Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale‐up from ideal lab‐scale bioreactors to ...large‐scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non‐ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single‐cell physiologies in bioprocesses are mandatory before scale‐up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L‐phenylalanine producing Escherichia coli strain FUS4 (pF81kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well‐mixed lab‐scale fed‐batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.
A CZE method was validated and implemented for fast and accurate in‐process determination of adenovirus concentrations of downstream process samples obtained during manufacturing of adenovirus ...vector‐based vaccines. An analytical‐quality‐by‐design approach was embraced for method development, method implementation, and method maintenance. CZE provided separation of adenovirus particles from sample matrix components, such as cell debris, residual DNA and proteins. The intermediate precision of the virus particle concentration was 6.9% RSD and the relative bias was 2.3%. In comparison, the CZE method is intended to replace a quantitative polymerase chain reaction method which requires three replicates in three analytical runs to achieve an intermediate precision of 8.1% RSD. Given that, in addition, the time from sampling till reporting results of the CZE method was less than 2 h, whereas quantitative polymerase chain reaction requires 3 days, it follows that the CZE method enables faster processing times in downstream processing.
•A fluorescence polarization-based at-line nanofractionation platform was developed.•Semi-HPLC and orthogonal separation were exploited to identify co-eluted compounds.•28 potential SARS-CoV-2 fusion ...inhibitors were identified.•4 potential dual-target inhibitors against influenza and SARS-CoV-2 were found.
In this study, a novel at-line nanofractionation platform was established for screening SARS-CoV-2 fusion inhibitors from natural products for the first time by combining HPLC-MS/MS with high-throughput fluorescence polarization (FP) bioassay. A time-course FP bioassay in 384 well-plates was conducted in parallel with MS/MS to simultaneously obtain chemical and biological information of potential fusion inhibitors in Lonicerae Japonicae Flos (LJF) and Lianhua Qingwen capsules (LHQW). Semi-preparative liquid chromatography and orthogonal HPLC separation were employed to enrich and better identify the co-eluted components. After comprehensive evaluation and validation, 28 potential SARS-CoV-2 fusion inhibitors were screened out and identified. Several compounds at low micromolar activity were validated by in vitro inhibitory assay, molecular docking, cytotoxicity test, and pseudovirus assay. Moreover, four potential dual-target inhibitors against influenza and COVID-19 were discovered from LJF using this method, offering novel insights for the development of future pharmaceuticals targeting epidemic respiratory diseases.
Viral vectors are an emerging, exciting class of biologics whose application in vaccines, oncology, and gene therapy has grown exponentially in recent years. Following first regulatory approval, this ...class of therapeutics has been vigorously pursued to treat monogenic disorders including orphan diseases, entering hundreds of new products into pipelines. Viral vector manufacturing supporting clinical efforts has spurred the introduction of a broad swath of analytical techniques dedicated to assessing the diverse and evolving panel of Critical Quality Attributes (CQAs) of these products. Herein, we provide an overview of the current state of analytics enabling measurement of CQAs such as capsid and vector identities, product titer, transduction efficiency, impurity clearance etc. We highlight orthogonal methods and discuss the advantages and limitations of these techniques while evaluating their adaptation as process analytical technologies. Finally, we identify gaps and propose opportunities in enabling existing technologies for real-time monitoring from hardware, software, and data analysis viewpoints for technology development within viral vector biomanufacturing.
Capacitance is used to monitor “biomass” in cell culture and fermentation processes. The present work explores various ways to support good manufacturing practices (GMP) use and create robust methods ...that can assure proper performance of capacitance equipment. An at‐line capacitance measurement method is particularly valuable as it can be used to correct or confirm online measurements from bioreactors. To obtain consistent predictions of online capacitance, the at‐line method requires appropriate control of culture temperature, vessel geometry, mixing, and timing. The at‐line method yields values that differ systematically from online values, but the variation is ≤11% for two cell lines tested. A cell line‐specific conversion factor addresses the difference and enables accurate predictions of online capacitance values. In addition to the at‐line method, verification of equipment performance is further enabled by using formaldehyde‐treated cell standards. The cell‐derived standards create a stabilized system for studying capacitance and recapitulate the performance of metabolically active cell culture. The formaldehyde‐treated cells, however, require a much greater conversion factor to predict online outputs. The stabilized system proves useful for evaluation and calibration of capacitance measuring systems. The at‐line method and stabilized cell‐derived suspensions support use of capacitance for process control in large scale mammalian cell culture.
