Strategies in Protein PEGylation

To better exploit the potential of PEGylation several strategies have been developed with the purpose of: (1) obtaining homogeneous products; (2) forming PEGylated conjugates with high retention of activity; and (3) performing PEGylation under gentle conditions that are compatible with easily degradable proteins.

Site-selective conjugation is always preferred as it allows easy purification and characterization of products and, most importantly, better retention of biological activity. As described above, selective PEGylation may be achieved taking advantage of a free cysteine, but this is possible only when this amino acid is present in the native protein or is introduced by genetic engineering. Alternatively, it is possible to take advantage of the lower pKa value of the a-amino group at the N-terminus with respect to the pKa of e-amine of lysines; in fact, performing the reaction under neutral or mildly acidic conditions, prevents the PEGylation at the level of lysine, but leaves the N-terminal amino group reactive.131 The most successful example of this strategy is the alkylation of r-metHuG-CSF with PEG-aldehyde, proposed by Kinstler. The reaction was carried out at pH 5.5 in the presence of sodium cyanoborohydride to reduce the Shiff's base initially formed.94'132 The conjugate obtained with a molecule of a 20kDa PEG showed an improved pharmacokinetic profile due to the reduced excretion by the kidneys. The PEG-G-CSF conjugate Pegfilgastrim has been on the market since 2002.

Site-specific PEGylation can also be achieved by exploiting the different accessibility of protein amino groups, as reported for a truncated form of growth hormone-releasing hormone (hGRF1-29). It was demonstrated that by using an appropriate solvent it is possible to alter the accessibility and reactivity of the three available amino groups. Nuclear magnetic resonance (NMR) and circular dichroism analysis indicated that the percentage of a-helix in hGRF1-29, which is only 20% in water, rises to 90% in structure-promoting solvents such as methanol/water or 2,2,2-trifluoroethanol (TFE), thus facilitating a region-selective modification. When PEGylation was performed in TFE the monoPEGylated conjugate at the level of Lys-12 reached 80% for all PEGylated isomers;133 however, the same reaction conducted in DMSO yielded an almost equimolar mixture of mono-PEGylated Lys-12 and Lys-21 isomers.134

Alternatively, specific PEGylation may be performed by blocking some of the reactive groups with a reversible protecting group as reported for insulin. This protein is formed by two polypeptide chains, A and B, and its three amino groups (Gly-A1, Phe-B1, and Lys-B29) are all candidates for PEGylation. Hinds proposed a site-directed PEGylation procedure involving the preliminary preparation of N-BOC (tert-butyl carbamate)-protected insulin.135 As an example, in order to synthesize N"B1-PEG-insulin the intermediate N^1, NB29-BOC-protected insulin was prepared prior to conjugation with PEG-SPA at the level of free N*B1. The final conjugate was obtained upon BOC removal with TFA treatment, forming the N"B1-PEG2000-insulin conjugate with 83% of the native insulin activity.

In general, in the PEGylation of an enzyme a requirement for high retention of the activity is that the PEG chains do not modify or obstruct the active site. Many strategies have been developed to achieve this goal: (1) the use of branched PEGs that, thanks to their higher hindrance with respect to linear polymers, have reduced accessibility to the active site (Figure 6); (2) to perform PEGylation in the presence of a substrate or an inhibitor that blocks polymer access to the active site; and (3) to conduct the modification after the enzyme is captured on an insoluble resin by substrates or inhibitors linked on it. In the last case, the obtained conjugate is eluted from the resin by changing the pH or adding denaturants, thus leading to a derivative that does not have linked PEG chains at the level of active site and its closer surroundings (Figure 11).136

A problem that may occur during protein PEGylation is the production of a low yield, especially when the modification is directed toward a buried or less accessible amino acid. This inconvenience is enhanced when the reaction is performed with high-molecular-weight PEGs due to the high steric hindrance. In the case of interferon beta (IFN-b), conjugation at cysteine 17 could only be achieved with a low-molecular-weight OPSS-PEG oligomer, but not with a high-molecular-weight polymer.137 Modification with high-molecular-weight PEGs could be successfully attempted via a two-step procedure: in the first reaction, the protein is modified with a short-chain heterobifunctional PEG oligomer, while in the second, the obtained conjugate is linked to a higher molecular weight PEG, possessing specific reactivity toward the terminal end of the first oligomer (Figure 12). The heterobifunctional PEG oligomer had

Amino group




Figure 11 PEGylation strategy for the protection of an enzyme active site from polymer conjugation: firstly the enzyme is loaded into an affinity resin functionalized with the appropriate ligand. The enzyme's active site binds the ligand, thus protecting the active site itself and the area close to it from PEG modification. After reaction under heterogeneous conditions the modified enzyme is eluted from the column.

High-molecular-weight PEG

Figure 12 Two-step tagging PEGylation strategy for a buried SH group in a protein. Smaller PEG molecules are more reactive than high-molecular-weight PEGs toward the buried cysteine. (Reproduced with permission from Pasut, G.; Guiotto, A.; Veronese, F. M. Exp. Op. Ther. Patents 2004, 14, 859-894.)

Figure 12 Two-step tagging PEGylation strategy for a buried SH group in a protein. Smaller PEG molecules are more reactive than high-molecular-weight PEGs toward the buried cysteine. (Reproduced with permission from Pasut, G.; Guiotto, A.; Veronese, F. M. Exp. Op. Ther. Patents 2004, 14, 859-894.)



R = lysine of protein, polymer, etc.

Figure 13 Reaction catalyzed by Tgase between a glutamine residue in a protein and an alkyl amine. (Reproduced with permission from Pasut, G.; Guiotto, A.; Veronese, F. M. Exp. Op. Ther. Patents 2004, 14, 859-894.)

a thiol reactive group at one end of the chain and a hydrazine group at the other (OPSS-PEG-Hz, 2 kDa). As mentioned above, hydrazine is still reactive at low pHs when a protein's amino groups are usually protonated and not reactive, thus preventing unwanted amino PEGylation of the protein. The INF-SS-PEG-Hz conjugate could therefore be selectively modified with PEG-aldehyde (30 kDa) by reductive alkylation. The overall yield was higher than 80%.

A recent study demonstrated that specific PEGylation at the lone, but not accessible, thiol groups of G-CSF could be achieved upon its exposure to partially denaturant conditions. After modification with OPSS-PEG, the native conformation of G-CSF was recovered by removal of the denaturant.138

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