Production of proteins in glutamine-free cell culture media
Inventors
Gawlitzek, Martin • Luo, Shun • Bevilacqua, Christina Teresa
Assignees
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Publication Number
US-12351641-B2
Publication Date
2025-07-08
Expiration Date
Abstract
The present invention relates generally to glutamine-free cell culture media supplemented with arginine. The invention further concerns the production of recombinant proteins, such as therapeutic IgG antibodies, in a Chinese hamster ovary (CHO) host cell in a production phase culture medium that is essentially free of glutamine and comprises arginine in arginine.
Core Innovation
The invention relates to producing a therapeutic IgG antibody in a Chinese hamster ovary (CHO) host cell during a production phase culture, wherein the production phase culture medium is essentially free of glutamine and comprises asparagine at a concentration in the range of 2.5 mM to 15 mM. The process includes CHO host cells expressing the therapeutic IgG antibody, including embodiments using dhfr− CHO cells and serum-free production phase culture media.
A central aspect is that glutamine-free production phase culture media are supplemented with asparagine, optionally with aspartic acid and/or glutamate, to support cell viability, culture longevity, specific productivity (Qp), and final therapeutic IgG antibody titer. The disclosure links reduced ammonia (NH4+) accumulation to lowered cytotoxicity and improved performance under glutamine-free conditions.
The disclosure includes experimental results and DOE analysis predicting maximal titer under glutamine-free conditions using Asn/Asp/Glu combinations, and reports application to particular therapeutic IgG antibodies. Examples identified in the document include Apomab (anti-DR5), anti-VEGF, and BR3-Fc immunoadhesin, with production in different culture formats such as shake-flask and DMEM/F12 formulations.
Claims Coverage
The partial claim set contains four independent claims. Across these independent claims, the coverage centers on culturing CHO host cells in a glutamine-essentially-free production phase culture medium containing asparagine at 2.5 mM to 15 mM, with antibody identity specified for three claims and selected from a group for one claim.
Glutamine-free production phase culture medium with asparagine for pertuzumab production in CHO
A process for producing a therapeutic IgG antibody in a Chinese hamster ovary (CHO) host cell expressing said therapeutic IgG antibody, wherein the process comprises culturing the CHO host cell in a production phase culture medium that is essentially free of glutamine, wherein the production phase culture medium comprises asparagine provided at a concentration in the range of 2.5 mM to 15 mM, wherein the therapeutic IgG antibody is pertuzumab.
Glutamine-essentially-free production phase medium with asparagine for group-selected IgG antibodies in CHO
A process for producing a therapeutic IgG antibody in a Chinese hamster ovary (CHO) host cell expressing said therapeutic IgG antibody, wherein the process comprises culturing the CHO host cell in a production phase culture medium that is essentially free of glutamine, wherein the production phase culture medium comprises asparagine provided at a concentration in the range of 2.5 mM to 15 mM, wherein the therapeutic IgG antibody is selected from the group consisting of an anti-CD4 antibody, an anti-EGF-like-domain 7 (EGFL7) antibody, an anti-IL13 antibody, an anti-DR5 antibody, an anti-BR3 antibody, an anti-beta 7 integrin subunit antibody, dacetuzumab, an anti-CD20 monoclonal, an anti-MET receptor tyrosine kinase antibody, an anti-neuropilin-1 (NRP1) antibody, an anti-OX40 ligand antibody, an anti-oxidized LDL (oxLDL) antibody, a HER dimerization inhibitor (HDI) antibody, and a rhuMAb IFN alpha antibody.
Glutamine-free production phase culture medium with asparagine for omalizumab production in CHO
A process for producing a therapeutic IgG antibody in a Chinese hamster ovary (CHO) host cell expressing said therapeutic IgG antibody, wherein the process comprises culturing the CHO host cell in a production phase culture medium that is essentially free of glutamine, wherein the production phase culture medium comprises asparagine provided at a concentration in the range of 2.5 mM to 15 mM, wherein the therapeutic IgG antibody is omalizumab.
Glutamine-free production phase culture medium with asparagine for ocrelizumab production in CHO
A process for producing a therapeutic IgG antibody in a Chinese hamster ovary (CHO) host cell expressing said therapeutic IgG antibody, wherein the process comprises culturing the CHO host cell in a production phase culture medium that is essentially free of glutamine, wherein the production phase culture medium comprises asparagine provided at a concentration in the range of 2.5 mM to 15 mM, wherein the therapeutic IgG antibody is ocrelizumab.
Across the independent claims, the inventive concept is the use of a production phase culture medium that is essentially free of glutamine, supplemented with asparagine at 2.5 mM to 15 mM, in CHO host cells expressing a therapeutic IgG antibody, with antibody coverage specified as pertuzumab, omalizumab, ocrelizumab, or selected from a defined group.
Stated Advantages
Improves cell viability.
Improves culture longevity.
Increases specific productivity (Qp).
Improves final therapeutic IgG antibody titer.
Reduces ammonia (NH4+) accumulation and lowers NH4+ cytotoxicity.
Documented Applications
Producing therapeutic IgG antibodies in CHO production phase culture using glutamine-free media supplemented with asparagine for antibodies including Apomab (anti-DR5), anti-VEGF, and BR3-Fc immunoadhesin.
Use in experimental formats including shake-flask and DMEM/F12 formulations for recombinant protein expression, with emphasis on therapeutic IgG antibodies.
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