Advanced Drug Delivery Reviews 54 (2002) 459–476
`
`www.elsevier.com / locate / drugdeliv
`
`Chemistry for peptide and protein PEGylation
`*
`M.J. Roberts , M.D. Bentley, J.M. Harris
`
`Shearwater Corporation,490 Discovery Drive, Huntsville, AL 35806, USA
`
`Received 17 December 2001; accepted 22 January 2002
`
`Abstract
`
`Poly(ethylene glycol) (PEG) is a highly investigated polymer for the covalent modification of biological macromolecules
`and surfaces for many pharmaceutical and biotechnical applications. In the modification of biological macromolecules,
`peptides and proteins are of extreme importance. Reasons for PEGylation (i.e. the covalent attachment of PEG) of peptides
`and proteins are numerous and include shielding of antigenic and immunogenic epitopes, shielding receptor-mediated uptake
`by the reticuloendothelial system (RES), and preventing recognition and degradation by proteolytic enzymes. PEG
`conjugation also increases the apparent size of the polypeptide, thus reducing the renal filtration and altering biodistribution.
`An important aspect of PEGylation is the incorporation of various PEG functional groups that are used to attach the PEG to
`the peptide or protein. In this paper, we review PEG chemistry and methods of preparation with a particular focus on new
`(second-generation) PEG derivatives, reversible conjugation and PEG structures.
`2002 Elsevier Science B.V. All rights
`reserved.
`
`Keywords: PEGylation; PEG-protein; PEG conjugation; PEG chemistry
`
`Contents
`
`1. Introduction ............................................................................................................................................................................
`2. Properties of PEG ...................................................................................................................................................................
`3. Chemistry of pegylation...........................................................................................................................................................
`3.1. First-generation PEG chemistry.........................................................................................................................................
`3.1.1. PEG chemistry for amine conjugation......................................................................................................................
`3.2. Second-generation PEGylation chemistry...........................................................................................................................
`3.2.1. PEG chemistry for amine conjugation......................................................................................................................
`3.2.2. PEG chemistry for cysteine modification .................................................................................................................
`3.2.3. PEG chemistry for oxidized carbohydrates or N-terminus .........................................................................................
`3.2.4. PEG chemistry for reversible PEGylation ................................................................................................................
`3.2.5. Heterobifunctional PEG chemistry ..........................................................................................................................
`3.3. PEG structures .................................................................................................................................................................
`4. Conclusions ............................................................................................................................................................................
`References ..................................................................................................................................................................................
`
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`462
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`466
`467
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`473
`474
`474
`
`*Corresponding author. Tel.: 11-256-704-7524; fax: 11-256-533-4201.
`E-mail address: mroberts@shearwatercorp.com (M.J. Roberts).
`
`0169-409X / 02 / $ – see front matter
`P I I : S 0 1 6 9 - 4 0 9 X ( 0 2 ) 0 0 0 2 2 - 4
`
`2002 Elsevier Science B.V. All rights reserved.
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`1. Introduction
`
`The use of proteins and peptides as human thera-
`peutics has expanded in recent years due to: (1)
`discovery of novel peptides and proteins, (2) a better
`understanding of the mechanism of action in vivo,
`(3)
`improvements in expression or synthesis of
`proteins and peptides that closely resemble fully
`human proteins and peptides, and (4) improvements
`in formulation or molecule-altering technologies that
`have the ability to deliver polypeptides in vivo with
`improved pharmacokinetic and pharmacodynamic
`properties. It was estimated that in the year 2000, as
`many as 500 biopharmaceutical products were under-
`going clinical trials, and the estimated annual growth
`rates
`of
`protein
`products
`(glycoproteins,
`un-
`glycosylated proteins and antibodies) will range from
`10 to 35% [1].
`Although more biopharmaceuticals are in develop-
`ment than ever before, many of these have problems
`that are typical of polypeptide therapeutics, including
`short
`circulating
`half-life,
`immunogenicity,
`proteolytic degradation, and low solubility. Several
`strategies have emerged as ways to improve the
`pharmacokinetic and pharmacodynamic properties of
`biopharmaceuticals, including: (1) manipulation of
`amino acid sequence to decrease immunogenicity
`and proteolytic cleavage, (2) fusion or conjugation to
`immunoglobulins and serum proteins, such as al-
`bumin, (3) incorporation into drug delivery vehicles
`for protection and slow release, and (4) conjugating
`to natural or synthetic polymers [2–6].
