Covalent Conjugation of Poly (Ethylene Glycol) To Proteins and Peptides: Strategies and Methods

Chapter 8
Covalent Conjugation of Poly(Ethylene Glycol)

to Proteins and Peptides: Strategies and Methods
Anna Mero, Chiara Clementi, Francesco M. Veronese,
and Gianfranco Pasut
Abstract
PEGylation, the covalent linking of PEG chains, has become the leading drug delivery approach for
proteins. This technique initiated its first steps almost 40 years ago, and since then, a variety of methods
and strategies for protein–polymer coupling have been devised. PEGylation can give a number of relevant
advantages to the conjugated protein, such as an important in vivo half-life prolongation, a reduction or
an abolishment of immunogenicity, and a reduction of aggregation. Furthermore, the technique has
demonstrated a great degree of versatility and efficacy – not only PEG–protein conjugates have reached
the commercial marketplace (with nine types of derivatives), but a PEG-aptamer and PEGylated lipo-
somes are now also available. Most of this success is due to the development of several PEGylation
strategies and to the large selection of PEGylating agents presently at hand for researchers. Nevertheless,
this technique still requires a certain level of familiarity and knowledge in order to achieve a positive
outcome for a PEGylation project. To draw general guidelines for conducting PEGylation studies is not
always easy or even possible because such experiments often require case-by-case optimization. On the
other hand, several common methods can be used as starting examples for the development of tailor-
made coupling conditions. Therefore, this chapter aims to provide a basic introduction to a wide range
of PEGylation procedures for those researchers who may not be familiar with this field.
Key words: Poly(ethylene glycol) (PEG), PEGylation, PEG–protein conjugate, Protein modification
1. Introduction
PEGylation, the covalent attachment of PEG moieties to a thera-

peutic agent, was first reported by Abuchowski et al. in the 1970s
(1) who demonstrated the usefulness of the strategy to improve
the therapeutic value of proteins and peptides. Since then,
PEGylation has been long studied in the literature and several
conjugates have already reached the marketplace. The most
Sonny S. Mark (ed.), Bioconjugation Protocols: Strategies and Methods, Methods in Molecular Biology, vol. 751,
DOI 10.1007/978-1-61779-151-2_8, © Springer Science+Business Media, LLC 2011
95
96 Mero et al.
prominent effect of PEGylation is a prolonged circulation time

for therapeutic conjugates due to a decreased rate of kidney clear-
ance and a reduction of proteolysis. Together, these advantages
have lead to lower doses of administration and increased compli-
ance by patients (2, 3).
PEG is synthesized by the anionic ring opening polymer-
ization of ethylene oxide initiated by nucleophilic attack of a
hydroxide or a methoxide ion to the epoxide ring. The polymer
can be obtained with a low polydispersity and a low content of
impurities. Several derivatives of PEG that vary in molecular
weight (300 Da to 40 kDa), structure (linear or branched), and
reactive moiety are currently available from a number of different
companies (e.g., Iris biotech, Laysan Bio, NOF, etc.). The choice
of an appropriate PEG derivative to use will depend on the
particular features of a given protein of interest, such as the
primary sequence, chemical reactivity of available functional
groups, molecular weight, biological activity, and function. In
this chapter, we outline several strategies for carrying out the
PEGylation of proteins and peptides, and also present a number
of methods for characterizing and purifying PEG–protein conju-
gates that have been used in our own laboratory or published in
the literature.
1.1. Characterization The development of an efficient and successful PEGylation study

of PEGylating Agents requires reliable methods for analyzing PEG reagents and PEG–
protein conjugates at various stages of the process. In fact, PEG
reagents contribute to a substantial portion of the manufacturing
costs associated with PEGylated proteins, and their purity impacts
the conjugation efficiency and overall product quality. The deter-
mination of the exact molecular weight, polydispersity index,
presence of reactive and nonreactive impurities, degree of activa-
tion, and the presence of PEG dimers in the raw material must all
be carefully evaluated.
1.1.1. NMR Spectroscopy To evaluate the degree of activation and to detect the presence of
of PEGylating Agents impurities (4), PEGs can be analyzed by both 1H- and 13C-NMR
spectroscopy (Fig. 1). This technique requires a few milligrams of
polymer and a suitable deuterated solvent such as dimethyl sul-
foxide (DMSO-d6), chloroform (CDCl3), or water (D2O).
Usually, CDCl3 or DMSO are preferred for the analysis of those
activated PEGs that can be hydrolyzed in D2O.
1.1.2. Determination The most utilized PEG for protein modification is methoxy-PEG
of PEG Diol Impurities (mPEG), where only one terminal end of the polymer can be
in Methoxy-PEG Batches activated while the other is capped with a methoxy group, pre-
venting undesired intra- or intermolecular crosslinking (5).
During the synthesis of mPEG, the anionic polymerization of
ethylene oxide initiated by CH3O– eventually contains PEG diol
Covalent Conjugation of Poly(Ethylene Glycol) to Proteins and Peptides 97
Fig. 1. NMR spectrum of Boc-PEG-NHS in CDCl3.
secondary by-products due to the presence of trace amounts

of water. In fact, OH− formed by water present in the reactor
can itself initiate the polymerization process, yielding a chain
that can grow at both ends. This process leads to the formation
of diol PEG impurities (HO–PEG–OH) with the peculiarity of a
double-MW value with respect to the MW of the mPEG batch.
Size-exclusion chromatography (SEC) analysis, as described in
Subheading 1.3.3 below, is suitable for the determination of such
PEG diol species in mPEG batches.
1.1.3. Activation Degree The degree of activation of most amino-reactive PEGs can be
of Amino-Reactive determined by using a spectroscopic assay based on the so-called
PEGylating Agents Glycyl-Glycine (Gly-gly) test. In this procedure, the degree of
PEG activation is determined by reacting an equimolar amount
of Gly-Gly with the activated PEG, followed by performing a
Snyder and Sobocinsky colorimetric assay of the unreacted
dipeptide (6). This assay uses 2,4,6-trinitrobenzenesulfonic acid
(TNBS), which reacts stoichiometrically with primary amino
groups in an alkaline medium to give a trinitrophenyl derivative
absorbing at 420 nm (Fig. 2).
1.1.4. Activation Degree To determine the degree of activation of thiol-reactive PEG

of Thiol-Reactive reagents (e.g., PEG-OPSS, PEG-Mal), the polymer is mixed with
PEGylating Agents an equimolar amount of a suitable molecule containing a free
thiol (e.g., cysteine (Cys) or glutathione (GSH)). The presence
of unreacted thiol groups is determined by the Ellman assay,
98 Mero et al.
Fig. 2. Reaction between 2,4,6-trinitrobenzensulfonic acid and a free primary amine group.
Fig. 3. Reaction between 5,5¢-dithiobis(2-nitrobenzoic acid) and a free thiol group.
Table 1
Hydrolysis half-lives of PEG-NHS species at pH 8, 25°C
PEG-NHS ester Active groups structure Half-life (min)
PEG–(CH2)4–CO2–NHS Succinimidyl valerate (SVA) 33.6

PEG–O–CO2–NHS Succinimidyl carbonate (SC) 20.4
PEG–O2C–(CH2)3–CO2–NHS Succinimidyl glutarate (SG) 17.6
PEG–O2C–(CH2)–CO2–NHS Succinimidyl succinate (SS) 9.8
PEG–O–CH2–CO2–NHS Succinimidyl carboxymethyl (SCM) 0.75
PEG–O–(CH2)–CO2–NHS Succinimidyl propionate (SPA) 16.5
which is based on the use of 5,5¢-dithiobis (2-nitrobenzoic acid)

(DTNB). In this assay, free thiols react with DTNB at neutral pH
to give 2-nitro-5-thiobenzoic acid (TNB), a derivative absorbing
at 412 nm (Fig. 3) (7).
1.1.5. Reactivity The reactivity of N-hydroxysuccinimide (NHS)-activated PEGs

of NHS-Activated PEGs can be easily evaluated by following the increase in UV absor-
bance at 260 nm due to the release of the NHS group when the
reaction is performed at room temperature in borate buffer.
The reactivity of several representative PEG-NHS species,
expressed in terms of the hydrolysis rate, is reported in Table 1 (8).
It is worth noting that the hydrolysis rate of such acylating
PEGs depends upon the nature of the chemical group adjacent to

the active ester. Another point to be highlighted is that in all
cases, the aminolysis rate is always higher than the hydrolysis rate
due to the higher nucleophilicity of free amino groups.
1.1.6. Mass Spectrometric PEG molecular weight determination can be rather difficult
Analyses of PEGylating because of polymer polydispersity. For such determinations,
Agents matrix-assisted laser-desorption ionization (MALDI) mass spec-
trometry (MS) has been the technique most often employed
because it produces mainly monocharged ions, thus generating
mass spectra of low complexity (9–11). The specific operating
conditions will depend upon the particular MALDI-TOF instru-
ment employed, but using an acceleration voltage of 20 kV with
linear detection is a suitable general method. The MALDI-TOF
mass spectrum of a PEG sample typically shows a monomodal
distribution of MW values with the main mass signals spaced apart
by Dm/z = 44, in agreement with the monomer unit mass of
the oxyethylene unit (44.053 g/mol) (Fig. 4) (12). Electrospray
ionization mass spectrometry (ESI-MS), a method widely used
for protein characterization, has a strong tendency to form mul-
tiply charged ions of the samples, thus hampering the analysis of
polydisperse PEG polymers with molecular masses above a few
kilodaltons.
1.1.7. Determination of The impurities found in raw PEG preparations can come from
Reactive, Low-Molecular either the process of synthesis of the polymer or the activation
Weight Impurities in PEG reaction, while others are formed by the degradation of the polymer
Batches (e.g., oxidation) or by cleavage of the chain itself. PEG-aldehyde,
for example, is easily oxidized in air and low-molecular weight
aldehydic impurities can often be found in the raw product.
Fig. 4. MALDI-TOF mass spectrum of PEG 6,000 Da.

