Method for purifying protein
View Patent ↗The present invention relates to protein purification. More particularly, a method for directly recovering an objective protein from a protein composition and purifying a protein with a desired quality in a rapid and efficient manner is provided. Further, a rapid purification method capable of efficiently removing impurities included in the protein composition is provided. Therefore, compared to the conventional purification methods, quality and yield of the protein can be remarkably improved.
1. A method for purifying antithrombin, comprising a first chromatographic process including the following processes of (a) to (c):
(a) a process of contacting a composition comprising the antithrombin and impurities comprising sialidase with an anion exchange carrier so that the antithrombin and the impurities are adsorbed to the anion exchange carrier;
(b) a process of washing the anion exchange carrier to which the antithrombin and the impurities are adsorbed using a first buffer weakening affinity of the impurities for the anion exchange carrier so as to remove the impurities and to inhibit the degradation of sialic acids by inhibiting the activity of sialidase, said first buffer containing 0.1 to 1 mol/L of glycine so as to remove the impurities, and said first buffer being one or more selected from the group consisting of phosphate, citrate, acetate, succinate, maleate, borate, Tris, HEPES, MES, PIPES, MOPS, TES, and Tricine; and
(c) a process of eluting the antithrombin from the anion exchange carrier by using a second buffer which is one or more selected from the group consisting of phosphate, citrate, acetate, succinate, maleate, borate, Tris, HEPES, MES, PIPES, MOPS, TES, and Tricine so as to purify antithrombin in which the number of bound sialic acids is 70% or more of the maximum number to be added, wherein the maximum number to be added is 8 mol/mol for Antithrombin α form or 6 mol/mol for Antithrombin β form.
2. The purification method according to claim 1 , wherein a recovery of the purified antithrombin is 50% or more.
3. The purification method according to claim 1 , further comprising one or more chromatographic processes after the first chromatographic process.
4. The purification method according to claim 1 , which further comprises a second chromatographic process, said second chromatographic process using any one selected from the group consisting of a mixed mode carrier, an anion exchange carrier, an anion exchange membrane, a cation exchange carrier, a cation exchange membrane, a hydrophobic interaction carrier, a size exclusion carrier, a gel filtration carrier, a reversed-phase carrier, a hydroxyapatite carrier, a fluorapatite carrier, a sulfated cellulose carrier, and a sulfated agarose carrier.
5. The purification method according to claim 1 , wherein the amount of glycine is 0.3-1 mol/L.