Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic structures.

Zero-mode waveguides (ZMW) have the potential to be powerful confinement tools for studying electron transfer dynamics at single molecule occupancy conditions. Flavin mononucleotide contains an isoalloxazine chromophore, which is fluorescent in the oxidized state (FMN) while the reduced state (FMNH2) exhibits dramatically lower light emission, i.e. a dark-state. This allows fluorescence emission to report the redox state of single FMN molecules, an observation that has been used previously to study single electron transfer events in surface-immobilized flavins and flavoenzymes, e.g. sarcosine oxidase, by direct wide-field imaging of ZMW arrays. Single molecule electron transfer dynamics have now been extended to the study of freely diffusing molecules using fluorescence measurements of Au ZMWs under single occupancy conditions. The Au in the ZMW serves both as an optical cladding layer and as the working electrode for potential control, thereby accessing single molecule electron transfer dynamics at µM concentrations. Consistent with expectations, the probability of observing single reduced molecules increases as the potential is scanned negative, E(appl) < E(eq), and the probability of observing emitting oxidized molecules increases at E(appl) > E(eq). Different single molecules exhibit different electron transfer properties as reflected in the position of E(eq) and the distribution of E(eq) among a population of FMN molecules. Two types of actively-controlled electroluminescence experiments were used: chronofluorometry experiments, in which the potential is alternately stepped between oxidizing and reducing potentials, and cyclic potential sweep fluorescence experiments, analogous to cyclic voltammetry, these latter experiments exhibiting a dramatic scan rate dependence with the slowest scan rates showing distinct intermediate states that are stable over a range of potentials. These states are assigned to flavosemiquinone species that are stabilized in the special environment of the ZMW nanopore.

Application of platform process development approaches to the manufacturing of Mabcalin™ bispecifics.

Bispecific biotherapeutics offer potent and highly specific treatment options in oncology and immuno-oncology. However, many bispecific formats are prone to high levels of aggregation and instability, leading to prolonged development timelines, inefficient manufacturing, and high costs. The novel class of Mabcalin™ molecules consist of Anticalin® proteins fused to an IgG and are currently being evaluated in pre-clinical and clinical studies. Here, we describe a robust high-yield manufacturing platform for these therapeutic fusion proteins providing data up to commercially relevant scales. A platform upstream process was established for one of the Mabcalin bispecifics and then applied to other clinically relevant drug candidates with different IgG target specificities. Process performance was compared in 3 L bioreactors and production was scaled-up to up to 1000 L for confirmation. The Mabcalin proteins' structural and biophysical similarities enabled a downstream platform approach consisting of initial protein A capture, viral inactivation, mixed-mode anion exchange polishing, second polishing by cation exchange or hydrophobic interaction chromatography, viral filtration, buffer exchange and concentration by ultrafiltration/diafiltration. All three processes met their target specifications and achieved comparable clearance of impurities and product yields across scales. The described platform approach provides a fast and economic path to process confirmation and is well comparable to classical monoclonal antibody approaches in terms of costs and time to clinic.