20110802_Shailesh T. Prajapati et al, IJAPR.pdf - international ...

Shailesh T. Prajapati., et al. / International Journal of Advances in Pharmaceutical Research 

IJAPR 

Available Online through 

www.ijapronline.org 

Review Article 

ISSN: 2230 – 7583 

PEGYLATION: AN APROACH FOR PROTEIN AND PEPTIDE DRUG DELIVERY SYSTEMS 

Shailesh T. Prajapati*, Amit N. Patel, Chhagan N. Patel 

Shri Sarvajanik Pharmacy College, Mehsana - 384 001, India. 

Received on 20 – 04 - 2011 Revised on 21 – 05- 2011 Accepted on 01 – 06 – 2011 

ABSTRACT 

A number of novel drug-delivery mechanisms have been developed to increase the utility of drugs having poor 

solubility, distribution and permeation, one of them is PEGylation. PEGylation defines the modification of a protein, 

peptide or non-peptide molecule by the linking of one or more polyethylene glycol (PEG) chains. PEGylated 

products generally have longer plasma half-lives and durations of bioactivity than their non PEGylated counterparts. 

Pegylation now play the important role in drug delivery and enhancing the potential of peptide and protein drugs. 

INTRODUCTION 

A number of novel drug-delivery mechanisms have 

been developed to increase the utility of drugs that 

are otherwise limited by suboptimal pharmacokinetic 

properties, such as poor absorption, distribution, and 

elimination. These include continuous-release 

injectable and liposomal systems, which alter the 

formulation of the drug, and PEGylation, which alters 

the drug molecule. [1] 

PEGylation defines the modification of a protein, 

peptide or non-peptide molecule by the linking of one 

or more polyethylene glycol (PEG) chains. This 

polymer is nontoxic, non-immunogenic, nonantigenic, 

highly soluble in water and FDA approved. 

The PEG-drug conjugates have several advantages: a 

prolonged residence in body, a decreased degradation 

by metabolic enzymes and a reduction or elimination 

of\protein immunogenicity. Thanks to these favorable 

properties, PEGylation now plays an important role 

in drug delivery, enhancing the potentials of peptides 

and proteins as therapeutic agents. [2] 

Corresponding Author: 

Dr. Shailesh T. Prajapati, 

Shri Sarvajanik Pharmacy College, 

Mehsana- 384 001, India; 

Mob:+91 9924456583, 

Email: stprajapati@gmail.com 

PEGylation was first described in the 1970s by 

Davies and Abuchowsky and reported in two key 

papers on albumin and catalase modification. This 

was an important milestone, because at that time it 

was not conceivable to modify an enzyme so 

extensively and still maintain its activity. Proteins 

were in fact considered very delicate entities and only 

few gentle modifications with low molecular-weight 

products were carried out, mainly to study SARs. [2] 

PEGylation is a new delivery technology that differs 

from traditional formulation in a number of ways. 

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. In 

PEGylated products, on the other hand, the 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. 

Consequently, PEGylation has to be considered early 

in the drug development process. [2] 

The advantages conferred by PEGylation include an 

increased molecule weight and hydrodynamic 

volume and a masking of the surface of the molecule 

with highly mobile PEG chains. PEGylation also 

reduces the rapid renal clearance of small proteins 

and makes it possible for liposomes to evade removal 

from the plasma by lipolytic enzymes and the 

reticuloendothelial system. As a result, pegylated 

agents generally have longer plasma half-lives and 

IJAPR / June 2011/ Vol. 2 / Issue. 6 / 252 - 262 252


durations of bioactivity than their nonpegylated 

counterparts and benefits of pegylated product given 

in table 1. [1] 

PROPERTIES OF PEG 

Polyethylene glycols are pH-neutral, nontoxic watersoluble 

polymers that consist of repeating ethylene 

oxide subunits, each with a molecular weight of 44, 

and two terminal hydroxyl groups. They are either 

linear (5 to 30 kd) or branched (40 to 60 kd) chain 

structures.PEG has Poly-dispersity i.e. Molecular 

weight distribution is narrow (1.01 – 1.1). The 

pharmacokinetic properties of PEGs vary according 

to their molecular weight and site of injection. The 

area under the time-concentration curve and the halflife 

of PEGs increase with their molecular weight. 

