Scale-up Studies Research Articles

Study of hydrodynamic stress in cell culture bioreactors via lattice-Boltzmann CFD simulations supported by micro-probe shear stress method

Mammalian cell cultivation in pharmaceutical industry can last up to units of weeks and requires proper transport of nutrients and oxygen for cell growth and production. Given the long time period, cells experience flow fields from all bioreactor’s zones, where the energy dissipation rate (ε) varies substantially. Shear sensitive micro-probes with size comparable to cells and Kolmogorov eddies are used for the determination of the maximum hydrodynamic stress (τmax) in bioreactors. For the very first time, the micro-probe method is applied successfully not only to laboratory (3 L) and pilot scale (80 L and 200 L), but also to industrial production scale bioreactor (12,500 L) with Rushton turbine and pitched-blade (RT-PB) impeller configuration. Experimentally obtained data are used for the validation of comprehensive CFD scale-up study, using the Lattice-Boltzmann large eddy simulation (LB-LES) method. Besides τmax, this work also focuses on the study of mixing time and flow field attributes.

Revolutionizing the Treatment of Idiopathic Pulmonary Fibrosis: From Conventional Therapies to Advanced Drug Delivery Systems.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease that has been well-reported in the medical literature. Its incidence has risen, particularly in light of the recent COVID-19 pandemic. Conventionally, IPF is treated with antifibrotic drugs-pirfenidone and nintedanib-along with other drugs for symptomatic treatments, including corticosteroids, immunosuppressants, and bronchodilators based on individual requirements. Several drugs and biologicals such as fluorofenidone, thymoquinone, amikacin, paclitaxel nifuroxazide, STAT3, and siRNA have recently been evaluated for IPF treatment that reduces collagen formation and cell proliferation in the lung. There has been a great deal of research into various treatment options for pulmonary fibrosis using advanced delivery systems such as liposomal-based nanocarriers, chitosan nanoparticles, PLGA nanoparticles, solid lipid nanocarriers, and other nanoformulations such as metal nanoparticles, nanocrystals, cubosomes, magnetic nanospheres, and polymeric micelles. Several clinical trials are also ongoing for advanced IPF treatments. This article elaborates on the pathophysiology of IPF, its risk factors, and different advanced drug delivery systems for treating IPF. Although extensive preclinical data is available for these delivery systems, the clinical performance and scale-up studies would decide their commercial translation.

Bioconversion of hemicellulosic fraction of wheat straw biomass to bioethanol by Scheffersomyces stipitis: A kLa-based scale-up study

The potent utilization of hemicellulosic fraction, which comprises one-third of carbohydrates in lignocellulosic biomass (LCB), is vital to strengthen the bioethanol production from it. Most studies on bioethanol production from LCB focused on cellulosic fraction only, or very low ethanol productivity is reported from hemicellulosic fraction. The present study investigated a process to convert the hemicellulosic fraction of wheat straw biomass to bioethanol effectively using Scheffersomyces stipitis. This work aimed to develop and demonstrate a systematic approach for scaling up the fermentation process, keeping the volumetric oxygen transfer coefficient (kLa) nearly constant in different scale bioreactors. It was found that 99.11±0.13% of synthetic xylose and 99.04±0.10% of lignocellulosic xylose were converted to bioethanol with a yield and productivity of 0.4706±0.0022 g/g and 1.5325±0.0020 g/Lh; 0.4290±0.0030 g/g and 0.7399±0.0043 g/Lh, respectively at an optimum kLa of 0.5776 (0.5735, 0.5817) min−1 and 0.6924 (0.6887, 0.6961) min−1, respectively in a 0.5 L bioreactor. After that, these optimum kLa were validated in 1 L and 5 L bioreactors. The successful kLa validation revealed that the hemicellulosic fraction of lignocellulosic biomass could also be well utilized for bioethanol production.

Analyzing of hydrodynamic stress and mass transfer requirements of a fermentation process carried out in a coaxial bioreactor: a scale-up study.

