As the biopharmaceutical industry grows, stable mammalian cell lines, especially CHO cells with human-like post-translational modifications, are essential for producing monoclonal antibodies, bispecific antibodies, recombinant proteins, and complex molecular drugs. However, as drug types become more complex and market scales expand, traditional cell line development methods are increasingly showing limitations: unstable expression levels, difficulty in fully controlling product quality, and overly long development cycles. For the industry, these issues directly translate into higher production risks, longer project timelines, and increased commercial costs.

Against this background, gene editing is becoming a key driving force for cell line construction, as a root-level approach to optimizing host cells. By precisely regulating genome structure, metabolic pathways, and post-translational modifications, researchers can not only significantly enhance protein expression levels but also greatly improve long-term stability and product consistency. This shift is not only a paradigm change in technology but also an inevitable trend to meet industrial demands and drive industry upgrading.

Gene editing-driven cell line optimization: Core competitiveness

In industrial cell line development, a well-established cell banking system (RCB→MCB→WCB) is the foundation for ensuring long-term stability and controllability. Even clones derived from the same monoclonal cell may undergo genetic mutations or phenotypic drift during extended culture, thus requiring stability verification over more than 80 generations. Traditional random integration methods rely on the random insertion of exogenous genes into the host genome, followed by extensive screening to obtain high-expressing clones. However, this approach is time-consuming, labor-intensive, and subject to position effects, leading to significant variability in expression levels.

In contrast, gene editing technology has ushered in the "designable" era of cell line optimization. Through precise integration and multigene regulation, it fundamentally improves the long-term stability and controllability of cell lines, significantly enhancing production efficiency and product consistency. In recent years, gene editing has become a core measure of competitiveness in determining whether a cell line development platform can stand out in the fierce industrial competition.

Targeted integration and site-specific expression

Using CRISPR/Cas9 together with recombinase systems such as Flp/FRT, Cre/loxP, and Bxb1/attP, researchers can insert target genes into transcriptionally active regions of the host genome, thereby achieving predictable and consistent expression levels. This method avoids the uncertainty of random integration, reduces the need for large-scale screening, and improves product uniformity.

Figure 1. Vector structure and insertion site determination workflow for analyzing gene integration patterns

This advantage is particularly significant for complex molecular drugs. For example, in the production of bispecific antibodies or multisubunit fusion proteins, an imbalance in exogenous gene fragments can lead to assembly failure or misfolding. Through targeted integration, the copy number and expression ratios of different fragments can be precisely controlled, significantly improving the probability of correct assembly. For the industry, this means being able to identify promising candidate clones with industrial potential early in development, greatly reducing the risk of late-stage failure.

Precise regulation of glycosylation

Although the natural glycosylation patterns of CHO cells are close to those of humans, differences still exist. Glycosylation directly determines the efficacy, safety, and half-life of antibody and protein drugs, and in the eyes of industry and regulatory agencies, its importance is on par with yield itself. Gene editing offers unprecedented opportunities to optimize glycosylation patterns.

For example, fucosylation regulation is an effective way to enhance antibody-dependent cellular cytotoxicity (ADCC). Antibodies produced by FUT8 knockout, which results in completely afucosylated molecules, show a 10–50 fold increase in ADCC activity, thereby strengthening antitumor efficacy.

Figure 2. FUT8 allele modification by replacing a 234 bp fragment (exon 2) containing the translation initiation site with a drug-resistance cassette flanked by loxP sites

Another strategy is the overexpression of N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the formation of bisecting GlcNAc structures, sterically hindering the addition of fucose. Additionally, regulating GDP-fucose transporter (SLC35C1) or using fucose analogs (such as 2-fluoro-fucose) can effectively reduce fucosylation levels.

Figure 3. Mechanisms of fucosylation inhibitors and their effects on enzyme activity and cellular GDP-fucose levels

Another modification is overexpressing human α(2,6)-sialyltransferase while knocking out sialidase genes (Neu1, Neu2, Neu3), increasing tetra-sialylation of rhEPO from 15% to over 40%, thereby extending its half-life in circulation.

Figure 4. Growth and rhEPO production characteristics of Neu1, Neu2, and Neu3 knockout cell lines in batch and fed-batch cultures

In actual production, adding the sialic acid precursor N-acetylmannosamine (ManNAc) or using sialidase inhibitors (such as 2,3-dehydro-2-deoxy-N-acetylneuraminic acid) can further increase sialylation levels. Studies show that such combination strategies can increase rhEPO tetra-sialylation from 15% to more than 40%. These modifications not only improve clinical efficacy but also enhance market competitiveness, since extended dosing intervals mean better patient compliance and broader therapeutic potential.

