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The heterotrophic cultivation of extremophilic archaea still heavily relies on complex media. However, complex media are associated with unknown composition, high batch-to-batch variability, potential inhibiting and interfering components, as well as regulatory challenges, hampering advancements of extremophilic archaea in genetic engineering and bioprocessing. For Metallosphaera sedula, a widely studied organism for biomining and bioremediation and a potential production host for archaeal ether lipids, efforts to find defined cultivation conditions have still been unsuccessful. This study describes the development of a novel chemically defined growth medium for M. sedula. Initial experiments with commonly used complex casein-derived media sources deciphered Casamino Acids as the most suitable foundation for further development. The imitation of the amino acid composition of Casamino Acids in basal Brock medium delivered the first chemically defined medium. We could further simplify the medium to 5 amino acids based on the respective specific substrate uptake rates. This first defined cultivation medium for M. sedula allows advanced genetic engineering and more controlled bioprocess development approaches for this highly interesting archaeon.

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Cell culture media play a critical role in cell growth and propagation by providing a substrate; media components can also modulate the critical quality attributes (CQAs). However, the inherent complexity of the cell culture media makes unraveling the impact of the various media components on cell growth and CQAs non-trivial. In this study, we demonstrate an end-to-end machine learning framework for media component selection and prediction of CQAs. The preliminary dataset for feature selection was generated by performing CHO-GS (-/-) cell culture in media formulations with varying metal ion concentrations. Acidic and basic charge variant composition of the innovator product (24.97 ± 0.54% acidic and 11.41 ± 1.44% basic) was chosen as the target variable to evaluate the media formulations. Pearson's correlation coefficient and random forest-based techniques were used for feature ranking and feature selection for the prediction of acidic and basic charge variants. Furthermore, a global interpretation analysis using SHapley Additive exPlanations was utilized to select optimal features by evaluating the contributions of each feature in the extracted vectors. Finally, the medium combinations were predicted by employing fifteen different regression models and utilizing a grid search and random search cross-validation for hyperparameter optimization. Experimental results demonstrate that Fe and Zn significantly impact the charge variant profile. This study aims to offer insights that are pertinent to both innovators seeking to establish a complete pipeline for media development and optimization and biosimilar-based manufacturers who strive to demonstrate the analytical and functional biosimilarity of their products to the innovator. KEY POINTS: • Developed a framework for optimizing media components and prediction of CQA. • SHAP enhances global interpretability, aiding informed decision-making. • Fifteen regression models were employed to predict medium combinations.

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The fibrous cap is formed by smooth muscle cells that accumulate beneath the plaque endothelium. Cap rupture is the main cause of coronary thrombosis, leading to infarction and sudden cardiac death. Therefore, the qualities of the cap are primary determinants of the clinical outcome of coronary and carotid atherosclerosis. In this mini-review, we discuss current knowledge about the formation of the fibrous cap, including cell recruitment, clonal expansion, and central molecular signaling pathways. We also examine the differences between mouse and human fibrous caps and explore the impact of anti-atherosclerotic therapies on the state of the fibrous cap. We propose that the cap should be understood as a neo-media to substitute for the original media that becomes separated from the surface endothelium during atherogenesis and that embryonic pathways involved in the development of the arteria media contribute to cap formation.