100 Mero et al.
MS analysis, reversed-phase high performance liquid chromatography

(RP-HPLC), or NMR spectroscopy are all useful methods to detect
the presence of these substances (13).
1.2. Protein PEGylation The PEGylation of proteins can be achieved either by a direct
chemical reaction between an amino acid residue and a suitable
PEGylating reagent, or by an enzyme-catalyzed linkage. Brief
descriptions of several strategies for conjugating PEG molecules
to proteins and peptides are presented below.
1.2.1. Random PEGylation The primary amino groups of proteins are good nucleophiles,
at Free Amino Groups and as such are exploited most frequently for PEG coupling by
reaction in mildly basic media (pH 8.0–9.5). Lysines, com-
monly located on protein surfaces, are relatively abundant and
easily accessible to reactive PEG reagents (e.g., activated PEG-
carboxylates and PEG-carbonates; see Fig. 5a, b). Random
PEGylation at these amino acid residues typically yields mix-
tures of different isomers and different degrees of modifica-
tion. To a lesser extent, such reactive PEGylating agents can
also target other types of nucleophilic groups found in pro-
teins such as the side chains of serine, threonine, tyrosine, and
histidine (14, 15). In this case, it is possible to cleave any
unstable bonds by treating the resultant conjugate mixture
with hydroxylamine, thus improving the homogeneity of the
final PEGylated product (16).
Fig. 5. Coupling of protein amines with activated PEG-carbonates (a1, a2), PEG-carboxylates (b) and PEG-aldehyde (c).
1.2.2. Protein N-Terminal In order to guarantee a higher degree of homogeneity of the product,
PEGylation it is possible to direct the PEG coupling reaction to take place only
at the N terminus of a protein. This selectivity is possible by taking
advantage of the different pKa values between the e-NH2 of lysine
and the a-NH2 of the N terminus. By lowering the pH of the reac-
tion mixture to ~5–6, all the e-amines in a protein will tend to be
protonated whereas the a-NH2 group will still be partially present
as a free base available for coupling with activated PEG molecules.
This method generally gives optimal results when less reactive
PEG-aldehydes are used. In these reactions, an unstable Schiff
base is initially obtained, which is in turn reduced to a stable
secondary amine (Fig. 5c). Several papers describe the modification
of primary amines with PEG-acetaldehyde and later with the more
stable PEG-propionaldehyde (17, 18). This conjugation method
has been successfully exploited for the preparation of several
PEG–protein conjugates; among these Neulasta®, an N-terminal
mono-PEGylated granulocyte colony-stimulating factor (G-CSF)
has demonstrated therapeutic and marketing success (19).
1.2.3. Thiol PEGylation Cysteine residues are valuable targets for achieving the site-specific
modification of proteins or peptides, and are present in the free
form at a relatively low natural abundance level compared to the
oxidized cystine species. Nevertheless, cysteines – if present – are
often found partially or fully buried within the structure of
proteins with limited accessibility to chemical reagents (20).
Under appropriate conditions, cysteine residues can be modified
selectively, rapidly, quantitatively, and either in a reversible or irre-
versible fashion (21). Furthermore, thanks to its relatively facile
coupling chemistry, there are several examples of the insertion of
cysteines by genetic engineering at desired positions in a protein
sequence for site-specific conjugation (22).
PEG-maleimide (PEG-Mal), PEG-vinyl sulfone (PEG-VS),
or PEG-iodo acetamide (PEG-IA) derivatives have been used to
obtain stable, irreversible thioether bonds between polymers
and proteins (Fig. 6). PEG-orthopyridyl disulfide (PEG-OPSS)
Fig. 6. Thiol-reactive PEGs (a) and conjugation of PEG to a free thiol group (b).
102 Mero et al.
is also widely used, and it forms a disulfide linkage with cysteine.

This linkage can be cleaved under reducing conditions, whereas
PEG-Mal gives stable conjugates. PEG-IA and PEG-VS are
both less reactive and infrequently used, whereas PEG-Mal and
PEG-OPSS yield quantitative protein modification.
Very recently, an interesting strategy was devised to direct
PEGylation to protein disulfide bridges as well; in this case, the
disulfide link is firstly reduced and then the resulting free thiols are
reacted with a special PEG monosulfone reagent to give a stable
three-carbon PEGylated bridge. This procedure, although very
promising, is not described here further because the relevant PEG
monosulfone is currently unavailable commercially. The reader,
however, may refer to the literature for further details (23).
1.2.4. PEGylation The direct coupling of PEG-NH2 to activated protein carboxylic

to Carboxylic Acid Groups groups cannot be easily performed because it typically yields
intra- or intermolecular linkages between the protein amines. An
original solution uses PEG-hydrazide that is reactive at low pH
toward carboxyl groups, but does not react with protein amino
groups that are protonated under such conditions (24).
1.2.5. PEGylation Aldehydic and keto groups, absent in natural proteins, can be
of Proteins Modified exploited for certain types of nucleophilic additions. Aldehyde
with Aldehydic and Keto functional groups can usually be introduced into proteins by the
Groups oxidation of an N-terminal threonine or serine residue using
sodium periodate. The introduced aldehydic groups can react
with an aminooxy-functionalized PEG chain to obtain a conjugate
at the N terminus of the protein (Fig. 7). In some cases, when the
N terminus is an amino acid other than threonine or serine, a
similarly reactive group can be introduced by metal-catalyzed
oxidation, although the conditions for this reaction are poten-
tially more damaging to proteins (25).
1.2.6. Selective Although proteins typically present a defined structural confor-

PEGylation in Structuring mation in aqueous solutions, peptides often have a random coil
or Denaturing Media structure. The presence of organic solvents or chaotropic salts
can promote structural rearrangements of the peptide and even-
tually modify the degree of solvent exposure and reactivity of
some residues. As a few examples of selective PEGylation in
the presence of organic solvents have been reported in the lit-
erature, they cannot be considered to be of general applicability.
Fig. 7. Conjugation of PEG to proteins containing aldehydic groups.

In fact, several parameters such as the solvent, PEG/peptide

molar ratio, peptide concentration, and the temperature need to
be evaluated on a case-by-case basis. For instance, it was reported
that in the presence of 60% (v/v) of dimethylformamide (DMF)
at high pH, insulin is selectively modified by a NHS-activated
PEG at LysB29 only, even though there are three potential sites
for conjugation that are present within the peptide sequence (viz.,
at GlyA1, PheB1, and LysB29) (26). Moreover, growth hormone
releasing factor (GRF), bearing the amino acid residues Lys12
and Lys21 within the N-terminal region, has been conjugated to
activated PEGs to yield equimolar amounts of two distinct mono-
PEGylated isomers (i.e., Lys12- and Lys21-conjugates) (27).
However, when the same reaction is conducted in trifluoro-
ethanol (TFE) (50% v/v), it was found that 90% of the reaction
mixture is composed of the derivative at Lys21. And finally, the
lone Cys17 of G-CSF – buried within a hydrophobic region –
could be PEGylated only under mild reversible denaturation con-
ditions that preserved the integrity of the disulfide bridges (28).
1.2.7. Amino PEGylation For the selective PEGylation of peptides, a different type of strategy
of Peptides by Reversible involves the reversible protection of specific residues. This proce-
Protection dure is possible for peptides only because they generally contain
just a few nucleophilic groups and are more stable than full-length
proteins toward harsh chemical treatments. This method involves
three steps: (1) protection by suitable reagents of the residues
known to be important for the activity and, eventually, the purifi-
cation of the desired isomers; (2) PEGylation at the level of the
lone unprotected, reactive target residue; and (3) removal of all
the protecting groups. This method is not suitable for proteins
because the harsh methods employed during the protection and/
or deprotection reactions can negatively affect protein integrity.
The protected peptides can be obtained by solid-phase synthesis
or by chemical modification. For example, a somatostatin analogue,
bearing one terminal a-amino group and one lysine residue,
was modified by selective pH-driven tert-butyloxycarbonyl (Boc)
protection of the first amino group, followed by PEGylation of
the second one and deprotection (29). In addition, GRF and
salmon calcitonin, prepared by solid-phase synthesis, were fluore-
nylmethyloxycarbonyl (Fmoc)-protected at the N terminus and
at one of the two internal lysine residues, and then selectively
conjugated to PEG (30, 31).
1.2.8. Glutamine As an alternative to chemical conjugation, promising selective

Enzymatic PEGylation methods have been proposed that use enzymes to catalyze the
covalent attachment of polymers to proteins. Among these, trans-
glutaminase (TGase)-mediated conjugation has drawn significant
attention for its high degree of site-specificity. TGase catalyzes an acyl
transfer between the g-carboxamide group of a glutaminyl residue
104 Mero et al.
Fig. 8. PEGylation mediated by transglutaminase (TGase).
(acyl donor) and a primary amine (acyl acceptor). The latter can
be selected among a variety of amines, including the e-amino
group of lysine or an appropriate PEG derivative bearing an amino
group (PEG-NH2) (Fig. 8) (32). Among the various prokaryotic
and eukaryotic TGases that have been explored for conjugation
applications, the most widely used enzyme is microbial TGase,
which has a number of advantageous properties over eukaryotic
TGases such as a calcium-independence and lower substrate
specificity requirements. These properties conveniently allow for
the use of TGase as a biochemical reagent on a large scale for
industrial applications. In the area of pharmaceutical biotechnology,
several proteins have been selectively modified by TGase; these
are recombinant human interleukin-2 (IL-2), G-CSF, and human
growth hormone. In these examples, the selective conjugation is
due to the TGase active site structure, which is accessible only to
those glutamines present within flexible regions of the protein
substrate (33).
1.3. PEG-Conjugate Usually PEG coupling reactions, especially random amino

