Overview of Antibody Drug Delivery

24 Pages·2017·1.34 MB·English

pharmaceutics
Review
Overview of Antibody Drug Delivery
SaharAwwad1,2,* ID andUkritAngkawinitwong1
1 UCLSchoolofPharmacy,LondonWC1N1AX,UK;[email protected]
2 NationalInstituteforHealthResearch(NIHR)BiomedicalResearchCentreatMoorfieldsEyeHospitalNHS
FoundationTrustandUCLInstituteofOphthalmology,LondonEC1V9EL,UK
* Correspondence:[email protected];Tel.:[phone]
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:27March2018;Accepted:29June2018;Published:4July2018
Abstract: Monoclonal antibodies (mAbs) are one of the most important classes of therapeutic
proteins, which are used to treat a wide number of diseases (e.g., oncology, inflammation and
autoimmune diseases). Monoclonal antibody technologies are continuing to evolve to develop
medicineswithincreasinglyimprovedsafetyprofiles,withtheidentificationofnewdrugtargets
beingonekeybarrierfornewantibodydevelopment. Therearemanyopportunitiesfordeveloping
antibody formulations for better patient compliance, cost savings and lifecycle management,
e.g.,subcutaneousformulations. However,mAb-basedmedicinesalsohavelimitationsthatimpact
their clinical use; the most prominent challenges are their short pharmacokinetic properties and
stabilityissuesduringmanufacturing,transportandstoragethatcanleadtoaggregationandprotein
denaturation. Thedevelopmentoflongactingproteinformulationsmustmaintainproteinstability
and be able to deliver a large enough dose over a prolonged period. Many strategies are being
pursued to improve the formulation and dosage forms of antibodies to improve efficacy and to
increasetherangeofapplicationsfortheclinicaluseofmAbs.
Keywords: antibodies;protein;pharmacokinetics;drugdelivery;stability
1. Introduction
Monoclonal antibodies (mAbs) are by far the largest class of therapeutic proteins and a key
driver in the biopharmaceutical growth [1]. Currently, humanized mAbs are the fastest growing
group in clinical trials [2]. Most therapeutics mAbs that are available share common functions
suchasblockingtargetreceptorsorligands,thusdecreasingtheoverallactivityofcertainpathway.
The Food and Drug Administration (FDA) approved the first mAb in 1986 (Orthoclone OKT3,
muromonab-CD3)forthepreventionofkidneytransplantrejection[2,3](laterwithdrawn)andmany
moreantibodiesandantibodyderivatives(e.g.,fragmentantigen-binding(Fab),fragmentcrystallisable
region(Fc)-fusionproteins,etc.)[4]havebeendevelopedsince[5].AmajorityofmAbtherapeuticshave
beenapprovedforoncology,rheumatoidandautoimmunediseases[4]. mAbshaverevolutionized
thetreatmentofophthalmicconditions(e.g.,useofanti-vascularendothelialgrowthfactor(VEGF)
medicinessuchasranibizumab(Lucentis®)inthetreatmentofwetage-relatedmaculardegeneration,
AMD) [6]. Monoclonal antibodies exhibit good safety profiles and enhanced efficacy; and have
been reported to show higher success rates in progression through early clinical development [7].
Approximately 70 mAb products have been predicted to be available in market by 2020 for the
treatmentofvariousdiseases[8].
Monoclonalantibodiesareimmunoglobulins(Ig)ofwhichtherearefiveclasses(IgA,IgD,IgE,
IgGandIgM)[9]. ThemostrelevantfortherapeuticsisIgG,whichhasfoursubclasses(IgG1,IgG2,
IgG3andIgG4)[10]. IgGsarebivalentmoleculeswithamolecularweightof~150kDa,whichcanbe
highlysitespecificwithaffinitiesrangingfromnano-topicomolar. IgGs(Figure1)havetwoseparate
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identicalheavy(H)andlight(L)chains[11]. TheN-terminaldomainofanIgGconsistsofavariable(V)
r(eVg)i orengwiointh wthitehc othmep cloemmepnletamrietny-tdareitteyr-mdeinteinrmgrineginiogn r(eCgDioRn) (tChaDtRb)i ntdhsatt obainsdpse ctoifi ac esppietocipfieco enpaitnotpigee onns.
Oanthtiegrendso.m Oathinesr wdoitmhianintsh ewIigthGinm tahke eIguGp mthaekceo unpst athnet c(Con)srteagniot n(Cs.) Trehgeiosntrsu. cTthuere storfuacntuIrgeG ofi sabnr IogaGd liys
dbrivoiaddeldy idnitvoidtheedF ianbto( 2t/h3e) Faanbd (F2c/3r)e gainodn sF(c1 r/e3g)i.oTnhse (t1w/3o).i dTehnet itcwaloF iadbesnetiaccahl cFoambsp eriasceho cfoamlipgrhitsec hoafi na
tlhigahtti scchlaoisne lythaasts oisc icaltoesdelbyy ansosno-ccioavteadle nbyti nntoenra-ccotivoanlsewnti tihnttehreahcteiaovnys cwhaitihn .tThhe ehreeaisvay scohlvaeinn.t -Tahcceerses iibs lea
isnotlevrecnhta-ainccdeisssuiblfiled ien(tSer-Sch)abient wdieseunlftihdee l(iSg-hSt) cbheatwineaenn dthhee laivgyhtc chhaainino afntdh ehFeaavby. Tchheaihne oafv tyhceh Faaibn.s Tahree
chleoasveyly cahsasioncsi aatreed cltohsreoluyg ahssnoocnia-tceodv athlernotuignhte nroacnt-icoonvsawleintht iinnttehreacFticornesg wiointh. Tinh tehhe iFncg reergeigoino.n Tlhine khsinthgee
Freagbisotno ltihneksF cthaen dFatbhse rteo atrhee( uFscu aanlldy )thtweroe saorlev e(unstuaaclcleys)s itbwleo dsiosluvlefindte ascbceetswsiebelen dthiseuhlfeiadveys cbheatwineseinn tthhee
hhienagvey rcehgaiionns. iTnh tehree hairnegael sreogiinotnra. -Tmheorlee caurlea raldsois uinltfirda-emswoleitchuinlare adcihsuclofindsetsa nwtitahnidn veaacrhia bcolensretagniot nanodf
bvoatrhiatbhlee rheegaivoyn aonf dbolitghh tthceh haeinavs.yT ahneds elidghistu clhfiadienss.a Trehnesoet dsoisluvelfnidt-easc caeress nibolte s.oMlvaennyt-aascpceescstisbolef.m MAabnsy,
ianscpluecdtisn gofe pmitAopbes,s pineccliuficdiitny,gi mepmituonpoeg esnpieccitiyfi,cpithya, rmimamcoukninoegteicniacnitdyi, mpmhaurnme-arceolaktiendeteifcf eacntodr fiumnmctuionnes-,
arerelaatecdti veeffaercetoars foufnrcetsieoanrsc,h atrhe aatcitsivfeo caurseeads oofn rtehseeacrocnht itnhuaet dise fvoocluusteidon oonf tahnet icboondtiineusefodr etvhoelruatpioenu toicf
uanseti[b1o2d].ies for therapeutic use [12].

