Consistent Product Quality Research Articles

Microevolutionary dynamics of eccDNA in Chinese hamster ovary cells grown in fed-batch cultures under control and lactate-stressed conditions

Chinese hamster ovary (CHO) cell lines are widely used to manufacture biopharmaceuticals. However, CHO cells are not an optimal expression host due to the intrinsic plasticity of the CHO genome. Genome plasticity can lead to chromosomal rearrangements, transgene exclusion, and phenotypic drift. A poorly understood genomic element of CHO cell line instability is extrachromosomal circular DNA (eccDNA) in gene expression and regulation. EccDNA can facilitate ultra-high gene expression and are found within many eukaryotes including humans, yeast, and plants. EccDNA confers genetic heterogeneity, providing selective advantages to individual cells in response to dynamic environments. In CHO cell cultures, maintaining genetic homogeneity is critical to ensuring consistent productivity and product quality. Understanding eccDNA structure, function, and microevolutionary dynamics under various culture conditions could reveal potential engineering targets for cell line optimization. In this study, eccDNA sequences were investigated at the beginning and end of two-week fed-batch cultures in an ambr®250 bioreactor under control and lactate-stressed conditions. This work characterized structure and function of eccDNA in a CHO-K1 clone. Gene annotation identified 1551 unique eccDNA genes including cancer driver genes and genes involved in protein production. Furthermore, RNA-seq data is integrated to identify transcriptionally active eccDNA genes.

Interpretable artificial neural networks for retrospective QbD of pharmaceutical tablet manufacturing based on a pilot-scale developmental dataset

As the pharmaceutical industry increasingly adopts the Pharma 4.0. concept, there is a growing need to effectively predict the product quality based on manufacturing or in-process data. Although artificial neural networks (ANNs) have emerged as powerful tools in data-rich environments, their implementation in pharmaceutical manufacturing is hindered by their black-box nature. In this work, ANNs were developed and interpreted to demonstrate their applicability to increase process understanding by retrospective analysis of developmental or manufacturing data. The in vitro dissolution and hardness of extended-release, directly compressed tablets were predicted from manufacturing and spectroscopic data of pilot-scale development. The ANNs using material attributes and operational parameters provided better results than using NIR or Raman spectra as predictors. ANNs were interpreted by sensitivity analysis, helping to identify the root cause of the batch-to-batch variability, e.g., the variability in particle size, grade, or substitution of the hydroxypropyl methylcellulose excipient. An ANN-based control strategy was also successfully utilized to mitigate the batch-to-batch variability by flexibly operating the tableting process. The presented methodology can be adapted to arbitrary data-rich manufacturing steps from active substance synthesis to formulation to predict the quality from manufacturing or development data and gain process understanding and consistent product quality.

The Kelvin Redefinition and Practical Primary Thermometry

In May 2019 four of the seven base units of the International System of Units (the SI) were redefined and are now founded on defined values of fundamental physical constants. One of these was the kelvin which is no longer defined by the triple point of water but instead through a fixed value of the Boltzmann constant. In this paper the kelvin redefinition is introduced and the implications for temperature traceability and practical temperature sensing discussed. This will include outlining new approaches for temperature traceability, as well as discussing the rise of in-process calibration through practical primary temperature sensing approaches (where, in principle, no sensor calibration is required). These forthcoming changes are likely to have significant impact on everyone in the temperature calibration chain, whilst the advent of in-process temperature calibration should lead to step change improvements in process control, energy efficiency and product quality consistency and will help facilitate autonomous production.

