Quality by design approach to improve quality and decrease cost of in vitro transcription of mRNA using design of experiments.

In vitro transcription (IVT) reaction is an RNA polymerase-catalyzed production of messenger RNA (mRNA) from DNA template, and the unit operation with highest cost of goods in the mRNA drug substance production process. To decrease the cost of mRNA production, reagents should be optimally utilized. Due to the catalytic, multicomponent nature of the IVT reaction, optimization is a multi-factorial problem, ideally suited to design-of-experiment approach for optimization and identification of design space. We derived a data-driven model of the IVT reaction and explored factors that drive process yield (in g/L), including impact of nucleoside triphosphate (NTP) concentration and Mg:NTP ratio on reaction yield and how to optimize the main cost drivers RNA polymerase and DNA template, while minimizing dsRNA formation, a critical quality attribute in mRNA products. We report a methodological approach to derive an optimum reaction design, with which cost efficiency of the reaction was improved by 44%. We demonstrate the validity of the model on mRNA construct of different lengths. Finally, we maximized the yield of the IVT reaction to 24.9 ± 1.5 g/L in batch, thus doubling the highest ever reported IVT yield.

Advanced 3D Electrospinning "Xspin" System: Fabrication of Bifiber Floating Oral Pharmaceutical Scaffolds for Controlled Drug Delivery.

Electrospinning has become a widely used and efficient method for manufacturing nanofibers from diverse polymers. This study introduces an advanced electrospinning technique, Xspin - a multi-functional 3D printing platform coupled with electrospinning system, integrating a customised 3D printhead, MaGIC - Multi-channeled and Guided Inner Controlling printheads. The Xspin system represents a cutting-edge fusion of electrospinning and 3D printing technologies within the realm of pharmaceutical sciences and biomaterials. This innovative platform excels in the production of novel fiber with various materials and allows for the creation of highly customized fiber structures, a capability hitherto unattainable through conventional electrospinning methodologies. By integrating the benefits of electrospinning with the precision of 3D printing, the Xspin system offers enhanced control over the scaffold morphology and drug release kinetics. Herein, we fabricated a model floating pharmaceutical dosage for the dual delivery of curcumin and ritonavir and thoroughly characterized the product. Fourier transform infrared (FTIR) spectroscopy demonstrated that curcumin chemically reacted with the polymer during the Xspin process. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) confirmed the solid-state properties of the active pharmaceutical ingredient after Xspin processing. Scanning electron microscopy (SEM) revealed the surface morphology of the Xspin-produced fibers, confirming the presence of the bifiber structure. To optimize the quality and diameter control of the electrospun fibers, a design of experiment (DoE) approach based on quality by design (QbD) principles was utilized. The bifibers expanded to approximately 10-11 times their original size after freeze-drying and effectively entrapped 87% curcumin and 84% ritonavir. In vitro release studies demonstrated that the Xspin system released 35% more ritonavir than traditional pharmaceutical pills in 2 h, with curcumin showing complete release in pH 1.2 in 5 min, simulating stomach media. Furthermore, the absorption rate of curcumin was controlled by the characteristics of the linked polymer, which enables both drugs to be absorbed at the desired time. Additionally, multivariate statistical analyses (ANOVA, pareto chart, etc.) were conducted to gain better insights and understanding of the results such as discern statistical differences among the studied groups. Overall, the Xspin system shows significant potential for manufacturing nanofiber pharmaceutical dosages with precise drug release capabilities, offering new opportunities for controlled drug delivery applications.

Optimised stress - intensification of pyocyanin production with zinc oxide nanoparticles.