For example, after intravenous administration in mice 

the half-life of 50kd PEG is substantially longer than 

that of 6 kd PEG (987vs 17.6 minutes); 50kd PEG is 

also retained longer at the injection site after 

subcutaneous or intramuscular injection than is 6 kd 

PEG. Polyethylene glycols appear to undergo 

oxidation by the cytochrome P450 enzyme system, 

and lowmolecular- weight PEGs are excreted into the 

bile. [1] 

PEGylation – MECHANISM OF ACTION 

After administration, when PEG comes in contact of 

aqueous environment, ethylene glycol sub-unit gets 

tightly attached to the water molecule. This binding 

to water renders them high mobility and hydration. 

Hydration and rapid motion causes PEGylated 

protein to function, as it causes PEG to sweep out a 

large volume which acts like a shield to protect the 

attached drug from enzymatic degradation and 

interaction with cell surface proteins. This increased 

size also helps to prevent rapid renal filtration and 

clearance sustaining the drug bioavailability. The 

high steric hindrance of branched-chain PEGs 

generally affords greater protection than do linear 

chain. [1] 

FACTORS AFFECTING PERFORMANCE OF 

PEGylated PRODUCT 

Molecular Weight 

Molecular weight less than 1000 Da of PEG broken 

down into sub-units, and have some toxicity, while 

Molecular weight greater than 1000 Da of PEG: does 

not demonstrate any toxicity in vivo. 

Molecular Weight upto 40,000 – 50,000 Da: used in 

clinical and approved pharmaceutical application. 

The molecular weight of PEG has a direct impact on 

the activity; Higher molecular weight PEGs tends to 

have higher in-vivo activity due to the improved 

pharmacokinetic profile like increasing half life as 

earlier discussed. [1] 

Structure 

Branched structure has more size than same 

molecular weight linear structure so; it’s also helps to 

prevent rapid renal filtration and clearance sustaining 

the drug bioavailability. The high steric hindrance of 

branched-chain PEGs generally affords greater 

protection than do linear chain. [1,4] 

Number of PEG chains 

Two or more lower molecular weight chains can be 

added to increase total molecular weight of PEG 

complex 

Specific location of PEG site of attachment to the 

molecule. 

Optimal PEGylation is product-specific, and can vary 

depending on the site of attachment, the chemistry 

used to create the conjugate, and the characteristics of 

the PEG used. Effective PEGylation of a drug may 

be achieved by attaching a single large PEG at a 

single site, a branched PEG at a single site, or several 

small PEG chains at several sites. [1] 

CHEMISTRY OF PEGYLATION 

To couple PEG to a molecule (i.e. polypeptides, 

polysaccharides, polynucleotide’s and small organic 

molecules) as shown in Figure 1, it is necessary to 

activate the PEG by preparing a derivative of the 

PEG having a functional group at one or both 

termini. The functional group is chosen based on the 

type of available reactive group on the molecule that 

will be coupled to the PEG. For proteins, typical 

reactive amino acids include lysine, cysteine, 

histidine, arginine, aspartic acid, glutamic acid, 

serine, threonine, tyrosine, N-terminal amino group 

and the C-terminal carboxylic acid. In the case of 

glycoproteins, vicinal hydroxyl groups can be 

oxidized with periodate to form two reactive formyl 

moieties. [3] 

The most common route for PEG conjugation of 

proteins has been to activate the PEG with functional 

groups suitable for reaction with lysine and N- 

terminal amino acid groups. Lysine is one of the most 

prevalent amino acids in proteins and can be upwards 

of 10% of the overall amino acid sequence. In 

reactions between electrophilically activated PEG 

and nucleophilic amino acids, it is typical that several 

amines are substituted. When multiple lysines have 

been modified, a heterogeneous mixture is produced, 

which is composed of a population of several 

polyethylene glycol molecules attached per protein 

molecule (‘PEGmers’) ranging from zero to the 

number of ´-and a-amine groups in the protein. [3] 

PEGylation technology is classified into two types: 

1. Early PEGylation technology (First 

generation PEGylation) 



2. Advanced PEGylation technology (Second 

generation PEGylation) 

1. First Generation PEGylation 

First generation PEGylation methods were fraught 

with difficulties. 

With first generation PEGylation, the PEG polymer 

was generally attached to ε amino group of lysine, 

and gave mixtures of PEG isomers with different 

molecular masses. The existence of these isomers 

makes it difficult to reproduce drug batches, and can 

contribute to the antigenecity of the drug and poor 

clinical outcomes. In addition, first generation 

methods mainly used linear PEG polymers of 12 kDa 

or less. Unstable bonds between the drug and PEG 

were also sometimes used, which leads to 

degradation of PEG-drug conjugate during 

manufacturing and injection 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 upto 10-15 

%. [1] 

Despite these limitations, several first generation 

PEGylated drugs receive regulatory approval. 

