Performing a Purification of IgM Antibodies with HiTrap™ IgM Purification HP

Extracted from Affinity Chromatography Vol. 1: Antibodies, 2016

HiTrap™ IgM Purification HP 1 ml columns are prepacked with a thiophilic adsorption medium (2-mercaptopyridine coupled to Sepharose^® High Performance). The binding capacity of HiTrap™ IgM Purification HP is 5 mg/ml of medium. The column can be used for purification of native human and human monoclonal IgM. The interaction between the protein and the ligand has been suggested to result from the combined electron donating- and accepting action of the ligand in a mixed-mode hydrophilic-hydrophobic interaction.

Protein A Sepharose^® chromatography media offer an alternative solution to HiTrap™ IgM Purification HP since some human monoclonal IgM, some IgM from normal and macroglobulinemic sera, and some monoclonal canine IgM and polyclonal IgA from pig, dog, and cat can bind to protein A.

Figure 1A shows the results from the purification of monoclonal α-Shigella IgM from hybridoma cell culture supernatant. Analysis by SDS-PAGE (Figure 1B) demonstrated a purity level of over 80%. Results from an ELISA (not shown) indicated high activity in the purified fraction.

Figure 1. (A) Purification of α -Shigella IgM on HiTrap™ IgM Purification HP. (B) SDS-PAGE of sample, flowthrough, and eluted pools was performed under reducing (left gel) and nonreducing (right gel) conditions. SDS-PAGE was performed on PhastSystem using PhastGel 4–15, silver staining.

Sample Preparation

Refer to Column packing and preparation for general considerations.

Buffer Preparation

Binding buffer: 20 mM sodium phosphate, 800 mM ammonium sulfate, pH 7.5

Elution buffer: 20 mM sodium phosphate, pH 7.5

Wash buffer: 20 mM sodium phosphate, pH 7.5 with 30% isopropanol

The sample must have the same concentration of ammonium sulfate as in the binding buffer (0.8 M). Slowly add small amounts of solid ammonium sulfate to the sample from the hybridoma cell culture until the final concentration is 800 mM. Stir slowly and continuously. Pass the sample through a 0.45 µm filter immediately before applying it to the column. Some monoclonal IgM might not bind to the column at a concentration of 800 mM ammonium sulfate. Binding can be improved by increasing the ammonium sulfate concentration to 1 M.

To avoid precipitation of IgM, it is important to add the ammonium sulfate slowly. An increased concentration of ammonium sulfate will cause more IgG to bind, which might be a problem if serum has been added to the cell culture medium. If there is IgG contamination of the purified IgM, the IgG can be removed by using HiTrap™ Protein A HP, HiTrap™ rProtein A FF, or HiTrap™ Protein G HP.

Ammonium sulfate can be replaced by 500 mM potassium sulfate. Most monoclonal IgM binds to the column in the presence of 500 mM potassium sulfate and the purity of IgM is comparable to the purity achieved with 800 mM ammonium sulfate.

Purification

See Affinity Purification Using HiTrap™ Columns for general instructions for purification using HiTrap™ columns.

1. Fill the syringe or pump tubing with distilled water. Remove the stopper and connect the column to the syringe (use the connector supplied).
2. Remove the snap-off end at the column outlet.
3. Wash out the ethanol with 5 mL of distilled water.
4. Equilibrate the column with 5 mL of binding buffer. The recommended flow rate is 1 mL/min*.
5. Apply the 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 during sample application.
6. Wash with 15 mL of binding buffer or until the absorbance reaches a steady baseline or no material remains in the effluent. Maintain a flow rate of 1 to 2 mL/min for washing.
7. Elute with 12 mL of elution buffer using a one-step or linear gradient though larger volumes are sometimes required to break the interaction.
8. After elution, regenerate the column by washing it with 7 mL of wash buffer and re-equilibrate the column with 5 mL of binding buffer. The column is now ready for a new purification of the same antibody.

* 1 mL/min corresponds to approximately 30 drops/min when using a syringe with a 1 mL HiTrap™ column.

Some monoclonal IgM might bind too strongly to the column matrix for complete elution. The remaining IgM will be eluted during cleaning, but the high concentration of isopropanol will cause precipitation of IgM. Perform an immediate buffer exchange (see Column packing and preparation) or dilute the sample to preserve the IgM. Lower concentrations of isopropanol can elute the IgM and decrease the risk of precipitation.

Reuse of HiTrap™ lgM Purification HP depends on the nature of the sample and should only be considered when processing identical samples to avoid cross-contamination.

To increase capacity, connect several HiTrap™ IgM Purification HP columns in series. HiTrap™ columns can be used with a syringe, a peristaltic pump, or connected to a liquid chromatography system.

Storage

Store in 20% ethanol at 2 °C to 8 °C.