Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand ‘azoCm’ was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a K D of 545 nM. As the aptamer cannot bind to the irradiated ligand ( λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.
Media preparation for perfusion cell culture processes contributes significantly to operational costs and the footprint of continuous operations for therapeutic protein manufacturing. In this study, definitions are given for the use of a perfusion equivalent nutrient feed stream which, when used in combination with basal perfusion medium, supplements the culture with targeted compounds and increases the medium depth. Definitions to compare medium and feed depth are given in this article. Using a concentrated nutrient feed, a 1.8-fold medium consumption (MC) decrease and a 1.67-fold increase in volumetric productivity (PR) were achieved compared to the initial condition. Later, this strategy was used to push cell densities above 100 × 10 6 cells/ml while using a perfusion rate below 2 RV/day. In this example, MC was also decreased 1.8-fold compared to the initial condition, but due to the higher cell density, PR was increased 3.1-fold and to an average PR value of 1.36 g L −1 day −1 during a short stable phase, and versus 0.46 g L −1 day −1 in the initial condition. Overall, the performance improvements were aligned with the given definitions. This multiple feeding strategy can be applied to gain some flexibility during process development and also in a manufacturing setup to enable better control on nutrient addition. K E Y W O R D S biopharmaceutical process, mammalian cell culture, monoclonal antibody, perfusion cell culture, recombinant protein 1 | INTRODUCTION Perfusion cell culture for the manufacturing of therapeutic recombinant proteins increases volumetric productivities compared to more traditional batch-like processes. This technology has been further developed in recent years while the industry continues to push for integrated and continuous manufacturing. 1-4 New economical and operational challenges are posed by different aspects of the continuous operation mode and some of the main limitations are linked to medium management. In fact, medium represents a significant portion of the perfusion cost of goods. 5-10 In terms of operation, handling of large medium volumes is also in contradiction to the general requirement for plant footprint reduction. 11-13 Thus, this study addresses some aspects of medium management for perfusion cell cultures by defining a concentrated feed that was used in parallel to the basal medium, and that only contained compounds that are depleted by the cellular activity. This dual feeding strategy was based on basal medium to maintain physiological conditions
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.