BACKGROUND: Pyocyanin is a blue pigment produced by Pseudomonas aeruginosa. Due to its unique redox properties over the last decade, it has gained more and more interest as a utile chemical. Nevertheless, it remains a rather costly reagent. It was previously shown that the production of pyocyanin can be enhanced by employing various methods. Among them are using statistical methods for planning the experiments or exposing bacterial cultures to stressors such as nanoparticles dosed in sublethal concentrations, e.g. zinc oxide nanoparticles. RESULTS: The Design of Experiment (DoE) methodology allowed for calculating the optimal process temperature and nanoparticle concentration to intensify pyocyanin production. Low concentrations of the nanoparticles (6.06 µg/mL) and a temperature of 32℃ enhanced pyocyanin production, whereas higher concentrations of nanoparticles (275.75 µg/mL) and higher temperature stimulated biomass production and caused the abolishment of pyocyanin production. Elevated pigment production in zinc oxide nanoparticles-supplemented media was sustained in the scaled-up culture. Conducted analyses confirmed that observed stimulation of pyocyanin production is followed by higher membrane potential, altered gene expression, generation of reactive oxygen species, and accumulation of zinc in the cell's biomass. CONCLUSIONS: Pyocyanin production can be steered using ZnO nanoparticles. Elevated production of pyocyanin due to exposure to nanoparticles is followed by the number of changes in physiology of bacteria and is a result of the cellular stress. We showed that the stress response of bacteria can be optimised using statistical methods and result in producing the desired metabolite more effectively.

Inorganic arsenic in seaweed: a fast HPLC-ICP-MS method without coelution of arsenosugars.

Seaweed is becoming increasingly popular in the Western diet as consumers opt for more sustainable food sources. However, seaweed is known to accumulate high levels of arsenic-which may be in the form of carcinogenic inorganic arsenic (iAs). Here we propose a fast method for the routine measurement of iAs in seaweed using HPLC-ICP-MS without coelution of arsenosugars that may complicate quantification. The developed method was optimised using design of experiments (DOE) and tested on a range of reference materials including TORT-3 (0.36 ± 0.03 mg kg-1), DORM-5 (0.02 ± 0.003 mg kg-1), and DOLT-5 (0.07 ± 0.007 mg kg-1). The use of nitric acid in the extraction solution allowed for the successful removal of interferences from arsenosugars by causing degradation to an unretained arsenosugar species, and a recovery of 99 ± 9% was obtained for iAs in Hijiki 7405-b when compared with the certified value. The method was found to be suitable for high-throughput analysis of iAs in a range of food and feed matrices including Asparagopsis taxiformis seaweed, grass silage, and insect proteins, and offers a cost-effective, fast, and robust option for routine analysis that requires minimal sample preparation. The method may be limited with regards to the quantification of dimethylarsenate (DMA) in seaweed, as the acidic extraction may lead to overestimation of this analyte by causing degradation of lipid species that are typically more abundant in seaweed than other marine matrices (i.e. arsenophospholipids). However, the concentrations of DMA quantified using this method may provide a better estimation with regard to exposure after ingestion and subsequent digestion of seaweed.

Development and validation of stability indicating assay method for mitapivat: Identification of novel hydrolytic, photolytic, and oxidative forced degradation products employing quadrupole-time of flight mass spectrometry.

Mitapivat is a novel, first-in-class orally active pyruvate kinase activator approved by the US Food and Drug Administration in 2022 for the treatment of hemolytic anemia. There is no literature available regarding the identification of degradation impurities of mitapivat. The present study deals with the degradation behavior of mitapivat under various stress conditions such as hydrolytic, photolytic, thermal, and oxidative stress. The multivariate analysis found that the independent variables, that is, molarity, temperature, and time, are interacting with each other to affect the degradation of mitapivat. A specific, accurate, and precise high-performance liquid chromatographic (HPLC) method was developed to separate mitapivat from its degradation products. The separation was achieved on the C-18 column (250 mm × 4.6 mm × 5 µm) using the combination of 0.1% formic acid buffer and acetonitrile in gradient elution profile. The method was validated as per the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use Q2(R2) guideline. LC-electrospray ionization-Quadrupole-time of flight was employed to identify degradation products. A total of seven novel degradation products of mitapivat were identified based on tandem mass spectrometry and accurate mass measurement. In-silico toxicity of mitapivat and its degradation products was qualitatively evaluated by the DEREK toxicity prediction tool.

Vegan grade medium component screening and concentration optimization for the fermentation of the probiotic strain Lactobacillus paracasei IMC 502® using Design of Experiments.