Purification PEGylation, yield a mixture of heterogeneous compounds and,
therefore, a purification step is always required. Even in the case
of a selective PEGylation reaction, a purification step is still needed
to eliminate unreacted proteins, excess amounts of polymer, and
various by-products. Several purification strategies may be used
depending upon the properties of both the protein and the con-
jugated PEG moiety. Dialysis or ultrafiltration of the reaction
mixture can be used to remove low-molecular weight compo-
nents or to exchange the solvent. The removal of unreacted PEG
polymers, non-conjugated proteins, and the separation of differ-
ent PEGylated products can be achieved by using more specific
and elaborate chromatographic techniques equipped with an
online detector. Proteins and their conjugates can usually be
monitored by measuring the UV absorbance at l = 214 or 280 nm
or by fluorescence detection (lEX = 295 nm, lEM = 310 nm). On
the contrary, unreacted PEGs, which are nearly transparent in the
UV spectrum, can be monitored by using refractive index (RI)
detectors (34) or light scattering techniques. Typically, the
amount of PEG polymers present in a sample can be estimated
by analyzing the collected fractions with an iodine assay (35).
In Subheadings 3.3 and 3.4, we describe several protocols that

can serve as a useful guideline for the purification and analysis of
most types of PEGylated proteins.
1.3.1. Ion Exchange When PEG is coupled to a protein’s amino groups, the resulting
Chromatography conjugate may have an isoelectric point (pI) different from that of
the starting native protein. Cation exchange chromatography
(CEX) is the method of choice for the separation of PEGylated
proteins because it exploits differences in charge at the protein
surface (36, 37). Effectively, PEG modifies the elution time of
proteins in ion exchange chromatography (IEX) separations,
either by coupling to the amines or shielding the charges on the
protein surface. Even in those situations where the net charge is
unaltered with respect to the starting protein, the presence of the
PEG moiety may decrease the interaction between the protein
and the chromatographic matrix, thus yielding a shorter elution
time for the PEG–protein conjugate compared to the starting
protein. In the case of CEX separations, the elution order of
PEG–protein conjugates is determined mainly by the number of
linked PEG chains: Highly PEGylated molecules tend to elute
first, followed by less PEGylated isomers and then by the unre-
acted protein (38); and any unreacted PEG that does not present
positive charges elutes in the column void volume.
Given that PEG–proteins tend to interact weakly with IEX
matrices, special consideration must be given to the buffer con-
ductivity and pH conditions. For this reason, it is convenient to
carry out an extensive dialysis of the PEGylation mixture against
the buffer that will be used for the CEX step in order to reach the
same ionic strength (as measured by conductometry) and pH
value of the column equilibration buffer. In addition, the reaction
mixture samples should be diluted and filtered to avoid high
back pressure and column fouling. Occasionally, a double-step
procedure may be suitable, where a first chromatographic step is
performed with a large-particle size resin to eliminate the free
polymer, followed by a second step in which a resin with higher
resolution is employed. The choice of buffers is a very important
step for successfully carrying out IEX separations. Usually the
equilibration buffer (A) has a low ionic strength, whereas the
elution buffer (B) contains a higher concentration of salt (NaCl).
In some cases, a change in pH can be also used between buf-
fers A and B. Small increases in the salt concentration or pH of
the buffer solution can effectively reduce the strength of the
interactions between PEG–proteins and the resin, causing the
conjugates to be eluted from the column before the un-PEGy-
lated proteins. A problem that is commonly encountered in the
purification of PEGylated proteins is the low capacity of the pores
of conventional IEX media; the effective diameter of these
pores can sometimes be too small to allow the penetration of
106 Mero et al.
PEG–protein complexes with a large surface area. Consequently,

the desired conjugate products can be lost in the column flow-through.
This is not generally considered to be an issue in analytical-scale
experiments where the sample loading is relatively low (around
1 mg of protein/mL of sample); for preparative-scale purifica-
tions, however, a higher loading (e.g., >6 mg/mL) of the PEG–
protein mixture may become a limiting factor.
Conventional strong cation exchange resins (e.g., Mono S™
(sulfo) or Mono SP™ (sulfopropyl) sepharose (GE Healthcare))
that are usually employed with standard fast protein liquid
chromatography (FPLC) or HPLC systems can be loaded at a
protein concentration of 5–10 mg/mL at a linear flow rate of
60 cm/h. MacroCap™ SP (GE Healthcare) is a new type of
strong cation exchange medium especially designed for the
purification of PEGylated proteins and other large biomolecules
under high sample loading conditions (12–15 mg/mL). The
matrix is highly porous and provides good mass transfer charac-
teristics and improved accessibility to the internal surface area for
the adsorption of large molecules.
PEGylated proteins can be also purified by anionic exchange
chromatography (AEX) (40). In this case, the PEG–protein
conjugate is bound to the column at a higher pH value than its
isoelectric point (pI), which results in a negative net charge on
the molecule. Once again, the charges at the surface of the protein
can be shielded by the presence of PEG. As in CEX, it is often
convenient to use a linear salt or pH gradient to elute the
PEGylated derivatives from the column.
1.3.2. Reversed-Phase Reversed-phase high performance chromatography (RP-HPLC) is

Liquid Chromatography often used for the characterization of PEGylated species due to its
high resolution and the possibility of coupling the technique with
an online mass spectrometer detector (41). Although the method is
very rapid, only a limited amount of sample material can be loaded
at once. Furthermore, PEGylated proteins often give wide peaks
due to polymer polydispersity, which compromises the resolution of
PEGylated species to different extents. The elution conditions
employed in RP-HPLC generally require high percentages of ace-
tonitrile (CH3CN) or methanol (MeOH), which can be a concern
for protein stability. Other parameters such as the column tempera-
ture, the elution gradient profile, and the mobile phase composition
must all be carefully evaluated on a case-by-case approach.
1.3.3. Size-Exclusion Size-exclusion chromatography (SEC) separates molecules based

Chromatography on differences in their hydrodynamic volumes. Since PEGylation is
often performed with the aim of increasing the size of proteins, it
is clear that SEC can be a useful technique for purifying PEG–
protein conjugates. SEC, however, has several limitations: it gener-
ally gives broad peaks with poor resolution for PEG-conjugates; it
is a low-throughput technique with relatively high costs, and thus

has limited applicability in large-scale processes; and it also cannot
separate positional isomers (which have the same mass and hydro-
dynamic volume). Furthermore, in those cases where there are
only small differences in size, unreacted PEG and protein mole-
cules can be co-eluted with monoPEGylated species. Conversely,
SEC is a useful method for removing low-molecular weight
impurities (e.g., by-products formed by the hydrolysis of function-
alized PEG, buffer salts, solvents, and other low-molecular mass
reagents), and also serves as an effective tool for obtaining an initial
evaluation of the degree of protein modification as well as for
determining the presence of aggregates. Typically, dextran- and
agarose-based SEC columns are often used in conjunction with
FPLC systems, with Sephadex G-75, G-50, and Superose 6 or 12
(42) being the most commonly used media to purify proteins. The
choice of a particular column type depends on the molecular
weight of the protein. Generally, buffers with a low percentage of
organic solvents are used to minimize the occurrence of hydro-
phobic interactions between proteins in the sample mixture and
the column matrix (43–45). One important factor in the use of
SEC for PEGylation applications is to take into account that the
apparent size of a PEG–protein conjugate is roughly five to ten
times larger than that of a globular protein with the same nominal
molecular weight.
1.3.4. Ultrafiltration/ One particularly important nonchromatographic step in the

Diafiltration production of pharmaceutical proteins is the ultrafiltration/diafil-
tration operation. Ultrafiltration/diafiltration are effective processes
for exchanging buffers between chromatographic steps and for
concentrating the conjugate products to achieve the desired
final concentration. Usually, membrane filters with the same
nominal molecular weight cutoff value as those used for the native
(unmodified) protein should also be employed to concentrate
monoPEGylated-protein samples since such types of PEGylated
products can potentially escape by the so-called “snake effect”
through membranes with larger porosities, causing substantial
losses of product (46–48). In the case of branched PEG or multi-
PEGylated conjugates, larger cutoffs may be used, since in this
case the “snake effect” of PEGs is not as likely to be an issue.
Regenerated cellulose and polyethersulfone membranes, with
their low protein binding and high protein retention characteris-
tics, are the most commonly used filter media and also provide
the highest rates of recovery for purified proteins (48). However,
polyethersulfone filters are much less efficient for processing
PEGylated protein species because of the hydrophobic nature of
their membrane surfaces. Indeed, the full application potential of
membrane-based technologies for PEGylated products is some-
what limited because the increased size, greater hydrophobicity,
108 Mero et al.
and lower electrostatic interactions of PEG-conjugates with

