Separation of human Fab fragments on negative mode Ni(II)-TREN-agarose chromatography

• Human Fab fragments were obtained in nonretained chromatographic fractions (negative chromatography).
• Proteins extracted from soybean seeds were adsorbed on Ni(II)-TREN-agarose.
• The selectivity of the Ni(II)-TREN-agarose was influenced by the nature of the buffer system and NaCl.

We evaluated the feasibility of using immobilized metal-ion affinity chromatography (IMAC) with nickel ion complexed with Tris(2-aminoethyl)amine (TREN) immobilized on agarose gel for purification of human Fab fragments by negative chromatography. Efficient purification of Fab fragments from digested human IgG (immunoglobulin G) (106.4% purity) was accomplished in Tris-HCl buffer at pH 7.5 without NaCl (based on total protein concentration and radial immunodiffusion of human Fab). A technological application of Ni(II)-TREN-agarose using non transgenic soybean protein extract spiked with human Fab fragments as feedstream was also studied. Experiments using Tris-HCl at pH 7.0 as loading buffer allowed the adsorption of almost all of the soybean proteins. Sixty-six percent of the loaded human Fab fragments were recovered in the flowthrough and washing fractions with about 90% purity. These results demonstrate that Ni(II)-TREN-agarose is a potential adsorbent for recombinant Fab fragment purification.