DFE-Based Affinity Chromatography: A Technique for Purification of 2F5 Monoclonal Antibodies from a Crude Sample Using High Ionic Strength-Based Elution Conditions

Overview

This video describes an affinity chromatography technique for purifying 2F5 monoclonal antibodies using the DsRed-2F5-Epitope or DFE ligands. The purified 2F5 monoclonal antibodies are essential drugs in immunotherapy.

Protocol

1. Testing the Purification of mAbs from Clarified Plants Extracts

1. Prepare 100 mL of clarified plant extract containing 2F55 or the supernatant from the preferred cell-based expression system, also containing 2F5.
2. Prepare equilibration buffer (20 mM sodium phosphate, 500 mM sodium chloride, pH 7.4), low-pH elution buffer (0.05 M citrate, 0.05 M sodium chloride, pH 4.0–3.25), and high-ionic-strength elution buffer (1.0–4.0 M magnesium chloride, 0.1 M HEPES, pH 8.0)
3. Flush the chromatography system with the buffers. Mount a DFE affinity column (Refer to the main protocol text) on the chromatography system and equilibrate with 5 CV of equilibration buffer at a flow rate of 1.0 mL min^-1. Monitor the UV absorbance at 280 nm.
   NOTE: Loading plant extract or cell culture supernatant onto the column can cause an increase in backpressure. Set a high-pressure alert at 0.2 MPa to avoid damage to the chromatography system or DFE column.
4. Load 80 mL of the clarified plant extract or supernatant (step 1.1) onto the column at a flow rate of 0.5 mL min-1 to guarantee a contact time of 2 min. Collect the flow-through samples in 2 mL fractions for breakthrough curve reconstruction. Store the flow-through samples at 4 °C if immediate sample analysis is not possible.
5. Wash the column with 6 CV of equilibration buffer. Collect a sample of the wash at the beginning, middle, and end of this step.
6. Elute mAb 2F5 with 5 CV of low-pH elution buffer or high-ionic-strength elution buffer (0.1 M HEPES, 1.25 M magnesium chloride, pH 8.0). Collect the DFE fraction when the UV 280 nm signal has increased to 5 mAU above the baseline.
   1. Optimize the elution buffer for each epitope–antibody pair. For 2F5, 1.25 M magnesium chloride achieved an optimal balance between product recovery and ligand stability.
      NOTE: The magnesium chloride solution is prone to precipitation. Therefore, dissolve the magnesium chloride in ~700 mL of water. Separately dissolve the HEPES in 100 mL of water and adjust the pH to 8.0. Add the dissolved magnesium chloride solution to the HEPES solution and add water to a final volume of 1.0 L. Do not adjust the pH after dissolving the magnesium chloride because this will cause precipitation.
7. Analyze all samples taken during steps 1.4–1.6 using the Bradford method, lithium dodecyl sulfate-polyacrylamide gel electrophoresis (LDSPAGE), and surface plasmon resonance (SPR) spectroscopy.

Materials

Name	Company	Catalog Number	Comments
DsRed	Fraunhofe IME	n/a	Standart
HiTrap NHS-activated Sepharose HP, 1 mL	GE Helthcare	17-0716-01	Chromatography columns
HEPES	Carl Roth GmbH	9105.3	Buffer component
Magnesium chloride	Carl Roth GmbH	KK36.2	Buffer component
PH 3110	WTW	2AA110	pH meter
Sephadex G-25 fine, cross linked dextran	GE Helthcare	17003301	Chromatography resin
Sodium chloride	Carl Roth GmbH	P029.2	Buffer component
Sodium citrate	Carl Roth GmbH	HN13.2	Buffer component