This article describes a method for purifying monoclonal antibodies using an FPLC column with immobilized Protein A. The process involves equilibrating the column, loading the sample, washing, and eluting the antibodies while monitoring UV absorbance.
Take an FPLC column containing a porous glass resin with immobilized Protein A — an antibody-binding bacterial protein. Equilibrate the column with a neutral pH buffer.
Add cell culture fluid harvested from Chinese hamster ovarian cells, containing monoclonal antibodies and contaminating proteins and DNA.
Protein A binds to the antibody's Fc region, immobilizing them.
Some contaminants non-specifically attach to the resin, while the rest flow through.
In the chromatogram, an increased UV absorbance during sample loading indicates a high protein concentration in the flow through.
Wash with the equilibration buffer to remove unbound contaminants, indicated by a decrease in the absorbance.
Flush with a high-salt buffer, disrupting the non-specific interactions and removing the contaminants, indicated by a small impurity peak.
Add a low-pH elution buffer to disrupt the Protein A-antibody interaction and elute the antibodies, which are collected by tracking the corresponding peak.
Neutralize the pH of the collected antibodies to prevent degradation.
Begin by opening the software attached to the purification system and placing 15-milliliter conical tubes to collect the purified antibody eluate, and 50-milliliter conical tubes to collect the flow-through during the high-salt wash into the fraction collector.
Next, add 0.22-micrometer pore-filtered harvested cell culture fluid to an empty 12-milliliter syringe with a capped nozzle. After removing the cap, insert the syringe nozzle into the manual injection port of the purification system. Twist the syringe to tighten it in place, and depress the plunger until the entire volume of the sample has been injected, and is visible in the attached 10-milliliter large-volume sample loop.
Select the saved method and click Run when prompted by the instrument software. When all of the antibody has been eluted, immediately neutralize the purified protein with 1 molar tris base to a pH of about 5.5.
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