respect to unmodified proteins tend to lead to increased fouling,
i.e., the largely irreversible adsorption and/or deposition of pro-
teins on and within membrane filter media (49).
1.4. PEG-Conjugate The first issue to be faced with the characterization of PEG-
Characterization conjugates deals with the accurate determination of the amount
of attached PEG. Several methods are available such as colori-
metric assays, SEC, electrophoresis, and mass spectrometry (MS).
However, these methods are not suitable for separating and
identifying positional isomers of PEG; for these purposes, IEX is
generally better suited. The identification of PEGylation sites
within proteins is based mainly on the use of standard protein
sequence analysis methods, with peptide mapping and mass
spectrometry both falling within the scope of such types of
useful techniques.
The simplest and most rapid methods available for conducting
preliminary characterizations of a PEG–protein conjugate are
colorimetric assays. In the case of amino-PEGylation products, the
Habeeb assay can be used to quantify the amount of unreacted
protein primary amines with TNBS reagent, thus allowing one to
indirectly calculate the number of bound PEG chains (50). In a
similar approach, Ellman’s assay can be used to determine the
presence of any remaining free cysteines following PEG–thiol
conjugation (51). Finally, the iodine assay, based on the noncova-
lent interaction of iodine with the PEG backbone, can be used to
obtain both qualitative and quantitative information about the
polymer (35). The preceding methods are all described in detail
in the following sections.
To characterize the protein conformation in the conjugates,
various spectroscopic methods may be used. In particular, circular
dichroism, fluorescence, and UV spectra can all be applied to
analyze PEG–protein conjugates – thanks to the fact that PEG is
transparent to light at UV–visible wavelengths. For the interpre-
tation of such spectra, the reader is referred to dedicated books
on protein characterization (52).
1.4.1. Bicinchoninic Acid The protein concentration in a non-PEGylated sample can usu-
Assay ally be determined by simply measuring the spectrophotometric
absorbance of the aromatic amino acids residues at l = 280 nm.
This approach may also be acceptable for measuring the concen-
tration of a PEG–protein conjugate sample after first confirming
that the absorbance of the protein is not altered by PEG coupling.
Alternatively, a colorimetric assay can be carried out to estimate
the protein concentration. The bicinchoninic acid (BCA) assay,
for example, is the most commonly used technique because it is
less affected than other types of dye-binding assays by the presence
of PEG. BCA protein assay kits are commercially available from
a number of different suppliers (e.g., Sigma-Aldrich, Bio-Rad,
Thermo Fisher, etc.); in these assays, the quantitative determination

of the protein concentration in a PEGylated sample relies on the
generation of a calibration curve using standard solutions of
the native protein (53).
1.4.2. Ion Exchange IEX, as previously discussed in Subheading 1.3.1, is the most
Chromatography widely used technique for the fractionation and purification of
PEGylated proteins on a preparative scale. IEX is also very useful
for analytical purposes because it allows the efficient separation of
positional isomers (54). In particular, our laboratory has found
that analytical strong cation exchange columns (e.g., TSKgel
SP-5PW, 7.5 mm × 7.5 cm (Tosoh); and HiLoad™ 16/10 SP
Sepharose™ HP (GE Healthcare)) can provide good results for
PEGylated samples.
1.4.3. Reversed-Phase RP-HPLC is a useful technique for the determination of purity

High Performance Liquid and species content of PEGylated protein samples. This fraction-
Chromatography ation method is based on differences in hydrophobicity of the
native and PEGylated proteins. PEG is an amphiphilic molecule,
and thus PEGylated proteins often exhibit higher retention times
on RP-HPLC columns than their un-PEGylated counterparts.
The increase is generally dependent on the length and mass of the
conjugated polymer (37).
RP-HPLC is not only an excellent and robust tool for the frac-
tionation of PEGylated and un-PEGylated species, but is also a
useful method to detect protein oxidation, deamidation, or cleavage
of the protein backbone (55). In addition, the technique can also be
used for the high-resolution analysis of protein fragments and
peptides (i.e., peptide fingerprinting) (19, 43), or to separate posi-
tional isomers (53) in the case of peptide PEGylation. In analytical
RP-HPLC applications, the PEG/protein ratio appears to be the
predominant factor affecting the resolution of PEGylated conju-
gates (37). Typically, reversed-phase columns containing packings
such as butyl (C4) or octadecyl (C18) can be employed for the
fractionation of PEGylated protein using an elution gradient of
H2O/CH3CN (either with or without trifluoroacetic acid (TFA)).
1.4.4. Size-Exclusion SEC, or gel filtration, is widely used to estimate the molecular
Chromatography weight (MW) of native (unmodified) proteins through the use of
a standard calibration curve. As PEGylated proteins show a larger
hydrodynamic volume than native proteins of the same nominal
MW, SEC (and, similarly, sodium dodecyl sulfate polyacrylamide
gel electrophoresis (SDS-PAGE)) cannot provide an accurate
determination of the exact MW of PEG–protein conjugates, but
can only be used to monitor a PEGylation reaction and character-
ize the homogeneity of the conjugate product. An interesting and
useful discussion on the effect of PEG on the apparent size of
conjugates and their behavior in SEC has recently appeared in the
literature (57).
110 Mero et al.
Several brands of gel filtration columns are commercially

available in the marketplace such as Superose 6 or 12 (GE
Healthcare), TSK-Gel (Tosoh), Zorbax GF-250 (Agilent), and
BioSep SEC (Phenomenex). Each type of column has a specified
MW fractionation range, and hence the selection of the most
appropriate column to use must be done on a case-by-case.
Typically, phosphate and HEPES buffers containing a salt (NaCl)
gradient are the most often used eluents in SEC applications. The
addition of water-miscible organic solvents (e.g., acetonitrile or
isopropanol) into the mobile phase can generally improve the
overall separation of PEG–protein conjugate mixtures by increasing
peak sharpness and reducing peak tailing due to nonspecific adhe-
sion of PEGs to the stationary phase (43–45).
SEC-HPLC is also useful for the determination of free PEG
in PEGylated samples, but since free PEG itself does not absorb
at UV wavelengths, it is necessary to use a refractive index (RI)
detector coupled with a second UV detector to reveal simultane-
ously the protein fractions, PEG, and other low molecular weight
by-products (e.g., NHS) that emerge from the column. It should
be noted, however, that a RI detector cannot be used for analyses
requiring a gradient elution because the changes in the eluent
composition modifies the baseline.
1.4.5. Sodium Dodecyl SDS-PAGE is a highly useful technique for determining the purity
Sulfate Polyacrylamide and molecular weight (MW) of native (unmodified) protein
Gel Electrophoresis samples. However, SDS-PAGE is not suitable as a direct method
to easily evaluate the MW of PEGylated proteins because proper
calibration standards are typically unavailable for such analyses.
During electrophoresis, the migration rate of PEG-conjugates
through the porous gel matrix is significantly slowed by the long
and heavily hydrated PEG chains. Consequently, PEGylated
proteins usually display apparent MW values on SDS-gels that do
not correlate with that of the free protein MW standards (54).
Generally, SDS-PAGE can only be used to qualitatively follow the
progress of a PEGylation reaction and the subsequent purifica-
tion procedures. For example, a significant shift in the SDS-PAGE
band position for a protein after PEGylation would provide
evidence in support of a success PEGylation experiment. Random
multi-PEGylation will typically yield several bands on an SDS-
PAGE gel, with each band migrating slower than that corre-
sponding to the native protein; on the contrary, only a single new
band should be expected to appear for monoPEGylated deriva-
tives. In some cases, using PEG molecules of different MWs
instead of native protein standards may enable a rough estimation
of the apparent MW of the conjugates (58, 59); however, it is
then necessary to use a specific iodine staining procedure to reveal
the PEG bands since PEG is negative toward conventional
protein stains. SDS-PAGE gels of varying degrees of crosslinking
can usually be employed for such analyses, but attention must be

paid to the full range of MWs actually present in a particular
PEGylated sample in order to accommodate the migration of all
the components in the mixture.
1.4.6. Mass Spectrometry To evaluate the molecular weight of PEGylated proteins, mass
of Conjugates spectrometry (MS) analyses are highly recommended. Typically,
MALDI-MS is employed for such determinations, even if these
types of spectra are sometimes complicated by PEG polydisper-
sity. For PEGylated proteins, the total MW is easily calculated as
the sum of the MW of the native protein and the MW of the conju-
gated PEG chains (60). Nevertheless, the MS technique does
present a few limits, such as (1) poor ionization efficiency with
large polymers, which can affect the detection sensitivity as well as
the accurate determination of the average MW and (2) degrada-
tion of the proteins during sample extraction and evaporation.
1.4.7. General Method The method used for the localization of the sites of PEG conjuga-
for Determination tion follows the strategy commonly employed for conventional
of PEGylation Sites protein sequence determinations. This approach is based on the
for Proteins Modified proteolytic digestion of the native (unmodified) protein and the
with Polydisperse PEGs conjugated protein, followed by a comparison of the two elution
patterns obtained by RP-HPLC. Subsequent analysis of each
peak in the RP-HPLC fingerprint by ESI-MS or MALDI-MS
allows the determination of peptide composition (61, 62) and the
site of amino acid PEGylation. The determination is carried out
by comparison of the fingerprint of the native protein and that of
the conjugate. The peptides that are missing in the conjugate
elution pattern represent those sequences that contain the poly-
mer. Typically, trypsin is the most commonly employed prote-
olytic enzyme; however, other proteolytic enzymes with different
digestion specificities may also be used depending on the sequence
of the protein under investigation. This is necessary in those cases
where the PEGylated peptide fragments contain more than one
potential site of PEGylation.
1.4.8. Simplified Method The method described above in Subheading 1.4.8 is an “indirect”

for Determination analysis because, due to the polydispersity of the polymer, the
of PEGylation Sites PEGylated peptide is identified by the disappearance of the corre-
for Proteins Modified sponding fragment. A simplified procedure for the identification of
with Monodisperse PEGs PEGylation sites can be applied if a monodisperse PEG is used for
conjugation. Monodisperse PEG polymers, only recently available
in the marketplace, are detectable by ESI-MS, as well. Unfortunately,
monodisperse PEGs are available only in low MW forms, making
them unsuitable for the half-life prolongation of proteins; however,
they can be useful for characterization purposes.
A monodisperse PEG was recently employed by our group
for the specific modification of several proteins of pharmaceutical
112 Mero et al.
interest mediated by the enzyme TGase (63). ESI-MS and tandem

mass spectrometry (MS/MS) of the digested peptides allowed for
a “direct” (as opposed to indirect, as above) characterization of
the peptide and the identification of the PEGylated site even if
more than one is present within the same peptide. It is important
to note that both the high-MW polydisperse PEG and the low-MW
monodisperse form of the polymer are linked to the same site in
the protein sequence since the specificity of the conjugation site
is dictated by the TGase enzyme (31, 61). Generally, the analysis
procedure requires only a very small amount of sample material
due to the possibility of determining both composition and
sequence in a single analysis.
2. Materials
2.1. Characterization 1. PEG reagents (see Note 1).

