FAQs – Stable cell lines for monoclonal antibody production

The demand for monoclonal antibodies keeps rising for multiple applications. This calls for increased standardization of the field and the continuous development of more cost-effective methods of production. Stable cell lines for monoclonal antibody production, particularly in Chinese hamster ovary (CHO) cells, continues to be the best way to produce these reagents on a large scale. On this page, we answer all most frequently asked questions about the process of stable cell line generation for monoclonal antibody production and describe ProteoGenix’s platform of production and its unique guarantees.

ProteoGenix’s approach to stable cell line generation for antibody production

How does your stable cell line generation for monoclonal antibody production work?

Our platform of stable cell line generation for monoclonal antibody production is designed to optimize production yields while minimizing development costs. The first step in every project comprises a developability assessment where antibody leads are transiently expressed in our proprietary system – XtenCHO™. This highly-productive cell line can express antibodies with glycosylation profiles and secondary structures comparable to stable cell lines such as CHO-S™. Plus, it produces antibodies in amounts that allow an extensive characterization of key antibody properties.

Based on this assessment, we develop a custom protocol for stable cell line generation and predict production yields. This plan and potential production hurdles are discussed with clients who will ultimately decide if they want to move forward with the project (go/no go). After this stage, stable expression and selection vectors are designed, synthesized, and transfected into competent cells.

Selection and amplification of positive clones are subsequently carried out. And positive clones are isolated and their stability and productivity evaluated. When single clones with robust levels of productivity are identified, the process is optimized and scaled up or transferred to the client’s facilities or a selected CMO.

What expression systems can you use for the stable production of monoclonal antibodies?

At ProteoGenix, we offer a high diversity of cell lines for stable cell line generation including CHO-DG44, CHO-S, a proprietary GS- CHO cell line, and HEK cells (HEK293T, etc.). We are also able to carry out stable cell line development starting from customer-provided cell lines.

Alternatively, given our 28+ years of experience in protein production, we can produce antibodies in a variety of non-mammalian systems including bacterial (Escherichia coli, Bacillus subtillis), yeast (Saccharomyces cerevisiae, Pichia pastoris), and insect/baculovirus systems. These systems may be particularly interesting for the production of antibody fragments or antibodies intended for non-therapeutic use.

What monoclonal antibody formats can you produce?

More than 100 projects were successfully completed using our platform of stable cell line generation for monoclonal antibody production with a productivity record of 8 g/L. These projects ranged from the production of full-length IgG, bispecific antibodies, Fc-fusion proteins, and antibody fragments including Fab, scFv, or VHH.

How long does it take to generate stable cell lines for monoclonal antibody production?

Our typical process of stable cell line generation for monoclonal antibody production starts at 4 to 6 months. The most laborious and time-intensive part of the process involves the selection of stable single clones and subsequent stability studies (upon request).

To learn more about ProteoGenix’s estimated lead times, read our dedicated article: How long does it take to generate stable cell lines for monoclonal antibody production?

What are the unique advantages of your stable cell line generation service?

You retain the intellectual property (IP) over the cell lines and protocols created at ProteoGenix. This simplifies protocol and cell transference to a CMO or your facilities for large-scale production. Our FTO (freedom-to-operate) approach to stable cell line generation ensures a smooth transition from the bench to large-scale bioreactors and greatly simplifies the process of licensing your antibodies for therapeutic or in vitro diagnostic applications.

Another advantage of our platform is the inclusion of an extensive early testing stage. The comprehensive characterization of antibody leads allows us to predict potential production hurdles and guarantee production yields. In this way, we can secure our clients’ investments while transferring all risks directly to us.

In addition to these advantages, as experts in antibody production and engineering, we offer a vast selection of upstream services including affinity maturation, antibody humanization, bispecific antibody development, antibody-drug conjugate development, antibody reformatting, among others. Our downstream capabilities include cell bank development (Research Cell Bank, Master and Working Cell Bank) and stability studies. By choosing to partner with us, you save time and reduce costs while maximizing the therapeutic efficacy of your antibody leads and minimizing foreseeable production risks.

What types of guarantees do you offer?

Due to our extensive preliminary developability study, we can guarantee production yields early on. This allows you to make informed decisions regarding the development of your antibody minimizing risks. At ProteoGenix, our dedicated project managers evaluate all antibody development projects on a case-by-case basis, allowing us to propose solutions that are fully adapted to your needs, timeframes, and available budget. Moreover, due to our milestone system, you will have access to all data regarding the progress of your project at all times.

Stable cell lines for monoclonal antibody production

What are the benefits of generating stable cell lines for monoclonal antibody production?

Stable cell lines imply the stable integration of antibody genes into the genome of a highly productive mammalian host. Since mammalian cells are unable to replicate exogenous DNA vectors (even in the presence of an origin of replication), stable integration is necessary to ensure these cells maintain their ability to secrete antibodies indefinitely.

Mammalian cell lines used for stable expression have the advantage of performing proper antibody folding and adequate post-translational modifications (glycosylation), two important processes with great impact on antibody’s reactivity, stability, and half-life. Plus, they have optimal productivities, superior batch-to-batch consistency, higher reproducibility, and are more amenable to scale-up.

To learn more about the benefits of producing these cell lines for monoclonal antibody production, read the complete article: What are the benefits of generating stable cell lines for monoclonal antibody production?

What are the main cell lines used for the stable production of monoclonal antibodies?

