Methods to control protein heterogeneity

Patent metadata

Field	Value
Publication number	US-9512214-B2
Application number	US-201414194305-A
Country	US
Kind code	B2
Filing date	Feb 28, 2014
Priority date	Sep 2, 2012
Publication date	Dec 6, 2016
Grant date	Dec 6, 2016

Abstract

The instant invention relates to the field of protein production and in particular to controlled protein heterogeneity compositions and processes for controlling the heterogeneity of proteins expressed in host cells.

First claim

What is claimed is:

1. A method for controlling the oligosaccharide distribution of a recombinantly-expressed immunoglobulin comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 2 and a light chain variable region comprising the sequence of SEQ ID NO: 7, comprising
supplementing during a production stage a cell culture medium used in the recombinant expression of said immunoglobulin with a yeast hydrolysate and/or a plant hydrolysate to achieve a yeast hydrolysate concentration in the medium of at least 11 g/L and/or a plant hydrolysate concentration of at least 7 g/L; and
assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin, thereby controlling the oligosaccharide distribution of the recombinantly-expressed immunoglobulin,
wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) present on the recombinantly-expressed immunoglobulin is decreased as compared to the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) of the immunoglobulin recombinantly-expressed in cell culture medium which is not supplemented with said yeast hydrolysate supplement and/or said plant hydrolysate supplement during the production stage; and/or
wherein the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the recombinantly-expressed immunoglobulin is increased as compared to the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) of the immunoglobulin recombinantly-expressed in cell culture medium which is not supplemented with said yeast hydrolysate supplement and/or said plant hydrolysate supplement during the production stage.

2. The method of claim 1 , wherein the immunoglobulin is adalimumab.

3. The method of claim 2 , wherein the method produces at least 2.0 g/L of adalimumab.

4. The method of claim 1 , wherein the yeast hydrolysate is selected from the group consisting of Bacto TC Yeastolate, HyPep Yeast Extract and UF Yeast Hydrolysate.

5. The method of claim 1 , wherein the plant hydrolysate is selected from the group consisting of a soy hydrolysate, a wheat hydrolysate, a rice hydrolysate, a cotton seed hydrolysate, a pea hydrolysate, a corn hydrolysate and a potato hydrolysate.

6. The method of claim 1 , wherein the plant hydrolysate is selected from the group consisting of BBL Phytone Peptone, HyPep 1510, SE50 MAF-UF, UF Soy Hydrolysate, Wheat Peptone E1, HyPep 4601 and Proyield WGE80M Wheat.

7. The method of claim 1 , wherein the cell culture medium is supplemented with the plant hydrolysate to achieve a plant hydrolysate concentration from 7 g/L to 15 g/L.

8. The method of claim 1 , wherein the cell culture medium is supplemented with the plant hydrolysate to achieve a plant hydrolysate concentration of 10 g/L to 15 g/L.

9. The method of claim 1 , wherein the cell culture medium is supplemented with the yeast hydrolysate and the plant hydrolysate to achieve a yeast hydrolysate to plant hydrolysate ratio of 0.25 to 1.55.

10. The method of claim 1 , wherein the recombinantly-expressed immunoglobulin is produced by a CHO cell line.

11. The method of claim 1 , wherein the cell culture medium comprises yeast and/or plant hydrolysate prior to supplementing the medium with the yeast hydrolysate and/or plant hydrolysate.

12. The method of claim 1 , wherein the cell culture medium is substantially free of yeast and/or plant hydrolysate prior to supplementing the medium with the yeast hydrolysate and/or plant hydrolysate.

13. The method of claim 1 , wherein assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin comprises assessing the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) present on the recombinantly-expressed immunoglobulin.

14. The method of claim 1 , wherein 64%-89% of the total N-linked oligosaccharides present on the immunoglobulin are of an agalactosyl fucosylated biantennary oligosaccharide (sum of NGA2F and NGA2F-GlcNac) form.

15. The method of claim 1 , wherein 64%-69% of the total N-linked oligosaccharides present on the immunoglobulin are of an agalactosyl fucosylated biantennary oligosaccharide (sum of NGA2F and NGA2F-GlcNac) form.

16. The method of claim 1 , wherein assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin comprises assessing the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) on the recombinantly-expressed immunoglobulin.

17. The method of claim 1 , wherein 8%-31% of the total N-linked oligosaccharides present on the immunoglobulin are of a galactose containing fucosylated biantennary oligosaccharide (sum of NA1F and NA2F) form.

18. The method of claim 1 , wherein 29%-31% of the total N-linked oligosaccharides present on the immunoglobulin are of a galactose containing fucosylated biantennary oligosaccharide (sum of NA1F and NA2F) form.

19. The method of claim 1 , wherein the method is a fed batch process.

20. The method of claim 1 , further comprising collecting and isolating the produced immunoglobulin.

21. The method of claim 1 , wherein the production stage initiates at an initial viable cell density of approximately 0.5×10 6 cells/mL.

22. The method of claim 1 , wherein the oligosaccharide distribution of the recombinantly-expressed immunoglobulin is assessed after the recombinantly-expressed immunoglobulin has been harvested from the cell culture medium.

23. The method of claim 1 , wherein the recombinantly-expressed immunoglobulin is expressed in a mammalian cell.

24. A method for controlling the oligosaccharide distribution of a recombinantly-expressed immunoglobulin comprising a heavy chain variable region comprising the sequence of SEQ ID NO: 2 and a light chain variable region comprising the sequence of SEQ ID NO: 7, comprising
supplementing a cell culture media used in the recombinant expression of said immunoglobulin with a yeast hydrolysate supplement and/or a plant hydrolysate supplement; and
assessing the oligosaccharide distribution of the recombinantly-expressed immunoglobulin, wherein the method produces at least 2.5 g/L of said immunoglobulin, thereby controlling the oligosaccharide distribution of said immunoglobulin,
wherein the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) present on the recombinantly-expressed immunoglobulin is decreased as compared to the level of agalactosyl fucosylated biantennary oligosaccharides (sum of NGA2F and NGA2F-GlcNac) of the immunoglobulin recombinantly-expressed in cell culture media which is not supplemented with said yeast hydrolysate supplement and/or said plant hydrolysate supplement; and/or
wherein the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) present on the recombinantly-expressed immunoglobulin is increased as compared to the level of galactose containing fucosylated biantennary oligosaccharides (sum of NA1F and NA2F) of the immunoglobulin recombinantly-expressed in cell culture media which is not supplemented with said yeast hydrolysate supplement and/or said plant hydrolysate supplement.

25. A method for controlling the oligosaccharide distribution of a recombinantly-expressed immunoglobulin comprising a heavy chain variable region comprisin

Assignees

• Abbvie Inc
• Abbvie Inc

Inventors

• Rives Lisa M
• Bengea Cornelia
• Zeng Xiaobei

Classifications

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N2510/02

  Cells for production · CPC title

• C07K2317/14

  Specific host cells or culture conditions, e.g. components, pH or temperature · CPC title

• C07K2317/41

  Glycosylation, sialylation, or fucosylation · CPC title

• C07K2317/21

  from primates, e.g. man · CPC title