[Preparation of a block copolymer-based temperature-responsive affinity chromatography stationary phase for antibody separation and purification].

Abstract

Antibodies play an essential role in cancer diagnosis and treatment because of the specificity for target biomolecules and reduction of side effects. However, antibodies separation and purification still face some challenges. Antibody elution from columns using a low-pH aqueous solution leads to aggregation or loss of activity of the antibody drugs. In this paper, a block copolymer-based temperature-responsive affinity chromatography (TRAC) stationary phase, SiO₂-P[NIPAM-b-4VP]-MEP using the block temperature-responsive copolymer poly(N-isopropylacrylamide-b-4-vinylpyridine) (P[NIPAM-b-4VP]) as the space arms and 4-mercaptoethyl pyridine (MEP) as the ligand was prepared for antibody separation. The TRAC column was tested using bovine serum albumin (BSA) and γ-globulin as model proteins, and the effects of salt concentration in the mobile phase and temperature on their separation were studied in detail. At 40 ℃, the TRAC stationary phase only selectively retained γ-globulin due to the specific affinity interaction between antibodies and the ligand MEP. At 5 ℃, γ-globulin can be eluted from the column with a mass recovery of 92.7% using a Tris-HCl buffer (pH 8.0) solution containing 0.6 mol/L NaCl. The adsorption capacity of γ-globulin on this stationary phase was (71.5 ±2.1) mg/g (n=3), which was twice that of a traditional temperature-sensitive affinity chromatography stationary phase SiO₂-PNIPAM-MEP. The stationary phase was also used to separate and purify immunoglobulin (IgG) in human serum in one step by altering the temperature and ion strength of the mobile phase, resulting in a purity of 97.4%±0.7%. Thus, this new technology has specific selectivity for antibodies, as well as mild and green elution conditions, ultimately resolving the problem of traditional affinity chromatography using acid elution, which can lead to the antibodies aggregation/inactivation. This technology has great application potential for the industrial production of antibody drugs.