An Information-Assisted Deep Reinforcement Learning Path Planning Scheme for Dynamic and Unknown Underwater Environment.

An autonomous underwater vehicle (AUV) has shown impressive potential and promising exploitation prospects in numerous marine missions. Among its various applications, the most essential prerequisite is path planning. Although considerable endeavors have been made, there are several limitations. A complete and realistic ocean simulation environment is critically needed. As most of the existing methods are based on mathematical models, they suffer from a large gap with reality. At the same time, the dynamic and unknown environment places high demands on robustness and generalization. In order to overcome these limitations, we propose an information-assisted reinforcement learning path planning scheme. First, it performs numerical modeling based on real ocean current observations to establish a complete simulation environment with the grid method, including 3-D terrain, dynamic currents, local information, and so on. Next, we propose an information compression (IC) scheme to trim the mutual information (MI) between reinforcement learning neural network layers to improve generalization. A proof based on information theory provides solid support for this. Moreover, for the dynamic characteristics of the marine environment, we elaborately design a confidence evaluator (CE), which evaluates the correlation between two adjacent frames of ocean currents to provide confidence for the action. The performance of our method has been evaluated and proven by numerical results, which demonstrate a fair sensitivity to ocean currents and high robustness and generalization to cope with the dynamic and unknown underwater environment.

Characterization of N-Terminal Asparagine Deamidation and Clipping of a Monoclonal Antibody.

This study presents a novel degradation pathway of a human immunoglobulin G (IgG) molecule featuring a light chain N-terminal asparagine. We thoroughly characterize this pathway and investigate its charge profiles using cation exchange chromatography (CEX) and capillary isoelectric focusing (cIEF). Beyond the well-documented asparagine deamidation into isoaspartic acid, aspartic acid, and succinimide intermediate, a previously unreported clipping degradation pathway is uncovered. This newly identified clipped N-terminal IgG variant exhibits a delayed elution in CEX, categorized as a "basic variant", while retaining the same main peak isoelectric point (pI) in cIEF. The influence of temperature and pH on N-terminal asparagine stability is assessed across various stressed conditions. A notable correlation between deamidation percentage and clipped products is established, suggesting a potential hydrolytic chemical reaction underlying the clipping process. Furthermore, the impact of N-terminal asparagine modifications on potency is evaluated through ELISA binding assays, revealing minimal effects on binding affinity. Sequence alignment reveals homology to a human IgG with the germline gene from Immunoglobulin Lambda Variable 6-57 (IGLV6-57), which has implications for amyloid light-chain (AL) amyloidosis. This discovery of the N-terminal clipping degradation pathway contributes to our understanding of immunoglobulin light chain misfolding and amyloid fibril deposition under physiological conditions.

CeO2/Cu2O/Cu Tandem Interfaces for Efficient Water-Gas Shift Reaction Catalysis.

Metal-oxide interfaces on Cu-based catalysts play very important roles in the low-temperature water-gas shift reaction (LT-WGSR). However, developing catalysts with abundant, active, and robust Cu-metal oxide interfaces under LT-WGSR conditions remains challenging. Herein, we report the successful development of an inverse copper-ceria catalyst (Cu@CeO2), which exhibited very high efficiency for the LT-WGSR. At a reaction temperature of 250 °C, the LT-WGSR activity of the Cu@CeO2 catalyst was about three times higher than that of a pristine Cu catalyst without CeO2. Comprehensive quasi-in situ structural characterizations indicated that the Cu@CeO2 catalyst was rich in CeO2/Cu2O/Cu tandem interfaces. Reaction kinetics studies and density functional theory (DFT) calculations revealed that the Cu+/Cu0 interfaces were the active sites for the LT-WGSR, while adjacent CeO2 nanoparticles play a key role in activating H2O and stabilizing the Cu+/Cu0 interfaces. Our study highlights the role of the CeO2/Cu2O/Cu tandem interface in regulating catalyst activity and stability, thus contributing to the development of improved Cu-based catalysts for the LT-WGSR.

Atomic-Interface Effect of Single-Atom Ru/CoOx for Selective Electrooxidation of 5-Hydroxymethylfurfural.