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BACKGROUND: Historically, complex media are used for the cultivation of Gluconobacter oxydans in industry and research. Using complex media has different drawbacks like higher costs for downstream processing and significant variations in fermentation performances. Synthetic media can overcome those drawbacks, lead to reproducible fermentation performances. However, the development of a synthetic medium is time and labour consuming. Detailed knowledge about auxotrophies and metabolic requirements of G. oxydans is necessary. In this work, we use a systematic approach applying the in-house developed µRAMOS technology to identify auxotrophies and develop a defined minimal medium for cultivation of G. oxydans fdh, improving the production process of the natural sweetener 5-ketofructose. RESULTS: A rich, defined synthetic medium, consisting of 48 components, including vitamins, amino acids and trace elements, was used as a basis for medium development. In a comprehensive series of experiments, component groups and single media components were individually omitted from or supplemented to the medium and analysed regarding their performance. Main components like salts and trace elements were necessary for the growth of G. oxydans fdh, whereas nucleotides were shown to be non-essential. Moreover, results indicated that the amino acids isoleucine, glutamate and glycine and the vitamins nicotinic acid, pantothenic acid and p-aminobenzoic acid are necessary for the growth of G. oxydans fdh. The glutamate concentration was increased three-fold, functioning as a precursor for amino acid synthesis. Finally, a defined minimal medium called 'Gluconobacter minimal medium' was developed. The performance of this medium was tested in comparison with commonly used media for Gluconobacter. Similar/competitive results regarding cultivation time, yield and productivity were obtained. Moreover, the application of the medium in a fed-batch fermentation process was successfully demonstrated. CONCLUSION: The systematic investigation of a wide range of media components allowed the successful development of the Gluconobacter minimal medium. This chemically defined medium contains only 14 ingredients, customised for the cultivation of G. oxydans fdh and 5-ketofructose production. This enables a more straightforward process development regarding upstream and downstream processing. Moreover, metabolic demands of G. oxydans were identified, which further can be used in media or strain development for different processes.

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Proposed herein is a systematic media design framework that combines multivariate statistical approaches with in silico analysis of a genome-scale metabolic model of Chinese hamster ovary cell. The framework comprises sequential modules including cell culture and metabolite data collection, multivariate data analysis, in silico modeling and flux prediction, and knowledge-based identification of target media components. Two monoclonal antibody-producing cell lines under two different media conditions were used to demonstrate the applicability of the framework. First, the cell culture and metabolite profiles from all conditions were generated, and then statistically and mechanistically analyzed to explore combinatorial effects of cell line and media on intracellular metabolism. As a result, we found a metabolic bottleneck via a redox imbalance in the TCA cycle in the poorest growth condition, plausibly due to inefficient coenzyme q10-q10h2 recycling. Subsequent in silico simulation allowed us to suggest q10 supplementation to debottleneck the imbalance for the enhanced cellular energy state and TCA cycle activity. Finally, experimental validation was successfully conducted by adding q10 in the media, resulting in increased cell growth. Taken together, the proposed framework rationally identified target nutrients for cell line-specific media design and reformulation, which could greatly improve cell culture performance.

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Developing media to sustain cell growth and production is an essential and ongoing activity in bioprocess development. Modifications to media can often address host or product-specific challenges, such as low productivity or poor product quality. For other applications, systematic design of new media can facilitate the adoption of new industrially relevant alternative hosts. Despite manifold existing methods, common approaches for optimization often remain time and labor-intensive. We present here a novel approach to conventional media blending that leverages stable, simple, concentrated stock solutions to enable rapid improvement of measurable phenotypes of interest. We applied this modular methodology to generate high-performing media for two phenotypes of interest: biomass accumulation and heterologous protein production, using high-throughput, milliliter-scale batch fermentations of Pichia pastoris as a model system. In addition to these examples, we also created a flexible open-source package for modular blending automation on a low-cost liquid handling system to facilitate wide use of this method. Our modular blending method enables rapid, flexible media development, requiring minimal labor investment and prior knowledge of the host organism, and should enable developing improved media for other hosts and phenotypes of interest.

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Chemically defined (CD) media are routinely used in the production of biologics in Chinese hamster ovary (CHO) cell culture and provide enhanced raw material control. Nutrient optimized CD media is an important path to increase cell growth and monoclonal antibody (mAb) productivity in recombinant CHO cell lines. However, nutrient optimization efforts for CD media typically rely on multifactorial and experimental design of experiment approaches or complex mathematical models of cellular metabolism or gene expression systems. Moreover, the majority of these efforts are aimed at amino acids since they constitute essential nutrients in CD media as they directly contribute to biomass and protein production. In this study, we demonstrate the utilization of multivariate data analytics (MVDA) coupled with amino acid stoichiometric balances (SBs) to increased cell growth and mAb productivity in efforts to support CD media development efforts. SBs measure the difference between theoretical demand of amino acids and the empirically measured fluxes to identify various catabolic or anabolic states of the cell. When coupled with MVDA, the statistical models were not only able to highlight key amino acids toward cell growth or productivity, but also provided direction on metabolic favorability of the amino acid. Experimental validation of our approach resulted in a 55% increase in total cell growth and about an 80% increase in total mAb productivity. Increased specific consumption of stoichiometrically balanced amino acids and decreased specific consumption of glucose was also observed in optimized CD media suggesting favorable consumption of desired nutrients and a potential for energy redistribution toward increased cellular growth and mAb productivity.

