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
• PEGylation process
• Generations
• Strategies
• Quality control considerations
• Application in NDDS
• PEGylated liposomes
• Limitations

3. Introduction
• The term ‘PEGylation’ can be defined as the covalent attachment of
polyethylene glycol (PEG) chain to bioactive substances.
• Protein and peptide drugs hold great promise as therapeutic agents.
However, many are degraded by proteolytic enzymes, can be rapidly
cleared by the kidneys, generate neutralizing antibodies and have a
short circulating half-life. Pegylation, the process by which polyethylene
glycol chains are attached to protein and peptide drugs, can overcome
these and other shortcomings By increasing the molecular mass of
proteins and peptides and shielding them from proteolytic enzymes,
PEGylation improves pharmacokinetics.

4. Contd..
• The FDA has approved PEG to use as a vehicle or base in foods,
cosmetics and pharmaceuticals, including injectable, topical, rectal and
nasal formulations.
• PEG shows very little toxicity and lacks immunogenicity.
• This technique was first described by Abuchowsky and co-workers in
1970 with the PEGylation of albumin and catalase.
• 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.
• PEGylated liposomes are also less extensively taken up by the
reticuloendothelial system and are less likely to leak drug while in
circulation.

5. Comparison with
other Technologies
• In PEGylated products,
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
 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 U.S.
 More than 350 Proteins and Peptides are undergoing clinical trails right
now.
 About a third of Drug candidates in clinical trails are Poly peptides.

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 proteolytic degradation.
 Opportunities for new delivery formats and dosing regimens.
 To Extend patent life of previously approved drugs.

8. How do the
PEGs Work?
PEGs actually work via three ways; by reducing kidney filtration,
increasing solubility due to the PEG hydrophilicity and decreasing
accessibility for proteolytic enzymes and antibodies. Among these three the
mechanism of reducing kidney filtration should be discussed.

9. Reducing Kidney
Filtration
PEG protein conjugate
Apparent Hydrodynamic Radius: 20 nm
• PEGylation significantly increases the
apparent size of the conjugated drug
compound.
• Renal glomerular capillary
diameter: 6-12 nm

10. Chemistry of
PEGylation
 Structure of PEG:
Ethylene
Glycol(EG) group
Numbers of
EG groups.
(Upto 1000)
𝐶2𝑛+2 𝐻4𝑛+6 𝑂 𝑛+2• Molecular Formula:
• Synthesized from the polymerization of ethylene oxide.
• Using chemical tools to link PEG molecules to native
proteins can yield conjugates with more favorable behavior.

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..
Methods for the activation of
PEG molecules:
A. Cyanuric chloride method.
B. A variation on the cyanuric
chloride.
Ca. Polyethylene glycol (PEG)-
succinimidyl succinate method.
Cb. Substitution of the
succinate residue by glutarate.
Cc. Substitution of the
aliphatic ester in Ca by an
amide bondD. Imidazole formate method
E. & F. Variations using phenylcarbonates of PEG
G. Succinimidyl carbonates of PEG
H. Succinimidyl active 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
PEGylation Process
 PEG polymerichydroxyl groups are reacted with, anhydrides, acid
chlorides, chloroformates and carbonates to form PEG Derivative.
 The most common reactive sites on polypeptides for attaching PEG
polymers are the α or ε amino groups of lysine or the N-terminal
amino-acid groups of other Amino acids.
 Mainly used linear PEG polymers with molecular masses of 12kDa 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.

17. Limitations of first
generation:
 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%

18. 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
HETEROBIFUNCTIONAL PEGs.

19. Contd...
 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 2nd-gen PEG- polymers is the use of branched
structures, in contrast to the solely linear structures found in !st-generation
PEGs20. Branched PEGs of increased molecular mass up to 60kda.

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

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 PEGylation can 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
 Polyethylene glycol (PEG) was genetically incorporated into a
polypeptide.
 Stop-anticodon-containing tRNAs were acylated with PEG-containing
amino acids and were then translated into polypeptides corresponding
to DNA sequences containing the stop codons.
 The PEG incorporation ratio decreased as the molecular weight of
PEG increased, and PEG with a molecular weight of 1000 Da was only
slightly incorporated.
 Although improvement is required to increase the efficiency of the
process, this study demonstrates the possibility of genetic PEGylation.

24. 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 formed:
I. The desired activated mPEG-X
II. Diactivated PEG that comes from PEG diol
III. 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.

25. Contd…
 The industry typically utilizes NMR to determine functionality, but this
technique does not allow measurement of the various PEGs.
 Advanced analytical techniques such as LC-MS allow us to separate and
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.

26. Applications of PEGylation
techniques in NDDS
 There are several applications of PEGylation technique in
Novel drug delivery system:
 In Protein Drug Delivery.
 In Brain Drug Delivery.
 In Colloidal Drug Delivery.
 In Gene Drug Delivery.
Among them protein drug delivery and brain drug delivery
should be broadly discussed:

27. In Protein Drug
Delivery:
 PEGASYS: PEGylated alpha-interferons for use in the treatment of
chronic hepatitis C and hepatitis-B(Hoffman-La Rochen).
 ADAGEN: received approval for the treatment of severe combined
immunodeficiency(SCID), a disease associated with an inherited
deficiency 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 chemotherapy induced
neutropenia(Amgen)

28. Contd…
 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
ACROMEGALY. Pegvisomant has been approved in Europe, and is
awaiting FDA approval in the US.

29. 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
disorders.
 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 brain, including the
striatum, hippocampus, and hypothalamus.
 Movement occurs without damage to the BBB.

30. 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 liposomes.
 PEGylated liposomes are also less extensively taken up by the
Reticulo-endothelial system and are less likely to leak drug while in
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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