PEGylation Technique and scope of it`s Applications

PEGylation Technique and scope of it’s
Applications in Drug Delivery Systems
M.Pharm II sem
Department of Pharmaceutics,
University College Of Pharmaceutical Sciences,
Kakatiya University, Warangal.
Basic Concepts…..
What is PEG?
How it is formed?
What are different types?
Why it is chosen?
Chemistry of PEGylation
PEGylation process
 PEGylation Technology
Applications of PEGylation technique in NDDS
Novel Applications
What is “PEGylation” ?
“PEGylation” is the covalent coupling of
Non-Toxic, Hydrophilic
Poly ethylene glycol (PEG) to active
Pharmaceutical ingredients Such as
Proteins , Peptides , Antibodies, colloids etc.
Who are the Pioneers ?
The Technology was developed from the pioneering
work carried out in the 1950’s and 1960’s on
the coupling of polymers to proteins, and by the 1970’s,
“Frank .F.Davis”, “ Dr. Abraham Abuchowski” and colleagues were
using PEG for protein modification.
The first PEG-Protein company was “Enzon” founded in 1981.
The first approved PEG-Drug Product was
PEG-Adenosine deaminase,Approved in 1990 by
Why is PEGylation a “Hot Topic”…
Non-toxic, non-immunogenic, highly soluble in
water and FDA approved
Since 1990 many PEGylated drugs have been
synthesized and approved including drugs for
cancer, Hepatitis, HIV, and MS
Low cost of manufacturing
Part of a multi-billion dollar molecular
medicines market
The need for PEGylation…
The Novel Proteins and Peptides have become important
new drugs with advent of a revolution in Biotechnology.
More than 80 Poly Peptide Drugs are marketed in The
More than 350 Proteins and Peptides are
undergoing clinical trails right now.
About a third of Drug candidates in clinical trails are
Poly peptides.
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 proteolytic degradation
Opportunities for new delivery formats and dosing regimens
 To Extend patent life of previously approved drugs
How do the PEGs Work…
PEGylation increases the half-life of the biomolecule in the body via
Reducing Kidney Filtration
•PEGylation significantly increases the apparent size of the conjugated
drug compound
Chemistry of PEGylation
Structure of PEG…
Molecular formula: C2n+2H4n+6On+2
Synthesized from the polymerization of ethylene oxide
Using chemical tools to link PEG molecules to native proteins can yield
conjugates with more favorable behavior
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)
Method for the activation of PEG molecules.
Conjugation Chemistry…
Conjugation Chemistry…
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
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.
The first generation PEGylation Process
PEG polymerichydroxyl groups are reacted with, anhydrides,
acid chlorides, chloroformates and carbonates to form PEG
The most common reactive sites on polypeptides for attaching
PEG polymers are the α or ε amino groups of lysine or the Nterminal amino-acid groups of other Amino acids.
Mainly used linear PEG polymers with molecular masses
of 12 k Da or less
Unstable bonds between the drug and PEG were also
sometimes used, which leads to degradation of the PEG–
drug conjugate during manufacturing and injection.
Isomerization of polymer
 Early PEGylation was performed with Methoxy –
PEG (m–PEG), which was contaminated with PEG
DIOL and which resulted in the cross linking of proteins
to form inactive aggregates.
Diol contamination Can reach up to 10-15%
The second generation PEGylation Process
Second-generation PEGylation strives to avoid the pitfalls
associated with mixtures of isomers, diol contamination,
unstable bonds and low-molecular mass m–PEG.
PEGylating site-specifically can minimize the loss of
biological activity and reduce Immunogenicity.
For instance, because there are far fewer cysteine residues
than lysine groups on polypeptides, the THIOL groups of
cysteine are ideal for specific modifications.
PEG derivatives include the incorporation of degradable linkages to
release drugs at targeted sites as well as the synthesis and use of
One method (of the many under investigation) for releasing drugs from
PEG employs a Para- or ortho -disulfide of benzyl urethane.
When subjected to mild reducing conditions, such as inside the
endosomes of cells, the drug breaks free .