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((mAbAbbr)e.vi(aAtibobnr:e vCiDatRio: nc:omCpDleRm:encotamriptyle-dmeetnertmariintyin-dge treergmioinn;i nCgOOre-g: iocanr;boCxOy Ote-r:micnaarlb; oCxHy: tceormnsitnaanlt;
CdoHm:caoinn,s htaenatvdyo cmhaaiinn,, CheLa: vcyoncshtaainnt, dCoLm:caoinn,s tliagnhttd cohmaiani;n F,alibg:h ftracghmainen;tF aabn:tifgreang-mbienndtianngt;i gFec:n f-rbaignmdienngt;
Fcrcy:sftraalglimsaebnlte crreygsitoalnli, sNabHle3r: eagmioinn,oN tHer3m:ainmailn eontder, mSi-nSa: ldeinsudl,fSid-Se: dbiosnudlfi; dVeHbo: nvda;riVaHbl:ev daroimabaliend, ohmeaaviny,
hcheaaviny; cVhLa:i nv;aVriLab:lvea drioamblaeind,o lmigahitn c,hliagihnt) chain)
The Fab structure allows different IgG molecules to identify different antigens [13]. Porter and
TheFabstructureallowsdifferentIgGmoleculestoidentifydifferentantigens[13]. Porterand
Edelman (1972) won the Noble Prize for elucidating the structure of antibodies by proteolytic
Edelman(1972)wontheNoblePrizeforelucidatingthestructureofantibodiesbyproteolyticdigestion
digestion using thiol proteases, e.g., papain. With the use of papain, Porter was able to cleave the IgG
usingthiolproteases,e.g.,papain. Withtheuseofpapain,PorterwasabletocleavetheIgGmolecule
molecule into two Fabs and an Fc. The Fab can bind to a specific antigen while the Fc is unable to
into two Fabs and an Fc. The Fab can bind to a specific antigen while the Fc is unable to block
block binding to a specific antigen [14,15]. Fabs have been used in the determination of antibody–
bindingtoaspecificantigen[14,15]. Fabshavebeenusedinthedeterminationofantibody–antigen
antigen interactions [16]. Fabs usually have better tissue penetration than full length IgGs and can
interactions [16]. Fabs usually have better tissue penetration than full length IgGs and can allow
allow interactions with enzyme sites (which IgGs can find difficult to access). However, Fabs do
interactionswithenzymesites(whichIgGscanfinddifficulttoaccess). However,Fabsdodisplay
display fast off-rates and poor retention times compared to IgGs on target as a result of the
fast off-rates and poor retention times compared to IgGs on target as a result of the monovalency
monovalency of Fab [17]. The FDA and the European Medicines Agency (EMA) have approved three
of Fab [17]. The FDA and the European Medicines Agency (EMA) have approved three Fabs i.e.,
cFearbtso liiz.ue.m, acberptoelgiozul(mCaimb zpiae®g)o,lr a(nCibimizzuima®a),b (rLauncibeinztuism®a)ba nd(Laubcceinxtiims®a) ba(Rndeo parboc®ix).imRaabn ib(iRzueomparbo®i)s.
Ranibizumab is a humanized Fab with specificity to all isoforms of VEGF [18], and has demonstrated
ahumanizedFabwithspecificitytoallisoformsofVEGF[18],andhasdemonstratedgoodsignsof
good signs of biological activity and acceptable safety when administered as an intravitreal injection
up to 6 months in patients with neovascular AMD [19–21]. Polyclonal Fabs have also been marketed
such as crotalidae polyvalent immune Fab (ovine) (CroFab®), digoxin immune Fab (DigiFab®) and
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biologicalactivityandacceptablesafetywhenadministeredasanintravitrealinjectionupto6months
inpatientswithneovascularAMD[19–21]. PolyclonalFabshavealsobeenmarketedsuchascrotalidae
polyvalentimmuneFab(ovine)(CroFab®),digoxinimmuneFab(DigiFab®)anddigoxinimmuneFab
(ovine)(Digibind®)[22]. ThebiologicalfunctionofapprovedFabsisrestricted to themonovalent
bindingoftheFabtoitstarget. Fabsconjugatedtoexogenousfunctionalmoietieshavebeeninclinical
development including twelve Fabs conjugated to cellular toxins, seven to radioisotopes, three to
cytokinesandoneFabconjugatedtoanenzymetargetingtoxicmetabolites. Fabscanbeproducedby
antibodydigestionusingproteolyticenzymesorbydirectexpressionfrombacteria,mammaliancells
oryeast[17].
The Fc domain can bind with Fcγ receptors (FcγR) to cause effector functions. The FcγR are
largely expressed on immune cells such as natural killer (NK) cells, macrophages and dendritic
cells [23]. The Fc domain endows mAbs with effector functions involving the stimulation of a
specific immune response to eliminate the mAb target molecule complex. This can include the
release of cytotoxic granules and apoptosis as a result of engagement of FcγRIIIa on NK cells
(antibody-dependentcell-mediatedcytotoxicity,ADCC);thephagocytosisofantibody-coatedtarget
moleculesbymacrophagesbyengagingFcγRIIa(antibody-dependentcell-mediatedphagocytosis,
ADCP) and the engagement of complement protein C1q (complement-dependent cytotoxicity,
CDC)[7,24,25].
Cetuximab,anepidermalgrowthfactorreceptor(EGFR)inhibitor,bindstoantigenonthesurface
oftumorcellsandexhibitsitsanti-tumoractivitythroughADCC[26].Theseeffectorfunctionscanhave
therapeuticbenefits,butmayalsocauseadverseeffectsinsomediseases. Forexample,itwasfound
thattheanti-canceragentstrastuzumabandrituximabfailedtopreventtumorgrowthinFcγ-deficient
mice [27], suggesting the significance of effector functions on the efficacy of mAbs. The ADCC
and CDC functions of rituximab and alemtuzumab can contribute to cell lysis and unintended
cytokinerelease[28]. Also,theFcγbindingpropertiesvaryamongtheIgGsubclasses. Forexample,
IgG1subclasscanbindtoallFcγR,whereasIgG4weaklybindstoFcγRIIbandmoderatelytoFcγRI[29].
Therefore,rationaldesignofboththeFabandFcdomainsisessentialtomodulatetherapeuticefficacy
andsafetyprofileofmAbs.
