Activated Cross-linked Agarose for the Rapid Development of Affinity Chromatography Resins - Antibody Capture as a Case Study

Monoclonal antibodies dominate the biopharmaceutical market and are typically purified by Protein A chromatography. The according resin is a major cost driver during production affecting the price of antibody-based therapeutics. Here we describe a new affinity ligand based on the fluorescent protein that is read as a carrier for the linear epitope of HIV neutralizing antibody 2F5.

In general our method allows the fast generation of affinity chromatography resins which can be used for the capture of monoclonal antibodies. We demonstrated this capability by capturing antibody 2F5 from crude plant extract. We optimized the capture conditions and solution buffer in order to improve the dynamic binding capacity and the resin reusability.

Our resin allows generally looser conditions than Protein A and has the potential to reduce purification costs of monoclonal antibodies. Furthermore, the resin can easily be adapted to other antibodies which bind to linear epitopes. To begin this procedure, adjust the concentration of the prepared affinity ligand solution so that is in the range of 5 to 15 grams per liter as defined by the design of experiments.

Store the solution on ice until the coupling reaction is ready. Next, fill one two milliliter syringe with the DFE solution. And prepare an adapter to mount the syringes on NHS-activated cross-link agarose columns with a bed volume of one milliliter.

For every 10 columns used for coupling, prepare 30 milliliters of deactivation solution, 30 milliliters of low pH solution, and one milliliter of storage solution. Then prepare 20 milliliters of one millimolar hydrochloric acid in a tube. And incubate it on ice for at least 20 minutes.

Prepare a precision scale to monitor the flow through fractions for all steps during the coupling reaction. After this, open a sealed NHS-activated crosslinked agarose column. Apply a drop of buffer onto the adapter inlet, to prevent air from entering the column.

Mount the syringe adapter at the column inlet. Wash the column with six milliliters of ice cold one millimolar hydrochloric acid, at a flow rate less that one milliliter per minute. Using a two milliliter syringe, immediately inject 1.5 milliliters of DFE solution at a flow rate less than one milliliter per minute.

And collect the flow through fraction on a precision scale for subsequent analysis. Seal the column at both ends and incubate for 15 to 45 minutes, at 22 degrees Celsius. Next inject six milliliters of the activation solution, followed by six milliliters of low pH solution, at a flow rate less than one milliliter per minute, remove non covalently bound ligands from the resin.

Inject six milliliters of the activation solution, and incubate the column for 15 minutes. After this, inject six milliliters of low pH solution into the column. Followed by six milliliters of the activation solution.

Then inject another six milliliters of low pH solution into the column. Inject two milliliters of storage solution into the column, and store it at four degrees Celsius. First prepare 100 milliliters of either the clarified plant extract containing 2F5, or the supernatant for the preferred cell based expression system, which also contains 2F5.

Next prepare the equilibration buffer, and the high iron X strength illusion 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 five CV of equilibration buffer, at a flow rate of one 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 5 milliliters per minute.

To guarantee a contact time of two minutes. Collect the flow through samples in two milliliter fractions or breakthrough curve reconstruction. Store the flow through samples at four degrees Celsius, if immediate sample analysis is not possible.

After this wash the column with 6CV of equilibration buffer. Collect a sample at the beginning, middle and end of the wash. Dilute the MAB2F5 with five volumes of high ionic strength illusion buffer.

Collect the DFE fraction when the UV signal at 280 nanometer has increased to five milli absorbance units above the baseline. Optimize the illusion buffer for each epitope-antibody pair, as outlined in the text protocol. The fusion protein DFE is expressed in transgenic tobacco plants, grown in a greenhouse.

The overall recovery of DFE is 23.5 milligrams per kilogram. With a purity of over 90%The absolute amount of DFE immobilized on the resin increases with the mass of DFE injected into the column, and plateaus at around 15 grams per liter, whereas the coupling yield declines continuously as more DFE is injected. The DFE molecules are seen to retain their red florescence, even after coupling.

The color intensity corresponds to the total amount of immobilized DFE. Therefore column color can be used as a simple quality control parameter to estimate the coupling efficiency, and column quality. The recombinant 2F5 antibody is transiently produced in Nicotiana benthamiana plants grown in a phytotron.

The capture of 2F5 from the crude plant extract, is tested using affinity columns, coupled with approximately seven milligrams of purified DFE. A magnesium chloride concentration of 1.25 molar is sufficient to elute 2F5 from the DFE affinity resin with a recovery close to 100%and a purity of approximately 97%which is comparable to Protein A resins. The DBC of the DFE affinity resin at 10%2F5 breakthrough, declines linearly over the course of the 25 cycles, to about 15%of the initial value.

We used a design of experiments approach to optimize the coupling capacity of our affinity ligand. These conditions can be fine tuned for other ligands in the future. Our method uses florescent protein to thread a sub carrier protein for the product specific ligand.

At the same time just add functions as a visual quality indicator of the mobilization procedure, providing an immediate and intuitive feedback to the operator.