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Monoclonal antibodies are the workhorse of the pharmaceutical industry due to their potential to treat a variety of different diseases while providing high specificity and efficiency. As a consequence, a variety of production processes have been established within the biomanufacturing industry. However, the rapidly increasing demand for therapeutic molecules amid the recent COVID-19 pandemic demonstrated that there still is a clear need to establish novel, highly productive, and flexible production processes. Within this work, we designed a novel discontinuous process by combining two intensification strategies, thus increasing inoculation density and media exchange via a fluidized bed centrifuge, to fulfill the need for a flexible and highly productive production process for therapeutic molecules. To establish this new process, firstly, a small-scale experiment was conducted to verify synergies between both intensification strategies, followed by a process transfer towards the proof-of-concept scale. The combination of these two-process intensification measures revealed overall synergies resulting in decreased process duration (−37%) and strongly enhanced product formation (+116%) in comparison to the not-intensified standard operation. This led to an impressive threefold increase in space-time yield, while only negligible differences in product quality could be observed. Overall, this novel process not only increases the ways to react to emergency situations thanks to its flexibility and possible short development times, but also represents a possible alternative to the current established processes due to high increases in productivity, in comparison to standard fed-batch operations.

Baculovirus expression vectors are successfully used for the commercial production of complex (glyco)proteins in eukaryotic cells. The genome engineering of single-copy baculovirus infectious clones (bacmids) in E. coli has been valuable in the study of baculovirus biology, but bacmids are not yet widely applied as expression vectors. An important limitation of first-generation bacmids for large-scale protein production is the rapid loss of gene of interest (GOI) expression. The instability is caused by the mini-F replicon in the bacmid backbone, which is non-essential for baculovirus replication in insect cells, and carries the adjacent GOI in between attTn7 transposition sites. In this paper, we test the hypothesis that relocation of the attTn7 transgene insertion site away from the mini-F replicon prevents deletion of the GOI, thereby resulting in higher and prolonged recombinant protein expression levels. We applied lambda red genome engineering combined with SacB counterselection to generate a series of bacmids with relocated attTn7 sites and tested their performance by comparing the relative expression levels of different GOIs. We conclude that GOI expression from the odv-e56 (pif-5) locus results in higher overall expression levels and is more stable over serial passages compared to the original bacmid. Finally, we evaluated this improved next-generation bacmid during a bioreactor scale-up of Sf9 insect cells in suspension to produce enveloped chikungunya virus-like particles as a model vaccine.