of PEGylating Agents 2. Deuterated DMSO-d6, CDCl3, or water (D2O).
3. Borate buffer, pH 8: 0.2 M borate buffer, pH 8.
4. Borate buffer, pH 9.3: 0.1 M borate buffer, pH 9.3.
5. Gly-Gly solution (2 mM): Dissolve 28.5 mg of glycyl-glycine
in 100 mL of 0.2 M borate buffer, pH 8.
6. TNBS (1% w/v solution in DMF).
7. Phosphate-ethylenediaminetetraacetic acid (EDTA) buffer:
0.1 M Sodium phosphate buffer, 1 mM EDTA, pH 7.
8. Cys or GSH solutions (2 mM): Dissolve 2.61 mg of Cys or
6.62 mg of GSH in 10 mL of phosphate-EDTA buffer, pH 7.
9. Ellman’s reagent (10 mM): Dissolve 4 mg of DTNB in 1 mL
of 0.1 M phosphate-EDTA buffer, pH 7.
10. MALDI-TOF MS matrix: Sinapinic acid (58), dihydroxy-
benzoic acid (65), or a-cyano-4-hydroxycinnamic acid (46)
mixed with acetonitrile/water (1:1, v/v).
2.2. Protein PEGylation 1. Glycine solution: Use 200 mL of glycine (250 mM) in water,
pH 7.4 for each milliliter of the reaction mixture.
2. NaCNBH3 solution: Prepare 20 mM NaCNBH3 in the same
buffer as that used in the reaction mixture.
3. Hydroxylamine solution: Prepare 2 mL NH2OH, pH 7.3 in
water.
2.3. Conjugate 1. CEX-buffer A: 10 mM phosphate buffer, 10 mM NaCl, pH 4.7.

Purification 2. CEX-buffer B: 10 mM phosphate buffer, 0.1 mM NaCl, pH 4.7.
3. RP-buffer A: 0.05% (v/v) TFA in water.
4. RP-buffer B: 0.05% (v/v) TFA in acetonitrile (CH3CN).
2.4. Conjugate 1. BCA Protein Assay Kit (Pierce): To prepare the BCA working
Characterization solution, mix 50 parts of reagent A (containing sodium car-
bonate, sodium bicarbonate, BCA, and sodium tartrate in
0.1 M sodium hydroxide) with one part of reagent B (con-
taining 4% CuSO4·5H2O) according to the manufacturer’s
instructions.
2. BaCl2 solution: 5% (w/v) barium chloride in 1N HCl.
3. Iodine solution: Dissolve 1.27 g of I2 in 100 mL of 2% (w/v)
KI in water.
4. Phosphate buffer: 20 mM phosphate buffer, pH 7.2.
5. Bicarbonate buffer: 4% (w/v) NaHCO3 buffer, pH 8.5.
6. Tris-Gdn buffer: 6 M guanidine hydrochloride, 50 mM Tris–
HCl, pH 9.0.
7. PepClean™ C-18 spin columns (Pierce, Rockford, IL).
3. Methods
3.1. Characterization 1. Dissolve the PEG sample (10–20 mg) (see Note 1) in 0.75 mL
of PEGylating Agents of deuterated solvent and perform the NMR analysis according
to the instrument manufacturer’s instructions.
3.1.1. NMR Spectroscopy
of PEGylating Agents In the 1H NMR spectrum, the integral values of the reactive
group signals can be compared with the integral values of the
backbone chain signals (–CH2–CH2–; e.g., PEG 5 kDa, 3.6 ppm,
491H) or with other characteristic signals of the polymer. For
example, Fig. 1 shows the NMR spectrum of commercial Boc-
PEG-NHS (5 kDa), where the H signals of the Boc group
(–C(CH3)3, 1.4 ppm, 9H) are compared with those arising from
the N-hydroxysuccinimide group (–NHS) (–CH2–CH2, 1.2 ppm,
4H) and the backbone chain signals. In this case, the analysis of
the integrals indicates that the polymer is activated with NHS at
only 60% of the maximum level, since the signal integration value
is 2.4 instead of the expected value of 4.
3.1.2. Analysis of PEG Diol 1. Equip an HPLC system with a suitable SEC column (see
Content in mPEG Batches Subheading 1.4.4 for a discussion on column selection).
Equilibrate the column with the desired elution buffer.
2. Solubilize the PEG sample (0.2 mM) in 1 mL of elution
buffer.
3. Load 20 mL of the sample solution onto the SEC column.
4. PEG does not absorb at wavelengths suitable for UV–visible
detection and can be revealed by employing a refractive index
detector, instead.
114 Mero et al.
Table 2
Preparation of test solutions for the TNBS assay
Blank PEG reaction mixture Gly-Gly standard solution
20 mL of TNBS 20 mL of TNBS 20 mL of TNBS

980 mL of Borate pH 9.3 955 mL of borate pH 9.3 955 mL of borate pH 9.3
25 mL of Sample A 25 mL of Sample G
3.1.3. Evaluation 1. Prepare Sample A (PEG reaction mixture) as follows: To 1 mL

of the Degree of Activation of 2 mM Gly-Gly solution, add 1 eq. (2 mmol) of the activated
of Amino-Reactive PEG polymer (e.g., PEG-NHS). The required amount of acti-
PEGylating Agents vated PEG to add depends on its MW; for example, if the MW
of PEG-NHS is 5 kDa, then 10.8 mg of the activated polymer
should be added to 1 mL of 2 mM Gly-Gly solution.
2. Let the mixture react for 30 min at room temperature under
continuous agitation.
3. Prepare 1 mL of 2 mM Gly-gly solution (sample G) as control.
4. Perform the TNBS assay in duplicate at room temperature
according to Table 2.
5. Incubate the reactions for 30 min.
6. Read the absorbance at l = 420 nm using a UV–visible
spectrophotometer.
The percentage of activation of the amino-reactive PEGs is
calculated by using the following formula:
% Activation = [1 - (AbsA - AbsB) / (AbsG - AbsB)] ´ 100%
AbsA = Absorbance of PEG reaction mixture
AbsG = Absorbance of Gly-Gly standard solution
AbsB = Absorbance of the blank solution.
3.1.4. Evaluation 1. Prepare Sample A (PEG reaction mixture) as follows: To

of the Degree of Activation 1 mL of 2 mM Cys or GSH solution, add 1 eq. of the thiol-
of Thiol-Reactive reactive PEG molecule. The required amount of thiol-
PEGylating Agents reactive PEG to add depends on its MW; for example, if the
MW of the PEG is 5 kDa, then 10.2 mg of polymer should
be added to 1 mL of 2 mM Cys or GSH solution.
2. Let the mixture to react for 30 min at room temperature
under continuous agitation.
3. Prepare 1 mL of 2 mM Cys or GSH (Sample C) as control.
4. Perform Ellman’s assay in duplicate at room temperature
according to Table 3.
5. Incubate the reactions for 15 min.
6. Read the absorbance at l = 412 nm using a UV–visible
spectrophotometer.
Table 3
Preparation of test solutions for Ellman’s assay
Blank PEG reaction mixture Cysteine standard solution
50 mL of Ellman’s reagent 50 mL of Ellman’s reagent 50 mL of Ellman’s reagent

1 mL of phosphate- 970 mL of phosphate- 970 mL of phosphate-
EDTA pH 7 EDTA pH 7 EDTA pH 7
30 mL of Sample A 30 mL of Cys or GSH
solution
The percentage of activation of thiol-reactive PEGs is

calculated by using the following formula:
% Activation = [1 - (AbsA - AbsB) / (AbsC - AbsB)] ´ 100%
AbsA = Absorbance of PEG reaction mixture

AbsC = Absorbance of cysteine standard solution
AbsB = Absorbance of the blank solution.
3.1.5. Half-Life 1. Prepare a solution of NHS-activated PEG (0.2–0.5 mM) in

Measurement of NHS- dioxane.
Activated PEGs 2. Add 50 mL of the PEG-NHS solution to 950 mL of 0.2 M
borate buffer (pH 8.0) and immediately read the absorbance
at 280 nm every 5 s until a plateau is reached. To evaluate the
aminolysis rate, add 50 mL of the PEG-NHS polymer solution
to 950 mL of a Gly-Gly solution (Gly-Gly/PEG-NHS polymer,
1:1 molar ratio) in 0.2 M borate buffer (pH 8.0).
3. The absorbance (l = 280 nm) at time point = 0 s is taken to be
the blank and corresponds to the NHS already present in the
mixture.
4. Plot the resulting absorbance data and determine the half-life
of the PEG-NHS reagent.
3.1.6. Mass Spectrometric 1. Dissolve PEG (20 mg) in 0.1% (v/v) TFA in water (5–10 mL).
Analyses of PEGylating 2. Mix one volume of saturated matrix solution with one volume
Agents of the PEG solution.
3. Load the matrix/PEG mixture (10–20 mL) onto the MALDI
sample plate and perform the MS analysis according to the
instrument manufacturer’s instructions.
3.1.7. Analysis 1. Dissolve PEG-aldehyde (0.25–1 mM) in water.