Lactobacillus paracasei IMC502® is a commercially successful probiotic strain. However, there are no reports that investigate growth medium composition in relation to improved biomass production for this strain. The major outcome of the present study is the design and optimization of a growth medium based on vegan components to be used in the cultivation of Lactobacillus paracasei IMC502®, by using Design of Experiments. Besides comparing different carbon sources, the use of plant-based peptones as nitrogen sources was considered. In particular, the use of guar peptone as the main nitrogen source, in the optimization of fermentation media for the production of probiotics, could replace other plant peptones (e.g. potato, rice, wheat, and soy) which are part of the human diet, thereby avoiding an increase in product and process prices. A model with R2 and adjusted R2 values higher than 95% was obtained. Model accuracy was equal to 94.11%. The vegan-optimized culture medium described in this study increased biomass production by about 65% compared to growth on De Man-Rogosa-Sharpe (MRS) medium. Moreover, this approach showed that most of the salts and trace elements generally present in MRS are not affecting biomass production, thus a simplified medium preparation can be proposed with higher probiotic biomass yield and titer. The possibility to obtain viable lactic acid bacteria at high density from vegetable derived nutrients will be of great interest to specific consumer communities, opening the way to follow this approach with other probiotics of impact for human health.

Mortality of older construction and craft workers employed at Department of Energy (DOE) nuclear sites: Follow-up through 2021.

BACKGROUND: To determine if construction and trades workers formerly employed at US Department of Energy (DOE) nuclear weapons sites are at significant risk for occupational diseases, we studied the mortality experience of participants in the Building Trades National Medical Screening Program (BTMed). METHODS: The cohort included 26,922 participants enrolled between 1998 and 2021 and 8367 deaths. Standardized mortality ratios were calculated based on US death rates. Cox models compared construction workers (n = 22,747; 7487 deaths) to two nonconstruction subpopulations: administrative, scientific and security workers (n = 1894; 330 deaths), and all other nonconstruction workers (n = 2218; 550 deaths). RESULTS: Mortality was elevated for all causes, all cancers, cancers of the trachea, bronchus, lung, kidneys, and lymphatic and hematopoietic system, mesothelioma, chronic obstructive pulmonary disease (COPD), asbestosis, transportation injuries, and other injuries, particularly accidental poisonings. There were 167 deaths from coronavirus disease 2019 (COVID-19), which was lower than expected using US death rates. Overall cause-specific mortality was significantly higher among construction workers than for internal comparison groups. CONCLUSIONS: Construction workers employed at DOE sites have a significantly increased risk for occupational illnesses. Apart from COVID-19 deaths, this update: (1) found that mortality among construction workers is significantly elevated compared to the US population and significantly higher than in the internal comparison populations, and (2) confirmed excess risk for these workers for first employment after 1990. Cancer mortality risks are similar to the cancers identified for DOE compensation from radiation exposures. The high lung cancer risk supports the value of early lung cancer detection. Continued medical surveillance is important.

Design of experiment-oriented development and validation of novel bioanalytical reverse phase high performance liquid chromatography method of palbociclib in rat plasma and tissues, and its application in pharmacokinetic studies.

The current research involved the development and validation of an easy, cost-effective, and sensitive bioanalytical reverse-phase high-performance liquid chromatography method for the assessment of palbociclib (PAL) in rat plasma and kidney, liver, spleen and heart. A response surface methodology-based Box-Behnken design was used to optimize critical chromatographic conditions such as buffer pH, organic phase concentration and flow rate to attain good sensitivity, tailing factor and retention time. The conditions were: pH of buffer, 4.5; organic phase concentration, 40%; Shimpac column with 1.0 ml/min flow rate. The responses were: tailing factor, 1.29 ± 0.03, sensitivity, 366,593 ± 8,592; and retention time, 4.5 ± 0.05 min. The samples were extracted by a protein precipitation method, and absolute recoveries were in the range of 88.99-95.08%. The linearity of the developed method was validated over the range 100-2,000 ng/ml (r2 ≥ 0.994) in all tested matrices. The developed bioanalytical method showed greater accuracy (0.98 and 4.01%) and precision (<4.88%). The method was optimized for the sensitive analysis of the PAL in rat plasma, and the kidney, liver, spleen and heart were effectively applied to pharmacokinetic studies.

Dairy goat doe-kid rearing systems: Farmers' motivations and a description of practices, benefits, and drawbacks.

