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

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

rProtein A Sepharose® Fast Flow (Figure 3.19) is an affinity medium with high binding capacity for monoclonal and polyclonal antibodies. The binding capacity of rProtein A Sepharose® Fast Flow is considerably higher than for nProtein A Sepharose® 4 Fast Flow. The recombinant protein A ligand of rProtein A Sepharose® Fast Flow has been specially engineered to favor an oriented  coupling giving a matrix with enhanced binding capacity. Ligand leakage is low. rProtein A Sepharose® Fast Flow is produced in E. coli and no human IgG affinity step is used during validated fermentation and purification processes, minimizing risk of human IgG contamination.

rProtein A is available as a bulk medium for packing in XK and Tricorn columns at laboratory scale. The low ligand leakage and high flow rate of the Sepharose® Fast Flow medium allow the  use of rProtein A Sepharose® Fast Flow for scaling up purification of monoclonal and polyclonal antibodies. The medium is also available in prepacked 1 ml and 5 ml HiTrap™ rProtein A FF columns, which allow convenient, one-step purification. Furthermore, purification capacity can be greatly increased by connecting columns in series.

 rProtein A Sepharose® Fast Flow

Fig 3.19. rProtein A Sepharose® Fast Flow is an affinity medium with high binding capacity for antibodies, enabling capture of up to 50 mg antibody/ml medium.

 

Column packing

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

A suitable packing method for 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 sodium citrate, pH 3.0 to 6.0

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

Use the protocol for Protein G Sepharose® 4 Fast Flow.

Materials

     
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