Example: Still in use today are Pegademase 

(ADAGEN ® ), a PEGylated form of the enzyme 

adenosine de-aminase for the treatment of Severe 

Combined Immuno-Deficiency (SCID) and 

Pegaspargase (ONCASPAR ® ), a PEGylated form of 

enzyme asparginase for the treatment of Leukemia. [5] 

2. Second Generation PEGylation 

Second generation PEGylation strives to avoid 

pitfalls associated with the first generation 

PEGylation. 

Overall goal of this technology is to create larger 

PEG polymers to improve the Pharmacokinetics and 

Pharmacodynamic effects seen with lower molecular 

mass PEGs. 

Newer pegylation methods create conjugates with 

strong linkages that are resistant to side-reactions and 

are able to withstand purification to remove dio1 

contaminants, thereby making it possible to use high 

molecular- weight PEGs . These methods attach an 

activated PEG to the drug molecule by incorporating 

part of the activating group as a link between the two 

entities. For example, PEGFILGRASTIM ® is formed 

by covalently attaching a 20 kd PEG chain through a 

stable secondary amine bond directly to the terminal 

amino group of the filgrastim molecule. In this way, 

the nitrogen atom to which the PEG chain is attached 

retains its surface charge, a factor that has been 

shown to be crucial in conserving the bioactivity of 

some molecules. [1] 

Amine PEGylation and N-terminal PEGylation 

Since most applications of PEG conjugation involve 

labile molecules, the coupling reaction generally 

requires mild chemical conditions. In case of 

polypeptides, the most common reactive groups 

involved in coupling are nucleophiles with the 

following decreasing rank order of reactivity: thiol, 

alpha amino group, epsilon amino group, 

carboxylate, hydroxylate. However, this order is not 

absolute, since it depends also on the reaction pH, 

furthermore other residues may react in special 

conditions, as the imidazole group of histidine. The 

thiol group is rarely present in proteins, furthermore 

it is often involved in active sites. The carboxylic 

groups cannot be easily activated without having. 

reaction with the protein amino groups, to yield intra 

or inter molecular cross linking. Therefore, amino 

groups, namely the alpha amino or the epsilon amino 

of lysine, are the usual sites of PEG linking. [4] 

PEGylating Agents used for amino PEGylation 

shown in Table 2. 

Carboxyl PEGylation 

PEG reagents react with carboxylic acid in the 

presence of coupling agents such as 

DCC (N,N'-dicyclohexylcarbodiimide) and EDIC (N- 

(3-dimethylaminopropyl)-N' ethylcarbodiimide, HCl 

salt). However, the procedure is successful only when 

amines are not present in the compound, as for 

instance in the case of non-peptide drugs. In peptides 

and proteins the risk of cross-linking is difficult to 

avoid. [3] PEGylating Agents used for Carboxyl 

PEGylation are shown in Table 3. 

PEGylation at the –SH (thiol) groups of Cysteine of 

polypeptides 

PEGylation of free cysteine residues in proteins is the 

main approach for site-specific modification because 

reagents that specifically react with cysteines have 

been synthesized, and the number of free cysteines on 

the surface of a protein is much less than that of 

lysine residues. In the absence of a free cysteine in a 

native protein, one or more free cysteines can be 

added by genetic engineering. PEGylating site 

specifically can minimize the loss of biological 

activity and reduce immunogenecity. [3] PEGylating 

Agents used for thiol PEGylation are shown in Table 

3. 

Hydroxyl PEGylation 

PEG-isocyanate is useful for hydroxyl group 

conjugation yielding a stable urethane linkage. 

However, its reactivity may be best exploited for 

non-peptide moieties such as drugs or hydroxylcontaining 

matrices to yield biocompatible surfaces. 

PEG-isocyanate is in fact highly reactive with amines 

also. [3] PEGylating Agents used for Hydroxyl 

PEGylation are shown in Table 3. 

Hetero-bifunctional PEGs 



As applications of PEG chemistry have become more 

sophisticated, there has been an increasing need for 

heterobifunctional PEGs, which are PEGs bearing 

dissimilar terminal groups. Such heterobifunctional 

PEGs bearing appropriate functional groups may be 

used to link two entities where a hydrophilic, flexible, 

and biocompatible spacer is needed. 

