Development of a highly concentrated perfusion medium supplement to decrease media demand leveraging a newly designed 250 mL single use perfusion bioreactor

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Upstream process intensification using frozen high cell density intermediates

Typical seed train operations start by thawing of a single vial followed by several expansion steps. Reaching sufficient absolute cell numbers for production bioreactor inoculation is time-consuming and reduces plant flexibility. Besides long ramp up times, open cell culture operations are a major source of process variability. High cell density cryopreservation (HCDC) is a method of freezing cells in bags instead of vials and at higher cell densities. This offers the advantage of decoupling expansion and production: both steps can be separated in space and time. Room classification could be decreased due to fully closed processing and reproducibility increased due to a reduction of manual handling steps. Furthermore, these frozen seed train intermediates allow global distribution from a central expansion facility to decentralized global production facilities. Besides from advantages in production, these HCDC bags can be used in process development to ensure equal starting points in experimental setups. In this study, we developed a single-use bag assembly that supports closed filling, freezing, thawing, and inoculation. Before using the bag application, relevant parameters for this process from filling to inoculation were evaluated in vials with different cell lines. We found that the DMSO concentration for optimal freezing must not be higher than 7,5%. Furthermore, direct freezing at -80 °C instead of using a controlled rate freezing method is possible. Maximum concentration of DMSO in cell cultures should not be higher than 0,5 % when cryopreserved cells in bags are used for inoculation. For the idea of seed train intensification, we tested increasing freezing cell densities from 10 to 100 million cells/mL showing comparable growth. Functionality test of this HCDC method in comparison to vials was demonstrated in 4,2 L bioreactors simulating a manufacturing process. Applicability of this cryopreservation technology has been demonstrated using different bioreactors, perfusion systems, and various CHO cell lines

Development of an N-1 perfusion medium to intensify seed train operation

Seed train expansion of cells before the final production step is often time-consuming and a major source of process variability. For the intensification of seed train operations there are several opportunities discussed across the biopharma industry today. One of the possibilities is to operate N-1 bioreactors in perfusion mode to shorten timelines and improve bioreactor utilization. In this work, we investigated the influence of using an expansion medium especially designed for N-1 perfusion to gain optimal results in the whole manufacturing campaign. For screening and designing an N-1 perfusion expansion medium, a scale down model which represents a typical production campaign, including the seed train, was established. Expansion medium prototypes were combined with different production media in the final production step, e.g. EX-CELL® Advanced HD Perfusion as medium designed for high-density perfusion, and Cellvento® 4CHO Medium and 4FEED as exemplary fed-batch process. After determining a suitable expansion medium formulation, the prototype was evaluated for solubility and streamlined to ensure a simple hydration and robust supply chain. Afterwards, results were confirmed using a simulated manufacturing process using benchtop bioreactor systems, showing that the positive impact of the expansion medium on the final yield is present both when using perfusion or fed-batch as final production stage. Our results indicate that using the right companion medium in seed train expansion - specifically designed for the purpose - can prepare the cells optimally for the final N-stage and increase productivity while using low CSPRs. Combining these findings with the application of a perfused N-1 step in the manufacturing campaign leads to a great opportunity for the intensification of the whole upstream process