In French dairy goat systems, kids are generally separated from their mother does shortly after birth. The main drivers of this practice are related to health-especially the prevention of the caprine arthritis encephalitis virus (CAEV) transmission-and economics. However, the separation of young ruminants from their does is being increasingly questioned by society and has raised concerns about the satisfaction of their behavioral needs. Some farmers choose to leave their female kids with their does. The aims of this study were to understand their motivations for leaving kids with their does, to describe how kids are reared in this case and how farmers perceive different aspects of the effects of this practice. Individual semi-structured interviews were carried out with 40 farmers who had implemented the kids-with-does practice for at least 1 yr. Compared with the national database describing the French population of dairy goat farmers, the characteristics of the interviewed farmers differed from those of the general population. They were younger and had done longer studies. Their farms were smaller, mainly with rare breeds, and milk was mainly processed on-farm under an organic certification system. They chose to implement this practice for different reasons: ethical considerations, improving integration of kids within the herd, saving time, and increasing comfort at work or improving the kids' growth, welfare, and health. The doe-kid rearing contact practices varied greatly between farms, with some kids staying with their does from a period of 45 d to never being separated; furthermore, some kids remained with their does all day, whereas others remained together part of the day or for a limited time, and daily contact evolved over time. Overall, farmers were satisfied as the benefits quoted were coherent with their motivations to implement this rearing practice. Most have decided to continue the practice, though usually with changes. However, as 40% of them had only 3 yr or less of experience of doe-kid rearing, some had not sufficient hindsight into long-term issues such as the transmission of CAEV. It is crucial to tackle challenges associated with this practice (i.e., potentially wild kids, health issues, and economic consequences stemming from a reduction in marketable milk).

Experimental design approach for the quantitative analysis of multicomponents by single marker and HPLC fingerprinting of Thunbergia laurifolia aqueous extract.

INTRODUCTION: Thunbergia laurifolia is used in traditional Thai medicine to reduce fever and treat mouth ulcers. However, the quantitative analysis of chemical markers has not yet been officially defined. OBJECTIVE: The objective of this study is to develop a high-performance liquid chromatography (HPLC) method using a design of experiment (DoE) for the quantitative analysis of multicomponents by single marker (QAMS) and fingerprinting of the T. laurifolia aqueous extract. MATERIALS AND METHODS: Critical variables were screened using a two-level fractional factorial design, followed by the optimization of the selected variables using a central composite design. The validated method was applied for quality assessment based on QAMS and fingerprinting of the extract. RESULTS: Optimum conditions of DoE for the analysis of caffeic acid, vicenin-2, and rosmarinic acid were determined. The relative correction factors for caffeic acid and vicenin-2 were calculated using rosmarinic acid as an internal reference standard, and their contents in 30 samples were determined. The differences between the external standard method (ESM) and QAMS were compared. No significant difference was observed in the quantitative determination, proving the consistency QAMS and ESM. HPLC fingerprints of T. laurifolia were established with 8 of 12 characteristic peaks that were structurally characterized using HPLC-diode array detection-electrospray ionization/tandem mass spectrometry. The similarity of the fingerprints in all samples was ≥0.74, and the pattern recognition of the characteristic peaks was satisfied. CONCLUSION: The proposed method efficiently detected multiple components of the T. laurifolia extract. Thus, the method is beneficial in providing references for enhancing the quality control of other herbal medicines.

Regression modelling strategies for projected and sustainable kraft pulping of wheat straw.

The demand for paper and paper-based packaging has seen a massive increase in past years, resulting in accelerated deforestation to meet the rising demand, negatively impacting the environment, and there is a need to look towards different non-woody raw materials. Kraft pulping (KP) is widely used in paper making, for which the chemical dose, temperature, time, and energy required must be optimized, for which many insignificant experimental trials are performed. An effort is made to solve this problem by developing the regression equations with the help of Excel using One Factor at a Time Analysis (OFAT), followed by carrying out design of experiments (DoE) using orthogonal approach and regression analysis in Minitab software. Life cycle Assessment (LCA) using the Open-LCA software estimates the effect of chemicals and energy required during pulping on human health, ecosystem quality, and resource depletion. Using regression analysis, the equations for predicting kappa number, yield (%), total energy consumed, and mechanical properties of the paper sheet showed a good fit with an R2 value in the range of 0.90-0.99. Apart from that, the mechanical properties, namely tensile index (41.43 Nm/g), tear index (6.96 mN m2/g), bending stiffness (0.5 mN m), and burst index (3.92 kPa m2/g) of the unbeaten sheet, were determined experimentally at optimized conditions. Based on the Open-LCA result, the optimized pulping conditions had less impact on human health, ecosystem quality, and resource depletion. Industries can use the model to predict the values of kappa number, yield, mechanical properties, and energy consumption without performing optimization experiments that may impact the industry's economy to a greater extent.

