Biotechnology Progress最新文献

Schistosoma mansoni infection and other neglected diseases pose significant challenges in diagnosis and treatment, particularly in resource-constrained regions. Despite being useful, traditional techniques lack sensitivity, offering frequent false-positive results, highlighting the emergence of innovative tools such as genosensors as a promising solution to this dilemma. In this work, we developed a simple electrochemical biosensor platform based on electropolymerized films of polythiophene acetic acid (PTAA) and a specific DNA probe for the detection of S. mansoni. Electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and atomic force microscopy (AFM) were used to assess the assembly process of the genosensor, as well as to evaluate biodetection assays. The developed biosensor was found to be effective in detecting the target analyte in pure and complex samples such as cerebrospinal fluid, urine, and plasma from infected patients at different concentrations. CV and EIS were extremely useful in the evaluation of the detection process based on the electron kinetics and charge transfer resistance (R_CT) in the interface of the biosensor, where the hybridization with the target single-stranded S. mansoni DNA resulted in the variation of these parameters. The genosensor exhibited high sensitivity and selectivity, with a limit of detection of 0.451 pg.μL⁻¹. As genosensors continue to evolve, they promise to revolutionize the field of neglected disease management, providing hope for improved healthcare outcomes worldwide.

Fed-batch recombinant therapeutic protein (RTP) production processes utilizing Chinese Hamster Ovary (CHO) cells can take a long period of time (>10 days). Within this period, not all critical features may be measured routinely, and in fact, some are only measured once the process is terminated, complicating decision making. As a consequence, utilizing routine current day bioreactor online data to aid in next day predictions is a promising strategy for model predictive control-based feeding strategies. The article details the development of a proposed soft sensor that merges current day bioreactor online data and offline historical sampling data to generate predictions about the next day of the production process. This approach demonstrated the ability to track product titer, cell growth, key metabolites, and cumulative glucose consumption across the 17-day process with low normalized root mean squared error (nRMSE = 0.24) and low normalized mean absolute error (nMAE = 0.18) as well as high linearity with respect to ground data (average R² = 0.97). It was also demonstrated that the same model architecture could effectively soft sense product titer and metabolic profiles (glucose, lactate, ammonia) without having sampling day's offline data as inputs to the model. This suggests that the proposed model could act as a true soft sensor of hard-to-determine variables such as the trimeric SARS-CoV-2 spike protein that relies on end-of-process measurements to acquire the data (labor-intensive semi-quantitative SDS-PAGE gels or ELISA assay). Instantaneous specific glucose consumption rates were also predicted and showed good agreement with experimental measurements, further offering opportunities for online glucose control.

Long-term functional hepatocyte and reproducible cultures are required in pharmaceutical industries to model chronic liver disorders and to perform associated drug testing. In this frame, we have investigated the behavior of the HepaSH cells when cultivated in liver Biochips, in 3D Spheroids, and in Petri for 20 days. HepaSH is a newly developed humanized hepatocyte harvested from chimeric mice. After the cells' harvesting and inoculation, the HepaSH were successfully maintained in cultures in Petri dishes, spheroids, and Biochips for 20 days. The immunostaining confirmed the expressions of albumin, CYP1A2, and CYP3A4 in all conditions. Furthermore, the CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities were successfully detected in all conditions after 20 days of cultures. Continuous production of albumin and biliary acids was detected in Biochips, Spheroids, and Petri, among which Biochip culture showed the highest albumin secretion level. The RNA sequencing analysis revealed that Biochips and Spheroids cultures enriched hepatic maturation, xenobiotic, lipid, small molecule, steroid, and alcohol metabolisms compared to Petri cultures. Overall, our data demonstrated the feasibility of cultivating the HepaSH cells in Petri, Biochips, and Spheroids for 20 days in the presented protocol, while keeping important liver functions. Biochip and Spheroids cultures show advantages in hepatic maturation, drug metabolism-related gene expression, and albumin secretion (in biochips) compared with conventional Petri culture.

