RESUMEN
The pharmaceutical industry employs various strategies to improve cell productivity. These strategies include process intensification, culture media improvement, clonal selection, media supplementation and genetic engineering of cells. However, improved cell productivity has inherent risk of impacting product quality attributes (PQA). PQAs may affect the products' efficacy via stability, bioavailability, or in vivo bioactivity. Variations in manufacturing process may introduce heterogeneity in the products by altering the type and extent of N-glycosylation, which is a PQA of therapeutic proteins. We investigated the effect of different cell densities representing increasing process intensification in a perfusion cell culture on the production of an IgG1-κ monoclonal antibody from a CHO-K1 cell line. This antibody is glycosylated both on light chain and heavy chain. Our results showed that the contents of glycosylation of IgG1-κ mAb increased in G0F and fucosylated type glycans as a group, whereas sialylated type glycans decreased, for the mAb whole protein. Overall, significant differences were observed in amounts of G0F, G1F, G0, G2FS1, and G2FS2 type glycans across all process intensification levels. G2FS2 and G2 type N-glycans were predominantly quantifiable from light chain rather than heavy chain. It may be concluded that there is a potential impact to product quality attributes of therapeutic proteins during process intensification via perfusion cell culture that needs to be assessed. Since during perfusion cell culture the product is collected throughout the duration of the process, lot allocation needs careful attention to process parameters, as PQAs are affected by the critical process parameters (CPPs). KEY POINTS: ⢠Molecular integrity may suffer with increasing process intensity. ⢠Galactosylated and sialylated N-glycans may decrease. ⢠Perfusion culture appears to maintain protein charge structure.
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Anticuerpos Monoclonales , Inmunoglobulina G , Cricetinae , Animales , Células CHO , Cricetulus , Perfusión , Polisacáridos/químicaRESUMEN
Gene therapy is a promising therapeutic approach for genetic and acquired diseases nowadays. Among DNA delivery vectors, recombinant adeno-associated virus (rAAV) is one of the most effective and safest vectors used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing lags behind the necessary dosages for clinical and commercial use, which embodies a concentrated reflection of low productivity of rAAV from host cells, difficult scalability of the rAAV-producing bioprocess, and high levels of impurities materialized during production. Those issues directly impact the price of gene therapy medicine in the market, limiting most patients' access to gene therapy. In this context, the current practices and several critical challenges associated with rAAV gene therapy bioprocesses are reviewed, followed by a discussion of recent advances in rAAV-mediated gene therapy and other therapeutic biological fields that could improve biomanufacturing if these advances are integrated effectively into the current systems. This review aims to provide the current state-of-the-art technology and perspectives to enhance the productivity of rAAV while reducing impurities during production of rAAV.
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Dependovirus , Vectores Genéticos , Humanos , Dependovirus/genética , Vectores Genéticos/genética , Terapia GenéticaRESUMEN
Previously, we identified six inhibitory metabolites (IMs) accumulating in Chinese hamster ovary (CHO) cultures using AMBIC 1.0 community reference medium that negatively impacted culture performance. The goal of the current study was to modify the medium to control IM accumulation through design of experiments (DOE). Initial over-supplementation of precursor amino acids (AAs) by 100% to 200% in the culture medium revealed positive correlations between initial AA concentrations and IM levels. A screening design identified 5 AA targets, Lys, Ile, Trp, Leu, Arg, as key contributors to IMs. Response surface design analysis was used to reduce initial AA levels between 13% and 33%, and these were then evaluated in batch and fed-batch cultures. Lowering AAs in basal and feed medium and reducing feed rate from 10% to 5% reduced inhibitory metabolites HICA and NAP by up to 50%, MSA by 30%, and CMP by 15%. These reductions were accompanied by a 13% to 40% improvement in peak viable cell densities and 7% to 50% enhancement in IgG production in batch and fed-batch processes, respectively. This study demonstrates the value of tuning specific AA levels in reference basal and feed media using statistical design methodologies to lower problematic IMs.
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Aminoácidos , Técnicas de Cultivo Celular por Lotes , Cricetinae , Animales , Cricetulus , Aminoácidos/metabolismo , Células CHO , Medios de Cultivo/química , Técnicas de Cultivo Celular por Lotes/métodosRESUMEN
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.
