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A novel approach has been proposed integrating sludge enrichment with intermittent gradient aeration to achieve partial nitrification (PN). Results indicated that this method suppressed nitrite-oxidizing bacteria (NOB) activity while maintained ammonia-oxidizing bacteria (AOB) activity, achieving an 82.87 % nitrite accumulation rate (NAR) during startup. During stable operation, specific ammonia oxidation rate and specific nitrite oxidation rate reached 18.44 and 2.67 mg N/g MLVSS/h, respectively. Shortening anoxic time from 30 to 20 min enhanced ammonia removal efficiency by 12.5 %, increasing NAR to 89.55 %. Further reduction to 5 min yielded 38.46 % and 91.88 %, respectively. AOB abundance enriched from 0.62 % to 22.28 %, with the AOB-to-NOB ratio jumping from 0.22 to 59.58. This research presented a rapid and effective strategy for achieving PN., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Batch fermentations of the wild type Yarrowia lipolytica MUCL 28849 were performed in a bench-top bioreactor to assess crucial operating conditions. A setup of carbon to nitrogen (mol/mol) ratio equal to 34, pH = 6.0 and 52 g/L of crude glycerol showed increased lipid production and complete glycerol consumption at t = 24 h, thus, selected for further process improvement. Α semi-continuous process was implemented, where a pH drop to 4.0 at 24 h, interrupted citric acid secretion without affecting lipid production. An in-situ membrane module was employed for membrane bioreactor fermentations, where yeast cells were successfully retained with minimum fouling. The membrane bioreactor fed-batch process, resulted in a high-cell-density culture reaching 49.8 g/L of dry biomass and 4.9 g/L of lipids. An unstructured model was developed and successfully simulated operation under all fermentation modes, distinguishing diverse physiological shifts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

This study conceptualizes the design of a small-scale system (250 mL-1 L) for the autotrophic cultivation of hydrogen-oxidizing bacteria, such as the representative strain Cupriavidus necator. The research aimed to systematically investigate the impact of bottle volume and gas composition, particularly oxygen concentration, on the growth and performance of C. necator during autotrophic cultivations. To this end, customized, pressure-tight, baffled glass bottles of various sizes (250, 500, and 1000 mL) and gas mixtures with varying oxygen concentrations (4%, 8%, and 12% v/v) were tested. Growth was monitored by measuring optical density. The maximum specific growth rate (µ max ), the biomass production rate (BPR), the volumetric gas-liquid mass transfer coefficient (k L a), and the oxygen transfer rate were calculated. Among the various combinations, the 1000-mL bottles demonstrated the highest µ max (0.13 h -1 ) and the second-highest BPR (0.074 g L -1  h -1 ) at an oxygen concentration of 8%, without the need to refill the headspace. The proposed small-scale system offers a swift and replicable method for concurrently investigating multiple autotrophic cultivations. In this regard, increasing the size of the bottle flask proved to be an efficient strategy to minimize the periodicity for gas refilling. Due to the inhibitory effect of oxygen, changing the liquid-gas volume ratio in hydrogen-driven shake flask cultivation had so far strongly influenced the growth rate. Our results provide a solid foundation for the scaling and optimization of small-scale cultivation of chemolithotrophic bacteria and will facilitate future parallelization and, hence, optimization of metabolic aspects., (© 2025 Wiley‐VCH GmbH.)

Background: Proteases are essential in various industries due to their unique substrate specificities and robustness in different operational conditions. Bacillus strains consist of a genotype favorable for rapid growth whilst secreting enzymes extracellularly, thereby simplifying recombinant protease production. Despite the widespread use of batch and fed-batch fermentations for their ease and robustness, these cultivation types are often marred by significant energy requirements and prolonged downtimes. The switch towards continuous cultivation methods promises reduced carbon footprints and improved equipment efficiency. Yet, research focusing on Bacillus strains is limited, therefore we aimed to establish a continuous cultivation as a competitive alternative to fed-batch., Results: Therefore, this study aimed to explore the potential of chemostat cultivations for producing a protease from Bacillus licheniformis utilizing a derepressed induction system, and comparing specific productivities and space-time yields to fed-batch cultivations. The continuous cultivations were described in a hybrid model, considering the effect of productivity as function of the applied dilution rate as well as the generation time. The workflow of this study demonstrates that screenings in a fed-batch mode and chemostat cultivations conducted at the same growth rate, result in different specific productivities for derepressible systems., Conclusion: The results of this study highlight that the feeding rate's impact on specific productivity varies significantly between fed-batch and chemostat cultivations. These differences suggest that fed-batch screenings may not be adequate for developing a continuous process using a derepressed promoter system in B. licheniformis. Although the space-time yield of fed-batch cultivations has not been surpassed by stable continuous operations-achieving only a third of the highest space-time yield observed in fed-batch-valuable mechanistic insights have been gained. This knowledge could facilitate the transition towards a more sustainable mode of cultivation for industrial protease production., Competing Interests: Declarations. Ethical approval: Not applicable. Consent for publication: Not applicable. Clinical trial number: Not applicable. Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: As a novel type of extracellular polysaccharide produced by Sphingomonas sp., welan gum has been widely applied in various fields because of its excellent properties. The study has improved the fermentation process., Results: The initial sucrose concentration, temperature and stirring speed were set to 20 g L -1 , 33 °C and 400 rpm, respectively, and 13.3 g L -1 sucrose was added at 24, 40 and 56 h. The temperature and stirring speed were then set at 28 °C and 600 rpm from 24 to 48 h and 28 °C and 600 rpm from 48 to 72 h, respectively. As a result, welan gum production, dry cell weight, sucrose conversion rate and viscosity were correspondingly increased to 38.60 g L -1 , 5.47 g L -1 , 0.64 g g -1 and 3779 mPa·s, respectively. In addition, the mechanism by which fermentation strategy promotes welan gum synthesis was investigated by transcriptome analysis., Conclusion: Improving respiration and ATP supply, reducing unnecessary protein synthesis, and alleviating competition between cell growth and welan gum synthesis contribute to promoting the fermentation performance of Sphingomonas sp., thus providing a practical strategy for efficient welan gum production. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Erythritol is a beneficial sugar alcohol that can be used as a sugar substitute for diabetic patients. Erythritol is a bioproduct produced by microorganisms as a response to high osmotic pressure and stress in the growth medium. High concentrations of carbon source substrate can increase the osmotic pressure and provide more nutrient supply for yeast growth and metabolism. Aside from that, an optimal carbon-to-nitrogen (C/N) ratio can also make the erythritol conversion pathway more favorable. Therefore, this research aims to determine the optimal concentrations of molasses as the carbon source, yeast extract as the nitrogen source, and the optimal carbon-to-nitrogen (C/N) ratio to achieve the highest erythritol productivity. The research also seeks to optimize NaCl concentrations and pH while comparing batch and fed-batch fermentation systems to determine which produces a higher erythritol yield. One-Factor-at-A-Time (OFAT) method was used to identify optimal production conditions. The study found that the highest erythritol concentration, 17.48 ± 0.86 g/L, was achieved using 200 g/L of molasses, 7 g/L of yeast extract (200/7), and 25 g/L of NaCl, with a yield mass of 0.262 ± 0.00 g/g and a volumetric productivity of 0.095 ± 0.021 g/Lh. The pH optimization revealed that the best erythritol production occurred within a pH of 5. Furthermore, fed-batch fermentation significantly increased erythritol concentration to 26.52 ± 1.61 g/L, with a yield mass of 0.501 ± 0.032 g/g and a volumetric productivity of 0.158 ± 0.01 g/Lh. These findings emphasize the importance of optimizing carbon source, nitrogen source and NaCl concentration, pH, and fermentation systems, particularly highlighting the benefits of fed-batch fermentation in maximizing erythritol production. These findings provide a solid foundation for improving erythritol yields for industrial applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Kembaren, Parikesit, Nataniel, Dharmawan, Dungus, Angelique and Aslanzadeh.)

