The Advantages of N-1 Process Development for Monoclonal Antibody Development

A key strategy for optimizing mAb production, improving productivity and consistency can include N-1 process development.

Monoclonal antibodies (mAbs) are a class of biopharmaceuticals that have been widely used for the treatment and diagnosis of various diseases, such as cancer, autoimmune disorders, and infectious diseases. However, the production of mAbs is often challenging and costly, due to the complex and sensitive nature of these molecules. Therefore, there is a need for developing efficient and robust processes that can ensure high quality and yield of mAbs.

One of the strategies that has been proposed to improve the productivity and consistency of mAb production is the N-1 process development. N-1 process development is a strategy to intensify the fed-batch production of monoclonal antibodies (mAbs) by implementing a perfusion operation at the N-1 stage, which is the last seed expansion step before the final production bioreactor.

What is the N-1 Stage in mAb development?

The N-1 stage in mAb development is the last subculture before inoculation into the production bioreactor. It is a pre-conditioning step that prepares the cells for optimal performance in the production bioreactor by providing optimal nutrient levels and avoiding nutrient limitations or toxic metabolite accumulations. The N-1 stage can also increase the inoculum size and density, reduce the variability and heterogeneity of cell populations, and enhance the cell-specific productivity and product quality. The N-1 stage can be performed using perfusion or fed-batch methods¹²³.

This approach involves optimizing the culture conditions and feeding strategies at the N-1 stage, which is the last subculture before inoculation into the production bioreactor. The N-1 stage can be considered as a pre-conditioning step that prepares the cells for optimal performance in the production bioreactor.

The advantages of N-1 process development for mAb production are:

• It can reduce the variability and heterogeneity of cell populations, by selecting cells with high viability, growth rate, and productivity.
• It can enhance cell-specific productivity (CSP) and product quality, by providing optimal nutrient levels and avoiding nutrient limitations. It can enhance the quality and consistency of the mAbs by reducing the accumulation of waste products, such as lactate and ammonia, and maintaining optimal pH and dissolved oxygen levels in the production bioreactor. This improves the potency and stability of the mAbs and reduces the risk of product degradation and aggregation.
• It can increase the inoculum size and density, by allowing higher cell expansion and concentration in the N-1 bioreactor. It can shorten the cell growth phase in the production bioreactor, resulting in higher peak cell density, higher productivity, and shorter culture duration. 
• It can shorten the production cycle time and reduce the operational costs, by eliminating or reducing the need for seed train expansion and media exchange.
• It can enable the use of high-throughput and automated technologies, such as single-use bioreactors, process analytical technologies (PAT), and in-line sensors, to monitor and control the perfusion rate, cell density, and nutrient levels in the N-1 bioreactor. This can simplify the process development and scale-up of the N-1 perfusion platform and ensure the robustness and reproducibility of the mAb production.
• All of these advantages help reduce the cost of goods sold (COGS) by improving the efficiency and capacity of the biomanufacturing process and reducing operational costs.

Several studies have demonstrated the benefits of N-1 process development for mAb production using different cell lines, media formulations, feeding strategies, and bioreactor scales. For example, Liu et al.⁴ reported that by optimizing the N-1 stage, they were able to increase the CSP by 40%, reduce the glycosylation heterogeneity by 50%, and decrease the seed train duration by 50% for a CHO cell line producing a mAb. Similarly, Carvalho et al.⁵ showed that by applying an N-1 perfusion strategy, they were able to achieve a 10-fold increase in inoculum density and a 2-fold increase in CSP for a HEK293 cell line producing a mAb.

What are the key considerations?

1. Increase culture duration and risk of contamination.
2. Requires experienced personnel for successful process operation.
3. Requires optimization of N-1 perfusion rate, final cell density of N-1 stage, and N stage initial seeding density.
4. Identify a feasible process for scaling up to manufacturing scale (2000L).

Conclusion

In conclusion, N-1 process development is a promising technique that can improve the efficiency and robustness of mAb production processes. By optimizing the culture conditions and feeding strategies at the N-1 stage, it is possible to achieve higher productivity and quality of mAbs, while reducing the variability and complexity of the production process.

EirGenix Case Study:

At EirGenix we recently worked on a client monoclonal antibody that needed intensification. The development flow/ strategy was from a conventional process to an intensive process, then an advanced process, to an ultra-high cell density (uHD) process with 50 L consistency runs. We were able to scale up to 2,000L batches. The conventional process was a 16-day fed batch, with no perfusion. We moved on to the intensive process which was 6 days N-1 with perfusion +14 days fed-batch). This increased the titer from the conventional process by 26%. Next the advanced process was 6 days N-1 with perfusion + 16 days fed-batch); increased 62 % of titer from the conventional process. The uHD process was 7 days N-1 with a higher perfusion rate + 14 days fed-batch with ultra-high initial seeding density. This increased the titer from the conventional process by 83%. Most importantly, the optimized processes did not impact the product quality compared to that of the conventional process.

Consider working with EirGenix!

Are you seeking a contract development and manufacturing partner that has it all – quality, reliability, flexibility, and reasonable pricing? At EirGenix, we offer all of this and top-notch customer service. Since 2012, EirGenix has offered mammalian and microbial biopharma development and production including cell line establishment, large-scale production process development, analytical method development, and cGMP quality system operations that are certified by the US FDA, Japan PMDA, Australian TGA, Taiwan FDA, and Europe EMA. You can virtually tour our site here. We would appreciate the opportunity to collaborate with you! Click on this link to contact our BD team.

Citations

¹ Development of an intensified fed-batch production platform with doubled titers using N-1 perfusion seed for cell culture manufacturing | Bioresources and Bioprocessing

² mAb Development: Trends and Technologies - BioProcess International (bioprocessintl.com)

³ Exploring the limits of conventional small-scale CHO fed-batch for accelerated on demand monoclonal antibody production | Bioprocess and Biosystems Engineering

⁴ https://www.tandfonline.com/doi/pdf/10.4161/mabs.2.5.12645

⁵ https://www.intechopen.com/chapters/51512