`Those in the biomedical, biotechnical and pharma-
`ceutical communities have become quite familiar
`with the improved pharmacological and biological
`properties that are associated with the covalent
`attachment of poly(ethylene glycol) or PEG to
`therapeutically useful polypeptides. For
`instance,
`PEG conjugation can shield antigenic epitopes of the
`polypeptide, thus reducing reticuloendothelial (RES)
`clearance and recognition by the immune system and
`also reducing degradation by proteolytic enzymes.
`PEG conjugation also increases the apparent size of
`the polypeptide,
`thus reducing renal filtration and
`altering biodistribution. Contributing factors that
`affect the foregoing properties are: (1) the number of
`PEG chains attached to the polypeptide, (2) the
`molecular weight and structure of PEG chains at-
`
`tached to the polypeptide, (3) the location of the
`PEG sites on the polypeptide and (4) the chemistry
`used to attach the PEG to the polypeptide.
`The importance of chemistry and quality of PEG
`reagents for peptide and protein modification has
`only been realized in the last several years as more
`and more PEG-conjugates have reached late phase
`clinical trials. The first few PEG-protein products,
`(cid:210) (cid:210)
`now on the market (Adagen , Oncospar , and PEG-
`Intron ), were developed using first generation PEG
`chemistry. One characteristic of first generation PEG
`chemistry is the use of low molecular weight linear
`PEGs ( # 12 kDa) with chemistry that may result in
`side reactions or weak linkages upon conjugation
`with polypeptides.
`The next generation of PEG-protein therapeutics,
`which will come to market in the next several years,
`uses second-generation PEG chemistries. Second-
`generation PEGylation was designed to avoid the
`problems of first generation chemistry, notably
`diactivated PEG impurities, restriction to low molec-
`ular weight mPEG, unstable linkages and lack of
`selectivity in modification. Readers are referred to
`several detailed reviews on different aspects of
`PEGylation [7–11]. In this paper, we review chemis-
`tries of both first- and second-generation, with an
`emphasis on newer PEGylation technologies,
`in
`order to provide an introduction to those chemistries
`that will be used in the following reviews.
`
`2. Properties of PEG
`
`In its most common form poly(ethylene glycol),
`PEG, is a linear or branched polyether terminated
`with hydroxyl groups and having the general struc-
`ture:
`
`HO–(CH CH O) –CH CH –OH
`2
`2
`2
`2
`n
`
`PEG is synthesized by anionic ring opening
`polymerization of ethylene oxide initiated by nu-
`cleophilic attack of a hydroxide ion on the epoxide
`ring. Most useful for polypeptide modification is
`monomethoxy PEG, mPEG, having the general
`structure:
`
`CH O–(CH CH O) –CH CH –OH
`3
`2
`2
`2
`2
`n
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`461
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`Monomethoxy PEG is synthesized by anionic ring
`opening polymerization initiated with methoxide
`ions. Commercially available mPEG contains a
`considerable amount of diol PEG due to the presence
`of trace amounts of water during polymerization.
`This diol PEG is also of relatively high molecular
`weight due to polymerization at both ends of the
`polymer. The amount of diol PEG can exceed 15%
`of the composition of mPEG. A solution to the
`problem of diol contamination has been developed in
`our laboratories [12]. In this work, a crude benzylox-
`y-PEG, containing diol impurity, is methylated and
`then hydrogenated to remove the benzyl group. Thus
`diol is converted to the inert dimethyl ether, which
`can be subsequently removed after activation and
`polypeptide attachment.
`BzO–PEG–OH 1 HO–PEG–OH fi fi
`OCH 1 CH O–PEG–OCH
`3
`3
`
`HO–PEG–
`
`3
`
`is to
`Another common route to remove diol
`convert the PEGs to PEG-carboxylic acids that can
`then be purified by ion-exchange chromatography
`[13]. PEG with various end groups can be prepared
`by use of suitable initiator and / or termination re-
`agents. Numerous functionalities can be introduced
`as end groups on PEG in this manner,
`including
`heterobifunctional products. For instance, Kataoka et
`al. synthesized a heterobifunctional PEG derivative
`containing aldehyde and thiol end groups [14].