Fluid hydrodynamic stress has a deterministic effect on the morphology of filamentous fungi. Although the coaxial mixer has been recognized as a suitable gas dispersion system for minimizing inhomogeneities within a bioreactor, its performance for achieving enhanced oxygen transfer while operating at a reduced shear environment has not been investigated yet, specifically upon scale-up. Therefore, the influence of the impeller type, aeration rate, and central impeller retrofitting on the efficacy of an abiotic coaxial system containing a shear-thinning fluid was examined. The aim was to assess the hydrodynamic parameters, including stress, mass transfer, bubble size, and gas hold-up, upon conducting a scale-up study. The investigation was conducted through dynamic gassing-in, tomography, and computational fluid dynamics combined with population balance methods. It was observed that the coaxial bioreactor performance was strongly influenced by the agitator type. In addition, coaxial bioreactors are scalable in terms of shear environment and oxygen transfer rate.

A perspective-driven and technical evaluation of machine learning in bioreactor scale-up: A case-study for potential model developments.

Bioreactor scale-up and scale-down have always been a topical issue for the biopharmaceutical industry and despite considerable effort, the identification of a fail-safe strategy for bioprocess development across scales remains a challenge. With the ubiquitous growth of digital transformation technologies, new scaling methods based on computer models may enable more effective scaling. This study aimed to evaluate the potential application of machine learning (ML) algorithms for bioreactor scale-up, with a specific focus on the prediction of scaling parameters. Factors critical to the development of such models were identified and data for bioreactor scale-up studies involving CHO cell-generated mAb products collated from the literature and public sources for the development of unsupervised and supervised ML models. Comparison of bioreactor performance across scales identified similarities between the different processes and primary differences between small- and large-scale bioreactors. A series of three case studies were developed to assess the relationship between cell growth and scale-sensitive bioreactor features. An embedding layer improved the capability of artificial neural network models to predict cell growth at a large-scale, as this approach captured similarities between the processes. Further models constructed to predict scaling parameters demonstrated how ML models may be applied to assist the scaling process. The development of data sets that include more characterization data with greater variability under different gassing and agitation regimes will also assist the future development of ML tools for bioreactor scaling.

Development of a high-throughput scale-down model in Ambr® 250 HT for plasmid DNA fermentation processes.

Recent advances in messenger ribonucleic acid (mRNA) vaccines and gene therapy vectors have increased the need for rapid plasmid DNA (pDNA) screening and production within the biopharmaceutical industry. High-throughput (HT) fermentor systems, such as the Ambr® 250 HT, can significantly accelerate process development timelines of pDNA upstream processes compared to traditional bench-scale glass fermentors or small-scale steam-in-place (SIP) fermentors. However, such scale-down models must be qualified to ensure that they are representative of the larger scale process similar to traditional small-scale models. In the current study, we developed a representative scale-down model of a Biostat® D-DCU 30 L pDNA fermentation process in Ambr® 250 HT fermentors using three cell lines producing three different constructs. The Ambr scale-down model provided comparable process performance and pDNA quality as the 30 L SIP fermentation process. In addition, we demonstrated the predictive value of the Ambr model by two-way qualification, first by accurately reproducing the prior trends observed in a 30 L process, followed by predicting new process trends that were then successfully reproduced in the 30 L process. The representative and predictive scale-down Ambr model developed in this study would enable a faster and more efficient approach to strain/clone/host-cell screening, pDNA process development and characterization studies, process scale-up studies, and manufacturing support.

Laboratory investigation and core flood demonstration of enhanced biogenic methane generation from lignite.

