Continuous culture in the age of single-use

Continuous culture offers a number of advantages, e.g. higher titres and constant culture environment and is the method of choice for challenging therapeutic proteins such as coagulation factors. Additional opportunities arise for the commercial production of monoclonal antibodies using so called concentrated fed-batch mode where the product is retained in the bioreactor and fresh medium is constantly perfused through the culture and combining it with single use production technologies. This approach is an effective way to reduce time to market as additional scale-up steps after phase III are not required to produce large amounts of product whilst benefiting from the widely discussed advantages of single use technologies. We will present our approach to single-use continuous manufacturing, how to deal with typical challenges and share data from a case study at 2000L scale

Bioprocess intensification for production of a Peste des petits ruminants virus (PPRV) vaccine

Peste des Petites Ruminants Virus (PPRV) is a highly contagious disease affecting small ruminants in Africa and Asian countries, with negative/significant economic impact. Aiming to eradicate the disease, targeted by the Food and Agriculture Organization for 2030, a novel and scalable PPRV vaccine production process is clearly needed. Built upon work previously done at iBET, a new production process is herein proposed using Vero cells growing on microcarriers, serum-free medium (SFM) and stirred-tank bioreactors (STB). This includes a new method for cells detachment from microcarriers, and perfusion culture for reducing turnaround time. The PPRV vaccine production process was developed in the 2L BIOSTAT® DCU-3 and the 20L BIOSTAT® Cplus STB (both from Sartorius) using Nigeria 75/1 strain. Engineering correlations (energy dissipation rate, shear stress and Kolmogorov Eddy size) were used to optimize culture conditions in the 2 L STB and to scale-up the process to the 20 L STB. Vero cells were adapted to grow in ProVeroTM-1 SFM (Sartorius). A new enzymatic and mechanical method for in situ cell detachment from microcarriers was designed. Perfusion was evaluated in the 2 L STB (equipped with internal spin-filter) in order to reduce seed-train preparation time. PPRV were clarified using depth filtration (Sartopure PP3, Sartorius). Process scalability was validated in the 20 L STB. Vero cells were adapted to ProVeroTM-1 SFM, reaching growth rates similar to serum-containing cultures (0.03 h-1). The new in situ cell detachment method was successfully implemented, with yields above 80%. A two-fold increase in maximum cell concentration was obtained using perfusion when compared to batch culture. Combining perfusion with the new in situ cell detachment method enabled the scale-up to 20 L STB directly from a 2 L STB, surpassing the need for a mid-scale platform and thus reducing seed-train preparation time. Infectious PPRV titers increase over culture time in both 2 L and 20 L STBs, reaching maximum values of 4.5-4.9x106 TCID50/mL at day 4-5 post-infection. The potential of depth filtration for PPRV clarification was confirmed; comparable PPRV recovery yields after clarification (85-90%) were obtained in both STBs. Overall, the novel and scalable vaccine production process herein proposed has the potential to assist the upcoming PPR Global Eradication Program (PPR GEP), to which iBET already contributes as partner in the PPR Global Research and Experts Network (PPR GREN), and thus support the One Health concept. Acknowledgments: This work was supported by Sartorius Stedim Biotech GmbH (Germany)

Automation of cell line development

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 μM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 μM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 μM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process