Uric acid (UA) is an antioxidant that has been reported to be a neuroprotective compound for injuries and diseases, and specifically, diseases of the central nervous system. However, uric acid is highly insoluble in aqueous solutions, and high levels in the serum lead to gout, which limits its use in humans. Here, we develop a novel drug delivery platform that will release uric acid in a sustained manner for application to neural tissue. We demonstrate that one-step incorporation of UA into an electrospun gelatin/hyaluronic acid nanofiber mat results in controlled release of UA in culture medium. Taking a unique approach, we made solutions of 12% gelatin and 1% hyaluronic acid in a formic acid solvent and added UA for production of nanofiber mats. We then dehydrothermally crosslinked the mats and tested for release of UA into physiological cell culture medium. To test whether the mats have any detrimental effects on healthy nervous system tissue, we cultured spinal cord explants on the mats extended and assessed extensions from the explants. We observed that comparable numbers and lengths of dendrites are extended from the spinal cord tissue, regardless of the amount UA content in the mats. Our results suggest that electrospun gelatin/hyaluronic acid nanofibers can be used as a platform for sustained uric acid delivery to neural tissue without detrimental effects.

Microbial decontamination is a critical concern in various sectors, from healthcare to food processing. Traditional decontamination methods, while effective to a degree, present limitations in terms of environmental impact, efficiency, and potential harm to the target material. This review investigates the emerging realm of non-thermal plasma (NTP) as a promising alternative for microbial decontamination, emphasizing its mechanisms, reactor designs and applications. The mechanism decomposed into physical, chemical and biological effects of plasma, are elaborated upon to provide a foundational understanding of the intrinsic principles of plasma decontamination. Except for the generation type of NTP, reactors and other parameters by which NTP achieves microbial decontamination, emphasizing the design considerations and parameters that influence its efficacy. Moreover, the latest applications of NTP in decontaminating air, water, and surfaces, supported by the latest research findings in each domain are explored. Additionally, the perspectives on the future research tendencies of NTP decontamination and disinfection are highlighted with potential avenues for exploration and innovation. Through this comprehensive review, the aim is to underscore the potential of NTP, particularly DBD plasma, as a versatile, efficient, and environmentally friendly method for microbial decontamination.

Tryptamines play diverse roles as neurotransmitters and psychoactive compounds found in various organisms. Psilocybin, a notable tryptamine, has garnered attention for its therapeutic potential in treating mental health disorders like depression and anxiety. Despite its promising applications, current extraction methods for psilocybin are labor-intensive and economically limiting. We suggest biocatalysis as a sustainable alternative, leveraging enzymes to synthesize psilocybin and other tryptamines efficiently. By elucidating psilocybin biosynthesis pathways, researchers aim to advance synthetic methodologies and industrial applications. This review underscores the transformative potential of biocatalysis in enhancing our understanding of tryptamine biosynthesis and facilitating the production of high-purity psilocybin and other tryptamines for therapeutic and research use.

Bispecific antibodies (bsAbs) can simultaneously bind two different antigens or epitopes. Their dual-targeting capability enables novel mechanisms of action, gaining therapeutic advantages over conventional monospecific mAbs. In recent years, the number of bsAbs grows rapidly and bsAbs under development are available in diverse formats. In particular, Fc-containing IgG-like bsAbs, which represent the major group, can be constructed in asymmetric or symmetric format. For asymmetric ones, whose assembly requires multiple distinct chains, although numerous strategies have been developed to promote desired chain pairing, product-related variants such as free chains, half molecules and mispaired species are usually present at various levels. For symmetric ones, increased level of aggregates and truncating variants is often associated with their production. In general, bsAbs pose greater challenges to the downstream team than regular mAbs. In the past few years, our team successfully developed the downstream process for over 70 bsAbs in greater than 30 different formats and accumulated substantial experience. This review introduces general strategies that we have used while purifying these challenging molecules.

