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
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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

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol: Displacement Chromatography of Monoclonal Antibody Charge Variants Using Source 15S Ion‐Exchange Resin
  • Support Protocol: Analysis of Cation‐Exchange Displacement Chromatography Fractions by Second Dimension–Analytical Cation‐Exchange HPLC
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol: 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.
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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.
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  •  FigureFigure 8.10.2 Displacement chromatogram of mAb preparation illustrating location and typical retention patterns of acidic and basic variants.
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  •  FigureFigure 8.10.3 Displacement chromatogram of mAb prep on a 4.6‐ × 250‐mm Source 15‐S cation‐exchange column.
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  •  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).
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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.
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