Protocatechuate acid (PCA) is a phenolic acid naturally synthesized by various organisms. Protocatechuic acid is synthesized by plants for physiological, metabolic functions, and self-defense, but extraction from plants is less efficient compared to the microbial culture process. The microbial synthesis of protocatechuic acid is sustainable and, due to its high yield, can save energy consumption when producing the same amount. To enhance PCA production using Corynebacterium glutamicum , a statistical optimization of the production medium was performed using full factorial design, the steepest ascent method, and the response surface method. The optimized production medium enabled a PCA production of over 5 g/L in a 72 h batch culture. However, PCA cytotoxicity affected the strain growth and PCA production rate, with an inhibitory concentration of approximately 5 g/L in the fermentation broth. Finally, continuous fermentation was operated for 150 h in the steady-state mode, maintaining the concentration of PCA below 5 g/L. The optimization method established in this study successfully increased PCA production levels, and the findings presented herein are anticipated to contribute to the industrialization of PCA production using C. glutamicum.

Food wastedigestatewas fed into a sequencing batch reactor (SBR) forHaloferax mediterranei(HM) to produce poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). This SBR was operated uninterruptedly for 450 days to test its stability, during which the cycle time and volume exchange ratiowere varied to understand their impacts on the PHBV fermentation performance under ranged organic loading rates (OLR). Results showed that 1) PHBV productivity was proportional to OLR of food wastedigestate; 2) substrate and product inhibitions were two limiting factors constraining substrate utilization and PHBV yields; 3) PHBV titer was dependent on the hydraulic retention time of the SBR while a volume exchange ratio lower than 0.5 is unfavorable due to the product inhibitor accumulation. This study for the first time demonstrated that the long-term stability of food waste-fed PHBV production byHMand revealed that inhibition effects could be barriers in SBR limiting the full-scale application of the technology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier Ltd.)

Chinese hamster ovary (CHO) cells are widely used to produce recombinant proteins, including monoclonal antibodies (mAbs), through various process modes. While fed-batch (FB) processes have been the standard, a shift toward high-density perfusion processes is being driven by increased productivity, flexible facility footprints, and lower costs. Ensuring the clearance of process-related impurities, such as host cell proteins (HCPs), is crucial in biologics manufacturing. Although purification processes remove most impurities, integrated strategies are being developed to enhance clearance of some high-risk HCPs. Current understanding of HCP expression dynamics in cell culture is limited. This study utilized data-independent acquisition (DIA) proteomics to compare the proteomic profiles of cell culture supernatants from 14 FB clones and three perfusion clones, all expressing the same mAb from the same host cell line. Results showed that perfusion processes enhance cell growth and productivity, exhibiting distinct proteomic profiles compared to FB processes. Perfusion processes also maintain a more comparable HCP abundance profile across clones, especially for 46 problematic HCPs monitored. Cluster analysis of FB proteomics revealed distinct abundance patterns and correlations with process parameters. Differential abundance analysis identified significant protein differences between the two processes. This is the first extensive study characterizing HCPs expressed by clones under different process modes. Further research could lead to strategies for preventing or managing problematic HCPs in biologics manufacturing., (© 2025 Merck Sharp & Dohme LLC. Biotechnology Journal published by Wiley‐VCH GmbH.)

Purpose: Perfusion cultures have been extensively used in the biotechnology industry to achieve high yields of recombinant products, especially those with stability issue. The WuXiUP™ platform represents a novel intensified perfusion that can achieve ultra-high productivity. This study describes a representative scale-down 24-deep well plate (24-DWP) cell culture model for intensified perfusion clone screening., Methods: Clonal cell lines were expanded and evaluated in 24-DWP semi-continuous culture. Cell were sampled and counted daily with the aid of an automated liquid handler and high-throughput cell counter. To mimic perfusion culture, 24-DWP plates were spun down and resuspended with fresh medium daily. Top clones were ranked based on growth profiles and productivities. The best performing clones were evaluated on bioreactors., Results: The selected clones achieved volumetric productivity (Pv) up to 5 g/L/day when expressing a monoclonal antibody, with the accumulative harvest Pv exceeding 60 g/L in a 21-day cell culture. Product quality attributes of clones cultured in 24-DWP were comparable with those from bioreactors. A high seeding strategy further shortened the clone screening timeline., Conclusion: In this study, a 24-DWP semi-continuous scale-down model was successfully developed to screen for cell lines suitable for intensified perfusion culture., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

There is a growing interest in perfusion or continuous processes to achieve higher productivity of biopharmaceuticals in mammalian cell culture, specifically Chinese hamster ovary (CHO) cells, towards advanced biomanufacturing. These intensified bioprocesses highly require concentrated feed media in order to counteract their dilution effects. However, designing such condensed media formulation poses several challenges, particularly regarding the stability and solubility of specific amino acids. To address the difficulty and complexity in relevant media development, the biopharmaceutical industry has recently suggested forming dipeptides by combining one from problematic amino acids with selected pairs to compensate for limitations. In this study, we combined one of the lead amino acids, L-tyrosine, which is known for its poor solubility in water due to its aromatic ring and hydroxyl group, with glycine as the partner, thus forming glycyl-L-tyrosine (GY) dipeptide. Subsequently, we investigated the utilization of GY dipeptide during fed-batch cultures of IgG-producing CHO cells, by changing its concentrations (0.125 × , 0.25 × , 0.5 × , 1.0 × , and 2.0 ×). Multivariate statistical analysis of culture profiles was then conducted to identify and correlate the most significant nutrients with the production, followed by in silico model-guided analysis to systematically evaluate their effects on the culture performance, and elucidate metabolic states and cellular behaviors. As such, it allowed us to explain how the cells can more efficiently utilize GY dipeptide with respect to the balance of cofactor regeneration and energy distribution for the required biomass and protein synthesis. For example, our analysis results uncovered specific amino acids (Asn and Gln) and the 0.5 × GY dipeptide in the feed medium synergistically alleviated the metabolic bottleneck, resulting in enhanced IgG titer and productivity. In the validation experiments, we tested and observed that lower levels of Asn and Gln led to decreased secretion of toxic metabolites, enhanced longevity, and elevated specific cell growth and titer. KEY POINTS: • Explored the optimal Tyr dipeptide for the enhanced CHO cell culture performance • Systematically analyzed effects of dipeptide media by model-guided approach • Uncovered synergistic metabolic utilization of amino acids with dipeptide., (© 2024. The Author(s).)

This study investigates the production of polyhydroxybutyrate (PHB) using the thermophilic bacterium Caldimonas thermodepolymerans in fed-batch fermentation. This research highlights the potential of thermophilic bacteria in biopolymer production due to their ability to operate at high temperatures, which reduces contamination risks and enhances energy efficiency. Optimal fermentation conditions were identified at a temperature of 50 °C, with the strain achieving a maximum specific growth rate (μ max ) of 0.57 h -1 and high biomass concentration of 63.1 g CDW /L. PHB production reached a peak concentration of 31.9 g/L with a productivity of 1.30 g PHB /L/h. The high cell density approach in fed-batch fermentation not only maximizes the productivity and yield of PHB, but also optimizes the production process, making it more suitable for industrial-scale applications. The findings highlight the potential of thermophilic bacteria as a sustainable solution for enhancing PHB production and advancing biodegradable polymer synthesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2025 Elsevier Ltd. All rights reserved.)

