Definitive Screening Design Research Articles

Analytical quality by design based on knowledge organization: A case study of developing an ultrahigh-performance liquid chromatography method for the detection of phenolic compounds.

Despite numerous successful cases, there are still some challenges in using analytical quality by design (AQbD) for the development of analytical methods. Knowledge organization helps to enhance the objectivity of risk assessment, reduce the number of preliminary exploratory experiments, identify potential critical method parameters (CMPs) and their scope. In the present study, we aimed to develop a simple, rapid, and robust analytical method for detecting phenolic compounds in Xiaochaihu capsule intermediates utilizing knowledge organization. Knowledge organization and AQbD were combined to obtain the initial analytical conditions through knowledge collection, extraction, reorganization, and analysis. The quantitative relationship between critical method attributes (CMAs) and CMPs was then established by a definitive screening design. The method operable design region was calculated using an exhaustive Monte Carlo approach based on the probability of reaching the standard. Robustness investigation and methodological validation were finally performed. Analytical target profiles, CMAs, potential CMPs, and initial analytical conditions were initially identified, and the optimized ranges of operating parameters were obtained. A UHPLC method was successfully established for the analysis of phenolic compounds in ginger-ginger pinellia percolate, and the method validation outcomes were also satisfactory. The developed method can be a reliable means to detect the phenolic compounds of Xiaochaihu capsule intermediates. Knowledge organization provides a new approach for making better use of prior knowledge, significantly enhancing the efficiency of analytical method development. The approach is versatile and can be similarly applied to the development of other methods.

Quantitative assessment and optimization of parallel contact model for flexible paddy straw: a definitive screening and central composite design approach using discrete element method

To simulate the bending behaviour of paddy straw at varied moisture contents after crop harvesting, we created a flexible paddy straw specimen model based on the Hertz–Mindlin with parallel contact bonding model using the discrete element model (DEM) approach. The research presented in this study aims to investigate a new approach called Definitive Screening Design (DSD) for parameterizing and screening the most significant parameters of the DEM model. This investigation will specifically focus on the three-point bending test as a means of parameterization, and the shear plate test will be used for validation purposes. In addition, the most influential DEM parameters were optimized using another Design of Experiments approach called Central Composite Design. The findings from the DSD indicated that parameters such as bonded disk scale, normal stiffness, and shear stiffness have the highest impact on the bending force, while the coefficient of static friction (Straw-Steel) has the least effect. The three bonding parameters were respectively calibrated with the loading rate (0.42, 0.5, and 0.58 mm s−1) and a good agreement between actual and simulated shear force at moisture content M1—35 ± 3.4%, M2—24 ± 2.2% and M3—17 ± 2.6%. Modelled stem helps simulate the straw with low error and increases the accuracy of the simulation. The validated model, with an average relative error of 5.43, 7.63, and 8.86 per cent, produced reasonable agreement between measured and simulated shear force value and loading rate.

Encapsulation of probiotics in freeze-dried calcium alginate and κ-carrageenan beads using definitive screening design: A comprehensive characterisation and in vitro digestion study

This research aimed to evaluate the encapsulation of the probiotic strain, Streptococcus thermophilus, in hydrogels employing sodium alginate (SA) with κ-carrageenan (κC) in gelation baths with varying salt concentrations (CaCl2 and KCl) followed by freeze-drying. The experimentation was conducted at varying levels of κC (0–0.5 % w/v) and SA (2–4 %). Freeze-dried hydrogels were evaluated based on encapsulation efficiency and loss of viability and further characterised. The study could successfully establish an encapsulation efficiency of 87.814 % and a viability loss of 1.201 log CFU·g−1 for the optimised samples. The SEM micrographs of the optimised Ca-alginate/κC hydrogels exhibited a much denser network with fewer pores. The influence of SA/κC in the beads was confirmed by FTIR and DSC, where distinct peak shifts were observed, which indicated the presence of κC and SA polymers. The probiotic survival under simulated gastrointestinal tract (GIT) conditions, performed in accordance with the INFOGEST protocol, indicated that the optimised Ca-alginate/κC beads had a lower rate of release in the gastric phase and a much higher rate of survival and release in the intestinal phase than the control sample. The swelling behaviour of beads varied due to varying pH in both gastric and intestinal phases, and the κC in the optimised beads affected the swelling ratio significantly.