The challenging task of designing biopharmaceutical downstream processes is initially to select the type of unit operations, followed by optimizing their operating conditions. For complex flowsheet optimizations, the strategy becomes crucial in terms of duration and outcome. In this study, we compared three optimization strategies, namely, simultaneous, top-to-bottom, and superstructure decomposition. Moreover, all strategies were evaluated by either using chromatographic Mechanistic Models (MMs) or Artificial Neural Networks (ANNs). An overall evaluation of 39 flowsheets was performed, including a buffer-exchange step between the chromatography operations. All strategies identified orthogonal structures to be optimal, and the weighted overall performance values were generally consistent between the MMs and ANNs. In terms of time-efficiency, the decomposition method with MMs stands out when utilizing multiple cores on a multiprocessing system for simulations. This study analyses the influence of different optimization strategies on flowsheet optimization and advices on suitable strategies and modeling techniques for specific scenarios.

This study aimed to propose a methodology for developing a mechanistic model for viral clearance of the minute virus of mice (MVM) on flow-through anion exchange (AEX) chromatography. Protein surface analysis was applied to investigate the possibility of molecular interaction between the recombinant biotherapeutic and MVM. The protein product-free Tris buffers were spiked with MVM, and the MVM elution profile from AEX chromatography was quantitatively analyzed using quantitative polymerase chain reaction (qPCR) for pooled fractions. GoSilico™ Chromatography Modeling Software was applied to develop the mechanistic models for MVM species. For evaluating the visual fit of the developed model, the R of intact MVM virions and uncoated capsids between the simulated and measured amount in each fraction are 0.880 and 0.948, respectively. Response surface plots of logarithmic reduction values (LRV) against pH and conductivity in loaded sample were generated to show the range for suitable loaded sample conditions for achieving a good LRV. To evaluate the applicability of the developed MVM elution model to a recombinant biotherapeutic, two demonstrations of AEX chromatography purification were performed with a loaded sample of a model monoclonal antibody. The peaks of the MVM species in the elution step of both runs were accurately simulated by the developed model. In addition, to assess the possibility of molecular interaction between the virus and the target protein significantly affecting the MVM elution behavior, the antibody's surface was evaluated in terms of hydrophobicity/hydrophilicity using surface analysis.

Off-the-shelf cell therapies hold significant curative potential for conditions, such as Parkinson's disease and heart failure. However, these therapies face unique cryopreservation challenges, especially when novel routes of administration, such as intracerebral or epicardial injection, require cryopreservation media that are safe for direct post-thaw administration. Current practices often involve post-thaw washing to remove dimethyl sulfoxide (MeSO), a cytotoxic cryoprotective agent, which complicates the development and clinical translation of off-the-shelf therapies. To overcome these obstacles, there is a critical need to explore MeSO-free cryopreservation methods. While such methods typically yield suboptimal post-thaw viability with conventional slow-freeze protocols, optimizing freezing profiles offers a promising strategy to enhance their performance. This comprehensive review examines the latest advancements in cryopreservation techniques across various cell therapy platforms, with a specific case study of iPSC-derived therapies used to illustrate the scalability challenges. By identifying key thermodynamic and biochemical phenomena that occur during freezing, this review aims to identify cell-type independent approaches to improve the efficiency and efficacy of cryopreservation strategies, thereby supporting the widespread adoption and clinical success of off-the-shelf cell therapies.

Alkanes are high-energy hydrocarbons that are foreseen as next generation biofuels. Cyanobacteria are known to naturally synthesize C15-C19 alkanes; however, the titers are too low to make this a commercially viable process. Therefore, to leverage these photosynthetic platforms for improved alkane production, here we engineered three novel isolates of Synechococcus elongatus PCC 11801, PCC 11802, and IITB6. The two gene AAR-ADO alkane biosynthesis pathway was constructed by cloning the genes for acyl-ACP reductase (aar) and aldehyde deformylating oxygenase (ado) from S. elongatus PCC 7942 under the regulation of P promoter from PCC 7942 and native promoters from PCC 11801 such as P, P, and P. The genes were separately cloned under two different promoters, creating a library of the engineered strains. The results indicated that the engineered strains of novel S. elongatus isolates produced significantly higher amounts of alkanes than the model strain PCC 7942. The highest alkane yield achieved was 4.1 mg/gDCW in BG-11, while the highest titer was 31.5 mg/L in 5X BG-11, with an engineered IITB6 strain (P:aar:T::P:ado:T). Overall, the study highlights the potential of newly isolated S. elongatus strains as efficient alkane production platforms.