Background: Oxygen provision is a bottleneck in conventional aerobic high cell density culturing (HCDC) of bacteria due to the low O 2 solubility in water. An alternative could be denitrification: anaerobic respiration using nitrogen oxides as terminal electron acceptors. Denitrification is attractive because NO 3 - is soluble in water, the end-product (N 2 ) is harmless, and denitrification is widespread among bacteria, hence suitable organisms for most purposes can be found. The pH must be controlled by injection of an inorganic acid to compensate for the pH increase by NO 3 - -consumption, resulting in salt accumulation if feeding the bioreactor with NO 3 - salt. We avoid this with our novel pH-stat approach, where the reactor is supplied with 5 M HNO 3 to compensate for the alkalization, thus sustaining NO 3 - -concentration at a level determined by the pH setpoint. Here we present the first feasibility study of this method, growing the model strain Paracoccus denitrificans anaerobically to high densities with glucose as the sole C-source and NO 3 - as the N-source and electron acceptor., Results: Our fed-batch culture reached 20 g cell dry weight L -1 , albeit with slower growth rates than observed in low cell density batch cultures. We explored reasons for slow growth, and the measured trace element uptake indicates it is not a limiting factor. Bioassays with spent medium excluded accumulation of inhibitory compounds at high cell density as the reason for the slow growth. The most plausible reason is that high metabolic activity led to CO 2 /H 2 CO 3 accumulation, thus suppressing pH, leading to a paucity in HNO 3 -feeding until N 2 -sparging had removed sufficient CO 2 . The three free intermediates in the denitrification pathway (NO 3 -  → NO 2 -  → NO → N 2 O → N 2 ) can all reach toxic concentrations if the electron flow is unbalanced, and this did occur if cells were glucose-limited. On the other hand, accumulation of polyhydroxyalkanoates occurred if the cells were NO 3 - -limited. Carefully balancing glucose provision according to the HNO 3 injected is thus crucial., Conclusions: This work provides a proof of concept, while also identifying CO 2 /H 2 CO 3 accumulation as a hurdle that must be overcome for further development and optimization of the method., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: A patent application, describing the basic concept of HCDC by denitrification has been filed (European Patent App No. 22700061.9 and US Patent App. 18/270,538, 2024)., (© 2024. The Author(s).)

Enzymatic production of nucleotide sugars on a multigram scale presents a challenge, as only a few processes have been reported for large-scale nucleotide sugar production. They rely primarily on batch synthesis and employ exceptional amounts of enzymes. This study introduces a novel approach for the multigram-scale production of nucleotide sugars with a continuous fed-batch membrane reactor. We successfully synthesized five main nucleotide sugars: UDP-Gal, UDP-GalNAc, UDP-GlcA, GDP-Man, and CMP-Neu5Ac on a multigram scale. Efficient biocatalyst utilization results in high performance, including space-time yield (STY, g*L -1 h -1 ), total turnover number (TTN, g product per g enzyme), and an efficient product formation rate (g/h) suitable for industrially relevant bioprocesses. The established continuous-fed batch reactor system produced up to 8.2 g CMP-Neu5Ac in three consecutive productions in less than 15 h with satisfying TTNs of 91 g Product /g Enzyme . Continuous production of UDP-GlcA over 28 h resulted in a final product amount of 14.8 g and TTN of 493 g P /g E . This process enables the production of nucleotide sugars with stable product formation, requiring minimal technical equipment for multigram quantities of nucleotide sugars at the laboratory scale. Notably, the system exhibited robustness and flexibility, allowing its application to various enzymatic nucleotide sugar synthesis cascades., Competing Interests: Declaration of Competing Interest This work encompasses an industrial-driven project. All prepared nucleotide sugar solutions were sent to our industrial partner for product purification. The details of the purification procedures cannot be fully disclosed. The described production of nucleotide sugars in a continuous fed-batch membrane reactor is subject to a patent application., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

This research delved into the effects of nutrient limitation on the level of sporulation and the cadmium adsorption capacity of the bacterium Bacillus sp. isolated from the rhizosphere of endemic soils in the Region of Valparaiso, Chile. The bacteria were subjected to nitrogen limitation in fed-batch mode and were compared to bacteria grown in batch culture without nutrient limitation. The cultures were carried out in a 3 L bioreactor with an external nitrogen supply of ammonium at a flow of 0.123 L h -1 . The specific maximum growth rate was 0.42 h -1 in batch and 0.45 h -1 in the exponential phase of the fed-batch. The analysis of sporulation did not show any significant difference between the biomass coming from the fed-batch and batch cultures. It was found that maximum cadmium adsorption capacity varied with culture strategy. The dry biomass grown without nutrient limitation exhibited a maximum adsorption capacity for cadmium of 65.0 mg Cd g -1 biomass . Conversely, the limited biomass achieved a lower cadmium adsorption capacity of approximately 36.0 mg Cd g -1 biomass . FTIR analysis showed that nitrogen limitation induced changes in the composition of the outer cell wall, specifically an increase of deacetlylated polysaccharides, reducing the relative amount of secondary amines and proteins from the peptidoglycan matrix. Amino groups from acetylated polysaccharides and proteins have been associated elsewhere with greater cadmium affinity, which could explain the poor results obtained with the nitrogen-restricted biomass. This study shows that new physiological states displaying different adsorption capabilities were effectively obtained by engineering the cell coverage of the bacteria using varying culture strategies. The fed-batch culture proved to be a valuable tool for studying PGPR strains for biosorption and other applications. Exploring diverse nutrient limitations and other pollutants in this bacterium and other members of the PGPR family offer great opportunities to tailor biosorption strategies based on specific conditions, ultimately contributing to sustainable environmental solutions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: In recent years, gene therapy drugs have been widely marketed, and their effectiveness and potential have been confirmed. Thus, increasing their production on an industrial scale is critical. Recombinant adeno-associated viruses (rAAVs) are optimal vectors for gene therapy applications, and the baculovirus expression vector system (BEVS), which is based on Sf9 cell culture, is a common tool for rAAV production., Methods: In this work, an Sf9 cell fed-batch process was developed using shake flasks. In the laboratory-scale bioreactor, four processes were selected as the key factors when carrying out the orthogonal experiment. On the basis of the equal P/V principle and considering the problem posed by air bubbles, a pilot-scale level bioreactor process was established., Results: Here, we describe a method in which a BEVS was used to produce rAAV vectors, with the cell density increasing to 22.8 × 10 6 cells/mL and the rAAV titre increasing to 20 × 10 11 VG/mL upon adding feed material. By resolving the problems associated with high-density cell culture and air bubbles, this process was successfully scaled to a 50 L pilot-scale level., Conclusions: This successful experiment not only provides a technological basis for further scale-up but also guarantees product capacity. We hope that this development process can provide reference data for studying cell culture-based drug production., (© 2024. The Author(s).)

The effect of gradients of elevated glucose and low dissolved oxygen in the addition zone of fed-batch E. coli thermoinduced recombinant high cell density cultures can be evaluated through two-compartment scale-down models. Here, glucose was fed in the inlet of a plug flow bioreactor (PFB) connected to a stirred tank bioreactor (STB). E. coli cells diminished growth from 48.2 ± 2.2 g/L in the stage of RP production if compared to control (STB) with STB-PFB experiments, when residence time inside the PFB was 25 s (34.1 ± 3.5 g/L) and 40 s (25.6 ± 5.1 g/L), respectively. The recombinant granulocyte-macrophage colony-stimulating factor (rHuGM-CSF) production decreased from 34 ± 7% of RP in inclusion bodies (IB) in control cultures to 21 ± 8%, and 7 ± 4% during the thermoinduction production phase when increasing residence time inside the PFB to 25 s and 40 s, respectively. This, along with the accumulation of acetic and formic acid (up to 4 g/L), indicates metabolic redirection of central carbon routes through metabolic flow and mixed acid fermentation. Special care must be taken when producing a recombinant protein in heat-induced E. coli , because the yield and productivity of the protein decreases as the size of the bioreactors increases, especially if they are carried at high cell density.

