CHOZN® GS-/- CHO Cell Line (ZFN-Edited, Glutamine Synthetase Knockout)

Overview

The CHOZN® GS-/- CHO cell line is a genetically engineered, clonally derived Chinese Hamster Ovary (CHO) host cell strain developed by Merck (formerly SAFC) using CompoZr® Zinc Finger Nuclease (ZFN) technology. This cell line features a biallelic knockout of the endogenous glutamine synthetase (GS) gene (GLUL), rendering it incapable of synthesizing glutamine de novo. As a result, it serves as a robust, selection-ready platform for recombinant protein production under glutamine-free, methionine sulfoximine (MSX)-free culture conditions. The ZFN-mediated editing introduces precise double-strand breaks (DSBs) at the GS locus, followed by error-prone non-homologous end joining (NHEJ), leading to frameshift mutations and functional gene ablation. This approach ensures high genomic specificity and minimal off-target effects compared to conventional random integration or RNAi-based knockdown strategies. The cell line is derived from the well-characterized ECACC-certified CHO K1 parental line and retains its favorable growth kinetics, suspension adaptability, and productivity stability in chemically defined, animal-component-free media—most notably EX-CELL® CD CHO Fusion medium.

Key Features

• First commercially available, fully validated GS-knockout CHO cell line—enabling MSX-free, glutamine-free selection workflows
• Engineered via proprietary CompoZr® ZFN technology for targeted, biallelic disruption of the GLUL gene with confirmed loss-of-function at the protein and enzymatic activity levels
• Clonally isolated and extensively characterized for genetic stability across ≥60 population doublings
• Compatible with standard industrial suspension bioreactor processes—including fed-batch and perfusion modes—in serum-free, chemically defined media
• Produced under cGMP-aligned manufacturing practices, including comprehensive mycoplasma, adventitious virus, and species-specific endogenous retrovirus testing
• Supplied with full traceability documentation: cell bank records, karyotype analysis, STR profiling, and GS enzyme activity assay reports

Sample Compatibility & Compliance

The CHOZN® GS-/- cell line is qualified for use in early-stage process development, clinical-grade cell line generation (CLD), and commercial manufacturing of therapeutic proteins—including monoclonal antibodies, Fc-fusion proteins, and bispecific formats. It complies with ICH Q5A(R2) and Q5D guidelines for characterization of master and working cell banks. All release testing adheres to USP , Ph. Eur. 5.2.3, and ISO 9001:2015 quality management standards. The cell line has undergone full adventitious agent testing per FDA Points to Consider and EMA Guideline on Virus Safety Evaluation, including PCR-based detection of 28 rodent-specific viruses and in vitro assays for retroviral infectivity. Its origin from ECACC CHO K1 ensures alignment with widely accepted reference standards in biopharmaceutical development.

Software & Data Management

While the CHOZN® GS-/- cell line itself does not include embedded software, Merck provides integrated digital support tools via the SAFC Bioprocess Resource Portal. Users gain access to downloadable SOPs, transfection protocols, selection timelines, and analytical method transfer packages—including qPCR primer sets for GS locus verification and ELISA-based GS activity quantification. All supporting documentation is version-controlled and audit-trail enabled in accordance with FDA 21 CFR Part 11 requirements for electronic records and signatures. Raw data packages—including STR profiles, karyograms, and whole-genome sequencing coverage reports (available upon request)—are delivered in standardized, machine-readable formats (e.g., FASTQ, VCF, PDF/A-2) compliant with ALCOA+ principles (Attributable, Legible, Contemporaneous, Original, Accurate, Complete, Consistent, Enduring, Available).

Applications

• Generation of stable, high-titer recombinant protein-producing clones without MSX supplementation
• Development of platform processes for rapid cell line construction and clone screening
• Comparative studies of GS versus DHFR selection systems in terms of titer, product quality (e.g., glycosylation profile, charge variant distribution), and process robustness
• Investigation of GS-independent metabolic adaptations during prolonged fed-batch cultivation
• Use as a chassis for further genome editing (e.g., knockout of apoptosis-related genes or overexpression of chaperones) due to its proven ZFN-editing compatibility
• Regulatory filing support: inclusion in IND/IMPD dossiers as a well-characterized, non-proprietary host system with published validation data

FAQ

Is CHOZN® GS-/- compatible with both transient and stable transfection methods?

Yes—it supports high-efficiency plasmid-based transient expression and is optimized for stable pool generation and single-cell cloning following linearized vector transfection.
What is the recommended selection marker co-expressed with the GOI?

The GS gene must be co-transfected or co-integrated with the gene of interest; no additional selection cassette (e.g., puromycin) is required for primary selection.
Does Merck provide a master cell bank (MCB) and working cell bank (WCB)?

Yes—each vial corresponds to a cryopreserved WCB aliquot; MCBs are available under custom agreement with full characterization and regulatory support documentation.
Can this cell line be adapted to other chemically defined media besides EX-CELL® CD CHO Fusion?

Empirical adaptation is possible; however, Merck validates performance only in EX-CELL® CD CHO Fusion and recommends initial qualification prior to media substitution.
How is genomic knockout confirmation performed post-thaw?

Users are advised to perform locus-specific PCR + Sanger sequencing or T7E1 assay at passage 2–3; Merck provides validated primer sequences and expected amplicon sizes in the technical manual.