Process mapping and optimization study of CHO cell cultures for mAb production using Ambr® 250 high-throughput parallel bioreactors.

Chinese hamster ovary (CHO) cells are widely used for manufacturing antibody drugs. We attempted to clone a novel high-expression promoter for producing monoclonal antibodies (mAbs) based on transcriptome analysis to enhance the transcriptional abundance of mAb genes. The efficacy of conventional promoters such as CMV and hEF1α decrease in the latter phase of fed-batch cell culture. To overcome this, we screened genes whose expression was maintained or increased throughout the culture period. Since CHO cells have diverse genetic expression depending on the selected clone and culture medium, transcriptome analysis was performed on multiple clones and culture media anticipated to be used in mAb manufacturing. We thus acquired the Hspa5 promoter as a novel high-expression promoter, which uniquely enables mAb productivity per cell to improve late in the culture period. Productivity also improved for various IgG subclasses under Hspa5 promoter control, indicating this promoter’s potential universal value for mAb production. Finally, it was suggested that mAb production with this promoter is correlated with the transcription levels of endoplasmic reticulum stress-related genes. Therefore, mAb production utilizing the Hspa5 promoter might be a new method for maintaining protein homeostasis and achieving stable expression of introduced mAb genes during fed-batch culture.

The present research investigates the effect of turning environments and parameters on surface integrity induced by turning of AA6061-T6. Specifically, the evolution of turning parameters of cutting speed, feed rate, and depth of cut was studied under dry, minimum quantity lubrication (MQL), and wet environments and their impact on surface roughness and residual stresses, as critical parameters for surface integrity, were quantified. Machining tests were carried out using a design of experiment (DoE) based on central composite design (CCD) under the three turning environments. The most effective turning parameters were identified for each environment using analysis of variance (ANOVA). Response surface method (RSM) was used to predict effective regression models for each turning environment for the average arithmetic surface roughness, the height peak from the valley, the axial surface residual stress, and the hoop surface residual stress. Then, using the predictive regression models, a multi-objective optimization study was performed to determine optimal turning parameters in each environment for improved surface integrity in low speed turning (LST) and high speed turning (HST) of AA6061-T6. The results showed that feed rate and cutting speed were the most effective parameters on surface integrity. It was also found that better surface integrity and higher material removal rate (MRR) were achieved using MQL mode, which is an environmental friendly and cost-saving turning environment.

Small molecule epigenetic modulators that modify epigenetic states in cells are useful tools for regulating gene expression by inducing chromatin remodeling. To identify small molecule epigenetic modulators that enhance recombinant protein expression in Chinese hamster ovary (CHO) cells, we examined eight histone deacetylase inhibitors (iHDACs) and six DNA methyltransferase inhibitors as chemical additives in recombinant CHO (rCHO) cell cultures. Among these, a benzamide-based iHDAC, CI994, was the most effective in increasing monoclonal antibody (mAb) production. Despite suppressing cell growth, the addition of CI994 to mAb-expressing GSR cell cultures at 10 μM resulted in a 2.3-fold increase in maximum mAb concentration due to a 3.0-fold increase in specific mAb productivity (qmAb ). CI994 increased mAb messenger RNA levels and histone H3 acetylation in GSR cells, and chromatin immunoprecipitation-quantitative polymerase chain reaction analysis revealed that CI994 significantly increased the histone H3 acetylation level at the cytomegalovirus promoter driving mAb gene expression, indicating that chromatin remodeling in the promoter region results in enhanced mAb gene transcription and qmAb . Similar beneficial effects of CI994 on mAb production were observed in mAb-expressing CS13-1.00 cells. Collectively, our findings indicate that CI994 increases mAb production in rCHO cell cultures by chromatin remodeling resulting from acetylation of histones in the mAb gene promoter.

A design of experiment (DOE) is used in many industrial sectors in the development and optimization of manufacturing processes. DOE is a more effective way to determine the impact of two or more factors on a response. In this paper, a Two-Level Fractional Factorial design was employed to develop the important factors that significantly affect new promising OT Rubber recycling machine using a DOEs analysis. Central composite design (CCD) and response surface methodology (RSM) were used to optimize the isolating screen size. The CCD considered five factors with two-level full factorial design. CCD experiments using RSM was proved to be an optimal tool for optimization.