A reduction in the cost of production and energy requirement is necessary for developing sustainable commercial bioprocesses. Bypassing sterilization, which is an energy and cost-intensive part of bioprocesses could be a way to achieve this. In this study, nonsterile cultivation of Yarrowia lipolytica was done on a synthetic medium containing acetic acid as the sole carbon source using two different strategies in the fed-batch mode. The contamination percentages throughout the process were measured using flow cytometry and complemented using brightfield microscopy. Maximum biomass and lipid yields of 0.57 (g biomass/g substrate) and 0.17 (g lipids/g substrate), respectively, and maximum biomass and lipid productivities of 0.085 and 0.023 g/L/h, respectively, were obtained in different fed-batch strategies. Feeding at the point of stationary phase resulted in better biomass yield and productivity with less than 2% contamination till 48 h. Feeding to maintain a minimum acetic level resulted in better lipid yield and productivity with less than 2% contamination during the complete process. The results of this study demonstrate the potential for cultivating Y. lipolytica in nonsterile conditions and monitoring the contamination throughout the process using flow cytometry., (© 2024 American Institute of Chemical Engineers.)

WuXiUP TM , WuXi Biologics' Ultra-high Productivity platform, is an intensified and integrated continuous bioprocess platform developed for production of various biologics including monoclonal antibodies, fusion proteins, and bispecific antibodies. This process technology platform has manifested its remarkable capability in boosting the volumetric productivity of various biologics and has been implemented for large-scale clinical material productions. In this paper, case studies of the production of different pharmaceutical proteins using two high-producing and intensified culture modes of WuXiUP TM and the concentrated fed-batch (CFB), as well as the traditional fed-batch (TFB) are discussed from the perspectives of cell growth, productivity, and protein quality. Both WuXiUP TM and CFB outperformed TFB regarding volumetric productivity. Additionally, distinctive advantages in product quality profiles in the WuXiUP TM process, such as reduced acidic charge variants and fragmentation, are revealed. Therefore, a simplified downstream purification process with only two chromatographic steps can be developed to deliver the target product at a satisfactory purity and an extremely-high yield., (© 2024 American Institute of Chemical Engineers.)

Purpose: Fed-batch cultures have rarely been used in single cell protein (SCP) research. This work evaluated multiple yeast species for suitability as SCP cultivated using glucose- and sucrose-based substrate and performed in-depth studies of fed-batch SCP cultivation kinetics for selected yeasts, including determination of specific crude nitrogen-to-protein conversion factors., Methods: SCP was cultivated using fully synthetic media in flask batch or bioreactor fed-batch cultures. Crude nitrogen and nucleic acid content were determined using the Dumas method and fluorescence assay kits, respectively., Results: C. utilis compared favorably to other yeasts in flask batch cultures in terms of process yield (0.52 ± 0.01 g x  g s -1 ) and crude nitrogen content (10.0 ± 0.5 and 9.9 ± 0.5% CDW for glucose and sucrose, respectively). This is the first time biomass composition data was reported for SCP cultivated in fed-batch mode. C. utilis crude nitrogen content was consistent across the tested conditions (protein content stabilized around 50% CDW in fed-batch), while that of the benchmark yeast S. cerevisiae was higher in batch cultures and at the beginning of fed-batch relative to the end (protein content decreased over time and stabilized around 43% CDW ). Total nucleic acid content of the yeasts was similar (6.8% CDW and 6.3% CDW , for C. utilis and S. cerevisiae, respectively), with crude nitrogen-to-protein conversion factors of 4.97 and 5.80., Conclusion: This study demonstrated the suitability of C. utilis as SCP, notably the robustness of its crude nitrogen content (as an indicator of protein content) across batch and fed-batch conditions, compared to that of the benchmark yeast S. cerevisiae., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Chinese hamster ovary (CHO) cells are widely used to manufacture biopharmaceuticals, most of all monoclonal antibodies (mAbs). Some CHO cell lines exhibit production instability, where the productivity of the cells decreases as a function of time in culture. To counter this, we designed a passaging strategy that, rather than maximizing the time spent in log-growth phase, mimics the first 7 days of a fed-batch production process. Cultures passaged using this method had lower net growth rates and were more oxidative throughout 6 weeks of passaging. Fed-batch cultures inoculated by cells passaged using this method had increased net growth rates, oxidative metabolism, and volumetric productivity compared to cells passaged using a conventional strategy. Cells from unstable cell lines passaged by this new method produced 80%-160% more mAbs per unit volume than cells passaged by a conventional method. This new method, named Super7, provides the ability to mitigate the impact of production instability in CHO-K1 cell lines without a need for further cell line creation, genetic engineering, or medium development., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

The performance of industrial strains has gradually improved with the rapid development of synthetic biotechnology. The production efficiency of traditional batch and fed-batch culture is limited and product quality varies since both are dynamic processes, whereas multi-stage continuous culture can maximise the production efficiency of specific fermentation processes and achieve consistent product quality. However, each single-stage fermentation under multi-stage continuous fermentation requires accurate steady-state control, and a model with adequate accuracy is required for designing and controlling a multi-stage continuous fermentation process. At present, there are few reports on kinetic models for the control of multi-stage continuous fermentation. In this work, we constructed a hybrid model for Saccharomyces cerevisiae multi-stage continuous culture, taking both oxygen limitation and Crabtree effect. The accuracy of the model was ∼80%, the advantages and limitations of the model are discussed and a potential improvement strategy is proposed., (© 2024 Wiley‐VCH GmbH.)

Bacterial nanocellulose was produced here using static, static intermittent-fed batch (SIFB) and rotary disc bioreactor (RDB) mode. Economical black tea broth media with symbiotic consortia of bacteria and yeast (SCOBY) was used towards feasible BNC production (instead of commercial NCIM 2526 strain and conventional HS media). The physicochemical characterization of BNC produced in all three modes via FE-SEM, ATR-FTIR, XRD and TGA results showed a highly porous morphology, mostly Iα form, good crystallinity and thermal stability, respectively. BNC crystallinity lies in the range of 68 % (RDB) to 79.4 % (static and SIFB). Water retention value (86 to 93 %) and moisture content (85 to 93 %) are high for BNC produced in all three modes. Commendable difference in the BNC yield, sugar consumption, conversion yield and residual sugar was observed using different methods. Highest BNC yield 29.4 ± 0.66 gL -1 was obtained under SIFB method as compared to static mode (13.6 ± 0.32 g L -1 ). Under RDB, a negligible amount of BNC i.e., 1.0 ± 0.2 g L -1 was produced. SCOBY with BTB medium was found unsuitable for BNC production under RDB and needs further investigation. Thus, this comparative study offers a way to produce a commendable amount of low-priced BNC for various techno-industrial usage., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: The production of surfactin, an extracellular accumulating lipopeptide produced by various Bacillus species, is a well-known representative of microbial biosurfactant. However, only limited information is available on the correlation between the growth rate of the production strain, such as B. subtilis BMV9, and surfactin production. To understand the correlation between biomass formation over time and surfactin production, the availability of glucose as carbon source was considered as main point. In fed-batch bioreactor processes, the B. subtilis BMV9 was used, a strain well-suited for high cell density fermentation. By adjusting the exponential feeding rates, the growth rate of the surfactin-producing strain, was controlled., Results: Using different growth rates in the range of 0.075 and 0.4 h -1 , highest surfactin titres of 36 g/L were reached at 0.25 h -1 with production yields Y P/S of 0.21 g/g and Y P/X of 0.7 g/g, while growth rates lower than 0.2 h -1 resulted in insufficient and slowed biomass formation as well as surfactin production (Y P/S of 0.11 g/g and Y P/X of 0.47 g/g for 0.075 h -1 ). In contrast, feeding rates higher than 0.25 h -1 led to a stimulation of overflow metabolism, resulting in increased acetate formation of up to 3 g/L and an accumulation of glucose due to insufficient conversion, leading to production yields Y P/S of 0.15 g/g and Y P/X of 0.46 g/g for 0.4 h -1 ., Conclusions: Overall, the parameter of adjusting exponential feeding rates have an important impact on the B. subtilis productivity in terms of surfactin production in fed-batch bioreactor processes. A growth rate of 0.25 h -1 allowed the highest surfactin production yield, while the total conversion of substrate to biomass remained constant at the different growth rates., (© 2024. The Author(s).)

