Characterisation of metal-chelate methacrylate monoliths

J Chromatogr A. 2006 Mar 17;1109(1):80-5. doi: 10.1016/j.chroma.2005.08.057. Epub 2005 Sep 12.

Abstract

Monoliths are attractive stationary phases for purification of large biomolecules like proteins because of their flow-unaffected properties. Isolation of histidine containing proteins to high purity can be efficiently performed using metal-chelate interactions within a single chromatographic step. In this work, we investigated properties of commercial metal-chelate methacrylate monoliths-Convective Interaction Media (CIM). Analytical CIM disk monolithic columns and CIM 8 ml monolithic columns were used for purification of tumor necrosis factor-alpha (TNF-alpha) analog LK-801 and green fluorescence protein with 6 histidine tag (GFP-6His). In both cases, purity over 90% was achieved. Dynamic binding capacity at 10% of breakthrough was around 17-18 mg/ml for LK-801 and around 30 mg/ml for GFP-6His. Adsorption isotherm revealed that the maximal capacity is achieved at protein concentration above 60 microg/ml. Dynamic binding capacity and resolution were found to be flow unaffected.

MeSH terms

  • Adsorption
  • Chelating Agents / chemistry*
  • Chromatography, High Pressure Liquid / instrumentation
  • Chromatography, High Pressure Liquid / methods
  • Copper / chemistry
  • Electrophoresis, Polyacrylamide Gel
  • Green Fluorescent Proteins / isolation & purification
  • Metals / chemistry*
  • Methacrylates / chemistry*
  • Tumor Necrosis Factor-alpha / isolation & purification

Substances

  • Chelating Agents
  • Metals
  • Methacrylates
  • Tumor Necrosis Factor-alpha
  • Green Fluorescent Proteins
  • Copper