Claims for Patent: 10,519,479

Summary for Patent: 10,519,479

Title:	Methods for modifying glycosylation using manganese
Abstract:	The present invention relates to a cell culture method for adjusting the proportion of G0, G1, G2, and high-mannose galactosylation variants in a population of a recombinant protein produced by a culture of cells in a cell culture medium by supplementing the cell culture medium with manganese at one or more days of the cell culture method duration.
Inventor(s):	Solacroup; Thomas (Blonay, CH), Stettler; Matthieu (Vucherens, CH), Jordan; Martin (Ecublens, CH), Broly; Herve (Chatel-St Denis, CH)
Assignee:	ARES TRADING S.A. (Aubonne, CH)
Application Number:	15/457,624
Patent Claims:	1. A cell culture method for adjusting the proportion of G0, G1, G2, and high-mannose glycosylation variants in a population of a recombinant protein produced by a culture of cells in a cell culture medium, the cell culture method comprising: i) initiating the cell culture method in a manganese-free, galactose-free cell culture medium; and ii) supplementing the cell culture medium with manganese (Mn) at one or more days of the cell culture method duration and wherein the cell culture medium remains galactose free for the cell culture method duration, thereby adjusting the proportion of G0, G1, G2, and high-mannose glycosylation variants in the population of the recombinant protein in comparison to a population of the recombinant protein produced in cell culture that is not supplemented with manganese and/or contains galactose. 2. The method of claim 1, wherein the Mn is supplemented in the form of MnCl.sub.2. 3. The method of claim 1 wherein the cell culture method is a fed-batch culture method. 4. The method of claim 1, wherein the Mn is added to the cell culture medium at one or more of day 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the cell culture method. 5. The method of claim 1, wherein Mn is added at day 3, day 5, day 7 and/or day 10 of the cell culture method. 6. The method of claim 1, wherein the Mn is not added on days 0, 1 or 2 of the cell culture method. 7. The method of claim 1, wherein Mn is added to the culture medium on day 3 of the cell culture method to a concentration of about 0.14 .mu.M to about 0.325 .mu.M. 8. The method of claim 1, wherein Mn is added to the culture medium on any one of day 5, day 7 or day 10 of the cell culture method to a concentration of about 0.75 .mu.M or about 0.29 .mu.M in addition to any Mn already present in the medium. 9. The method of claim 1, wherein the protein is a TNF.alpha. binding protein. 10. The method of claim 9, wherein the TNF.alpha. binding protein is etanercept, adalimumab, infliximab, or a biosimilar thereof. 11. The method of claim 9, wherein the TNF.alpha. binding protein is an antibody. 12. The method of claim 11, wherein the antibody has: (i) a light chain sequence having at least 90% identity with SEQ ID NO:1 and a heavy chain sequence having at least 90% identity with SEQ ID NO:2; (ii) a light chain sequence having at least 95% identity with SEQ ID NO:1 and a heavy chain sequence having at least 95% identity with SEQ ID NO:2; (iii) a light chain sequence of SEQ ID NO:1 and a heavy chain sequence of SEQ ID NO:2; (iv) a light chain sequence comprising SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7; and a heavy chain sequence comprising SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8; (v) a light chain sequence comprising SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, wherein Xaa in SEQ ID NO:3 is Threonine or Alanine, and a heavy chain sequence comprising SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8; (vi) a light chain sequence comprising SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, and a heavy chain sequence comprising SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, wherein Xaa of SEQ ID NO: 4 is Tyrosine or Asparagine; or (vii) a light chain sequence comprising SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 7, wherein Xaa in SEQ ID NO:3 is Threonine or Alanine, and a heavy chain sequence comprising SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, wherein Xaa of SEQ ID NO: 4 is Tyrosine or Asparagine. 13. The method of claim 1, wherein the cell culture medium is serum-free and/or protein-free. 14. The method of claim 1, further comprising supplementing with asparagine and/or iron. 15. A method of making a recombinant protein in a cell culture, comprising: i) initiating the cell culture method in a manganese-free, galactose-free cell culture medium; ii) growing a host cell in a cell culture medium, iii) expressing the recombinant protein in the host cell, iv) supplementing the cell culture medium with manganese at one or more days of the cell culture method duration and wherein the cell culture medium remains galactose free for the cell culture method duration, thereby adjusting the proportion of G0, G1, G2, and high-mannose glycosylation variants in the population of the recombinant protein in comparison to a population of the recombinant protein produced in cell culture that is not supplemented with manganese and/or contains galactose, and v) purifying the recombinant protein from the cell culture.

Details for Patent 10,519,479

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Janssen Biotech, Inc.	REMICADE	infliximab	For Injection	103772	08/24/1998		2040-01-24
Immunex Corporation	ENBREL	etanercept	For Injection	103795	11/02/1998		2040-01-24
Immunex Corporation	ENBREL	etanercept	For Injection	103795	05/27/1999		2040-01-24
Immunex Corporation	ENBREL	etanercept	Injection	103795	09/27/2004		2040-01-24