Custom Cell Line Development

Our custom cell line development platform for monoclonal antibody production is designed to optimize production yields while minimizing development costs. The first step in each project is 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™. It also produces antibodies in quantities that allow extensive characterization of key antibody properties.

Based on this assessment, we develop a custom protocol for stable cell line generation and predict production yields. Stable expression and selection vectors are then designed, synthesized, and transfected into competent cells.

Selection and amplification of positive clones are then performed. Positive clones are isolated and evaluated for stability and productivity. If single clones with robust productivity are identified, the process is optimized and scaled up or transferred to the customer’s facilities or a selected CMO.

Proprietary CHO-K1, as well as CHO-DG44, CHO-S, and HEK cell lines adapted for suspension culture, are available through ProteoGenix’s stable cell line generation service. We can also perform stable cell line development using 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 subtilis), 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.

More than 100 projects were successfully completed using our platform of stable cell line generation for monoclonal antibody production with a productivity record of 10 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.

Our typical process of custom cell line development 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).

ProteoGenix offers two types of guarantees to minimize the risks associated with your investment:

1. A yield guarantee, which is estimated based on the sequence to be produced at the beginning of the project. This guarantee secures your investment and provides a good estimate of what we believe we can achieve. In general, we are often able to achieve yields significantly higher than our initial estimate.
2. Our service is divided into several “Working Packages,” which are typically concluded with Go/No Go decisions. At the end of each working package, you will have the option to decide whether to continue the service or stop, paying only for the work completed!

Custom cell line development is a mandatory step in the successful clinical development of monoclonal antibodies. At ProteoGenix, we recommend the extensive early testing of antibody leads before committing to large-scale production.

Early developability assessment is a well-established and successful approach used to ensure an antibody meets the best stability, reactivity, and effectiveness before developing a custom and optimized production protocol. In this way, we can bypass many stable expression hurdles and ensure optimal production at reasonable costs. Antibody characteristics such as aggregation, affinity and avidity, specificity and selectivity, and glycosylation profile are known to have the greatest impact on developability and can be analyzed in silico and further measured on recombinant antibodies produced in our facilities.

Full-length monoclonal antibodies are often produced in mammalian expression systems such as Chinese hamster ovary (CHO) or mouse myeloma (NS0) cells. At ProteoGenix, we specialize in CHO cell lines offering a large diversity of hosts including our proprietary CHO-K1 cell line and also CHO-DG44 (a cell line lacking both alleles of DHFR, suitable for metabolic selection) or CHO-S, among others.
After over 28 years of experience in protein production in different expression systems (mammalian, bacterial, yeast, insect), CHO-based stable production is our system of choice when expressing antibodies. CHO cells are more tolerant to changes in pH, temperature, pressure, and oxygen concentrations. Plus, they are more amenable to gene amplification leading to production levels significantly higher than those achieved by other production systems. Plus, CHO cells produce proteins with glycosylation profiles similar to humans resulting in lower immunogenicity and optimal therapeutic efficacy.

In contrast, antibody fragments devoid of glycans may be produced faster at high titers in simpler systems such as bacterial cells (e.g. Escherichia coli or Bacillus subtills) known for their high turnover rates. The simpler genetic background of bacteria makes them more amenable to manipulation and scale-up. However, antibodies in E. coli are produced in the cytoplasm or periplasm requiring harsher extraction methods.

In addition, our service includes a comprehensive early testing phase. The comprehensive characterization of antibody leads allows us to predict potential production hurdles and guarantee production yields. In this way, we can protect our clients’ investments while transferring all risks directly to us.
Furthermore, you retain the intellectual property (IP) of the cell lines and protocols generated at ProteoGenix. This simplifies protocol and cell transfer to a CMO or your facilities for large-scale production. Our FTO (freedom-to-operate) 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.

As experts in antibody production and engineering, we also offer a wide range of upstream services, including affinity maturation, antibody humanization, bispecific antibody development, antibody-drug conjugate development, and antibody reformatting. Our downstream capabilities include research cell bank development and stability studies. By partnering with us, you can save time and reduce costs while maximizing the therapeutic efficacy of your antibody leads and minimizing foreseeable production risks.

Ensuring monoclonality is critical in the development of a stable cell line, particularly to guarantee consistent yield, reproducibility, and quality of the final products. At ProteoGenix, we offer two methods to isolate monoclonal cells:

1. 1. Limiting Dilution Method

Limiting dilution is the most well-known method for isolating monoclonal cells. It involves diluting the cell suspension into wells so that the probability of having a single cell per well is high. While this method is relatively cost-effective to implement, it has several drawbacks:

1. 1. • Lack of control: Since the method relies on probabilities, some wells may end up empty, while others may contain two or more cells, compromising the isolation.
      • High level of human intervention: This process requires considerable manual effort, increasing the chances of variability and error.
      • Difficulty in documenting monoclonality: Observing a single cell under the microscope can be challenging. Due to rapid cell division, it’s often difficult to determine whether observed colonies originate from a single clone or multiple different clones. This lack of clear documentation can be a significant disadvantage, particularly when submitting an IND (Investigational New Drug) application for therapeutic antibody development.