Background: Vibrio natriegens, a halophilic marine γ-proteobacterium, holds immense biotechnological potential due to its remarkably short generation time of under ten minutes. However, the highest growth rates have been primarily observed on complex media, which often suffer from batch-to-batch variability affecting process stability and performance. Consistent bioprocesses necessitate the use of chemically defined media, which are usually optimized for fermenters with pH and dissolved oxygen tension (DOT) regulation, both of which are not applied during early-stage cultivations in shake flasks or microtiter plates. Existing studies on V. natriegens' growth on mineral media report partially conflicting results, and a comprehensive study examining the combined effects of pH buffering, sodium concentration, and medium osmolality is lacking., Results: This study evaluates the influence of sodium concentration, pH buffering, and medium osmolality on the growth of V. natriegens under unregulated small-scale conditions. The maximum growth rate, time of glucose depletion, as well as the onset of stationary phase were observed through online-monitoring the oxygen transfer rate. The results revealed optimal growth conditions at an initial pH of 8.0 with a minimum of 300 mM MOPS buffer for media containing 20 g/L glucose or 180 mM MOPS for media with 10 g/L glucose. Optimal sodium chloride supplementation was found to be between 7.5 and 15 g/L, lower than previously reported ranges. This is advantageous for reducing industrial corrosion issues. Additionally, an osmolality range of 1 to 1.6 Osmol/kg was determined to be optimal for growth. Under these optimized conditions, V. natriegens achieved a growth rate of 1.97 ± 0.13 1/h over a period of 1 h at 37 °C, the highest reported rate for this organism on a mineral medium., Conclusion: This study provides guidelines for cultivating V. natriegens in early-stage laboratory settings without pH and DOT regulation. The findings suggest a lower optimal sodium chloride range than previously reported and establish an osmolality window for optimal growth, thereby advancing the understanding of V. natriegens' physiology. In addition, this study offers a foundation for future research into the effects of different ions and carbon sources on V. natriegens., (© 2024. The Author(s).)

Due to their proteinaceous structure, monoclonal antibodies (mAbs) are susceptible to irreversible aggregation, with harmful consequences on drug efficacy and patient safety. To mitigate this risk in modern biopharmaceutical processes, it is critical to comply with current good manufacturing practices (cGMP) and pursue operating strategies minimizing irreversible aggregation whilst also maximizing mAb throughput. These conflicting objectives are targeted in this study by formulating and analyzing an integrated dynamic model accounting for both cultivation and aggregation of mAbs from a Chinese Hamster Ovary (CHO) cell line. Two manipulated dynamic variables are considered here in simulation studies: firstly temperature manipulation within a batch reactor, and secondly feed flow manipulation within a series of isothermal fed-batch reactors. Following this, dynamic optimization investigations have been conducted, firstly with the single objective of maximizing mAb throughput and secondly with multiple (two) objectives of maximizing mAb throughput while also minimizing irreversible aggregate content, simultaneously. The study provides key insight into tradeoffs of how simultaneous temperature and feed flowrate manipulation affects mAb throughput and aggregation inside bioreactors., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Cultivating cells in shake flasks is a routine operation that is largely unchanged since its inception. A glass or plastic Erlenmeyer vessel with the primary gas exchange taking place across various porous plugs is used with media volumes typically ranging from 100 mL to 2 L. Oxygen limitation and carbon dioxide accumulation in the vessel is a major concern for studies involving shake flask cultures. In this study, we enhance mass transfer in a conventional shake flask by replacing the body wall with a permeable membrane. Naturally occurring concentration gradient across the permeable membrane walls facilitates the movement of oxygen and carbon dioxide between the flask and the external environment. The modified flask called the breathable flask, has shown a 40% improvement in mass transfer coefficient (k L a) determined using the static diffusion method. The prokaryotic cell culture studies performed with Escherichia coli showed an improvement of 28%-66% in biomass and 41%-56% in recombinant product yield. The eukaryotic cell culture study performed with Pichia pastoris expressing proinsulin exhibited a 40% improvement in biomass and 115% improvement in protein yield. The study demonstrates a novel approach to addressing the mass transfer limitations in conventional shake flask cultures. The proposed flask amplifies its value by providing a membrane-diffusion-based sensing platform for the integration of low-cost, noninvasive sensing capabilities for real-time monitoring of critical cell culture parameters like dissolved oxygen and dissolved carbon dioxide., (© 2024 Wiley Periodicals LLC.)

The fast-growing Chinese hamster lung (CHL)-YN cell line was recently developed for monoclonal antibody production. In this study, we applied a serum-free fed-batch cultivation process to immunoglobulin (Ig)G1-producing CHL-YN cells, which were then used to design a dynamic glucose supply system to stabilize the extracellular glucose concentration based on glucose consumption. Glucose consumption of the cultures rapidly oscillated following three phases of glutamine metabolism: consumption, production, and re-consumption. Use of the dynamic glucose supply prolonged the viability of the CHL-YN-IgG1 cell cultures and increased IgG1 production. Liquid chromatography with tandem mass spectrometry-based target metabolomics analysis of the extracellular metabolites during the first glutamine shift was conducted to search for depleted compounds. The results suggest that the levels of four amino acids, namely arginine, aspartate, methionine, and serine, were sharply decreased in CHL-YN cells during glutamine production. Supporting evidence from metabolic and gene expression analyses also suggest that CHL-YN cells acquired ornithine- and cystathionine-production abilities that differed from those in Chinese hamster ovary-K1 cells, potentially leading to proline and cysteine biosynthesis., (© 2024 The Author(s). Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Rapidly expanding biopharmaceutical market demands more cost-effective platforms to produce protein therapeutics. To this end, novel approaches, such as perfusion culture or concentrated fed-batch, have been explored for higher yields and lower manufacturing costs. Although these new approaches produced promising results, but their wide-spread use in the industry is still limited. In this study, a dialysis rolled scaffold bioreactor was presented for long-term production of monoclonal antibodies with reduced media consumption. Media dialysis can selectively remove cellular bio-wastes without losing cells or produced recombinant proteins. The dialysis process was streamlined to significantly improve its efficiency. Then, extended culture of recombinant CHO cells for 41 days was successfully demonstrated with consistent production rate and minimal media consumption. The unique configuration of the developed bioreactor allows efficient dialysis for media management, as well as rapid media exchange to harvest produced recombinant proteins before they degrade. Taken together, it was envisioned that the developed bioreactor will enable cost-effective and long-term large-scale culture of various cells for biopharmaceutical production., (© 2024 Wiley‐VCH GmbH.)

Purpose: Simultaneous membrane-based feeding and monitoring of the oxygen transfer rate shall be introduced to the newly established perforated ring flask, which consists of a cylindrical glass flask with an additional perforated inner glass ring, for rapid bioprocess development., Methods: A 3D-printed adapter was constructed to enable monitoring of the oxygen transfer rate in the perforated ring flasks. Escherichia coli experiments in batch were performed to validate the adapter. Fed-batch experiments with different diffusion rates and feed solutions were performed., Results: The adapter and the performed experiments allowed a direct comparison of the perforated ring flasks with Erlenmeyer flasks. In batch cultivations, maximum oxygen transfer capacities of 80 mmol L -1  h -1 were reached with perforated ring flasks, corresponding to a 3.5 times higher capacity than in Erlenmeyer flasks. Fed-batch experiments with a feed reservoir concentration of 500 g glucose L -1 were successfully conducted. Based on the oxygen transfer rate, an ammonium limitation could be observed. By adding 40 g ammonium sulfate L -1 to the feed reservoir, the limitation could be prevented., Conclusion: The membrane-based feeding, an online monitoring technique, and the perforated ring flask were successfully combined and offer a new and promising tool for screening and process development in biotechnology., (© 2024. The Author(s).)

