Fed-batch Process Research Articles

Nostoxanthin Biosynthesis by Sphingomonas Species (COS14-R2): Isolation, Identification, and Optimization of Culture Conditions.

Nostoxanthin, a yellow pigment, belongs to the xanthophyll group of carotenoids found in various species of bacteria and cyanobacteria. Several species of Sphingomonas can produce appropriate carotenoids for survive in various environments. This comprise nostoxanthin, a significant carotenoid. The study isolated the Sphingomonas species strain COS14-R2 from the Cosmos bipinnatus and identified it through the whole-genome sequence. The strain consists of a circular chromosome with a length of 3,677,457 base pairs.s ecThe genome consists of three carotenoid biosynthesis genes, specifically crtB (phytoene synthase), crtI (phytoene desaturase), and crtY (Lycopene beta-cyclase), which are involved in the synthesis of nostoxanthin. The strain has a circular, undulated colony morphology with a deep yellow color. It demonstrates optimal growth in liquid media at 25 to 35°C and exhibits a high tolerance for pH levels between 5 and 11 and requires adequate quantities of carbon and nitrogen. We observed the highest concentration of nostoxanthin was recorded at 35°C, pH of 7.5, glucose concentration of 40g L-1, and a yeast extract concentration of 5g L-1 during dark incubation. The fed-batch fermentation process produced nostoxanthin at a concentration of 217.22 ± 9.60mg L-1, with a selectivity of 72.32% and a productivity of 2.59g/L/h. The dry biomass extract was purified using column chromatography. The LC-MS/MS analysis of the purified fraction indicated that the molecular weight of nostoxanthin is 600.5098m/z. The DPPH assay result of 75.5 ± 0.33% indicates nostoxanthin is highly effective in scavenging free radicals.

Pilot-scale process development for recombinant adeno-associated virus (rAAV) production based on high-density Sf9 cell culture

BackgroundIn 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.MethodsIn 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.ResultsHere, we describe a method in which a BEVS was used to produce rAAV vectors, with the cell density increasing to 22.8 × 106 cells/mL and the rAAV titre increasing to 20 × 1011 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.ConclusionsThis 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.

Advancing biopharmaceutical manufacturing: economic and sustainability assessment of end-to-end continuous production of monoclonal antibodies

Monoclonal antibodies (mAbs) have become essential therapeutics for treating various diseases. The robust, cost-effective, and sustainable production of mAbs is crucial due to their growing clinical and commercial demand. Advances in bioprocessing, such as improved cell lines, perfusion bioreactors, multicolumn chromatography, and automation, can significantly increase productivity, making treatments more accessible. Streamlining the production process also aligns with environmental sustainability by reducing waste and energy consumption. This study quantifies the economic and environmental impacts of incorporating recent advances into end-to-end continuous bioprocessing of mAbs. The results demonstrate that, compared with an optimized best-in-class fed-batch process (with 15 g/l titer and multicolumn chromatography), continuous manufacturing can reduce the total annual production costs, facility footprint, plastic waste, and CO2 emissions by up to 23%, 51%, 57%, and 54%, respectively, in a producing clinical and commercial lots. Additionally, uncertainty analysis indicates that these gains are even more substantial under demand fluctuations.

From molasses to purified α-ketoglutarate with engineered Corynebacterium glutamicum

α-ketoglutarate (AKG) is a valuable dicarboxylic acid with multiple applications in the food, pharmaceutical and chemical industries. Its chemical synthesis is associated with toxic by-products, low specificity and high energy input. To create a more environmentally friendly and sustainable alternative, a microbial production process for AKG was developed. Four potential producer strains were generated by metabolic engineering of Corynebacteriumglutamicum and characterized on defined glucose/sucrose media as well as molasses, a side stream from sugar beet processing. While strain C.glutamicum PO6-iolT1Δgdh ΔgltB mscCG’ ΔodhA was not able to grow on defined media it outperformed all predecessor variants on molasses. Successful scale-up into a fed-batch bioreactor process with molasses yielded 96.2 g AKG with a conversion yield of 0.64 gg−1. Finally, downstream processing by liquid–liquid extraction with ethyl acetate enabled product purification with an extraction efficiency of 87 % and an AKG purity of > 93 %.

Model-based study of Yarrowia lipolytica cultivation on crude glycerol under different fermentation modes: Development of a membrane bioreactor process.

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.