1. Automated methods: VIPS™ Technology

Regulatory agencies today increasingly prefer automated methods, such as those provided by VIPS™ (Verified In-Situ Plate Seeding) technology, due to several key advantages over limiting dilution:

• Automation with minimal human intervention: The VIPS™ system is fully automated, reducing human error and intervention.
• Definitive proof of monoclonality: Unlike limiting dilution, which offers only a probabilistic assurance, VIPS™ provides visual proof of monoclonality. This proof is available immediately after seeding the suspension, as opposed to the delay in limiting dilution. This certainty is crucial for IND submissions.
• Real-time monitoring of cell growth: The system tracks cell growth in real-time, ensuring consistent and reliable results.
• Significant time savings: Since VIPS™ is fully automated, the process of generating monoclonal cells is approximately 50% faster than with limiting dilution.

Whether for therapeutic, diagnostic, or veterinary projects, certain development steps prove to be particularly critical. In addition to the generation and engineering of your antibody, the development of a stable cell line for the production of your antibody is a step not to be overlooked. Indeed, this step will partially determine the final production cost of your product, particularly through the production yield of your antibody and the simplicity of the purification process. To best determine whether it is relevant for you to outsource the development of your stable cell line, here are several criteria to consider:

• Expertise: Stable cell line development is a process that requires a certain level of expertise, from the analysis of the initial sequence to the selection of the best stable clones. At ProteoGenix, we consider the analysis of your antibody’s sequence to assess its developability and manufacturability. If elements such as liability sequences, solubility risks, and aggregation risks are identified, ProteoGenix can offer optimized sequences without altering the initial properties of your biomolecule. This step aims to optimize the physicochemical properties of your antibody but also to facilitate future purification steps. Furthermore, the selection of the best pools and stable clones is based on complex multifactorial analysis, such as the yield and quality of the final product.
• Equipment: Due to its complexity, stable cell line development requires a large amount of equipment, whether for the isolation of monoclonal cells, the monitoring of cell growth, or the characterization of the final product. This non-exhaustive list includes, among others, a mass spectrometer, a Biacore, an HPLC system, a single-cell seeder coupled with imaging… These pieces of equipment are crucial for making decisions based on complete information. A decision based on partial characterization could lead to additional costs that may be difficult to recover later.
• Limiting financial risk: Developing a stable cell line in-house will mobilize your resources without any guarantee of success. Using a service provider can mitigate this risk. For example, at ProteoGenix, thanks to our yield guarantee and the implementation of several go/no-go checkpoints throughout the stable cell line development process, we allow you to access stable cell line development without risk. We can offer these guarantees for several reasons:
  • We have produced over 1,500 proteins and 5,000 antibodies, and we have developed over 100 stable cell lines. This experience allows us to trust our process and offer you unparalleled guarantees.
  • We have developed our own AI platform to estimate the manufacturability of your biomolecule. Through an in silico pre-study, we can, with only your sequence, have a good idea of our chances of success. To top it off, our tool can propose sequence optimizations to improve the manufacturability of your biomolecule, and the different sequences can be tested using our high-throughput antibody production platform.
• Timeline: Stable cell line development usually spans long periods, ranging from 3 months to a year. The shortest timelines are typically achieved by CROs that implement proprietary platforms designed to reduce development times while maintaining or even improving the quality of the final product. At ProteoGenix, to achieve this goal of reducing timelines, our team of bioinformaticians has developed a platform to analyze and improve the manufacturability of your sequences. Our research team is also constantly optimizing our processes to meet your expectations. Our most recent improvement involved the purchase of a VIPS system, allowing us to reduce monoclonal selection times by around 50% compared to a classic limiting dilution protocol.
• Innovation: As mentioned earlier, stable cell line development is a core service of certain service providers. As such, a service provider like ProteoGenix constantly strives to push the performance of its service further. In addition to the examples already mentioned, our researchers have also developed a proprietary cell line specially designed for high antibody expression. By combining all these technologies, ProteoGenix is able to develop a stable cell line that expresses high-quality antibodies with high yields, requiring minimal adaptation to your processes.
• Bonus: Full control of the antibody generation process: Developing a stable cell line is the final step in antibody development before the manufacturing phase. It cannot be stressed enough that entrusting your entire antibody development project to a CRO (from antibody generation to stable cell line development) is the best way to ensure good developability and manufacturability. Indeed, by entrusting your project to ProteoGenix from the start, you can be sure that everything will be anticipated to reach your final goal, whether for a therapeutic, diagnostic, or veterinary project.