Column Chromatography of Antibodies and Fragments

John A. Smith1

1 University of Alabama at Birmingham, Birmingham, Alabama
Publication Name:  Current Protocols in Immunology
Unit Number:  Appendix 3I
DOI:  10.1002/0471142735.ima03is21
Online Posting Date:  May, 2001
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Abstract

Since both size‐exclusion (SE) chromatography (also known as gel filtration) and ion‐exchange (IEX) chromatography are the fundamental methods on which antibody and fragment purifications are based, they are dealt with in detail in this appendix. The procedures for preparing the column matrix, for pouring the chromatography column, and for estimating the appropriate size of the column for the amount of antibody and fragment that are to be purified are all described.

     
 
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Table of Contents

  • Basic Protocol 1: Size‐Exclusion Chromatography
  • Support Protocol 1: Selection of a Size‐Exclusion Gel Matrix and Column
  • Basic Protocol 2: Ion‐Exchange Chromatography
  • Support Protocol 2: Selecting an IEX Gel Matrix and Column
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Size‐Exclusion Chromatography

  Materials
  • SE gel matrix (see protocol 2 and Table 3.0.1)
  • recipeSE buffer (see recipe)
  • SE protein standards (see Table 3.0.2)
  • Buchner or sintered‐glass funnel
  • SE chromatography column (see protocol 2)
  • Buffer reservoir
    Table 0.i.1   MaterialsFractionation Ranges of Commonly Used Gel Filtration Matrices   Molecular Weights of Protein Standards for Gel Filtration c   Molecular Weights of Protein Standards for Gel Filtration

    Gel type a (molecular weight) Fractionation range b Specific application
    Sephadex G‐200 5 × 103–2.5 × 105 IgG
    Sephacryl S‐200 Superfine 5 × 103–2.5 × 105 IgG
    Bio‐Gel A‐0.5m 104–5 × 105 IgG
    Ultrogel AcA 34 2 × 104–3.5 × 105 IgG
    Bio‐Gel P‐200 3 × 104–2 × 105 IgG
    Bio‐Gel A‐1.5m <104–1.5 × 106 IgM
    Sephacryl S‐300 104–1.5 × 106 IgM
    Sepharose 6B 104–4 × 106 IgM
    Toyopearl TSK HW55(s) 105–2 × 106 IgM
    Ultrogel AcA 22 105–1.2 × 106 IgM
    Protein Molecular weight
    Cytochrome c 11,700
    Myoglobin (sperm whale) 16,800
    Trypsinogen (PMSF‐treated) 24,000
    Carbonic anhydrase 29,000
    Ovalbumin 45,000
    Hemoglobin 64,500
    Bovine serum albumin 66,000
    Transferrin 74,000
    IgG 158,000
    Fibrinogen 341,000
    Ferritin 470,000
    Thyroglobulin 670,000

     aSephadex, Sephacryl and Sepharose are available from Pharmacia LKB. Bio‐Gel A is available from Bio‐Rad. Ultrogel AcA is available from IBF Biotechnics. Toyopearl is available from TosoHaas.
     bThe fractionation range indicates the molecular size of a protein expected to elute at an elution volume equal to the bed volume (number on left) and the molecular size of a protein expected to be totally excluded from the column and to elute at the void volume (number on right). The fractionation ranges given are for dilute aqueous buffers and will differ for separations carried out under denaturing conditions (e.g., 6 M guanidine˙Cl, 8 M urea, or detergents).
    Table 0.i.2   MaterialsFractionation Ranges of Commonly Used Gel Filtration Matrices   Molecular Weights of Protein Standards for Gel Filtration c   Molecular Weights of Protein Standards for Gel Filtration

    Gel type a (molecular weight) Fractionation range b Specific application
    Sephadex G‐200 5 × 103–2.5 × 105 IgG
    Sephacryl S‐200 Superfine 5 × 103–2.5 × 105 IgG
    Bio‐Gel A‐0.5m 104–5 × 105 IgG
    Ultrogel AcA 34 2 × 104–3.5 × 105 IgG
    Bio‐Gel P‐200 3 × 104–2 × 105 IgG
    Bio‐Gel A‐1.5m <104–1.5 × 106 IgM
    Sephacryl S‐300 104–1.5 × 106 IgM
    Sepharose 6B 104–4 × 106 IgM
    Toyopearl TSK HW55(s) 105–2 × 106 IgM
    Ultrogel AcA 22 105–1.2 × 106 IgM
    Protein Molecular weight
    Cytochrome c 11,700
    Myoglobin (sperm whale) 16,800
    Trypsinogen (PMSF‐treated) 24,000
    Carbonic anhydrase 29,000
    Ovalbumin 45,000
    Hemoglobin 64,500
    Bovine serum albumin 66,000
    Transferrin 74,000
    IgG 158,000
    Fibrinogen 341,000
    Ferritin 470,000
    Thyroglobulin 670,000

     cProtein standards are commercially available in kits, either combined or as individual proteins.