In this study, we present a hybrid modeling framework that integrates piecewise Partial Least Squares (PLS) regression with Dynamic Flux Balance Analysis (dFBA) to simulate and optimize Chinese Hamster Ovary (CHO) cell fed-batch culture. Twenty-four Ambr15 experiments were conducted to systematically vary feed and inoculum compositions. Time-resolved metabolite, biomass, and Monoclonal antibodies (mAb) concentrations were collected and modeled. The hybrid model achieved high prediction accuracy (Normalized Mean Squared Error (NMSE) < 0.15 for most metabolites) and provided interpretable flux profiles. Multivariate analysis revealed consistent metabolic signatures tied to media formulation, where specific feed–inoculum combinations drove shifts in glycolysis, TCA cycle flux, and nitrogen metabolism. These insights demonstrate the model’s capacity to capture key metabolic adaptations and support data-driven media optimization in CHO cell culture. • A piecewise hybrid model developed and validated for CHO fed-batch culture. • Bioreactor experiments were performed with 24 different media blends. • A balanced production\consumption of key metabolites results in higher mAb titer. • Specific feed–inoculum blends drive shifts in glycolytic and TCA cycle fluxes. • Hybrid model results in good predictions for varying media composition (NMSE<0.15).

The aim of this research is to study the machinability aspects of hardened AISI 4340 High Strength Low Alloy (HSLA) steel (50 ± 2 HRC (Hardness Rockwell C)). The experimental investigation using coated carbide inserts is carried out during the dry hard milling process in a sustainable environment. The input parameters in the study are speed, feed rate and depth of cut and the responses are Average surface Roughness (Ra) and Material Removal Rate (MRR) that are selected through screening. Central Composite Design (CCD) in response surface methodology has been utilized as the experimental design technique with twenty experiments. Analysis of variance has been employed to examine the momentous machining parameters and responses. A mathematical model has been developed to optimize the surface roughness and material removal rate. It has been observed that the most significant factor for Ra is feed rate while for MRR depth of cut is the most significant factor. The results show that the minimum value of Ra ~ 0.098 μm is achieved at speed ~ 1000 RPM, feed rate ~ 300 mm/min and depth of cut ~ 0.2 mm while the maximum value of MRR ~ 6.35 cm3/min is attained at feed rate ~ 500mm/min and depth of cut ~ 0.4 mm regarding less or no effect of speed ~ 500-1000 RPM. The average forecast error for the validation information has been observed to be 3.35%. for Ra and 3.2% for MRR. Further, it is investigated that good surface finish like grinding and dimensional accuracy can be achieved with coated carbide tools.

Recent optimizations of cell culture processes have focused on the final seed scale-up step (N - 1 stage) used to inoculate the production bioreactor (N-stage bioreactor) to enable higher inoculation cell densities (2-20 × 106 cells/mL), which could shorten the production culture duration and/or increase the volumetric productivity. N - 1 seed process intensification can be achieved by either non-perfusion (enriched-batch or fed-batch) or perfusion culture to reach those higher final N - 1 viable cell densities (VCD). In this study, we evaluated how different N - 1 intensification strategies, specifically enriched-batch (EB) N - 1 versus perfusion N - 1, affect cell growth profiles and monoclonal antibody (mAb) productivity in the final N-stage production bioreactor operated in fed-batch mode. Three representative Chinese Hamster Ovary (CHO) cell lines producing different mAbs were cultured using either EB or perfusion N - 1 seeds and found that the N-stage cell growth and mAb productivities were comparable between EB N - 1 and perfusion N - 1 conditions for two of the cell lines but were very different for the third. In addition, within the two similar cell growth cell lines, differences in cell-specific productivity were observed. This suggests that the impact of the N - 1 intensification process on production was cell-line dependent. This study revealed that the N - 1 intensification strategy and the state of seeds from the different N - 1 conditions may affect the outcome of the N production stage, and thus, the choice of N - 1 intensification strategy could be a new target for future upstream optimization of mAb production.

Dynamic Simulation and Visualisation of pH-Modulated Fed-batch Fermentation for mAb Production from CHO Cell Cultures

Chinese hamster ovary (CHO) cells remain the industry standard for producing numerous therapeutic proteins, particularly monoclonal antibodies (mAbs). However, achieving higher recombinant protein titers remains an ongoing challenge and a fundamental understanding of the cellular mechanism driving improved bioprocess performance remains elusive. To directly address these challenges and achieve substantial improvements, a more in-depth understanding of cellular function within a bioprocess environment may be required. Over the past decade, significant advancements have been made in the building of genome-scale metabolic models (GEMs) for CHO cells, bridging the gap between high information content 'omics data and the ability to perform in silico phenotypic predictions. Here, time-course transcriptomics has been employed to constrain culture phase-specific GEMs, representing the early exponential, late exponential, and stationary/death phases of CHO cell fed-batch bioreactor culture. Temporal bioprocess data, including metabolite uptake and secretion rates, as well as growth and productivity, has been used to validate flux sampling results. Additionally, high mAb-producing solutions have been identified and the metabolic signatures associated with improved mAb production have been hypothesized. Finally, constraint-based modeling has been utilized to infer specific amino acids, cysteine, histidine, leucine, isoleucine, asparagine, and serine, which could drive increased mAb production and guide optimal media and feed formulations.

