Isolation of Monoclonal Antibody Charge Variants by Displacement Chromatography

C. Patrick McAtee1, Jacob Hornbuckle2

1 SACHEM, Inc., Austin, Texas, 2 Emergent Biosolutions, Seattle, Washington
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 8.10
DOI:  10.1002/0471140864.ps0810s69
Online Posting Date:  August, 2012
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

This unit discusses the important parameters in designing and optimizing a separation of monoclonal antibody (mAb) charge variants from process streams by ion‐exchange displacement chromatography, including sample preparation and selection of matrix, column, and appropriate buffer. A protocol is provided for determination of optimal column binding and displacement conditions, including cleaning and regeneration of the displacement columns. Curr. Protoc. Protein Sci. 69:8.10.1‐8.10.13. © 2012 by John Wiley & Sons, Inc.

Keywords: displacement chromatography; ion‐exchange chromatography; monoclonal antibodies; charge variants; immunogenicity; quality by design

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Displacement Chromatography of Monoclonal Antibody Charge Variants Using Source 15S Ion‐Exchange Resin
  • Support Protocol 1: Analysis of Cation‐Exchange Displacement Chromatography Fractions by Second Dimension–Analytical Cation‐Exchange HPLC
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Displacement Chromatography of Monoclonal Antibody Charge Variants Using Source 15S Ion‐Exchange Resin

  Materials
  • Appropriate buffers and solvents (see Strategic Planning)
  • Column and resin (chromatography matrix; see Strategic Planning)
  • Regeneration buffer (e.g., 2.0 M NaCl or other suitable salt)
  • Loading buffer without sample
  • Protein solution for purification, dissolved in loading buffer
  • Displacer in loading buffer
  • Buffer for displacer removal and column cleaning (e.g., REGENERATE, available from SACHEM)
  • Column cleaning solutions:
    • Base (e.g. 100 mM KOH)
    • Acid [e.g., 15% acetic acid or 100 mM betaine (pH = 2.0)]
    • Nonionic detergent (e.g., NP‐40, Triton X‐100)
    • Buffered 2 M NaCl
  • Chromatographic system (see Strategic Planning)
NOTE: Longer columns tend to provide better recoveries. The aspect ratio (length to width) should be at least 20:1. Flow rates are low, so there are rarely problems with high backpressure.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 8.10.1 Calculation of column binding capacity from displacement chromatogram of mAb preparative run on 4.6‐ × 250‐mm Source S‐15 column.
  •   FigureFigure 8.10.2 Displacement chromatogram of mAb preparation illustrating location and typical retention patterns of acidic and basic variants.
  •   FigureFigure 8.10.3 Displacement chromatogram of mAb prep on a 4.6‐ × 250‐mm Source 15‐S cation‐exchange column.
  •   FigureFigure 8.10.4 Two‐dimensional chromatogram of selected fractions from a displacement chromatography run of a mAb preparation on a 4.6‐ × 250‐mm Source 15‐S cation‐exchange column run in second dimension utilizing elution mode (same column).

Videos

Literature Cited

Literature Cited
   Beck, A., Wurch, T., Bailly, C., and Corvaia, N. 2010. Strategies and challenges for the next generation of therapeutic antibodies. Nature Rev. Immunol. 10:345‐352.
   Dillon, T.M., Ricci, M.S., Vezina, C.M., Flynn, G.C., Liu, Y.D., Rehder, D.S., Plant, M., Henkle, B., Li, Y., Deechongkit, S., Varnum, B., Wypych, J., Balland, A., and Bondarenko, P.V. 2008. Structural and functional characterization of disulfide isoforms of the human IgG2 subclass. J. Biol. Chem. 283:16206‐16215.
   Khawli, L.A., Goswami, S., Hutchinson, R., Kwong, Z.W., Yang, J., Wang, X., Yao, Z., Sreedhara, A., Cano, T., Tesar, D., Nijem, I., Allison, D.E., Wong, P.Y., Kao, Y.‐H., Quan, C., Joshi, A., Harris, R.J., and Motchnik, P. 2010. Charge variants in IgG1:Isolation, characterization, in vitro binding properties and pharmacokinetics in rats. mAbs 2:613‐624.
   Rathore, A.S. and Winkle, H. 2009. Quality by design for biopharmaceuticals. Nature Biotech. 27:26‐34.
   Vlasak, J. and Ionescu, R. 2008. Heterogeneity of monoclonal antibodies revealed by charge‐sensitive methods. Curr. Pharm. Biotech. 9:468‐481.
   Wypych, J., Li, M., Guo, A., Zhang, Z., Martinez, T., Allen, M.J., Fodor, S., Kelner, D.N., Flynn, G.C., Liu, Y.D., Bondarenko, P.V., Ricci, M.S., Dillon, T.M., and Balland, A. 2008. Human IgG2 antibodies display disulfide‐mediated structural isoforms. J. Biol. Chem. 283:16194‐16120.
   Zhang, T., Bourret, J., and Cano, T. 2011. Isolation and characterization of therapeutic antibody charge variants using cation exchange displacement chromatography. J. Chromatogr. A 1218:5079‐5086.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library