Напрями удосконалення операційного менеджменту в контексті забезпечення сталого розвитку організації

The article outlines that improvement of operational management to ensure sustainable development of an enterprise is an integral part of success in modern business environment. Taking into account the aspects of sustainability allows enterprises to achieve efficiency, reduce costs, improve product quality and customer satisfaction, as well as reduce negative impact on the environment, while contributing to the social development of society. The essence of the category of operational management was investigated in the article. It has been established that compliance with the policy of sustainable development correlates with the field of functional activity of this type of management in the context of increasing globalisation, digitalisation and riskiness of economic activity. Key environmental practices that contribute to strengthening the economic and financial sustainability of organisations, primarily the use of environmentally friendly technologies, reduction of emissions and pollution, efficient use of resources and waste recycling to ensure environmental sustainability were analysed. The authors outline main directions of improvement of the operational management system with consideration of the sustainable development policy. An important step in this process is the analysis and determination of key performance indicators, which allows enterprises to focus on achieving specific goals and assess the effectiveness of their operations in the direction of sustainable development. The next stage in ensuring economic sustainability is the implementation of a sustainability-oriented quality management system, which includes defining quality standards, procedures for monitoring and improving product quality in compliance with environmental norms and standards. The implementation of a sustainability-based quality management system contributes to the production of consistent quality products, reduction of waste and improved customer satisfaction. The authors see another area of operational management improvement in optimising the supply chain and establishing cooperation with suppliers who support sustainability policies. Improving supply chain management processes ensures efficiency and sustainability in the supply chain, reduces risks and optimises logistics. Process improvement and innovation play a significant role in the implementation of sustainable development policy in the organisation's activities, as it involves continuous process improvement, introduction of the latest technologies and innovations aimed at preserving the environment, ensuring labour safety and meeting social needs. The article emphasises that operating on the basis of social responsibility with improved attitude towards staff, consumers and other stakeholder groups emphasises the organisation's understanding of its role in the social development of society and strengthens its competitive position in the market.

Methodology for adaptive decision--making research on manufacturing process of traditional Chinese medicine based on deep reinforcement learning

The manufacturing process of traditional Chinese medicine is subject to material fluctuation and other uncertain factors which usually cause non-optimal state and inconsistent product quality. Therefore, it is necessary to design and collect the quality-rela-ted physical parameters, process parameters, and equipment parameters in the whole manufacturing process of traditional Chinese medicine for digitization and modeling of the process. In this paper, a method for non-optimal state identification and self-recovering regulation was developed for active quality control in the manufacturing process of traditional Chinese medicine. Moreover, taking vacuum belt drying process as an example, a DQN algorithm-based intelligent decision model was established and verified and the implementation process was also discussed and studied. Thus, the process parameters-based self-optimization strategy discovery and path planning of optimal process control were rea-lized in this study. The results showed that the deep reinforcement learning-based artificial intelligence technology was helpful to improve the product quality consistency, reduce production cost, and increase benefit.

Comparative Assessment of the Binding and Neutralisation Activity of Bispecific Antibodies Against SARS-CoV-2 Variants.

Neutralising antibodies against SARS-CoV-2 are a vital component in the fight against COVID-19 pandemic, having the potential of both therapeutic and prophylactic applications. Bispecific antibodies (BsAbs) against SARS-CoV-2 are particularly promising, given their ability to bind simultaneously to two distinct sites of the receptor-binding domain (RBD) of the viral spike protein. Such antibodies are complex molecules associated with multi-faceted mechanisms of action that require appropriate bioassays to ensure product quality and manufacturing consistency. We developed procedures for biolayer interferometry (BLI) and a cell-based virus neutralisation assay, the focus reduction neutralisation test (FRNT). Using both assays, we tested a panel of five BsAbs against different spike variants (Ancestral, Delta and Omicron) to evaluate the use of these analytical methods in assessing binding and neutralisation activities of anti-SARS-CoV-2 therapeutics. We found comparable trends between BLI-derived binding affinity and FRNT-based virus neutralisation activity. Antibodies that displayed high binding affinity against a variant were often followed by potent neutralisation at lower concentrations, whereas those with low binding affinity also demonstrated reduced neutralisation activity. The results support the utility of BLI and FRNT assays in measuring variant-specific binding and virus neutralisation activity of anti-SARS-CoV-2 antibodies.

Effect of Nanoemulsion Containing Enterocin GR17 and Cinnamaldehyde on Microbiological, Physicochemical and Sensory Properties and Shelf Life of Liquid-Smoked Salmon Fillets.

