Commercialization of a 2nd generation intensified perfusion process during life cycle management

Continuous biomanufacturing provides many advantages for the production of therapeutic proteins through process integration, automation and intensification. Sanofi is currently developing robust cell culture processes using ATF perfusion technology to achieve improved volumetric productivity with consistent product quality. This presentation is a case study on how we applied intensified process technology on product commercialization. Using QbD approach, we successfully implemented an intensified perfusion process coupled with continuous capturing on a commercial product life cycle management. For the 2nd generation process, entire production and capturing stage is fully integrated and automated. The new perfusion process comprises of high cell density and achieves significant increase in volumetric productivity, which allows a substantial footprint reduction and increases flexibility in a new facility. More importantly, product quality was remarkably comparable with the 1st generation process. Dramatic improvement in process robustness and consistency were demonstrated as well. Facilitated by computational fluid dynamics (CFD) simulation, we successfully scaled up to commercial scale. In support of technology transfer and manufacturing, process control strategy was drafted based on the results of bioreactor characterization using univariate and multivariate studies

Process scale up and characterization of an intensified perfusion process

Continuous biomanufacturing provides many advantages for the production of therapeutic proteins through process integration, automation and intensification. Sanofi currently has developed a robust and integrated continuous biomanufacturing platform to achieve improved volumetric productivity and consistent product quality. Process intensification reduces the physical footprint as well as capital and operating expenses of manufacturing facilities. This presentation is a case study on the implementation of the intensified process for commercialization of a biotherapeutic product. Using a QbD approach, we successfully implemented an intensified perfusion process coupled with continuous product capture for a commercial product. High cell densities have resulted in a significant increase in volumetric productivity, which allows a substantial footprint reduction and increases flexibility in the commercial facility. To understand the impact of process parameters on critical quality attributes (CQAs), univariate and multivariate studies were conducted in small scale bioreactors. Mix model repeated measurement was applied in the data analysis to incorporate time-dependent information into the predictive model. This was followed by Monte Carlo simulation to determine proven acceptable ranges (PARs) for critical process parameters in support of process control strategy (PCS). Facilitated by computational fluid dynamics (CFD) simulation, we successfully scaled up the process to commercial scale. In this presentation, challenges associated with application of QbD approach for a perfusion process and the advantages of an intensified perfusion process will be discussed

Application of 13C flux analysis to determine impacts of media alterations on industrial CHO cell metabolism

Industrial bioprocesses place extraordinary demands on the metabolism of host cells to meet the biosynthetic requirements for maximal growth and protein production. Identifying host cell metabolic phenotypes that promote high recombinant protein titer is a major goal of the biotech industry. 13C metabolic flux analysis (MFA) provides a rigorous approach to quantify these metabolic phenotypes by applying stable isotope tracers to map the flow of carbon through intracellular metabolic pathways. We have conducted a series of 13C MFA studies to examine the metabolic impacts of altering the composition of a proprietary chemically defined growth medium on CHO cell metabolism. CHO cell cultures characteristically produce excess ammonia and lactate as byproducts, both of which are toxic at high concentrations. Whereas lactate is often consumed during stationary growth phase in CHO cell cultures, ammonia continues to accumulate in the extracellular media throughout the course of cell growth due mainly to glutamine catabolism. For CHO cells that utilize glutamine, rational media design can alleviate ammonia stress from the cell culture. However, manipulating carbon sources in the growth medium can also have negative effects on cellular metabolism such as decreased culture growth, viability, recombinant protein productivity, or longevity. This study highlights a rationally engineered cell culture medium that successfully reduces culture ammonia levels by 40% while maintaining the original metabolic phenotype. First, the basal media developed in-house by Sanofi was chemically altered to cause CHO cells to produce significantly less ammonia byproduct. This low ammonia-producing media variant was experimentally developed by altering the ratio of carbon sources in the media to strategically reduce flux through metabolic pathways that result in ammonia production while supplementing complementary, non-ammonia producing pathways to balance metabolism. This altered media variant successfully decreased the ammonia concentration in industrial CHO while maintaining culture growth, viability, and specific productivity. Parallel 13C MFA studies were performed on IgG-producing CHO cells grown identically in three media variants: the basal control media, the low-ammonia media, and the low-ammonia media supplemented with basal ammonia levels. The latter media was used to control for any direct effects of changing ammonia concentrations on cellular metabolism. 13C labeling studies utilizing [U-13C5]glutamine and [1,213C2]glucose were carried out in parallel for each condition. From the comparison of the 13C flux analysis across the three media types, we have concluded that the media alterations did not have a significant impact on the intracellular metabolism of CHO cultures. This suggests that Sanofi can use their newly developed media formulation to decrease toxic ammonia buildup in IgG-producing CHO cell lines without significantly altering host metabolic phenotype or productivit

Delivering steady-state product quality with an intensified and integrated perfusion cell culture process

Continuous biomanufacturing provides many important strategic advantages for the production of protein therapeutics through process integration, simplification and intensification. To achieve upstream process intensification, Sanofi is currently developing robust cell culture processes that can achieve ultra-high cell densities and productivities (“push to high”) while minimizing cell-specific perfusion rates (“push to low”). We have applied ATF perfusion technology and improved the cell culture environment to achieve high cell densities and volumetric productivities with minimal ATF filter fouling. Meanwhile, we have employed high-throughput screening strategies to increase medium depth and reduce medium requirements. We will describe results as well as ongoing efforts to further intensify this continuous cell culture platform and realize even more of its significant upward potential. Continuous biomanufacturing also has the potential to deliver robust, steady-state product quality, resulting in enormous operational flexibility. Instead of traditionally defining batches by unit operation, product can be batched in time (first-in, first-out), removing downstream processing constraints and minimizing production cycle times. In this presentation, we use both theoretical models and experimental data to evaluate the effects of perfusion on product quality, considering the impact of perfusion-specific controllable parameters (e.g., perfusion rate, bleed rate, target viable cell density) on product quality. We also compare and contrast product quality attributes between perfusion and fed-batch processes and examine the feasibility of maintaining a process and product quality at steady state while presenting relevant, real-world case studies

Overcoming process intensification challenges to deliver a manufacturable and competitive integrated continuous biomanufacturing platform

Groups in both industry and academia have achieved high densities and productivities in perfusion cell culture processes. At Sanofi, we have demonstrated perfusion densities greater than 100 million cells/mL (with associated high productivities) at a cell-specific perfusion rate of only 20 pL/cell/day. This process intensification reduces the footprint of upstream unit operations as well as capital and operating expenses of manufacturing facilities. The continuous nature of perfusion cell culture also creates opportunities for integration of continuous downstream operations, leading to further process intensifications and volume reductions. In this presentation, we will discuss our work on several upstream challenges that must be overcome to create a manufacturable, continuous bioprocessing platform. These will include (1) mitigation strategies for the large shear forces accompanying the high sparge rates necessary to sustain a high-density culture, (2) efforts to minimize the economic and logistical burden of media cost and consumption in perfusion cell culture, (3) the challenge of maintaining consistent product quality over long durations and (4) scale-up of these intensified processes to 50-L and 500-L manufacturing-scale systems. We can address each of these areas to create an efficient, competitive cell culture platform that generates high cell viabilities and excellent product quality at manufacturing scales. We will demonstrate real-world examples of both enzyme and antibody-producing processes, showing that such a platform can reliably deliver good results across diverse products