Heterobifunctional PEG can be used in a variety of 

ways that includes linking macromolecules to 

surfaces (for immunoassays, biosensors or various 

probe applications), targeting of drugs, liposomes and 

viruses to specific tissues, liquid phase peptide 

synthesis and many others. Preferred end groups for 

hetero-bifunctional PEGs are NHS esters, maleimide, 

vinyl sulfone, pyridyl disulfide, amine, and 

carboxylic acids. [3] 

Branched structures 

Second generation PEGylation uses branched 

structures of PEG, in contrast to the solely linear 

structures found in first generation PEGs. Branched 

PEGs of greatly increased molecular masses – upto 

60 kDa or more, as compared with the 12 kDa or less 

found in the first generation PEGs – have been 

prepared. A branched PEG ‘acts’ as if it were much 

larger than a corresponding linear PEG of the same 

molecular mass. Branched PEGs are also better at 

cloaking the attached polypeptide drug from the 

immune system and proteolytic enzymes, thereby 

reducing its antigenecity and likelihood of 

destruction. [1,3] 

Specific PEGylation by enzymes or by reversible 

protection 

The specific conjugation of PEG to the amide group 

of glutamines or to the hydroxyl group of serines and 

threonines is only possible under mild conditions 

using enzymes. Sato discovered that glutamine in 

proteins can be the substrate of the transglutaminase 

enzymes, if an amino PEG is used as the nucleophilic 

donor. Through a transglutamination reaction the 

enzyme links PEG to the protein at the level of the 

glutamine residue as shown in Figure 2. [2] Now a 

days, PEG conjugates with different enzyme like 

arginines n histaminase are also available. 

LIMITATIONS IN TH USE OF PEG 

PEG is obtained by chemical synthesis and, like all 

synthetic polymers, it is polydisperse, which means 

that the polymer’s batch is composed of molecules 

having different number of monomers, yielding a 

Gaussian distribution of the molecular weights. This 

leads to a population of drug conjugates, which 

might have different biological properties, mainly in 

body-residence time and immunogenicity. 

Polydispersivity problem must be still taken into 

consideration, especially when dealing with low 

molecular weight drugs, either peptide or nonpeptide 

drugs, where the mass of linked PEG is more 

relevant for conveying the conjugate’s 

characteristics, mainly those related to the molecular 

size. A second problem for the use of this polymer 

relates to the excretion from the body. As for other 

polymers, PEGs are usually excreted in urine or 

feces but at high molecular weights they can 

accumulate in the liver, leading to macromolecular 

syndrome. [2] 

APPLICATION OF PEGYLATION 

PEG as Diagnostic Carrier 

In vivo non invasive diagnosis is done by using 

tracers detected through magnetic resonance or 

radioactivity. Usually they are administered in a 

chelated form using compounds that can give 

specific biodistribution, stability or targeting. 

PEGylation increases the body-residence time of 

paramagnetic chelates that will be cleared more 

slowly than the unmodified molecules through the 

kidney or liver, thus allowing more detailed images 

by magnetic resonance. C225 is a monoclonal 

antibody directed against the epidermal growth factor 

receptor, which was conjugated to a 

heterobifunctional PEG bearing a radiometal chelator 

(diethylenetriaminepentaacetic acid, DTPA) at one 

terminus. The conjugate DTPA–PEG–C225 retained 

66% of binding affinity, and, more importantly, 

when labeled with Indium-111 (111In) it showed 

narrower steady-state distribution than the non- 

PEGylated 111In–DTPA–C225, because of reduced 

nonspecific binding. Therefore, in case of protein 

targeted diagnostic, PEG could help to collect better 

defined images by limiting the background noise due 

to nonspecific protein–protein interaction. [2] 

PEG oligonucleotides 

Mainly antisense oligonucleotides and are now under 

active investigation as new potential drugs because 

of their extremely high selectivity in target 

recognition. All of them, however, share the 

problems of short half-life in vivo because of either 

low stability towards the eso- and endo-nucleases 

(present in plasma and inside the cells) or their rapid 

excretion caused by their small size. Furthermore, 

their negative charge prevents an easy penetration 

into the cells. A PEG molecule, bound to the 

hydroxyl group of a nucleic acid (directly or through 

a spacer link), was found to increase the stability 

towards enzyme degradation,prolong the plasma 

permanence and enhance the penetration into cells by 

masking the negative charges of oligonucleotides. A 

PEGylated aptamer, the 28mer oligomeraptanib, has 

already been approved by FDA for the treatment of 

age-related macular degeneration of retina. In this 

product, a branched PEG of 40 kDa was attached to 

the oligonucleotides through a pentamino linker. [2] 



PEGylated conjugate as Anticancer agent [7] 

PEG conjugates with low molecular weight 

anticancer drugs 

PEG has been successful for protein modification but 

in the case of low molecular weight drugs it presents 

a crucial limit, the low drug payload accompanying 

the available methoxy or diol forms of this polymer. 

