PEGylation technique

The term ‘PEGylation’ can be defined as the covalent attachment of polyethylene glycol (PEG) chain to bioactive substances.

1. PEGylation
Technique

2. Contents
•Introduction
•Comparison with other technologies
•Need of PEGylation
•Purpose of PEGylation
•Mechanism of work
•Derivatives
•PEGylationprocess
•Generations
•Strategies
•Quality control considerations
•Application in NDDS
•PEGylatedliposomes
•Limitations

3. Introduction
•Theterm‘PEGylation’canbedefinedasthecovalentattachmentof
polyethyleneglycol(PEG)chaintobioactivesubstances.
•Proteinandpeptidedrugsholdgreatpromiseastherapeuticagents.
However,manyaredegradedbyproteolyticenzymes,canberapidly
clearedbythekidneys,generateneutralizingantibodiesandhavea
shortcirculatinghalf-life.Pegylation,theprocessbywhichpolyethylene
glycolchainsareattachedtoproteinandpeptidedrugs,canovercome
theseandothershortcomingsByincreasingthemolecularmassof
proteinsandpeptidesandshieldingthemfromproteolyticenzymes,
PEGylationimprovespharmacokinetics.

4. Contd..
•TheFDAhasapprovedPEGtouseasavehicleorbaseinfoods,
cosmeticsandpharmaceuticals,includinginjectable,topical,rectaland
nasalformulations.
•PEGshowsverylittletoxicityandlacksimmunogenicity.
•ThistechniquewasfirstdescribedbyAbuchowskyandco-workersin
1970withthePEGylationofalbuminandcatalase.
•LiposomesarePEGylatedtoprolongtheirbloodcirculationtime.
Comparedwithclassicalliposomes,PEGylatedcounterpartsshow
increasedhalf-life,decreasedplasmaclearance,andashiftin
distributioninfavourofdiseasedtissues.
•PEGisincorporatedintothelipidbilayeroftheliposome,forminga
hydratedshellthatprotectsitfromdestructionbyproteins.
•PEGylatedliposomesarealsolessextensivelytakenupbythe
reticuloendothelialsystemandarelesslikelytoleakdrugwhilein
circulation.

5. Comparison with
other Technologies
•In PEGylatedproducts,
API is chemically
modified in a durable
fashion, and the drug is
not released from a
formulation but has a
permanent action and is
in fact classed as a new
API.
Pegylation
•Other formulated products
such as tablets, liquids and
capsules, the formulation
process is reversible, the
drug becomes active after
its release from the
formulation and the API
remains unchanged.
Other delivery
systems

6. Need of
PEGylation
TheNovelProteinsandPeptideshavebecomeimportantnewdrugs
withadventofarevolutioninBiotechnology.
Morethan80PolyPeptideDrugsaremarketedinTheU.S.
Morethan350ProteinsandPeptidesareundergoingclinicaltrailsright
now.
AboutathirdofDrugcandidatesinclinicaltrailsarePolypeptides.

7. The purpose of
PEGylation
To improve drug solubility.
To reduce dosage frequency, without diminished efficacy
with potentially reduced toxicity.
To extend circulating life.
To Increase drug stability.
To enhance protection from proteolyticdegradation.
Opportunities for new delivery formats and dosing regimens.
To Extend patent life of previously approved drugs.

8. How do the
PEGs Work?
PEGsactuallyworkviathreeways;byreducingkidneyfiltration,
increasingsolubilityduetothePEGhydrophilicityanddecreasing
accessibilityforproteolyticenzymesandantibodies.Amongthesethreethe
mechanismofreducingkidneyfiltrationshouldbediscussed.

9. Reducing Kidney
Filtration
PEG protein conjugate
Apparent Hydrodynamic Radius: 20 nm
•PEGylationsignificantlyincreasesthe
apparentsizeoftheconjugateddrug
compound.
•Renal glomerular capillary
diameter: 6-12 nm

10. Chemistry of
PEGylation
Structure of PEG:
Ethylene
Glycol(EG) group
Numbers of
EG groups.
(Upto1000)
??????
2??????+2??????
4??????+6??????
??????+2
•Molecular Formula:
•Synthesizedfromthepolymerizationofethyleneoxide.
•UsingchemicaltoolstolinkPEGmoleculestonative
proteinscanyieldconjugateswithmorefavorablebehavior.

11. Contd..
•PEG is not ready for conjugation reactions by itself…
1.Needs a capped terminus with unreactive moiety.
2.Other end has reactive moiety that is covalently with reactive
partner (protein, peptide, other compounds).

