Abstract
Process intensification strategies are needed in the field of therapeutic protein production for higher productivities, lower cost of goods and improved facility utilization. This work describes an intensification approach, which connects a tangential-flow-filtration (TFF) based pre-stage perfusion process with a concentrated fed-batch production culture inoculated with an ultra-high seeding density (uHSD). This strategy shifted biomass production towards the pre-stage, reaching up to 45 × 106 cells/mL in perfusion mode. Subsequently, production in the intensified fed-batch started immediately and the product titer was almost doubled (1.9-fold) in an equivalent runtime and with comparable product quality compared to low-seeded cultures. Driven by mechanistic modelling and next-generation sequencing (NGS) the process had been optimized by selecting the media composition in a way that minimized cellular adaptation between perfusion and production culture. As a main feature, lactate feeding was applied in the intensified approach to promote cell culture performance and process scalability was proven via transfer to pilot-scale i.e., 20 L pre-stage perfusion and 80 L production reactor. Moreover, an earlier shift from a growth associated to a production stage associated gene expression pattern was identified for uHSD cultures compared to the reference. Overall, we showed that the described intensification strategy yielded in a higher volumetric productivity and is applicable for existing or already approved molecules in common, commercial fed-batch facilities. This work provides an in-depth molecular understanding of cellular processes that are detrimental during process intensification.
Highlights
Monoclonal antibodies and other derived therapeutic proteins are currently one of the most important product classes in the pharmaceutical industry and will likely remain relevant for many years to come [1, 2]
Few genes were differentially expressed for inoculation samples. These results strongly suggest that there is no immediate change in gene expression patterns between control and ultrahigh seeding density (uHSD) culture conditions within the first two process days
Our study provides an in-depth analysis of the process performance, product quality and gene expression during cultivation of Chinese Hamster Ovary (CHO) production cells in N − 1 prestage perfusion as well as in the production bioreactor with and without ultra-high seeding density
Summary
Monoclonal antibodies (mAbs) and other derived therapeutic proteins are currently one of the most important product classes in the pharmaceutical industry and will likely remain relevant for many years to come [1, 2]. Increasing market demands for monoclonal antibody therapeutics and the pressure for cost-reduction in their production [4, 5] have led to new manufacturing approaches and improved facility utilization strategies e.g., continuous production [6, 7]. For implementation of upcoming process intensification technologies, pharmaceutical companies can aim for scale out by building additional manufacturing capacities or for intensification in existing pharmaceutical production plants. In the case of fully continuous production processes, an implementation in existing fed-batch facilities is often impossible due to open technical and regulatory questions such as higher complexity and increased media amounts [7,8,9]. A smart step for the development and maintenance of marketable antibody portfolios is to search for intensification strategies, which debottleneck production chains using available capacities whenever possible and which are implementable in existing facilities, such as N − 1 perfusion [10]
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