of Low-Molecular Weight 2. At determined time points, withdraw 100 mL and treat the
Impurities of PEG-Aldehyde sample aliquot with 2,4-dinitrophenylhydrazine (DNPH/
PEG-aldehyde, 1:1 eq.).
116 Mero et al.
3. Fractionate the resulting reaction mixture by RP-HPLC on

a C8 column (100 × 2.1 mm; flow rate = 0.8 mL/min; mobile
phase = H2O/CH3CN/H3PO4, 1:1:1).
4. Collect the fractions, lyophilize, and analyze by RP-HPLC on
a C18 column (flow rate = 0.2 mL/min; mobile phase = H2O/
CH3CN) using an ESI-MS detector operating in the negative
ion mode to identify low-MW impurities.
5. As an alternative to the above procedure (Steps 1–4), dissolve
the PEG-aldehyde in MeOH and infuse the sample into an
ion mobility/quadrupole/time-of-flight mass spectrometer
at a flow rate of 5 mL/min to analyze for the presence of trun-
cated PEG-aldehyde molecules.
3.2. Protein PEGylation 1. Prepare the protein solution (1–5 mg/mL) in 0.1 M borate
buffer, pH 8.0–9.0.
3.2.1. Random PEGylation
at Free Amino Groups 2. Determine the exact concentration of the protein by UV
of a Protein absorption using its molar extinction coefficient (66).
3. Add the amino-reactive PEG – in small amounts – to the protein
solution under gentle stirring. An excess of activated PEG is
usually required. The optimum ratio of PEG to each protein
amino group may range from 1 to 10, depending on the
particular PEG and the reactivity of the amino groups on
the protein. Table 4 lists some examples of protein conjuga-
tion experiments that have reported in the literature using
different ratios of PEG/protein, PEGs with different MWs
and different protein concentrations.
4. Incubate the reaction mixture at room temperature for 1–5 h.
5. Quench the reaction with glycine solution and stir for 1 h.
6. Eventually add 1 mL of hydroxylamine solution and stir for
30 min.
Table 4
PEGylation reaction conditions and yields as reported in the literature
for different molar ratios of NHS-activated PEG to protein NH2 groups
Protein
Reaction concentration Protein MW PEG MW PEG/protein
conditions (mg/mL) (kDa) (kDa) molar ratio Yield (%) References
Aqueous, pH 8.5 10 21 5 1–3 85 (42)

Aqueous, pH 8.5 4 23 5 5 50 (67)
Aqueous, pH 8.5 1.5 13.7 5 2.5 55 (67)
Aqueous, pH 8.5 6 141 10 3 50 (68)
Aqueous, pH 8.5 2 17.3 5 8 60 (42)
Aqueous, pH 9 5 19.3 40 3 60 (69)
7. Dialyze the resulting solution to eliminate low-molecular

weight products.
8. Purify the PEG–protein conjugates as described in
Subheading 3.3, and characterize the products as described in
Subheading 3.4.
3.2.2. PEGylation at the 1. Prepare a protein solution at 1–5 mg/mL (as determined by

N Terminus of a Protein absorbance spectrophotometry) in a buffer with pH
Using PEG-Aldehyde ~5.0–6.0.
2. Add PEG-aldehyde (see Note 2) to the protein solution at
the desired molar ratio. Depending on the protein properties,
a great excess of PEG-aldehyde may be needed. It is advisable
to test several different PEG/protein molar ratios to optimize
the reaction (e.g., 10–50 eq. with respect to the amount of
protein molecules).
3. Incubate the reaction mixture for 1 h, and then add NaCNBH3
solution (20 mM) (add 50 eq. of NaCNBH3 per 1 eq. of PEG).
4. Incubate the reaction mixture further at 4°C for 24 h under
gentle stirring.
5. Quench the reaction with glycine solution and stir for 1 h.
Subheading 3.4.
3.2.3. Thiol PEGylation 1. Prepare a protein solution of 1–5 mg/mL in 0.1 M phosphate-

EDTA buffer, pH 7.2.
2. Add PEG-OPSS (see Note 3) or PEG-maleimide PEG-MAL
(see Note 4) to the protein solution at a molar ratio 1:1 or
2:1 with respect to the amount of free thiols present. If
PEG-VS is used, an excess of 2–10 eq. is recommended.
3. Incubate the reaction mixture at 4°C for 4–24 h (depending
on the PEG derivatives used) under gentle stirring.
4. Monitor the disappearance of the free thiols in the reaction
mixture by Ellman’s assay (see Subheading 3.4.7).
Subheading 3.3, and characterize the products as described
in Subheading 3.4.
3.2.4. PEGylation at Protein 1. Prepare a protein solution at 1–5 mg/mL in 0.1 M phos-
Carboxylic Groups phate buffer, pH 4.0–5.0 (see Note 5);
2. Add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (2–5 eq.
with respect to the amount of protein carboxylic acid groups)
and PEG-hydrazide (10–50 eq. with respect to the amount of
protein molecules).
118 Mero et al.
3. Incubate the reaction mixture at 4°C for 24 h under gentle

stirring.
Subheading 3.4.
3.2.5. PEGylation 1. Prepare a solution of protein containing N-terminal serine or

of Proteins Containing threonine residues at 1–5 mg/mL in 1% (w/v) ammonium
Introduced Aldehydic bicarbonate buffer, pH 8.
Groups 2. Add a tenfold molar excess of sodium periodate for 10 min.
3. Add a 2,000-fold molar excess of ethylene glycol to stop the
oxidation reaction, and then dialyze the mixture against an
acidic buffer solution.
4. Add aminooxy-PEG (10–50 eq.) and adjust the pH of the
reaction mixture to 3.6.
5. Incubate the reaction mixture for 20 h at room temperature.
Subheading 3.4.
3.2.6. Selective PEGylation 1. Dissolve the peptide (2 mg/mL) in a suitable mixture of H2O
of Peptides in Structuring and a water-miscible organic solvent (e.g., H2O/DMF, 2:3
or Denaturing Media v/v; or H2O/TFE, 1:1 v/v);
2. Bring the pH of the peptide solution to 9–10.
3. Add PEG-NHS (1–3 eq. with respect to the peptide) to the
peptide solution.
4. Incubate the reaction mixture for 30 min under gentle stirring.
5. Quench the reaction mixture with glycine solution and con-
tinue stirring for 1 h.
Subheading 3.4.
3.2.7. Amino PEGylation 1. Protect the most reactive amine groups of the peptide with a
of Peptides by Reversible suitable procedure (e.g., using t-Boc or Fmoc groups);
Protection 2. Purify the desired products by chromatography (e.g., RP-HPLC).
3. Dissolve the protected peptide in DMF, DMSO, or other sol-
vent at a final concentration of 5–10 mg/mL.
4. Add an activated amino-reactive PEG at an excess of 2–10 eq.
over the peptide.
5. Incubate the reaction mixture at room temperature for 4 h
under gentle stirring.
6. Quench the reaction with glycine solution and continue stir-
ring for 1 h.
7. Dialyze the solution against water to remove the organic

solvent and lyophilize.
8. Deprotect the conjugate using a suitable method; for example,
Boc removal can be carried out by dissolution in TFA, while the
Fmoc group can be cleaved in 20% (v/v) piperidine in DMF.
Subheading 3.4.
3.2.8. Enzymatic 1. Prepare a protein solution at 1–5 mg/mL in 0.1 M phos-

PEGylation Using phate buffer, pH 7.0.
Microbial Transglutaminase 2. Add an excess of 2–10 eq. of PEG-NH2 with respect to the
amount of protein.
3. Add TGase at an enzyme:substrate ratio of 1:75 (w/w).
4. Incubate the reaction mixture at room temperature for 4 h
under gentle stirring.
5. Quench the reaction with a few drops of acetic acid at pH 3.
Subheading 3.4.
3.3. Conjugate After quenching the PEGylation reaction mixture, dialysis is typi-
Purification cally performed using regenerated cellulose membranes against
buffers with low ionic strength at 4°C (see Note 6) for 2 days with
continuous stirring. As an alternative to dialysis, ultrafiltration/dia-
filtration can also be used to exchange the buffer components of
the reaction. For both procedures, it is always important to verify
that the protein remains stable during all the processing steps. After
dialysis or ultrafiltration/diafiltration, the removal of unreacted
PEG, unreacted protein, and the separation of the different
PEGylated species can be achieved by using several different chro-
matographic techniques, as described in the following sections.
3.3.1. Ultrafiltration/ 1. Equilibrate the membrane filter in isopropanol for 45 min to

Diafiltration remove any wetting/storage agents. Following this, wash the
membrane with water and equilibrate it in the same buffer as
that used in the PEG–protein solution mixture.
2. Add the PEG–protein solution and allow the system to equil-
ibrate before pressurizing.
3. Fill the stirring cell with the feed solution and connect it to a
reservoir containing pure buffer.
4. Pressurize the system with air and monitor the filtrate flux over
time; use small adjustments of the pressure to maintain a con-
stant flux. Samples can be removed periodically from both the
collected filtrate and the stirred cell to analyze the solute concen-
tration by SEC-HPLC in order to detect any loss of protein.
120 Mero et al.
3.3.2. Cationic Exchange 1. Equilibrate a cation ion exchange column with CEX-buffer A
Chromatography (see Note 7) according to the manufacturer’s instructions.
For a TSKgel SP-5PW column (21.5 mm × 15 cm, 10 mm), it
is recommended to use a flow rate of 5–8 mL/min.
2. Load the dialyzed reaction mixture onto the column.
3. Elute the PEGylated products by slowly increasing the elu-
tion gradient with CEX-buffer B.
4. Analyze the eluted fractions for the presence of PEG by per-
forming an iodine assay, and for the presence of protein by
monitoring the UV absorption. Collect and pool the frac-
tions containing the PEGylated products.
5. Concentrate and exchange the buffer against CEX-buffer A.
Keep the solution at 4°C for short-term storage or at −20°C
for long-term storage (see Note 8).
3.3.3. Reversed Phase 1. Connect C4 (or C18) RP-HPLC column to the HPLC system.
HPLC 2. Equilibrate the C4 column (250 × 21.1 mm, 10 mm) with
RP-buffer A at a flow rate of 8 mL/min.
3. A column temperature of ~45°C is recommended for run-
ning the RP-HPLC procedure.
4. Load the dialyzed reaction mixture onto the column at
5–10 mg/mL (total protein concentration).
5. Elute the column initially with RP-buffer A.
6. Continue the elution using a moderately shallow gradient
(1–2% per min) with RP-buffer B (see Note 9).
7. Collect and pool the fractions containing protein (as detected
by monitoring the UV absorbance). Check for the presence
of PEG by performing an iodine assay.
8. Concentrate and exchange the buffer against an appropriate
saline solution and keep at 4°C for short-term storage or
−20°C for long-term storage (see Note 8).
3.3.4. Size-Exclusion 1. Connect a suitable SEC column to the HPLC system.