Heterobifunctional PEGs contain dissimilar terminal groups, which are
advantageous for applications in immunoassays, biosensors and probes to
link macromolecules to surfaces, as well as for the targeting of drugs,
liposomes or viruses to specific tissues.
Another improvement in second-generation PEG- polymers is the use of
branched structures, in contrast to the solely linear structures found in
first-generationPEGs20. Branched PEGs of greatly increased molecular
mass up to 60kda.
Quality control considerations
 PEG quality is important to achieve reproducible PEGylation.
 Traditional PEG systems are polydispersed.
 The starting material for activated PEGs is mPEG-OH. The
mPEG-OH contains small amounts of PEG diol. When the
mPEG-OH is activated for conjugation, several PEGs can be
1. The desired activated mPEG-X
2. Di-activated PEG that comes from PEG diol
3. Any mPEG-OH that has not been activated
 It is important to understand the concentration of these
various PEGs as they have a direct impact on the quality of
your conjugate.
The industry typically utilizes NMR to determine functionality,
but this technique does not allow measurement of the various
 Advanced analytical techniques such as LC-MS allow us to
separateand quantify the various PEGs.
This is illustrated by the different elusion times in the LC of
each of these PEGs as shown in the accompanying chart.
Traditional PEGylation Vs CelaSYS
Traditional PEGylation
• chain-like structure
• polydisperse
• cross-links possible
• structurally determined
• fluctuations in quality
at any time
• limited optimization
• branched structure
• monodisperse
• cross-links impossible
• consistently high,
• reproducible quality
• various drug-specific
Optimization possibilities
The PEGylation process was further developed to
determine the optimal PEG/Protein ratio.
Optimization of the PEG gave good PEGylation
efficiency with no residual un modified protein.
High reproducibility of PEGylation achieved by
performing the PEGylation for 2 hrs at a pH of 9.5
and subsequently performing one step
chromatographic purification.
PEGylation Technology
Three different strategies of PEGylation Technology
Chemical PEGylation Technology
 Enzymatic PEGylation Technology
Genetic PEGylation Technology
Chemical PEGylation Technology
Use of established chemistry procedures.
Reactions occur in high yields.
Broad applicability.
Reactions are not highly specific.
Side reactions can occur and PEGylation can be
Enzymatic/Genetic PEGylation Technology.
Highly specific
Few side-reactions
Restricted to a limited number of
Process requires a recognition site
Enzyme has to be separated at the end
of the process.
Applications of
PEGylation techniques in NDDS
In Protein Drug Delivery
In Brain Drug Delivery
In Colloidal Drug Delivery
In Gene Drug Delivery
In Protein Drug Delivery:
 PEGASYS: PEGylated alpha-interferons for use in the treatment of
chronic hepatitis C and hepatitis-B(Hoffman-La Rochen)
ADAGEN: receivedapproval for the treatment of severe combined
immunodeficiency(SCID), a disease associated with an
inheriteddeficiency of adenosine deaminase36. Before the availability
 PEG-Intron: PEGylated alpha-interferons for use in the treatment of
chronic hepatitis C and hepatit B(Schering-Plough / Enzon)
Oncaspar: PEGylated L-asparaginase for the treatment of acute
lymphoblastic leukemia in patients who are hypersensitive to the native
unmodified form of L-asparaginase (Enzon).
 This drug was recently approved for front line use.
 Neulasta: PEGylated recombinant methionyl human granulocyte
colony stimulating factor for severe cancer chemotherapyinduced
Pegfilgrastim (Neulasta), which was approved in 2002, is a pegylated
form of the earlier drug filgrastim (Neupogen). Both contain
recombinant methionyl human G-CSF,which is known as filgrastim.
The drugs stimulate the production of the infection fighting white
blood cells (neutrophils) that are depleted by cancer chemotherapy.
Whereas filgrastim requires daily injections for about 14 days,
pegfilgrastim requires one injection per chemotherapy cycle.
A pegylated form of human growth hormone antagonist called
pegvisomant (Somavert) is being developed for the treatment of
Pegvisomant has been approved in Europe, and is awaiting FDA
approval in the US.
Pharmacokinetic profiles for interferon (IFN)-α2a and
40 kDa polyethylene glycol (PEG)–IFN-α2a.