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BACKGROUND: Nonribosomal peptide synthases (NRPS) can synthesize functionally diverse bioactive peptides by incorporating nonproteinogenic amino acids, offering a rich source of new drug leads. The bacterium Escherichia coli is a well-characterized production host and a promising candidate for the synthesis of nonribosomal peptides, but only limited bioprocess engineering has been reported for such molecules. We therefore developed a medium and optimized process parameters using the design of experiments (DoE) approach. RESULTS: We found that glycerol is not suitable as a carbon source for rhabdopeptide production, at least for the NRPS used for this study. Alternative carbon sources from the tricarboxylic acid cycle achieved much higher yields. DoE was used to optimize the pH and temperature in a stirred-tank reactor, revealing that optimal growth and optimal production required substantially different conditions. CONCLUSIONS: We developed a chemically defined adapted M9 medium matching the performance of complex medium (lysogeny broth) in terms of product concentration. The maximum yield in the reactor under optimized conditions was 126 mg L-1, representing a 31-fold increase compared to the first shaking-flask experiments with M9 medium and glycerol as the carbon source. Conditions that promoted cell growth tended to inhibit NRPS productivity. The challenge was therefore to find a compromise between these factors as the basis for further process development.

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The thermoacidophilic Crenarchaeon Sulfolobus acidocaldarius is an important model organism for Archaea and genetic systems are well established. To date, the organism is routinely cultivated on complex media based on protein hydrolysates and no common defined medium is established. In this work we address this lack of a standardized defined medium and replaced the complex protein hydrolysate with sodium glutamate as primary substrate. Starting from an existing medium formulation we stepwise managed to improve the medium regarding formation of precipitates, buffer capacity, concentration of basal salts and trace elements, and optimized growth rates. The differences on the cellular level between the original medium and our new formulation, called VD Medium, were investigated by comparative gene expression analysis and significant differences were discussed. The final formulation of the VD Medium contains 1.75 g/L Na-glutamate, 3 g/L D-glucose and 0.5 g/L citric acid as carbon sources. Using the described medium for the cultivation of S. acidocaldarius DSM 639 in shake flasks yields 1.1 g/L dry cell weight (OD600 = 1.7) after a typical incubation time of 95 h with an overall biomass yield of 0.33 gDCW/gsubstrate.

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Perfusion is a cell culture mode that is gaining popularity for the manufacture of monoclonal antibodies and their derivatives. The cell culture media supporting perfusion culture need to support higher cell densities than those used in fed-batch culture. Therefore, when switching from a fed-batch to a perfusion mode, a new medium need to be developed which supports high cell densities, high productivity, and favorable product quality. We have developed a method for deriving perfusion culture media based on existing fed-batch media and feeds. We show that we can obtain culture media that successfully support perfusion cultures in a single-use rocking bioreactor system at cell-specific perfusion rates below 25 pL-1 cell-1 day-1 . High productivities and favorable product quality are also achievable.

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Ranibizumab is a biotherapeutic Fab fragment used for the treatment of age-related macular degeneration and macular oedema. It is currently expressed in the gram-negative bacterium, Escherichia coli. However, low expression levels result in a high manufacturing cost. The protein expression can be increased by manipulating nutritional requirements (carbon source, nitrogen source, buffering agent), process parameters (pH, inducer concentration, agitation, temperature), and the genetic make-up of the producing strain. Further, understanding the impact of these factors on product quality is a requirement as per the principles of Quality by Design (QbD). In this paper, we examine the effect of various media components and process parameters on the expression level and quality of the biotherapeutic. First, risk analysis was performed to shortlist different media components based on the literature. Next, experiments were performed to screen these components. Eight components were identified for further investigation and were examined for their effect and interactions using a Fractional Factorial experimental design. Sucrose, biotin, and pantothenate were found to have the maximum effect during Fab production. Furthermore, cyanocobalamin glutathione and biotin-glutathione were the most significant interactions observed. Product identification was performed with Liquid Chromatography⁻Mass Spectrometry (LC-MS), the expression level was quantified using Bio-layer Interferometry, Reverse Phase-HPLC, and SDS-PAGE, and product quality were measured by RP-HPLC. Overall, a five-fold enhancement of the target protein titer was obtained (from 5 mg/L to 25 mg/L) using the screened medium components vis-a-vis the basal medium, thereby demonstrating the efficacy of the systematic approach purported by QbD.