Integrated Approach of Life Cycle Assessment and Experimental Design in the Study of a Model Organic Reaction: New Perspectives in Renewable Vanillin-Derived Chemicals.

This work is focused on performing a quantitative assessment of the environmental impacts associated with an organic synthesis reaction, optimized using an experimental design approach. A nucleophilic substitution reaction was selected, employing vanillin as the substrate, a phenolic compound widely used in the food industry and of pharmaceutical interest, considering its antioxidant and antitumoral potential. To carry out the reaction, three different solvents have been chosen, namely acetonitrile (ACN), acetone (Ace), and dimethylformamide (DMF). The syntheses were planned with the aid of a multivariate experimental design to estimate the best reaction conditions, which simultaneously allow a high product yield and a reduced environmental impact as computed by Life Cycle Assessment (LCA) methodology. The experimental results highlighted that the reactions carried out in DMF resulted in higher yields with respect to ACN and Ace; these reactions were also the ones with lower environmental impacts. The multilinear regression models allowed us to identify the optimal experimental conditions able to guarantee the highest reaction yields and lowest environmental impacts for the studied reaction. The identified optimal experimental conditions were also validated by experimentally conducting the reaction in those conditions, which indeed led to the highest yield (i.e., 93%) and the lowest environmental impacts among the performed experiments. This work proposes, for the first time, an integrated approach of DoE and LCA applied to an organic reaction with the aim of considering both conventional metrics, such as reaction yield, and unconventional ones, such as environmental impacts, during its lab-scale optimization.

Optimizing neural network algorithms for submerged membrane bioreactor: A comparative study of OVAT and RSM hyperparameter optimization techniques.

Hyperparameter tuning is an important process to maximize the performance of any neural network model. This present study proposed the factorial design of experiment for screening and response surface methodology to optimize the hyperparameter of two artificial neural network algorithms. Feed-forward neural network (FFNN) and radial basis function neural network (RBFNN) are applied to predict the permeate flux of palm oil mill effluent. Permeate pump and transmembrane pressure of the submerge membrane bioreactor system are the input variables. Six hyperparameters of the FFNN model including four numerical factors (neuron numbers, learning rate, momentum, and epoch numbers) and two categorical factors (training and activation function) are used in hyperparameter optimization. RBFNN includes two numerical factors such as a number of neurons and spreads. The conventional method (one-variable-at-a-time) is compared in terms of optimization processing time and the accuracy of the model. The result indicates that the optimal hyperparameters obtained by the proposed approach produce good accuracy with a smaller generalization error. The simulation results show an improvement of more than 65% of training performance, with less repetition and processing time. This proposed methodology can be utilized for any type of neural network application to find the optimum levels of different parameters.

Design of Experiments-Driven Optimization of Spray Drying for Amorphous Clotrimazole Nanosuspension.

This study employed a Quality by Design (QbD) approach to spray dry amorphousclotrimazole nanosuspension (CLT-NS) consisting of Soluplus® and microcrystallinecellulose. Using the Box-Behnken Design, a systematic evaluation was conducted toanalyze the impact of inlet temperature, % aspiration, and feed rate on the criticalquality attributes (CQAs) of the clotrimazole spray-dried nanosuspension (CLT-SDNS). In this study, regression analysis and ANOVA were employed to detect significantfactors and interactions, enabling the development of a predictive model for the spraydrying process. Following optimization, the CLT-SD-NS underwent analysis using Xraypowder diffraction (XRPD), Fourier transform infrared spectroscopy (FTIR), Dynamic Scanning Calorimetry (DSC), and in vitro dissolution studies. The resultsshowed significant variables, including inlet temperature, feed rate, and aspiration rate,affecting yield, redispersibility index (RDI), and moisture content of the final product. The models created for critical quality attributes (CQAs) showed statistical significanceat a p-value of 0.05. XRPD and DSC confirmed the amorphous state of CLT in theCLT-SD-NS, and FTIR indicated no interactions between CLT and excipients. In vitrodissolution studies showed improved dissolution rates for the CLT-SD-NS (3.12-foldincrease in DI water and 5.88-fold increase at pH 7.2 dissolution media), attributed torapidly redispersing nanosized amorphous CLT particles. The well-designed studyutilizing the Design of Experiments (DoE) methodology.