This intrinsic limitation had for many years 

prevented the development of a small drug-PEG 

conjugate. A few studies have been conducted to 

overcome the low PEG loading by either branching 

the end chain groups or coupling on them small 

Dendron structures. 

Pegamotecan ® (Enzon Pharmaceuticals, Inc.) is a 

prodrug obtained by coupling two molecules of 

camptothecin to a diol PEG of 40 kDa. The drug is 

linked through an ester bond involving the C-20 

hydroxyl group and a carboxylic group of PEG. The 

aim of this approach was double, to increase the drug 

half-life in blood by PEGylation and to stabilize by 

acylation the active lactone configuration of 

camptothecin. 

PEG-irinotecan: The architecture of new multi-arm 

PEGs was also exploited for the preparation of PEGirinotecan 

(NKTR-102) by Nektar Therapeutics. The 

drug has been covalently bound to a four arms PEG. 

In preclinical studies NKTR-102 plasma half-life 

was evaluated in a mouse model taking into 

consideration the active metabolite SN-38, released 

from irinotecan. The conjugate showed prolonged 

pharmacokinetic profiles with a half-life of 15 days 

when compared to 4 h with free irinotecan. 

PEG-docetaxel : PEGylated docetaxel (NKTR-105) 

has been prepared with the same multi-arm PEG 

technology. The derivative has shown good 

preclinical activity in colon and lung cancer 

xenograft models.This product has just entered phase 

1 clinical studies enrolling approximately 30 patients 

with refractory solid tumours who have failed all 

prior available therapies. 

PEG-Protein conjugates 

In PEGylation of protein conjugate two different 

approaches can be identified based on the type of 

protein studied: 

Heterologous protein, Usually the main limit of these 

proteins is the immunogenicity rather than a short 

pharmacokinetic. Therefore, both PEG molecular 

weight and coupling chemistry should ensure a wide 

shielding of the protein surface or, at least, the 

immunogenic sites. Basically, in these cases low 

molecular weight PEGs (5– 10 kDa) and random 

amine coupling are used. It is important to note that 

all the enzymes studied possess small substrates; 

these can cross the PEG layer, around the protein, 

and easily reach the active site. Conversely, active 

site approach of large and hindered substrates would 

be prevented this compromising the enzyme activity. 

This would suggest that PEGylation may be not a 

suitable approach for immunogenic enzymes having 

big substrates. 

Endogenous protein, For these biopharmaceutical 

drugs the prolongation of body circulation half-life is 

the driving force in seeking for a polymer conjugate. 

Most of the endogenous proteins act through a 

receptor-mediated activity. This dictates the strategy 

for an optimum PEGylation approach, namely a site 

specific conjugation to generate monoPEGylated 

isomers. In particular the site of polymer attachment 

must be far from the receptor recognition area. In this 

case, it is mandatory the use of high molecular 

weight polymers to reach the PEG mass for the 

desired half-life prolongation. 

PEG-antibody fragment angiogenesis inhibitor 

(CDP791) 

Vascular endothelial growth factor receptor-2 

(VEGFR-2) is involved in the formation of new 

blood vessels in tumours (angiogenesis), allowing 

cancer cells to receive nutrients and to maintain 

growth. Therefore, a molecule able to block VEGFR- 

2 can interfere with the development of tumour 

vasculature. CDP791 is a PEGylated diFab antibody 

that binds the VEGFR-2, with a Kd of 49pM, 

preventing the activation by VEGF ligands. The 

unconjugated CDP791 antibody fragment is affected 

by a too fast in vivo clearance, because it has a 

reduced mass due to the absence of Fc region. This 

problem was overcome by PEGylation of the 

cysteine present at the C-terminus. 

PEG-interferon-alpha conjugates 

Several clinical studies are evaluating the 

effectiveness of PEGinterferon-α2b (PEG- 

INTRON®), presently used for the treatment of 

hepatitis B and C, as adjuvant therapy in certain 

anticancer protocols. The native interferon-α2b is one 

of the most studied agents for adjuvant therapy in 

stage IIb and stage III melanoma. Improvements in 

the recurrence-free survival have been shown when 

interferon- α2b therapy was prolonged for 12–15 

months. This long therapy, consisting in a daily drug 

administration, can particularly compromise the 

patient compliance. This can be highly improved 

using the PEGylated form of interferon-α2b, a 

monoPEGylated derivative obtained by conjugating 

the protein with a linear 12 kDa amino reactive 

PEGylating agent. The conjugate maintains the 

therapeutic level of interferon-α2b by a weekly, self 

administered, dose schedule and its safety has been 

studied in several cancers. 