RESUMEN
Due to the favorable attributes of Chinese hamster ovary (CHO) cells for therapeutic proteins and antibodies biomanufacturing, companies generate proprietary cells with desirable phenotypes. One key attribute is the ability to stably express multi-gram per liter titers in chemically defined media. Cell, media, and feed diversity has limited community efforts to translate knowledge. Moreover, academic, and nonprofit researchers generally cannot study "industrially relevant" CHO cells due to limited public availability, and the time and knowledge required to generate such cells. To address these issues, a university-industrial consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) has acquired two CHO "reference cell lines" from different lineages that express monoclonal antibodies. These reference cell lines have relevant production titers, key performance outcomes confirmed by multiple laboratories, and a detailed technology transfer protocol. In commercial media, titers over 2 g/L are reached. Fed-batch cultivation data from shake flask and scaled-down bioreactors is presented. Using productivity as the primary attribute, two academic sites aligned with tight reproducibility at each site. Further, a chemically defined media formulation was developed and evaluated in parallel to the commercial media. The goal of this work is to provide a universal, industrially relevant CHO culture platform to accelerate biomanufacturing innovation.
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Anticuerpos Monoclonales , Reactores Biológicos , Cricetinae , Animales , Cricetulus , Células CHO , Reproducibilidad de los Resultados , Técnicas de Cultivo Celular por Lotes/métodosRESUMEN
This work presents a compact model for the equipment capability limit of a common configuration of pharmaceutical lyophilizers, a product chamber separated from the condenser by a duct and isolation valve, at a wide range of design parameters. The equipment capability limit is one of the most important characteristics determining the lyophilization design space for a particular product, container, and equipment combination. Experimental measurements of equipment capability are time-consuming and expensive, especially at the production scale. Numerical modeling using computational fluid dynamics may reduce the number of experiments and provide insights into the physics of the process with high resolution. The computational fluid dynamics (CFD) modeling has been used in this work to develop a compact model for lyophilizer equipment capability. This eliminates the need for end users to create a full CFD model of the equipment and process. Full CFD and compact model simulations for laboratory and pilot-scale lyophilizers have been compared with tunable diode laser absorption spectroscopy measurements of the water vapor mass flow during ice slab tests. The compact model results average deviation from the experimental data is within 10%, whereas the full CFD simulations are within 5%. The compact model is based on several key parameters which are the main characteristics of a lyophilizer affecting the equipment capability curve. These parameters are discussed, and their effect on the modeling results is shown.
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Hidrodinámica , Tecnología Farmacéutica , Diseño de Equipo , Liofilización , Análisis EspectralRESUMEN
In this study, the novel high-speed tablet film coating process in the continuous manufacturing was investigated. The influence of key process variables (inlet air flow rate, inlet air temperature, and suspension spray rate) were investigated using a Box-Behnken experimental design method. Statistical regression models were developed to predict the outlet air temperature and relative humidity, the coating efficiency, the tablet moisture content, and coating uniformity. The effects of the three key process variables were comprehensively investigated based on mathematical analysis, contour plots, and interaction plots. The results indicate that all the process responses are affected by changing the inlet air flow rate, temperature, and suspension spray rate. A design space (DS) in terms of failure probability was determined based on specifications for tablet moisture content (< 3.5%) and coating uniformity (tablet weight standard deviation < 4 mg for tablet weight of 200 mg) using Monte Carlo simulations. Independent experiments were carried out and successfully validated the robustness and accuracy of the determined DS for the investigated tablet film coating process. All the data were generated using an industrial pilot-scale novel high-speed tablet coating unit from a continuous manufacturing line. The work facilitates the quality by design implementation of continuous pharmaceutical manufacturing.
