DFE-Based Affinity Chromatography: A Technique for Purification of 2F5 Monoclonal Antibodies from a Crude Sample Using High Ionic Strength-Based Elution Conditions
Transcript
First, prepare 100 milliliters of either the clarified plant extract containing 2F5 or the supernatant from the preferred cell-based expression system, which also contains 2F5. Next, prepare the equilibration buffer and the high-ionic-strength elution buffer, as outlined in the text protocol. Flush the chromatography system with the buffers.
Mount a DFE affinity column on the chromatography system, and equilibrate with 5 CV of equilibration buffer at a flow rate of 1 milliliter per minute. Monitor the UV absorbance at 280 nanometers. Then, load 80 milliliters of either the clarified plant extract or the supernatant onto the column at a flow rate of 0.5 milliliters per minute to guarantee a contact time of 2 minutes. Collect the flow-through samples in 2-milliliter fractions for breakthrough curve reconstruction.
Store the flow-through samples at 4 degrees Celsius, if immediate sample analysis is not possible. After this, wash the column with 6 CVs of equilibration buffer. Collect a sample at the beginning, middle, and end of the wash. Elute the mAb 2F5 with 5 volumes of high-ionic-strength elution buffer. Collect the DFE fraction when the UV signal at 280 nanometers has increased to 5 milliabsorbance units above the baseline.