PEG-Interferon-α2a, a conjugate obtained by linking 

a branched PEG 40 kDa to the protein and marketed 

as PEGASYS®, is used in clinic to treat hepatitis as 

PEG-INTRON®. The higher polymer molecular 



weight of PEGASYS® (40 kDa versus 12 kDa of 

PEG-INTRON®) and the higher stability of the 

PEG-protein linkages (i.e. His residues are involved 

in this case) allowed for the prolonging of the in vivo 

half-life to 65 h with respect to the 27–37 h of PEG- 

INTRON®. 

PEGylation: the in vitro activity is reduced to about 

7% of that of native interferon, this being the 

weakness of stable polymer conjugation, but this 

limitation is more than counterbalanced by the 

enhanced in vivo half-life of the conjugate. 

PEG-granulocyte colony stimulating factor 

Granulocyte colony stimulating factor (G-CSF) is 

used as adjuvant therapy to treat granulocytes 

depletion during chemotherapy. The fast blood 

clearance of the free drug was addressed by 

PEGylation. Different PEG coupling approaches 

were conducted but the most successful one consisted 

of a reductive alkylation with PEG aldehyde 

performed at acidic pH. Under this condition, a 

monoPEGylated conjugate was preferentially 

obtained in which the polymer was linked to the 

protein N-terminal α amino group. The PEG 20 kDa 

conjugate showed an improved pharmacokinetic 

profile as consequence to the reduced kidney 

excretion. 

The PEG-G-CSF conjugate (Pegfilgastrim, 

Neulasta®) was approved for human use in 2002 for 

the first and subsequent cycleadministration against 

febrile neutropenia in patients with nonmyeloid 

malignancies receiving myelosuppressive 

chemotherapy associated with a 30%–40% risk of 

febrile neutropenia. 

PEG conjugates with enzymes 

PEG conjugated Asparaginase 

several leukemic lymphoblasts cells rely on the 

serum supply of asparagine, for their growth, 

because they lack the enzyme asparagine synthetase. 

Asparaginase, the enzyme that converts asparagine 

into aspartate and ammonia, has therefore been 

proposed as a therapeutic agent for acute 

lymphoblastic leukaemia (ALL). FDA approval for 

PEG-asparaginase (Rhone-Poulenc Rorer as 

Oncaspar®) was granted in 1994 for treatment of 

patients with ALL who are hypersensitive to the two 

native isoforms of the enzyme. PEGylated 

asparaginase has been used in combination with 

several traditional anticancer molecules, often in a 

multiagent regimen, including for example one or 

more of the following drugs: cyclophosphamide, 

daunorubicin, vincristine, cytarabine, prednisone, 

etc. In these studies the PEGylated enzyme was well 

tolerated, showing hyperbilirubinaemia and 

hyperglycaemia as the most common adverse effects. 

Arginine deiminase and Arginase 

In literature two types of arginine degrading enzymes 

are reported, and both have been suggested as 

antitumour agents: i) arginine deiminase (ADI), 

which degrades arginine in citrulline and 

ammonia, ii) arginase (ARG) that catalyses the 

conversion of arginine in ornithine and urea. This 

enzyme was shown to be even more powerful than 

asparaginase in killing human leukaemia cells. 

arginine depleting enzymes can be useful in treating 

these tumours. Indeed, arginine deficiency inhibits 

tumour growth, angiogenesis and nitric oxide 

synthesis. 

Chitosan–PEG nanocapsules as new carriers for 

oral peptide delivery 

Chitosan–PEG nanocapsules and the control PEGcoated 

nanoemulsions were obtained by the solvent 

displacement technique. Their size was in the range 

of 160–250 nm. Their zeta potential was greatly 

affected by the nature of the coating, being positive 

for chitosan–PEG nanocapsules and negative in the 

case of PEG-coated nanoemulsions. The presence of 

PEG, whether alone or grafted to chitosan, improved 

the stability of the nanocapsules in the 

gastrointestinal fluids. Using the Caco-2 model cell 

line it was observed that the pegylation of chitosan 

reduced the cytotoxicity of the nanocapsules. Finally, 

the results of the in vivo studies showed the capacity 

of chitosan–PEG nanocapsules to enhance and 

prolong the intestinal absorption of salmon 

calcitonin. Additionally, they indicated that the 

pegylation degree affected the in vivo performance of 

the nanocapsules. Therefore, by modulating the 

pegylation degree of chitosan, it was possible to 

obtain nanocapsules with a good stability, a low 

cytotoxicity and with absorption enhancing 

properties. [8] 

Gene Delivery 

Polyethyleneglycol modified polyethylenimine for 

improved CNS gene transfer 

One problem of using polycation DNA complexes, 

especially in an in vivo study, is their poor solubility. 

