Conference Program

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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

Leveraging Sanofi intensified ICB platform to enable early process development for a labile and hard-to-express molecule

Within the biopharmaceutics industry, tremendous progress has been made in the implementation of early development antibody platforms to achieve high volumetric productivity and consistent product quality for novel therapies. More recently, development of new modalities provide opportunities for advancing exciting new therapeutic possibilities. However, many of these modalities present new upstream and downstream development challenges, e.g., low expression, labile molecules, low recovery, and unreliable product quality. The resulting additional development requirements increase the timelines for demonstrating Proof of Concept and may even prohibit certain therapeutic candidates from reaching the clinic at all. The Sanofi ICB platform provides opportunities to increase productivity and improve product quality, enabling manufacture of new entities previously inaccessible. Here, we present a case study of such a situation, in which the ICB platform is applied to an early-stage, labile, hard to express molecule produced from non-CHO mammalian cells. A combination of upstream and downstream high-throughput technologies have been incorporated to rapidly define a process sufficient for first-in-human studies. Process intensification enables adequate material generation within an acceptable number of batches for both development and clinical manufacturing. This case study demonstrates the strategy of using intensified perfusion platform for non-antibody modalities to support a diverse portfolio for our evolving industry

Demonstration of a commercial scale end-to-end continuous purification process

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