Chromatography 2. Equilibrate the SEC column with an appropriate saline buffer
for 1 h at a flow rate of 1 mL/min (for a 10 × 300 mm
column).
3. Load the dialyzed reaction mixture at 5–10 mg/mL (total
protein concentration).
4. Elute the product with the same buffer used for column
equilibration.
5. Collect and pool the fractions containing protein (as detected
by monitoring the UV absorbance). Check for the presence
of PEG by performing an iodine assay.
6. Concentrate the product by ultrafiltration and keep at 4°C

for short-term storage or −20°C for long-term storage
(see Note 8).
3.4. Conjugate 1. Prepare a series of unmodified (i.e., non-PEGylated) protein

Characterization samples with known protein concentrations (0.2–1.2 mg/
mL) at a final volume of 50 mL.
3.4.1. BCA Protein Assay
2. Prepare triplicate samples (50 mL each) of the PEGylated pro-
tein with unknown concentrations.
3. To each sample, add 1 mL of BCA working reagent.
4. Incubate the samples for 30 min at 37°C and then read the
absorbance at l = 562 nm.
5. Prepare a standard curve by plotting the measured absorbance
values versus protein concentration. Using the standard curve,
determine the protein concentration of the PEGylated samples.
3.4.2. Ion Exchange 1. Pre-equilibrate the column (7.5 × 75 cm, 5-mm particle size)
Chromatography with CEX-buffer A at a flow rate of 1 mL/min.
2. Load 200 mL of the PEG–protein conjugates (1 mg/mL
protein) dissolved in CEX-buffer A.
3. Elute the products with CEX-buffer B using a shallow elution
gradient.
4. Analyze the eluate with a UV–visible or fluorescence detector
at a suitable wavelength.
5. In case further analysis of the samples is desired (e.g., SDS
electrophoresis, mass spectrometry), collect the fractions
containing the conjugate and use dialysis or ultrafiltration to
change the buffer.
6. Lyophilize the conjugate products and store at −20°C until
use (see Note 8).
3.4.3. Reversed-Phase 1. Pre-equilibrate the column with RP-buffer A at a flow rate of

HPLC 1 mL/min.
2. Load 20 mL of the PEG–protein conjugates (0.1 mg/mL of
protein) solubilized or diluted in RP-buffer A.
3. Elute the products with RP-buffer B using an appropriate
elution gradient (formic acid can be used instead of TFA if a
LC/MS detector is employed).
4. Analyze the eluate with a UV–visible or fluorescence detector
at a suitable wavelength.
5. In case further analysis of the samples is desired (e.g., SDS
electrophoresis, mass spectrometry), collect the fractions
containing the conjugate and use dialysis or ultrafiltration to
change the buffer.
122 Mero et al.
6. Lyophilize the conjugate products and store at −20°C until

use (see Note 8).
3.4.4. Size-Exclusion 1. Equilibrate the size-exclusion column and the refractive index
Chromatography (RI) detector with elution buffer for 1 h at a flow rate of
1 mL/min.
2. Load 20 mL of the PEG–protein conjugates (0.1–0.5 mg/
mL protein).
3. Elute the product with the same elution buffer used to equili-
brate the column in Step 1.
4. To analyze the eluate, the system can be connected to two
channels: a UV–visible or fluorescence detector and a RI
detector.
3.4.5. SDS-Polyacrylamide 1. Run the PEG–protein conjugate sample on an SDS-PAGE

Gel Electrophoresis gel according to the electrophoresis apparatus manufacturer’s
and PEG–Protein Detection instructions.
2. After separating the PEG–protein conjugates by electropho-
resis, soak the resulting SDS-PAGE gel in 20 mL of perchlo-
ric acid (0.1 M) for 15 min.
3. Add 5 mL of BaCl2 solution and 2 mL of iodine solution. The
brown-stained PEG bands should appear within a few minutes.
4. After 10–15 min, replace the staining solution with H2O and
incubate the gel for another 15 min.
5. The iodine-stained gel can also be further stained with
Coomassie blue for the detection of proteins.
3.4.6. Mass Spectrometry 1. Dissolve the PEG–protein conjugate sample (20 mg) in a 0.1%
of PEG–Protein Conjugates (v/v) TFA aqueous solution (5–10 mL).
2. Mix a saturated matrix solution with the PEG–protein sample
solution in the ratio of 1:1 (v/v).
3. Load the mixture (10–20 mL) onto the sample plate and
perform the MS analysis procedure after solvent evaporation.
3.4.7. Determination 1. Separately dissolve the native and PEGylated proteins

of PEGylation Sites (200 mg) in Tris-Gdn buffer (pH 9.0) at a final protein con-
for Proteins Modified centration of 1 mg/mL.
with a Polydisperse PEG 2. Add tris (2-carboxyethyl) phosphine to the protein solution
at a final concentration of 5 mM. Incubate the reaction mix-
ture for 1 h at 37°C.
3. Add iodoacetamide (25 mM) to the reduced protein solu-
tion and incubate the reaction mixture for 30 min at 37°C
in the dark.
4. Purify the protein samples by RP-HPLC using a C18 column.
Dry the collected fractions.
5. Dissolve the reduced and S-carboxamidomethylated samples of

native and PEGylated protein in 8 M urea. Next, dilute the
samples further in phosphate buffer to obtain a final protein con-
centration of 0.8 mg/mL and a final urea concentration of 0.8 M.
6. Add trypsin at an enzyme/substrate (E/S) ratio of 1:50
(w/w) and let the proteolysis reaction proceed at 37°C over-
night (see Note 10).
7. Fractionate (e.g., use an analytical C4 or C18 column) both
the native and PEGylated protein digestion mixtures by
RP-HPLC. Collect the products by monitoring the eluate
by UV absorbance (l = 214 nm).
8. Compare the elution patterns of the peptides obtained from
the modified and native digests. The identity of the peptides
that are missing in the PEGylated protein digest can be estab-
lished by analysis of the corresponding peaks in the non-PEGy-
lated digest. For this purpose, mass spectrometry is employed.
9. The elution pattern obtained from the modified (PEGylated)
protein may sometimes show new peaks corresponding to the
PEGylated peptides. Their identity can be revealed by
MALDI-TOF mass spectrometry, even though this may not
be an easy task (due to the polydispersity of PEG).
3.4.8. Determination Follow Steps 1–6 in Subheading 3.4.7 above, and then continue
of PEGylation Sites with the following procedure:
for Proteins Modified
1. Desalt the native and PEGylated protein digestion mixtures
with a Monodisperse PEG
using a PepClean™ C-18 spin column and analyze the prod-
ucts directly by ESI-MS.
2. For both the native protein and PEGylated protein samples,
identify all the resulting peptide fragments. Some of the pep-
tides obtained in the PEGylated sample may show an increase
in mass corresponding to the conjugation of a single chain of
polymer.
3. If more than one available site for conjugation is present
within the same fragment, a tandem mass spectrometry (MS/
MS) analysis is performed to determine which particular
amino acid is modified.
3.4.9. Degree of Protein 1. Prepare the native (unmodified) protein sample and the
Modification by the Habeeb PEGylated derivative at the same protein concentration (0.2–
Assay 0.8 mg/mL) in phosphate buffer, pH 7.2.
2. Prepare the TNBS reagent and perform the assay in test tubes
(in duplicate) at room temperature according to Table 5.
3. Incubate all the samples in a water bath at 40°C for 2 h, and
then add 250 mL of 10% (w/v) SDS and 125 mL of 1N HCl.
4. Read the absorbance of the solution at l = 335 nm using a
spectrophotometer.
124 Mero et al.
Table 5
Preparation of test solutions for the Habeeb assay
Blank Native protein Pegylated protein
250 mL phosphate pH 7.2 250 mL of protein sample 250 mL of PEGylated sample

250 mL bicarbonate pH 8.5 250 mL bicarbonate pH 8.5 250 mL bicarbonate pH 8.5
250 mL TNBS 250 mL TNBS 250 mL TNBS
Table 6
Preparation of test solutions for the indirect Ellman’s assay
Blank Native protein Pegylated protein
50 mL of Ellman’s reagent 50 mL of Ellman’s reagent 50 mL of Ellman’s reagent