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Composición de Medicamentos/métodos , Comprimidos , Modelos Estadísticos , Proyectos Piloto , TemperaturaRESUMEN
Freeze-drying is a deceptively complex operation requiring sophisticated design of a robust and efficient process that includes understanding and planning for heterogeneity across the batch and shifts in parameters due to vial or lyophilizer changes. A software tool has been designed to assist in process development and scale-up based on a model that includes consideration of the process heterogeneity. Two drug formulations were used to test the ability of the new tool to develop a freeze-drying cycle and correctly predict product temperatures and drying times. Model inputs were determined experimentally, and the primary drying heterogeneous freeze-drying model was used to design drying cycles that provided data to verify the accuracy of model-predicted product temperature and primary drying time. When model inputs were accurate, model-predicted primary drying times were within 0.1 to 15.9% of experimentally measured values, and product temperature accuracy was between 0.2 and 1.2°C for three vial locations, center, inner edge, and outer edge. However, for some drying cycles, differences in vial heat transfer coefficients due to changes in shelf and product temperature as well as altered product resistance due to product temperature-dependent microcollapse increased inaccuracy (up to 28.6% difference in primary drying time and 5.1°C difference in product temperature). This highlights the need for careful determination of experimental conditions used to calculate model inputs. In future efforts, full characterization of location- and shelf temperature-dependentKv as well as location- and product temperature-dependentRp will enhance the accuracy of the predictions by the model within the user-friendly software.
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Desecación , Laboratorios , Liofilización , Programas Informáticos , Tecnología Farmacéutica , TemperaturaRESUMEN
N-Linked glycosylation is a cellular process transferring sugars from glycosyl donors to proteins or lipids. Biopharmaceutical products widely produced by culturing mammalian cells such as Chinese hamster ovary (CHO) cells are typically glycosylated during biosynthesis. For some biologics, the N-linked glycan is a critical quality attribute of the drugs. Nucleotide sugars are the glycan donors and impact the intracellular glycosylation process. In current analytical methods, robust separation of nucleotide sugar isomers such as UDP glucose and UDP galactose remains a challenge because of their structural similarity. In this study, we developed a strategy to resolve the separation of major nucleotide sugars including challenging isomers based on the use of ion-pair reverse phase (IP-RP) chromatography. The strategy applies core-shell columns and connects multiple columns in tandem to increase separation power and ultimately enables high-resolution detection of nucleotide sugars from cell extracts. The key parameters in the IP-RP method, including temperature, mobile phase, and flow rates, have been systematically evaluated in this work and the theoretical mechanisms of the chromatographic behavior were proposed. Graphical abstract.
Asunto(s)
Cromatografía de Fase Inversa/métodos , Azúcares de Nucleósido Difosfato/análisis , Animales , Células CHO , Cricetulus , Glicosilación , Isomerismo , Azúcares de Nucleósido Difosfato/aislamiento & purificaciónRESUMEN
The variability of trace metals in cell culture media is a potential manufacturing concern because it may significantly affect the production and quality of therapeutic proteins. Variability in trace metals in CHO cell culture has been shown to impact critical production metrics such as cell growth, viability, nutrient consumption, and production of recombinant proteins. To better understand the influence of excess supplementation, zinc and copper were initially supplemented with 50-µM concentrations to determine the impact on the production and quality of ß-glucuronidase, a lysosomal enzyme, in a parallel bioreactor system. Ethylenediaminetetraacetic acid (EDTA), a metal chelator, was included as another treatment to induce a depletion of trace metal bioavailability to examine deficiency. Samples were drawn daily to monitor cell growth and viability, nutrient levels, ß-glucuronidase activity, and trace zinc flux. Cell cycle analysis revealed the inhibition of sub-G0/G1 species in zinc supplemented cultures, maintaining higher viability compared to the control, EDTA-, and copper-supplemented cultures. Enzyme activity analysis in the harvests revealed higher specific activity of ß-glucuronidase in reactors supplemented with zinc. A confirmation run was conducted with supplementations of zinc at concentrations of 50, 100, and 150 µM. Further cell cycle analysis and caspase-3 analysis demonstrated the role of zinc as an apoptosis suppressor responsible for the enhanced harvest purity of ß-glucuronidase from zinc-supplemented bioreactors.
