A number of peptides have been used to effectively treat diseases, such as Adagen (Sigma-Tau Pharmaceuticals), which treats severe combined immunodeficiency. However, the use of peptides continues to be hampered by their extremely short half-life. Peptides are small and are often cleared by the kidneys or mononuclear phagocyte system within minutes of administration (1). They are also susceptible to degradation by proteolytic enzymes.

The problems associated with peptides can be overcome by PEGylation, that is, linking peptides to polyethylene glycol (PEG). Once linked to a peptide, each PEG subunit becomes tightly associated with two or three water molecules, which have the dual function of rendering the peptide more soluble in water and making its molecular structure larger. As the kidneys filter substances according to size, the addition of PEG's molecular weight prevents the premature renal clearance undergone by small peptides. PEG's globular structure also acts as a shield to protect the peptide from proteolytic degradation, and reduces the immunogenicity of foreign peptides by limiting their uptake through the dendritic cells (1, 2). PEG itself is not immunogenic or toxic, and allows for lower doses and less-frequent administrations. In some instances, PEG can increase the circulating half-life of a peptide drug by more than 100 times (1, 3). This saves money and resources, promotes patient compliance, and reduces the development of toxicity, tolerance and allergic reactions compared with their non-PEGylated counterparts (4). In addition to improving the pharmacokinetic and pharmacodynamic properties of peptide drugs once inside the body, PEGylation can also aid drug delivery because PEGylated petides act as permeation enhancers for nasal drug delivery. Synthetic PEGylated glycoproteins have been used in lieu of viruses for targeted gene delivery (5,6).

Despite the benefits associated with PEGylation, there are many factors that must be considered, including the peptide's primary and secondary structures, molecular weight, the number and placement of the linked PEG moieties, PEG's size and shape, and conjugation chemistry. All of these can impact the final product's physical, chemical, and biological properties.

Originally, monomethoxy PEG (mPEG), which has relatively clean chemistry because of the simplicity of its monofunctionality (CH₃O-(CH₂CH₂O)n-CH₂CH₂-OH), was used for polypeptide modification. This "first-generation" PEG chemistry was used to create Adagen and Oncaspar (Enzon), where mPEG was linked to the N-terminal amino group or the alpha or epsilon amino groups of a lysine residue.

Lysines are among the more common amino acids comprising proteins—sometimes comprising 10% of a protein's overall amino acid sequence. Before PEG can be conjugated to an amino group, its terminal hydroxyl group must be activated by adding a functional group that is reactive with the peptide. This can be achieved by either acylating the PEG or alkylating the PEG. Most first-generation PEG chemistries used acylation, which generates a neutral amide. Alkylation, on the other hand, maintains a positively charged amine.

Most acylated PEGs are hydroxysuccinimidyl esters (-OSu) of carboxylated PEGs. The distance between the active ester (-COOSu) and the terminal ether in PEG can vary by up to four methylene units, and can greatly affect the reaction rate of PEG with both water and amino groups. Other acylating chemistries include activating the terminal PEG hydroxyl group with chloroformates or carbonylimidazole. These reagents tend to have a slower reaction rate than –Osu activated PEGs, and enable the reaction to be more easily stopped at the desired degree of modification, after attachment to the desired number of lysines.

If a positive charge is important for biological activity, then alkylation is the method of choice. One of the more common alkylated PEGs is PEG-aldehyde, which provides a permanent linkage after Shiff base formation and cyanoborohydride reduction. However, the reaction rate for the Shiff base formation is very slow—sometimes taking more than 24 h. When generating alkylating PEGs, the pH of the reaction is also critical for selectivity.

The problem. After these first-generation PEG-protein derivatives came to market, it quickly became evident that the technology's potential would only be fully realized when further methods of conjugation were developed. One major hindrance was the polydispersivity inherent in the PEG molecule; the presence of some PEGdiol—PEG dimmers, or PEG molecules that interact with each other to form complexes—even in monomethoxylated PEG, can yield unwanted cross-linked conjugates. Moreover, these firstgeneration chemistries are limited to low-molecular weight PEG, can generate unstable linkages, and are difficult to selectively modify. A population of modified peptides can contain a mixture of molecules with PEG attached to different lysines, as well as molecules with different numbers of linked PEGs. This variability in modification diminishes the purity of the finished product, impedes reproducibility, and can have pharmacological ramifications.