Membrane chromatography devices are a viable alternative to packed-bed resins and enable highly productive purification cascades for monoclonal antibodies and Fc-fusion proteins. In this study, ion exchange and protein A membrane chromatography performances were assessed and compared with their resin counterparts. Protein A dynamic binding capacities were higher than 50 g/L for two of the tested membranes and with a residence time of 0.2 min. For polishing, it was observed that aggregate clearance was generally less performant with membrane separation when compared to resins with similar ligands. However, the comparable yield and increased productivity of membranes could be enough to consider their implementation. In addition, lifetime studies demonstrated that the performance of membranes remained robust over cycles. One hundred cycles were reached for most of the tested membranes with no impact on the process performance nor product quality. Finally, purification cascades were fully operated with membranes, from capture to polishing, reaching good levels of host cells proteins (less than 50 ppm) and aggregates (equal to or less than 1%). The outcome of this study demonstrated that resin chromatography could be fully replaced by membranes for monoclonal antibody and Fc-fusion protein purification processes.

The bio-pharmaceutical industry heavily relies on mammalian cells for the production of bio-therapeutic proteins. The complexity of implementing and high cost-of-goods of these processes are currently limiting more widespread patient access. This is driving efforts to enhance cell culture productivity and cost reduction. Upstream process intensification (PI), using perfusion approaches in the seed train and/or the main bioreactor, has shown substantial promise to enhance productivity. However, developing optimal process conditions for perfusion-based processes remain challenging due to resource and time constraints. Model-based optimization offers a solution by systematically screening process parameters like temperature, pH, and culture media to find the optimum conditions in silico. To our knowledge, this is the first experimentally validated model to explain the perfusion dynamics under different operating conditions and scales for process optimization. The hybrid model accurately describes Chinese hamster ovary (CHO) cell culture growth dynamics and a neural network model explains the production of mAb, allowing for optimization of media exchange rates. Results from six perfusion runs in Ambr® 250 demonstrated high accuracy, confirming the model's utility. Further, the implementation of dynamic media exchange rate schedule determined through model-based optimization resulted in 50% increase in volumetric productivity. Additionally, two 5 L-scale experiments validated the model's reliable extrapolation capabilities to large bioreactors. This approach could reduce the number of wet lab experiments needed for culture process optimization, offering a promising avenue for improving productivity, cost-of-goods in bio-pharmaceutical manufacturing, in turn improving patient access to pivotal medicine.

Healthcare-associated infections (HAIs) pose significant challenges to global health due to pathogen complexity and antimicrobial resistance. Biosensors utilizing antimicrobial peptides offer innovative solutions. Hylarana picturata Multiple Active Peptide 1 (Hp-MAP1), derived from Temporin-PTA, exhibits antibacterial properties sourced from the skin secretions of the Malaysian fire-bellied frog. An innovative sensing layer was developed for the electrochemical biorecognition of diverse pathogens: Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Staphylococcus aureus. Electrochemical impedance spectroscopy differentiated microorganisms based on distinct electrochemical responses. The sensor layer, composed of functionalized multi-walled carbon nanotubes (MWCNTs) associated with Hp-MAP1, exhibited varying levels of charge transfer resistance (R) for different microorganisms. Gram-negative species, especially P. aeruginosa, displayed higher R values, indicating better impedimetric responses. Excellent LODs were observed for P. aeruginosa (0.60), K. pneumoniae (0.42), E. coli (0.67), and S. aureus (0.59), highlighting the efficacy of the MWCNTs/Hp-MAP1 biosensor in microbial identification. The MWCNTs/Hp-MAP1 biosensor platform presents a promising and effective microbial identification strategy with potential healthcare applications to mitigate HAIs and enhance patient care.

