Two-step Purification of Mouse Monoclonal IgG1 for Diagnostic Use

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

The goal of this example was purification of a monoclonal antibody to achieve a level of purity sufficient for in vitro diagnostic use. The two-step procedure combined hydrophobic interaction chromatography (HIC) for the capture step and SEC for polishing.

Target molecule:  Mouse monoclonal IgG1 anti-IgE.

Source material:  Hybridoma cell culture.

Clarification:  Sample was filtered and ammonium sulfate added to 50 mM. This was to enhance binding to the HIC column, not to precipitate the monoclonal antibody.



HIC purification was chosen for the capture step because the antibody binds very strongly to the medium (Phenyl Sepharose® High Performance) and most fetal calf serum proteins pass through the column as shown in Figure 6.9. The sample was concentrated into a smaller volume for polishing.


Screening of HIC media using HiTrap™ HIC Selection Kit is recommended to select the medium that gives optimal results. See Ordering information or refer to Hydrophobic Interaction and Reversed Phase Chromatography: Principles and Methods, 11001269 for more information.


Buffer conditions should be checked to select the concentration of ammonium sulfate that gives the highest binding selectivity for the antibody and avoids binding albumin.


Binding buffer: 20 mM potassium phosphate, 500 mM ammonium sulfate, pH 7.0  

Elution buffer: 20 mM potassium phosphate, pH 7.0

  1. Equilibrate column in binding buffer.
  2. Apply sample.
  3. Wash the column with binding buffer until the absorbance at 280 nm has returned to baseline.
  4. Use the elution buffer to create a linear gradient (10 column volumes) from 0.5 to 0 M ammonium sulfate.
  5. Wash with 2 to 3 column volumes of 100% elution buffer.
  6. Re-equilibrate with 2 to 3 column volumes of binding buffer.

Capture of mouse IgG1 on HiLoad 16/10 Phenyl Sepharose HP

Figure 6.9. Capture of mouse IgG1 on HiLoad 16/10 Phenyl Sepharose HP.


Intermediate purification

No intermediate step is required as the capture step gives a purity level > 95%.



A final purity of > 99% was achieved using Superdex 200 prep grade (Figure 6.10).

  1. Equilibrate column in phosphate buffered saline, pH 7.5.
  2. Apply sample (maximum sample volume 1% to 2% of total column volume).
  3. Elute sample in one column volume of buffer. Collect fractions.
  4. Wash with 2 to 3 column volumes of buffer.

Polishing of mouse monoclonal IgG1 anti-IgE

Figure 6.10. Polishing of mouse monoclonal IgG1 anti-IgE using Superdex 200 prep grade.


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