Development of a Modular Biosensor System for Rapid Pathogen Detection

Progress in the field of pathogen detection relies on at least one of the following three qualities: selectivity, speed, and cost-effectiveness. Here, we demonstrate a proof of concept for an optical biosensing system for the detection of the opportunistic human pathogen Pseudomonas aeruginosa while addressing the abovementioned traits through a modular design. The biosensor detects pathogen-specific quorum sensing molecules and generates a fluorescence signal via an intracellular amplifier. Using a tailored measurement device built from low-cost components, the image analysis software detected the presence of P. aeruginosa in 42 min of incubation. Due to its modular design, individual components can be optimized or modified to specifically detect a variety of different pathogens. This biosensor system represents a successful integration of synthetic biology with software and hardware engineering

Assessment of a novel high-throughput process development platform for biopharmaceutical protein production

Plants can complement dominant expression systems such as Escherichia coli and Chinese hamster ovary (CHO) cells with additional production capacity in response to emerging infectious diseases, but compared to these hosts few high-throughput screening tools that facilitate biopharmaceutical development have been available in plants. To advance this situation, high-throughput techniques were implemented during cloning, expression, purification and quantification, thus increasing the screening throughput across the entire development process. This concept was applied to a transient expression in Nicotiana spp., which allows to establish production processes in as little as 3 weeks and thus quickly react to changing demands. In combination with statistical design of experiments, the established high-throughput screening tools allowed to rapidly clone and test libraries of expression cassette elements such as promotors, 5′ UTRs and signal sequences and identify combinations thereof that maximize target protein accumulation. This strategy was successfully employed to produce interleukins, polyphosphate kinases, IgG3 antibodies, biofilm degrading enzymes and endolysins selected for a multilayered strategy directed against methicillin-resistant Staphylococcus aureus (MRSA). Notably, IgG3 accumulation levels achieved by systematically screening expression cassette elements were threefold higher than the literature. Using the same strategy, dispersin B accumulation levels equivalent to E. coli were reached. Further improvement can be expected in the future by expanding the set of expression cassette elements used for screening, particularly with synthetic promotors, 3′ UTRs and terminators. Plant-derived target proteins were functional, except for a reduced enzymatic activity of polyphosphate kinases, indicating that Nicotiana spp. can supply recombinant proteins to counter emerging MRSA. Using the high-throughput screening tools established herein, additional plant-made proteins can be rapidly assessed for their usefulness against MRSA in the future.In accordance with a recent approach in biopharmaceutical development, data generated with the different target proteins were used to identify parameters that can guide the selection of candidate proteins and even optimization strategies. For instance, the target protein origin had a major impact on the ideal expression compartment, thus allowing to pre-select suitable expression compartments and reduce the screening workload. Assessing intrinsic protein stability parameters allowed to sort out unsuitable candidate proteins, albeit currently limited to comparisons within the same protein class. A parameter that can guide the optimization of plant cell cultivation media is the medium osmolality, essentially controlling the uptake of water into plant cells. Characterization of host cell proteins that persist after chromatography allowed to derive purification strategies that facilitate their removal

Cellulose-based filter aids increase the capacity of depth filters during the downstream processing of plant-derived biopharmaceutical proteins

Downstream processing (DSP) is a major cost factor during the production of biopharmaceutical proteins. Clarification can account for approximate to 40% of these costs, especially when a large amount of dispersed particulate material is generated, such as during the extraction of intracellular proteins from plants. Filter capacity can be increased (and DSP costs reduced) by using flocculants. Here we show that cellulose-based filter aids can enhance the positive effect of flocculants by improving depth filter capacity even further. A design-of-experiments (DoE) approach was used to identify the optimal size and concentration of filter aids, at different values of pH and conductivity, for the clarification of tobacco leaf extracts during the production of a monoclonal antibody and a fluorescent protein. Filter aids approximate to 28 or approximate to 100 m in length at concentrations of approximate to 10 and approximate to 5 g L-1 respectively were most efficient in combination with a strong cationic flocculant, but were ineffective without the flocculant. The filter aids increased depth filter capacity by 35-fold compared to an additive-free extract reaching approximate to 1000 L m(-2) without affecting the target proteins. Thus, filter aids can be used to reduce production costs of plant-derived biopharmaceuticals while the DoE approach enabled the identification of robust process conditions