Technology scale-up and transfer are a fundamental and critical part of process development in biomanufacturing. Important bioreactor hydrodynamic characteristics such as working volume, overhead gas flow rate, volumetric power input (P/V), impeller type, agitation regimen, sparging aeration strategy, sparger type, and ka must be selected based on key performance indicators (KPI) to ensure a smooth and seamless process scale-up and transfer. Finding suitable operational setpoints and developing an efficient feeding regimen to ensure process efficacy and consistency are instrumental. In this investigation, process development of a cumate inducible Chinese hamster ovary (CHO) stable pool expressing trimeric SARS-CoV-2 spike protein in 1.8 L benchtop stirred-tank bioreactors is detailed. Various dissolved oxygen levels and aeration air caps were studied to determine their impact on cell growth and metabolism, culture longevity, and endpoint product titers. Once hydrodynamic conditions were tuned to an optimal zone, various feeding strategies were explored to increase culture performance. Dynamic feedings such as feeding based on current culture volume, viable cell density (VCD), oxygen uptake rate (OUR), and bio-capacitance signals were tested and compared to standard bolus addition. Increases in integral of viable cell concentration (IVCC) (1.25-fold) and protein yield (2.52-fold), as well as greater culture longevity (extension of 5 days) were observed in dynamic feeding strategies when compared to periodic bolus feeding. Our study emphasizes the benefits of designing feeding strategies around metabolically relevant signals such as OUR and bio-capacitance signals.

Optimizing a biopharmaceutical chromatographic purification process is currently the greatest challenge during process development. A lack of process understanding calls for extensive experimental efforts in pursuit of an optimal process. In silico techniques, such as mechanistic or data driven modeling, enhance the understanding, allowing more cost-effective and time efficient process optimization. This work presents a modeling strategy integrating quantitative structure property relationship (QSPR) models and chromatographic mechanistic models (MM) to optimize a cation exchange (CEX) capture step, limiting experiments. In QSPR, structural characteristics obtained from the protein structure are used to describe physicochemical behavior. This QSPR information can be applied in MM to predict the chromatogram and optimize the entire process. To validate this approach, retention profiles of six proteins were determined experimentally from mixtures, at different pH (3.5, 4.3, 5.0, and 7.0). Four proteins at different pH's were used to train QSPR models predicting the retention volumes and characteristic charge, subsequently the equilibrium constant was determined. For an unseen protein knowing only the protein structure, the retention peak difference between the modeled and experimental peaks was 0.2% relative to the gradient length (60 column volume). Next, the CEX capture step was optimized, demonstrating a consistent result in both the experimental and QSPR-based methods. The impact of model parameter confidence on the final optimization revealed two viable process conditions, one of which is similar to the optimization achieved using experimentally obtained parameters. The multiscale modeling approach reduces the required experimental effort by identification of initial process conditions, which can be optimized.

The immobilization of free enzymes is crucial for enhancing their stability in different environments, enabling reusability, and expanding their applications. However, the development of a straightforward immobilization method that offers stability, high efficiency, biocompatibility, and modifiability remains a significant challenge. Silk fibroin (SF) is a good carrier for immobilized enzymes and drugs. Here, we employed urease as a model enzyme and utilized our developed technology called unidirectional nanopore dehydration (UND) to efficiently dehydrate a regenerated SF solution containing urease in a single step, resulting in the preparation of a highly functionalized SF membrane immobilizing urease (UI-SFM). The preparation process of UI-SFM is based on an all-water system, which is mild, green and able to efficiently and stably immobilize urease in the membranes, maintaining 92.7% and 82.8% relative enzyme activity after 30 days of storage in dry and hydrated states, respectively. Additionally, we performed additional post-treatments, including stretching and cross-linking with polyethylene glycol diglycidyl ether (PEGDE), to obtain two more robust immobilized urease membranes (UI-SFMs and UI-SFMc). The thermal and storage stability of these two membranes were significantly improved, and the recovery ratio of enzyme activity reached more than 90%. After 10 repetitions of the enzymatic reaction, the activity recovery of UI-SFMs and UI-SFMc remained at 92% and 88%, respectively. The results suggest that both UND-based and post-treatment-developed membranes exhibit excellent urease immobilization capabilities. Furthermore, the enzyme immobilization method offers a straightforward and versatile approach for efficient and stable enzyme immobilization, while its flexible modifiability caters to diverse application requirements.

