Performing a Purification of IgG Antibodies with nProtein A Sepharose® 4 Fast Flow

Extracted from Affinity Chromatography Vol. 1: Antibodies, GE Healthcare, 2016
 

nProtein A Sepharose® 4 Fast Flow (Figure 3.14) is native protein A coupled to Sepharose® 4 Fast Flow. The medium is designed for recovery and purification of monoclonal antibodies from cell culture supernatants, serum, and ascites at both laboratory and process scale. Leakage of  ligand from the Sepharose® Fast Flow matrix is low. Moreover, nProtein A Sepharose® 4 Fast Flow has high mechanical and chemical stability, withstanding high concentrations of hydrogen bond disrupting agents such as urea, guanidine hydrochloride, and sodium thiocyanate. The low  ligand leakage and high flow rate of the Sepharose® Fast Flow medium allow the use of nProtein A Sepharose® 4 Fast Flow for scale-up of monoclonal and polyclonal antibody purification.

nProtein A Sepharose® 4 Fast Flow

Fig 3.14. nProtein A Sepharose® 4 Fast Flow is an affinity medium for capture of monoclonal and polyclonal antibodies from cell culture supernatants, serum, and ascites.

 

Column packing

Refer to Column packing and preparation for general guidelines on column packing.

A suitable packing method for nProtein A Sepharose® 4 Fast Flow is described in Protein G Sepharose® 4 Fast Flow.

 

Sample preparation

Refer to Desalting and buffer exchange for general considerations.

Centrifuge samples (10 000 × g for 10 min) to remove cells and debris. Filter through a 0.45 µm filter.

 

IgG from many species has a medium to strong affinity for protein A at physiological pH. Sample pH should be between 6.0 and 9.0 before applying to the column. If required, adjust sample conditions to the pH and ionic strength of the binding  buffer by either buffer exchange on a desalting column (see Desalting and buffer exchange) or dilution  and pH adjustment.

 

Buffer preparation

Binding buffer: 0.02 M sodium phosphate, pH 7.0

Elution buffer: 0.1 M citric acid, pH 3.0

Neutralizing buffer: 1 M Tris-HCl, pH 9.0

Water and chemicals used for buffer preparation should be of high purity. Filter buffers through a 0.45 µm filter before use.

 

Purification

Follow the protocol described in Protein G Sepharose® 4 Fast Flow.

Materials

     
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