Method for purifying antibody
The present invention relates to a method of purifying an antibody with high purity and high quality by removing impurities by sequential use of a cation-exchange column, a culture supernatant multilayer filter and an anion-exchange column without using a protein-A column that is an affinity chromatography column which is generally used for antibody purification.
1. A method for purifying an antibody, the method comprising:
(a) loading a sample, which comprises the antibody and at least one host cell protein (HCP) and has a pH of 5.5-7.0 and a conductivity of 5-7 mS/cm, into an equilibrated cation-exchange column, washing the cation-exchange column, and then eluting the antibody bound to the column with elution buffer;
(b) passing the eluate of step (a) through a multilayer filter, and collecting the filtrate; and
(c) passing the filtrate of step (b) through an anion-exchange column, and collecting the flow-through.
2. The method according to claim 1 , wherein the antibody has an isoelectric point of 7-10.
3. The method according to claim 1 , wherein the antibody is Bevacizumab or Adalimumab.
4. The method according to claim 1 , wherein the content of host cell protein (HCP) in the antibody eluate of step (a) is 95-99.5% lower than that in the loaded sample.
5. The method according to claim 1 , wherein the cation exchange column has a functional group selected from the group consisting of carboxymethyl (CM), sulfoethyl (SE), sulfopropyl (SP), phosphate (P) and sulfonate (S).
6. The method according to claim 5 , wherein the cation exchange column having sulfonate as a functional group comprises a cross-linked polymethacrylate resin.
7. The method according to claim 6 , wherein step (a) comprises:
(i) loading the sample comprising the antibody onto the cation exchange column equilibrated with an equilibration buffer comprising 10-50 mM phosphate (pH 5.5-7.0);
(ii) washing the cation exchange column with a buffer comprising 20-40 mM sodium chloride and 10-50 mM phosphate (pH 6.0-7.0); and
(iii) eluting the antibody with an elution buffer comprising 50-200 mM sodium chloride and 10-50 mM phosphate (pH 6.0-7.0).
8. The method according to claim 1 , wherein the multilayer filter has a product water conductivity of 8.66-10.5 μS/cm or 19.34-53.2 μS/cm post autoclave and pure water flush of 10 liters per ft 2 (100 L/m 2 ) of surface area of the multilayer filter.
9. The method according to claim 1 , wherein the antibody eluate of step (b) is subjected to viral inactivation before it is passed through the multilayer filter.
10. The method according to claim 1 , wherein the antibody eluate of step (b) is adjusted to a pH of 5.5-7.0 before it is passed through the multilayer filter.
11. The method according to claim 1 , wherein the content of host cell protein (HCP) in the filtrate of step (b) is 1,200-2,500 times lower than that in the antibody eluate of step (a).
12. The method according to claim 1 , wherein the anion exchange column in step (c) has a functional group of a quartenary amine and further comprises a cross-linked agarose base matrix.
13. The method according to claim 1 , wherein the content of host cell protein (HCP) in the protein purified by the method is 0.001-10 ppm.
14. The method according to claim 1 , wherein the method comprises no additional column chromatography step.