AnotherfunctionoftheFcistorecycletheantibodywhileitcirculatesintheblood. Thespecific
transport of IgG from mother to offspring through the placenta is carried out by the neonatal Fc
receptor(FcRn)[30,31]. FcRnisahistocompatibilityclassI-relatedproteinthatinteractswithIgGand
albumininapHdependentmanner[32]. Inrodents,theacidicpH(pH6.5)allowsforhighbinding
betweentheFcportionoftheIgGandFcRn. FcRntranscytosesIgGafterbindingandreleasesonthe
neonatalsideatneutralpH(pH7.4). Inhumans,theIgGtransferoccursacrossthesyncytiotrophoblast
oftheplacenta. TheFcRnbindstothematernalIgGafterendosomeacidificationandreleasestheIgG
inthefetalcirculationatphysiologicalpH[30](Figure2). TheinhibitionofFcRnup-regulationorthe
FcRneliminationpathwayresultsinanincreasedeliminationandprolongationofthehalf-lifeofan
IgG[33]FcRntransportsitsligandsacrossacellularmonolayerprovidingarouteforproteinsinto
thebloodstream(acrosstheepithelialbarriers)[34]. FcRnhasbeenfoundinmucosalsitesinadult
mammaliansandanumberofstudieshavediscussedtheroleofFcRn-mediatedIgGintheintestine
andgastrointestinaltract(GIT)[34–39]. TheroleofFcRnhasalsobeendescribedinpulmonarydrug
delivery[34,40,41],wherethequantityandqualityofIgGintheairwaysareregulatedbyFcRn[40].
However,FcRnrecyclinghasbeenreportednottocontributetothevitrealhalf-lifeofanIgGwhen
injected into the back of the eye [42]. The expression level of FcRn in the eye and the systemic
epitheliummightcontributetotheFcRnnotplayingamajorroleintheeyewhencomparedtothe
centralcompartment[43].
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Figure 2. A schematic diagram illustrating neonatal Fc receptor (FcRn) recycling pathway: (1)
Figure 2. A schematic diagram illustrating neonatal Fc receptor (FcRn) recycling pathway:
Immunoglobulin G (IgGs) are internaliszed into endocytic vesicles. (2) Endosome becomes acidic
(1)ImmunoglobulinG(IgGs)areinternaliszedintoendocyticvesicles.(2)Endosomebecomesacidic
rreessuullttiinngg iinn tthhee bbiinnddiinngg ooff FFcc ddoommaaiinn ttoo FFccRRnn.. ((33)) BBoouunndd IIggGG aarree rreeccyycclleedd bbaacckk ttoo tthhee cceellll mmeemmbbrraannee
aanndd ((44)) IIggGG ddiissssoocciiaatteess aatt nneeuuttrraall ppHH ((77..44)) ffrroomm FFccRRnn aanndd iiss rreelleeaasseedd bbaacckk iinnttoo tthhee bblloooodd..
The development of therapeutic mAbs involves several complex processes including mAb
The development of therapeutic mAbs involves several complex processes including mAb
screening, engineering, production and purification. The productivity of each step has been
screening,engineering,productionandpurification. Theproductivityofeachstephasbeenimproved
improved as technology has evolved in recombinant techniques, bioprocesses and affinity-based
astechnologyhasevolvedinrecombinanttechniques,bioprocessesandaffinity-basedpurification.
purification. Only mAb screening and engineering will be discussed here; more comprehensive
OnlymAbscreeningandengineeringwillbediscussedhere;morecomprehensivereviewsofupstream
reviews of upstream and downstream mAb production are detailed elsewhere [44–47].
anddownstreammAbproductionaredetailedelsewhere[44–47].
Generally, mAbs are generated by means of animal immunization as a part of screening process.
Generally,mAbsaregeneratedbymeansofanimalimmunizationasapartofscreeningprocess.
Hybridomas are engineered cells that are capable of producing mAbs [48] and were developed by
HybridomasareengineeredcellsthatarecapableofproducingmAbs[48]andweredevelopedby
George Kohler and Cesar Milstein who won a Nobel Prize for their discovery. The main advantage
GeorgeKohlerandCesarMilsteinwhowonaNobelPrizefortheirdiscovery. Themainadvantageof
of hybridoma technology is its ability to grow continuously as well as its ability to produce a large
hybridomatechnologyisitsabilitytogrowcontinuouslyaswellasitsabilitytoproducealargeamount
amount of pure antibody [48,49]. The original hybridoma technology was established more than 35
ofpureantibody[48,49]. Theoriginalhybridomatechnologywasestablishedmorethan35yearsago,
years ago, which involved the use of hemagglutinating virus of Japan (HVJ) or Sendai and
whichinvolvedtheuseofhemagglutinatingvirusofJapan(HVJ)orSendaiandpoly(ethyleneglycol)
poly(ethylene glycol) (PEG) for fusing antigen-sensitized B lymphocytes and myeloma cells [50].
(PEG)forfusingantigen-sensitizedBlymphocytesandmyelomacells[50].
Briefly, animals (usually mice or rats) are immunized by injection with a soluble antigen that is
Briefly, animals(usuallymiceorrats)areimmunizedbyinjectionwithasolubleantigenthat
mixed with an adjuvant. The antigen-specific producing plasma cells from the spleen are then
is mixed with an adjuvant. The antigen-specific producing plasma cells from the spleen are then
isolated and are fused with a cancerous immune cell (called a myeloma cell) using fusing reagents
isolatedandarefusedwithacancerousimmunecell(calledamyelomacell)usingfusingreagents
such as HVJ or PEG [50]. The fused cells are then cultured in a growth medium supplemented with
such as HVJ or PEG [50]. The fused cells are then cultured in a growth medium supplemented
hypoxanthine-aminopterin-thymidine (HAT) medium. The cells that survive are the ones that
with hypoxanthine-aminopterin-thymidine (HAT) medium. The cells that survive are the ones
express hypoxanthine guanine phosphoribosyl transferase (HGPRT) and are called hybridoma cells.
thatexpresshypoxanthineguaninephosphoribosyltransferase(HGPRT)andarecalledhybridoma
Hybridoma cells become visible after 4 days and are grown for about 3 weeks. The culture
cells. Hybridoma cells become visible after 4 days and are grown for about 3 weeks. The culture
supernatant is eventually screened for secretion of the desired antibody (assays include Western blot
supernatantiseventuallyscreenedforsecretionofthedesiredantibody(assaysincludeWesternblot
and enzyme-linked immunosorbent assay (ELISA)) [50]. Given the rapid proliferation of myeloma
andenzyme-linkedimmunosorbentassay(ELISA))[50].Giventherapidproliferationofmyelomacells
cells and the production of specific antibodies, these two properties lead to the feasibility to produce
andtheproductionofspecificantibodies,thesetwopropertiesleadtothefeasibilitytoproducemAbs
mAbs in vitro. Plasma and myeloma cells can also be fused using an electrical field (pearl-chain
invitro. Plasmaandmyelomacellscanalsobefusedusinganelectricalfield(pearl-chainformation)
formation) or laser radiation in order to enhance fusion efficiency [50].
orlaserradiationinordertoenhancefusionefficiency[50].