Chinese hamster ovary (CHO) cells are the most widely used for therapeutic antibody production. In cell line development, engineering secretion processes such as folding-related protein upregulation is an effective way of constructing cell lines with high recombinant protein productivity. However, there have been few studies on the transport of recombinant proteins between the endoplasmic reticulum (ER) and the Golgi apparatus. In this study, Sar1A, a protein involved in COPII vesicle formation, was focused on to improve antibody productivity by enhancing COPII vesicle-mediated antibody transport from the ER to the Golgi apparatus, and to clarify its effect on the secretion process. The constructed Sar1A-overexpressing CHO cell lines were batch-cultured, in which they showed an increased specific antibody production rate. The intracellular antibody accumulation and the specific localization of the intracellular antibodies were investigated by chase assay using a translation inhibitor and observed by immunofluorescence-based imaging analysis. The results showed that Sar1A overexpression reduced intracellular antibody accumulation, especially in the ER. The effects of the engineered antibody transport on the antibody's glycosylation profile and the unfolded protein response (UPR) pathway were analyzed by liquid chromatography-mass spectrometry and UPR-related gene expression evaluation, respectively. Sar1A overexpression lowered glycan galactosylation and induced a stronger UPR at the end of the batch culture. Sar1A overexpression enhanced the antibody productivity of CHO cells by modifying their secretion process. This approach could also contribute to the production of not only monoclonal antibodies but also other therapeutic proteins that require transport by COPII vesicles., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

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 × 10 6 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., (© 2024 American Institute of Chemical Engineers.)

The strain Gluconobacter oxydans LMG 1385 was used for the bioconversion of crude glycerol to dihydroxyacetone. The suitability of fed-batch cultures for the production of dihydroxyacetone was determined, and the influence of the pH of the culture medium and the initial concentration of glycerol on maximizing the concentration of dihydroxyacetone and on the yield and speed of obtaining dihydroxyacetone by bioconversion was examined. The feeding strategy of the substrate (crude glycerol) during the process was based on measuring the dissolved oxygen tension of the culture medium. The highest concentration of dihydroxyacetone P K = 175.8 g·L -1 and the highest yield Y P/Sw = 94.3% were obtained when the initial concentration of crude glycerol was S 0 = 70.0 g·L -1 and the pH of the substrate was maintained during the process at level 5.0.

Secretion levels required of industrial Chinese hamster ovary (CHO) cell lines can challenge endoplasmic reticulum (ER) homeostasis, and ER stress caused by accumulation of misfolded proteins can be a bottleneck in biomanufacturing. The unfolded protein response (UPR) is initiated to restore homeostasis in response to ER stress, and optimization of the UPR can improve CHO cell production of therapeutic proteins. We compared the fed-batch growth, production characteristics, and transcriptomic response of an immunoglobulin G 1 (IgG 1 ) producer to its parental, non-producing host cell line. We conducted differential gene expression analysis using high throughput RNA sequencing (RNASeq) and quantitative polymerase chain reaction (qPCR) to study the ER stress response of each cell line during fed-batch culture. The UPR was activated in the IgG 1 producer compared to the host cell line and our analysis of differential expression profiles indicated transient upregulation of ATF6α target mRNAs in the IgG 1 producer, suggesting two upstream regulators of the ATF6 arm of the UPR, ATF6β and WFS1, are rational engineering targets. Although both ATF6β and WFS1 have been reported to negatively regulate ATF6α, this study shows knockdown of either target elicits different effects in an IgG 1 -producing CHO cell line. Stable knockdown of ATF6β decreased cell growth without decreasing titer; however, knockdown of WFS1 decreased titer without affecting growth. Relative expression measured by qPCR indicated no direct relationship between ATF6β and WFS1 expression, but upregulation of WFS1 in one pool was correlated with decreased growth and upregulation of ER chaperone mRNAs. While knockdown of WFS1 had negative impacts on UPR activation and product mRNA expression, knockdown of ATF6β improved the UPR specifically later in fed-batch leading to increased overall productivity., (© 2024. The Author(s).)

Submerged cultivation of edible filamentous fungi (Agaricomycetes) in bioreactors enables maximum mass transfer of nutrients and has the potential to increase the volumetric productivity of fungal biomass compared to solid state cultivation. These aspects are paramount if one wants to increase the range of bioactives (e.g. glucans) in convenient time frames. In this study, Trametes versicolor (M9911) outperformed four other Agaricomycetes tested strains (during batch cultivations in an airlift bioreactor). This strain was therefore further tested in semi-continuous cultivation. Continuous and semi-continuous cultivations (driven by the dilution rate, D) are the preferred bioprocess strategies for biomass production. We examined the semi-continuous cultivation of T. versicolor at dilution rates between 0.02 and 0.1 h -1 . A maximum volumetric productivity of 0.87 g/L/h was obtained with a D of 0.1 h -1 but with a lower total biomass production (cell dry weight, CDW 8.7 g/L) than the one obtained at lower dilution rates (12.3 g/L at D of 0.04 and vs 13.4 g/L, at a D of 0.02 h -1 ). However, growth at a D of 0.1 h -1 resulted in a very short fermentation (18 h) which terminated due to washout (the specific D exceeded the maximum growth rate of the fungal biomass). At a D of 0.04 h -1 , a CDW of 12.3 g/L was achieved without compromising the total residence time (184 h) of the fermentation. While the D of 0.04 h -1 and 0.07 h -1 achieved comparable volumetric productivities (0.5 g/L/h), the total duration of the fermentation at D of 0.07 h -1 was only 85 h. The highest glucan content of cells (27.8 as percentage of CDW) was obtained at a D of 0.07 h -1 , while the lowest glucan content was observed in T. versicolor cells grown at a D of 0.02 h -1 . KEY POINTS: • The highest reported volumetric productivity for fungal biomass was 0.87 g/L/h. • Semi-continuous fermentation at D of 0.02 h-1 resulted in 13.4 g/L of fungal biomass. • Semi-continuous fermentation at D of 0.07 h-1 resulted in fungal biomass with 28% of total glucans., (© 2024. The Author(s).)

The archaeon Sulfolobus acidocaldarius has emerged as a promising thermophilic model system. Investigating how thermophiles adapt to changing temperatures is a key requirement, not only for understanding fundamental evolutionary processes but also for developing S. acidocaldarius as a chassis for bioengineering. One major obstacle to conducting experimental evolution with thermophiles is the expense of equipment maintenance and energy usage of traditional incubators for high-temperature growth. To address this challenge, a comprehensive experimental protocol for conducting experimental evolution in S. acidocaldarius is presented, utilizing low-cost and energy-efficient bench-top thermomixers. The protocol involves a batch culture technique with relatively small volumes (1.5 mL), enabling tracking of adaptation in multiple independent lineages. This method is easily scalable through the use of additional thermomixers. Such an approach increases the accessibility of S. acidocaldarius as a model system by reducing both initial investment and ongoing costs associated with experimental investigations. Moreover, the technique is transferable to other microbial systems for exploring adaptation to diverse environmental conditions.

