Method of producing recombinant ADAMTS13 in cell culture

Patent metadata

Field	Value
Publication number	US-9458222-B2
Application number	US-201113179399-A
Country	US
Kind code	B2
Filing date	Jul 8, 2011
Priority date	Jul 8, 2010
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

Among other aspects, the present invention relates to cell culture conditions for producing high molecular weight vWF, in particular, highly multimericWF with a high specific activity and ADAMTS13 with a high specific activity. The cell culture conditions of the present invention can include, for example, a cell culture medium with an increased copper concentration and/or cell culture supernatant with a low ammonium (NH 4 + ) concentration. The present invention also provides methods for cultivating cells in the cell culture conditions to express high molecular weight vWF and rA13 having high specific activities.

First claim

What is claimed is:

1. A method for producing a recombinant ADAMTS 13 (rA13) composition, the method comprising the steps of:
(a) providing a protein free, chemically-defined, basal cell culture media;
(b) supplementing the basal cell culture media with copper to provide a final copper concentration of from 2 μg/L to 4 μg/L;
(c) providing one or more mammalian cells comprising a nucleic acid encoding a rA13 protein;
(d) culturing the one or more mammalian cells in the copper supplemented cell culture media such that rA13 is expressed and excreted from the cells into a culture supernatant, wherein the NH 4 + content of the cell culture supernatant is maintained at a concentration below 10 mM; and
(e) recovering at least a portion of the culture supernatant,
wherein the mammalian cell density is maintained at less than 4.0×10 6 cells per mL during the step of culturing the one or more cells; and
wherein at least 1500 Units FRETS-VWF73 activity per liter supplemented basal cell culture media per day is present in the recovered culture supernatant.

2. The method of claim 1 , wherein the mammalian cells are CHO cells.

3. The method of claim 1 , wherein culturing the one or more mammalian cells comprises batch cultivation of the cells.

4. The method of claim 1 , wherein culturing the one or more mammalian cells comprises continuous cultivation of the cells.

5. The method of claim 4 , wherein the continuous cultivation of mammalian cells is performed in chemostatic mode.

6. The method of claim 4 , wherein the continuous cultivation of mammalian cells is performed in perfusion mode.

7. The method of claim 1 , wherein the one or more mammalian cells is cultured in at least 100 L of the supplemented basal cell culture media.

8. The method of claim 1 , wherein the mammalian cell density is maintained at between 3.0×10 6 cells per mL and 4.0×10 6 cells per mL during the step of culturing the one or more cells.

9. The method of claim 1 , wherein at least 2000 Units FRETS-VWF73 activity per liter supplemented basal cell culture media per day is present in the recovered culture supernatant.

10. The method of claim 1 , wherein the NH 4 + content of the mammalian cell culture supernatant is maintained at a concentration below 5 mM.

Assignees

• Grillberger Leopold
• Reiter Manfred
• Fleischanderl Daniel
• Bramberger Gregor
• Baxalta Inc
• Baxalta GmbH

Inventors

• Grillberger Leopold
• Reiter Manfred
• Fleischanderl Daniel
• Bramberger Gregor

Classifications

• C12Y304/24087

  ADAMTS13 endopeptidase (3.4.24.87) · CPC title

• C07K1/00

  General methods for the preparation of peptides {, i.e. processes for the organic chemical preparation of peptides or proteins of any length} · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title

• A61P33/06

  Antimalarials · CPC title

• A61P7/04

  Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents · CPC title