An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Overview

This video demonstrates a technique for purifying monoclonal antibodies from the culture supernatant obtained from an antibody-producing cell culture. The process involves using a matrix of crosslinked agarose beads with conjugated Protein A, which immobilizes the antibodies in the supernatant on the beads, allowing for effective separation from contaminants. By applying an elution buffer with low pH, the protein A-antibody interaction is loosened, facilitating the collection of antibody-containing fractions.

Protocol

1. Antibody Purification by Affinity Chromatography Using an Automated Fast Protein Liquid Chromatography (FPLC) System

NOTE: The following procedure can generally be applied to most automated systems. Purification can be performed at room temperature or at 4 °C (if FPLC system is kept in a cool room). A series of scouting tests can be performed to identify the optimal purification conditions including appropriate column matrix, binding buffer, elution buffer and pH to ensure maximum recovery of purified antibody from conditioned media (refer to Results section). The optimal conditions are dependent on the antibody or protein being purified. Purifications were performed on an automated FPLC system. Purifications were performed at room temperature using a 5 ml Protein A column.

1. Prepare the following buffers using ultrapure water and adjust to the recommended pH then filter through a 0.22 µm filter.
   1. Prepare 1 L of phosphate buffered saline (PBS) pH 7.4 (binding buffer) by mixing the following: 0.14 M NaCl, 0.0027 M KCl, 0.01 M Na₂HPO₄ and 0.001 M KH₂PO₄. Adjust pH if necessary before filtering the buffer.
   2. Prepare 500 ml of 0.1 M glycine-HCl pH 2.7 (elution buffer). Adjust pH before filtering the buffer.
   3. Prepare 50 ml of 1 M Tris pH 9.0 (neutralization buffer).
2. Ensure all system power and communication connections with computer are made. Devices should be visible in the software 'System Control' module. Ensure UV cell is set at 280 nm wavelength. Optional: Calibrate pH meter if connected and to be used.
3. Immerse inlet tubes of A, B and sample pump in ultrapure water to wash the system. Purge the pumps with a syringe if there is air in the tubing or a suspicion of air in the system. Wash the system with water by manual operation via the system controller or via an automated method. Observe that pressure, conductivity, UV280 tracing and pH remain consistent and that the pressure limit for the column is not exceeded as per manufacturer's specification.
   NOTE: Abnormalities may indicate air or blockage in the system that should be addressed before proceeding.
4. In the system controller module, start the flowrate at 1 ml/min manually via pump A in either load (bypassing sample loop) or inject (through the sample loop) position to begin connecting the column. Disconnect system tubing at the column position inlet and remove the stopper connected to the column inlet.
   1. Allow water to flow from the system tubing dropwise on top of the column then attach the system tubing to the column. Having the column inlet and inlet fitting overflowing with drops of water ensures a connection free of air bubbles.
5. Attach the column outlet at the downstream outlet and verify all fittings are tightly fastened. With the column now attached to the system, do not exceed limits for maximum pressure limit and flowrate as outlined in column specifications.
6. Wash column with 5 column volumes (CV) of water. If necessary, continue column wash until UV280 tracing has stabilized.
7. Immerse inlet tubes of A in Binding buffer, B in Elution buffer and sample pump in Binding buffer to equilibrate the system in correct buffers. Fill the inlet tubes with buffer using PumpWash by manual operation.
8. In the 'Method Editor' module of the software, use the method wizard to setup an affinity chromatography method for the column that is intended to be used.
   NOTE: Automated FPLC systems come with methods pre-filled with the recommended settings (i.e. flowrate and pressure limit) and run steps based on the column (manufacturer, matrix and size) selected for purification.
   1. Use the following run steps for Protein A affinity chromatography:
      1. Equilibrate system with 5 CV of Binding buffer and collect flowthrough into waste container.
      2. Load sample onto the column (volume to be loaded is specified manually in the method) and collect flowthrough into a separate container.
      3. Wash system with 5 CV of Binding buffer and collect flowthrough into a separate container.
      4. Elute column with 5 CV isocratic fractionation using Elution buffer and collect purified antibody sample as fractions by fraction collector.
      5. Equilibrate system with 5 CV of Binding buffer and collect flowthrough into waste container.
9. Once the method has been setup, specify the volume of conditioned media to be applied to the column and save the method.
   NOTE: The specified volume in the method should be 5-10 ml less than the actual volume to avoid introduction of air during the sample run. The specified volume used in this protocol is 90-120 ml.
10. Submerge the sample pump tubing into the vessel containing the conditioned media.
11. Prepare a separate container to collect sample flowthrough via the specified outlet tubing.
    NOTE: It is important to collect the flowthrough in the case an error occurs during the run or the binding capacity of the column is exceeded requiring the flowthrough to be reapplied to the column or the purification repeated.
12. Prepare collection tubes in the fraction collector. Add 100 µl of neutralization buffer per 1 ml fraction volume. The FPLC system will automatically elute the fractions into the collection tubes.
13. In the 'System Control' module of the software, open the method to be run.
    NOTE: The method run is initiated in a series of pages that include checking the variables of the method, fraction collector setup and defining result file name and storage location.
14. Click START to initiate the run. The run can be monitored in the 'System Control' module.
15. At the completion of the purification run, check the resulting chromatogram in the 'Evaluation' module in the system software.
16. Combine all protein-containing fractions into a separate tube, buffer exchange and concentrate in PBS using a centrifugal filter device with 30 kDa molecular weight cut off. Perform centrifugation steps according to manufacturer's instructions.
17. Measure antibody concentration using bicinchoninic acid assay (BCA) according to manufacturer's instructions.
18. If another purification run is to be performed, prime the sample pump tubing in Binding buffer and repeat steps 1.9-1.14.
19. At the completion of purification runs, immerse inlet tubes of A, B and sample pump in ultrapure water and wash the system and column as performed in step 3.
20. Submerge A, B and sample pump tubing in 20% ethanol and repeat wash procedure for storage of the system and column.
21. Disconnect the column from the downstream outlet and replace column stopper, then disconnect column at the inlet and replace stopper, reconnect tubing to the system. Store the column at 4 °C.

Materials

Name	Company	Catalog Number	Comments
Phosphate Buffered Saline (PBS) Tablets, pH 7.4, 100 ml	09-2051-100	Astral Scientific
HiTrap Protein A High Performance, 1 x 5 ml column	GE17-0403-01	Sigma-Aldrich
AKTApurifier 100	28406266	GE Healthcare	Automated FPLC system, which can include a P-960 sample pump and Frac-920 fraction collector.
Glycine-HCl	G2879	Sigma-Aldrich
Citric Acid, monohydrate	BIOC2123	Astral Scientific
Sodium Citrate, trisodium salt dihydrate	BIOCB0035	Astral Scientific
1 M Tris-HCl solution pH 9.0	BIOSD8146	Astral Scientific
Amicon Ultra Centrifugal Filters (30 MWCO)	UFC803008/UFC903008	Merck Millipore	Used to buffer exchange and concentrate purified protein.
Pierce Bicinchoninic Acid (BCA) Assay Kit	23227	Thermo Fisher Scientific
BLItz System	45-5000	fortéBIO	Instrument used for bio-layer interferometry (BLI) measurements.
Protein A biosensors	18-5010	fortéBIO