The science of cell line development for biologics: Improving stability and yield

March 17, 2025 by Palak Patel (15 minute read)

Mammalian cell line development is essential to biologics manufacturing, ensuring stable, high-yield expression of therapeutic proteins. With expanding biologics pipelines, the industry is continuously innovating to improve productivity, speed to patient, and scalability. Among the most widely used cell lines for biologics production are Chinese Hamster Ovary (CHO) cells, which have become the gold standard for monoclonal antibody and recombinant protein production. Their adaptability, scalability, and ability to achieve high titers make them essential for developing monoclonal antibodies and other complex biologics.

Cell line development steps for monoclonal antibody production

Developing a stable, high-yield cell line requires multiple steps, including the genetic modification of mammalian cells to integrate the gene encoding the protein of interest into the host genome. This is followed by rigorous screening, characterization, and banking to ensure high productivity and product stability.

Key stages in monoclonal antibody cell line development include:

Stage	Description
1. Selection of host cell line	Choosing an appropriate cell line that is amenable to genetic manipulation and capable of high-yield protein production.
2. Transfection	This initial step involves integrating the gene of interest into the host genome. Transfection can be achieved through physical methods like electroporation or chemical methods such as lipofection or methods using calcium phosphate.
3. Stable pool generation	Post-transfection, cells incorporating the gene of interest are selected using selection markers and antibiotic markers. Common systems used include methotrexate (MTX) or the glutamine synthetase (GS) system.
4. Single-cell cloning	Ensuring the monoclonality of cell lines is crucial. This step involves isolating single cells to establish monoclonal cell lines, which guarantee consistent production of the target protein. Regulatory authorities require stringent standards of monoclonality. Advanced equipment like the Beacon from Berkeley Lights utilizes microfluidics and Opti Electro Positioning (OEP) technology to move cells in and out of nanopens, significantly reducing development timelines compared to traditional methods like limiting dilution.
5. Screening and isolation	This step involves assessing a large number of clones for yield, quality, and manufacturability. High-throughput equipment like the ambr250 system is typically used to screen multiple clones efficiently.
6. Cell line stability studies	The stability of established cell lines is evaluated to ensure that clones can maintain consistent titer and quality of the product over multiple generations.
7. Master cell banking and characterization	Master cell banks of the lead clones are created to ensure an adequate source of cells for future large-scale production. These banks are comprehensively characterized to meet regulatory requirements.

Development of cell lines for bispecific antibodies

Developing cell lines for bispecific antibodies is particularly challenging due to the inherent complexity of these molecules. Bispecific antibodies are designed to bind two different antigens simultaneously, adding both structural and functional complexities.

Efficiently expressing bispecific antibodies requires cells to produce two different heavy chains and two different light chains, which must correctly pair to form functional bispecific molecules. Incorrect pairing often results in product-related impurities, such as homodimers, which are less effective and difficult to remove during purification due to their similar physical and chemical properties.

The role of CHO cell lines in biologics manufacturing

CHO cells are the predominant mammalian cell lines used for producing therapeutic proteins and monoclonal antibodies. First isolated in 1956, CHO cells have been extensively optimized to create subclones that result in higher yields and improved product quality. Initially existing as adherent cell lines, they have been adapted to suspension culture, allowing them to grow in a suspension environment without the need for solid media.

Some widely used CHO cell lines include:

CHO-K1: CHO-K1 remains one of the most widely used CHO cell lines due to its adaptability, genetic stability, and scalability in biologics manufacturing. However, not all CHO-K1 cell lines are created equal. Advances in cell line engineering have led to next-generation CHO-K1 variants that optimize titer expression, gene stability, and process scalability.

For example, Thermo Fisher Scientific’s CHO-K1 high-titer cell line leverages proprietary transposase technology to enhance gene integration and achieve titers of up to 8 g/L. This next-generation CHO-K1 platform improves expression stability and process efficiency, accelerating IND submission timelines and reshaping expectations for biologics development.
CHO-S: CHO-S is a suspension-adapted variant of Chinese Hamster Ovary cells, making it highly suitable for large-scale bioreactor production. Its capability for proper protein folding and post-translational modifications, coupled with high-yield, renders it ideal for industrial-scale production of biotherapeutics and research in cellular biology. Additionally, CHO-S is widely accepted by regulatory agencies for the production of biopharmaceuticals, further solidifying its role in the industry.
CHO-DG44: This CHO variant, characterized by a double deletion of the dihydrofolate reductase (DHFR) gene, enables the use of Methotrexate (MTX) for selection and gene amplification. This attribute facilitates high-level expression of recombinant proteins. Like other CHO cell lines, CHO-DG44 is leveraged for large-scale production due to its high-yield, proper protein folding, and effective post-translational modifications.
CHO-DXB11: Also deficient in DHFR, CHO-DXB11 is utilized for similar purposes as CHO-DG44. It holds historical significance as the first CHO cell line used in the biotechnology era for the production of recombinant mammalian proteins. CHO-DXB11 was pivotal in producing large quantities of human tissue plasminogen activator (TPA). However, its utility was somewhat limited by the potential for reversion of DHFR activity under mutagenic conditions.

These CHO variants have distinct characteristics that make them well-suited for a range of biotechnology and biopharmaceutical applications. Selecting the right cell line depends on multiple factors, including growth characteristics, gene amplification capabilities, protein production efficiency, and regulatory compliance.

Advancing biologics through cell line innovation

Cell line development is a fundamental step in biologics manufacturing, shaping the efficiency, scalability, and success of monoclonal antibody and recombinant protein production. From host cell selection and genetic modification to screening and banking, each stage plays a critical role in ensuring high-yield, stable expression.

CHO cell lines continue to set the industry standard, with advancements in engineering enabling higher titers, improved gene stability, and more efficient pathways to IND submission. Whether for monoclonal antibodies, bispecifics, or other complex biologics, selecting the right cell line is key to optimizing production and meeting regulatory standards.

As cell line development evolves, next-generation CHO-K1 platforms, such as Thermo Fisher Scientific’s high-titer CHO-K1 cell line, are pushing the boundaries of productivity and process efficiency. With innovations in transposase technology and gene integration, these advancements are reshaping expectations for biologics development, offering more scalable and efficient solutions for today’s fast-moving pipelines.

Explore how CHO-K1 can optimize your process.

Learn More about advances in CHO-K1 cell line engineering.

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