The spoilage of liquid-smoked salmon represented a serious restriction for shelf life, due to the loss of taste, smell, color and consistency in product quality. The objective of this study was to investigate the feasibility of applying a nanoemulsion delivery system co-encapsulated enterocin Gr17 and essential oils (EOs) to the refrigerated storage of liquid-smoked salmon. The synergistic inhibiting effects of enterocin Gr17 and EOs were evaluated, a nanoemulsion delivery system with the optimal combination was developed, and the evolution of the microbiological, physicochemical, and sensory properties of liquid-smoked salmon fillets were analyzed during a 49-day period of refrigerated storage. The results showed that the combination of enterocin Gr17 and cinnamaldehyde essential oil (CEO) displayed the strongest synergistic inhibiting effect on foodborne pathogens. A nanoemulsion system incorporating enterocin Gr17 and CEO was successfully developed and presented a broad spectrum of activity against most of the tested bacteria. A nanoemulsion system incorporating enterocin Gr17 and CEO (CO-NE) could significantly inhibit the growth of microflora, suppress the accumulation of total volatile basic nitrogen (TVB-N) and thiobarbituric acid reactive substance (TBARS), and maintain better color, texture, and sensory profiles during smoked salmon storage at 4 °C. Overall, from a microbiological, physicochemical, and sensory point of view, the CO-NE treatment could extend the shelf life to 42 days and maintain the relatively low TVB-N value (≤15.38 mg/100 g), TBARS value (≤2.51 mg MDA/kg), as well as a relatively high sensory score (≥5.83) during the whole storage period. Hence, a nanoemulsion system incorporating enterocin Gr17 and CEO could be a promising bio-preservative technology and alternative to the conventional processes used for improving the safety and quality of chilled liquid-smoked salmon.

In-line monitoring of protein concentration with MIR spectroscopy during UFDF.

Rapid increase of product titers in upstream processes has presented challenges for downstream processing, where purification costs increase linearly with the increase of the product yield. Hence, innovative solutions are becoming increasingly popular. Process Analytical Technology (PAT) tools, such as spectroscopic techniques, are on the rise due to their capacity to provide real-time, precise analytics. This ensures consistent product quality and increased process understanding, as well as process control. Mid-infrared spectroscopy (MIR) has emerged as a highly promising technique within recent years, owing to its ability to monitor several critical process parameters at the same time and unchallenging spectral analysis and data interpretation. For in-line monitoring, Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR) is a method of choice, as it enables reliable measurements in a liquid environment, even though water absorption bands are present in the region of interest. Here, we present MIR spectroscopy as a monitoring tool of critical process parameters in ultrafiltration/diafiltration (UFDF). MIR spectrometer was integrated in the UFDF process in an in-line fashion through a single-use flow cell containing a single bounce silicon ATR crystal. The results indicate that the one-point calibration algorithm applied to the MIR spectra, predicts highly accurate protein concentrations, as compared with validated offline analytical methods.

Walking on thin ice: controlled freezing & thawing of pharmaceutical active molecules

Cryopreservation of active pharmaceutical ingredients, cells, or tissues is fundamental to maintain the quality of the respective product. Ice formation, re-crystallization, and other phenomena that occur during the freezing and thawing process can cause significant damage. These ice-induced injuries can be minimized by appropriate molecule design and the use of chemical cryoprotectants or bio-substances in combination with suitable equipment and physical conditions during the freezing and thawing process. It is important to adapt both freezing and thawing processes to the characteristics of the protein or active pharmaceutical ingredient that requires a cold chain to maintain its biological and chemical stability during storage and shipment. Controlled freezing and thawing facilitates consistent product quality during the fluid management processes involved in manufacturing. By preventing damage to active ingredients, the supply of medications is optimized. 

