Method for production of recombinant human iduronate 2-sulfatase

Patent metadata

Field	Value
Publication number	US-9279109-B2
Application number	US-201213979973-A
Country	US
Kind code	B2
Filing date	Jan 23, 2012
Priority date	Jan 25, 2011
Publication date	Mar 8, 2016
Grant date	Mar 8, 2016

Abstract

Disclosed is a method for production of recombinant human iduronate 2-sulfatase (rhI2S) (the 26th to 550th amino acids of SEQ ID NO: 10) in a large scale, with a high purity, and mannose 6-phosphate residues. The method comprises the steps of (a) culturing rhI2S (the 26th to 550th amino acids of SEQ ID NO: 10)-producing mammalian cells in a serum-free medium, (b) collecting culture supernatant, (c) subjecting the culture supernatant to cation-exchange column chromatography, (d) to dye affinity column chromatography, (e) to anion-exchange column chromatography, and (f) to a column chromatography employing as solid phase a material having affinity for phosphate group, and (g) to gel filtration column chromatography, in the order.

First claim

The invention claimed is:

1. A method for producing recombinant human wild-type iduronate 2-sulfatase (rhI2S) consisting of the 26 th to 550 th amino acids of the amino acid sequence set forth in SEQ ID NO:10, wherein the method comprises:
(a) culturing rhI2S-producing mammalian cells in a serum-free medium so as to allow the cells to secrete the rhI2S in the medium, wherein the cells are those that have been transformed to express the gene encoding the rhI2S by introduction of the gene using an expression vector in which the gene is incorporated,
(b) collecting culture supernatant by removing the cells from the culture that is obtained in step (a) above,
(c) subjecting the culture supernatant collected in step (b) above to cation-exchange column chromatography to collect rhI2S-active fractions,
(d) subjecting the fractions collected in step (c) above to dye affinity column chromatography to collect rhI2S-active fractions,
(e) subjecting the fractions collected in step (d) above to anion-exchange column chromatography to collect rhI2S-active fractions,
(f) subjecting the fractions collected in step (e) above to a column chromatography employing as a solid phase, a material having affinity for phosphate group to collect rhI2S-active fractions, and
(g) subjecting the fractions collected in step (1) above to gel filtration column chromatography to collect rhI2S-active fractions, in the respective order.

2. The method according to claim 1 , wherein the cation exchanger employed in the cation-exchange column chromatography is a weak cation exchanger.

3. The method according to claim 2 , wherein the weak cation exchanger has phenyl groups, amide bonds and carboxyl groups.

4. The method according to claim 1 , wherein the dye employed in the dye affinity column chromatography is a blue triazine dye.

5. The method according to claim 1 , wherein the material having affinity for phosphate group is selected from the group consisting of fluoroapatite and hydroxyapatite.

6. The method according to claim 5 , wherein the material having affinity to phosphate group is fluoroapatite.

7. The method according to claim 1 , wherein the mammalian cells are CHO cells transfected with an expression vector which is designed to express rhI2S under the regulation of EF-1(alpha) promoter.

8. A method for purifying recombinant human wild-type iduronate 2-sulfatase (rhI2S) consisting of the 26 th to 550 th amino acids of the amino acid sequence set forth in SEQ ID NO:10 from contaminants in a sample, wherein the rhI2S has an oligosaccharide chain linked thereto containing one or more mannose 6-phosphate residues, the method comprising:
(a) applying the sample to a chromatography column which employs fluoroapatite as a solid phase,
(b) flowing a first mobile phase through the column to wash the column while letting the rhI2S be adsorbed by the column, and
(c) eluting the rhI2S from the column by flowing a second mobile phase through the column, wherein the second mobile phase contains a phosphate at a higher concentration than the first mobile phase.

Assignees

• Mihara Kazutoshi
• Kawasaki Atsuko
• Ootsuki Kouta
• Hatano Yuukichi
• Kamei Shoichiro
• Sugimura Atsushi
• Japan Chem Res

Inventors

• Mihara Kazutoshi
• Kawasaki Atsuko
• Ootsuki Kouta
• Hatano Yuukichi
• Kamei Shoichiro
• Sugimura Atsushi

Classifications

• A61P3/00

  Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00) · CPC title

• C12Y301/06013

  Iduronate-2-sulfatase (3.1.6.13) · CPC title

• C12N9/16 Primary

  acting on ester bonds (3.1) · CPC title

• C07K1/16

  by chromatography · CPC title