Despite the advance of hybridoma methods, the technique encounters challenges such as low
Despitetheadvanceofhybridomamethods,thetechniqueencounterschallengessuchaslow
yield and contamination [51]. Performing under controlled in vitro conditions, the combination of
yieldandcontamination[51]. Performingundercontrolledinvitroconditions,thecombinationof
genetic engineering and in vitro selection strategy provides more scalable and high-throughput
geneticengineeringandinvitroselectionstrategyprovidesmorescalableandhigh-throughputmAbs
mAbs production that is suitable for commercial development [52]. Phage display is a widely used
productionthatissuitableforcommercialdevelopment[52]. Phagedisplayisawidelyusedinvitro
in vitro selection for screening therapeutic mAbs [53]. Adalimumab, an anti-tumor necrosis factor
(TNF) drug, was the first phage-display-derived mAb approved for clinical use [51].
The phage display technique can produce expressed antibody motifs, which are often Fab or
single-chain variable fragments (scFv) [54]. The pool of the antibody phages creates an antibody
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selectionforscreeningtherapeuticmAbs[53]. Adalimumab,ananti-tumornecrosisfactor(TNF)drug,
Pharmaceutics 2018, 10, x FOR PEER REVIEW 5 of 24
wasthefirstphage-display-derivedmAbapprovedforclinicaluse[51].
The phage display technique can produce expressed antibody motifs, which are often Fab or
library, which is used in the selection process or ‘panning’ step to screen the antibody candidates
single-chainvariablefragments(scFv)[54].Thepooloftheantibodyphagescreatesanantibodylibrary,
based on the binding property to target molecules. The selected phage will be re-amplified in
whichisusedintheselectionprocessor‘panning’steptoscreentheantibodycandidatesbasedon
Escherichia coli where the new antibody phage is generated. The panning steps are repeated for 2–3
thebindingpropertytotargetmolecules. Theselectedphagewillbere-amplifiedinEscherichiacoli
rounds potentially increasing the number of antigen-specific antibody phage clones. The resulting
wherethenewantibodyphageisgenerated. Thepanningstepsarerepeatedfor2–3roundspotentially
antibody genes can be sub-cloned to produce other antibody formats such as scFv-Fc or IgG [53]. The
increasingthenumberofantigen-specificantibodyphageclones. Theresultingantibodygenescanbe
advantage of mAbs obtained from phage display technique is the improved affinity in the picomolar
sub-clonedtoproduceotherantibodyformatssuchasscFv-FcorIgG[53]. TheadvantageofmAbs
range whereas the mAb affinity was reported to be approximately 100 pM by in vivo immunization
obtainedfromphagedisplaytechniqueistheimprovedaffinityinthepicomolarrangewhereasthe
[55].
mAbaffinitywasreportedtobeapproximately100pMbyinvivoimmunization[55].
Apart from screening process, genetic engineering also plays a crucial role in producing human-
Apart from screening process, genetic engineering also plays a crucial role in producing
mAb structures to reduce overall immunogenicity of generated mAbs from animals. Chimeric mAbs
human-mAb structures to reduce overall immunogenicity of generated mAbs from animals.
display approximately 60–70% human homology and are produced by combining the gene encoding
ChimericmAbsdisplayapproximately60–70%humanhomologyandareproducedbycombiningthe
murine Fv and human Fc [46]. A murine CDR can be grafted onto a human framework to generate
geneencodingmurineFvandhumanFc[46]. AmurineCDRcanbegraftedontoahumanframework
humanized mAbs (90–95% human) [56]. Without the murine CDR, a fully human mAb (100% human)
togeneratehumanizedmAbs(90–95%human)[56]. WithoutthemurineCDR,afullyhumanmAb
can also be generated. Moreover, genetic engineering has been examined to generate small mAb-
(100%human)canalsobegenerated. Moreover,geneticengineeringhasbeenexaminedtogenerate
derived fragments such as recombinant Fab, scFv and bispecific antibodies (Figure 3) [46].
smallmAb-derivedfragmentssuchasrecombinantFab,scFvandbispecificantibodies(Figure3)[46].
Recombinant Fabs contain one heterodimer of CH1 and VH covalently linked with CL and VL
RecombinantFabscontainoneheterodimerofCH1andVHcovalentlylinkedwithCLandVLdomains
domains while the Fc portion is deleted. ScFv technology uses one VH and VL sequence responsible
whiletheFcportionisdeleted. ScFvtechnologyusesoneVHandVLsequenceresponsibleforantigen
for antigen domain binding domains connected with a linker sequence or bispecific mAbs, which
domain binding domains connected with a linker sequence or bispecific mAbs, which allow dual
allow dual targeting. They can be used to bind to various antigens (haptens, proteins and pathogens)
targeting. Theycanbeusedtobindtovariousantigens(haptens,proteinsandpathogens)andcan
and can be used alone or as fusions in ELISA [17]. ScFv are smaller in size as compared to Fabs
be used alone or as fusions in ELISA [17]. ScFv are smaller in size as compared to Fabs resulting
resulting in better tissue penetration and pharmacokinetic profiles, however, they have also been
inbettertissuepenetrationandpharmacokineticprofiles,however,theyhavealsobeenreportedto
reported to have fast off-rates and poor retention times [17]. The modification of the length of the
havefastoff-ratesandpoorretentiontimes[17]. ThemodificationofthelengthofthescFvlinkercan
scFv linker can create derivatized multivalent scFv such as bivalent scFv (diabodies) and trivalent
createderivatizedmultivalentscFvsuchasbivalentscFv(diabodies)andtrivalentscFv(tribodies)that
scFv (tribodies) that enable multi-mAb binding [46]. In one study, a 5-fold increase in half-life and
enablemulti-mAbbinding[46]. Inonestudy,a5-foldincreaseinhalf-lifeand30-foldincreaseintumor
30-fold increase in tumor uptake were observed with a site-specific polysialylated anti-
uptakewereobservedwithasite-specificpolysialylatedanti-carcinoembryonicantigen(CEA)scFvas
carcinoembryonic antigen (CEA) scFv as compared to an unmodified scFv [57]. So far, no multivalent
comparedtoanunmodifiedscFv[57]. Sofar,nomultivalentmAbproductiscommerciallyavailable.
mAb product is commercially available.

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ffrraaggmmeenntt aannttiiggeenn--bbiinnddiinngg ((FFaabb)),, ((bb)) SSiinnggllee--cchhaaiinn vvaarriiaabbllee ffrraaggmmeenntt ((ssccFFvv)) aanndd ((cc)) bbiissppeecciiffiicc mmAAbb
ppllaattffoorrmmss.. TThhee llaatttteerrc caannc ocommpprirsiesetw twooco cvoavleanletnlytllyin lkinedkehde theerotegreongoeunsoIugsG Ig(IGg G(I2g)Gor2)h oert ehroetgeernooguesnoFuabs
Fdaobm daoinmsa(iFn(sa b(F’)(2a)b.’)2).
