Full metadata record
DC Field | Value | Language |
---|---|---|
dc.contributor.author | 이은규 | - |
dc.date.accessioned | 2019-03-06T05:57:59Z | - |
dc.date.available | 2019-03-06T05:57:59Z | - |
dc.date.issued | 2016-09 | - |
dc.identifier.citation | JOURNAL OF CHROMATOGRAPHY A, v. 1457, Page. 88-96 | en_US |
dc.identifier.issn | 0021-9673 | - |
dc.identifier.issn | 1873-3778 | - |
dc.identifier.uri | https://www.sciencedirect.com/science/article/pii/S0021967316307944 | - |
dc.identifier.uri | https://repository.hanyang.ac.kr/handle/20.500.11754/100525 | - |
dc.description.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. | en_US |
dc.description.sponsorship | This work was supported by the National Research Foundation of Korea (NRF) Grant funded by the Korean Government (MSIP) (No. 2014R1A2A2A03004266). | en_US |
dc.language.iso | en_US | en_US |
dc.publisher | ELSEVIER SCIENCE BV | en_US |
dc.subject | Exenatide | en_US |
dc.subject | Mono-PEGylation | en_US |
dc.subject | Positional isomers | en_US |
dc.subject | PEG-maleimide | en_US |
dc.subject | Cation exchange chromatography | en_US |
dc.subject | Peptide analogs | en_US |
dc.title | Separation of mono- and di-PEGylate of exenatide and resolution of positional isomers of mono-PEGylates by preparative ion exchange chromatography | en_US |
dc.type | Article | en_US |
dc.relation.volume | 1457 | - |
dc.identifier.doi | 10.1016/j.chroma.2016.06.035 | - |
dc.relation.page | 88-96 | - |
dc.relation.journal | JOURNAL OF CHROMATOGRAPHY A | - |
dc.contributor.googleauthor | Nguyen, Ngoc-Thanh Thi | - |
dc.contributor.googleauthor | Lee, Jae Sun | - |
dc.contributor.googleauthor | Yun, Soi | - |
dc.contributor.googleauthor | Lee, E. K. | - |
dc.relation.code | 2016003346 | - |
dc.sector.campus | E | - |
dc.sector.daehak | COLLEGE OF ENGINEERING SCIENCES[E] | - |
dc.sector.department | DEPARTMENT OF BIONANO ENGINEERING | - |
dc.identifier.pid | eklee | - |
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.