Optimization of environmentally benign micro-drilling process with nanofluid minimum quantity lubrication using response surface methodology and genetic algorithm

The machinability and tribological characteristics of aluminum alloys with improved elevated temperature properties using rapidly solidified powder

Reducing the temperature rise during turning operation improves the quality of the product and reduces tool wear. Experiments are conducted as per the Design of Experiments (DoE) of Response Surface Methodology (RSM) to predict the temperature rise by varying the cutting parameters such as cutting speed, feed rate and depth of cut. In the present study, the experiment was conducted on Aluminium Al 6061 by coated carbide tool. A second order mathematical model in terms of machining parameters was developed for temperature rise prediction using RSM. This model gives the factor effects of the individual process parameters. Values of Prob> F less than 0.05 indicate model terms are significant. The cutting speed is the most important parameter that cause the temperature of the turning process compared to the other factors such as feed rate and depth of cut. Validation results show good agreement between the actual process output and the predicted temperature rise.

Introduction: Among alloys of medium-carbon and high-strength steel, EN 24 steel is characterised by its nickel-chromium-molybdenum composition. EN 24 steel is highly suitable for application in heavy-duty projects due to its notable resilience to damage, especially when exposed to low temperatures. With the objective of minimising surface irregularities, this research endeavours to enhance the milling process of EN 24 steel by employing coated tungsten carbide (WC) tool inserts.Methods: Feed rate, cutting speed, depth of cut, and cutting fluid are all crucial process factors in the experimental investigation. Four distinct levels are applied to each factor. The research utilises the Design of Experiments (DOE)-based Central Composite Design of Response Surface Methodology. To predict output parameters, mathematical models are developed utilising analysis of variance (ANOVA) for optimisation purposes.Results and discussions: Through the utilisation of multi-objective optimisation, the optimal combination for tungsten carbide inserts was determined, which provided surface irregularities of 0.301 µm. Cutting speed (CS) of 149.507 m/min, feed rate (FR) of 340.27 mm/min, depth of cut (DOC) of 0.599 mm, and cutting fluid (CF) of 12.50 L/min are the optimal parameters. The surface morphologies of the machined workpiece at particular parameter values can be discerned through scanning electron microscope (SEM) analysis, yielding significant insights. The optimal parameters that have been identified provide practical recommendations for improving the milling method of EN 24 steel when tungsten carbide inserts are utilised. Understanding the milling process in its entirety is facilitated by SEM analysis of surface morphologies and microstructures under particular cutting conditions. The morphology and surface irregularities of the machined workpiece are evaluated using profilometry, which provides additional insight into surface integrity. The discourse investigates the potential applications and implications of the results, as well as suggests directions for further study concerning the enhancement of milling processes for similar steel alloys.

Discharge of heavy metals into the environment by wastewater from production and application processes is nowadays getting more and more serious due to the rapid development of such processes. Produced water during oil production and wastewater form metal plating and ore dressing are few examples to mention. In this research, removal of chromium ions from aqueous solutions has been studied by foam fractionation process using cetyl trimethyl ammonium bromide as surfactant. Effect of processing time, gas flow rate, initial surfactant concentration, and initial pH on removal efficiency has been investigated. Preliminary screening tests were performed, and initial surfactant concentration and processing time were recognized as the variables with a significant effect on removal efficiency. Response surface methodology based on central composite design as a design of experiment method was employed to design experiments to study the effect of important variables more precisely. Five levels for each significant variable were considered and design of experiment. An accurate correlative model is developed for the estimation of removal efficiency under different operating conditions in foam fractionation process with a maximum relative error of 1.674%. Four sets of validation tests were also conducted, and the results of validation tests were shown to be in accordance with those obtained on tests dictated by design of experiment. Optimum operating conditions were found to be with an initial surfactant concentration of 175 ppm and 45 min of processing time that yielded 97.74% removal of Cr(VI) from a solution of 10 ppm initial concentration.

It is well known that machining parameters have strong effect on the properties of surface roughness. Many investigations have done on this area. This work studies of combining effect of turning process parameters (cutting speed, feed rate, and depth of cut) with on surface roughness. Where, nose radius of the tool and machining parameters have taken as input variables and the surface roughness (Ra) as response or output. Three experiments were conducted; they were used to investigate the surface roughness resulted by tool corner radiuses of the values: 0.4mm, 0.8mm, and 1.2mm Response surface methodology (R.S.M) is applied as a tool to show the cause and effect of output (response) and input control and relationship between them as a two or three dimensional hyper surface. A three factor with five level central composite rotatable factors design was used. The results showed that R.S.M is a strong tool and capability tool to predict the effect of machining parameters on surface roughness. Results improved that the five level factorial designs can be employed for developing statistical models to predict surface roughness by controllable machining parameters. Results showed that the combined effect of cutting speed at its higher level, feed rate and depth of cut at their lower values, and large nose radius can result in better surface roughness.