Protein A/G Affinity

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Overview

Protein A and G chromatography media are commonly used in antibody purification due to the high binding affinity and specificity of Protein A or G with the Fc region of the antibody. Key attributes for these methods are simplicity, purity, and yield. Purification can be completed quickly on any chromatography system, using basic gravity fed columns to more sophisticated HPLC systems. This section provides an overview of Protein A and G chromatography with general considerations

Related Topics in Chromatography: Affinity Chromatography, Affinity Purification of Tagged Recombinant Proteins, and Affinity Chromatography: Activated Supports.

General Considerations for Affinity Purification

Immobilized Protein A and G from Staphylococcus aureus have been used for many years to purify antibodies from a variety of species (Hjelm et al. 1972). The high selectivity and stability of protein A and G have made them a popular choice for the purification of antibodies from a wide range of sample sources, including serum, ascitic fluid, and hybridoma cell culture supernatants. Mammalian antibodies are categorized into five major classes: IgA, IgD, IgE, IgG, and IgM. IgG is the predominant class of antibody in serum and is generated in large amounts during the secondary immune response. The IgG class of antibody is further divided into subclasses that vary depending upon the species and the properties of the heavy chain component. There are four subclasses of IgG in humans (IgG1, IgG2, IgG3, IgG4) and in mice (IgG1, IgG2a, IgG2b, IgG3). The affinity of protein A for IgG varies considerably between species and IgG subtypes and has been extensively characterized (Duhamel et al. 1979, Schwartz 1990). In humans, protein A binds with high affinity to IgG1, IgG2, and IgG4, but poorly to IgG3. Among the four IgG subtypes in mice, protein A has the weakest affinity for IgG1 while protein G has affinity for all four IgG subclasses. Bio-Rad offers a suite of products for Protein A chromatography: UNOsphere SUPrA™ media, and Affi-Gel® and Affi-Prep® protein A media.

Antibody binding affinity to Protein A and Protein G*

Species IgG Class Protein A Protein G
Chicken egg IgY
Cow IgG +
Dog IgG + +
  IgM +
Goat IgG + +++
  IgM
Horse IgG +++ +++
Rabbit IgG +++ +++
  IgM
Rat IgG + ++
  IgM
Sheep IgG +++ +++
  IgM
Mouse IgG1 + ++
  IgG2a ++ ++
  IgG2b ++ ++
  IgG3 + ++
  IgM ++ +
  IgA ++ ++
Human lgG1 +++ +++
  IgG2 +++ +++
  IgG3 +++
  IgG4 +++ +++
  IgA +
  IgM +
  IgE +

—, No binding; +, weak binding; ++, moderate binding; +++, strong binding.
* Data obtained from Handbook of Affinity Chromatography by David
S. Hage (ISBN 0824740572). Chapter 14 "Affinity Chromatography in
Antibody and Antigen Purification" by Terry M. Phillips.

Protein A and G Binding and Elution

The binding of antibodies to protein A is mediated, at neutral or alkaline pH values, through hydrophobic interactions involving a highly conserved histidine residue located in the protein A binding site of IgG. The elution of IgG from immobilized protein A is commonly achieved by lowering the pH using an acidic buffer. Protein A-purified antibodies are then typically neutralized with a base, dialyzed against a neutral buffer, or desalted using a gel-filtration column to avoid acid-mediated hydrolysis and denaturation.

Protein G exhibits a higher binding affinity than Protein A. It binds optimally at acidic pH and, due to its higher binding affinity, requires harsher elution conditions, pH 3.0 or lower, which is inherently detrimental to the activity of the purified antibody. These effects can be minimized, though not entirely eliminated, through rapid neutralization of the collected fractions. The strength of the Protein G/IgG interaction can also result in IgG carry over. To avoid cross contamination, Protein G columns should be specific to the antibody being purified. Due to retained IgG, Protein G has a shorter column life due to the loss in binding capacity after a few purifications. (P. Gagnon, ISBN 0-9653515-9-9).

References

Duhamel RC et al., pH gradient elution of human IgG1, IgG2 and IgG4 from protein A-sepharose, J Immunol Methods 31(3–4), 211 (1979).

Gagnon P (1996). Purification tools for monoclonal antibodies (Tucson: Validated Biosystems Inc).

Hjelm H et al., ProteinA Staphylococcus aureus. Its isolation by affinity chromatography and its use as an immunosorbent for isolation of immunoglobulins, FEBS Lett 28(1), 73–76 (1972).

Schwartz L (1990), In bacterial immunoglobulin-binding proteins, Vol. 2, M. Boyle, ed. (San Diego: Academic Press), p. 309.

Related Content

Literature
Number Description Download
5712 The Profinia Protein Purification System Simplifies Antibody Purification With Protein A, Rev A Click to download
5925 Affinity Purification Buffer Kit Product Information Sheet, Rev B Click to download
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