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

Overview

Journal MAbs

Specialty Allergy & Immunology

Date 2019 May 3

PMID 31043114

Citations 13

Authors

Pao-Chun Lin

Kah Fai Chan

Irene A Kiess

Joselyn Tan

Wahyu Shahreel

Sze-Yue Wong

Zhiwei Song

Affiliations

Soon will be listed here.

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. 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.

Citing Articles

Generating and characterizing a comprehensive panel of CHO cells glycosylation mutants for advancing glycobiology and biotechnology research.

Haryadi R, Chan K, Lin P, Tan Y, Wan C, Shahreel W Sci Rep. 2024; 14(1):23068.

PMID: 39367021 PMC: 11452509. DOI: 10.1038/s41598-024-73722-z.

SiMPl-GS: Advancing Cell Line Development via Synthetic Selection Marker for Next-Generation Biopharmaceutical Production.

Yoon C, Lee E, Kim D, Joung S, Kim Y, Jung H Adv Sci (Weinh). 2024; 11(38):e2405593.

PMID: 39105414 PMC: 11481413. DOI: 10.1002/advs.202405593.

From Efficiency to Yield: Exploring Recent Advances in CHO Cell Line Development for Monoclonal Antibodies.

Majumdar S, Desai R, Hans A, Dandekar P, Jain R Mol Biotechnol. 2024; 67(2):369-392.

PMID: 38363529 DOI: 10.1007/s12033-024-01060-6.

Chemical and Genetic Modulation of Complex I of the Electron Transport Chain Enhances the Biotherapeutic Protein Production Capacity of CHO Cells.

Kretzmer C, Reger K, Balassi V, Pham Q, Johns M, Peters S Cells. 2023; 12(22).

PMID: 37998396 PMC: 10670226. DOI: 10.3390/cells12222661.

Glutamine synthetase (GS) knockout (KO) using CRISPR/Cpf1 diversely enhances selection efficiency of CHO cells expressing therapeutic antibodies.

Srila W, Baumann M, Riedl M, Rangnoi K, Borth N, Yamabhai M Sci Rep. 2023; 13(1):10473.

PMID: 37380701 PMC: 10307828. DOI: 10.1038/s41598-023-37288-6.

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