1 mL of phosphate pH 7.2 970 mL of phosphate pH 7.2 970 mL of phosphate pH 7.2
30 mL of protein sample 30 mL of PEGylated sample
The degree (%) of amine substitution is calculated as follows:
% Substitution = [1 - (AP - AB)/(AN - AB)] ´ 100%
AP = Absorbance of the PEGylated protein

AB = Absorbance of the blank
AN = Absorbance of the native protein.
3.4.10. Degree of Cysteine 1. Prepare the native (unmodified) protein sample and the
Modification by the Indirect PEGylated derivative at the same protein concentration
Ellman’s Assay (0.2–0.8 mg/mL) in phosphate buffer, pH 7.2.
2. Prepare the Ellman’s reagent and perform the assay in test
tubes (in duplicate) at room temperature according to
Table 6.
3. Incubate all the samples for 15 min.
4. Read the absorbance at l = 412 nm using a
spectrophotometer.
The percentage of free SH groups is calculated by applying
the following formula:
% Free thiol groups = [(AP - AB)/(AN - AB)] ´ 100
AP = Absorbance of the PEGylated sample

AB = Absorbance of the blank
AN = Absorbance of the native protein.
Table 7
Preparation of test solutions for the iodine assay
Blank Sample
525 mL of Milli-Q water 500 mL of Milli-Q water

250 mL of BaCl2 solution 250 mL of BaCl2 solution
250 mL of iodine solution 250 mL of iodine solution
25 mL of PEG solution
3.4.11. Qualitative Test A rapid, qualitative analysis of the total PEG content in a sample
for the Presence of PEG can be performed as follows:
by the Iodine Assay
1. To a clean tube, add 975 mL of deionized water, 250 mL of
BaCl2 solution, and 250 mL of iodine solution.
2. To the above mixture, add 25 mL of the PEG–protein conju-
gate sample solution.
3. The test is positive if the final mixture forms a dark precipi-
tate, or if it shows increased absorbance at l = 535 nm.
3.4.12. Quantitative Test A quantitative analysis of the total PEG content in a sample can
for the Amount of PEG be performed as follows (see Note 11):
by the Iodine Assay
1. Prepare the blank, PEG standard solutions (0.2–0.5 mg/
mL PEG) and unknown sample solutions according to
Table 7.
2. Incubate the solutions for 15 min and then read the absor-
bance at l = 535 nm.
3. Generate a calibration curve by plotting the measured absor-
bance values (535 nm) versus the known concentration val-
ues of the PEG standards.
4. The amount of PEG present in the unknown sample solu-
tions can be determined from comparison of the measured
absorbance values against the standard curve generated in
Step 3.
4. Notes
1. A limitation of the use of PEG is its hygroscopicity: If not

stored under dry conditions, activated PEGs are easily hydro-
lyzed. It is useful to always verify the degree of activation of
new batches of activated PEGs that have been obtained
126 Mero et al.
commercially. Activation values between 70 and 90% are

acceptable, but they must be taken into consideration when
determining the excess amount of PEG needed for a desired
conversion yield. To minimize the levels of deactivation due
to hydrolysis, store activated PEG reagents at −20°C under
nitrogen and warm the bottle to room temperature before
opening.
2. A major limitation of PEG aldehyde derivatives is their sus-
ceptibility to air oxidation. Low-temperature storage under
an inert atmosphere is mandatory, even if such conditions
may not always be effective.
3. When PEG-OPSS is used, careful attention should be paid to
avoid the presence of any thiol-reducing agents in all steps of
conjugate preparation and purification.
4. When PEG-Mal is used, avoid pH conditions above 7.5 since
at higher pH, reaction with primary amine groups can also
take place (although at a slower rate compared with free thiol
groups).
5. Different aqueous buffers may be employed (e.g., phosphate,
borate, HEPES, etc.), but do not use tris (hydroxymethyl)
aminomethane (Tris) or any other primary amine-containing
buffer components because they will compete with proteins
in the PEG coupling reaction.
6. Dialysis is effective if the outside buffer is often changed and
the dialysis volume is 500- to 1,000-fold greater than the vol-
ume of the sample.
7. The mobile phase in CEX can be a phosphate, acetate, or
citrate buffer solution. Specific buffers are provided in
Subheading 2.3 as examples, but the optimal buffer to use
needs to be considered case-by-case depending on the par-
ticular protein studied.
8. Some proteins are also stable at room temperature provided
that maintenance of sterility is guaranteed.
9. For RP-HPLC buffer B, CH3CN is typically used, but in
some cases a mixture of CH3CN/MeOH or only MeOH may
also be suitable.
10. Different enzymes may be used to digest the protein samples;
trypsin, chymotrypsin, and V8-protease are the most com-
monly employed.
11. Iodine assays for the quantitative determination of PEG must
be performed after purification of the conjugates from free,
unbounded PEG molecules.
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Covalent Conjugation of Poly (Ethylene Glycol) To Proteins and Peptides: Strategies and Methods

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Covalent Conjugation of Poly (Ethylene Glycol) To Proteins and Peptides: Strategies and Methods

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Chapter 8

Covalent Conjugation of Poly(Ethylene Glycol)

PEGylation, the covalent attachment of PEG moieties to a thera-

prominent effect of PEGylation is a prolonged circulation time

1.1. Characterization The development of an efficient and successful PEGylation study

Fig. 1. NMR spectrum of Boc-PEG-NHS in CDCl3.

secondary by-products due to the presence of trace amounts

1.1.4. Activation Degree To determine the degree of activation of thiol-reactive PEG

Fig. 3. Reaction between 5,5¢-dithiobis(2-nitrobenzoic acid) and a free thiol group.

PEG-NHS ester Active groups structure Half-life (min)

PEG–(CH2)4–CO2–NHS Succinimidyl valerate (SVA) 33.6

which is based on the use of 5,5¢-dithiobis (2-nitrobenzoic acid)

1.1.5. Reactivity The reactivity of N-hydroxysuccinimide (NHS)-activated PEGs

PEGs depends upon the nature of the chemical group adjacent to

Fig. 4. MALDI-TOF mass spectrum of PEG 6,000 Da.

MS analysis, reversed-phase high performance liquid chromatography

is also widely used, and it forms a disulfide linkage with cysteine.

1.2.4. PEGylation The direct coupling of PEG-NH2 to activated protein carboxylic

1.2.6. Selective Although proteins typically present a defined structural confor-

Fig. 7. Conjugation of PEG to proteins containing aldehydic groups.

In fact, several parameters such as the solvent, PEG/peptide

1.2.8. Glutamine As an alternative to chemical conjugation, promising selective

Fig. 8. PEGylation mediated by transglutaminase (TGase).

1.3. PEG-Conjugate Usually PEG coupling reactions, especially random amino

In Subheadings 3.3 and 3.4, we describe several protocols that

PEG–protein complexes with a large surface area. Consequently,

1.3.2. Reversed-Phase Reversed-phase high performance chromatography (RP-HPLC) is

1.3.3. Size-Exclusion Size-exclusion chromatography (SEC) separates molecules based

is a low-throughput technique with relatively high costs, and thus

1.3.4. Ultrafiltration/ One particularly important nonchromatographic step in the

and lower electrostatic interactions of PEG-conjugates with

Thermo Fisher, etc.); in these assays, the quantitative determination

1.4.3. Reversed-Phase RP-HPLC is a useful technique for the determination of purity

Several brands of gel filtration columns are commercially

can usually be employed for such analyses, but attention must be

1.4.8. Simplified Method The method described above in Subheading 1.4.8 is an “indirect”

interest mediated by the enzyme TGase (63). ESI-MS and tandem

2.1. Characterization 1. PEG reagents (see Note 1).

2.3. Conjugate 1. CEX-buffer A: 10 mM phosphate buffer, 10 mM NaCl, pH 4.7.

Blank PEG reaction mixture Gly-Gly standard solution

20 mL of TNBS 20 mL of TNBS 20 mL of TNBS

3.1.3. Evaluation 1. Prepare Sample A (PEG reaction mixture) as follows: To 1 mL

3.1.4. Evaluation 1. Prepare Sample A (PEG reaction mixture) as follows: To

Blank PEG reaction mixture Cysteine standard solution

50 mL of Ellman’s reagent 50 mL of Ellman’s reagent 50 mL of Ellman’s reagent

The percentage of activation of thiol-reactive PEGs is

AbsA = Absorbance of PEG reaction mixture

3.1.5. Half-Life 1. Prepare a solution of NHS-activated PEG (0.2–0.5 mM) in

3.1.7. Analysis 1. Dissolve PEG-aldehyde (0.25–1 mM) in water.

3. Fractionate the resulting reaction mixture by RP-HPLC on

Aqueous, pH 8.5 10 21 5 1–3 85 (42)

7. Dialyze the resulting solution to eliminate low-molecular

3.2.2. PEGylation at the 1. Prepare a protein solution at 1–5 mg/mL (as determined by

3.2.3. Thiol PEGylation 1. Prepare a protein solution of 1–5 mg/mL in 0.1 M phosphate-

3. Incubate the reaction mixture at 4°C for 24 h under gentle

3.2.5. PEGylation 1. Prepare a solution of protein containing N-terminal serine or

7. Dialyze the solution against water to remove the organic

3.2.8. Enzymatic 1. Prepare a protein solution at 1–5 mg/mL in 0.1 M phos-

3.3.1. Ultrafiltration/ 1. Equilibrate the membrane filter in isopropanol for 45 min to

3.3.4. Size-Exclusion 1. Connect a suitable SEC column to the HPLC system.

6. Concentrate the product by ultrafiltration and keep at 4°C

3.4. Conjugate 1. Prepare a series of unmodified (i.e., non-PEGylated) protein

3.4.3. Reversed-Phase 1. Pre-equilibrate the column with RP-buffer A at a flow rate of