High-Density Perfusion Cultures of the Marine Bacterium Rhodovulum sulfidophilum for the Biomanufacturing of Oligonucleotides.

Therapeutic oligonucleotides (ONs) are typically manufactured via solid-phase synthesis, characterized by limited scalability and huge environmental footprint, limiting their availability. Biomanufactured ONs have the potential to reduce the immunogenic side-effects, and to improve the sustainability of their chemical counterparts. Rhodovulum sulfidophilum was demonstrated a valuable host for the extracellular production of recombinant ONs. However, low viable cell densities and product titer were reported so far. In this work, perfusion cell cultures were established for the intensification of ON biomanufacturing. First, the perfusion conditions were simulated in 50mL spin tubes, selected as a scale-down model of the process, with the aim of optimizing the medium composition and process parameters. This optimization stage led to an increase in the cell density by 44% compared to the reference medium formulation. In addition, tests at increasing perfusion rates were conducted until achieving the maximum viable cell density (VCDmax), allowing the determination of the minimum cell-specific perfusion rate (CSPRmin) required to sustain the cell culture. Intriguingly, we discovered in this system also a maximum CSPR, above which growth inhibition starts. By leveraging this process optimization, we show for the first time the conduction of perfusion cultures of R. sulfidophilum in bench-scale bioreactors. This process development pipeline allowed stable cultures for more than 20 days and the continuous biomanufacturing of ONs, testifying the great potential of perfusion processes.

Soft sensor based on Raman spectroscopy for the in-line monitoring of metabolites and polymer quality in the biomanufacturing of polyhydroxyalkanoates.

Polyhydroxyalkanoates (PHA) are among the most promising bio-based alternatives to conventional petroleum-based plastics. These biodegradable polyesters can in fact be produced by fermentation from bacteria like Cupriavidus necator, thus reducing the environmental footprint of the manufacturing process. However, ensuring consistent product quality attributes is a major challenge of biomanufacturing. To address this issue, the implementation of real-time monitoring tools is essential to increase process understanding, enable a prompt response to possible process deviations and realize on-line process optimization. In this work, a soft sensor based on in situ Raman spectroscopy was developed and applied to the in-line monitoring of PHA biomanufacturing. This strategy allows the collection of quantitative information directly from the culture broth, without the need for sampling, and at high frequency. In fact, through an optimized multivariate data analysis pipeline, this soft sensor allows monitoring cell dry weight, as well as carbon and nitrogen source concentrations with root mean squared errors (RMSE) equal to 3.71, 7 and 0.03 g/L, respectively. In addition, this tool allows the in-line monitoring of intracellular PHA accumulation, with an RMSE of 14 gPHA/gCells. For the first time, also the number and weight average molecular weights of the polymer produced could be monitored, with RMSE of 8.7E4 and 11.6E4 g/mol, respectively. Overall, this work demonstrates the potential of Raman spectroscopy in the in-line monitoring of biotechnology processes, leading to the simultaneous measurement of several process variables in real time without the need of sampling and labor-intensive sample preparations.