The development of single-atom catalysts with effective interfaces for biomass conversion is a promising but challenging research area. In this study, a Ru1/CoOx catalyst was successfully fabricated with the impregnation method, which featured Ru single atoms on a cobalt oxide substrate. The Ru1/CoOx catalyst showed superior performance in the selective electrooxidation of 5-hydroxymethylfurfural (HMF) to produce 2,5-furandicarboxylic acid (FDCA), a high value-added product. The introduction of Ru single atoms with an ultralow loading of ∼0.5 wt % was revealed to accelerate the electroredox of Co2+/Co3+/Co4+ and improve the intrinsic activity of the CoOx substrate with an FDCA selectivity of 76.5%, which is better than that of the pristine CoOx electrocatalysts (62.7%). The interfacial synergistic effect of the Ru1/CoOx interface clarified that Ru single atoms can enhance the adsorption of HMF at the Ru1/CoOx interface, which promoted the rate-determining step of the selective C-H bond activation for FDCA production. This finding provides valuable insights into the rational design of single-atom catalysts with functional interfaces for biomass upgrading.

Intermolecular Dearomative 1,2-Amination/Carbonylation via Nucleophilic Addition of Simple Amines to Arene π-Bonds.

The incorporation of the privileged amino functionality is of paramount importance in organic synthesis. In contrast to the well-developed amination methods for alkenes, the dearomative amination of arenes is largely underexplored due to the inherently inert reactivity of arene π-bonds and selectivity challenges. Herein, we report an intermolecular dearomative aminofunctionalization via direct nucleophilic addition of simple amines to chromium-bound arenes. This multicomponent 1,2-amination/carbonylation reaction enables rapid access to complicated alicyclic compounds containing amino and amide functionalities from benzene derivatives under CO-gas-free conditions, which also represents the first application of nitrogen-based nucleophiles in η6 -coordination-induced arene dearomatizations.

CuC(O) Interfaces Deliver Remarkable Selectivity and Stability for CO2 Reduction to C2+ Products at Industrial Current Density of 500 mA cm-2.

The electrocatalytic CO2 reduction reaction (CO2 RR) is an attractive technology for CO2 valorization and high-density electrical energy storage. Achieving a high selectivity to C2+ products, especially ethylene, during CO2 RR at high current densities (>500 mA cm-2 ) is a prized goal of current research, though remains technically very challenging. Herein, it is demonstrated that the surface and interfacial structures of Cu catalysts, and the solid-gas-liquid interfaces on gas-diffusion electrode (GDE) in CO2 reduction flow cells can be modulated to allow efficient CO2 RR to C2+ products. This approach uses the in situ electrochemical reduction of a CuO nanosheet/graphene oxide dots (CuOC(O)) hybrid. Owing to abundant CuOC interfaces in the CuOC(O) hybrid, the CuO nanosheets are topologically and selectively transformed into metallic Cu nanosheets exposing Cu(100) facets, Cu(110) facets, Cu[n(100) × (110)] step sites, and Cu+ /Cu0 interfaces during the electroreduction step, the faradaic efficiencie (FE) to C2+ hydrocarbons was reached as high as 77.4% (FEethylene  ≈ 60%) at 500 mA cm-2 . In situ infrared spectroscopy and DFT simulations demonstrate that abundant Cu+ species and Cu0 /Cu+ interfaces in the reduced CuOC(O) catalyst improve the adsorption and surface coverage of *CO on the Cu catalyst, thus facilitating CC coupling reactions.

Development of a novel, label-free N-glycan method using charged aerosol detection.

Monoclonal antibodies (mAbs) are complex glycoproteins that are developed for treatment of various therapeutic indications such as cancer and autoimmune diseases. MAbs are glycosylated at conserved asparagine residues (N-X-S/T) of the Fc region at amino acid position 297 of the heavy chain. Glycans are important in governing the functions of efficacy and serum half-life of protein therapeutics and are part of the critical quality attribute panel for release testing. Traditionally, N-linked glycans are released from glycoproteins after denaturation and enzymatic digestion with PNGase F, followed by fluorescent labeling of the liberated glycans. The labeled glycans are then separated using hydrophilic liquid chromatography (HILIC) with fluorescence detection to generate chromatographic profile. Despite decades of use, this strenuous process remains unchanged, utilizing toxic reagents and extended sample preparation time. As an intervention, this report showcases a novel, label-free approach to detect and quantify N-glycans without using fluorescent labeling. Separation of glycans using mixed-mode PGC column along with detection of non-derivatized glycans using charged aerosol detector, the overall turnaround time can be greatly reduced with significant cost savings. The label-free method provides similar quantitative results as the conventional fluorescent labeled method, confirming the validity of the method for product release.

