Developing Next-Gen Cell Lines Using Targeted Integration

Rapid growth in the development of biologics during the past two decades has irrevocably changed the landscape of drug discovery and development. As of 2024, biologics accounted for 65% of FDA newly approved drugs in the United States, and the global market for biologics is predicted to reach $961.51 billion by 2032 (1).

Around 70% of today’s biologics are produced using Chinese hamster ovary (CHO) cells (2) CHO cell lines are preferred for recombinant protein production due to their ability to perform human protein-like post-translational modifications (PTMs). A critical step in therapeutic protein production is the generation of a CHO clonal cell line with high productivity and desired product profile. Conventionally, this step relied on uncontrolled integration of the expression cassette into the host genome, a serendipitous method that necessitated a labor-intensive workflow for screening clones to identify stable, high-performing cell lines.

In recent years, progress has been made with more controlled integration technologies, including transposase-mediated semi-targeted systems (3). Several commercially available transposase systems are widely used in biopharmaceutical R&D (e.g., Sleeping Beauty, Ascenion, Munich, Germany); PiggyBac, (Hera BioLabs, Lexington, Ky.); Leap-In, ATUM, Newark, Calif.); and DirectedLuck, ProBioGen, Berlin, Germany). The cell line development (CLD) workflow employing these systems involves two main stages: first, host cells are co-transfected with a transposon vector housing the transgene and transposase messenger RNA, and, second, a stable cell pool is generated, which exhibits greater homogeneity in transgene integration, thereby simplifying the downstream screening process.

Although this approach facilitated transgene integration in a semi-targeted, more controlled manner, the ultimate goal remained achieving fully controlled transgene integration at specific predefined spots in the host genome. With advancements in genome editing and availability of the CHO genome, targeted integration (TI) has emerged to fulfill that ambition. TI allows precise transgene insertion into the CHO genome, resulting in recombinant cell lines with predictable transgene copies and genome location, more predictable productivity, and assured cell line stability (Figure 1).

TI host cell line generation and expression

The establishment of an effective TI CLD platform begins with the generation of a TI host cell line harboring a landing pad capable of efficient recombinase-mediated cassette exchange. Identification of suitable TI host cells involves a sophisticated screening process, including the evaluation of cell growth, metabolic profiles, titer performance, and product quality attributes.

With this approach, it was possible to establish a robust TI host cell line and develop a full TI system (e.g., WuXia TrueSite, Wuxi Biologics, China) (4) that routinely achieves pool titers around 6.0 g/L and lead clone productivity around 8.0 g/L under platform fed-batch processes for monoclonal antibody (mAb) molecules (Figure 2). The titer could be further increased to over 10.0 g/L by process optimization, such as intensification or temperature shift (data not shown). In addition to mAbs, the system can be applied to fusion proteins and symmetric/asymmetric antibodies.

Predictable quality consistency and stability

Product quality consistency among pool and clonal cell lines is a critical consideration for employing pool-derived non-good manufacturing practice (non-GMP) materials to accelerate investigational new drug (IND) timelines. Comparative analysis revealed that key quality attributes, such as size exclusion chromatography–liquid chromatography main peak, charge variant profiles, and N-glycan patterns, were highly comparable between the TI CLD platform (WuXia TrueSite) clones and parental pools (Figure 3A). These findings support the strategic use of the proprietary TI CLD platform pools for non-GMP (or even early clinical) material production, significantly accelerating development timelines while maintaining product quality standards.

Conventional uncontrolled integration cell lines often faced challenges with productivity decline during cell banking and scale-up processes. To systematically evaluate the expression stability of the TI CLD production clones, extended fed-batch cultures were conducted across 60 population doubling levels (PDLs), without selection pressure. This stability assessment included 48 clones representing three mAbs and one asymmetric antibody. All TI production cell lines demonstrated robust genetic and quality stability, and more than 99% of clones remained stable, with less than 20% titer reduction after 60 PDLs (Figure 3B).

This performance profile supports the reliable use of these TI cell lines for large-scale production up to more than 20,000 L.

How are CMC timelines reshaped?

