Double-Knockout CHO Cell Line for Higher Therapeutic Protein Yield

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Solution Overview

Problem

Existing CHO cell lines face challenges in achieving high expression yields and scale-up for therapeutic proteins, particularly monoclonal antibodies, due to limitations in gene amplification systems using GS and DHFR as selectable markers, which result in low protein production and impurity issues.

Innovation Solution

A double-knockout CHO cell line is generated by disrupting the Glutamine Synthetase (GS) and Dihydrofolate Reductase (DHFR) genes using CRISPR/Cas9, Zinc Finger Nuclease, or TALEN, followed by selective cloning and optimization with MSX and MTX, to enhance protein expression and reduce impurities.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If GS and DHFR genes are used as selectable markers for gene amplification, then cell selection is achieved, but protein expression yield remains low

Engineering Contradiction:

Improvecell selection capabilityVSAvoidprotein expression yield

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent extracts and removes the GS and DHFR genes from the CHO cell genome using CRISPR/Cas9 technology. By eliminating these genes that were previously used as selectable markers, the invention prevents them from limiting protein expression yield while maintaining their original selection function through alternative mechanisms

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

Instead of using GS and DHFR genes to select cells and hoping for high expression, the invention inverts the approach by first knocking out these genes to remove expression limitations, then using alternative selection methods to identify high-expressing clones

Inventive Principle:

Principle #13The other way round (Inversion)

2Reliability

If GS gene is used as selectable marker with MSX treatment, then recombinant protein expression is selected, but protein yield decreases over long-term culturing

Engineering Contradiction:

Improverecombinant protein selectionVSAvoidculturing duration

Core Design Contradiction:

ReliabilityVSDuration of action of moving object

Solution Approach 1:

The patent removes the GS gene from the cell lineage, eliminating the dependency on MSX treatment for selection. This extraction allows long-term culturing without the progressive yield decrease associated with GS-based selection systems

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The GS gene is knocked out preliminarily before long-term culturing begins. This preliminary action prevents the accumulation of expression limitations that would otherwise develop during extended culture periods

Inventive Principle:

Principle #10Preliminary action

3Reliability

If DHFR system is used for selection with MTX, then transfectants are selected, but isolation of single cell line takes several months

Engineering Contradiction:

Improvetransfectant selectionVSAvoidisolation time

Core Design Contradiction:

ReliabilityVSLoss of time

Solution Approach 1:

The patent extracts and removes the DHFR gene, eliminating the need for prolonged MTX-based selection processes. This enables faster isolation of stable transfectant lines without the months-long timeline required by traditional DHFR systems

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention changes the selection parameter from DHFR/MTX dependency to a CRISPR-mediated knockout system, fundamentally altering the selection timeline and enabling much faster isolation of single cell lines

Inventive Principle:

Principle #35Parameter changes

4Productivity

If gene amplification is used to increase expression levels, then protein yield improves, but light chain impurities increase

Engineering Contradiction:

Improveprotein expression yieldVSAvoidlight chain impurities

Core Design Contradiction:

ProductivityVSObject-generated harmful factors

Solution Approach 1:

The patent converts the harmful effect of GS/DHFR gene presence (which limits expression and causes impurities) into a benefit by systematically removing these genes. This elimination prevents the formation of light chain impurities while maintaining high expression yields through alternative selection strategies

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

Data Source

PatentUS12529048B2Double knock-out CHO cell line method of its generation and producing therapeutic proteins therefrom

Publication Date: 2026.01.20 ENZENE BIOSCIENCES LTD

AI summary

The present disclosure relates to providing an engineered CHO cell line wherein the two essential metabolic genes are knocked out. Particularly, the present invention relates to a double knockout CHO cell line (DHFR−/− and GS−/−) with disrupted Dihydrofolate Reductase (DHFR) and Glutamine Synthetase (GS) genomic loci. The double knockout CHO cell line (DHFR−/− and GS−/−) being suitable for expression of monoclonal antibodies, dimeric therapeutic proteins, Fab, single chain fragments, or the like. The present disclosure also provides method of generation of a double knock out CHO cell line (DHFR−/− and GS−/−) using gene selection and/or manipulating techniques such as CRISPR/Cas9 system, Zinc Finger Nuclease, TALEN, or the like. The present disclosure further provides method of selection of clones and production of therapeutic proteins of interest with increased titre.