N-1 Perfusion Platform Development Using a Capacitance Probe for Biomanufacturing.

Fed-batch process intensification with a significantly shorter culture duration or higher titer for monoclonal antibody (mAb) production by Chinese hamster ovary (CHO) cells can be achieved by implementing perfusion operation at the N-1 stage for biomanufacturing. N-1 perfusion seed with much higher final viable cell density (VCD) than a conventional N-1 batch seed can be used to significantly increase the inoculation VCD for the subsequent fed-batch production (referred as N stage), which results in a shorter cell growth phase, higher peak VCD, or higher titer. In this report, we incorporated a process analytical technology (PAT) tool into our N-1 perfusion platform, using an in-line capacitance probe to automatically adjust the perfusion rate based on real-time VCD measurements. The capacitance measurements correlated linearly with the offline VCD at all cell densities tested (i.e., up to 130 × 106 cells/mL). Online control of the perfusion rate via the cell-specific perfusion rate (CSPR) decreased media usage by approximately 25% when compared with a platform volume-specific perfusion rate approach and did not lead to any detrimental effects on cell growth. This PAT tool was applied to six mAbs, and a platform CSPR of 0.04 nL/cell/day was selected, which enabled rapid growth and maintenance of high viabilities for four of six cell lines. In addition, small-scale capacitance data were used in the scaling-up of N-1 perfusion processes in the pilot plant and in the GMP manufacturing suite. Implementing a platform approach based on capacitance measurements to control perfusion rates led to efficient process development of perfusion N-1 for supporting high-density CHO cell cultures for the fed-batch process intensification.

Improved Titer in Late-Stage Mammalian Cell Culture Manufacturing by Re-Cloning.

Improving productivity to reduce the cost of biologics manufacturing and ensure that therapeutics can reach more patients remains a major challenge faced by the biopharmaceutical industry. Chinese hamster ovary (CHO) cell lines are commonly prepared for biomanufacturing by single cell cloning post-transfection and recovery, followed by lead clone screening, generation of a research cell bank (RCB), cell culture process development, and manufacturing of a master cell bank (MCB) to be used in early phase clinical manufacturing. In this study, it was found that an additional round of cloning and clone selection from an established monoclonal RCB or MCB (i.e., re-cloning) significantly improved titer for multiple late phase monoclonal antibody upstream processes. Quality attributes remained comparable between the processes using the parental clones and the re-clones. For two CHO cells expressing different antibodies, the re-clone performance was successfully scaled up at 500-L or at 2000-L bioreactor scales, demonstrating for the first time that the re-clone is suitable for late phase and commercial manufacturing processes for improvement of titer while maintaining comparable product quality to the early phase process.

The Sea Route Planning for Survey Vessel Intelligently Navigating to the Survey Lines.

Marine surveying is an important part of marine environment monitoring systems. In order to improve the accuracy of marine surveying and reduce investment in artificial stations, it is necessary to use high-precision GNSS for shipborne navigation measurements. The basic measurement is based on the survey lines that are already planned by surveyors. In response to the needs of survey vessels sailing to the survey line, a method framework for the shortest route planning is proposed. Then an intelligent navigation system for survey vessels is established, which can be applied to online navigation of survey vessels. The essence of the framework is that the vessel can travel along the shortest route to the designated survey line under the limitation of its own minimum turning radius. Comparison and analysis of experiments show that the framework achieves better optimization. The experimental results show that our proposed method can enable the vessel to sail along a shorter path and reach the starting point of the survey line at the specified angle.

Mechanistic insights into inter-chain disulfide bond reduction of IgG1 and IgG4 antibodies.