Enhancing early-stage cell culture process development efficiency using an integrated approach of high-throughput miniaturized bioreactors and definitive screening design

Rapidly expanding pipelines in biopharmaceutical industry and increasingly stringent requirements from regulatory agencies call for a more efficient and rational study design at early-stage cell culture process development. Recent developments in high-throughput bioreactor system and design of experiments (DoE) enable cell culture process development sector to meet such demand by integrating both technological advances. In this study, we compared varied forms of fractional factorial designs (FFDs) and definitive screening design (DSD) covering key process parameters such as temperature shift target, temperature shift timing, feed strategy, pH, dissolved oxygen, harvest time and additives in Ambr® 15 bioreactors to better understand the initial cell culture process and thus optimize it. Results showed that DSD was more effective and efficient in detecting both main and quadratic effects comparing to FFDs. Comparison of varied available model development strategies revealed that two-stage forward regression combined with corrected Akaike's information criterion (AICc) and least absolute shrinkage and selection operator (LASSO) regression combined with self-validated ensembled models (SVEM) offered superior predictive capability. Moreover, key DoE regularities was evaluated for their validity. In conclusion, DSD is a suitable experimental design for the early-stage cell culture process development if quadratic effects might exist. Statistical models derived based on DSD-based dataset could be applied for predictive optimization of antibody producing bioprocess while regularities such as effect heredity and effect hierarchy might be relaxed considering higher-order interaction effects are more likely to existed for a cell-based biological system.

Innovative optimization for enhancing Pb2+ biosorption from aqueous solutions using Bacillus subtilis.

Toxic heavy metal pollution has been considered a major ecosystem pollution source. Unceasing or rare performance of Pb2+ to the surrounding environment causes damage to the kidney, nervous, and liver systems. Microbial remediation has acquired prominence in recent decades due to its high efficiency, environment-friendliness, and cost-effectiveness. The lead biosorption by Bacillus subtilis was optimized by two successive paradigms, namely, a definitive screening design (DSD) and an artificial neural network (ANN), to maximize the sorption process. Five physicochemical variables showed a significant influence (p < 0.05) on the Pb2+ biosorption with optimal levels of pH 6.1, temperature 30°C, glucose 1.5%, yeast extract 1.7%, and MgSO4.7H2O 0.2, resulting in a 96.12% removal rate. The Pb2+ biosorption mechanism using B. subtilis biomass was investigated by performing several analyses before and after Pb2+ biosorption. The maximum Pb2+ biosorption capacity of B. subtilis was 61.8 mg/g at a 0.3 g biosorbent dose, pH 6.0, temperature 30°C, and contact time 60 min. Langmuir's isotherm and pseudo-second-order model with R2 of 0.991 and 0.999 were suitable for the biosorption data, predicting a monolayer adsorption and chemisorption mechanism, respectively. The outcome of the present research seems to be a first attempt to apply intelligence paradigms in the optimization of low-cost Pb2+ biosorption using B. subtilis biomass, justifying their promising application for enhancing the removal efficiency of heavy metal ions using biosorbents from contaminated aqueous systems.

