Chemistry for peptide and protein PEGylation

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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.

Introduction

The use of proteins and peptides as human therapeutics 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 undergoing clinical trials, and the estimated annual growth rates of protein products (glycoproteins, unglycosylated proteins and antibodies) will range from 10 to 35% [1].

Although more biopharmaceuticals are in development 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 albumin, (3) incorporation into drug delivery vehicles for protection and slow release, and (4) conjugating to natural or synthetic polymers [2], [3], [4], [5], [6].

Those in the biomedical, biotechnical and pharmaceutical 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 attached 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, 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 molecular weight mPEG, unstable linkages and lack of selectivity in modification. Readers are referred to several detailed reviews on different aspects of PEGylation [7], [8], [9], [10], [11]. In this paper, we review chemistries 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.

Section snippets

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 structure:HO–(CH2CH2O)n–CH2CH2–OH

PEG is synthesized by anionic ring opening polymerization of ethylene oxide initiated by nucleophilic attack of a hydroxide ion on the epoxide ring. Most useful for polypeptide modification is monomethoxy PEG, mPEG, having the general structure:CH3O–(CH2CH2O)n–CH2CH2–OH

Monomethoxy PEG is synthesized by anionic ring opening

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 functional group at one or both termini. The functional group is chosen based on the type of available reactive group on the molecule that will be coupled to the PEG. For proteins, typical reactive amino acids include lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine, threonine,

Conclusions

The array of PEG chemistries reviewed here are among dozens being used for clinical development of PEGylated peptides and proteins. The transition from first-generation chemistries to second-generation chemistries is taking place at a rapid pace and future demands for PEG reagents will lead to new reagents for novel applications in the biopharmaceutical industry. Novel PEG chemistry for site-specific modification, as well as control of PK/PD parameters, will be synthesized when the needs arise.

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