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The extent and pattern of glycosylation on therapeutic antibodies can influence their circulatory half-life, engagement of effector functions, and immunogenicity, with direct consequences to efficacy and patient safety. Hence, controlling glycosylation patterns is central to any drug development program, yet poses a formidable challenge to the bio-manufacturing industry. Process changes, which can affect glycosylation patterns, range from manufacturing at different scales or sites, to switching production process mode, all the way to using alternative host cell lines. In the emerging space of biosimilars development, often times all of these aspects apply. Gaining a deep understanding of the direction and extent to which glycosylation quality attributes can be modulated is key for efficient fine-tuning of glycan profiles in a stage appropriate manner, but establishment of such platform knowledge is time consuming and resource intensive. Here we report an inexpensive and highly adaptable screening system for comprehensive modulation of glycans on antibodies expressed in CHO cells. We characterize 10 media additives in univariable studies and in combination, using a design of experiments approach to map the design space for tuning glycosylation profile attributes. We introduce a robust workflow that does not require automation, yet enables rapid process optimization. We demonstrate scalability across deep wells, shake flasks, AMBR-15 cell culture system, and 2 L single-use bioreactors. Further, we show that it is broadly applicable to different molecules and host cell lineages. This universal approach permits fine-tuned modulation of glycan product quality, reduces development costs, and enables agile implementation of process changes throughout the product lifecycle.

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CHO cells are the most prevalent platform for modern bio-therapeutic production. Currently, there are several CHO cell lines used in bioproduction with distinct characteristics and unique genotypes and phenotypes. These differences limit advances in productivity and quality that can be achieved by the most common approaches to bioprocess optimization and cell line engineering. Incorporating omics-based approaches into current bioproduction processes will complement traditional methodologies to maximize gains from CHO engineering and bioprocess improvements. In order to highlight the utility of omics technologies in CHO bioproduction, the authors discuss current applications as well as limitations of genomics, transcriptomics, proteomics, metabolomics, lipidomics, fluxomics, glycomics, and multi-omics approaches and the potential they hold for the future of bioproduction. Multiple omics approaches are currently being used to improve CHO bioprocesses; however, the application of these technologies is still limited. As more CHO-omic datasets become available and integrated into systems models, the authors expect significant gains in product yield and quality. While individual omics technologies provide incremental improvements in bioproduction, the authors will likely see the most significant gains by applying multi-omics and systems biology approaches to individual CHO cell lines.

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We observed that removing pantothenate (vitamin B5), a precursor to co-enzyme A, from the growth medium of Saccharomyces cerevisiae engineered to produce ß-farnesene reduced the strain׳s farnesene flux by 70%, but increased its viability, growth rate and biomass yield. Conversely, the growth rate and biomass yield of wild-type yeast were reduced. Cultivation in media lacking pantothenate eliminates the growth advantage of low-producing mutants, leading to improved production upon scale-up to lab-scale bioreactor testing. An omics investigation revealed that when exogenous pantothenate levels are limited, acyl-CoA metabolites decrease, ß-oxidation decreases from unexpectedly high levels in the farnesene producer, and sterol and fatty acid synthesis likely limits the growth rate of the wild-type strain. Thus pantothenate supplementation can be utilized as a "metabolic switch" for tuning the synthesis rates of molecules relying on CoA intermediates and aid the economic scale-up of strains producing acyl-CoA derived molecules to manufacturing facilities.

