Performing a Separation with IgG Sepharose 6 Fast Flow

Extracted from Affinity Chromatography Principles and Methods , GE Healthcare, 2007

Purification Option

Product Binding capacity/ml medium Maximum operating flow
IgG Sepharose 6 Fast Flow 2 mg protein A at pH 7.5 400 cm/h*

* See Appendix 4 to convert linear flow (cm/h) to volumetric flow rate. Maximum operating flow is calculated from measurement in a packed column with a bed height of 10 cm and i.d. of 5 cm.

Purification Example

Figure 29 shows automatic on-line monitoring of the production of a secreted fusion protein during fermentation. The fusion protein, ZZ-IGF-1 is insulin-like growth factor 1 fused with a derivative of protein A (designated ZZ), expressed in E. coli.

Chromatogram of a sample taken at one time point during fermentation

Fig. 29. A) Chromatogram of a sample taken at one time point during fermentation. B) Results from automatic monitoring of the product concentration during fermentation. Concentration of ZZ-IGF-1 is determined by integration of the ZZ-IGF-1 peak obtained during each chromatographic analysis. Bacterial density is measured manually at A600 nm.

Performing a Separation

Binding buffer: 0.05 M Tris-HCl, 0.15 M NaCl, 0.05% Tween 20, pH 7.6

Wash buffer: 5 mM ammonium acetate, pH 5.0

Elution buffer: 0.5 M acetic acid, adjusted to pH 3.4 with ammonium acetate

Neutralization buffer: 1 M Tris-HCl, pH 9.0

  1. Pack the column (see Appendix 3, Column packing and preparation for affinity chromatography) and wash with at least 5 column volumes of binding buffer.
  2. Equilibrate the column with approximately 5 column volumes of binding buffer.
  3. Wash with 2–3 column volumes of acetic acid followed by 5 column volumes of binding buffer.
  4. Apply the sample.
  5. Wash with 10 column volumes binding buffer.
  6. Wash with 2 column volumes of wash buffer or until no material appears in the eluent (determined by UV absorbance at A280 nm).
  7. Elute with 2–5 column volumes of elution buffer.*
  8. Immediately re-equilibrate the column with binding buffer until the eluent reaches pH 7.0 (the IgG may denature if left at a lower pH).

* Since elution conditions are quite harsh, it is recommended to collect fractions into neutralization buffer (60 µl – 200 µl 1 M Tris-HCl, pH 9.0 per ml fraction), so that the fnal pH of the fractions will be approximately neutral.

This method, while giving a concentrated eluate, can only be used if the fusion product is stable under the acid conditions.

An alternative eluent is 0.1 M glycine-HCl, pH 3.0. Chaotropic agents may also be used  for elution.

Media Characteristics

  Ligand Composition pH stability* Particle size
IgG Sepharose 6 Fast Flow Human polyclonal IgG IgG coupled to Sepharose Fast Flow by the cyanogen bromide method.    Short term 3–10 Long term 3–10 90 µm

* Long term refers to the pH interval over which the medium is stable over a long period of time without adverse effects on its subsequent chromatographic performance. Short term refers to the pH interval for regeneration, cleaning-in-place and sanitization procedures.

Chemical Stability

Avoid reducing agents such as 2-mercaptoethanol or DTT since they may disrupt disulphide bonds within the IgG ligand.


Wash with 5 column volumes of 20% ethanol at neutral pH and store at +4 to +8 °C.



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