A Quality by Design Approach for Optimizing Solid Lipid Nanoparticles of Bedaquiline for Improved Product Performance.

Bedaquiline (BQ) solid lipid nanoparticles (SLNs), which have previously been formulated for parenteral administration, have a risk of patient non-compliance in treating tuberculosis. This research presents a strategy to develop BQ SLNs for oral delivery to improve patient adherence, The upper and lower levels for the formulation excipients were generated from screening experiments. Using 4 input factors (BQ, lecithin, Tween 80, and PEG), a full factorial design from 3 × 2x2 × 2 experiments was randomly arranged to investigate 3 response variables: Particle size distribution (PSD), polydispersity index (PdI), and zeta potential (ZP). High shear homogenization was used to mix the solvent and aqueous phases, with 15% sucrose as a cryoprotectant. The response variables were assessed using a zeta sizer while TEM micrographs confirmed the PSD data. Solid-state assessments were conducted using powdered X-ray diffraction and scanning electron microscopy (SEM) imaging. A comparative invitro assessment was used to determine drug release from an equivalent dose of BQ free base powder and BQ-SLN, both packed in hard gelatin capsules. The sonicated formulations obtained significant effects for PSD, PdI, and ZP. The p-values (0.0001 for PdI, 0.0091 for PSD) for BQ as an independent variable in the sonicated formulation were notably higher than those in the unsonicated formulation (0.1336 for PdI, 0.0117 for PSD). The SEM images were between 100 - 400 nm and delineated nanocrystals of BQ embedded in the lipid matrix. The SLN formulation provides higher drug levels over the drug's free base; a similarity factor (f2 = 18.3) was estimated from the dissolution profiles.

Design and Evaluation of Microemulsion-Based Drug Delivery Systems for Biofilm-Based Infection in Burns.

Normal skin is the first line of defense in the human body. A burn injury makes the skin susceptible to bacterial infection, thereby delaying wound healing and ultimately leading to sepsis. The chances of biofilm formation are high in burn wounds due to the presence of avascular necrotic tissue. The most common pathogen to cause burn infection and biofilm is Pseudomonas aeruginosa. The purpose of this study was to create a microemulsion (ME) formulation for topical application to treat bacterial burn infection. In the present study, tea tree oil was used as the oil phase, Tween 80 and transcutol were used as surfactants, and water served as the aqueous phase. Pseudo ternary phase diagrams were used to determine the design space. The ranges of components as suggested by the design were chosen, optimization of the microemulsion was performed, and in vitro drug release was assessed. Based on the characterization studies performed, it was found that the microemulsion were formulated properly, and the particle size obtained was within the desired microemulsion range of 10 to 300 nm. The I release study showed that the microemulsion followed an immediate release profile. The formulation was further tested based on its ability to inhibit biofilm formation and bacterial growth. The prepared microemulsion was capable of inhibiting biofilm formation.

Sol-gel and Pechini niobium modified: synthesis, characterization and application in the 2,4-D herbicide degradation.

In this work, a comparison was made between the synthesis of niobium-based materials (Nb2O5), both in terms of material characterization and catalytic performance. The methods used were chemical mixtures: modified sol-gel and Pechini. The materials were calcined at different temperatures (753, 873 and 993K) and characterized by the following techniques: photoacousticspectroscopy (PAS), zero charge point (pHPZC), scanning electron microscopy (SEM/EDS), thermogravimetric analysis (TGA/DTG) and X-ray diffraction (XRD). The photocatalytic process was carried out to evaluate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) under UV radiation (250 W mercury vapor lamp) and different experimental conditions. In addition, to better understand the influence of parameters such as pH, catalyst concentration (0.2, 0.5 and 0.8 g L-1) and calcination temperature, a Design of Experiments (DoE) was used. The results indicated that despite having similar structures and phases in the XRD analysis, the morphology presents two distinct surfaces, due to the preparation method. Differences in the synthesis method affected the catalytic activity in the parameters studied. Although the zero charge point values are close (6.18-6.36), we observed differences in the band gap depending on the calcination temperature. In the optimal condition studied, the catalyst prepared by the sol-gel method obtained the best results.