Therapeutic monoclonal antibodies (mAbs), primarily immunoglobin G1 (IgG1) and IgG4 with an engineered CPPC motif in its hinge region, are predominant biologics. Inter-chain disulfide bonds of IgG mAbs are crucial to maintaining IgG integrity. Inter-chain disulfide bond-reduced low molecular weight (LMW) is considered as one of quality attributes of IgG drug substance and is observed in drug substance manufacturing. In this study, we demonstrate that IgG1 and IgG4 are susceptible to the reducing agent TCEP differently and they follow different pathways to form LMWs. Our study shows that IgG1 is more sensitive to TCEP than IgG4. Both therapeutic IgG1 and human blood plasma IgG1 follow a heavy-heavy-light chain (HHL) pathway, featured with HHL and HH as intermediate species. Human blood plasma IgG4 with a CPSC motif in its hinge region follows heavy-light chain (HL) pathway, featured with HL as the intermediate species. However, therapeutic IgG4 follows a hybrid pathway with the HL pathway as the primary and the HHL pathway as the secondary. These experimental observations are further explained using solvent accessibility of inter-chain disulfide bonds obtained from computational modeling and molecular dynamics simulations. Findings from this study provide mechanistic insights of LMW formation of IgG1 and IgG4, which suggest selection of IgG1 or IgG4 for bispecific antibodies and cysteine-based antibody-drug conjugates. KEY POINTS: • Experimentally discovered preferable disulfide bond reduction pathways between IgG1 and IgG4 antibodies, driven by the different solvent accessibilities of these disulfide bonds. • Computationally explained the solvent accessibility aided by molecular dynamics simulations. • Provided insights in developing robust biologics process and designing bispecific antibodies and cysteine-based antibody-drug conjugates.

Recent advancements in mass spectrometry for higher order structure characterization of protein therapeutics.

Molecular characterization of higher order structure (HOS) in protein therapeutics is crucial to the selection of candidate molecules, understanding of structure-function relationships, formulation development, stability assessment, and comparability studies. Recent advances in mass spectrometry (MS), including native MS, hydrogen/deuterium exchange (HDX)-MS, and fast photochemical oxidation of proteins (FPOP) coupled with MS, have provided orthogonal ways to characterize HOS of protein therapeutics. In this review, we present the utility of native MS, HDX-MS and FPOP-MS in protein therapeutics discovery and development, with a focus on epitope mapping, aggregation assessment, and comparability studies. We also discuss future trends in the application of these MS methods to HOS characterization.

A Metabolomics Approach to Increasing Chinese Hamster Ovary (CHO) Cell Productivity.

Much progress has been made in improving the viable cell density of bioreactor cultures in monoclonal antibody production from Chinese hamster ovary (CHO) cells; however, specific productivity (qP) has not been increased to the same degree. In this work, we analyzed a library of 24 antibody-expressing CHO cell clones to identify metabolites that positively associate with qP and could be used for clone selection or medium supplementation. An initial library of 12 clones, each producing one of two antibodies, was analyzed using untargeted LC-MS experiments. Metabolic model-based annotation followed by correlation analysis detected 73 metabolites that significantly correlated with growth, qP, or both. Of these, metabolites in the alanine, aspartate, and glutamate metabolism pathway, and the TCA cycle showed the strongest association with qP. To evaluate whether these metabolites could be used as indicators to identify clones with potential for high productivity, we performed targeted LC-MS experiments on a second library of 12 clones expressing a third antibody. These experiments found that aspartate and cystine were positively correlated with qP, confirming the results from untargeted analysis. To investigate whether qP correlated metabolites reflected endogenous metabolic activity beneficial for productivity, several of these metabolites were tested as medium additives during cell culture. Medium supplementation with citrate improved qP by up to 490% and more than doubled the titer. Together, these studies demonstrate the potential for using metabolomics to discover novel metabolite additives that yield higher volumetric productivity in biologics production processes.

Untargeted proteomics reveals upregulation of stress response pathways during CHO-based monoclonal antibody manufacturing process leading to disulfide bond reduction.