PEGylated Nanoparticles for brain delivery:
The blood–brain barrier (BBB) is formed by special endothelial
cells sealed with tight junctions.
Blocks many compounds that might be of therapeutic value
Disrupting the BBB carries high risks for patients.
Polymer nanoparticles, such as n-hexadecylcyanoacrylate
(PHDCA), show promise as a way to transport drugs across the
BBB.Animal studies show that PEG–PHDCA penetrates into the
brain to a significantly greater extent than PHDCA alone.
PEG–PHDCA distributes into deep areas of the brain,
including the striatum ,hippocampus, and hypothalamus.
movement occurs without damage to the BBB
PEGylated liposomes
LIPOSOME is a Phospholipid capsule that protect enclosed drug
from degradation.
Liposomes are pegylated to prolong their blood circulation time.
Compared with classical liposomes, pegylated counterparts show
increased half-life, decreased plasma clearance, and a shift in
distribution in favour of diseased tissues.
PEG is incorporated into the lipid bilayer of the liposome, forming a
hydrated shell that protects it from destruction by proteins.
For the antitumour drug doxorubicin, peglyation of the
liposome brings an eightfold increase in plasma half-life of the
liposome compared to an unmodified liposome.
Pegylated liposomes are also less extensively taken up by the
Reticulo endothelial system and are less likely to leak drug while in
PEGylated targeted nanoparticles for drug/gene delivery and
imaging in pancreatic cancer
Quantum dots for optical
imaging and drug delivery
Biodegradable polymeric
nanoparticles for drug delivery
Biodegradable calcium phosphate
nanoparticles for gene delivery
PEG-based hydrogels
•PEG can be chemically cross linked to form polymer
networks that swell and form gels.
•The biocompatibility = ideal for wound-healing applications.
•In 2000, the FDA approved surgical sealant Focal Seal to
prevent air leaks in the lungs following the removal of lung
tumors and other chest surgeries.
• FocaSeal uses a PEG that is applied as a liquid, and then
transformed into a water proof hydrogel seal by irradiation.
•The sealant protects wound sites from leaking during tissue
healing, and then naturally degrades and dissolves.
Spray Gel:
 Prevents
post-operative adhesion formation.
Internal wounds often develop adhesions—a type of scar tissue—
that cause severe pain
Spray Gel is sprayed onto the wound site and acts as a protective
barrier during healing.
This material also degrades and dissolves at a programmed rate.
Other PEG-based hydro gels under development deliver
encapsulated drugs as implants.
Degradable linkages between hydro gels and incorporated drugs
allow drugs to be slowly and specifically released in the body.
In Gene Drug Delivery:
 Gene delivery vectors do not possess the basic pharmacokinetic
properties required for systemic applications.
 Polyplexes, lipoplexes and lipopolyplexes all have potential for gene
delivery to organs such as lung, liver and spleen.
 PEGylation, to enhance the circulation lifetimes of these particles.
SPLP, which possesses long circulation lifetimes and which preferentially
delivers plasmid to distal tumor sites following intravenous injection, with
associated gene expression.
Enhanced levels of gene expression may be achieved by
modifying the lipid composition.
The use of PEG-Cer molecules with optimized dissociation rates may
result in enhanced in vivo activity
Novel Applications
These are just a few of the biomedical applications of pegylation
undergoing investigation.
Other molecules including small-molecule drugs, cofactors,
oligonucleotides, lipids, saccharides and biomaterials, can also be
pegylated as well.
Other candidates include
PEGylated insulin with a lengthened circulation time and
reduced immunogenicity.
PEGylated antibody fragments for immunotherapy or tumor
PEGylatedN superoxide dismutase for the treatment of
ischaemia/reperfusion injury or burns.
The benefits of pegylated catalase, uricase, honeybee
venom, haemoglobin, pyrrolidone and dextran are also
under investigation.
PEGylated Nan particles to cross the blood–brain
barrier or using pegylated DNA-containing liposomes
with tethered antibodies to provide targeted gene
J.Milton Harris* & Robert B. Chess‡
Long-circulating vectors for the systemic delivery of genes
David B Fenske*1, Ian MacLachlan2 & Pieter R Cullis

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