`Polymerization was initiated with 3,3-diethoxy-1-
`propanol, which forms a propionaldehyde after acid
`hydrolysis, and the polymerization was terminated
`with methansulfonyl chloride with successive con-
`version to ethyldithiocarbonate and a free thiol.
`Compared with other polymers, PEG has a rela-
`tively narrow polydispersity (M /M ) in the range of
`w
`n
`1.01 for low molecular weight PEGs (,5 kDa) to
`1.1 for high molecular weight PEGs (.50 kDa).
`The unique ability of PEG to be soluble in both
`aqueous solutions and organic solvents makes it
`suitable for end group derivatization and chemical
`conjugation to biological molecules under mild
`physiological conditions. Studies of PEG in solution
`have shown that PEG typically binds 2–3 water
`molecules per ethylene oxide unit. Due to both the
`high flexibility of
`the backbone chain and the
`binding of water molecules, the PEG molecule acts
`
`as if it were five to 10 times as large as a soluble
`protein of comparable molecular weight. These
`factors have been suggested as the reason that PEG
`exhibits the ability to precipitate proteins [15],
`exclude proteins and cells from surfaces [16], reduce
`immunogenicity and antigenicity [17] and prevent
`degradation by mammalian cells and enzymes [18].
`Low molecular weight oligomers of PEG (,400
`Da) have been shown to be degraded in vivo by
`alcohol dehydrogenase to toxic metabolites, however
`the lack of toxicity of PEGs with a molecular weight
`above 1000 Da has been revealed over many years of
`use in foods, cosmetics and pharmaceuticals [18].
`PEG is rapidly cleared in vivo without structural
`change and clearance is dependent on molecular
`weight. Below a molecular weight of about 20 kDa
`the molecule is cleared in the urine, and higher
`molecular weight PEGs are cleared more slowly in
`the urine and feces. PEG is only weakly immuno-
`genic even at high molecular weights. Antibodies to
`PEG have been generated when attached to a highly
`immunogenic molecule under
`an immunization
`protocol with Freund’s adjuvant [19–21]. There are
`no known situations in which anti-PEG antibodies
`have been generated under ‘normal’ clinical adminis-
`tration of a PEG-modified protein.
`
`3. Chemistry of pegylation
`
`To couple PEG to a molecule (i.e. polypeptides,
`polysaccharides, polynucleotides and small organic
`molecules) it is necessary to activate the PEG by
`preparing a derivative of the PEG having a func-
`tional 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.
`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-termi-
`nal amino acid groups. Lysine is one of the most
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`prevalent amino acids in proteins and can be up-
`wards 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 that 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. For
`a protein molecule that has a single PEG attached by
`this nonspecific modification method,
`the poly-
`ethylene glycol moiety may be attached at a number
`of different amine sites. Therefore there is the
`potential for a large number of positional isomers (P)
`as the degree of modification increases:
`
`N!
`P 5 (N 2 k)! 3 k!
`]]]]
`
`where N is the number of possible sites and k is the
`number of sites modified. The extent of modification
`is important
`in determining the pharmacological
`properties of the bioconjugate. Typically, a higher
`degree of modification will extend the circulation
`half-life and reduce the likelihood of antigenicity
`[22]. Each positional isomer of the heterogeneous
`mixture is likely to have an influence on whether the
`conjugate is active or whether an antibody will bind
`an antigenic epitope. The heterogeneity in lysine
`substitution and in PEG molecular weights is of
`some concern for PEG-protein pharmaceuticals, and
`it
`is generally necessary to demonstrate that
`the
`pattern for a particular pharmaceutical can be mea-
`sured and is reproducible. Many of the important
`benefits of PEGylation can be controlled by proper
`conjugation of various molecular weight PEGs to the
`protein at specific locations on the protein’s surface.
`The monofunctionality of methoxyPEG makes it
`particularly suitable for protein and peptide modi-
`fication because it yields reactive PEGs that do not
`produce crosslinked polypeptides, as long as diol
`PEG has been removed. As we will see in the
`discussion of second generation PEGylation,
`it
`is
`also possible in some instances to reduce or elimi-
`nate heterogeneity in the position of substitution.
`
`3.1. First-generation PEG chemistry
`
`3.1.1. PEG chemistry for amine conjugation
`Since most applications of PEG conjugation in-
`volve labile molecules, the coupling reactions require
`mild chemical conditions. In the case of polypep-
`tides, the most common reactive groups involved in
`coupling are the alpha or epsilon amino groups of
`lysine. In Fig. 1 is listed a wide range of first
`generation PEG derivatives used for protein PEGyla-
`tion of either the alpha or epsilon amino groups.