Monoclonal antibody (mAb) interchain disulfide bond reduction can cause a loss of function and negatively impact the therapeutic's efficacy and safety. Disulfide bond reduction has been observed at various stages during the manufacturing process, including processing of the harvested material. The factors and mechanisms driving this phenomenon are not fully understood. In this study, we examined the host cell proteome as a potential factor affecting the susceptibility of a mAb to disulfide bond reduction in the harvested cell culture fluid (HCCF). We used untargeted liquid-chromatography-mass spectrometry-based proteomics experiments in conjunction with a semi-automated protein identification workflow to systematically compare Chinese hamster ovary (CHO) cell protein abundances between bioreactor conditions that result in reduction-susceptible and reduction-free HCCF. Although the growth profiles and antibody titers of these two bioreactor conditions were indistinguishable, we observed broad differences in host cell protein (HCP) expression. We found significant differences in the abundance of glycolytic enzymes, key protein reductases, and antioxidant defense enzymes. Multivariate analysis of the proteomics data determined that upregulation of stress-inducible endoplasmic reticulum (ER) and other chaperone proteins is a discriminatory characteristic of reduction-susceptible HCP profiles. Overall, these results suggest that stress response pathways activated during bioreactor culture increase the reduction-susceptibility of HCCF. Consequently, these pathways could be valuable targets for optimizing culture conditions to improve protein quality.

Fast and high-resolution purification of a PEGylated protein using a z2 laterally-fed membrane chromatography device.

PEGylated proteins comprise a class of value-added biopharmaceuticals. High-resolution separation techniques are required for the purification of these molecules. In this study, we discuss the application of a newly developed z2 laterally-fed membrane chromatography (or z2LFMC) device for carrying out high-resolution purification of a PEGylated protein drug. The device used in the current study contained a stack of anion exchange (Q) membranes. The membrane bed-height of this z2LFMC device being small, it could be operated at very high flow rates, at relatively low back pressures. The primary goal was to speedily and efficiently separate a mono-PEGylated protein from impurities present in the PEGylation reaction mixture. A resin-based anion exchange column having the same ligand and bed-volume was used as the control device. The purification performance of the z2LFMC device and the control column were compared terms of resolution, recovery and purity. The z2LFMC device outperformed the control column in terms of every metric compared in this study. Higher purity (85.4% as opposed to 77.9%) and higher recovery (28% greater) of the target mono-PEGylated protein were obtained using the z2LFMC device at 20-time higher speed. These results clearly demonstrate that the z2LFMC device could be a faster and more efficient alternative to resin-based columns for purification of biopharmaceuticals.

Mimicking Low pH Virus Inactivation Used in Antibody Manufacturing Processes: Effect of Processing Conditions and Biophysical Properties on Antibody Aggregation and Particle Formation.

Low pH virus inactivation (VI) step is routinely used in antibody production manufacturing. In this work, a mimic of the VI step was developed to focus on evaluating adverse effects on product quality. A commercially available lab-scale glass reactor system was utilized to assess impacts of process and solution conditions on process-induced monoclonal antibody particle formation. Flow imaging was found to be more sensitive than light obscuration in detecting microparticles. NaOH as a base titrant increased protein microparticles more than Tris. Both stirring and NaCl accelerated particle formation, indicating that interfacial stress and protein colloidal stability were important factors. Polysorbate 80 was effective at suppressing particle formation induced by stirring. In contrast, trehalose led to higher microparticle levels suggesting a conformational stabilizer may have other adverse effects during titration with stirring. Additionally, conformational and colloidal stability of antibodies were characterized to investigate the potential roles of antibody physicochemical properties in microparticle formation during VI. The stability data were supportive in rationalizing particle formation behaviors, but they were not predictive of particle formation during the mimicked viral inactivation steps. Overall, the results demonstrate the value of testing various solution and processing conditions in a scaled-down system prior to larger-scale VI bioprocesses.

Online monitoring and control of upstream cell culture process using 1D and 2D-LC with SegFlow interface.