The removal of host cell proteins (HCPs) is crucial in biopharmaceutical production, as residual impurities can impact product safety and efficacy. While a number of studies have demonstrated that depth filtration can provide significant HCP removal, there is little information on its effectiveness in removing specific HCPs. This study examines the application of liquid chromatography-mass spectrometry (LC-MS) to track HCP removal during depth filtration, providing a detailed analysis of HCP behavior with two commercial depth filters. Our findings reveal significant variability in HCP breakthrough behavior, with transmission patterns showing minimal correlation with either the protein isoelectric point or hydrophobicity, highlighting the unique behavior of individual HCPs. Both the X0SP and X0HC depth filters achieved almost complete removal of Lipoprotein Lipase, and the X0SP filter also effectively removed Lysosomal Acid Lipase (LAL), both known to degrade polysorbate in monoclonal antibody formulations. However, neither filter provided significant removal of Alpha-enolase, Carboxypeptidase D, Glutathione S-transferase, or Phospholipase B-like 2. The X0SP filter showed equal or better removal for 18 out of 20 problematic HCPs, with greater HCP removal seen at lower conductivity. This work provides a detailed framework for understanding and optimizing depth filtration processes, offering insights into the effectiveness of depth filters for removal of problematic HCPs.

Artemisinin is a sesquiterpene lactone extracted from the medicinal plant Artemisia annua L. (sweet wormwood). It has traditionally been utilized in artemisinin-based combination therapies (ACTs) for the malarial parasite, including drug-resistant strains. Natural artemisinin extraction is costly with low yields. Due to its effectiveness, there is a significant rise in the demand for artemisinin production. In vitro cell suspension culture offers a cost-effective and viable technique for artemisinin production. Therefore, this study aimed to optimize a protocol for cell suspension culture of A. annua L. to enhance biomass and artemisinin production. A successful cell suspension culture was initiated from induced callus. The highest cell biomass was obtained in suspension cultures grown with an initial inoculum size of 0.1 g of mixed type cell aggregates, in media with a pH of 6.2 and a rotation speed of 90 rpm. Macronutrient concentrations influenced both biomass and artemisinin content, with optimal biomass achieved at 19 mM KNO₃ and 1.56 mM KH₂PO₄. The absence of these nutrients resulted in the highest artemisinin levels. Different LED wavelengths also significantly influenced biomass and artemisinin production. Red + blue LED increased cell biomass, while the highest artemisinin content was observed under red LED. The upscaling of the culture indicated a variation in biomass yield pattern, but the highest growth index was achieved in the 500 mL Erlenmeyer flask. This study successfully established a cell suspension culture for A. annua L., demonstrating the influence of macronutrients and red LED on biomass and artemisinin production, providing insights for potential large-scale production.

For bioprocesses producing live virus, such as enterovirus Coxsackievirus A21, viral titer (infectivity basis) decay rates can exceed 30% within a day. Consequently, harvest timing is paramount. To optimize titer at harvest, a continuous viral product titer model was generated to elucidate kinetics. The model leveraged experimentally determined viable cell density, cell-specific viral productivity, and viral specific decay rates. Next, three separate online process analytical technology (PAT) harvest triggers were developed to predict maximal viral titer. Finally, the PAT harvest triggers were tested alongside traditional time-based harvests. The harvest triggers utilized common bioprocessing tools – dissolved oxygen (DO) and capacitance probes – to track DO and viable cell volume (VCV) and derived a third parameter, cell-specific oxygen uptake rate. Harvesting with PAT triggers allowed for significantly improved batch-to-batch consistency. The standard deviation of harvest yield was reduced by 41% (DO), 56% (OUR) and 71% (capacitance) as compared to the industry standard time-based harvest. Even when a process deviation in inoculated cell density occurred, causing a significant shift in viral titer kinetics, the PAT harvest triggers yielded greater than 87% of peak titer. By comparison, the time-based harvest yielded 16%.