They may immediately precipitate out of a solution 

when prepared at a higher concentration. 

Polyethylene glycol (PEG) modification 

(PEGylation) often can improve the solubility of 

macromolecules, minimize aggregation of 

particulates and reduce their interaction with proteins 

in the physiological fluid. PEGylation of PEI reduced 

surface charge of PEI/DNA particles, increased their 

dispersion ability at high concentrations, decreased 

plasma protein binding and erythrocyte aggregation, 



prolonged blood circulation and reduced systemic 

toxicity & increased invivo transgene expression of 

PEI. The study provides the in vivo evidence that an 

appropriate degree of PEG modification is decisive in 

improving gene transfer mediated by PEGylated 

polymers. [9] 

Small interfering RNA (siRNA) delivery 

Small interfering RNA was conjugated with 

poly(ethylene glycol) (PEG) at four different terminal 

ends (sense 3′, sense 5′, antisense 3′, and antisense 5′) 

via cleavable disulfide and noncleavable thioether for 

gene silencing efficiencies. The PEGylation site at 

the four siRNA termini and PEG molecular weight 

were not critical factors to significantly affect gene 

silencing activities. Cleavable siRNA-PEG 

conjugates showed comparable gene silencing 

activities to naked siRNA, and exhibited sequencespecific 

degradation of a target mRNA. Interestingly, 

noncleavable siRNA-PEG conjugates were processed 

by Dicer, enabling to exert RNAi effect without 

showing a target sequence-specific manner. 

However, only cleavable siRNA-PEG conjugates 

significantly reduced the extent of INF-α release as 

compared to noncleavable siRNA-PEG conjugates, 

suggesting that they can be potentially used for 

therapeutic siRNA applications. [10] 

Dendrimer 

in drug delivery mostly in antiviral and cancer 

therapy. [11] 

Insulin PEGylation 

Despite the robust structure of 

polyamidoamine(PAMAM)dendrimers, they are not 

stable when complexed with surfactants. 

Modification of PAMAM dendrimers by grafting 

PEG chains on the surface of PAMAM substantially 

improves its colloidal stability in the presence of 

sodiumdodecylsulfate(SDS). Michael addition 

reaction was employed to synthesize PEGylated- 

PAMAM by activating MPEG with 4- 

nitrophenylchloroformate.The PEGylated-PAMAM 

dendrimers did not aggregate in the presence of upto 

100mM SDS as the complexes were sterically 

stabilized by PEGchains. ITC and zetapotential 

measurements revealed that the binding mechanism 

of SDS and PEGylated-PAMAM was induced by 

electrostatic interaction and polymer-induced 

micellization of SDS on PEG chains. The interaction 

of PEGylated-PAMAM and amphiphilic molecules, 

such as SDS was elucidated, and this provided a 

useful basis for the application PEGylated-PAMAM 

A novel long-acting insulin based on the following 

properties: (i) action as a prodrug to preclude supra 

physiological concentrations shortly after injection; 

(ii) maintenance of low-circulating level of 

biologically active insulin for prolonged period; and 

(iii) high solubility in aqueous solution. A 

spontaneously hydrolyzable prodrug was thus 

designed and prepared by conjugating insulin through 

its amino side chains to a 40 kDa polyethylene glycol 

containing sulfhydryl moiety (PEG40-SH), 

employing recently developed hetero-bifunctional 

spacer 

9-hydroxymethyl-7(amino-3- 

maleimidopropionate)-fluorene-Nhydroxysucinimide 

(MAL-Fmoc-0Su). A conjugate 

trapped in the circulatory system and capable of 

releasing insulin by spontaneous chemical hydrolysis 

has been created. PEG40-Fmoc-insulin is a watersoluble, 

reactivatable prodrug with low biological 

activity. Upon incubation at physiological conditions, 

the covalently linked insulin undergoes spontaneous 

hydrolysis at a slow rate and in a linear fashion, 

releasing the nonmodified immunologically and 

biologically active insulin with a t1/2 value of 30h. A 

single subcutaneous administration of PEG40-Fmocinsulin 

to healthy and diabetic rodents facilitates 

prolonged glucose-lowering effects 4- to 7-fold 

greater than similar doses of the native hormone. The 

beneficial pharmacological features endowed by 

PEGylation are thus preserved. In contrast, 

nonreversible, ‘‘conventional” pegylation of insulin 

led to inactivation of the hormone. [12] 

PEGylated derivatives of rosin (PD) used as 

sustained release film forming materials for 

controlled release formulation. The mechanism of 

drug release from these coated systems however 

followed class II transport (n>1.0). [13] 

Recently approved pegylated products shown in 

Table 4. 