Cost-effective method of simultaneous removal of copper and phosphate on environmentally friendly nanomaterial

Environmentally friendly and economically viable methods are essential in the selection of materials and techniques for the synthesis of nano- -zero-valent iron. Plants, with their high polyphenol content and antioxidant capacity, have found application in eco-friendly synthesis processes. The defi-nitive screening design (DSD) monitored four key process parameters for the concurrent removal of copper and phosphate: copper concentration (ranging from 1 to 9 mg L-1), phosphate concentration (ranging from 1 to 9 mg L-1), initial pH values (ranging from 2 to 10), and the dosage of nano-zero-valent iron (ranging from 2 to 16 mL). The analysis results provide valuable insights into the significant individual factors influencing the process, along with the potential for their interactions. The model also proposes process optimization to attain maximum removal efficiency, and subsequent verification confirmed its superiority among the alternatives. Mechanisms such as sorption, reduction, complexation, electrostatic attraction, and ligand exchange play pivotal roles in the effective removal of copper and phosphate using nano-zero-valent iron. In summary, this research yields several benefits: the utilization of environment-ally sustainable materials, a substantial reduction in experimental complexity, coupled with the ease of the entire procedure, simultaneous and highly efficient copper and phosphate removal, favorable pH levels and, notably, no require-ment for additional treatment.

Optimisation of Surface Roughness of FDM Fabricated Parts: Application of Definitive Screening Design and Genetic Algorithm Techniques

Optimisation of Surface Roughness of FDM Fabricated Parts: Application of Definitive Screening Design and Genetic Algorithm Techniques

Characterizing a design space for a twin-screw wet granulation process: A case study of extended-release tablets

Twin-screw wet granulation is an emerging continuous manufacturing technology for solid oral dosage forms. This technology has been successfully employed for the commercial manufacture of immediate-released tablets. However, the higher polymer content in extended-release (ER) formulations may present challenges in developing and operating within a desired design space. The work described here used a systematic approach for defining the optimum design space by understanding the effects of the screw design, operating parameters, and their interactions on the critical characteristics of granules and ER tablets. The impacts of screw speed, powder feeding rate, and the number of kneading (KEs) and sizing elements on granules and tablets characteristics were investigated by employing a definitive screening design. A semi-mechanistic model was used to calculate the residence time distribution parameters and validated using the tracers. The results showed that an increase in screw speed decreased the mean residence time of the material within the barrel, while an increase in the powder feeding rate or number of KEs did the opposite and increased the barrel residence time. Screw design and operating parameters affected the flow and bulk characteristics of granules. The screw speed was the most significant factor impacting the tablet's breaking strength. The dissolution profiles revealed that granule characteristics mainly influenced the early phase of drug release. This study demonstrated that a simultaneous optimization of both operating and screw design parameters was beneficial in producing ER granules and tablets of desired performance characteristics while mitigating any failure risks, such as swelling during processing.

Investigation and Prediction of Tensile, Flexural, and Compressive Properties of Tough PLA Material Using Definitive Screening Design.

The material extrusion fused deposition modeling (FDM) technique has become a widely used technique that enables the production of complex parts for various applications. To overcome limitations of PLA material such as low impact toughness, commercially available materials such as UltiMaker Tough PLA were produced to improve the parent PLA material that can be widely applied in many engineering applications. In this study, 3D-printed parts (test specimens) considering six different printing parameters (i.e., layer height, wall thickness, infill density, build plate temperature, printing speed, and printing temperature) are experimentally investigated to understand their impact on the mechanical properties of Tough PLA material. Three different standardized tests of tensile, flexural, and compressive properties were conducted to determine the maximum force and Young's modulus. These six properties were used as responses in a design of experiment, definitive screening design (DSD), to build six regression models. Analysis of variance (ANOVA) is performed to evaluate the effects of each of the six printing parameters on Tough PLA mechanical properties. It is shown that all regression models are statistically significant (p<0.05) with high values of adjusted and predicted R2. Conducted confirmation tests resulted in low relative errors between experimental and predicted data, indicating that the developed models are adequately accurate and reliable for the prediction of tensile, flexural, and compressive properties of Tough PLA material.

QbD-Guided Traditional Chinese Medicine Manufacturing Process: Development and Optimization of Fluid-Bed Granulation and Drying Processes for Xiaochaihu Capsules.

