Characterization of the Reversed-Phase Chromatographic Behavior of PEGylated Peptides Based on the Poly(ethylene glycol) Dispersity.

The separation mechanism of PEGylated peptides in reversed-phase high-performance liquid chromatography (RP-HPLC) is complex, because the PEGylated molecules exhibit physicochemical properties that are different from those of the parent molecules and have heterogeneous structure. Since most separation studies have focused on the hydrophobicity of the peptide relative to poly(ethylene glycol) (PEG), the role of PEG in the separation of PEGylated peptides on RP-HPLC is not clear. To elucidate the effect of the attached PEG on the retention of PEGylated peptides on RP-HPLC, the mono-PEGylated forms of collagen pentapeptide and octreotide were fractionated drop-by-drop from the outlet of the HPLC system and each drop was subjected to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This approach demonstrated that the dispersity of the attached PEG leads to the peak broadness of PEGylated peptides in RP-HPLC and the elution order inside the HPLC peak of PEGylated peptides was dependent on the attached PEG chain length. The retention time of PEGylated peptide increased as the attached PEG chain increased in length. When uniform PEG was conjugated to octreotide, its well-resolved positional isomers of the mono-PEGylated forms showed narrow peaks comparable to native peptide peak under the same HPLC conditions, which confirmed the effect of the attached PEG dispersity on the chromatographic behavior of PEGylated peptides. In conclusion, this study indicates that the chromatographic behavior of PEGylated peptides is affected by the PEG chain length dispersity as well as the peptide's hydrophobicity.