Separation of mono- and di-PEGylate of exenatide and resolution of positional isomers of mono-PEGylates by preparative ion exchange chromatography.

Exenatide is a synthetic version of the 39-mer peptide of Exendin-4, which is an FDA-approved therapeutic against Type II diabetes mellitus. However, exenatide has a very short in-serum half-life and PEGylation have been performed to improve its in-serum stability. PEGylation often yields multivalent binding to non-specific residues, and the desired species should be carefully separated by chromatographies. In this study, we first devised an aqueous-phase, two-step PEGylation process. This consists of thiolation of Lys 12 and 27 residues followed by attachment of PEG-maleimide (10kD) to thiol groups. This process yields various species: mono-PEGylates with positional isomers, di-PEGylate, and other higher MW substances. A prep-grade cationic exchange chromatography (HiTrap SP) at pH 3.0 partially separated mono- and di-PEGylates based on the molar ratio of conjugated PEG and peptide and thus molecular weight of the conjugates. To further investigate the chromatographic separation of positional isomers of mono-PEGylates, we prepared two kinds of exenatide analogs by point mutation; K12C and K27C. Each analog was mono-PEGylated with very high yield (>95%). When a mixture of the two positional isomers of mono-PEGylates was applied to HiTrap SP chromatography, K12C-PEGylate and K27C-PEGylate eluted separately at 0.22M and 0.33M NaCl, respectively. When the proportions of acid and its conjugate base of the amino acid residues adjacent to the PEGylation site at pH 3.0 were analyzed, K27C-PEGylate shows stronger positive charge than K12C-PEGylate, and we propose the residence time difference between the two mono-PEGylates could be due to the charge difference. ELISA result shows that the immuno-binding activity of both analogs and their mono-PEGylates are well maintained. Furthermore, both mono-PEGylates of the analogs show higher than 50-fold improved anti-trypsin stability. We expect that mono-PEGylates of the exenatide analogs are alternatives to the conventional C40-PEG.