Capacitance is used to measure “biomass” online in cell culture processes. The present work describes new tools to provide assurance of properly functioning capacitance equipment including an at‐line measurement method and using fixed cells as a cell‐like standard. The studies confirm the practical use of capacitance for controlling processes while not requiring an absolute correspondence to viable cell densities.
Three experimental setups, based on near infrared technology (NIR), were tested for rapid “at-line” assessment of sunflower oil adulteration by mineral oil. Experimental setups included a commercial ...portable NIR, coupled to both reflexion (S1) and immersion probes (S2), and a prototype of a multichannel Quasi Imaging Visible NIR spectrometer (QIVN) coupled to an immersion probe (S3). Independent calibration and validation samples sets were prepared with mineral oils (MOs) content up to 10% (w/w), and calibrations were developed using partial least square (PLS) regressions. Root mean square error of prediction (RMSEP) ranges from 0.23 to 1.26% (w/w) MOs, depending on the NIR setup.
The best performances were obtained with S1, which provides satisfactory calibrations, and low number of false positives starting from levels of mineral oil around 1%. S3 still provides acceptable calibrations, and could be practically used to detect mineral oil at concentrations higher than 2.5% (w/w) MOs.
•Three NIR setups were tested for “at-line” detection of sunflower oil adulterated with mineral oil.•Our results agree with those obtained “off-line” with a benchtop instrument by other Authors.•The setup 1, with a reflexion probe, provided an RMSEC of 0.25% (w/w) MOs and R2cal better than 0.99.
•Fabrication of potentiometric sensor that meets the green analytical chemistry principles.•Novel implementation of ISE for at-line monitoring of the oxidative degradation kinetics of an oxidizable ...drug.•Real-time observation for continuous profile of the oxidative behavior of BR under various H2O2 concentration and temperature.•Increased accuracy in rate constants estimation superior to the off-line methods.•The proposed sensor was capable of determination the drug in dosage form without any prior separation step.
Kinetics studies are important in quality control to assure the safety and efficacy of the pharmaceutical compounds. Recently, potentiometric ion-selective electrodes (ISEs) breakthrough kinetics study due to their ability to provide real-time measurements which meet “green analytical chemistry” (GAC) principles. A polyvinyl chloride (PVC) matrix membrane was fabricated to monitor bromhexine (BR) oxidative degradation kinetics using tetraphenylborate as a cation exchanger. At-line monitoring of the oxidative degradation kinetics of BR was performed; by continuous measuring the decrease in the sensor potential over time. So a real-time observation and a continuous profile were obtained for the oxidation of BR under various H2O2 concentrations and temperature. This kinetic study determines the oxidation activation energy that was 6.4 kcal mol−1. To expand the application of this sensor, BR was determined in bulk powder and dosage form in the presence of both co-formulated drug (acefylline piperazine) and dosage form additives. The proposed sensor had been characterized according to IUPAC recommendations and a linear dynamic range was 5 × 10−6 to 1 × 10−3 M and other sensor parameters had been calculated.
•An at-line nanofractionation screening platform was established for NA inhibitors for the first time.•It involves parallel oseltamivir-sensitive/resistant neuraminidase (NAS/NAR) bioassays.•20 ...potential inhibitors were identified from Lonicera japonica thunb.•The proposed platform shows potential for rapid quality control of Lonicera japonica Thunb.
In this study, an advanced at-line nanofractionation based screening platform was developed to screen potential neuraminidase inhibitors (NAIs) from Lonicera japonica Thunb by involving two parallel bioassays, for determining both oseltamivir-sensitive neuraminidase (NAS) and oseltamivir-resistant neuraminidase (NAR) inhibitory activities. 20 potential NAIs with both NAS and NAR inhibitory effects were screened from Lonicera japonica Thunb and identified by mass spectrometer, including 11 phenolic acids, 8 flavonoids and one iridoid glycoside. The proposed at-line nanofractionation based screening platform for NAIs was also used to rapidly screen nine batches of water extracts of Lonicera japonica Thunb or its similar species. Clear differences in the number and content of active components were easily observed, demonstrating that the proposed method possesses great potential for the quality control of herb medicines.
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