Over the last several decades, coalbed methane (CBM) has emerged as an important energy source in developing nations like India as well as worldwide and is expected to play a significant role in the energy portfolio of the future. The current scenario of rapid exhaustion of fossil fuels is leading to the need to explore alternative and efficient fuel resources. The present study demonstrates enhanced methane production per gram of lignite (lowest-rank coal). Optimization of the bioconversion of lignite to methane revealed 55°C temperature and 1.5g/L NaCl concentration as ambient conditions for the process. A scale-up study in the optimized condition showed 2,800mM methane production per 25g of lignite in anaerobic conditions. Further, Fourier transform Infrared (FTIR) and Gas Chromatography Mass Spectrometry (GCMS) analysis showed bioconversion of lignite into simpler intermediate substrates required for methane production. The results highlighted that the bacterial action first converts lignite into volatile fatty acids, which subsequently get converted into methane. Further, the exploration of indigenous microbial consortia in Tharad well (THAA) mainly comprises the order Methanosarcinales and Methanomicrobiales. The pathogenicity of the microbial consortium THAA was declared safe for use in mice via the oral route by The Energy and Resources Institute (TERI), India. The study demonstrated the development of indigenous consortia (TERI THAA), which can potentially enhance methane production from the lowest coal grade under extreme conditions in Indian coal beds.

Estudio de factibilidad técnica para el establecimiento de un proceso industrial en la elaboración de grageas de almendra con chocolate amargo

This article assumes that a cocoa producing company also manufactures chocolate dragees. In order to carry out a scale-up study of an industrial process in the production of almond dragees with bitter chocolate, a literature review was carried out on the technical development of the process, considering the necessary machinery and the physical and biochemical changes that occur in the production of the product. The literature on the market behavior of the chocolate industry was reviewed, as well as the raw materials necessary for the production of the dragee (sugar, cocoa and almonds). Understanding the changes that occur at each stage of the process is crucial to ensure product quality. Although this study is an approximation of what is needed to start a chocolate company, it is important to conduct commercial and financial feasibility studies to ensure success in production and marketing.

Polymer nanoparticles of [(1-methyl-(S)-4,5-dihydroorotyl)-histidyl-prolinamide] as a potential central nervous system antidepressant formulation and their scale-up studies

Polymer nanoparticles of [(1-methyl-(S)-4,5-dihydroorotyl)-histidyl-prolinamide] as a potential central nervous system antidepressant formulation and their scale-up studies

Continuum modeling of high-temperature (>1000 °C) heat extraction from a moving-bed oxidation reactor for thermochemical energy storage

There is a growing need for efficient and reliable energy storage technologies to expand renewable energy adoption and transit to a decarbonized clean energy future. Thermochemical energy storage (TCES) through reversible gas-solid reactions has exhibited considerable potential. Up to now, TCES has primarily been studied at the material level and within lab-scale reactors. As such, accurate numerical models are needed for further reactor design and scale-up. In this work, a transient three-dimensional heat and mass transfer continuum model was developed to simulate the transport phenomena coupled with exothermic oxidation of magnesium‑manganese-oxide particles in a high-temperature moving-bed reactor for TCES. The reactor included direct heat extraction at temperatures >1000 °C. The predicted temperature distributions, oxygen concentration profiles, and steady-state reactor energy extraction efficiency were in good agreement with measured values from a corresponding experimental setup. The performance of the reactor was further investigated through two parametric studies (varied particle flow rates and gas extraction ratios), which demonstrated a strong dependence of reactor efficiency (varied between 15 %–40 %) on both operating parameters. The present heat and mass transfer model will be valuable in further reactor design and scale-up studies, as well as for selection of operating conditions to maximize efficiency.

Production of biopesticide from Melia azedarach Linn extract obtained by supercritical fluid extraction for the control of Tetranychus urticae

Compounds derived from plant extracts, such as those from the fruits and leaves of Melia azedarach Linn (chinaberry tree), which contain azadirachtin, have been utilized for crop pest control. However, these natural compounds are susceptible to degradation during extraction and in field conditions. To address this, extraction processes involving supercritical fluid and microencapsulation via spray drying may be employed to obtain and preserve biopesticidal molecules. This study focuses on producing a biopesticide from M. azedarach L. fruit extract using supercritical CO2, and microencapsulation via spray drying with a blend of cheese whey and gum Arabic. The extract was obtained from chinaberry fruits rich in azadirachtin (736.92 μg/L) using supercritical CO2 at 250 bar and 50 °C for 120 min, with a yield of 3.44%. The extract was then encapsulated using cheese whey and gum Arabic (70:30) as encapsulating agents at extract:encapsulating agents ratios of 1:10 and 1:15. Mortality rates of Tetranychus urticae Koch adult females and their eggs, in contact and residual assays with both non-encapsulated and encapsulated extract, did not significantly differ (p > 0.05) from the commercial acaricide abamectin. This study introduces a potential new biopesticide that utilizes chinaberry extract obtained through supercritical CO2 extraction and encapsulation for industrial scale-up studies, and this approach holds promise for the production of an effective biopesticide for crops affected by two-spotted spider mites.