Figure 5. Involvement of GlcN, Gal, and ManNAc in nucleotide sugar biosynthesis affecting glycosylation; GlcN increases ManNAc and CMP-sialic acid levels, while glucose and glutamine regulate UDP-Gal

Delayed apoptosis and enhanced stability

Under high-density culture conditions, cell survival time is a key determinant of total yield. By knocking out pro-apoptotic genes such as Bax and Bak, researchers significantly delayed apoptosis, extending production cycles and increasing protein accumulation. When combined with process optimizations such as perfusion or fed-batch strategies, these anti-apoptotic modifications allow for long-term cell viability and substantially improve productivity. This translates into higher output within the same culture period, reducing costs and improving commercial feasibility.

Figure 6. Schematic of sgRNA target sites in BAK and BAX genes and Western blot results of KO clones on day 3

Figure 7. Proportion of sialylated glycans in batch (A) and fed-batch (B) cultures of CHO-EPO cells and 5X KO clones

Engineering challenges in complex protein expression systems

With bispecific antibodies, fusion proteins, and multi-domain proteins becoming the focus of R&D and production, expression regulation faces greater challenges. These molecules often consist of multiple subunits, and an imbalance in expression ratios or insufficient folding efficiency can easily lead to chain mispairing, aggregation, and reduced yield, severely affecting process feasibility and product quality.

The key to solving this lies in the precise regulation of multigene expression within the cell line. By constructing stable multigene expression frameworks in the genome, researchers can achieve equimolar expression of light chains (LC), heavy chains (HC), and functional domains within a single cell, improving correct assembly rates. Studies show that gene order alone can significantly affect expression balance. For example, in bispecific antibody cell line development, adopting a "HC-prioritized expression" strategy can improve yield by about 30% compared to "LC-prioritized" and increase correct pairing rates from 60% to over 85%.

Figure 8. Performance comparison of host clones in stable expression pools for IgG4 mAb and Etanercept production

In addition, co-expression of molecular chaperones is another effective approach to improve solubility and folding rates of complex proteins. Overexpressing enhanced chaperones such as protein disulfide isomerase (PDI) and immunoglobulin binding protein (BiP) in host cells significantly improves folding efficiency, often raising correct conformation rates by 40–60%. Moreover, introducing ER folding and redox regulators such as HSP70 and ERO1 can effectively relieve folding stress caused by high-level expression.

Figure 9. Changes in proteasome-related and antibody assembly/secretion-suppressing gene/protein expression under high-density culture

Industrial value of modern construction strategies

In the highly competitive biopharmaceutical industry, cell line optimization has become central to industrial production. It directly determines whether projects can proceed efficiently, development cycles can be shortened, and risks can be effectively managed. Cell line construction and gene expression regulation are not merely laboratory-level scientific issues but decisive factors in industrial success. The introduction of gene editing into industrial cell line optimization has not replaced other modern strategies but instead complements targeted integration, transposon systems, and high-throughput screening technologies. Whether through targeted integration for predictable expression, glycosylation regulation for enhanced clinical efficacy, or apoptosis suppression for extended production cycles, gene editing is fundamentally reshaping the industrial value of cell lines.

Creative Biogene — Next-generation cell line construction platform

In biopharmaceutical R&D and production, rapidly and efficiently obtaining stable, high-yielding cell lines is critical to project success. At the same time, reducing development costs, improving process efficiency, and ensuring product quality are challenges every R&D team must face. Creative Biogene is dedicated to providing complete, reliable cell line construction services, offering a solid foundation for both research and industrial production.

Service types

• Biosimilar Cell Line Development
• Bioproduction Stable Cell Line Development
• High-performance CHO Stable Cell Line Development
• CAR-NK Cell Development Services
• CAR-T Cell Development Services
• cGMP Cell Line Production Process

Technical highlights and advantages

Wide variety of host cells: H1299, 2F-2B, 4T1, 786-O, 9607, THP-1, MCF7/ADR, A549, V79, CHO, HepG2, and more.

Flexible vector design: Support for 2A, IRES, multiple promoters, fusion expression; multiple promoter, reporter, and tag options available.

End-to-end technical support: From stable cell line construction to recombinant protein production, one-stop service covering the entire process.

Efficiency and cost-effectiveness: Advanced equipment and mature technology platforms for short-cycle, cost-effective development.

Toward future industrial applications

Creative Biogene focuses not only on monoclonal screening and validation but also on maximizing productivity and stability through strategies such as gene editing, automated screening, and small-scale pre-culture, providing strong support for industrial production. Our services help clients shorten R&D cycles while ensuring long-term stability, high productivity, and quality control of cell lines, safeguarding the success of next-generation biopharmaceutical projects.