Traditional methods of producing Xiaochaihu (XCH) capsules, a traditional Chinese medicine, are time-consuming, costly, and labor-intensive, which is not conductive to modernizing TCM. To address the challenges, new fluid-bed granulation and drying processes with water as the binder were developed and optimized guided by the principles of Quality by Design (QbD) in this study. Ishikawa diagram was applied to conduct a preliminary risk assessment, followed by 6-factor definitive screening design (DSD) serving as a QbD statistical tool to develop and optimize the new processes. Multiple potential factors and interactions were studied with a small number of experiments using the DSD. This study identified critical process parameters (CPPs), established quadratic regression models to reveal CPP-critical quality attributes (CQAs) connections within the DSD framework, and defined a dependable design space. Processes conducted by parameter combinations in the design space produced qualified granules with production yield and raw material utilization higher than 90% and moisture content lower than 4%. Furthermore, quantitative analysis of baicalin of all the granules ensured qualified contents of active pharmaceutical ingredient. The newly developed processes for XCH capsules, with advantages of shorter time, environmental friendliness, and decreased cost, exemplify the effective application of QbD and design of experiments (DoE) methodologies in the modernization of TCM manufacturing processes.

On-Line Monitoring of Enzymatic Degumming of Soybean Oil Using Near-Infrared Spectroscopy.

Degumming is an oil refinement process in which the naturally occurring phospholipids in crude vegetable oils are removed. Enzymatic degumming results in higher oil yield and more cost-efficient processing compared to traditional degumming processes using only water or acid. Phospholipase C hydrolyses phospholipids into diglycerides and phosphate groups during degumming. The diglyceride content can therefore be considered a good indicator of the state of the enzymatic reaction. This study investigates the use of near-infrared (NIR) spectroscopy and chemometrics to monitor the degumming process by quantifying diglycerides in soybean oil in both off-line and on-line settings. Fifteen enzymatic degumming lab scale batches originating from a definitive screening design (with varying water, acid, and enzyme dosages) were investigated with the aim to develop a NIR spectroscopy prediction method. By applying tailored preprocessing and variable selection methods, the diglyceride content can be predicted with a root mean square error of prediction of 0.06% (w/w) for the off-line set-up and 0.07% (w/w) for the on-line set-up. The results show that the diglyceride content is a good indicator of the enzyme performance and that NIR spectroscopy is a suitable analytical technique for robust real-time diglyceride quantification.

Optimized Single-Step Recovery of Lipophilic and Hydrophilic Compounds from Raspberry, Strawberry and Blackberry Pomaces Using a Simultaneous Ultrasound-Enzyme-Assisted Extraction (UEAE).

An ultrasound-enzyme-assisted extraction (UEAE) was optimized to extract, simultaneously, the hydrophilic and lipophilic compounds from three berry pomaces (raspberry, strawberry and blackberry). First, an enzyme screening designated a thermostable alkaline protease as the most suitable enzyme to recover, in an aqueous medium, the highest yields of polyphenols and oil in the most efficient way. Secondly, the selected enzyme was coupled to ultrasounds (US) in sequential and simultaneous combinations. The simultaneous US-alkaline enzyme combination was selected as a one-single-step process and was then optimized by definitive screening design (DSD). The optimized parameters were: US amplitude, 20% (raspberry pomace) or 70% (strawberry and blackberry pomaces); pH, 8; E/S ratio, 1% (w/w); S/L ratio, 6% (w/v); extraction time, 30 min; temperature, 60 °C. Compared to conventional extractions using organic solvents, the UEAE extracted all the polyphenols, with around 75% of the active polyphenols (measured by the DPPH● method) and up to 75% of the initial oil from the berry pomaces. Characterized lipophilic compounds were rich in polyunsaturated fatty acids (PUFAs), tocols and phytosterols. The polyphenolics were analyzed by UPLC-MS/MS; characteristic ellagitannins of the Rosaceae family (sanguiin H-6 or agrimoniin, sanguiin H-10, …) and ellagic acid conjugates were found as the major components.