Antibodies that are currently licensed for therapeutic applications are mainly produced by Chinese hamster ovary (CHO) or NS0 (mouse myeloma) cell lines. Additionally, a small percentage of antibodies are produced by Escherichia coli, particularly those consisting of small antibody fragments. CHO cells are one of the most successful hosts for the production of antibodies at a large-scale.

To learn more about the cell lines used for stable expression, read the complete article: What are the main cell lines used for the stable production of monoclonal antibodies?

What are the advantages of using Chinese hamster ovary (CHO) cell lines for the stable expression of monoclonal antibodies?

Antibody production by CHO cell lines accounts for at least 71% of all antibodies licensed for therapeutic use. What makes these cell lines so successful? CHO cells were first established in the late 1950s and ever since expanded for multiple applications. Nowadays, protocols for stable cell line generation in CHO are optimized for their unique genetic background. For this reason, CHO cells are now considered one of the most robust systems with increased tolerance to changes in pH, temperature, pressure, and oxygen concentration.

Additionally, well-established strategies of gene amplification can be used in conjugation with stable gene integration. This allows a stepwise increase of production levels in stable CHO cells, making it even more desirable for large-scale production.

What are the most promising alternatives to CHO-based monoclonal antibody production?

The two most important challenges of working with CHO cells for monoclonal antibody production are the long lead times (4-8 months) and the slight differences in glycan composition of CHO-produced antibodies. CHO cells produce α-gal and NGNA glycans which may cause adverse reactions in humans (rare).

For this reason, researchers continue pushing the boundaries of recombinant antibody production by working with alternative systems. Two of the most important alternatives to CHO cells are yeast (specifically Pichia pastoris) and plant cells or tissue. Yeast is easier to manipulate than mammalian cells and can replicate plasmids, but unable of performing human-like glycosylation. This ability can be engineered into yeast cells, which a costly and laborious process. Plants, on the contrary, have often been suggested as a low-cost production system. However, researchers are still struggling with low transfection efficiencies which have limited the success of this approach for the production of biopharmaceuticals.

What are the most commonly used selection markers for stable cell line generation?

Mammalian cells used for stable cell line generation can be selected using either metabolic or resistance markers. Mammalian systems are typically selected using metabolic markers Dihydrofolate reductase (DHFR) or Glutamine synthetase (GS). Cell lines may be either deficient in either of these enzymes (e.g. CHO-DG44 has been engineered to lose DHFR genes and NS0 is naturally deficient in GS) or their endogenous enzymes are inhibited before selection.

Alternatively, resistance markers can be used; the most commonly used for mammalian cell line selection include Blasticidin, G418/Geneticin, Hygromycin B, Puromycin, Zeocin, etc. Unlike metabolic-based selection, resistance-based selection does not require gene knockout or inhibition, only the identification of a “kill curve” (growing cells in different concentrations of antibiotic).

What are the main differences between DHFR and GS selection?

For DHFR-based selection, DHFR- cells are grown in the presence of hypoxanthine and thymidine (HT) prior to transfection. After co-transfection of antibody genes and exogenous DHFR, HT is withdrawn from the culture medium thus creating a selective pressure. Methotrexate (MTX) can be used in this system for gene amplification leading to improved productivity.

In contrast, for GS-based selection, it is sufficient to remove glutamine from the medium to exert sufficient selective pressure. The GS system has been gaining ground over other systems due to its simplicity. For this reason, although GS-NS0 has for long been considered the gold standard of GS-based selection, multiple CHO cell lines lacking GS are currently being developed.

Stable production of monoclonal antibodies – technologies and trends

What are the main applications of stable cell lines for monoclonal antibody production?

The development of stable cell lines is a laborious and time-intensive process, for this reason, it is typically reserved for monoclonal antibodies with commercial value such as those used for therapeutic applications or in vitro diagnostic (IVD) devices. On occasion, stable cell lines may be generated for the production of research antibodies used in routine analysis.

What is the difference between transient and stable monoclonal antibody production?

Transient expression of antibodies is frequently used to screen drug candidates or characterize antibody leads before stable cell line development. But is the difference between the two approaches?

As the name implies, transient expression comprises the temporary modification of mammalian cells. Since these cells are unable to replicate exogenous DNA independently from their genome, the copy number of non-integrated expression vectors declines with each growth cycle. In contrast, stable expression requires the integration of antibody-encoding genes into the genome of the expression host. Integration is considered a rare event, for this reason, the most laborious part of the process comprises the selection of positive single clones from transfected stable pools.

Unlike stable expression, transient production does not require a selection step; instead, it uses reporter genes (e.g. luciferase) to measure and potentially improve transfection efficiencies.

Discover more differences between transient and stable expression in our dedicated article: What is the difference between transient and stable monoclonal antibody production?

What transfection strategies are used for stable cell line generation?

Several methods of transfection can be used to deliver expression and selection vectors into mammalian cells. The most commonly used methods are based on viral particles (e.g. lentivirus) or chemical and physical methods to increase membrane permeability and allow DNA vectors (negatively charged) to cross cell membranes (also negatively charged). The most commonly used chemical transfection methods include the use of calcium phosphate, cationic polymers, lipids, etc. In contrast, electroporation has also been successfully used to transfect mammalian cells.

What strategies can be used for the stable integration of antibody genes?

Mammalian genomes are complex and divided into regions with variable expression levels. For this reason, random integration can often produce clones with low antibody expression. To bypass this issue, many antibody developers are turning to site-directed gene integration for stable cell line development. Common strategies for site-directed integration include CRISPR/Cas9, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). The greatest bottleneck in site-directed integration is the need for in-depth knowledge of the expression’s host genome.