An Affinity Chromatography Technique for the Purification of Monoclonal Antibodies

Abstract

Source: Elgundi, Z., et al. Laboratory Scale Production and Purification of a Therapeutic Antibody. J. Vis. Exp. (2017).

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 t....

Materials

List of materials used in this article

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