Artificial Intelligence (AI) technology is spearheading a new industrial revolution, which provides ample opportunities for the transformational development of traditional fermentation processes. During plasmid fermentation, traditional subjective process control leads to highly unstable plasmid yields. In this study, a multi-parameter correlation analysis was first performed to discover a dynamic metabolic balance among the oxygen uptake rate, temperature, and plasmid yield, whilst revealing the heating rate and timing as the most important optimization factor for balanced cell growth and plasmid production. Then, based on the acquired on-line parameters as well as outputs of kinetic models constructed for describing process dynamics of biomass concentration, plasmid yield, and substrate concentration, a machine learning (ML) model with Random Forest (RF) as the best machine learning algorithm was established to predict the optimal heating strategy. Finally, the highest plasmid yield and specific productivity of 1167.74 mg L -1 and 8.87 mg L -1 /OD 600 were achieved with the optimal heating strategy predicted by the RF model in the 50 L bioreactor, respectively, which was 71% and 21% higher than those obtained in the control cultures where a traditional one-step temperature upshift strategy was applied. In addition, this study transformed empirical fermentation process optimization into a more efficient and rational self-optimization method. The methodology employed in this study is equally applicable to predict the regulation of process dynamics for other products, thereby facilitating the potential for furthering the intelligent automation of fermentation processes., (© 2024 Wiley‐VCH GmbH.)

The biopharmaceutical industry is replacing fed-batch with perfusion processes to take advantage of reduced capital and operational costs due to the operation at high cell densities (HCD) and improved productivities. HCDs are achieved by cell retention and continuous medium exchange, which is often based on the cell-specific perfusion rate (CSPR). To obtain a cost-productive process the perfusion rate must be determined for each process individually. However, determining optimal operating conditions remain labor-intensive and time-consuming experiments, as investigations are performed in lab-scale perfusion bioreactors. Small-scale models such as microwell plates (MWPs) provide an option for screening multiple perfusion rates in parallel in a semi-perfusion mimic. This study investigated two perfusion rate strategies applied to the MWP platform operated in semi-perfusion. The CSPR-based perfusion rate strategy aimed to maintain multiple CSPR values throughout the cultivation and was compared to a cultivation with a perfusion rate of 1 RV d -1 . The cellular performance was investigated with the dual aim (i) to achieve HCD, when inoculating at conventional and HCDs, and (ii) to maintain HCDs, when applying an additional manual cell bleed. With both perfusion rate strategies viable cell concentrations up to 50 × 10 6  cells mL -1 were achieved and comparable results for key metabolites and antibody product titers were obtained. Furthermore, the combined application of cell bleed and CSPR-based medium exchange was successfully shown with similar results for growth, metabolites, and productivities, respectively, while reducing the medium consumption by up to 50% for HCD cultivations., (© 2024 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG 1 )-producing cells were generated, and the IgG 1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Currently, the predominant method for the industrial production of 1,3-dihydroxyacetone (DHA) from glycerol involves fed-batch fermentation. However, previous research has revealed that in the biocatalytic synthesis of DHA from glycerol, when the DHA concentration exceeded 50 g·L -1 , it significantly inhibited microbial growth and metabolism, posing a challenge in maintaining prolonged and efficient catalytic production of DHA. In this study, a new integrated continuous production and synchronous separation (ICSS) system was constructed using hollow fiber columns and perfusion culture technology. Additionally, a cell reactivation technique was implemented to extend the biocatalytic ability of cells. Compared with fed-batch fermentation, the ICSS system operated for 360 h, yielding a total DHA of 1237.8 ± 15.8 g. The glycerol conversion rate reached 97.7 %, with a productivity of 3.44 g·L -1 ·h -1 , representing 485.0 % increase in DHA production. ICSS system exhibited strong operational characteristics and excellent performance, indicating significant potential for applications in industrial bioprocesses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Chinese hamster ovary (CHO) cells are widely used for the industrial production of therapeutic monoclonal antibodies (mAbs). To meet the increasing market demands, high productivity, and quality are required in cell culture. One of the critical attributes of mAbs, from a safety perspective, is mAb fragmentation. However, methods for preventing mAbs fragmentation in CHO cell culture are limited. In this study, we observed that the antibody fragment content increased with increasing titers in fed-batch cultures for all three cell lines expressing recombinant antibodies. Adding copper sulfate to the culture medium further increased the fragment content, suggesting the involvement of reactive oxygen species (ROS) in the fragmentation process. Though antioxidants may be helpful to scavenge ROS, several antioxidants are reported to decrease the productivity of CHO cells. Among the antioxidants examined, we observed that the addition of catechin or (-)-epigallocatechin gallate to the culture medium prevented fragmentation content by about 20% and increased viable cell density and titer by 30% and 10%, respectively. Thus, the addition of catechins or compounds of equivalent function would be beneficial for manufacturing therapeutic mAbs with a balance between high titers and good quality., (© 2024 American Institute of Chemical Engineers.)

This study assessed Rhodotorula paludigena CM33's growth and β-carotene production in a 22-L bioreactor for potential use as an aquatic animal feed supplement. Optimizing the feed medium's micronutrient concentration for high-cell-density fed-batch cultivation using glucose as the carbon source yielded biomass of 89.84 g/L and β-carotene concentration of 251.64 mg/L. Notably, using sucrose as the carbon source in feed medium outperforms glucose feeds, resulting in a β-carotene concentration of 285.00 mg/L with a similar biomass of 87.78 g/L. In the fed-batch fermentation using Sucrose Feed Medium, R. paludigena CM33 exhibited high biomass production rates (Q x ) of 0.91 g/L.h and remarkable β-carotene production rates (Q p ) of 2.97 mg/L.h. In vitro digestibility assays showed that R. paludigena CM33, especially when cultivated using sucrose, enhances protein digestibility affirming its suitability as an aquatic feed supplement. Furthermore, R. paludigena CM33's nutrient-rich profile and probiotic potential make it an attractive option for aquatic nutrition. This research highlights the importance of cost-effective carbon sources in large-scale β-carotene production for aquatic animal nutrition., (© 2024. The Author(s).)

Dynamic flux balance analysis (FBA) allows estimation of intracellular reaction rates using organism-specific genome-scale metabolic models (GSMM) and by assuming instantaneous pseudo-steady states for processes that are inherently dynamic. This technique is well-suited for industrial bioprocesses employing complex media characterized by a hierarchy of substrate uptake and product secretion. However, knowledge of exchange rates of many components of the media would be required to obtain meaningful results. Here, we performed spent media analysis using mass spectrometry coupled with liquid and gas chromatography for a fed-batch, high-cell density cultivation of Escherichia coli BL21(DE3) expressing a recombinant protein. Time course measurements thus obtained for 246 metabolites were converted to instantaneous exchange rates. These were then used as constraints for dynamic FBA using a previously reported GSMM, thus providing insights into how the flux map evolves through the process. Changes in tri-carboxylic acid cycle fluxes correlated with the increased demand for energy during recombinant protein production. The results show how amino acids act as hubs for the synthesis of other cellular metabolites. Our results provide a deeper understanding of an industrial bioprocess and will have implications in further optimizing the process., (© 2024 Wiley Periodicals LLC.)