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Rice straw is one of the potential economic feedstock for biobutanol production through ABE fermentation. However, the rice straw hydrolysate-based fermentation medium needs to be supported with nutritional elements. In this study, an attempt is made to optimize the rice straw hydrolysate-based fermentation medium employing Clostridium acetobutylicum MTCC 481 using Taguchi design of experiments (DOE) statistical model. Initially, a set of 12 nutrient components viz. MgNO3·6H2O, FeNO3, NH4NO3, yeast extract, PABA, biotin, PABA + biotin mixture, CaCl2, KCl, NaCl, MgSO4 and CH3COONa were screened through classical (one-variable-at-a-time) method. Based on the results, four components (PABA, yeast extract, MgSO4 and CH3COONa) were found to have significant impact, and were further subjected to statistical optimization through Taguchi DOE method. These experiments revealed that RSH supported with 3 g L-1 of yeast extract and 4 mg L-1 PABA to RSH was the most optimum fermentation medium. Experiments using 2 L bioreactor with this optimum fermentation medium showed nearly complete utilization of soluble sugars with the production of 8.7 g L-1 of total solvents and 6 g L-1 of butanol. The experimental data were fitted to kinetic models reported in the literature to determine the kinetic parameters of the fermentation process. An interesting result was revealed from this analysis that the under optimized fermentation medium, the kinetic parameters for both shake flask and bioreactor level were similar. This essentially means that effect of scale of operation is rendered insignificant when fermentation medium is under optimum conditions.

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The standard culture system for in vitro cartilage research is based on cells in a three-dimensional micromass culture and a defined medium containing the chondrogenic key growth factor, transforming growth factor (TGF)-ß1. The aim of this study was to optimize the medium for chondrocyte micromass culture. Human chondrocytes were cultured in different media formulations, designed with a factorial design of experiments (DoE) approach and based on the standard medium for redifferentiation. The significant factors for the redifferentiation of the chondrocytes were determined and optimized in a two-step process through the use of response surface methodology. TGF-ß1, dexamethasone, and glucose were significant factors for differentiating the chondrocytes. Compared to the standard medium, TGF-ß1 was increased 30%, dexamethasone reduced 50%, and glucose increased 22%. The potency of the optimized medium was validated in a comparative study against the standard medium. The optimized medium resulted in micromass cultures with increased expression of genes important for the articular chondrocyte phenotype and in cultures with increased glycosaminoglycan/DNA content. Optimizing the standard medium with the efficient DoE method, a new medium that gave better redifferentiation for articular chondrocytes was determined.

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Complex B vitamins as Biotin and Riboflavin are required by living organisms, not only for growth but also for metabolite production, and the feed market classifies them as growth promoters. Since Brazil will soon be one of the world's biggest animal protein producers, feed production is a large consumer of vitamins and micronutrients. The industry requires 10 mg riboflavin/0.2 mg biotin per kilogram of feed; a ratio of 40 ~ 50:1. Although few studies have been conducted specifically on riboflavin production using factorial design and surface response method as an optimization strategy, it is a common practice in biotechnology with many research reports available. However, there are no reports on the use of statistical design for biotin production. This study set out to evaluate medium composition influence on biotin and riboflavin production using a statistical design. There are no studies relating biotin and riboflavin production by Candida sp LEB 130. In this preliminary study to improve the simultaneous production of biotin and riboflavin, the maximum riboflavin/biotin ratio of 8.3 µg/mL was achieved with medium component concentrations of: sucrose 30 g/L, KH2PO4 2 g/L, MgSO4 1 g/L and ZnSO4 0.5mL/L.

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Complex B vitamins as Biotin and Riboflavin are required by living organisms, not only for growth but also for metabolite production, and the feed market classifies them as growth promoters. Since Brazil will soon be one of the world's biggest animal protein producers, feed production is a large consumer of vitamins and micronutrients. The industry requires 10 mg riboflavin/0.2 mg biotin per kilogram of feed; a ratio of 40 ~ 50:1. Although few studies have been conducted specifically on riboflavin production using factorial design and surface response method as an optimization strategy, it is a common practice in biotechnology with many research reports available. However, there are no reports on the use of statistical design for biotin production. This study set out to evaluate medium composition influence on biotin and riboflavin production using a statistical design. There are no studies relating biotin and riboflavin production by Candida sp LEB 130. In this preliminary study to improve the simultaneous production of biotin and riboflavin, the maximum riboflavin/biotin ratio of 8.3 µg/mL was achieved with medium component concentrations of: sucrose 30 g/L, KH2PO4 2 g/L, MgSO4 1 g/L and ZnSO4 0.5mL/L.