The biopharmaceutical industry is transitioning from currently deployed batch-mode bioprocessing to a highly efficient and agile next-generation bioprocessing with the adaptation of continuous bioprocessing, which reduces capital investment and operational costs. Continuous bioprocessing, aligned with FDA's quality-by-design platform, is designed to develop robust processes to deliver safe and effective drugs. With the deployment of knowledge-based operations, product quality can be built into the process to achieve desired critical quality attributes (CQAs) with reduced variability. To facilitate next-generation continuous bioprocessing, it is essential to embrace a fundamental shift-in-paradigm from "quality-by-testing" to "quality-by-design," which requires the deployment of process analytical technologies (PAT). With the adaptation of PAT, a systematic approach of process and product understanding and timely process control are feasible. Deployment of PAT tools for real-time monitoring of CQAs and feedback control is critical for continuous bioprocessing. Given the current deficiency in PAT tools to support continuous bioprocessing, we have integrated Infinity 2D-LC with a post-flow-splitter in conjunction with the SegFlow autosampler to the bioreactors. With this integrated system, we have established a platform for online measurements of titer and CQAs of monoclonal antibodies as well as amino acid analysis of bioreactor cell culture.

High-resolution purification of a therapeutic PEGylated protein using a cuboid packed-bed device.

PEGylated proteins which are a class of protein-synthetic polymer conjugates that have shown significant promise in the area of biotherapeutics are difficult to purify. A cuboid packed-bed device was used to purify a mono-PEGylated therapeutic protein from impurities such as high molecular weight (HMW) species (e.g., tri- and/or di-PEGylated forms), and low molecular weight (LMW) species such as unreacted protein and polyethylene glycol (or PEG). The separation efficiency of this device was compared with that of an equivalent cylindrical column. The effects of operating conditions such as flow rate, buffer composition, elution gradient, and column loading were systematically compared. An equivalent column with the same bed volume, same resin and same bed height was served as control. In mono-PEGylated protein purifications experiments, the cuboid packed-bed device exhibited sharper peaks and gave better resolution at all conditions examined in this study. The purity of mono-PEGylated protein in the samples collected from the cuboid packed-bed device and the column were comparable, i.e., 98.1% and 97.9% respectively. The recovery of mono-PEGylated protein in the pooled eluate from the cuboid packed-bed device was 31.7% greater than that recovered in the pooled eluate from the column. Therefore, significantly higher recovery of mono-PEGylated protein was obtained with the cuboid packed-bed device while maintaining the same purity specification as obtained with the column.

Online/at-line measurement, analysis and control of product titer and critical product quality attributes (CQAs) during process development.

Process analytical technology (PAT) has been defined by the Food and Drug Administration as a system for designing, analyzing, and controlling manufacturing through timely measurements to ensure final product quality. Based on quality-by-design (QbD) principles, real-time or near-real-time data monitoring is essential for timely control of critical quality attributes (CQAs) to keep the process in a state of control. To facilitate next-generation continuous bioprocessing, deployment of PAT tools for real-time monitoring is integral for process understanding and control. Real-time monitoring and control of CQAs are essential to keep the process within the design space and align with the guiding principles of QbD. The contents of this manuscript are pertinent to the online/at-line monitoring of upstream titer and downstream product quality with timely process control. We demonstrated that an ultra-performance liquid chromatography (UPLC) system interfaced with a UPLC-process sample manager (UPLC-PSM) can be utilized to measure titer and CQAs directly from bioreactors and downstream unit operations, respectively. We established online titer measurements from fed-batch and perfusion-based alternating tangential flow bioreactors as well as product quality assessments of downstream operations for real-time peak collection. This integrated, fully automated system for online data monitoring with feedback control is designed to achieve desired product quality.

A novel approach enables imaged capillary isoelectric focusing analysis of PEGylated proteins.

PEGylation has been used as a strategy to enhance pharmacokinetic properties of therapeutic proteins by pharmaceutical industry. Imaged CIEF (iCIEF) is the current industry standard technology for pI determination and charge variant quantification of proteins and antibodies. However, the charge variants of PEGylated proteins merge into one broad peak during iCIEF, most likely due to masking of proteins by the surrounding PEG chain as well as the increased hydrodynamic volume due to PEGylation. Here, we report our novel matrix formula with a combination of glycine and taurine that significantly improved the separation of charge variants in PEGylated proteins. As a result, it is no longer necessary to conduct IEF of proteins prior to PEGylation, which does not reflect the changes caused by PEGylation and purification processes. The novel matrix (glycine and taurine) enables iCIEF analysis of PEGylated proteins in their real conjugated states.