Chinese Hamster Ovary (CHO) cells have rapidly become a cornerstone in biopharmaceutical production. Recently, a reinvigoration of perfusion culture mode in CHO cell cultivation has been observed. However, most cell lines currently in use have been engineered and adapted for fed-batch culture methods, and may not perform optimally under perfusion conditions. To improve the cell's resilience and viability during perfusion culture, we cultured a triple knockout CHO cell line, deficient in three apoptosis related genes BAX, BAK, and BOK in a perfusion system. After 20 days of culture, the cells exhibited a halt in cell proliferation. Interestingly, following this phase of growth arrest, the cells entered a second growth phase. During this phase, the cell numbers nearly doubled, but cell specific productivity decreased. We performed a proteomics investigation, elucidating a distinct correlation between growth arrest and cell cycle arrest and showing an upregulation of the central carbon metabolism and oxidative phosphorylation. The upregulation was partially reverted during the second growth phase, likely caused by intragenerational adaptations to stresses encountered. A phase-dependent response to oxidative stress was noted, indicating glutathione has only a secondary role during cell cycle arrest. Our data provides evidence of metabolic regulation under high cell density culturing conditions and demonstrates that cell growth arrest can be overcome. The acquired insights have the potential to not only enhance our understanding of cellular metabolism but also contribute to the development of superior cell lines for perfusion cultivation., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Mammalian cells remain the mainstay of biological production host. In industry, cultivating and harvest strategies are sorted in batch mode (e.g., batch, fed-batch, concentrated fed-batch and intensified fed-batch) and continuous mode (e.g., perfusion). To retrieve greater productivity and better product quality, especially for the sensitive products prone to fragmentation, culture modes with various modifications are innovated (e.g., intensified perfusion culture [IPC]). In our study, we demonstrated that the fragmentation of Fc-fusion product (Molecule A) is time-dependent in traditional fed-batch (TFB) culture. The fragmentation proportion increased from 3.8% to 12.4% for Clone A, 0.8% to 1.7% for Clone B and 0.9% to 2.0% for Clone C from Day 10 to Day 14. By applying a novel bioprocess, IPC, which allows continuous feeding of the fresh medium and constant removal of the spent medium without bleeding cells to maintain a defined constant viable cell density, the fragmentation was reduced to 0.3% while the productivity was increased from 2.96 g/L to 15.51 g/L for Clone A. To validate whether the fragmentation reduction is product-sensitive, plasmids carrying the DNA sequences of two other Fc-fusion molecules (Molecule B and Molecule C) were transfected into the host. The results showed consistent fragmentation reducing effect by using IPC. Furthermore, the cultivation scale was expanded to 50 L and 1000 L. A minimum fragmentation level below 0.1% was observed for Molecule C. Our study revealed the capability of IPC in reducing Fc-fusion protein fragmentation and the reproducibility when scaling up while maintaining high productivity., (© 2023 American Institute of Chemical Engineers.)

An important consideration for biopharmaceutical processes is the cost of goods (CoGs) of biotherapeutics manufacturing. CoGs can be reduced by dramatically increasing the productivity of the bioreactor process. In this study, we demonstrate that an intensified process which couples a perfused N-1 seed reactor and a fully automated high inoculation density (HID) N stage reactor substantially increases the bioreactor productivity as compared to a low inoculation density (LID) control fed-batch process. A panel of six CHOK1SV GS-KO® CHO cell lines expressing three different monoclonal antibodies was evaluated in this intensified process, achieving an average 85% titer increase and 132% space-time yield (STY) increase was demonstrated when comparing the 12-day HID process to a 15-day LID control process. These productivity increases were enabled by automated nutrient feeding in both the N-1 and N stage bioreactors using in-line process analytical technologies (PAT) and feedback control. The N-1 bioreactor utilized in-line capacitance to automatically feed the bioreactor based on a capacitance-specific perfusion rate (CapSPR). The N-stage bioreactor utilized in-line Raman spectroscopy to estimate real-time concentrations of glucose, phenylalanine, and methionine, which are held to target set points using automatic feed additions. These automated feeding methodologies were shown to be generalizable across six cell lines with diverse feed requirements. We show this new process can accommodate clonal diversity and reproducibly achieve substantial titer uplifts compared to traditional cell culture processes, thereby establishing a baseline technology platform upon which further increases bioreactor productivity and CoGs reduction can be achieved., (© 2023 Lonza. Biotechnology Progress published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)

Automation of metabolite control in fermenters is fundamental to develop vaccine manufacturing processes more quickly and robustly. We created an end-to-end process analytical technology and quality by design-focused process by replacing manual control of metabolites during the development of fed-batch bioprocesses with a system that is highly adaptable and automation-enabled. Mid-infrared spectroscopy with an attenuated total reflectance probe in-line, and simple linear regression using the Beer-Lambert Law, were developed to quantitate key metabolites (glucose and glutamate) from spectral data that measured complex media during fermentation. This data was digitally connected to a process information management system, to enable continuous control of feed pumps with proportional-integral-derivative controllers that maintained nutrient levels throughout fed-batch stirred-tank fermenter processes. Continuous metabolite data from mid-infrared spectra of cultures in stirred-tank reactors enabled feedback loops and control of the feed pumps in pharmaceutical development laboratories. This improved process control of nutrient levels by 20-fold and the drug substance yield by an order of magnitude. Furthermore, the method is adaptable to other systems and enables soft sensing, such as the consumption rate of metabolites. The ability to develop quantitative metabolite templates quickly and simply for changing bioprocesses was instrumental for project acceleration and heightened process control and automation., One-Sentence Summary: Intelligent digital control systems using continuous in-line metabolite data enabled end-to-end automation of fed-batch processes in stirred-tank reactors., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Effective microbial bioprocessing relies on maintaining ideal cultivation conditions, highlighting the necessity for tools that monitor and regulate cellular performance and robustness. This study evaluates a fed-batch cultivation control system based on at-line flow cytometry monitoring of intact yeast cells having a fluorescent transcription factor-based redox biosensor. Specifically, the biosensor assesses the response of an industrial xylose-fermenting Saccharomyces cerevisiae strain carrying the TRX2p-yEGFP biosensor for NADPH/NADP+ ratio imbalance when exposed to furfural. The developed control system successfully detected biosensor output and automatically adjusted furfural feed rate, ensuring physiological fitness at high furfural levels. Moreover, the single-cell measurements enabled the monitoring of subpopulation dynamics, enhancing control precision over traditional methods. The presented automated control system highlights the potential of combining biosensors and flow cytometry for robust microbial cultivations by leveraging intracellular properties as control inputs., One-Sentence Summary: An automated control system using flow cytometry and biosensors enhances microbial bioprocessing by regulating cellular performance in response to the environmental stressor furfural., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Most pharmaceutical biotechnology companies use stirred-tank bioreactors (STR) for recombinant protein manufacturing. These bioreactors are used at a variety of different scales ranging from bench to production scales, with working volumes from 10 mL to 25,000 L. Bench-scale STRs are commonly used to culture mammalian cells for process development, to troubleshoot production scale bioreactors using scale-down models (SDM), or to conduct fundamental research. In this chapter, we describe the operations of a bench-scale STR for the production of recombinant proteins with suspension-adapted Chinese hamster ovary (CHO) cells. These operations include bioreactor setup and configuration, batching media, inoculation of the seed cell culture, production phase, and harvest of cell-free fluids., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The mammalian cell culture process is a key step in commercial therapeutic protein production and needs to be monitored and controlled due to its complexity. Raman spectroscopy has been reported for cell culture process monitoring by analysis of many important parameters. However, studies on in-line Raman monitoring of the cell culture process were mainly conducted on small or pilot scale. Developing in-line Raman analytical methods for commercial-scale cell culture process monitoring is more challenging. In this study, an in-line Raman analytical method was developed for monitoring glucose, lactate, and viable cell density (VCD) in the Chinese hamster ovary (CHO) cell culture process during commercial production of biosimilar adalimumab (1500 L). The influence of different Raman measurement channels was considered to determine whether to merge data from different channels for model development. Raman calibration models were developed and optimized, with minimum root mean square error of prediction of 0.22 g L -1 for glucose in the range of 1.66-3.53 g L -1 , 0.08 g L -1 for lactate in the range of 0.15-1.19 g L -1 , 0.31 E6 cells mL -1 for VCD in the range of 0.96-5.68 E6 cells mL -1 on test sets. The developed analytical method can be used for cell culture process monitoring during manufacturing and meets the analytical purpose of this study. Further, the influence of the number of batches used for model calibration on model performance was also studied to determine how many batches are needed basically for method development. The proposed Raman analytical method development strategy and considerations will be useful for monitoring of similar bioprocesses., (© 2024 Wiley-VCH GmbH.)