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Apoptosis/efectos de los fármacos , Medios de Cultivo/química , Glucuronidasa/biosíntesis , Zinc/farmacología , Animales , Técnicas de Cultivo Celular por Lotes , Reactores Biológicos , Células CHO , Técnicas de Cultivo de Célula , Proliferación Celular/efectos de los fármacos , Cobre/farmacología , CricetulusRESUMEN
Continuous manufacturing of oral-dosage drug products is increasing the need for rigorous process understanding both from a process design and control perspective. The purpose of this study is to develop a methodology that analyzes the effects of upstream process parameters on continuous tablet compaction and then correlates associated upstream variables to the final tablet attributes (e.g. relative density and hardness). The impact of three process parameters (system throughput, blender speed, and compaction force) on tablet attributes is investigated using a full factorial experimental design. As expected, the compaction force was found to be the most significant process parameter. However, importantly, throughput was discovered to have a non-negligible impact which was previously unaccounted for. This impact is proposed to be related to differing levels of powder pre-compression. An empirical model for this relationship is regressed and incorporated into a flowsheet model. The flowsheet model is then used to develop an in silico design space which is compared favorably to that built from experiments. Moreover, in the future, the in silico design space based on the validated flowsheet model can provide better manufacturing flexibility and make control strategy development simpler.
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Química Farmacéutica/métodos , Modelos Estadísticos , Modelos Teóricos , Tecnología Farmacéutica/métodos , Simulación por Computador , Composición de Medicamentos/métodos , Dureza , Fenómenos Mecánicos , Preparaciones Farmacéuticas/administración & dosificación , Preparaciones Farmacéuticas/química , Polvos , Presión , ComprimidosRESUMEN
Trace metals are supplied to chemically-defined media (CDM) for optimal Chinese hamster ovary (CHO) cell culture performance during the production of monoclonal antibodies and other therapeutic proteins. However, lot-to-lot and vendor-to-vendor variability in raw materials consequently leads to an imbalance of trace metals that are supplied to CDM. This imbalance can yield detrimental effects rooted in several primary mechanisms and pathways including oxidative stress, apoptosis, lactate accumulation, and unfavorable glycan synthesis. Recent research endeavors involve supplying zinc, copper, and manganese to CDM in excess to further maximize culture productivity and product quality. These treatments significantly impact critical quality attributes and furthermore highlight the degree to which trace metal availability can affect CHO cell culture performance. This review highlights the role of trace metal variability, supplementation, and interplay on key cellular mechanisms responsible for overall culture performance and the production and quality of therapeutic proteins.
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Técnicas de Cultivo de Célula , Medios de Cultivo , Oligoelementos , Animales , Anticuerpos Monoclonales/biosíntesis , Células CHO , Cricetinae , Cricetulus , Medios de Cultivo/química , Medios de Cultivo/farmacología , Oligoelementos/química , Oligoelementos/farmacologíaRESUMEN
In this study, a novel three-compartmental population balance model (PBM) for a continuous twin screw wet granulation process is developed, combining the techniques of PBM and regression process modeling. The developed model links screw configuration, screw speed, and blend throughput with granule properties to predict the granule size distribution (GSD) and volume-average granule diameter. The granulator screw barrel was divided into three compartments along barrel length: wetting compartment, mixing compartment, and steady growth compartment. Different granulation mechanisms are assumed in each compartment. The proposed model therefore considers spatial heterogeneity, improving model prediction accuracy. An industrial data set containing 14 experiments is applied for model development. Three validation experiments show that the three-compartmental PBM can accurately predict granule diameter and size distribution at randomly selected operating conditions. Sixteen combinations of aggregation and breakage kernels are investigated in predicting the experimental GSD to best judge the granulation mechanism. The three-compartmental model is compared with a one-compartmental model in predicting granule diameter at different experimental conditions to demonstrate its advantage. The influence of the screw configuration, screw speed and blend throughput on the volume-average granule diameter is analyzed based on the developed model.
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Química Farmacéutica/métodos , Modelos Teóricos , Tecnología Farmacéutica/métodos , Tamaño de la Partícula , Reproducibilidad de los ResultadosRESUMEN
A tablet film coating and drying process was assessed by an experimentally validated thermodynamic balance model. Mass conservation equations were derived for the process air and the aqueous coating solution. Thermodynamic behavior of the solution was described by evaporation at the tablet surface and penetration into the tablet. Energy balance equations including heat loss to the atmosphere were coupled to the mass conservation equation. Experimental data using the ConsiGma™ coater (GEA, Belgium) were used for both parameter estimation and model validation. The results showed the proposed model can investigate primitive outlet variables and further internal variables representing evaporation and penetration. A sensitivity analysis revealed that evaporation depended more on the input parameters while penetration hinges on the tablet properties, particularly on the tablet volume affecting the tablet porosity.