Typically, the lead clone for GMP clinical manufacturing is also used to produce materials for critical preclinical studies, such as good lab practice (GLP) toxicology, to minimize risk. However, the lengthy cell-line development process to identify this clone can create a bottleneck. Instead, utilizing a stable pool (or a pool of clones) accelerates toxicology study material production by bypassing single-clone selection. This approach allows toxicology studies to commence earlier, enabling parallel development and significantly shortening the first-in-human timeline. In response to the COVID-19 pandemic, several developers (e.g., Ei Lilly and Company, Vir Biotechnology, WuXi Biologics) accelerated timelines by using non-clonal CHO pools to produce GLP-Tox and early-phase clinical material—a strategy known as ‘deferred cloning’ (5). Companies, including Genentech, a Roche company, and Merck, known as MSD outside of the US and Canada, further demonstrated this approach's utility for both mAbs and complex molecules (6,7).

With similar goals of accelerated timelines in mind, the proven stability of the TI CLD platform (WuXia TrueSite) cell line eliminates the need for cell-line stability studies from the critical path. Furthermore, its consistency of product quality supports a streamlined approach whereby non-GMP materials can be generated from TI pools. Clone selection for master cell bank generation can be based on matching pool product quality attributes, and GMP production can proceed with selected clones.

Through these and other cross-functional efforts, the proprietary TI CLD platform is being applied to achieve a targeted six-month IND timeline. Specifically, the platform enables acceleration of MCB establishment to nine to 10 weeks, cutting overall development time in half compared with the conventional 12-month timeline (Figure 4). The accelerated approach is expected to be applicable not only to pandemic response proteins, but also to standard antibody production. While regulatory acceptance for non-pandemic programs remains a hurdle, the inherent genomic plasticity of CHO cells supports the argument that genetically defined stable pools, such as TI, might be suitable for early-stage clinical manufacturing.

A next-generation approach

The TI CLD platform has demonstrated superior development efficiency, including reduced clone screening requirements, simplified process development (by leveraging well-characterized host properties), and elimination of redundant characterization steps. These features enable biologics developers to significantly accelerate molecules into clinical development. By combining the latest academic advancements with industrial-scale manufacturing expertise, this next-generation approach to CLD enables biomanufacturers to provide reduced technical risks, accelerated development cycles, and superior process economics.

References

1. Dey, M. Biopharmaceuticals Statistics by Global Market Size, Trends and Facts. Market Research Report, sci-tech-today.com. May 29, 2025.
2. Majumdar, S.; Desai, R.; Hans, A; et al From Efficiency to Yield: Exploring Recent Advances in CHO Cell Line Development for Monoclonal Antibodies. Molecular Biotechnol. 2025, 67 (2), 369-392.
3. Zeh, N.; Schmidt, M.; Schulz, P.; et al. The New Frontier in CHO Cell Line Development: From Random to Targeted Transgene Integration Technologies. Biotechnol. Adv. 2024, 75, 108402.
4. WuXI Biologics. WuXi Biologics Launches WuXia 4.0, Targeted Integration Cell Line Platform TrueSite TI to Accelerate Biologics Development with High Titer and Superior Stability. Press release, Sept. 25, 2025.
5. McGovern, Ā. T.; Salisbury, C. M.; Nyberg, G. B. The Pandemic and Resilience for the Future: AccBio 2021. Biotechnol. Prog. 2022, 38 (1), e3207. DOI: 10.1002/btpr.3207
6. Barnard G. C.; Zhou, M.; Shen, A.; et al. Utilizing Targeted Integration CHO Pools to Potentially Accelerate the GMP Manufacturing of Monoclonal and Bispecific Antibodies. Biotechnol. Prog. 2024, 40 (1), e3399. DOI: 10.1002/btpr.3399
7. Pan, J.; McPhee, J.; Dow, A.; et al. Utilizing Non-Clonal CHO Cell Derived Materials for Preclinical Studies of Complex Molecules. BMC Biotechnol. 2025, 25, 33. DOI: 10.1186/s12896-025-00968-4

About the authors

Tao Sun, PhD, is senior principal scientist; Xiaoyue Chen, PhD, is executive director; and Fei Chen, PhD, is head; all at Cell Line Development, WuXi Biologics. Hang Zhou, PhD, is head of Bioprocess Research & Development, WuXi Biologics.