Dataset Information

Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologics.

ABSTRACT: Chinese hamster ovary (CHO) cells are the biopharmaceutical industry's primary means of manufacturing therapeutic proteins, including monoclonal antibodies. The major challenge in cell line development for the production of recombinant biopharmaceuticals lies in generating and isolating rare high-producing stable clones, amongst thousands of low-producing or unstable clones, in a short period of time. One approach to accomplish this is to use the glutamine synthetase (GS) selection system, together with the GS inhibitor, methionine sulfoximine (MSX). However, MSX can only increase protein productivity to a limited extent. Often productivity will drop when MSX is removed from the system. We evaluated a congenital GS mutation, R324C, which causes glutamine deficiency in human as an attenuated selection marker for CHO cell line generation. We also created a panel of GS mutants with diminished GS activity. Our results demonstrated that using attenuated GS mutants as selection markers significantly increased antibody production of stably transfected pools. Furthermore, these stably transfected pools sustained high productivity levels for an extended period of time, whereas cells transfected with wild-type GS lost considerable protein productivity over time, particularly after MSX was removed. In summary, the use of attenuated GS as a selection marker in CHO cell line development bypasses the need for MSX, and generates stable clones with significantly higher antibody productivity.Abbreviations: CHO: Chinese hamster ovary; CMV: Cytomegalovirus; DHFR: Dihydrofolate reductase; GFP: Green fluorescent protein; GOI: gene-of-interest; GS: Glutamine synthetase; IRES: internal ribosomal entry site; MSX: Methionine sulfoximine; MTX: Methotrexate; psGS: pseudoGS; RVDs: Repeated variable di-residues; TALENs: transcription activator-like effector nucleases; VCD: Viable cell density; ZFNs: zinc finger nucleases.

SUBMITTER: Lin PC

PROVIDER: S-EPMC6601560 | biostudies-literature | 2019 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologics.

Lin Pao-Chun PC Chan Kah Fai KF Kiess Irene A IA Tan Joselyn J Shahreel Wahyu W Wong Sze-Yue SY Song Zhiwei Z

mAbs 20190604 5

Chinese hamster ovary (CHO) cells are the biopharmaceutical industry's primary means of manufacturing therapeutic proteins, including monoclonal antibodies. The major challenge in cell line development for the production of recombinant biopharmaceuticals lies in generating and isolating rare high-producing stable clones, amongst thousands of low-producing or unstable clones, in a short period of time. One approach to accomplish this is to use the glutamine synthetase (GS) selection system, toget ...[more]

PMID: 31043114

Similar Datasets

Project description:Hemophilia B patients suffer from an inherited blood-clotting defect and require regular administration of blood-clotting factor IX replacement therapy. Recombinant human factor IX produced in cultured CHO cells is nearly identical to natural, plasma-derived factor IX and is widely used in clinical practice. Development of a biosimilar recombinant human factor IX for medical applications requires the generation of a clonal cell line with the highest specific productivity possible and a high level of specific procoagulant activity of the secreted factor IX. We previously developed plasmid vectors, p1.1 and p1.2, based on the untranslated regions of the translation elongation factor 1 alpha gene from Chinese hamster. These vectors allow one to perform the methotrexate- driven amplification of the genome-integrated target genes and co-transfect auxiliary genes linked to various resistance markers. The natural open reading frame region of the factor IX gene was cloned in the p1.1 vector plasmid and transfected to CHO DG44 cells. Three consecutive amplification rounds and subsequent cell cloning yielded a producer cell line with a specific productivity of 10.7 ± 0.4 pg/cell/day. The procoagulant activity of the secreted factor IX was restored nearly completely by co-transfection of the producer cells by p1.2 plasmids bearing genes of the soluble truncated variant of human PACE/furin signal protease and vitamin K oxidoreductase from Chinese hamster. The resulting clonal cell line 3B12-86 was able to secrete factor IX in a protein-free medium up to a 6 IU/ml titer under plain batch culturing conditions. The copy number of the genome- integrated factor IX gene for the 3B12-86 cell line was only 20 copies/genome; the copy numbers of the genome-integrated genes of PACE/furin and vitamin K oxidoreductase were 3 and 2 copies/genome, respectively. Factor IX protein secreted by the 3B12-86 cell line was purified by three consecutive chromatography rounds to a specific activity of up to 230 IU/mg, with the overall yield > 30%. The developed clonal producer cell line and the purification process employed in this work allow for economically sound industrial-scale production of biosimilar factor IX for hemophilia B therapy.

Dataset Information

Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologics.

Publications

Attenuated glutamine synthetase as a selection marker in CHO cells to efficiently isolate highly productive stable cells for the production of antibodies and other biologics.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets