Packing Tricorn 10/100 columns with MabSelect™ or MabSelect™ SuRe

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

Preparing the suspension

  • Suspension solution: 50 mM phosphate buffer, 150 mM sodium chloride, pH 7.0 Packing solution: 150 mM sodium chloride
  • 10 ml MabSelect™ or MabSelect™ SuRe (corresponds to 14 ml MabSelect™ or MabSelect™ SuRe/20% ethanol suspension)
  • Sintered glass filter funnel (medium grade, G3 type)
  • Filtering flask
  1. Equilibrate all materials to room temperature.
  2. Mount the glass filter funnel onto the filtering flask.
  3. Pour 14 ml of MabSelect™ or MabSelect™ SuRe/20% ethanol suspension into the funnel and wash with 2 × 20 ml distilled water followed by 2 × 20 ml packing solution.
  4. Transfer the sedimented medium from the funnel into a beaker and add 9.5 ml packing solution. This will give a 51% medium suspension.

Assembling and packing the column

Equipment needed:

  • Tricorn 10/100 column
  • Tricorn 10 Coarse Filter Kit
  • Tricorn Packing Connector 10-10
  • Tricorn Glass Tube 10/100
  • Tricorn 10 bottom unit with a 10 mm filter mounted
  • Pump P-901 or similar
  • 20 ml pipettes
  1. Details of column parts and packing equipment can be found in the instructions supplied with the column. Before packing, ensure that all parts are clean and intact.
  2. Wet a bottom coarse filter from the Tricorn 10 Coarse Filter Kit in 70% ethanol. Insert into the column.
  3. Insert the filter holder into the column tube. Ensure that the keyed part of the filter holder fits into the slot on the threaded section on the column tube. Push the filter holder into place.
  4. Screw the end cap onto the column tube. Insert a stop plug into the bottom unit.
  5. Screw Tricorn Packing Connector 10-10 onto the top of the column tube. The packing connector must be fitted with suitable O-rings (included with Tricorn Packing Connector 10-10).
  6. Mount the column and packing unit vertically.
  7. Screw Tricorn Glass Tube 10/100 into the upper fitting of Tricorn Packing Connector 10-10.
  8. Transfer the resuspended 51% suspension of MabSelect or MabSelect SuRe into the glass tube in a continuous motion using a 20 ml pipette.

Pipetting the suspension down the column wall minimizes formation of air bubbles.

  1. Attach the Tricorn 10 Bottom Unit mounted with a 10 mm filter to the top of the glass tube. The filter will distribute an even flow during packing. Place a beaker beneath the column tube and connect a pump to the top of the packing unit. Remove the stop plug from the bottom of the column tube.
    Packing is performed in two steps:
    For MabSelect™, pack at 2.5 ml/min (191 cm/h) for 20 min, followed by 10 ml/min (764 cm/h) for 2 min.
    For MabSelect™ SuRe, pack at 0.8 ml/min (61 cm/h) for 20 min, followed by 10 ml/min (764 cm/h) for 2 min.

Ensure that back pressure does not exceed the pressure limits (< 5 MPa) of the column during packing.

  1. Switch off and disconnect the pump, re-fit the stop plug into Tricorn 10 Bottom Unit. Take the column from the stand and remove Tricorn 10/100 Glass Tube and packing connector over a sink. Remount the column vertically.
  2. If necessary, remove excess medium by resuspending the top of the packed bed and remove with a Pasteur pipette or spatula. For a 10 cm bed height, the surface of the packed bed should be leveled with the lower end of the glass tube threads. Top-off the column with packing solution.
  3. Wet a top coarse filter from the Tricorn 10 Coarse Filter Kit, the bottom of the adapter, and O-ring in 70% ethanol. Place the top coarse filter at the center of the adapter with the glossy side towards the adapter. Mount the adapter unit onto the column tube. Connect the pump.
  4. Remove the stop plug and continue to pack at 10 ml/min (764 cm/h) for 2 min.
  5. Mark the position of the bed surface on the column. Stop the pump, re-fit the stop plug into the bottom of the column. Reposition the adapter to approximately 1 mm below the marked position.
  6. Wash the column with 15 ml of distilled water at 5 ml/min (382 cm/h) before checking packing quality.

Materials

     
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