`First-generation chemistries are generally plagued by
`PEG impurities, restriction to low molecular weights,
`unstable linkages, and lack of selectivity in modi-
`fication. Examples of first-generation PEG deriva-
`tives include: (a) PEG dichlorotriazine, (b) PEG
`tresylate, (c) PEG succinimidyl carbonate, (d) PEG
`benzotriazole carbonate, (e) PEG p-nitrophenyl car-
`bonate, (f) PEG trichlorophenyl carbonate, (g) PEG
`carbonylimidazole and (h) PEG succinimidyl succi-
`nate.
`The initial work of Davis et al. used cyanuric
`chloride to prepare activated PEG for attachment to
`proteins [6,17]. The PEG dichlorotriazine (Fig. 1a)
`derivative can react with multiple nucleophilic func-
`tional groups such as lysine, serine, tyrosine, cys-
`teine, and histidine, which results in displacement of
`one of the chlorides and produces a conjugate with
`retained charge in the form of a secondary amine
`linkage [23]. The remaining chloride is less suscep-
`tible to reactions with nucleophilic residues. Un-
`fortunately,
`the reactivity is sufficient
`to allow
`crosslinking of protein molecules containing addi-
`tional nucleophilic residues. To solve this problem,
`Inada et al. synthesized 2,4-bis(methoxypolyethylene
`glycol)-6-chloro-s-triazine
`(mPEG -chlorotriazine)
`2
`as shown in Fig. 2 [24]. The lower reactivity of the
`remaining chlorine translates into a more selective
`modification of lysine and cysteine residues without
`further side reactions.
`Another alkylating reagent used to nonspecifically
`modify multiple amino groups to form secondary
`amine linkages to proteins, viruses and liposomes is
`PEG tresylate (Fig. 1b) [25]. Although more specific
`to amino groups than PEG dichlorotriazine,
`the
`chemistry of conjugation and the conjugation prod-
`ucts are not unique and well defined. For example,
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`
`463
`
`Fig. 1. First-generation amine reactive PEG derivatives.
`
`Fig. 2. Branched PEG (PEG2) based on PEG-triazine.
`
`Gais et al. have shown that PEG-tresylate conjuga-
`tion to small molecule amines can produce a product
`that contains a degradable sulfamate linkage [26].
`Therefore, a heterogeneous mixture that results from
`
`attaching PEG-tresylate to proteins may contain a
`population of conjugates with degradable linkages.
`Most first-generation PEG chemistries are those
`that produce conjugates through acylation. Two
`widely used first-generation activated mPEGs are
`succinimidyl carbonate (SC-PEG in Fig. 1c) [27,28]
`and benzotriazole carbonate (BTC-PEG in Fig. 1d)
`[29]. SC-PEG and BTC-PEG react preferentially
`with lysine residues to form a carbamate linkage, but
`are also known to react with histidine and tyrosine
`residues. SC-PEG is slightly more stable to hy-
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`drolysis than BTC-PEG with a half-life of 20.4 min
`at pH 8 and 25 8C compared to the 13.5 min
`hydrolysis half-life of BTC-PEG under the same
`conditions [30]. It has recently been observed that
`SC-PEG and BTC-PEG couple to histidine residues
`of a-interferon at slightly acidic conditions to form a
`hydrolytically unstable imidazolecarbamate linkage
`[31]. The weak linkage could be used to advantage
`in preparation of controlled-release formulation, or it
`could be a disadvantage if conjugate instability were
`not desired.
`Other PEG acylating reagents which produce
`urethane linked proteins include p-nitrophenyl car-
`bonate (pNPC-PEG in Fig. 1e), trichorophenyl car-
`bonate (TCP-PEG in Fig. 1f) and carbonylimidazole
`(CDI-PEG in Fig. 1g) [32,33]. These reagents are
`prepared
`by
`reacting
`chloroformates
`or
`car-
`bonylimidazole with the terminal hydroxyl group on
`mPEG, and these have much lower reactivity than
`either the SC-PEG or BTC-PEG. Generally,
`the
`slower the reaction the more specific the reagent is to
`certain amino acid groups of the protein. In this way,
`some selectivity is achieved. The extent and rate of
`modification can easily be followed in the case of
`pNPC-PEG and TCP-PEG by monitoring the
`phenolate-ion leaving-group by colorimetric analysis.
