Recombinant cell clones having increased stability and methods of making and using the same

Patent metadata

Field	Value
Publication number	US-RE46860-E
Application number	US-201615203558-A
Country	US
Kind code	E1
Filing date	Jul 6, 2016
Priority date	Jun 20, 1997
Publication date	May 22, 2018
Grant date	May 22, 2018

Abstract

Disclosed are a stable recombinant cell clones which are stable in serum- and protein-free medium for at least 40 generations, a biomass obtained by multiplying the stable cell clone under serum- and protein-free culturing conditions, and a method of preparing recombinant proteins by means of the biomass. Furthermore, the invention relates to a method of recovering stable recombinant cell clones.

First claim

What is claimed is:

1. A method for obtaining a stable recombinant mammalian cell clone that produces a recombinant product and is stable under production conditions in serum- and protein-free medium for at least 40 generations, the method comprising:
providing a recombinant original mammalian cell clone, wherein the recombinant original mammalian cell clone has a selection marker, cultivating the recombinant original cell clone on serum-containing medium, adapting the cells to serum- and protein-free medium with neither selection pressure for the selection marker nor selection for a polypeptide factor that replaces serum components, testing the cell culture after adaptation for stable product-producers with neither selection pressure for the selection marker nor selection for a polypeptide factor that replaces serum components, and cloning a stable product-producer-cell clone in serum- and protein-free conditions with neither selection pressure for the selection marker nor selection for a polypeptide factor that replaces serum components.

2. The method according to claim 1 , wherein the stable product-producer cell clone obtained is present in isolated form after the step of cloning.

3. The method according to claim 1 , wherein the recombinant cell clone comprises a nucleic acid encoding a recombinant polypeptide or protein.

4. The method according to claim 1 , wherein the recombinant product is Factor VIII.

5. The method according to claim 1 , wherein the recombinant product is Factor IX.

6. The method according to claim 1 , wherein the recombinant product is Factor VII.

7. The method according to claim 1 , wherein the recombinant product is von Willebrand factor (vWF).

8. The method according to claim 1 , wherein the original mammalian cell clone is a CHO cell clone.

9. A method for preparing a recombinant product, the method comprising:
culturing a stable recombinant mammalian cell clone that produces a recombinant product, wherein the cell clone is stable in serum- and protein-free medium for at least 40 generations with neither selection pressure for a selection marker nor selection pressure for an amplification marker in the cell clone, and expressing the recombinant product in serum- and protein-free medium so as to obtain a cell culture; and recovering the expressed recombinant product from the cell culture, wherein the recombinant product is selected from the group consisting of: Factor VIII, Factor IX, Factor VII, and von Willebrand Factor (vWF).

10. The method according to claim 9, wherein the stable recombinant mammalian cell clone is a CHO cell clone.

11. The method according to claim 9, wherein the serum- and protein-free medium comprises one or more amino acids selected from the group consisting of: L-asparagine, L-cysteine, L-cystine, L-proline, L-tryptophan and L-glutamine.

12. The method according to claim 9, wherein the serum- and protein-free medium comprises soybean peptone having a molecular weight of ≤1000 Dalton.

13. The method according to claim 9 further comprising
providing a stable recombinant mammalian cell clone that expresses a recombinant product and is stable in serum- and protein-free medium for at least 40 generations with neither selection pressure for a selection marker nor selection pressure for an amplification marker in the cell clone.

14. The method of claim 9, wherein the stable recombinant mammalian cell clone is produced by:
providing a recombinant original mammalian cell clone, wherein the recombinant original mammalian cell clone has a selection marker and an amplification marker, cultivating the recombinant original cell clone on serum-containing medium to create a cell culture, adapting the cell culture to serum- and protein-free medium with neither selection pressure for the selection marker nor selection pressure for the amplification marker, testing the cell culture after adaptation for stable product-producers with neither selection pressure for the selection marker nor selection pressure for the amplification marker, and isolating a stable product-producer-cell clone in serum- and protein-free conditions with neither selection pressure for the selection marker nor selection pressure for the amplification marker to obtain a stable recombinant mammalian cell clone.

Assignees

• Baxalta Inc
• Baxalta GmbH

Inventors

• Reiter Manfred
• Mundt Wolfgang
• Dorner Friedrich

Classifications

• C12N5/0043

  Medium free of human- or animal-derived components · CPC title

• C12N5/0075 Primary

  using microcarriers · CPC title

• C12N2500/76

  from plants · CPC title

• C12N2531/00

  Microcarriers · CPC title

• C12N2500/32

  Amino acids · CPC title