Recombinant adeno-associated virus (rAAV) is a widely used delivery vehicle in gene therapy. A scalable production technology is essential for its wide clinical applications. We have taken a synthetic biology approach to generate HEK293-based cell lines which harbor integrated genetic elements encoding essential AAV and adenoviral helper components and can be induced to produce rAAV. Through cycles of cell line enhancement, a high rAAV productivity could be achieved. The cell lines, like their parental HEK293, grew adherently. For scalable production, cell cultivation in suspension is highly desirable. A producer cell line GX6B was adapted to suspension growth in serum-free medium (named GX6Bs). However, it had substantially reduced virus titer. Returning GX6Bs cells to adherent culture conditions using adherent medium and cultured stationarily brought the productivity back to close to the level of adherent GX6B. A survey of the transcriptome revealed that induction and rAAV production elicited a wide range of cellular changes in various functional classes, including host immune defense response and nucleosome organization. The response was more subdued in suspension-growing GX6Bs. Upon reverting to adherent growth, the cellular transcriptome change regained its vigor to be more similar to that seen in GX6B. The GX6Bs maintained in suspension serum-free conditions were then reverted to the adherent culture medium but under an agitated culture environment to keep suspension growth for rAAV production. The productivity returned to within 25%–50% of GX6B. This work demonstrated the feasibility of the suspension culture of synthetic cell lines for the expansion and production of rAAV.

In recent years, accelerating Chemistry, Manufacturing, and Controls (CMC) workflows for clinical entry has become a critical focus in biologics development. Advances in the development of cell lines, cell culture processes, and analytical technologies have enabled the generation of more homogeneous stable pool populations with increased productivity. Leveraging the experience gained from the COVID-19 product development, the strategic use of stable cell pools or a pool of clones for early-stage non-GMP material generation and process development has proven transformative in significantly reducing the CMC timeline to investigational new drug (IND). This study provides a comprehensive comparison of bioprocess performance and product quality attributes of materials produced from stable pools or a pool of clones (toxicology study materials) versus those from clonally derived cells (GMP clinical batches) across six First-in-Human (FIH) programs involving mAbs, bsAb, and Fc-fusion proteins. The results demonstrate a strong alignment and the feasibility of using protein materials from stable pools or a pool of clones in toxicology studies. In conclusion, utilizing non-clonal CHO cell-derived material for preclinical studies offers a strategic approach that can be broadly applied to complex molecules across various disease areas, even under standard regulatory filings, accelerating the path to clinical trials.

Antioxidant supplementation to serum-free culture media is a common strategy to enhance productivity through oxidative stress alleviation. In this study, it was hypothesized that certain antioxidants can improve the specific productivity of a CHO-GS cell line expressing a bi-specific antibody. A fed-batch (FB) screening study investigated several antioxidants and revealed rosmarinic acid (RoA) and retinyl acetate (RAc), to a lesser extent, improved cell productivity. Contrary to the previous literature reports, the addition of RoA and/or RAc resulted in slower cell growth and reduced peak viable cell density, counteracting the enhanced specific productivity. We hypothesized that supplementing RoA/RAc after the exponential growth phase would increase titer through enhanced specific productivity without substantially impeding cell growth. This hypothesis was tested in three different ways: (1) supplementing RoA/RAc to the feed, rather than the basal media, in the FB process; (2) implementing the intensified fed-batch (iFB) process mode which started with high seeding VCD, bypassing the exponential cell growth phase; (3) supplementing RoA/RAc to the production phase perfusion media, rather than the growth phase perfusion media, in the perfusion-based continuous manufacturing (CM) process. All three methods were proven effective in titer improvement, which supported the hypothesis. Additionally, RoA/RAc significantly impacted product quality, with variations depending on the process mode and components. Overall, their supplementation led to decreased N-glycan mannose percentage and increased product fragmentation and aggregation. These changes do not fully align with the previous reports, highlighting that the supplementation strategy needs to be evaluated carefully based on cell line and expressed molecule type.