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Química Farmacéutica/métodos , Desecación/métodos , Comprimidos Recubiertos/química , Cinética , Porosidad , Agua/químicaRESUMEN
A new integrated continuous biomanufacturing platform for continuous production of antibodies at fixed cell volumes and cell concentrations for extended periods with immediate capture is presented. Upstream antibody production has reached technological maturity, however, the bottleneck for continuous biomanufacturing remains the efficient and cost-effective capture of therapeutic antibodies in an initial chromatography step. In this study, the first successful attempt at using one-column continuous chromatography (OCC) for the continuous capture of therapeutic antibodies produced through alternating tangential flow perfusion is presented. By performing upstream media optimizations, the upstream perfusion rate was reduced to one vessel volume per day (vv/d), increasing antibody titer and reducing the volume of perfusate. In addition, process improvements were performed to increase productivity by 80% over previously reported values. In addition, a real-time method for evaluating column performance to make column switching decisions was developed. This improved productivity coupled with the use of a single-column improved process monitoring and control in OCC compared to multi-column systems. This approach is the first report on using a single column for the implementation of an integrated continuous biomanufacturing platform and offers a cost-effective and flexible platform process for the manufacture of therapeutic proteins.
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Anticuerpos Monoclonales/aislamiento & purificación , Reactores Biológicos , Biotecnología/instrumentación , Cromatografía/instrumentación , Animales , Reactores Biológicos/economía , Células CHO , Recuento de Células , Tamaño de la Célula , Cromatografía/economía , Cricetulus , Perfusión/economía , Perfusión/instrumentación , Resinas Sintéticas/química , Proteína Estafilocócica A/químicaRESUMEN
A modified shrinking core model (MSCM) has been used to describe the mechanism for the degradation of Protein A resin particles taking place under continuous chromatographic operation. The model is based on the hypothetical shrinkage of the boundary layer of the resin particles, which house the active Protein A ligands within their pores. The caustic during the sanitization phase of chromatography has been determined to cause the Protein A ligand degradation. Protein A resins provided by manufacturers possess unique caustic stability, which has been used in MSCM to appraise the ligand degradation. The kinetic model utilized semiempirical parameters including diffusion constant, rate constant, stoichiometric factor, and reaction order. The parameters were estimated from column breakthrough experiments to simulate continuous Protein A chromatography for three distinct resins. The reaction order has been identified as the key parameter for predicting the degradation kinetics. The recorded reaction orders vary for three different resins with the resin B showing the highest reaction order of 4 and lowest being 1.65 for the resin C. The model can predict the effects of caustic on resin performance and displayed that minimal degradation of the resins A and B occurred, when exposed to 0.1 N and 0.2N NaOH, retaining up to 96% binding capacity after 240 cycles. The adsorption study conducted for the resin B demonstrated the dynamic physical and chemical changes transpiring through the life cycle of the resin, further supported the degradation model. The performance data demonstrate that the resin B exhibits the desirable performance, with higher reaction order indicating slower resin degradation, higher binding capacities, and increased sustenance of this binding capacity for extended duration. The degradation model can be extended to build effective cleaning strategies for continuous downstream processing.
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Cromatografía de Afinidad , Proteína Estafilocócica A/química , Staphylococcus aureus/química , Adsorción , Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía de Afinidad/métodos , Cinética , Ligandos , Modelos Biológicos , Modelos Químicos , ProteolisisRESUMEN
Continuous manufacturing techniques are increasingly being adopted in the pharmaceutical industry and powder blending is a key operation for solid-dosage tablets. A modeling methodology involving axial and radial tanks-in-series flowsheet models is developed to describe the residence time distribution (RTD) and blend uniformity of a commercial powder blending system. Process data for a six-component formulation processed in a continuous direct compression line (GEA Pharma Systems) is used to test the methodology. Impulse tests were used to generate experimental RTDs which are used along with parameter estimation to determine the number of axial tanks in the flowsheet. The weighted residual from the parameter estimation was less than the χ2 value at a 95% confidence indicating a good fit between the model and measured data. In-silico impulse tests showed the tanks-in-series modeling methodology could successfully describe the RTD behavior of the blenders along with blend uniformity through the use of radial tanks. The simulation output for both impulse weight percentage and blend uniformity were within the experimentally observed variance.