Analyzing Surface Roughness Variations in Material Extrusion Additive Manufacturing of Nylon Carbon Fiber Composites.

In recent years, fused deposition modeling (FDM) based on material extrusion additive manufacturing technology has become widely accepted as a cost-effective method for fabricating engineering components with net-shapes. However, the limited exploration of the influence of FDM process parameters on surface roughness parameters, i.e., Ra (average surface roughness), Rq (root mean square surface roughness), and Rz (maximum height of the profile) across different sides (bottom, top, and walls) poses a challenge for the fabrication of functional parts. This research aims to bridge the knowledge gap by analyzing surface roughness under various process parameters and optimizing it for nylon carbon fiber printed parts. A definitive screening design (DSD) was employed for experimental runs. The Pareto chart highlighted the significant effects of layer height, part orientation, and infill density on all surface roughness parameters and respective sides. The surface morphology was analyzed through optical microscopy. Multi-response optimization was performed using an integrated approach of composited desirability function and entropy. The findings of the present study hold significant industrial applications, enhancing the quality and performance of 3D printed parts. From intricate prototypes to durable automotive components, the optimized surfaces contribute to production of functional and visually appealing products across various sectors.

A novel process for developing a quantitative chromatographic fingerprint analysis method based on analytical quality by design: A case study of Xiaochaihu capsules

In this study, an improved Analytical Quality by Design (AQbD) process was proposed to establish quantitative fingerprints of Chinese medicines. A quantitative fingerprint method for the strong polar components in Xiaochaihu capsules was developed as an example. Different method evaluation indicators were used to characterize this method, and these indicators were measured at different stages of quantitative fingerprint development. Through this method, the difficulty in determining common peaks in the early stage of quantitative fingerprint development is eliminated. The method parameters that needed to be optimized were the mobile phase gradient conditions, column temperature and flow rate through the Ishikawa diagram. Single-factor experiments were used to determine some parameters. Definitive screening design (DSD) experiments were used to optimize critical method parameters (CMPs). The common peaks were determined, and indices were optimized. The critical method attributes (CMAs) were the resolution of guanosine, the percentage of common peak to the total peak area, retention time of the last peak and the common peak number. A stepwise regression method was used to build quantitative models for critical method attributes and critical method parameters. The method operable design region (MODR) was calculated by the experimental error simulation method and successfully verified. After the analysis conditions were optimized, four components, uridine, adenine, 5-hydroxymethylfurfural, and guanosine, were quantitatively determined. There were 7 common peaks in the fingerprints. The common peak area accounted for 90.64%. Both fingerprint and quantitative detection analysis methods were methodologically validated. The new AQbD process proposed in this study can be potentially applied to develop other Chinese medicines fingerprint methods.

Optimization of the Crystallization Process of TFA-MOD ErBCO Films on IBAD-Substrate Under Low-Pressure Conditions via DSD Approach

Crystallization in a sub-atmospheric reactor could achieve higher production rate while dramatically reducing the flow rate of high-purity carrier gas. Due to enhanced supersaturation conditions at low total pressure, it is necessary to find the optimum processing window to avoid misoriented growth. This is extensive and incomplete when using one-factor-at-a-time experiments as significant parameters are often correlated. <p xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">We prepared ErBa₂Cu₃O_7-<i>_δ</i> (ErBCO) films by the TFA-MOD route with low-fluorine solutions on technical IBAD template with CeO₂ top buffer layer in an industrial reel-to-reel furnace. We used a novel design-of-experiment (DOE) technique, the Definitive Screening Design (DSD), to identify significant factors for improving critical temperature and microstructure of the films. Within the investigated range, lower crystallization temperature as well as lower total pressure are beneficial. With the new process window, the self‑field critical current density at 77 K was successfully increased from almost zero to 1 MA/cm². This shows that DSD is an attractive approach to optimize the CSD process under low-pressure conditions to enhance the growth rate or possibly achieve a reduction of carrier gas flow rate by a factor <i>p</i>_tot/<i>p</i>_atm of 3.3 for equal output as the atmospheric reactor. Further improvement of <i>J</i>_c is necessary and possible.