`The remaining first-generation PEG reagent
`is
`succinimidyl succinate (SS-PEG in Fig. 1h) [34].
`SS-PEG is prepared by reaction of mPEG with
`succinic anhydride, followed by activation of the
`carboxylic acid to the succinimidyl ester. The poly-
`mer backbone contains a second ester linkage that
`remains after the conjugation reaction with a protein.
`This linkage is highly susceptible to hydrolysis after
`the polymer has been attached to the protein. Not
`only does this hydrolysis lead to loss of the benefits
`of PEG attachment, but the succinate tag that re-
`mains on the protein after hydrolysis can act as a
`hapten and lead to immunogenicity of the remaining
`protein [35].
`Techniques used to form first generation PEG
`derivatives are generally straightforward and involve
`reacting the PEG polymer with a group that
`is
`reactive with hydroxyl groups, typically anhydrides,
`chlorides, chloroformates and carbonates. With the
`exception of
`the work by Bentley et al.,
`these
`techniques lack the ability to produce pure mono-
`functional PEG derivatives of high molecular weight
`
`[12]. Since the diol content of high molecular weight
`PEGs can reach 15%, high-molecular-weight, first-
`generation PEG chemistry is inefficient for protein
`conjugation. The ability to generate an intermediate
`that can be purified from diactivated species renders
`second-generation chemistry a valuable tool
`for
`protein modification.
`
`3.2. Second-generation PEGylation chemistry
`
`3.2.1. PEG chemistry for amine conjugation
`Second-generation PEGylation chemistry has been
`designed to avoid the above noted problems of diol
`contamination, restriction to low molecular weight
`mPEG, unstable linkages, side reactions and lack of
`selectivity in substitution. One of the first examples
`of second-generation chemistry is mPEG-propional-
`dehyde [36]. mPEG-propionaldehyde is easier to
`prepare and use than PEG-acetaldehyde because the
`acetaldehyde is very susceptible to dimerization via
`aldol condensation. A key property of mPEG-
`priopionaldehyde, as disclosed by Kinstler et al. in
`work on PEGylation of G-CSF, sTNF-RI, and con-
`sensus IFN, is that under acidic conditions (approxi-
`mately pH 5), aldehyde is largely selective for the
`N-terminal a-amine because of the lower pK of the
`a
`a-amine compared to other nucleophiles [37–39].
`The conjugation of electrophilic PEGs to amino acid
`residues on proteins is highly dependent on the
`nucleophilicity of each amino acid residue. Nu-
`cleophilic attack will only take place when the pH of
`the protein solution is near or above the residue’s
`pK . Therefore the reactivity of each residue also
`a
`depends on neighboring amino acid residues. Al-
`though complete selectivity is not observed,
`the
`extensive heterogeneity frequently seen with lysine
`chemistry is greatly reduced. Coupling of aldehydes
`to primary amines proceeds through a Schiff base,
`which is reduced in situ to give a stable secondary
`amine linkage as shown in Fig. 3.
`An alternative approach to using PEG-aldehyde is
`to use the acetal derivative of PEG-propionaldehyde
`or PEG-acetaldehyde [40]. The aldehyde hydrate of
`the acetal derivatives can be generated in situ by acid
`hydrolysis (Fig. 4). The pH of the solution can then
`be adjusted to values sufficient for protein modi-
`fication with the same mechanism as the free alde-
`hyde derivative in Fig. 3. The benefit of using the
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`465
`
`Fig. 3. Reductive amination using PEG-propionaldehyde.
`
`Fig. 4. In situ generation of PEG-aldehyde hydrate for use in reductive amination.
`
`acetal derivative over the free propionaldehyde or
`acetaldehyde is longer storage stability and higher
`purity.
`Active esters of PEG carboxylic acids are the most
`used acylating agents for protein modification. Ac-
`tive esters react with primary amines near physiolog-
`ical conditions to form stable amides as shown in
`Fig. 5. Generating the carboxylic acid intermediate
`allows the PEG to be purified from unsubstituted or
`disubstituted impurities by ion-exchange chromatog-
`raphy [41]. Purities of greater than 97% are routinely
`obtainable by this method. Activation of PEG-car-
`boxylic acids to the succinimidyl active esters is
`accomplished by reacting the PEG-carboxylic acid
`with N-hydroxysuccinimide (NHS or HOSu) and a
`carbodiimide.