Development strategy of Marigold flower farming integrated with Trigona bees in the ecotourism area of ancient tree “kayu putih”

Marigold flower farming community is a group of farmers who carry out the processing production of marigold flower agricultural products. This study was to identify internal and external conditions, as well as determine strategies that can be used in developing an integrated marigold flower farm with Trigona bees. This research was conducted in the “Kayu Putih” Ancient Tree Ecotourism Area at Tabanan Regency in the Gumitir Flower Farmer Community, with a total of 26 respondents. The alternative strategy formulation process goes through two stages, namely: The data collection stage; and the Analysis phase. Based on the research results, the internal conditions in the development of Integrated Marigold Flower Farming with Trigona Bees are the availability of production facilities, short marketing chain, Religious facilities, Health products, lack of manpower, lack of skills, cultivation knowledge. While the external environment in the development of Marigold Flower Farming Integrated with Trigona Bees, namely: local government attention; establishing partnership relationship; development of technology and information; There are lots of bee-feeding flowers; competitive product price; changes in the weather; pest attack; Decreased tourist visits. These results place this farm in the position of cell V. The strategy used in cell V is a hold and maintain strategy. This strategy can be done through sales promotion with social media or extensive sales promotion, for example, by providing additional bonus products for purchases in large quantities and providing tour packages by adding flower and honey harvesting activities in the garden. Another strategy that can be done is to provide better services, such as continuous product availability, consistent product quality, timely and ordered delivery, and friendliness to customers and tourists when buying and visiting the Ecotourism Area of Ancient Tree “Kayu Putih”.

Risk-Based Control Strategies of Recombinant Monoclonal Antibody Charge Variants.

Since the first approval of the anti-CD3 recombinant monoclonal antibody (mAb), muromonab-CD3, a mouse antibody for the prevention of transplant rejection, by the US Food and Drug Administration (FDA) in 1986, mAb therapeutics have become increasingly important to medical care. A wealth of information about mAbs regarding their structure, stability, post-translation modifications, and the relationship between modification and function has been reported. Yet, substantial resources are still required throughout development and commercialization to have appropriate control strategies to maintain consistent product quality, safety, and efficacy. A typical feature of mAbs is charge heterogeneity, which stems from a variety of modifications, including modifications that are common to many mAbs or unique to a specific molecule or process. Charge heterogeneity is highly sensitive to process changes and thus a good indicator of a robust process. It is a high-risk quality attribute that could potentially fail the specification and comparability required for batch disposition. Failure to meet product specifications or comparability can substantially affect clinical development timelines. To mitigate these risks, the general rule is to maintain a comparable charge profile when process changes are inevitably introduced during development and even after commercialization. Otherwise, new peaks or varied levels of acidic and basic species must be justified based on scientific knowledge and clinical experience for a specific molecule. Here, we summarize the current understanding of mAb charge variants and outline risk-based control strategies to support process development and ultimately commercialization.

Nanoplasmonic Avidity-Based Detection and Quantification of IgG Aggregates.

Production of therapeutic monoclonal antibodies (mAbs) is a complex process that requires extensive analytical and bioanalytical characterization to ensure high and consistent product quality. Aggregation of mAbs is common and very problematic and can result in products with altered pharmacodynamics and pharmacokinetics and potentially increased immunogenicity. Rapid detection of aggregates, however, remains very challenging using existing analytical techniques. Here, we show a real-time and label-free fiber optical nanoplasmonic biosensor system for specific detection and quantification of immunoglobulin G (IgG) aggregates exploiting Protein A-mediated avidity effects. Compared to monomers, IgG aggregates were found to have substantially higher apparent affinity when binding to Protein A-functionalized sensor chips in a specific pH range (pH 3.8-4.0). Under these conditions, aggregates and monomers showed significantly different binding and dissociation kinetics. Reliable and rapid aggregate quantification was demonstrated with a limit of detection (LOD) and limit of quantification (LOQ) of about 9 and 30 μg/mL, respectively. Using neural network-based curve fitting, it was further possible to simultaneously quantify monomers and aggregates for aggregate concentrations lower than 30 μg/mL. Our work demonstrates a unique avidity-based biosensor approach for fast aggregate analysis that can be used for rapid at-line quality control, including lot/batch release testing. This technology can also likely be further optimized for real-time in-line monitoring of product titers and quality, facilitating process intensification and automation.

Quality-by-control of intensified continuous filtration-drying of active pharmaceutical ingredients.

