Method for the production of a glycosylated immunoglobulin
AssigneesHoffman-La Roche Inc., Chugai Seiyaku Kabushiki Kaisha
InventorsReinhard Franze, Chikashi Hirashima, Thomas Link, Yoshinori Takagi, Shinya Takuma, Yuriko Tsuda
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Abstract
Herein is reported a method for the production of an immunoglobulin comprising the following steps: a) providing a eukaryotic cell comprising a nucleic acid encoding the immunoglobulin, b) cultivating the eukaryotic cell in a cultivation medium wherein the amount of glucose available in the cultivation medium per time unit is kept constant and limited to less than 80% of the amount that could maximally be utilized by the cells in the cultivation medium per time unit, and c) recovering the immunoglobulin from the culture.
Claims22
A method for production of a composition comprising Tocilizumab protein with mannose-5 glycostructure (M5) attached to Asn297 of the Tocilizumab protein, wherein the area % fraction of M5 is in a range from 2.8 to 10% of the area sum comprising M5, G(0), G(1), and G(2) glycostructure attached to Asn297 of the Tocilizumab protein, wherein the area % fraction is determined by a method comprising liquid chromatography, comprising:
- a. cultivating recombinant Chinese Hamster Ovary (CHO) cells comprising one or more nucleic acids encoding the Tocilizumab protein in a cultivation medium under continuous or fed-batch cultivation conditions to produce the composition, wherein initial CHO cell density comprises at least 105 cells/ml and a pH set-point of the cultivation is between 7.0 and 7.2, and an amount of glucose available in the cultivation medium is in a range from 80% to 10% of the amount that could maximally be utilized by the CHO cells in the cultivation medium;
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- b. recovering the composition from the cultivation medium.
A method for production of a composition comprising Tocilizumab protein with mannose-5 glycostructure (M5) attached to Asn297 of the Tocilizumab protein, wherein the area % fraction of M5 is in a range from 2.8 to 10% of the area sum comprising M5, G(0), G(1), and G(2) glycostructure attached to Asn297 of the Tocilizumab protein, wherein the area % fraction is determined by a method comprising liquid chromatography, comprising:
- a. cultivating recombinant Chinese Hamster Ovary (CHO) cells comprising one or more nucleic acids encoding the Tocilizumab protein in a cultivation medium under fed-batch cultivation conditions to produce the composition, wherein initial CHO cell density comprises at least 105 cells/ml and a pH set-point of the fed-batch cultivation is between 7.0 and 7.2, and degree of glucose limitation during the fed-batch cultivation is in the range from 0.8 to 0.2;
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- b. recovering the composition from the cultivation medium.
Dependencies are inferred from the claim text.