`The first carboxylic acid derivative of PEG not
`containing a degradable linkage to the PEG back-
`bone, as in SS-PEG, was carboxymethylated PEG
`
`this
`[42]. The succinimidyl ester of
`(CM-PEG)
`compound (SCM-PEG) is extremely reactive (hy-
`drolysis t
`of 0.75 min at pH 8 and 25 8C) and is
`1 / 2
`therefore difficult to use. To take advantage of the
`intermediate purification step and have an active
`ester that had more favorable kinetics for protein
`modification, Harris et al. prepared propionic acid
`(PEG–O–CH CH –COOH)
`and
`butanoic
`acid
`2
`2
`(PEG–O–CH CH CH –COOH) derivatives of PEG
`2
`2
`2
`(Fig. 5(1)) [13]. Changing the distance between the
`active ester and the PEG backbone by the addition of
`methylene units had a profound influence on the
`reactivity towards amines and water. For example,
`SBA-PEG, which has two additional methylene
`groups, has a longer hydrolysis half-life of 23 min at
`pH 8 and 25 8C. SPA-PEG, which has one additional
`methylene group, has a hydrolysis half-life of 16.5
`min at pH 8 and 25 8C.
`Reactivity of PEG active esters towards amines
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`
`Fig. 5. PEG NHS esters. (1) PEG NHS esters based on propionic and butanoic acids and (2) a-branched PEG NHS esters based on
`propionic and butanoic acids.
`
`and water can be further decreased by introducing an
`a-branching moiety to the carboxylic acid as shown
`in Fig. 5(2). An a-methyl branched PA-PEG deriva-
`tive has a hydrolysis half-life of 33 min at pH 8 and
`25 8C.
`
`3.2.2. PEG chemistry for cysteine modification
`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 [43].
`The advantage of this approach is that
`it makes
`possible site-specific PEGylation at areas on the
`protein that will minimize a loss in biological
`activity but decrease immunogenicity. This strategy
`is not without its shortcomings. The addition of free
`cysteines by genetic engineering increases the possi-
`bility of incorrect disulfide formation and protein
`dimerization.
`PEG derivatives such as PEG-maleimide (Fig.
`6(1)), vinylsulfone (Fig. 6(2)), iodoacetamide (Fig.
`6(3)), and orthopyridyl disulfide (Fig. 6(4)) have
`been developed for PEGylation of cysteine residues,
`with each derivative having its own advantages and
`disadvantages [43–46]. PEG-vinylsulfone (PEG-VS)
`reacts slowly with thiols to form a stable thioether
`linkage to the protein at slightly basic conditions (pH
`7–8) but will proceed faster if the pH is increased.
`
`Although PEG-VS is stable in aqueous solutions, it
`may react with lysine residues at elevated pH. Unlike
`PEG-VS, PEG-maleimide (PEG-MAL) is more reac-
`tive to thiols even under acidic conditions (pH 6–7),
`but it is not stable in water and can undergo ring
`opening or addition of water across the double bond.
`PEG-iodoacetamide (PEG-IA)
`reacts slowly with
`free thiols by nucleophilic substitution, creating a
`stable thioether linkage. The reaction should be done
`in slight molar excess of PEG-IA in a dark container
`to limit the generation of free iodine that may react
`with other amino acids. The thioether linkage be-
`tween the PEG-MAL and protein is stable, but slow
`cleavage of one of the amide linkages can occur by
`hydrolysis. Orthopyridyl disulfide-PEG (PEG-OPSS)
`reacts specifically with sulfhydryl groups under both
`acidic and basic conditions (pH 3–10) to form a
`disulfide bond with the protein. Disulfide linkages
`are also stable, except in a reducing environment
`when the linkage is converted to thiols.
`Scientists in our laboratories recently prepared a
`highly active, long circulating and stable conjugate
`of IFN-b using a two-step method with PEG-OPSS
`[47]. The tertiary structure of IFN-b was determined
`by Karpusas et al. who showed that the free cysteine
`residue at position 17 was proximal to the surface
`but hidden [48]. In this case, the available thiol was
`not accessible to high molecular weight PEG that
`would be needed for improved pharmacokinetics.