Sustainable Process to Recover Metals from Waste PCBs Using Physical Pre-Treatment and Hydrometallurgical Techniques

Printed Circuit Boards (PCBs) are an essential component of electronic devices. The digitalization and upgrading of gadget generates lots of PCB-containing electronic waste. Conserving resources and protecting the environment requires the recycling of such e-waste. This paper focuses on the recovery of metals from waste PCBs using physical pre-treatment and hydrometallurgical processes. Initially, the waste PCBs were pre-treated and beneficiated to separate the metallic and non-metallic fractions. The metallic concentrate obtained was leached using nitric acid (a strong oxidative agent) to dissolve the metals. The system was fully jacketed with a scrubber and condenser to prevent the emission of toxic gases into the environment. The process parameters, such as the effect of acid concentration, pulp density, temperature, time, etc., were studied, optimized, and scientifically validated. The kinetics of leaching fitted well with the following shrinking core models: XB = kc.t for Cu, (1 − (1 − XB)1/2) for Ni, and 1 − 3(1 − XB)2/3 + 2(1 − XB) for Pb. The activation energy was 19.42 kJ/mol. The tin left in the residue was treated separately. The developed process is useful for recovering metals from waste PCBs and has the potential to be commercialized after conducting scale-up studies.

Applicability of β-lactamase entrapped agarose discs for removal of doripenem antibiotic: reusability and scale-up studies

Applicability of β-lactamase entrapped agarose discs for removal of doripenem antibiotic: reusability and scale-up studies

Scale-up studies for production of α-amylase by A. Tamarii MTCC5152 and development of enzyme product

α-amylases are one of the most vital commercially produced starch hydrolyzing enzymes. Increasing enzyme production is becoming one of the most important goals in biotechnological processes. In this study, the production of α-amylase by A.tamarii MTCC5152 in flask level studies is experimentally validated by scale-up studies in Koji room under controlled conditions using wheat bran, a low cost agro-industrial waste as substrate. The production of amylase was increased by optimising various culture conditions such as inoculum size, incubation period and moisture content. It was found that A.tamarii MTCC5152 produced highest titre of α-amylase activity (668.01 u/g), after 4 days of incubation period at 5% inoculum concentration and 80% moisture content. The results of the comparative analysis of protease and amylase production demonstrated 15% increase in α- amylase and 20% increase in protease production compared to flask level studies. Studies conducted for the development of enzyme products as liquid enzyme concentrate and dry enzyme powder showed that addition of hydrophilic agents such as poly ethylene glycol (PEG- 6000) was found to be an effective way to increase the activity and enzyme yield in the preparation of a dry enzyme powder using A.tamarii MTCC5152. The ammonium sulfate precipitated enzyme at saturation, lyophilized by freeze drying gave the best recovery (96%) of the enzyme activity.

Extraction, separation and purification of fatty acid ethyl esters for biodiesel and DHA from Thraustochytrid biomass.

A novel approach for in situ transesterification, extraction, separation, and purification of fatty acid ethyl esters (FAEE) for biodiesel and docosahexaenoic acid (DHA) from Thraustochytrid biomass has been developed. The downstream processing of Thraustochytrids oil necessitates optimization, considering the higher content of polyunsaturated fatty acids (PUFA). While two-step methods are commonly employed for extracting and transesterifying oil from oleaginous microbes, this may result in oxidation/epoxidation of omega-3 oil due to prolonged exposure to heat and oxygen. To address this issue, a rapid single-step method was devised for in situ transesterification of Thraustochytrid oil. Through further process optimization, a 50% reduction in solvent requirement was achieved without significantly impacting fatty acid recovery or composition. Scale-up studies in a 4L reactor demonstrated complete FAEE recovery (99.98% of total oil) from biomass, concurrently enhancing DHA yield from 16% to nearly 22%. The decolorization of FAEE oil with fuller's earth effectively removed impurities such as pigments, secondary metabolites, and waxes, resulting in a clear, shiny appearance. High-performance liquid chromatography (HPLC) analysis indicated that the eluted DHA was over 94.5% pure, as corroborated by GC-FID analysis.

