Cell culture media and methods

Patent metadata

Field	Value
Publication number	US-10066200-B2
Application number	US-201615198237-A
Country	US
Kind code	B2
Filing date	Jun 30, 2016
Priority date	Dec 22, 2011
Publication date	Sep 4, 2018
Grant date	Sep 4, 2018

Abstract

Compositions and methods are described for preparing media, feeds, and supplements. Such methods and medias may display increased stability of labile components and may use, for example, microsuspension and/or encapsulation technologies, chelation, and optionally, coating and/or mixing the labile compounds with anti-oxidants. The compositions may withstand thermal and/or irradiation treatment and have reduced virus number. These techniques may result in product with extended shelf-life, extended release of their internal components into culture, or in product that can be added aseptically into a bioreactor using minimal volumes. The compositions and methods may optimize the bioproduction workflow and increase efficiency.

First claim

What is claimed is:

1. A method of making a recombinant polypeptide or virus, the method comprising:
a) preparing a cell culture medium, feed or supplement comprising a microsuspension by:
(i) adding a minimal volume of an aqueous solution to a dry powder medium, feed or supplement to make a paste;
(ii) mixing the paste vigorously to prepare a microsuspension;
(iii) optionally, adding an effective amount of an anti-oxidant to the microsuspension to form a mixture;
(iv) encapsulating the microsuspension of (ii), or the mixture of (iii), in a capsular material to form a bead;
(v) drying the bead to form the cell culture medium, feed or supplement,

b) dissolving the cell culture medium, feed or supplement of (v) in a solvent to form a liquid cell culture medium,
c) contacting said liquid cell culture medium with a cell expressing the recombinant polypeptide or virus,
d) culturing said cell in said liquid cell culture medium under conditions favoring the expression of the recombinant polypeptide or virus.

2. The method of claim 1 , wherein the cell culture medium comprises a labile substance.

3. The method of claim 1 , wherein the capsular material that is selected from the group consisting of alginate, poly-L-lactic acid, chitosan, agarose, gelatin, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives and carrageenan.

4. The method of claim 1 , wherein the bead is coated with a coating solution.

5. The method of claim 4 , wherein the coating solution is selected form the group consisting of poly-glycolic acid, PLGA (poly-lactic-co-glycolic acid), collagen, polyhydroxyalkanoates (PHA), poly-ε-caprolactone, poly-ortho esters, poly-anhydrides, poly-phosphazenes, poly-amino acids, polydimethylsiloxane, polyurethanes, poly-tetrafluoroethylene, polyethylene, polysulphone, poly-methyl methacrylate, poly-2-hydroxyethylmethacrylate, polyamides, polypropylene, poly-vinyl chloride, polystyrene, poly-vinyl pyrrolidone, poly-L-lysine and polyornithine.

6. The method of claim 4 , wherein the bead further comprises a reactive species that is chelated using a chelator.

7. The method of claim 6 , wherein the reactive species is a cation, a metal ion or a trace element.

8. The method of claim 6 , wherein the chelating moieties are selected from the group consisting of EDTA, citrate, succinate, cyclodextrin, clatharates, dendrimers and amino acids.

9. The method of claim 1 , wherein the cell culture medium, feed or supplement is irradiated.

10. The method of claim 9 wherein the irradiation is with gamma-rays.

11. The method of claim 10 , wherein the gamma-rays are 25-100 kGy.

12. The method of claim 1 wherein the cell is a mammalian cell.

13. The method of claim 1 , wherein the cell culture medium, feed or supplement is protein free.

14. The method of claim 1 , wherein the powdered cell culture medium used for the microsuspension is AGT (advanced granulation technology cell culture medium).

15. A method of culturing a cell comprising:
a) preparing a medium, feed or supplement comprising a microsuspension by:
(i) adding a minimal volume of an aqueous solution to a dry powder medium, feed or supplement to make a paste;
(ii) mixing the paste vigorously to prepare a microsuspension;
(iii) optionally, adding an effective amount of an anti-oxidant to the microsuspension to form a mixture;
(iv) encapsulating the microsuspension of (ii), or the mixture of (iii), in a capsular material to form a bead;
(v) drying the bead to form the medium, feed or supplement comprising the microsuspension,

b) dissolving the medium, feed or supplement of (v) in a solvent to form a liquid cell culture medium,
c) contacting said liquid cell culture medium with said cell to be cultured.

16. The method of culturing a cell of claim 15 wherein, the medium, feed or supplement comprising the microsuspension is stored at ambient temperatures.

17. The method of culturing a cell of claim 15 wherein the cell is a mammalian cell.

18. The method of culturing a cell of claim 17 wherein the mammalian cell is selected from the group consisting of Chinese Hamster Ovary (CHO) cells, 293, BHK, Vero, PerC6, MDBK and MDCK cells.

19. The method of culturing a cell of claim 18 wherein the mammalian cell is a CHO cell.

20. The method of culturing a cell of claim 15 wherein the medium, feed or supplement is irradiated.

Assignees

• Life Technologies Corp

Inventors

• Fike Richard
• Barrett Shawn
• Jiang Zhou
• Judd David

Classifications

• C12N2533/74

  Alginate · CPC title

• C12N2500/38

  Vitamins · CPC title

• C12N5/0018 Primary

  Culture media for cell or tissue culture (media for specific animal cell type C12N5/06) · CPC title

• C12N2500/30

  Organic components · CPC title

• C12N2533/78

  Cellulose · CPC title