Development of separation process for PEGylated positional isoforms based on the intrinsic interaction with specific structure on the solid-liquid interface

• Project/Area Number: 26820363
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biofunction/Bioprocess
• Research Institution: Yamaguchi University
• Principal Investigator: Yoshimoto Noriko 山口大学, 医学(系)研究科(研究院), 助教 (40432736)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2014: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: PEG化タンパク質 / イオン交換クロマトグラフィー / 異性体分離 / chromatography / PEGylation / PEGylated protein / 変性 / リフォールディング

Outline of Final Research Achievements

The mechanism for the retention on ion exchange chromatography was analyzed with respect to the positional isoforms of PEGylated proteins by the liner salt salt gradient and pH gradient methods. The distribution coefficients of positional isoforms depended on pKa of modified lysine residue. The isoform eluted earlier when the modified lysine had low pKa values. The steric effect was found to be important in the separation of isoforms compared to their difference in charge on the basis of the retention behaviors of denatured proteins and PEGylated DNA. The diffusion coefficients of PEGylated proteins were determined in bulk solution and in the ion exchange resin to clarify the mass transfer properties in column.

Research Products

• [Int'l Joint Research] リュビアナ大学(スロベニア)
• [Journal Article] Monolith disk chromatography separates PEGylated protein positional isoforms within minutes at low pressure (2015)
  • Author(s): Yu Isakari, Ales Podgornik, Noriko Yoshimoto, Shuichi Yamamoto
  • Journal Title: Biotechnology Journal Volume: 11 Issue: 1 Pages: 100-106
  • DOI: 10.1002/biot.201500294
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] モノリスイオン交換クロマトグラフィーにおけるPEG化タンパク質異性体の分離機構 (2016)
  • Author(s): 吉本 則子
  • Organizer: 化学工学会 第81年会
  • Place of Presentation: 関西大学 千里山キャンパス （大阪府、吹田市）
  • Year and Date: 2016-03-14
• [Presentation] Optimization of monomer separation processes by electrostatic-interaction chromatography (2015)
  • Author(s): Shuichi Yamamoto , Noriko Yoshimoto , Yu Isakari , Ales Podgornik
  • Organizer: 249th ACS National Meeting & Exposition
  • Place of Presentation: USA, Atlanta
  • Year and Date: 2015-03-22
• [Presentation] On-Column (solid-phase) Pegylation and Separation of Pegylated Proteins (2014)
  • Author(s): Yu Isakari, Noriko Yoshimoto, Shuichi Yamamoto and Ales Podgornik
  • Organizer: 14AIChE Annual meeting
  • Place of Presentation: USA, Atlanta
  • Year and Date: 2014-11-18
• [Presentation] Diffusion Coefficients of DNAs and Pegylated Proteins for Estimating the Performance of Chromatography (2014)
  • Author(s): Masataka Hamachi, Yu Isakari, Noriko Yoshimoto, Shuichi Yamamoto and Ales Podgornik
  • Organizer: 14AIChE Annual meeting
  • Place of Presentation: USA, Atlanta
  • Year and Date: 2014-11-18
• [Presentation] Predicting the Efficiency of Capture Chromatography of Proteins (2014)
  • Author(s): Noriko Yoshimoto, Yu Isakari, and Shuichi Yamamoto
  • Organizer: ICSST14
  • Place of Presentation: 奈良県、奈良市、奈良県新公会堂
  • Year and Date: 2014-10-31
• [Presentation] Analysis of chromatographic separation for PEGylated proteins on the basis of their diffusion behaviours in porous particle (2014)
  • Author(s): Yu Isakari, Noriko Yoshimoto, Shuichi Yamamoto
  • Organizer: 2nd International Symposium on Multiscale Multiphase Process Engineering (MMPE2)
  • Place of Presentation: Germany, Hamburg
  • Year and Date: 2014-09-15