Optimization and synthesis of a novel sorbent composite based on magnetic chitosan-amine-functionalized bimetallic MOF for the simultaneous dispersive solid-phase microextraction of four aflatoxins in real water, herbal distillate, and food samples.

Aflatoxins (AFs), an important category of pollutants, are formed in many foods and adversely affect human health. Therefore, their determination is critical to ensuring human food health. An efficient dispersive solid-phase microextraction technique was developed as a simple and straightforward sample preparation technique for determination of four aflatoxins using a high-performance liquid chromatography (HPLC) fluorescence detector. A novel efficient, green sorbent for extracting AFs was synthesized based on hydrothermal and chemical strategies. The amounts of three sorbent components were optimized using a mixture design (simplex lattice design), including 14 experiments. The optimal amount of amino-bimetallic Fe/Ni-MIL-53 nanospheres, chitosan, and magnetic Fe3O4 nanoparticles as sorbent components was 0.87, 0.67, and 0.47 g, respectively. Also, various factors affecting the process of AF determination were studied and optimized in two successive experimental designs, including the definitive screening design and the Box-Behnken design. Under optimal conditions, the linear ranges for measuring aflatoxin B1, aflatoxin B2, aflatoxin G1, and aflatoxin G2 were 0.05-82.6, 0.07-86.4, 0.08-85.7, and 0.07-89.5 ng mL-1, respectively. The relative standard deviations under inter-day and intra-day conditions for measuring AFs at three analyte concentrations were determined in triplicate analysis and were in the ranges of 3.7-4.6% and 4.9-6.1% for water sample analysis, respectively. The qualitative detection limits for determining AFs were between 0.01 and 0.05 ng mL-1. The pre-concentration factor of the method for measuring AFs ranged from 739.7 to 802.1. The proposed method was used for determining AFs in several real samples, including herbal distillate, black tea, corn, and real water samples. The relative recovery and standard deviation were 87.8-97.8% and 4.10-6.82%, respectively.

Efficient optimization approaches for microwave assisted extraction of high-quality antioxidant compounds from Salvia officinalis L.: UHPLC-HRMS differential analysis of phenolic profiles obtained by ultrasound and microwave extraction

The study aims to optimize MAE of total phenolic compounds (TPC) and antioxidant capacity from Salvia officinalis L. leaves using a definitive screening design (DSD) and I-optimal design. UHPLC-HRMS analysis was used to identify and compare the composition of MAE and UAE optimal extracts. The results showed that DSD and I-optimal design were successfully applied for the optimization of MAE targeting phenolics and other antioxidants from S. officinalis L. with the following optimum conditions: 60% ethanolic solvent, time of 4.75 min, power of 600 W, and L/S ratio of 50 mL g−1. The UHPLC-MS analysis results allowed the identification of more than 80 compounds, and the differential analysis indicated that the MAE yielded a higher level of 181 substance peaks, while the UAE yielded a higher level of 87 substance peaks. This study provides valuable information for selecting the appropriate extraction technique when targeting specific compounds.

Optimizing the Functional Properties of Starch-Based Biodegradable Films.