Bispecific antibodies are intended to bind to different antigens or epitopes by combining the
Bispecific antibodies are intended to bind to different antigens or epitopes by combining the
specificities of two antibodies [58] One hope is to develop bispecific antibodies to address
specificitiesoftwoantibodies[58]. Onehopeistodevelopbispecificantibodiestoaddressapplications
applications where they can exploit spatial–temporal relationships that are not possible by using a
wheretheycanexploitspatial–temporalrelationshipsthatarenotpossiblebyusingacombination
combination or mixture of antibodies. There are several potential advantages to bispecific antibodies:
or mixture of antibodies. There are several potential advantages to bispecific antibodies: (i) they
(i) they can redirect specific immune cells to the tumor cells to enhance tumor killing; (ii) they can
enable simultaneous blocking of two different mediators/pathways that exert unique or overlapping
functions in pathogenesis; and (iii) they can potentially increase binding specificity by interacting
with two different cell-surface antigens instead of one. It is generally difficult to ensure
Pharmaceutics2018,10,83 6of24
canredirectspecificimmunecellstothetumorcellstoenhancetumorkilling; (ii)theycanenable
simultaneousblockingoftwodifferentmediators/pathwaysthatexertuniqueoroverlappingfunctions
in pathogenesis; and (iii) they can potentially increase binding specificity by interacting with two
differentcell-surfaceantigensinsteadofone. Itisgenerallydifficulttoensuremultifunctionalproteins
suchasbispecificantibodiesareisolatedinapureandsufficientamountduringearlydevelopment
andatadecentscaleforproduction[59].
Inessence,fullyhumanmAbscanalsobeproducedbyperformingahybridomaapproachina
transgenicmouseinwhichthemurineIggeneisknockedoutandisreplacedwithahuman-derived
segment,thusenablingtheproductionofentirelyhumanmAbs[60]. Thistransgenicapproachcan
avoidahumananti-mouseantibody(HAMA)response. Owingtotheircapabilitiestoproducefully
humanmAbs,phage-displayandtransgenicmice-derivedmAbsgainrelativelyhighsuccessratein
thelaterstagesofclinicaltrials[51].
2. GeneralLimitationsandFormulationChallenges
2.1. PharmacokineticLimitations
While most mAbs can display prolonged circulation times by FcRn-mediated recycling,
many therapeutic proteins have short invivo half-lives, which is usually a matter of hours to a
fewdays[61]. Forexample,bevacizumabisamAbthatisusedunlicensedforneovascularizedocular
tissuesanditisacost-effectiveanti-VEGFmedicinethatisclinicallyequivalenttoranibizumab[62,63].
Anintravitrealdoseofbevacizumab(1.25mg,50µL)andranibizumab(0.5mg,50µL)yieldshalf-life
valuesof6.7–10.0and7.2–9.0daysrespectively[64–67]. Theshortinvivohalf-liferesultsinfrequent
drugdosing,whichcanincreasethechancesofundesirablesideeffects[68]andcanresultinhighcosts
andpoorercompliancetobothpatientsandhealthcaresystems[69]. Ingeneral,smallermodified
antibodies(e.g.,withouttheFc)haveshorterinvivohalf-liveswhenadministeredviaotherroutes[70].
MostmAbsarelimitedintheirabilitytopenetrateandaccumulateintissuesduetotheirlarge
size. Thesolutionsizecanhaveadirectimpactonthepharmacokineticpropertiesandaffectclinical
efficacy. Therapeuticproteinscanbepassivelydistributedfromthebloodcirculationtoperipheral
tissuesbyconvectivetransportthroughfenestraeporesoncapillarywalls,orthroughthetranscellular
pathwayviaendothelialcells[71]. Thelattermechanismhighlightsthepossibilityforactivetransport
ofmAbs,whichcanincludecellularuptakeofproteinsinsurroundingfluid(fluid-phasepinocytosis),
receptor-mediated endocytosis (FcγR-mediated) and phagocytosis by immune cells [71]. In terms
of mass transport, passive diffusion underpins the permeation or absorption of drugs across cell
membranesanddependsonthesize,hydrophilicity–hydrophobicityandchargeofthecompounds.
However,passivediffusionislimitedforbiotherapeuticsowingtotheirlargesolutionsizeandcharge.
Giventhatsubcutaneousadministrationisadesiredrouteofadministration,macromoleculesare
likelytoberestrictedtotheinterstitialspaceafterinjectionowingtotheirlargesize[72]. Ingeneral,
biotherapeuticscanreachbloodcirculationbytwopathways:viabloodcapillariesorlymphaticvessels.
Absorptionthroughbloodcapillariesreliesonpassivetransportandisrestrictedtocompoundswith
a molecular weight cut-off (MWCO) of up to 16 kDa [73]. Hence, most biotherapeutics will not
be able to be transported via capillary routes, but rather rely on the lymphatic system where the
protein reaches the systemic circulation at the thoracic duct [74]. The distribution of therapeutic
proteinsislimitedtoplasmaratherthantissue[75]. Asaconsequence,deliveringproteinstomaintain
effectivetherapeuticconcentrationattargettissuesischallenging. Forexample,bevacizumaband
ranibizumabaregivenintravitreally. Owingtothesizeofbothanti-VEGFmedicines,itisunlikely
to deliver a significant level of both drugs to the posterior segment of the eye by systemic
administration, where the blood retina barrier (BRB) is a major barrier for drug transport [76].
The subcutaneous administration of trastuzumab is enabled by the use of recombinant human
hyaluronidase(rHuPH20)[77],whichbehavesasapermeationenhancer. Subcutaneoustrastuzumab
has a fixed dose of 600 mg administered 3-weekly and avoids weight-based infusion dosing [78].
Pharmaceutics2018,10,83 7of24
rHuPH20enhancestheinfusionratesandpenetrationofmoleculesupto200nmindiameterupto
20-foldwithoutelicitinginflammation,vascularpermeability,immune-genicorallergicreactions[79].
Enzymaticdegradation,eitheratthesiteofadministrationorwhileincirculation,canfurther
reducethepharmacokineticsofmostclassesofproteins. Protein-basedmedicinesusuallyexerttheir
actionextracellularly(e.g.,cellsurfacereceptorinteractionandligandbinding). Itisgenerallyknown
thatproteinsandmAbsarepronetoundergoenzymaticdegradationandunfoldingespeciallyinthe
GIT.Oraldeliverysystemsforthesebiologicallyactivecompoundsarehencechallengingtodevelop,
unlike most small molecule drugs. Thus, the routes of administration of biologics are normally
parenteralinjection(intravenous,subcutaneous,intramuscularorintradermal). Mostproteinsand
mAbs are now formulated for subcutaneous injection [74]. Even if parenteral application is used,
macromoleculedrugscanstillsufferfrompre-systemicdegradationbyenzymessuchasproteasesand
hydrolase,sincetheseareabundantthroughoutthebody[80].
