Steady state kinetic analyses of nitroalkane oxidase mutants

Nitroalkane oxidase (NAO) catalyzes the oxidation of neutral nitroalkanes to aldehydes and ketones with oxygen consumption and the production of hydrogen peroxide and nitrite. The enzyme is a flavoprotein from the fungus Fusarium oxysporum. The active site base, Asp402, abstracts one proton from the substrate to give a carbanion which then attacks the flavin adenine dinucleotide (FAD). The three dimensional crystal structure of NAO shows that Arg409 is 3.6 Å from Asp402. When Arg409 is mutated to Lys, the rate constant for proton abstraction decreases 100-fold. The three-dimensional structure of NAO also reveals the existence of a tunnel which extends from the protein exterior and terminates at the FAD N5 atom and the residues Asp402 and Phe401. We mutated amino acids in the tunnel into tryptophan, phenylalanine and leucine. The L99W, S276W and S276A enzymes showed the biggest decreases in both kcat and kcat/Km; these amino acids are closest to the FAD molecule and the active site. Mutation of amino acids farther away from the active site showed very small changes in the kinetic parameters. Ser276 is hydrogen bonded to Asp402 in the wild-type enzyme. When this amino acid is mutated to alanine or tryptophan, k3, the rate constant for proton abstraction, decreases around 35 fold. Asp402, Arg409 and Ser276 constitute a catalytic triad in the active site of nitroalkane oxidase, and both Arg409 and Ser276 are important for positioning Asp402 and catalysis

Biomanufacturing and testbed development for the continuous production of monoclonal antibodies

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Continuous Online Titer Monitoring in CHO Cell Culture Supernatant Using a Herringbone Nanofluidic Filter Array

Online monitoring of monoclonal antibody product titers throughout biologics process development and production enables rapid bioprocess decision-making and process optimization. Conventional analytical methods, including high-performance liquid chromatography and turbidimetry, typically require interfacing with an automated sampling system capable of online sampling and fractionation, which suffers from increased cost, a higher risk of failure, and a higher mechanical complexity of the system. In this study, a novel nanofluidic system for continuous direct (no sample preparation) IgG titer measurements was investigated. Tumor necrosis factor α (TNF-α), conjugated with fluorophores, was utilized as a selective binder for adalimumab in the unprocessed cell culture supernatant. The nanofluidic device can separate the bound complex from unbound TNF-α and selectively concentrate the bound complex for high-sensitivity detection. Based on the fluorescence intensity from the concentrated bound complex, a fluorescence intensity versus titer curve can be generated, which was used to determine the titer of samples from filtered, unpurified Chinese hamster ovary cell cultures continuously. The system performed direct monitoring of IgG titers with nanomolar resolution and showed a good correlation with the biolayer interferometry assays. Furthermore, by variation of the concentration of the indicator (TNF-α), the dynamic range of the system can be tuned and further expanded