CONCLUSION 

PEGylation improves the biopharmaceutical 

properties of drugs that increase stability, resistant to 

proteolytic inactivation, decrease to nonexistent 

immunogenicity, increase circulatory lives and low 

toxicity. These type of alter properties improve the 

efficacy of protein and peptide drug delivery. 



Table 1. Potential benefits of PEGylated products 

Greater biologic activity 

Greater passive tumour targeting of liposomes 

Longer circulating half-life 

Lower peak plasma concentrations 

Smaller fluctuations in plasma concentrations 

Less enzymatic degradation 

Less immunogenicity and antigenicity 

Greater solubility 

Less-frequent administration 

Greater patient adherence and improved quality of life 

PEG reagents 

PEG-NHS 

PEG-aldehyde 

PEG-isocyanate 

PEG epoxide 

PEG-isothiocyanate 

PEG-COOH 

PEG-NPC 

PEG-acrylate 

Table 2. PEGylating agent for Amino PEGylation [6] 

PEGylation 

The N-hydroxysuccinimide (NHS) activated ester of PEG carboxylic 

acid can react with the amino group of lysine. The coupling requires 

only mild conditions, pH 7-9, low temperature (5-25ºC) for short 

period of time. The formed amide bond is physiologically stable. 

Reductive amination with primary amines to produce secondary 

amines, in the presence of reducing agents such as sodium 

borohydride and sodium cyanoborohydride. pH is important for 

reductive amination. 

Reaction with amine to produce a stable urethane linkage. 

Nucleophilic addition 

React with amine to produce a stable thiourea linkage. 

Usually the acid needs to be activated, such as NHS ester. 

Amine reacts with NPC functionalized PEG under proper conditions. 

Michael addition between amine and acrylate ester 

Table 3. PEGylating agent for Carboxyl, Thiol and Hydroxyl PEGylation [6] 

PEG reagents for Carboxyl PEGylation 

PEG-amine 

PEG-hydrazide 

Amide formation under DCC or EDIC coupling conditions 

After activated by EDIC at mild acidic pH, the carboxyl group of proteins 

readily react with PEG-hydrazide, while the amino groups present in all 

reagents remain inactive in this particular conditions. 



PEG reagents for Thiol (-SH) PEGylation 

PEG-Maleimide 

PEG-OPSS 

PEG-vinylsulfone 

Michael addition, thiols react with the C=C bond in the maleimic ring to 

form a physiological stable linkage. The best reaction condition is at pH 8. 

Disulfide S-S bond formation, which can be reversed by reducing agents 

such as sodium borohydride and thioethanolamine. 

Michael addition, thiols react with the C=C bond to form 

a physiological stable linkage. 

PEG-thiol 

Oxidative disulfide S-S bond formation. 

PEG reagents for Hydroxyl PEGylation 

PEG-isocyanate 

PEG-NPC 

Hydroxyl groups react with PEG-NCO, however special considerations are 

required. 

Hydroxyl groups react with NPC to from a carbonate linkage. 

PEG-epoxide PEG-epoxide reacts with hydroxyls best at pH 8.5-9.5. 

Table 4. Approved PEGylated Products [5] 

Brand name Product Company Indication 

PEGasys 

PEG-INF α-2a 

(interferon) 

Hoffmann-La 

Roche 

Hepatitis 

PEG-Intron 

PEG-INF α-2b 

Enzon 

Hepatitis 

(interferon) 

Neulasta 

PEG- filgrastim(granulocyte 

colony stimulating factor) 

Amgen 

Neutropenia 

Adagen PEG-adenosine deaminase Enzon Immunodeficiency 

Oncaspar PEG-asparginase Enzon Cancer 

Somavert PEG-visomant Pfizer Acromegaly 

PEG-hirudin PEG-recombinant hirudin Abbot Thrombosis(phase III) 

PEG- monoclonal 

antibody 

PEG-CDP 870 Pfizer Rheumatoid arthritis 

(phase III) 

PEG-Axokine PEG-cilliary nurotrophic 

factor 

Regeneron 

Obesity (phase III) 



Figure 1. PEGylation process in general 

Figure 2. Specific PEGylation of Glutamine by transglutaminase enzyme. 

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technology and chemistry.mht updated on 20 

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12. Shechter Y, Mironchik M, Rubinraut S, 

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