Design space determination to optimize DNA complexation and full capsid formation in transient rAAV manufacturing.

Recombinant adeno-associated virus (rAAV) vectors are a promising platform for in vivo gene therapies. However, cost-effective, well-characterized processes necessary to manufacture rAAV therapeutics are challenging to develop without an understanding of how process parameters (PPs) affect rAAV product quality attributes (PQAs). In this work, a central composite orthogonal experimental design was employed to examine the influence of four PPs for transient transfection complex formation (polyethylenimine:DNA [PEI:DNA] ratio, total DNA/cell, cocktail volume, and incubation time) on three rAAV PQAs related to capsid content (vector genome titer, vector genome:capsid particle ratio, and two-dimensional vector genome titer ratio). A regression model was established for each PQA using partial least squares, and a design space (DS) was defined in which Monte Carlo simulations predicted < 1% probability of failure (POF) to meet predetermined PQA specifications. Of the three PQAs, viral genome titer was most strongly correlated with changes in complexation PPs. The DS and acceptable PP ranges were largest when incubation time and cocktail volume were kept at mid-high setpoints, and PEI:DNA ratio and total DNA/cell were at low-mid setpoints. Verification experiments confirmed model predictive capability, and this work establishes a framework for studying other rAAV PPs and their relationship to PQAs.

Estimation of measurement uncertainty for the quantification of protein by ID-LC-MS/MS.

The emergence of mass spectrometry (MS)-based methods to quantify proteins for clinical applications has led to the need for accurate and consistent measurements. To meet the clinical needs of MS-based protein results, it is important that the results are traceable to higher-order standards and methods and have defined uncertainty values. Therefore, we outline a comprehensive approach for the estimation of measurement uncertainty of a MS-based procedure for the quantification of a protein biomarker. Using a bottom-up approach, which is the model outlined in the "Guide to the Expression of Uncertainty of Measurement" (GUM), we evaluated the uncertainty components of a MS-based measurement procedure for a protein biomarker in a complex matrix. The cause-and-effect diagram of the procedure is used to identify each uncertainty component, and statistical equations are derived to determine the overall combined uncertainty. Evaluation of the uncertainty components not only enables the calculation of the measurement uncertainty but can also be used to determine if the procedure needs improvement. To demonstrate the use of the bottom-up approach, the overall combined uncertainty is estimated for the National Institute of Standards and Technology (NIST) candidate reference measurement procedure for albumin in human urine. The results of the uncertainty approach are applied to the determination of uncertainty for the certified value for albumin in candidate NIST Standard Reference Material® (SRM) 3666. This study provides a framework for measurement uncertainty estimation of a MS-based protein procedure by identifying the uncertainty components of the procedure to derive the overall combined uncertainty.

How much have adverse occupational health outcomes among construction workers improved over time? Evidence from 25 years of medical screening.

BACKGROUND: Construction workers have always had a high risk of occupational illnesses. We used 25 years of data from a medical screening program serving older construction workers to determine how much health outcomes have improved over the past 60 years. METHODS: We investigated changes in relative risk for chest radiographs consistent with pneumoconiosis, COPD by spirometry, lung cancer mortality, and audiometry-assessed hearing impairment among workers participating in a medical screening program. Results were stratified by decade of first construction employment: before 1960, 1960-1969, 1970-1979, 1980-1989, and after 1990. Poisson and Cox regression analyses assessed relative risk by decade adjusted for age, sex, smoking, and years of construction trade work. RESULTS: Subjects were 94% male and, on average, 60 years old with 25 years of construction work. When compared to workers employed before 1960, those first employed after 1990 experienced the following reductions in model-adjusted relative risks: chronic obstructive pulmonary disease, 32%; all pneumoconiosis, 68%; parenchymal abnormalities, 35%; pleural abnormalities, 71%; hearing impairment, 20%; and lung cancer mortality, 48%. Risks started to decline in the 1960s with greatest reductions among workers first employed after 1970. CONCLUSIONS: This study demonstrates the positive impact that adoption of occupational health protections have had over the past 60 years. The greatest risk reductions were observed for outcomes with strong regulatory and legal incentives to reduce exposures and associated risks, such as those associated with inhalation hazards (asbestos and silica), while lowest improvement was for hearing impairment, for which little regulatory enforcement and few prevention incentives have been adopted.