Vesicular stomatitis virus (VSV) has been increasingly demonstrated as a promising viral vector platform. As the interest over this modality for vaccine and gene therapy applications increases, the need for intensified processes to produce these vectors emerge. In this study, we develop fed-batch-based operations to intensify the production of a recombinant VSV-based vaccine candidate (rVSV-SARS-CoV-2) in suspension cultures of HEK293 cells. A feeding strategy, in which a commercial concentrated medium was added to cultures based on cell growth through a fixed cell specific feeding rate (CSFR), was applied for the development of two different processes using Ambr250 modular bioreactors. Cultures operated in hybrid fed-batch/perfusion (FB/P) or fed-batch (FB) were able to sustain infections performed at 8.0 × 10 cells/mL, respectively resulting in 3.9 and 5.0-fold increase in total yield (Y) and 1.7 and 5.6-fold increase in volumetric productivity (VP) when compared with a batch reference. A maximum viral titer of 4.5 × 10 TCID/mL was reached, which is comparable or higher than other processes for VSV production in different cell lines. Overall, our study reports efficient fed-batch options to intensify the production of a rVSV-based vaccine candidate in suspension HEK293 cells.

It is important to increase manufacturing speed to make medicines more widely available. One bottleneck for CHO-based drug substance release is the in vitro viral (IVV) cell-based assay on unprocessed bulk. To increase process speed, we evaluate the suitability of replacing the IVV cell-based assay with next-generation sequencing (NGS). First, we outline how NGS is currently used in the pharmaceutical industry, and how it may apply to CHO virus testing. Second, we examine CHO virus contamination history. Since prior virus contaminants can replicate in the production bioreactor, we perform a literature search and classify 159 viruses as high, medium, low, or unknown risk based on their ability to infect CHO cells. Overall, the risk of virus contamination during the CHO manufacturing process is low. Only six viruses were reported to have contaminated CHO bioprocesses over the past several decades, and were primarily caused by fetal bovine serum or cell culture components. These virus contamination events can be mitigated through limitation and control of raw materials, combined with virus testing and virus clearance technologies. The list of CHO infectious viruses provides a starting framework for virus safety risk assessment and NGS development. Furthermore, ICH Q5A (R2) includes NGS as a molecular method for adventitious agent testing, paving a path forward for modernizing CHO virus testing.

Bacteriocins are ribosomally synthesized peptides with the innate ability to kill or inhibit growth of other bacteria. In recent years, bacteriocins have received increased interest, as their antimicrobial activity enhances food safety and shelf life by combatting pathogens such as Listeria monocytogenes. They also have application potential as an active pharmaceutical compound to combat multidrug-resistant pathogens. As new bacteriocins continue to be discovered, accelerated workflows for screening, identification, and process development have been developed. However, antimicrobial activity measurement is often still limited with regards to quantification and throughput. Here, we present the use of a non-linear calibration model to infer nisin concentrations in cultivation supernatants of Lactococcus lactis ssp. lactis B1629 using readouts of pHluorin2 fluorescence-based antimicrobial activity assays.

Short-chain esters, particularly isobutyl acetate and isoamyl acetate, hold significant industrial value due to their wide-ranging applications in flavors, fragrances, solvents, and biofuels. In this study, we demonstrated the biosynthesis of acetate esters using Yarrowia lipolytica as a host by feeding alcohols to the yeast culture. Initially, we screened for optimal alcohol acyltransferases for ester biosynthesis in Y. lipolytica. Strains of Y. lipolytica expressing atf1 from Saccharomyces cerevisiae, produced 251 or 613 mg/L of isobutyl acetate or of isoamyl acetate, respectively. We found that introducing additional copies of ATF1 enhanced ester production. Furthermore, by increasing the supply of acetyl-CoA and refining the culture conditions, we achieved high production of isoamyl acetate, reaching titers of 3404 mg/L. We expanded our study to include the synthesis of a range of acetate esters, facilitated by enriching the culture medium with various alcohols. This study underscores the versatility and potential of Y. lipolytica in the industrial production of acetate esters.