Continuous manufacturing and closed-loop quality control are emerging technologies that are pivotal for next-generation pharmaceutical modernization. We develop a process control framework for a continuous carousel for integrated filtration-drying of crystallization slurries. The proposed control system includes model-based monitoring and control routines, such as state estimation and real-time optimization, implemented in a hierarchical, three-layer quality-by-control (QbC) framework. We implement the control system in ContCarSim, a publicly available carousel simulator. We benchmark the proposed control system against simpler methods, comprising a reduced subset of the elements of the overall control system, and against open-loop operation (the current standard in pharmaceutical manufacturing). The proposed control system demonstrates superior performance in terms of higher consistency in product quality and increased productivity, proving the benefits of closed-loop control and of model-based techniques in pharmaceutical manufacturing. This study represents a step forward toward end-to-end continuous pharmaceutical processing, and in the evolution of quality-by-design toward quality-by-control.

Accomplishing Scalability by Using Plate‐Based Freeze and Thaw Technologies

AbstractControlling the freezing behavior of bulk drug substance opens doors to process reproducibility and consistent quality of the final drug product by maintaining uniform conditions for the biopharmaceuticals during freezing, and consequently for frozen storage and shipment. This simplifies the commercial bulk production, particularly when manufacturing different drug substances at different sensitivities and temperature set points and enables scalability. The leverage of the ice front growth speed had a significant impact on controllability and, as a result, on the protein quality.

Multiattribute Monitoring of Charge-Based Heterogeneity of Recombinant Monoclonal Antibodies Using 2D HIC-WCX-MS.

Charged heterogeneity of monoclonal antibody (mAb) products is regarded as a critical quality attribute (CQA) depending on its impact on the safety and efficacy profile of the product. Hence, manufacturers are expected to perform a comprehensive characterization of the charge heterogeneity to ensure that the manufactured product meets its specifications. Further, monitoring is also expected during the product lifecycle to demonstrate consistency in product quality. However, conventional analytical methods for characterization of hydrophobic and charge variants are nonvolatile salt-based and require manual fraction collection and desalting steps before analysis through mass spectrometry can be performed. In the present study, a workflow of a two-dimensional liquid chromatography method using mass spectrometry (MS)-compatible buffers coupled with native mass spectrometry was performed to characterize hydrophobic variants in the first dimension and charge variants in the second dimension without any need for manual fractionation. This novel two-dimensional (2D) hydrophobic interaction chromatography (HIC)-weak cation-exchange chromatography (WCX)-MS workflow identified 10 variants in mAb A, out of which 2 variants are exclusive to the 2D orthogonal method. Similarly, for mAb B, a total of 11 variants are identified, including 5 variants exclusive to the 2D orthogonal workflow. When compared to stand-alone, HIC resolved only 4 variants for both mAbs and WCX resolved 7 variants for mAb A and 6 variants for mAb B. In addition, the proposed method allows direct characterization of hydrophobic/charge variant peaks through native mass spectrometry in a single-run workflow.

The Effects of Phosphate Impurity on Recovered LiNi0.6Co0.2Mn0.2O2 Cathode Material via a Hydrometallurgy Method.

From portable electronics to electric vehicles, lithium-ion batteries have been deeply integrated into our daily life and industrial fields for a few decades. The booming field of battery manufacturing could lead to shortages in resources and massive accumulation of battery waste, hindering sustainable development. Therefore, hydrometallurgy-based approaches have been widely used in industrial recycling to recover cathode materials due to their high efficiency and throughput. Impurities have always been a great challenge for hydrometallurgical recycling, introducing challenges to maintain the consistency of product quality because of potential unintended effects caused by impurities. Herein, after comprehensive investigation, we first report the impacts of phosphate impurity on a recycled LiNi0.6Co0.2Mn0.2O2 ("NCM622") cathode via a hydrometallurgy method. We demonstrate that a passivation layer of Li3PO4 is formed at grain boundaries during sintering, which significantly raises the activation barrier and hinders lithium diffusion. In addition, the distinct degradation of cathode electrochemical properties is observed from poor particle morphology and high cation mixing as a result of phosphate impurity. Cathode powders with 1 at. % phosphate impurity retain a capacity of 146 mAh/g after 100 cycles at 0.33C, 6% less than that of a virgin cathode. Furthermore, cathodes with higher phosphate concentrations perform even worse in electrochemical tests. Therefore, phosphate impurities are detrimental to the hydrometallurgical recycling of NCM cathode materials and need to be excluded from the recycling process.

Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode.

Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids’ full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an “on/off” retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method.

Effects and mechanisms of animal-free hydrolysates on recombination protein yields in CHO cells.

Chinese hamster ovary (CHO) cells are the commonly used cell lines for producing recombinant therapeutic proteins (RTPs) because they possess post-translational modifications similar to human cells. Culture media are necessary for cell growth, and their quality affects the yields and quality of RTPs. Due to safety concerns for the complex purification of RTPs, the development of serum-free media (SFM) is necessary for CHO cells. To meet the need for CHO cells with higher cell density and RTP productivity with consistent product quality in large-scale suspension cultures, the optimization of SFM through adding some enzymatic animal-free hydrolysates (AFHs) is preferred. The AFHs can improve cell culture performance and product yield of RTPs without affecting their quality. Here, the effect and mechanism of various AFHs in improving CHO cell culture performance and protein expression are reviewed. KEY POINTS: • AFHs that improve the recombinant protein yield of CHO cells are reviewed. • AFHs improve recombinant protein yield via influencing cell performance. • The AFHs do not affect the quality of recombinant protein in CHO cells. • AFHs can provide nutrients, block cell cycle, and reduce oxidative stress.

Survey on the Advantages of Metal 3D Printing Using Laser Sintering Compared to Traditional Metal Manufacturing Methods

AbstractAdditive manufacturing (AM) is a niche process that has seen extensive growth and adoption in the past decade in the industrial sector. Among the AM techniques present on the market today there are identified only a few methods allowing the 3D printing of metallic materials to be of special interest for their applications in various domains like the automotive, aeronautical, and healthcare industries. The most important questions needing answers are how to produce components from metallic materials that have superior qualities like versatility, wear resistance, ease of customization, and ease of integration with existing components. Compared to traditional metalworking methods where it has been studied how the metal behaves and morphs during the process and where it is known exactly what to do in order to modify the properties of the resulting component into a favor metal, AM changes everything, forcing a researcher to start from the beginning. While the technology is starting to be used more and more, the need to find production methods leads to consistent quality products and with outstanding material properties. New methods need to be developed to help attune the properties of the 3D printed parts to the requirements of their respective uses. The present study refers to 3D metal additive manufacturing (MAM), providing an overview on the process itself and its possible results as well as hurdles with added methods to overcome them.

Implementation of a high cell density fed-batch for heterologous production of active [NiFe]-hydrogenase in Escherichia coli bioreactor cultivations

BackgroundO2-tolerant [NiFe]-hydrogenases offer tremendous potential for applications in H2-based technology. As these metalloenzymes undergo a complicated maturation process that requires a dedicated set of multiple accessory proteins, their heterologous production is challenging, thus hindering their fundamental understanding and the development of related applications. Taking these challenges into account, we selected the comparably simple regulatory [NiFe]-hydrogenase (RH) from Cupriavidus necator as a model for the development of bioprocesses for heterologous [NiFe]-hydrogenase production. We already reported recently on the high-yield production of catalytically active RH in Escherichia coli by optimizing the culture conditions in shake flasks.ResultsIn this study, we further increase the RH yield and ensure consistent product quality by a rationally designed high cell density fed-batch cultivation process. Overall, the bioreactor cultivations resulted in ˃130 mg L−1 of catalytically active RH which is a more than 100-fold increase compared to other RH laboratory bioreactor scale processes with C. necator. Furthermore, the process shows high reproducibility of the previously selected optimized conditions and high productivity.ConclusionsThis work provides a good opportunity to readily supply such difficult-to-express complex metalloproteins economically and at high concentrations to meet the demand in basic and applied studies.