TheeliminationofmAbtherapeuticscanbeacceleratedbyimmunogenicityandthedevelopment
ofantibody-drugantibodies(ADAs),particularlywiththoseproteinsderivedfromanimals. ADAscan
alsocauseacutehypersensitivityorinfusionreactions.Furthermore,ADAscanalsocompetitivelybind
totheactiveregionofthetherapeuticproteinsuchasthereceptor-bindingsitetoneutralizetheantibody
drug,thereforecompromisingefficacy. ADAscanalsounpredictablychangedrugpharmacokinetic
properties,biologicaleffectsandthetoxicityprofile[81]. HumanizedandfullyhumanmAbsandother
therapeuticproteinsarelessimmunogenicinhumanscomparedwithnon-humanderivedproteins
(e.g.,murineantibodies),albeithumanizedproteinscouldalsocauseanimmuneresponseinhuman
beings[82]. Usinganimalstopredictthehumanresponsetoacandidateproteinisanotherchallengein
thedevelopmentofproteintherapeutics[83]. Animalsdevelopanimmuneresponseagainstantigens
ofinterest,however,thelevelofimmunogenicitybetweenanimalmodelsandhumansisrelatively
different. Itisreportedthatimmunogenicityisover-estimatedinconventionalanimalmodelsmaking
them unreliable to predict patient immunogenicity [84,85] owing to different mechanisms in the
immuneresponsebetweenhumansandanimals[12]. Consequently,theimmuneresponsesobtained
fromanimalstudiesareequivocaltopredictclinicalconsequences(e.g.,productionofneutralizing
antibodiesandneo-epitopesonmodifiedproteins)[85,86]andtranslateintohumanstudies.
Likemostclassesoftherapeuticproteins,antibody-basedmedicinesgenerallytendtobecleared
morequicklywhenusedinahighdoseasaresultofpossibleaggregation[26]. However,thisalso
depends on the precise clearance mechanism of the protein molecule. High protein dose will
have a faster elimination if phagocytosis in the reticuloendothelial systems (RES) plays a major
role. Incontrast,theeliminationratewillbedecreasedwithhighdosesifendocytosisisthemajor
elimination process [87]. As endocytosis is a receptor-mediated process, this suggests that the
saturationofreceptorscaninfluencethebioavailabilityofproteindrugstargetingsurfacereceptors.
Thisphenomenonisknownas‘antigensink’andiscommonlyfoundwithmAbstargetinginternalizing
receptors[88]. Forexample,trastuzumab,ananti-humanepidermalgrowthfactorreceptor(HER)2
antibody,waseliminatedslowlywithahighdose[89]. Asmentionedearlierinthissection,afrequent
dosing regimen is required to maintain the drug at therapeutic levels, which can have negative
implicationsintheclinic. Aninfusionreactionisacommonadverseeffectassociatedwithparenteral
treatment. Forexample,astudyshowedthatpatientsdevelopedacutereactionswithincreasingdoses
ofinfliximabwith61%ofpatientsexperiencinganacutereactionatthefifthdose[90].
2.2. FormulationChallenges
BiologicalmacromoleculesincludingmAbshavethree-dimensional(3D)structuresknownasthe
tertiarystructure. Anintricatebalanceofintra-andintermolecularinteractionsbetweenaminoacid
functionalgroupsandexternalenvironmentsdictatesthefoldedstructure. Arangeofnon-covalent
interactionsiscrucialtomaintainthenativefoldedstructure,e.g.,electrostaticinteractions,vander
Waals interactions of the backbone and side chain residues, hydrogen bonding and hydrophobic
interactions[91]. Asthefoldedstructureisindynamicequilibrium,anyfactorsshiftingtheinteraction
Pharmaceutics2018,10,83 8of24
balance can cause the structure to change, which can contribute to an unstable state of the large
molecules. For example, in aqueous solution more soluble amino acid residues become exposed
andinteractwithaccessiblesolventmoleculeswhilenon-polarresiduesareencapsulatedforminga
hydrophobiccore.
Proteinfoldingisafunctionoftheaminoacidsequenceandgivesthebiologicallyactiveform
oftheprotein. However,proteininthenativestructurecanbeunfoldedviaanintermediatestateor
undergodirectunfoldingtoadenaturedstate. Whentheintermediateorunfoldingspeciesisformed,
the variants are prone to assemble to form more stable complexes such as aggregation, owing to
their higher free energy. Aggregates are composed of more than one monomer in any form and
are strengthened by either covalent or non-covalent bonds. The type of monomer association can
influencethereversibilityoftheaggregates;forexample,aggregatesofnativemonomerclusterscan
bedissociatedbydilution[92]. Precipitationorirreversibleaggregatesmayresultfromthenucleation
ofdifferentmonomers[93]. Anumberofstressfactorscanleadtoaggregation,themostcommon
onesinmanufacturingbeingtemperature,mechanicalandfreeze/thawstress[26]. Hightemperatures
canleadtoconformationaldestabilizationorpartial/completeunfolding[26]andachangeinpHcan
leadtoaggregation. Forexample,astudycomparedthestabilityprofilesofIgG1andIgG4. IgG4was
reportedtoformmoresolubleaggregatesthanIgG1atlowerpHandhightemperatureduetoreduced
conformationalstabilityfromlowerunfoldingtemperatureandchangesintertiarystructures[94].
As the tertiary structure of biologics are susceptible to physical stress from the environment,
structuralalterationofmAbscanemergeatanystageduringthemanufacturingprocess,frominitial
proteinexpressionthroughprocessingtostorage[95]. Structuraltransformationcanbeascribedto
thepresenceofnon-physiologicalconditionsduringtheprocessesthatmaydrivetheadaptationof
structural variants in the finished product. It is a major issue and challenge in biopharmaceutical
development that is distinctly different to the production of small molecule drugs where such
problemsdonotarise. Forinstance, stressessuchasbufferchoice, manufacturingtechniquesand
choiceofcontainerscanpotentiatethisissue[96]. Astudyreportedthestabilityofamodelmurine
IgG3invariousbuffersolutionsusingultraviolet/visiblespectroscopy(UV-VIS)andsize-exclusion
chromatography(SEC).Theuseofacetatewasreportedtoformvisibleprecipitates,whereasarginine
and histidine were shown to improve stability for a long-term storage and multiple freeze/thaw
cyclesofIgG3[97]. AstudydemonstratedhowlyophilizationledtoapHshiftinproteinsolutions
resultinginachangeinproteinactivity[98]. Adjustingtheconcentrationofbuffersaltscouldtherefore
help prevent activity changes. The pH of lyophilizate is also critical to control the aggregation of
recombinantvaccineantigensandoftenadditionalstabilizerssuchastrehaloseisrequiredtomaintain
theintegrityoftheantigen[99].
Additionally,thepresenceofnon-nativeproteinsinanyfinishedmAb-basedbiopharmaceutical
productsmustbeavoided.Aggregationcancausenon-uniformdosingwhendrawingproteinsolutions
fromvials[100]. Unstablebiologicscancontributetoalackoftherapeuticeffectoradverseeffects.
Forexample,theaggregationofIFN–βcanstimulatetheproductionofneutralizinganti-drugAbs
(NAb),whichblockstheIFNreceptorbinding,thusloweringclinicalefficacy[101]. Theunwanted
effectscanalsoarisefrominducedNAbsonthenormalfunctionofendogenousproteins,especiallyin
thecaseoftherapeutichormonesorcytokines. Notably,pure-redcellaplasia(PRCA),asuddensevere
anemia, was developed in patients treated with Eprex® rh erythropoietin [102]. The complication
emerged as a result of NAb induced against Eprex® blocking endogenous erythropoietin [103].
