Performing a Purification of IgG Antibodies with HiTrap® MabSelect and HiTrap® MabSelect Xtra

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

This section describes a general procedure for purification of MAbs using HiTrap® MabSelect and HiTrap® MabSelect Xtra prepacked columns.

Figure 3.24 shows capture of mouse monoclonal IgG2a by AC using HiTrap® MabSelect followed by an SEC polishing step. The purified MAb is seen in lane 4 of the SDS-PAGE gel.

Capture of mouse monoclonal IgG2a by AC using HiTrap MabSelect

Fig 3.24. (A) Purification of IgG2a using HiTrap® MabSelect 1 ml in an automated, two-step purification on AKTAxpress Mab; (B) SDS-PAGE analysis on ExcelGel SDS Gradient 8-18, reducing conditions.

 

Successful purification of human monoclonal IgG1 in one step using HiTrap® MabSelect Xtra is shown in Figure 3.25. Highly pure MAb is seen in lane 4 of the SDS-PAGE gel.

Purification of human monoclonal IgG1 in one step using HiTrap MabSelect Xtra

Sample preparation

Refer to Chapter 2 (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 and 9 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 (Chapter 2, Desalting and buffer exchange) or dilution and pH adjustment.

 

Buffer preparation

Binding buffer: 0.02 M sodium phosphate, 0.15 M sodium chloride, pH 7.2

Elution buffer: 0.1 M sodium citrate, pH 3.0 to 3.6

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

See Appendix 4 for general instructions for purification using HiTrap® columns.


Binding buffer
: 0.02 M sodium phosphate, 0.15 M sodium chloride, pH 7.2

Elution buffer: 0.1 M sodium citrate, pH 3.0 to 3.6

Neutralizing buffer: 1 M Tris-HCl, pH 9.0

  1. Prepare collection tubes by adding 60 to 200 µl of 1 M Tris-HCl, pH 9.0 per milliliter of fraction to be collected.

To preserve the activity of acid-labile IgG, we recommend adding 60 to 200 µl of 1 M Tris-HCl pH 9.0 to collection tubes, which ensures that the final pH of the sample will be approximately neutral.

  1. Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (use the connector supplied), laboratory pump, or chromatography system “drop to drop” to avoid introducing air into the system.
  2. Remove the snap-off end at the column outlet.
  3. Wash out the ethanol with 3 to 5 column volumes of distilled water.
  4. Equilibrate the column with at least 5 column volumes of binding buffer. Recommended flow rates are 1 ml/min (1 ml column) and 5 ml/min (5 ml column)*.
  5. Apply the pretreated sample using a syringe fitted to the Luer connector or by pumping it onto the column. For optimal results, use a flow rate of 0.2 to 1 ml/min (1 ml column) and 0.5 to 5 ml/min (5 ml column) during sample application.
  6. Wash with binding buffer (generally at least 5 to 10 column volumes) until the absorbance reaches a steady baseline or no material remains in the effluent. Maintain a flow rate of 1 to 2 ml/min (1 ml column) and 5 to 10 ml/min (5 ml column) for washing.
  7. Elute with elution buffer using a one-step or linear gradient. For step elution, 5 column volumes are usually sufficient. For linear gradient elution, 10 to 20 column volumes are usually sufficient. Maintain a flow rate of 1 to 2 ml/min (1 ml column) and 5 to 10 ml/min (5 ml column) for elution. For purification using a syringe, elute with 2 to 5 column volumes of binding buffer.

* 1 ml/min corresponds to approximately 30 drops/min when using a syringe with a 1 ml HiTrap® column; 5 ml/min corresponds to approximately 120 drops/min when using a 5 ml HiTrap® column.

Stepwise elution allows the target antibody to be eluted in a more concentrated form, thus decreasing buffer consumption and shortening cycle times. Decreasing the flow rate might be necessary due to the high concentrations of protein in the eluted pool. When optimizing elution conditions, determine the highest pH that allows efficient elution of antibody from the column. This will prevent denaturing sensitive antibodies due to low pH.

  1. After elution, regenerate the column by washing it with 3 to 5 column volumes of binding buffer. The column is now ready for a new purification.
  2. If required, perform cleaning-in-place (see Cleaning-in-place below).

Desalt and/or transfer purified IgG fractions to a suitable buffer using a desalting column (see Chapter 2, Desalting and buffer exchange).

Reuse of HiTrap® columns depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination.

 

Cleaning-in-place (CIP)

For cleaning-in-place protocols for the removal of unwanted precipitated or denatured contaminants and hydrophobically bound substances, see Appendix 5 (Column packing and preparation) and the Instructions for HiTrap® MabSelect/HiTrap® MabSelect Xtra (28408414).

 

Storage

Store in 20% ethanol at 2°C to 8°C.

Materials

     
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