Automated bioprocess feedback operation in a high throughput facility via the integration of a mobile robotic lab assistant

Biotechnological processes development is challenging due to the sheer variety of process parameters. For efficient upstream development parallel cultivation systems have proven to reduce costs and associated timelines successfully, while offering excellent process control. However, the degree of automation of such small scale systems is comparably low and necessary sample analysis requires manual steps. Although the subsequent analysis can be performed in a high-throughput manner, the integration of analytic devices remains challenging. Especially, when cultivation and analysis laboratories are spatially separated. Mobile robots offer a potential solution, but the implementation in research laboratories is not widely adopted. Our approach demonstrates the integration of a small scale cultivation system into a liquid handling station for an automated sample procedure. The samples are transferred via a mobile robotic lab assistant and subsequently analysed by a high-throughput analyzer. The process data is stored in a centralized database. The mobile robotic workflow guarantees a flexible solution for device integration and facilitates automation. Restrictions regarding spatial separation of devices are circumvented, enabling a modular platform throughout different laboratories. The presented cultivation platform is evaluated based on industrial relevant E. coli BW25113 high cell density fed-batch cultivation. Here its suitability for accelerating bioprocess development is proven. The necessary magnesium addition for reaching high cell densities in mineral salt medium is automated via a feedback operation loop. The feedback operation loop demonstrates the possibility for advanced control options. This study sets the foundation for a fully integrated facility with different cultivation scales sharing the same data infrastructure, where the mobile robotic lab assistant physically connects the devices.

Technologies for studying functional neural networks of the human brain based on data of nuclear functional magnetic tomography

Nuclear functional magnetic resonance imaging (fMRI) is one of the most popular methods for studying the functional activity of the human brain. In particular, this method is used in medicine to obtain information about the state of the functional networks of the patient’s brain. However, the process of processing and analysis of experimental fMRI data is complex and requires the selection of the correct technique, depending on the specific task. Practice has shown that different processing methods can give slightly different results for the same set of fMRI data. There are a number of alternative specialized software packages for processing and analysis, but the methodology still needs improvement and development. We are working in this direction: we analyze the effectiveness of existing methods; we develop our own methods; we create software services for processing and analysis of fMRI data on the basis of the distributed modular platform “Digital Laboratory”, with the involvement of the supercomputer NRC “Kurchatov Institute”. For research we use experimental fMRI data obtained on the scanner Siemens Verio Magnetom 3T at the Kurchatov Institute. One of our tasks within the framework of this project is to improve the technology for studying large-scale functional areas of the cerebral cortex at rest. To build a hierarchical model of interaction of large-scale neural networks, a verified binding of functional areas to anatomy is required. Today, there are a number of generally accepted atlases of the functional areas of the human cerebral cortex, which, nevertheless, are constantly being finalized and refined. This article presents the results of our study of the Glasser atlas for the consistency of voxels within one region and the connectivity metrics of voxel dynamics.

A modular platform for automated cryo-FIB workflows

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

MiniCarb: A Passive, Occultation-Viewing, 6U CubeSat for Observations of CO2, CH4, and H2O

We present the final design, environmental testing, and launch history of MiniCarb, a 6U CubeSat developed through a partnership between NASA Goddard Space Flight Center and Lawrence Livermore National Laboratory. MiniCarb’s science payload, developed at Goddard, was an occultation-viewing, passive laser heterodyne radiometer for observing methane, carbon dioxide, and water vapor in Earth’s atmosphere at ~1.6 microns. MiniCarb’s satellite, developed at Livermore, implemented their CubeSat Next Generation Bus plug-and-play architecture to produce a modular platform that could be tailored to a range of science payloads. Following the launch on December 5, 2019, MiniCarb traveled to the International Space Station and was set into orbit on February 1, 2020 via Northrop Grumman’s (NG) Cygnus capsule which deployed MiniCarb with tipoff rotation of about 20 deg/sec (significantly higher than the typical rate of 3 deg/sec from prior CubeSats), from which the attitude control system was unable to recover resulting in a loss of power. In spite of this early failure, MiniCarb had many successes including rigorous environmental testing, successful deployment of its solar panels, and a successful test of the radio and communication through the Iridium network. This prior work and enticing cost (approximately $2M for the satellite and $250K for the payload) makes MiniCarb an ideal candidate for a low-cost and rapid rebuild as a single orbiter or constellation to globally observe key greenhouse gases.

Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies

Neutralizing antibodies (nAbs) that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S-protein) are promising therapeutics for COVID-19. However, natural bivalent nAbs suffer from limited potency and are vulnerable to SARS-CoV-2 variants with mutated RBDs. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The diabody-Fc-Fab format consists of a central Fc with a bivalent diabody fused to its N-terminus and two Fabs fused to its C-terminus. The diabody and Fab modules do not interfere with each other, and thus, any diabody can be combined with any Fab in a facile manner. We engineered a diabody-Fc-Fab that contained the paratopes of two distinct nAbs derived from a phage-displayed library of synthetic Abs. The tetravalent nAb was purified in high yields with methods used to produce conventional IgGs, and it exhibited favorable biophysical characteristics comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the S-protein trimer at least 100-fold more tightly than the bivalent IgGs (apparent KD <1 pM). Most importantly, the tetravalent nAb exhibited extremely high potencies in neutralization assays across a panel of pseudoviruses representing seven natural SARS-CoV-2 variants (IC50 <5 ng/mL), including several that resisted IgGs and are known to evade approved IgG drugs. Taken together, our results showed that the tetravalent diabody-Fc-Fab is a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that far exceed those of conventional bivalent IgGs.