The NAb was induced by aggregation driven by the presence of formulation ingredients such as
polysorbate80[104]andsiliconeoilinpre-filledsyringes[105,106].
mAbsareproducedbyrecombinanttechnologiesoreukaryoticorganismse.g.,Chinesehamster
ovary(CHO)cells. Eukaryoticcellscanbeengineeredtoexpresstheproteininitsglycosylatedform.
Producingfullsizeantibodiesineukaryoticorcellsisextremelylaborintensive,costly,andproblems
arisewithcomplexintellectualproperty(IP)issuesassociatedwithantibodycompositionandCDR,
and the use of technical processes that are needed. Production and purification processes can be
Pharmaceutics2018,10,83 9of24
expensive,andcoupledtoresearchandthenmarketingcostscanresultingenerallyhighdrugcosts,
whichultimatelylimitspatientaccess[7,107,108].
3. PotentialStrategiestoOvercomeChallengesinAntibody-BasedTherapies
3.1. UseofExcipientstoStabilizeFormulations
Excipients have been used to increase the stability of a wide range of protein and
peptide based formulations [109] by reducing protein dynamics and motion, increasing the
conformationalstabilityofmAbsespeciallyathighconcentrationsandinhibitinginterface-dependent
aggregation[110–112]. Excipientsusuallyinhibitaggregationandprotectstheproteinbyadsorbing
to the air–liquid interface; for example, the use of surfactants (e.g., polysorbate 20 and 80),
carbohydrates(e.g.,cyclodextrinderivatives)andaminoacids(e.g.,arginineandhistidine)canhelp
preventaggregationbythismechanism[103]. However,theuseofpolysorbate80canleadtomicelle
formationandhence,increasethechanceofimmunogenicity[103]. Inonestudy,cyclodextrinwas
reportedtostabilizecommerciallyavailableantibody-baseddrugsinahydrogelformulation[113].
Some of the generally recognized as safe (GRAS) excipients include pluronic F68, trehalose,
glycine and amino acids such as arginine, glycine, glutamate and histidine, which are found in
anumberofcommercialproteintherapeuticproducts[110,114]. Forexample,Avastin®(bevacizumab,
25 mg/mL) contains trehalose dehydrate, sodium phosphate and polysorbate 20. Excipients in
subcutaneousHerceptin® (trastuzumab, 600mg)arerHuPH20, histidinehydrochloride, histidine,
trehalosedehydrate,polysorbate20,methionineandwaterforinjection[115]. Anumberofstudies
havestudiedtheuseofexcipientsinthestabilizationofmAbs[114,116,117]. Thechoiceofexcipients
requiresthoroughscreeningandoptimizationtopreventaggregation[103]. Oneformulationexcipient
stabilizingaparticularantibodymightnotbesuitableforanotherantibodyduetodifferencesintheir
sequence[114,118].
3.2. ProductionofProteinScaffolds
There is an increased focus on making more stable proteins so they can be formulated and
usedinlong-actingforms. TherearefundamentalstabilityissueswithmAbs,withthehingeregion
beingthebiggestproblem. Severalproteinfamilieswithnon-IgGarchitecturehavebeendeveloped
with novelbinding motifsand are known asengineeredprotein scaffolds [12]. A scaffold isoften
definedasasinglechainpolypeptideframeworkthatcontainsahighlystructuredcoreassociated
with variable portions of high conformational tolerance allowing insertions, deletions and other
substitutions. MostscaffoldshavebeendevelopedagainstvalidatedtargetsincludingTNF-α,CD20,
VEGF,CD19andCD3[7]. ScaffoldstendtobelowerinmolecularweightthanmAbsandwhilethey
cansharesomestructuralfeaturesofanantibody,scaffoldshavealsobeendescribedthatareunrelated
tomAbs[7]. Proteinscaffoldshavebeenreportedtohaveenhancedsolubilityandthermalstability
andbettertissuepenetration[107]. Italsooffersasinglepolypeptidechainformatandhighbacterial
expressionforcheapproduction[119]. Manyspecialistshavedevelopedtheirownindividualprotein
scaffoldniches. ScaffoldscanbeeitherIgG-(e.g.,nanobody,scFv,singledomains)ornon-IgG-like
molecules(affibodies,anticalins,DARPins,dual-affinityretargetingmolecules)andbothcategories
haveyieldedsmallermoleculesthatpossessmuchoftheepitopebindingandspecificityproperties
ofmAbs. Forexample,non-IgG-likemoleculespossesshigherstability,cysteine-freesequencesand
flexiblepharmacokineticproperties[120].
Proteinscaffoldsdohavesomeadvantagesovercurrentproteintherapeutics,howevertheyalso
displaysomelimitationstoo. Asexogenousproteins,scaffoldproteinsarepotentiallyimmunogenic.
Mostproteintherapeuticsthathavebeendevelopedduringthelast30–40yearshavebeennatural
occurring proteins (e.g., cytokines, blood factors) or mAbs, which are very similar to endogenous
IgGs. Minimizingimmunogenicityisonemajorconcernwiththedevelopmentofnon-endogenous
therapeuticproteins. Forexample,affibodiesarederivedfromproteinA,whichisabacterialprotein.
Pharmaceutics2018,10,83 10of24
Theygenerateanimmuneresponseafteradministrationtohumanpatients. Therefore,affibodiesare
targetedtobeusedasmolecularimagingagentsforcancerdetection,insteadoftherapeuticagents[107].
Pharmaceutics 2018, 10, x FOR PEER REVIEW 10 of 24
Thoughmostproteinscaffoldsarenowbeingderivedfromnaturallyoccurringhumanproteins,itstill
doesnotcompletelyeliminateundesirableimmuneresponses[107]. Inaddition,thoughtherehasbeen
though there has been much investment to develop protein scaffolds, there is only one product
muchinvestmenttodevelopproteinscaffolds,thereisonlyoneproduct(Kalbitorescallantide/DX-88),
(Kalbitor escallantide/DX-88), which has been successfully registered for clinical use [107].
whichhasbeensuccessfullyregisteredforclinicaluse[107].
3.3. mAb Formulations to Prolong the Duration of Action
3.3. mAbFormulationstoProlongtheDurationofAction
Formulation strategies have also been investigated to increase the duration of action of proteins,
Formulationstrategieshavealsobeeninvestigatedtoincreasethedurationofactionofproteins,
e.g., the preparation of controlled release systems (Figure 4). Here, more general approaches for the
e.g.,thepreparationofcontrolledreleasesystems(Figure4). Here,moregeneralapproachesforthe
extension of protein action will be described that are also investigated for the development of longer
extensionofproteinactionwillbedescribedthatarealsoinvestigatedforthedevelopmentoflonger
acting mAb formulations.
actingmAbformulations.

Figure4.Commonformulationstrategiesusedtoprolongproteinrelease.
Figure 4. Common formulation strategies used to prolong protein release.