`The approach that was ultimately adopted was to
`couple a low molecular weight di-OPSS PEG (Mw
`2000) to the interferon and then couple a PEG thiol
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`MPI EXHIBIT 1044 PAGE 8
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`MPI EXHIBIT 1044 PAGE 8
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`M.J. Roberts et al. / Advanced Drug Delivery Reviews 54(2002)459–476
`
`467
`
`Fig. 6. Thiol reactive PEGs. (1) PEG maleimide, (2) PEG vinyl sulfone, (3) PEG iodoacetamide, and (4) PEG orthopyridyl disulfide.
`
`to the remaining terminal OPSS group. The disulfide
`linkage between PEG and the protein was found to
`be stable in plasma circulation (unpublished data).
`
`3.2.3. PEG chemistry for oxidized carbohydrates
`or N-terminus
`Oxidation of carbohydrate residues or N-terminal
`serine or threonine is an alternative method for
`site-directed PEGylation of proteins. Carbohydrates
`can be oxidized with enzymes, such as glucose
`oxidase, or chemically with sodium periodate. Oxi-
`dation of the carbohydrate residues generates multi-
`ple reactive aldehyde groups, which can be reacted
`with either PEG-hydrazide to produce a hydrazone
`linkage or with PEG-amine to produce a reversible
`Schiff base (Fig. 7) [49]. The hydrazone linkage may
`be reduced with sodium cyanoborohydride to a more
`stable alkyl hydrazide and the Schiff’s base may be
`reduced to form a secondary amine. Reductive
`alkylation with PEG-amine is problematic because
`the amino groups of a protein possess similar
`reactivity to PEG-amines and thus may form cross-
`
`linked aggregates. PEG-hydrazides are more useful
`in these situations. Under acidic conditions (approx.
`pH 5), amino groups of the protein are predominant-
`ly protonated, but because the PEG-hydrazide is a
`weaker base (pK approx. 3) than primary amines
`a
`(pK approx. 10),
`the reaction is selective to the
`a
`PEG-hydrazone formation. Multiple attachment sites
`are generated using this method, but the modification
`site is specific to the carbohydrate.
`Another approach to site-specific conjugation is to
`take advantage of the presence of a N-terminal serine
`or threonine, which can be converted by periodate
`oxidation to a glyoxylyl derivative. Gaertner et al.
`oxidized the N-terminal serine of IL-8 to form a
`glyoxylyl derivative, which they conjugated to
`aminooxy and hydrazide PEG derivatives [50].
`
`3.2.4. PEG chemistry for reversible PEGylation
`Most PEGylation chemistry is designed to create a
`conjugate that contains a stable linkage to the
`protein. In most cases having a stable linkage to the
`protein is beneficial because of the suitability for
`
`MPI EXHIBIT 1044 PAGE 9
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`468
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`M.J. Roberts et al. / Advanced Drug Delivery Reviews 54(2002)459–476
`
`Fig. 7. Attachment of poly(ethylene glycol) to oxidized carbohydrates of glycoproteins.
`
`long-term storage, easier purification and availability
`of prefilled syringes. It is also generally observed
`that stable linkages to a protein can reduce the
`activity, possibly due to the presence of the PEG
`chain at the active or binding site of the protein or
`steric crowding at the active or binding site. Also the
`PEG molecular weight has a direct impact on the
`activity; higher molecular weight PEG conjugates
`tend to have lower in vitro activity but have higher in
`vivo activity due to the improved pharmacokinetic
`profile [51]. The objective of most PEG conjugation
`techniques is to increase the circulation half-life
`without altering activity. In the development of PEG-
`Intron , Enzon used a degradable linkage between
`the PEG and protein to improve the pharmacokinetic
`half-life but minimize loss of activity by releasing
`native
`interferon alpha-2b [52]. PEG-Intron is
`formed by conjugation of PEG-SC to interferon
`alpha-2b at low pH (around pH 5). The conjugation
`leads to a population of PEG conjugates coupled to
`34His
`(Fig. 8). In this case, the PEG is coupled to the
`d1N position of the imidazole ring in histidine to
`form a carbamate linkage and the PEG was found to
`be released from the protein over time. Note should
`be taken when comparing PEG-Intron to the branch-
`ed
`PEG
`-interferon
`alpha-2a
`conjugate
`40 kDa
`(Pegasys ) that the PEG-Intro