12. Contd..
Methodsfortheactivationof
PEGmolecules:
A.Cyanuricchloridemethod.
B.Avariationonthecyanuric
chloride.
Ca.Polyethyleneglycol(PEG)-
succinimidylsuccinatemethod.
Cb.Substitutionofthe
succinateresiduebyglutarate.
Cc.Substitutionofthe
aliphaticesterinCabyan
amidebond
D. Imidazole formatemethod
E. & F. Variations using phenylcarbonatesof PEG
G. Succinimidylcarbonates of PEG
H. Succinimidylactive ester of PEG

13. Derivatives

14. Contd..

15. PEGylation
process
Functionalization of the PEG polymer at one or both
terminals
PEGS that are activated at each terminus with the same
reactive moiety are known as "homobifunctional"
If the functional groups present are different, then the PEG
derivative is referred as "heterobifunctional" or
"heterofunctional."
The chemically activated derivatives of the PEG polymers are
prepared to attach the PEG to the desired molecule.

16. The first generation
PEGylationProcess
PEGpolymerichydroxylgroupsarereactedwith,anhydrides,acid
chlorides,chloroformatesandcarbonatestoformPEGDerivative.
ThemostcommonreactivesitesonpolypeptidesforattachingPEG
polymersaretheαorεaminogroupsoflysineortheN-terminal
amino-acidgroupsofotherAminoacids.
MainlyusedlinearPEGpolymerswithmolecularmassesof12kDaor
less.
UnstablebondsbetweenthedrugandPEGwerealsosometimesused,
whichleadstodegradationofthePEG–drugconjugateduring
manufacturingandinjection.

17. Limitationsof first
generation:
Isomerizationofpolymer.
EarlyPEGylationwasperformedwithMethoxy–PEG
(mPEG),whichwascontaminatedwithPEGDIOLand
whichresultedinthecrosslinkingofproteinstoform
inactiveaggregates.
DiolcontaminationCanreachupto10-15%

18. The second generation
PEGylationProcess
Second-generationPEGylationstrivestoavoidthepitfallsassociated
withmixturesofisomers,diolcontamination,unstablebondsand
low-molecularmassm–PEG.
PEGylatingsite-specificallycanminimizethelossofbiological
activityandreduceImmunogenicity.
Forinstance,becausetherearefarfewercysteineresiduesthanlysine
groupsonpolypeptides,theTHIOLgroupsofcysteineareidealfor
specificmodifications.
PEGderivativesincludetheincorporationofdegradablelinkagesto
releasedrugsattargetedsitesaswellasthesynthesisanduseof
HETEROBIFUNCTIONAL PEGs.

19. Contd...
Onemethod(ofthemanyunderinvestigation)forreleasingdrugsfrom
PEGemploysaPara-orortho-disulfideofbenzylurethane.
Whensubjectedtomildreducingconditions,suchasinsidethe
endosomesofcells,thedrugbreaksfree.
HeterobifunctionalPEGscontaindissimilarterminalgroups,whichare
advantageousforapplicationsinimmunoassays,biosensorsandprobesto
linkmacromoleculestosurfaces,aswellasforthetargetingofdrugs,
liposomesorvirusestospecifictissues.
Anotherimprovementin2nd-genPEG-polymersistheuseofbranched
structures,incontrasttothesolelylinearstructuresfoundin!st-generation
PEGs20.BranchedPEGsofincreasedmolecularmassupto60kda.

20. Strategies
Three different strategies of PEGylationTechnology:
1)Chemical PEGylationTechnology
2)Enzymatic PEGylationTechnology
3)Genetic PEGylationTechnology

21. Chemical PEGylation
Technology
Highlights:
Use of established chemistry procedures.
Reactions occur in high yields.
Broad applicability.
Disadvantages:
•Reactions are not highly specific.
•Side reactions can occur and PEGylationcan be incomplete.

22. Enzymatic PEGylation
Technology
Highlights:
Highly specific.
Few side-reactions
Disadvantages:
•Restricted to a limited number of applications.
•Process requires a recognition site.
•Enzyme has to be separated at the end of the process.

23. Genetic PEGylation
Technology
Polyethyleneglycol(PEG)wasgeneticallyincorporatedintoa
polypeptide.
Stop-anticodon-containingtRNAswereacylatedwithPEG-containing
aminoacidsandwerethentranslatedintopolypeptidescorresponding
toDNAsequencescontainingthestopcodons.
ThePEGincorporationratiodecreasedasthemolecularweightof
PEGincreased,andPEGwithamolecularweightof1000Dawasonly
slightlyincorporated.
Althoughimprovementisrequiredtoincreasetheefficiencyofthe
process,thisstudydemonstratesthepossibilityofgeneticPEGylation.

24. Quality control
considerations
PEGqualityisimportanttoachievereproduciblePEGylation.
TraditionalPEGsystemsarepolydispersed.
ThestartingmaterialforactivatedPEGsismPEG-OH.ThemPEG-
OHcontainssmallamountsofPEGdiol.WhenthemPEG-OHis
activatedforconjugation,severalPEGscanbeformed:
I.ThedesiredactivatedmPEG-X
II.DiactivatedPEGthatcomesfromPEGdiol
III.AnymPEG-OHthathasnotbeenactivated
ItisimportanttounderstandtheconcentrationofthesevariousPEGs
astheyhaveadirectimpactonthequalityofyourconjugate.

