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

Title
Separation of mono- and di-PEGylate of exenatide and resolution of positional isomers of mono-PEGylates by preparative ion exchange chromatography
Author
이은규
Keywords
Exenatide; Mono-PEGylation; Positional isomers; PEG-maleimide; Cation exchange chromatography; Peptide analogs
Issue Date
2016-09
Publisher
ELSEVIER SCIENCE BV
Citation
JOURNAL OF CHROMATOGRAPHY A, v. 1457, Page. 88-96
Abstract
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 (10 kD) 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 0.22 M and 0.33 M 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. (C) 2016 Elsevier B.V. All rights reserved.
URI
https://www.sciencedirect.com/science/article/pii/S0021967316307944https://repository.hanyang.ac.kr/handle/20.500.11754/100525
ISSN
0021-9673; 1873-3778
DOI
10.1016/j.chroma.2016.06.035
Appears in Collections:
COLLEGE OF ENGINEERING SCIENCES[E](공학대학) > BIONANO ENGINEERING(생명나노공학과) > Articles
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