrix
determinedusingradiolabeledtracersbydissectionstudiesfol-
The matrix containing the analyte(s) of interest has a major lowedbygammaor liquid scintillationcountingandautoradi-
impact onthestudydesign andchoiceofanalytical method.If ography.Suchanapproach,however,istediousandrequiresa
one is interested in measuring multiple analytes in a single largenumberofanimalstoensurethereproducibilityandreli-
matrix (e.g., both the cytotoxic drug and the antibody compo- abilityoftheresults.Thecontinuingdevelopmentofhigh-reso-
nents of an ADC),the use of separate analytical modalities for lution PET and SPECT scanners for small animals and the
each analyte may be required. For distribution studies, the availability of suitable isotopes (e.g., 64Cu, 89Zr, 111In, 131I) are
matrixisusuallytissue,whichposessomeanalyticalandstudy providinganalternativewhichsimplifiesconsiderablythemea-
design issues. Determinationoftheconcentrationoftherapeu- surement for the kinetics and biodistribution of radiolabeled
tic proteins in tissue using certain methods (e.g.,, immunoas- biotherapeutics. The major advantages of radionuclide-based
says, mass spectrometry) can be more difficult than in liquid molecular imaging techniques (SPECT and PET) are that they
matricessuchasplasmaorserum,oftenresultinginlowersen- are very sensitive (down to the picomolar level), quantitative,
sitivityandtime-consuming methoddevelopment foreachtis- and there is no tissue penetration limit. Another advantage is
sue.108,111,123,124 For the investigation of therapeutic protein thatSPECThasgoodspatialresolution(0.35mmvs.One.5mm
elimination, one willlikelycollecturine,bile,or feces. Inaddi- forPET)allowingdifferentiationoftraceruptakeonthesubor-
tiontoconsiderationsrelatedtocollection,eachofthesematri- ganlevel.Itisimportanttokeepinmindthatalthoughnosin-
ces has characteristics that may determine the analytical gle technology currently provides all the answers one would
method used for detection of the analyte. Beyond analytical like,however,integratingdifferentmodalitiesintootherinvivo
aspects,thechoiceofmatrixwillalsodeterminehowastudyis methodologies (e.g. QWBA, microautoradiography) can
conducted.Bloodsamplingformeasurementofblood,plasma, enhance our quantitative understanding of intra-tissue distri-
or serum is quite routine. Collection of excreta (bile, urine, bution.Anillustrativeexampleisthedistributionoftherapeutic