A definitive screening design was used in order to evaluate the effects of starch, glycerol and montmorillonite (MMT) concentrations, as well as the drying temperature, drying tray type and starch species, on packaging film's functional properties. Optimization showed that in order to obtain films with the minimum possible thickness, the maximum elongation at break, the maximum tensile strength, as well as reduced water vapor permeability and low opacity, a combination of factors should be used as follows: 5.5% wt starch concentration, 30% wt glycerol concentration on a dry starch basis, 10.5% wt MMT concentration on a dry starch basis, 45 °C drying temperature, chickpea as the starch species and plexiglass as the drying tray type. Based on these results, starch films were prepared, and fresh minced meat was stored in them for 3 days. It was shown that the incorporation of MMT at 10.5% wt on a dry starch basis in the packaging films led to a decreased mesophilic and psychrotrophic bacteria growth factor compared to commercial packaging. When assessed for their biodegradability, the starch films disintegrated after 10 days of thermophilic incubation under simulated composting conditions. Finally, to prove their handling capability during industrial production, the starch films were rewound in a paper cylinder using an industrial-scale rewinding machine.

Development of probiotic-loaded calcium alginate-maltodextrin microparticles based on electrohydrodynamic technique

Maintaining the viability of probiotic bacteria in food products for intestinal delivery remains a challenge. Consequently, the objective of this study was to encapsulate Lactobacillus rhamnosus (NCDC 18) in calcium alginate–maltodextrin hydrogel systems by combining electrospraying and freeze-drying techniques. The microparticles were produced using varying concentrations of sodium alginate (SA) (2, 3, and 4%) in conjunction with maltodextrin (MD) (0, 2, and 4%). A definitive screening design (DSD) was used to design experiments and explain the effect of independent variables (concentrations of SA, calcium chloride, and MD and electrospraying voltage) on the response variables: loss of viability, encapsulation efficiency, drying yield, Carr's index, and Hausner ratio. The obtained microparticles were characterized by employing Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), and Differential Scanning Calorimetry (DSC). The addition of MD significantly increased encapsulation efficiency up to 75.19% and decreased viability loss up to 2.539 log CFU/mL in microparticles. The morphological analysis revealed the particle surface structure and variation in porosity in response to varying variables. The FTIR spectra confirmed that the addition of SA, MD, and probiotics caused synergistic changes in the functional bonding. The XRD analysis could further elucidate the interactions of SA with MD in the microcapsules. Our study could confirm that MD acted as a cryoprotectant and decreased the concavities and pores on the surface of the microparticles, thereby enhancing their probiotic-related properties.

The synergetic effect from the combination of different adsorption resins in batch and semi-continuous cultivations of S. Cerevisiae cell factories to produce acetylated Taxanes precursors of the anticancer drug Taxol.

In situ product recovery is an efficient way to intensify bioprocesses as it can perform adsorption of the desired natural products in the cultivation. However, it is common to use only one adsorbent (liquid or solid) to perform the product recovery. For this study, the use of an in situ product recovery method with three combined commercial resins (HP-20, XAD7HP, and HP-2MG) with different chemical properties was performed. A new yeast strain of Saccharomyces cerevisiae was engineered using CRISPR Cas9 (strain EJ2) to deliver heterologous expression of oxygenated acetylated taxanes that are precursors of the anticancer drug Taxol ® (paclitaxel). Microscale cultivations using a definitive screening design (DSD) were set to get the best resin combinations and concentrations to retrieve high taxane titers. Once the best resin treatment was selected by the DSD, semi-continuous cultivation in high throughput microscale was performed to increase the total taxanes yield up to 783 ± 33 mg/L. The best T5α-yl Acetate yield obtained was up to 95 ± 4 mg/L, the highest titer of this compound ever reported by a heterologous expression. It was also observed that by using a combination of the resins in the cultivation, 8 additional uncharacterized taxanes were found in the gas chromatograms compared to the dodecane overlay method. Lastly, the cell-waste reactive oxygen species concentrations from the yeast were 1.5-fold lower in the resin's treatment compared to the control with no adsorbent aid. The possible future implications of this method could be critical for bioprocess intensification, allowing the transition to a semi-continuous flow bioprocess. Further, this new methodology broadens the use of different organisms for natural product synthesis/discovery benefiting from clear bioprocess intensification advantages.