Gigavalent display of proteins on monodisperse polyacrylamide hydrogels as a versatile modular platform for functional assays and protein engineering

The robust modularity of biological components that are assembled into complex functional systems is central to synthetic biology. Here we apply modular 'plug and play' design principles to a microscale solid phase protein display system that enables protein purification and functional assays for biotherapeutics. Specifically, we capture protein molecules from cell lysates on polyacrylamide hydrogel display beads ('PHD beads'), made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA). Anchors form covalent bonds with fusion proteins bearing cognate tag recognition (SNAP and Halo-tags) in specific, orthogonal and stable fashion. Given that these anchors are copolymerised throughout the 3D structure of the beads, proteins are also distributed across the entire bead sphere, allowing attachment of ~109 protein molecules per bead (∅ 20 μm). This mode of attachment reaches a higher density than possible on widely used surface-modified beads, and additionally mitigates surface effects that often complicate studies with proteins on beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either non-covalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher and SnpTag). Proteins can be displayed in their monomeric forms, but also reformatted as a multivalent display (using secondary capture modules that create branches) to test the contributions of avidity and multivalency towards protein function. Finally, controlled release of modules by irradiation of light is achieved by incorporating the photocleavable protein PhoCl: irradiation severs the displayed protein from the solid support, so that functional assays can be carried out in solution. As a demonstration of the utility of valency engineering, an antibody drug screen is performed, in which an anti-TRAIL-R1 scFv protein is released into solution as monomers-hexamers, showing a ~50-fold enhanced potency in the pentavalent format. The ease of protein purification on solid support, quantitative control over presentation and release of proteins and choice of valency make this experimental format a versatile, modular platform for large scale functional analysis of proteins, in bioassays of protein-protein interactions, enzymatic catalysis and bacteriolysis.

A modular in vitro flow model to analyse blood-surface interactions under physiological conditions

Newly developed materials for blood-contacting devices need to undergo hemocompatibility testing to prove compliance with clinical requirements. However, many current in vitro models disregard the influence of flow conditions and blood exchange as it occurs in vivo. Here, we present a flow model which allows testing of blood-surface interactions under more physiological conditions. This modular platform consists of a triple-pump-chip and a microchannel-chip with a customizable surface. Flow conditions can be adjusted individually within the physiological range. A performance test with whole blood confirmed the hemocompatibility of our modular platform. Hemolysis was negligible, inflammation and hemostasis parameters were comparable to those detected in a previously established quasi-static whole blood screening chamber. The steady supply of fresh blood avoids secondary effects by nonphysiological accumulation of activation products. Experiments with three subsequently tested biomaterials showed results similar to literature and our own experience. The reported results suggest that our developed flow model allows the evaluation of blood-contacting materials under physiological flow conditions. By adjusting the occurring wall shear stress, the model can be adapted for selected test conditions.

Strain-Promoted Azide-Alkyne Cycloaddition-Based PSMA-Targeting Ligands For Multimodal Intraoperative Tumor Detection of Prostate Cancer

Purpose: Strain-promoted azide-alkyne cycloaddition (SPAAC) is a straightforward and multipurpose conjugation strategy. Use of SPAAC to link different functional elements to prostate specific membrane antigen (PSMA) ligands would facilitate the development of a modular platform for PSMA-targeted imaging and therapy of prostate cancer (PCa). As a first proof-of-concept for the SPAAC chemistry platform we synthesized and characterized four dual-labeled PSMA ligands for intraoperative radiodetection and fluorescence imaging of PCa. Methods: Ligands were synthesized using solid phase chemistry and contained a chelator for 111In or 99mTc labeling. The fluorophore IRDye800CW was conjugated using SPAAC chemistry or conventional N-hydroxysuccinimide (NHS)-ester coupling. LogD values were measured and PSMA-specificity of these ligands was determined in LS174T-PSMA cells. Tumor targeting was evaluated in BALB/c nude mice with subcutaneous LS174T-PSMA and LS174T wildtype tumors using µSPECT/CT imaging, fluorescence imaging, and biodistribution studies. Results: SPAAC chemistry increased lipophilicity of the ligands (range LogD: -2.4 to -4.4). In vivo, SPAAC chemistry ligands showed high and specific accumulation in s.c. LS174T-PSMA tumors up to 24 hours after injection, enabling clear visualization using µSPECT/CT and fluorescence imaging. Overall, no significant differences between the SPAAC chemistry ligands and their NHS-based counterparts were found (2 h p.i., p > 0.05), while 111In-labeled ligands outperformed the 99mTc ligands. Conclusion: Here we demonstrate that our newly developed SPAAC-based PSMA ligands show high PSMA-specific tumor targeting. Use of click-chemistry in PSMA ligand development opens up the opportunity for fast, efficient and versatile conjugations of multiple imaging moieties and/or drugs.