3.3.1. MicroparticulateAssociatedFormulations
3.3.1. Microparticulate Associated Formulations
Microparticles have been examined for the long-term delivery of proteins including mAbs
andpMeipctriodpeasr.tTichlees mhaovste cboemenm eoxnammianteedri faolru tsheed loinngm-tiecrrmop daretliicvleerfyo ormf purloatteioinnss iinsctlhuedihnygd mroAlybtsic aanlldy
dpeepgrtiaddeasb. leThceo -pmoolystm ceormcamlloend pmoaltye(rliaaclt icu-sceod- gilny comliiccraocpidar)ti(cPlLe GfoAr)m. Puelapttiiodnes loisa dtehde mhyicdrrooplyatritcicallelys
hdaegvreabdeaebnlee cxote-pnosilvyemlyerd ceaslclreidb epdolsyu(clahctaisc-Ecox-egnlyatciodleic, Saacnidd)o (sPtaLtGinA®),. VPievpittridole® ,loRaidsepde rmdailc®roCpoarntsictale®s,
Zhaovlaed bexeenan edxtLeunspirvoenly. dAesscoripbpedos esudchto atsh eErxaepneauttidice,p Sepantiddoesst,attihne®,e Vncivaiptsroull®a,t iRonispoefrdparlo®t eCinosnsitnat®o,
PZLolGaAdemx aicnrdo sLpuhperroesn.i sAqsu oiptepcohseadll etnog tihnegraapneduntioc tpyeeptticdliensi,c tahlley eanpcpaprosvueladtidoun eotfo ptrhoeteliancsk inotfop ProLtGeiAn
smtaicbriolistpy.heLreoss siso qfutietert ciahrayllesntrguicntgu raenda nndotb yioetl ocgliinciaclaallcyt iavpitpyrocvaendo dcucue rtou tphoen lapcrko olof npgreodteiinn csutabbaitliiotyn.
wLoitshs obfi otelortgiaicrayl sfltruuicdtusruen adnedr bpiohlyosgiioclaolg aicctailvictoyn cdaint ioocncsu.r uPpLoGnA prpoololynmgeedrs inacruebhaytidorno wphitohb bicio,lhoegniccael,
dfliuffiderse untncdlears spehsyosfioplorogtiecianl cmonoldeictuiolness. ePnLcGapAs uploaltyemdebrys eamreu hlsyidornopohropbhica,s ehesnecpea, rdaitfifoenreanrte cplarsosnees toof
spurorftaecine amdosloercputlieosn e,nacgagprseuglaattieodn ,bdye enmatuulrsaiotino no,ro pxhidaaseti osnepaanrdaticolena avraeg eprloeande intog stuorfaacloes asdinsoorpvteiroanll,
aacgtgivreitgyat[i1o2n1,] .dOenftaetnu,rtahtieorne,i soxaibduatrisotnr ealneads eclweaivthagPeL lGeaAd-ibnags etdo fao rlomssu liant ioovnearnaldl acoctnivtriotyll i[n1g21t]h. eObfuternst,
rtheleeraes eisc aa nbuinrcstr eraesleeatshee wduitrha tPioLnGoAf-dbarusegdr efloeramseu.lTathioenb uanrsdt rceolneatrsoellcianng btheein bfluuresnt creedleabsyem caann yinfcarcetaosres
sthuec hduasratthioenp oofly dmruegr croelmeapsoes. iTtihoen b[u12rs2t] ,reenlecaaspes cualnat bioen inpfrlouceenscse,da nbdy mdrayninyg famcteotrhso sdu[c1h2 a3s]. the polymer
compNoosivtieolne n[1c2a2p]s, uelnactaiopnsutleacthionno lporgoycecsosu, ladndb edurysiendg tmoeotvheordco [m12e3]t.h eabove-mentionedlimitations.
AzerNooovrdele ernrcealepassuelaotfiobne vteacchiznuomloagbyf croomuldp obley u(csaepdr tool aocvtoernceo)m(PeC tLhe) ealbeocvtreo-smpeunntifiobneerds lwimasitaactihoinesv.e Ad
ozevreor otwrdoerm reolnetahsse owfi tbhevtahceizcuamreafub lfrcoomnt rpoollyo(fcappHroolafctloonade)e d(PCbeLv)a ecliezcutrmoaspbuann fdibeenrsc awpasus laacthioienvbedy
eolveecrtr otwhyod rmodonynthasm wiciathto mthiez actaiornef(uEl HcDonAt)ro[1l 2o4f] .pTHhe osfu ploearidoerda dbveavnatcaigzeumofatbh eaEnHd DeAnciaspastutrliabtuiotend btyo
tehleecptrroohcyesdsroadlloywnaimngice antcoampsizualatitoionn (EatHaDmAb)i e[n12t4te].m Tpheer asutupreersi,owr ahdicvhacnatangper oecf ltuhdee EtHheDrmA aisll ya-titnridbuucteedd
dtoe gtrhaed aptrioocne.ssA anlluowmibnegr oenfcoatphseurlsattuiodni eastu asminbgiemnitc rtoempapretircalteusrfeosr, twhheicdhe licvaenr yporefcalundtieb otdheiersmhaallvye-
binedeuncreedp odretgerda.dIantioonn.e As tnuudmy,baerp ooft eonttheinr tsrtauvditireesa ulsainntgi- mVEicGroFpfaorrtmicluelsa ftoiorn th(ed idmeelirvicermy oolfe acnutlieb/odduieasl
dhaAvbe bcoenenta rienpinogrtetwd.o Ind oifnfee rsetnutdyan, ati p-VotEeGntF indtoramvaitirneaaln atnibtio-dViEesGF(d fAorbm)ualtataticohne (dditmoearihcu mmoalencuIgleG/d1uFacl
rdeAgbio nco)nutsaiinnginmg ictwroop adritfifcelreesnotf aPnotliy-VAEcGtivFe ™domhyadinro agnetlibcoo-dpioesly (mdeArbw) aasttraecphoerdt etdo tao shhuomwana 6IgmGo1n Fthc
rreelgeiaosne) [u12si5n].gI mnaicnrootphaerrtisctluedsy o,fi nPfloilxyimAcatbivwe™as ehnycdarposguella tceod-pinotloymmeicr rwosapsh reerpesorutseidn gtoe isthhoewrl yao 6p hmiloiznetdh
prealretaicsue la[t1e25a]n. tiIbno daynootrhaern asqtuudeyo,u sinsfolilxuitmioanb owf aans tiebnocdayp,saulslaotekdn oiwntno ams tihcreorsmpahlelrye-sin duusicnegd peihthaseer
slyeopparhaitliiozend( TpIPaSrt)icteuclhanteo laongtyi.bTohdey EoLrI SaAn eaxqpuereiomuesn stoclountfiiornm eodf bainotliobgoidcayl, aactlsivoi tkynoofwinnfl iaxsim tahberamgaailnlys-t
induced phase separation (TIPS) technology. The ELISA experiment confirmed biological activity of
infliximab against TNF-α. TIPS technology avoids the long-term exposure of proteins to oil/water
interfaces and can prevent leaching and protein denaturation [126].