A New Microwave-Assisted Protocol for Cellulose Extraction from Eucalyptus and Pine Tree Wood Waste.

An enormous interest in the development of efficient protocols for cellulose extraction has been demonstrated in the last few years, although usually based on non-sustainable chemical and thermal approaches. In this work, we propose a new and more sustainable method for cellulose extraction from eucalyptus and pine tree wood waste products exclusively performed using microwave-assisted radiation. The methodology includes three main steps: (i) alkaline treatment; (ii) bleaching I, using H2O2; and (iii) bleaching II, an acidic treatment. Samples obtained in each step were characterized by Fourier-transform Infrared (FTIR) spectroscopy, powder X-ray diffraction (PXRD), thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results were compared with the structural and thermal profile of the starting materials, a commercially available microcrystalline cellulose and with an industrial paper pulp sample. Results confirmed that for both types of wood wastes, cellulose was retained during the extraction procedures and that the removal of hemicellulose and lignin was mainly achieved in the last step, as seen by the FTIR spectra and TGA curves. The developed protocol is innovative, as it constitutes an easy and quick approach for extracting cellulose from eucalyptus and pine tree wood waste. Mild chemical and thermal conditions are used during the three extraction steps (microwave irradiation, aqueous solutions, maximum of 120 °C in a total of 3 h). Moreover, environmentally friendly purification steps are applied based on the use of water and ethanol. This approach offers the possibility of a future scale-up study to potentially apply the developed protocol to the extraction of cellulose on an industrial scale.

Functional parametric influence on glutaminase free l-asparaginase production by Amycolatopsis thermoflava SFMA-103

l-Asparaginase (l-ASNase) is a well known chemotherapeutic enzyme, to treat different neoplasms, especially in treating Acute Lymphoblastic Leukaemia (ALL) of children and adults. However, production of glutaminase free l-ASNase is the central area of research to produce cytotoxicity free L-ASNases in the cancer therapeutics. The present study explored the potential glutaminase free l-ASNase production from rare actinobacterium, Amycolatopsis thermoflava SFMA-103 isolated from agricultural field soils. The attention of the enzyme production was sequentially optimized using various methods starting from One-factor-at-a-time (OFAT) to Taguchi orthogonal array (OA) experimental design (DOE). Four critical factors, pH, temperature, arabinose and yeast extract were identified initially, hence, three levels with a layout of L9 (34) were used and further processed with Qualitek-4 software. Upon validation an enhanced enzyme production of 68.55% was observed (from 10 IU −1 min−1 to 31.8 IU −1 min−1). As far as our knowledge extends, glutaminase-free l-ASNase production is the first time report from Amycolatopsis thermoflava SFMA-103. Further, scale-up studies were performed using bench scale bioreactor (5L capacity) with the statistically optimized conditions (aeration of 0.5 VVM and agitation of 150 rpm at 40 °C) resulted in an improved enzyme production to the level of 132.69% (61.6 IU −1 min−1) with an incubation period of 66 h. Taguchi methods for scale-up studies of l-ASNase proves useful in bioprocess optimization with minor experimental set up, saving cost and time consumption is low.