A reduction in the cost of production and energy requirement is necessary for developing sustainable commercial bioprocesses. Bypassing sterilization, which is an energy and cost-intensive part of bioprocesses could be a way to achieve this. In this study, nonsterile cultivation of Yarrowia lipolytica was done on a synthetic medium containing acetic acid as the sole carbon source using two different strategies in the fed-batch mode. The contamination percentages throughout the process were measured using flow cytometry and complemented using brightfield microscopy. Maximum biomass and lipid yields of 0.57 (g biomass/g substrate) and 0.17 (g lipids/g substrate), respectively, and maximum biomass and lipid productivities of 0.085 and 0.023 g/L/h, respectively, were obtained in different fed-batch strategies. Feeding at the point of stationary phase resulted in better biomass yield and productivity with less than 2% contamination till 48 h. Feeding to maintain a minimum acetic level resulted in better lipid yield and productivity with less than 2% contamination during the complete process. The results of this study demonstrate the potential for cultivating Y. lipolytica in nonsterile conditions and monitoring the contamination throughout the process using flow cytometry.

Human serum albumin (HSA) is currently used as a plasma expander (PE) to increase blood volume during hypovolemic conditions, such as blood loss. However, its effectiveness is suboptimal in septic shock and burn patients due to their enhanced endothelial permeability, resulting in HSA extravasation into the tissue space leading to edema, and deposition of toxic HSA-bound metabolites. Hence, to expand HSA's applicability toward treating patients with compromised endothelial permeability, HSA has been previously polymerized to increase its molecular size thus compartmentalizing the polymerized HSA (PolyHSA) molecules in the vascular space. Previous studies bracketed PolyHSA between 100 kDa and 0.2 μm. In this research, PolyHSA was synthesized at two cross-link densities 43:1 and 60:1 (i.e., molar ratios of glutaraldehyde to HSA) and subsequently fractionated via tangential flow filtration (TFF) into two narrower brackets: bracket A (500 kDa and 0.2 μm) and bracket B (50-500 kDa). PolyHSA within the same size bracket at different cross-link densities exhibited similar solution viscosity, zeta potential, and osmolality but differed in hydrodynamic diameter. At the same cross-link density, the PolyHSA A bracket showed higher viscosity, lowered zeta potential, and a larger hydrodynamic diameter compared with the PolyHSA B bracket while maintaining osmolality. Interestingly, PolyHSA 43:1 B, PolyHSA 60:1 A, and PolyHSA 60:1 B brackets exhibited colloid osmotic pressure similar to HSA, indicating their potential to serve as PEs.

The biodistribution of many therapeutics is controlled by the immune system. In addition, some molecules are cytotoxic when not encapsulated inside of larger cellular structures, such as hemoglobin (Hb) encapsulation inside of red blood cells (RBCs). To counter immune system recognition and cytotoxicity, drug delivery systems based on red blood cell membrane fragments (RBCMFs) have been proposed as a strategy for creating immunoprivileged therapeutics. However, the use of RBCMFs for drug delivery applications requires purification of RBCMFs at large scale from lysed RBCs free of their intracellular components. In this study, we were able to successfully use tangential flow filtration (TFF) to remove >99% of cell-free Hb from lysed RBCs at high concentrations (30%-40% v/v), producing RBCMFs that were 2.68 ± 0.17 μm in diameter. We were also able to characterize the RBCMFs more thoroughly than prior work, including measurement of particle zeta potential, along with individual TFF diacycle data on the cell-free Hb concentration in solution and time per diacycle, as well as concentration and size of the RBCMFs. In addition to purifying RBCMFs from lysed RBCs, we utilized a hypertonic solution to reseal purified RBCMFs encapsulating a model protein (Hb) to yield resealed Hb-encapsulated RBC ghosts (Hb-RBCGs). TFF was then compared against centrifugation as an alternative method for removing unencapsulated Hb from Hb-RBCGs, and the effects that each washing method on the resulting Hb-RBCG biophysical properties was assessed.