High-performance monolith affinity chromatography for fast quantitation of immunoglobulin G

J Chromatogr A. 2009 Mar 27;1216(13):2676-82. doi: 10.1016/j.chroma.2008.07.063. Epub 2008 Jul 26.

Abstract

High-performance monolith affinity chromatography employing protein A resins has been introduced previously for the fast purification of IgG from different sources. Here we describe the design and evaluation of a fast and specific method for quantitation of IgG from purified samples as well as crude supernatant from Chinese hamster ovary (CHO) cells. We used a commercially available affinity monolith with protein A as affinity ligand (CIM protein A HLD disk). Interferences of CHO host cell proteins with the quantitation of IgG from CHO supernatant were eliminated by a careful choice of the equilibration buffer. With this method developed, it is possible to quantify IgG within 5 min in a concentration range of 23-250 microg/ml. The calibration range of the method could be extended from 4 to 1000 microg/ml by adjusting the injection volume. The method was successfully validated by measuring the low limit of detection and quantification, inter- and intra-day precision and selectivity.

Publication types

  • Research Support, Non-U.S. Gov't
  • Validation Study

MeSH terms

  • Animals
  • CHO Cells
  • Chromatography, Affinity / methods*
  • Chromatography, High Pressure Liquid / methods*
  • Cricetinae
  • Cricetulus
  • Humans
  • Immunoglobulin G / analysis*
  • Sensitivity and Specificity
  • Staphylococcal Protein A / chemistry

Substances

  • Immunoglobulin G
  • Staphylococcal Protein A