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Química Farmacéutica/métodos , Simulación por Computador , Modelos Químicos , Fuerza Compresiva , PolvosRESUMEN
Chinese hamster ovary (CHO) cells have been widely used for producing many recombinant therapeutic proteins. Constraint-based modeling, such as flux balance analysis (FBA) and metabolic flux analysis (MFA), has been developing rapidly for the quantification of intracellular metabolic flux distribution at a systematic level. Such methods would produce detailed maps of flows through metabolic networks, which contribute significantly to better understanding of metabolism in cells. Although these approaches have been extensively established in microbial systems, their application to mammalian cells is sparse. This review brings together the recent development of constraint-based models and their applications in CHO cells. The further development of constraint-based modeling approaches driven by multi-omics datasets is discussed, and a framework of potential modeling application in cell culture engineering is proposed. Improved cell culture system understanding will enable robust developments in cell line and bioprocess engineering thus accelerating consistent process quality control in biopharmaceutical manufacturing.
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Ingeniería Metabólica/métodos , Análisis de Flujos Metabólicos/métodos , Redes y Vías Metabólicas/fisiología , Modelos Biológicos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/uso terapéutico , Animales , Células CHO/metabolismo , Simulación por Computador , Cricetulus , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
In omics studies aimed at the early detection and diagnosis of cancer, bioinformatics tools play a significant role when analyzing high dimensional, complex datasets, as well as when identifying a small set of biomarkers. However, in many cases, there are ambiguities in the robustness and the consistency of the discovered biomarker sets, since the feature selection methods often lead to irreproducible results. To address this, both the stability and the classification power of several chemometrics-based feature selection algorithms were evaluated using the Monte Carlo sampling technique, aiming at finding the most suitable feature selection methods for early cancer detection and biomarker discovery. To this end, two data sets were analyzed, which comprised of MALDI-TOF-MS and LC/TOF-MS spectra measured on serum samples in order to diagnose ovarian cancer. Using these datasets, the stability and the classification power of multiple feature subsets found by different feature selection methods were quantified by varying either the number of selected features, or the number of samples in the training set, with special emphasis placed on the property of stability. The results show that high consistency does not necessarily guarantee high predictive power. In addition, differences in the stability, as well as agreement in feature lists between several feature selection methods, depend on several factors, such as the number of available samples, feature sizes, quality of the information in the dataset, etc. Among the tested methods, only the variable importance in projection (VIP)-based method shows complementary properties, providing both highly consistent and accurate subsets of features. In addition, successive projection analysis (SPA) was excellent with regards to maintaining high stability over a wide range of experimental conditions. The stability of several feature selection methods is highly variable, stressing the importance of making the proper choice among feature selection methods. Therefore, rather than evaluating the selected features using only classification accuracy, stability measurements should be examined as well to improve the reliability of biomarker discovery.
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Biomarcadores de Tumor , Detección Precoz del Cáncer/métodos , Espectrometría de Masas , Neoplasias/diagnóstico , Algoritmos , Biología Computacional/métodos , Bases de Datos Factuales , Femenino , Humanos , Neoplasias Ováricas/diagnóstico , Reproducibilidad de los Resultados , Tamaño de la MuestraRESUMEN
A new biomanufacturing platform combining intracellular metabolic engineering of the oleaginous yeast Yarrowia lipolytica and extracellular bioreaction engineering provides efficient bioconversion of plant oils/animal fats into high-value products. However, predicting the hydrodynamics and mass transfer parameters is difficult due to the high agitation and sparging required to create dispersed oil droplets in an aqueous medium for efficient yeast fermentation. In the current study, commercial computational fluid dynamic (CFD) solver Ansys CFX coupled with the MUSIG model first predicts two-phase system (oil/water and air/water) mixing dynamics and their particle size distributions. Then, a three-phase model (oil, air, and water) utilizing dispersed air bubbles and a polydispersed oil phase was implemented to explore fermenter mixing, gas dispersion efficiency, and volumetric mass transfer coefficient estimations (kL a). The study analyzed the effect of the impeller type, agitation speed, and power input on the tank's flow field and revealed that upward-pumping pitched blade impellers (PBI) in the top two positions (compared to Rushton-type) provided advantageous oil phase homogeneity and similar estimated kL a values with reduced power. These results show good agreement with the experimental mixing and kL a data.