25. Contd…
TheindustrytypicallyutilizesNMRtodeterminefunctionality,butthis
techniquedoesnotallowmeasurementofthevariousPEGs.
AdvancedanalyticaltechniquessuchasLC-MSallowustoseparateand
quantifythevariousPEGs.
ThisisillustratedbythedifferentelusiontimesintheLCofeachof
thesePEGsasshownintheaccompanyingchart.

26. Applications of PEGylation
techniques in NDDS
ThereareseveralapplicationsofPEGylationtechniquein
Noveldrugdeliverysystem:
InProteinDrugDelivery.
InBrainDrugDelivery.
InColloidalDrugDelivery.
InGeneDrugDelivery.
Amongthemproteindrugdeliveryandbraindrugdelivery
shouldbebroadlydiscussed:

27. In Protein Drug
Delivery:
PEGASYS:PEGylatedalpha-interferonsforuseinthetreatmentof
chronichepatitisCandhepatitis-B(Hoffman-LaRochen).
ADAGEN:receivedapprovalforthetreatmentofseverecombined
immunodeficiency(SCID),adiseaseassociatedwithaninherited
deficiencyofadenosinedeaminase36.Beforetheavailability.
PEG-Intron:PEGylatedalpha-interferonsforuseinthetreatmentof
chronichepatitisCandhepatitB(Schering-Plough/Enzon)
Oncaspar:PEGylatedL-asparaginaseforthetreatmentofacute
lymphoblasticleukemiainpatientswhoarehypersensitivetothenative
unmodifiedformofL-asparaginase(Enzon).
Thisdrugwasrecentlyapprovedforfrontlineuse.
Neulasta:PEGylatedrecombinantmethionylhumangranulocyte
colonystimulatingfactorforseverecancerchemotherapyinduced
neutropenia(Amgen)

28. Contd…
Pegfilgrastim(Neulasta),whichwasapprovedin2002,isaPEGylated
formoftheearlierdrugfilgrastim(Neupogen).Bothcontain
recombinantmethionylhumanG-CSF,whichisknownasfilgrastim.
Thedrugsstimulatetheproductionoftheinfectionfightingwhiteblood
cells(neutrophils)thataredepletedbycancerchemotherapy.
Whereasfilgrastimrequiresdailyinjectionsforabout14days,
pegfilgrastimrequiresoneinjectionperchemotherapycycle.
Apegylatedformofhumangrowthhormoneantagonistcalled
pegvisomant(Somavert)isbeingdevelopedforthetreatmentof
ACROMEGALY .PegvisomanthasbeenapprovedinEurope,andis
awaitingFDAapprovalintheUS.

29. PEGylatedNanoparticles
for brain delivery
Theblood–brainbarrier(BBB)isformedbyspecialendothelial
cellssealedwithtightjunctions.
Blocksmanycompoundsthatmightbeoftherapeuticvalue
disorders.
DisruptingtheBBBcarrieshighrisksforpatients.
Polymernanoparticles,suchasn-hexadecylcyanoacrylate
(PHDCA),showpromiseasawaytotransportdrugsacrossthe
BBB.
AnimalstudiesshowthatPEG–PHDCApenetratesintothe
braintoasignificantlygreaterextentthanPHDCAalone.
PEG-PHDCAdistributesintodeepareasofbrain,includingthe
striatum,hippocampus,andhypothalamus.
MovementoccurswithoutdamagetotheBBB.

30. PEGylated
liposomes
LIPOSOMEisaPhospholipidcapsulethatprotectencloseddrug
fromdegradation.
LiposomesarePEGylatedtoprolongtheirbloodcirculationtime.
Comparedwithclassicalliposomes,PEGylatedcounterpartsshow
increasedhalf-life,decreasedplasmaclearance,andashiftin
distributioninfavourofdiseasedtissues.
PEGisincorporatedintothelipidbilayeroftheliposome,forming
ahydratedshellthatprotectsitfromdestructionbyproteins.
Fortheantitumourdrugdoxorubicin,PEGlyationoftheliposome
bringsaneightfoldincreaseinplasmahalf-lifeoftheliposome
comparedtoanunmodifiedliposomes.
PEGylatedliposomesarealsolessextensivelytakenupbythe
Reticulo-endothelialsystemandarelesslikelytoleakdrugwhilein
circulation.

31. Limitation of
PEGylation
•PEG has limited conjugation capacity.
•possibility of side products due to chemical reactions.
•Loss of activity.
•Require specific enzyme for processing.
•Each drug require separate PEGylation.
•Cost benefit ratio.

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References

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