Support Protocol 1: Selection of a Size‐Exclusion Gel Matrix and Column

  Materials
  • IEX gel matrix (see protocol 4)
  • recipeIEX buffer (see recipe)
  • IEX chromatography column (see protocol 4)
  • Gradient mixer (Pharmacia LKB GM‐1 or equivalent)
  • Conductivity meter (e.g., Bio‐Rad conductivity monitor; optional)
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Figures

  •   FigureFigure a0.3I.1 Configurations for conventional columns. (A) An open buffer reservoir attached to a column. The rate of flow is controlled by hydrostatic pressure, measured as the distance in centimeters between the surface of the solution in the reservoir and the end of the outlet tubing. (B) A closed buffer reservoir (a so‐called Mariotte flask) attached to a column. (C) An open buffer reservoir attached to a column. The rate of flow is controlled by a peristaltic pump. (D) A closed buffer reservoir and sample solution reservoir attached to the bottom of a column. The direction of flow of the column is from the bottom to the top. This configuration has the advantage of maintaining long column life, since the gel matrix will not be compressed.
  •   FigureFigure a0.3I.2 Gradient mixer for forming salt gradients and pH gradients used during ion‐exchange (IEX) chromatography of proteins. The apparatus shown is a Pharmacia Gradient Mixer GM‐1, which can be used for preparing gradients of 500 ml or less. If a gradient elution is used for a given IEX separation, a gradient apparatus should be substituted for the buffer reservoir in one of the column configurations shown in Figure . A salt gradient is produced by filling the reservoirs with buffers at the same pH and different ionic strength. The mixing chamber contains the lower ionic strength buffer, and the other chamber contains the higher ionic strength buffer. A linear salt gradient can be produced with this apparatus. A pH gradient is produced by filling the reservoirs with buffers at the same ionic strength and different pH. A gradient of increasing pH is used for cationic separation, and a gradient of decreasing pH is used for anionic separations. A continuous, nonlinear pH gradient can be produced with this apparatus. The buffer chambers are joined by a channel controlled by a valve. The outflow from the mixing chamber is also controlled by a valve.

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Literature Cited

Literature Cited
   Andrews, P. 1970. Estimation of molecular size and molecular weights of biological compounds by gel filtration. In Methods of Biochemical Analysis, (D. Glick, ed.), Vol. 18, pp. 1‐53. Wiley‐Interscience, New York.
   Fischer, L. 1980. Gel‐Filtration Chromatography. Elsevier, Amsterdam.
   Himmelhoch, S.R. 1971. Chromatography of proteins on ion‐exchange adsorbents. Methods Enzymol. 22:273‐286.
   Peterson, E.A. and Sober, H.A. 1956. Chromatography of proteins. I. Cellulose ion‐exchange adsorbents. J. Am. Chem. Soc. 78:751‐758.
   Porath, J. and Flodin, P. 1959. Gel filtration: A method for desalting and group separation. Nature (Lond.) 183:1657‐1659.
   Scopes, R.K. 1982. Ion exchangers—principles, properties and uses. In Protein Purification: Principles and Practice, pp. 75‐101. Springer‐Verlag, New York.
Key References
   Bio‐Rad Price List L. Bio‐Rad, Richmond, California.
  Contains a detailed description of gel filtration and available chromatographic matrices for separation and desalting.
   Fischer, 1980. See above.
  An excellent summary of the theory and practice of gel‐filtration chromatography.
   FPLC Ion Exchange and Chromatofocusing: Principles and Methods. Pharmacia, Uppsala, Sweden.
  Manufacturer's guide to FPLC.
   Gel Filtration: Theory and Practice. Pharmacia, Uppsala, Sweden.
  Practical guide to gel‐filtration chromatography.
   Ion‐Exchange Chromatography: Principles and Methods. Pharmacia, Uppsala, Sweden.
  Detailed guide to the theory and practice of ion‐exchange chromatography.
   LKB Ultrogel (Instruction Manual). LKB‐Produkter AB, Bromma, Sweden.
  A useful guide to the operational details of gel‐filtration chromatography.
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