Fabrication of biocompatible chitosan/graphene based nanocomposite for the competitive adsorption of heavy metal ions from wastewater in binary and ternary systems: Scale-up and upgradation studies

Coexistence of multiple metal ions in the wastewater alters the adsorption behavior compared to single metal ion system using the existing adsorbents which are chemically driven, requires harsh operating conditions and additional energy. The present study evaluates the performance of Chitosan/Graphene based nanocomposite for the competitive adsorption of heavy metals in single, binary and ternary systems. Maximum removal efficiency was 89.35 % (As), 90.41 % (Cu), 93.22 % (Ni), 96.47 % (Cd), 95.45 % (Pb) and 88.65 % (Cr) under optimum conditions (pH 4, Temperature = 25 °C, Equilibrium Time = 90 min, Adsorbent Dosage = 1.25 g and Initial Metal Ion Concentration = 150 mg/L) in a single system. A detailed investigation on the competitive adsorption of Cadmium, Copper, and Chromium ions was performed in binary. Maximum adsorption capacity was 1.55 mg/g and 1.48 mg/g (CuCd); 2.98 mg/g and 3.21 mg/g (CuCr) and 2.21 mg/g and 1.90 mg/g (CdCr system) at 4 mM concentration. The constructed mathematical models accurately forecast the adsorption behavior and shows a good match with the experimental data. Also, fixed-bed column studies were performed for the large-scale upgradation and optimum flow rate and bed height was 1 L/h and 15 cm at 25 °C. The study proposes the fabrication of natural-polymer based composite which can significantly reduce the dependency on synthetic and chemical-based adsorbents which poses severe negative impacts such as high-cost, poor selectivity and rapid saturation due to their low recyclability or sludge generation. Such application can effectively transform wastewater into contamination free water that could be valuable to society.

Physical Factors Affecting the Scale-Up of Vegetative Insecticidal Protein (Vip3A) Production by Bacillus thuringiensis Bt294

Vip3A (vegetative insecticidal protein) is a representative member of the Vip3 family, which is widely used for lepidopteran pest control. This Vip3A protein, a non-growth-associated protein, is an effective bioinsecticide against insect pests, but there is relatively little information about its production processes at large scales. Hence, the effects of environmental factors on Vip3A production by Bacillus thuringiensis Bt294 (antifoam agents, shaking speeds, agitation and aeration rates), as well as controlling physical conditions such as the lowest point of dissolved oxygen and controlling of culture pH, were observed in shaking flasks and bioreactors. The results showed that antifoam agents, flask types and shaking speeds had significant effects on Vip3A and biomass production. Cultivation without pH control and DO control in 5 L bioreactors at lower agitation and aeration rates, which was not favorable for biomass production, resulted in a high Vip3A protein production of 5645.67 mg/L. The scale-up studies of the Vip3A protein production in a pilot-scale 750 L bioreactor gave 3750.0 mg/L. Therefore, this study demonstrated the significant effects of agitation, aeration rates and culture pH on Vip3A production by B. thuringiensis Bt294. Balancing of physical conditions was necessary for obtaining the highest yield of Vip3A by slowing down the production rate of biomass. Moreover, this Vip3A protein has high potential as a bioinsecticide for lepidopteran pest control in organic crops. This information will be important for significantly increasing the Vip3A protein concentration by the bacterium and will be useful for field application at a lower cost.

Effects of green and innovative pretreatment techniques on kinetic parameters of sunflower seed husk

The effects of green pretreatment processes, such as ultrasonic process (US) and deep eutectic solvent (DES), applied to the biomass on kinetic parameters are as important as their effects on characteristic properties. Process conditions and course of reaction progression depend on the knowledge about kinetic parameters, activation energy and reaction model of thermal degradation in scale-up studies. Therefore, in this study, the change in the kinetic parameters with US applied with both distilled water and DES (glycerol:sodium acetate) at 100 W power and 20 kHz frequency for 10 minutes to sunflower seed husk (SSH) was revelaed. Isoconversional methods Kissinger-Akahira-Sunose (KAS) and Flynn- Wall-Ozawa (FWO) were preferred for the evaluation of activation energy of SSH and pretreated SSH at 15°C/min, 20°C/min and 25°C/min heating rates. US pretreatment with DES instead of water resulted in increment of average activation energy values (Ea) from 113.13 to 143.65 kJ/mol in the KAS method. Ea values for all SSH samples changed in the range of 87.72-143.65 kJ/mol and higher Ea values was obtained with KAS method for pretreated SSH samples. Consequently, the use of DES in US pretreatment was more effective to change kinetic parameters of SSH compared to water.