Comparative evaluation of the extracellular production of a polyethylene terephthalate degrading cutinase by Corynebacterium glutamicum and leaky Escherichia coli in batch and fed-batch processes

BackgroundWith a growing global population, the generation of plastic waste and the depletion of fossil resources are major concerns that need to be addressed by developing sustainable and efficient plastic recycling methods. Biocatalytic recycling is emerging as a promising ecological alternative to conventional processes, particularly in the recycling of polyethylene terephthalate (PET). However, cost-effective production of the involved biocatalyst is essential for the transition of enzymatic PET recycling to a widely used industrial technology. Extracellular enzyme production using established organisms such as Escherichia coli or Corynebacterium glutamicum offers a promising way to reduce downstream processing costs.ResultsIn this study, we compared extracellular recombinant protein production by classical secretion in C. glutamicum and by membrane leakage in E. coli. A superior extracellular release of the cutinase ICCGDAQI was observed with E. coli in batch and fed-batch processes on a litre-scale. This phenomenon in E. coli, in the absence of a signal peptide, might be associated with membrane-destabilizing catalytic properties of the expressed cutinase. Optimisations regarding induction, expression temperature and duration as well as carbon source significantly enhanced extracellular cutinase activity. In particular, in fed-batch cultivation of E. coli at 30 °C with lactose as carbon source and inducer, a remarkable extracellular activity (137 U mL−1) and cutinase titre (660 mg L−1) were achieved after 48 h. Literature values obtained with other secretory organisms, such as Bacillus subtilis or Komagataella phaffii were clearly outperformed. The extracellular ICCGDAQI produced showed high efficacy in the hydrolysis of PET textile fibres, either chromatographically purified or unpurified as culture supernatant. In less than 18 h, 10 g L−1 substrate was hydrolysed using supernatant containing 3 mg cutinase ICCGDAQI at 70 °C, pH 9 with terephthalic acid yields of up to 97.8%.ConclusionExtracellular production can reduce the cost of recombinant proteins by simplifying downstream processing. In the case of the PET-hydrolysing cutinase ICCGDAQI, it was even possible to avoid chromatographic purification and still achieve efficient PET hydrolysis. With such production approaches and their further optimisation, enzymatic recycling of PET can contribute to a more efficient and environmentally friendly solution to the industrial recycling of plastics in the future.

Knock-out of the major regulator Flo8 in Komagataella phaffii results in unique host strain performance for methanol-free recombinant protein production

Flo8 is a main transcriptional regulator of flocculation and pseudohyphal growth in yeast. Disruption of FLO8 in the popular recombinant protein production host Komagataella phaffii (Pichia pastoris) prevents pseudohyphal growth and reduces cell-to-surface adherence, making the mutant an interesting platform for research and industry. However, knowledge of the physiological impact of the mutation remained scarce. In-depth analysis of transcriptome data from FLO8-deficient K. phaffii revealed that Flo8 affects genes involved in cell cycle, mating, respiration, and catabolite repression additionally to flocculation targets. One gene with considerably increased expression in flo8 was GTH1, encoding a high-affinity glucose transporter in K. phaffii. Its promoter (PG1) was previously established as a strong, glucose-regulatable alternative to methanol-induced promoters. PG1 and its improved derivatives PG1-3, D-PGS4 and D-PGS5, proved to be promising candidates for controlling recombinant protein production in the FLO8-deficient background. In small-scale screenings, PG13-controlled intracellular EGFP levels were 2.8-fold higher, and yields of different secreted recombinant proteins were up to 4.8-fold increased. The enhanced productivity of the flo8 mutant in combination with the PG1 variants was transferrable to glucose-limited fed-batch processes and could largely be attributed to higher transcriptional activity of the promoter, leading to a much higher productivity per chromosomally integrated gene copy. K. phaffii flo8 has many advantageous characteristics, such as reduced surface growth and increased transcriptional strength of glucose-regulatable promoters. These features turn the flo8 strain into a valuable new base strain for various experimental designs and establish flo8 as an excellent strain background for methanol-free recombinant protein production processes.

The influence of growth rate-controlling feeding strategy on the surfactin production in Bacillus subtilis bioreactor processes

BackgroundThe 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.ResultsUsing 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 YP/S of 0.21 g/g and YP/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 (YP/S of 0.11 g/g and YP/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 YP/S of 0.15 g/g and YP/X of 0.46 g/g for 0.4 h-1.ConclusionsOverall, 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.

CHO stable pool fed-batch process development of SARS-CoV-2 spike protein production: Impact of aeration conditions and feeding strategies.

Technology scale-up and transfer are a fundamental and critical part of process development in biomanufacturing. Important bioreactor hydrodynamic characteristics such as working volume, overhead gas flow rate, volumetric power input (P/V), impeller type, agitation regimen, sparging aeration strategy, sparger type, and kLa must be selected based on key performance indicators (KPI) to ensure a smooth and seamless process scale-up and transfer. Finding suitable operational setpoints and developing an efficient feeding regimen to ensure process efficacy and consistency are instrumental. In this investigation, process development of a cumate inducible Chinese hamster ovary (CHO) stable pool expressing trimeric SARS-CoV-2 spike protein in 1.8 L benchtop stirred-tank bioreactors is detailed. Various dissolved oxygen levels and aeration air caps were studied to determine their impact on cell growth and metabolism, culture longevity, and endpoint product titers. Once hydrodynamic conditions were tuned to an optimal zone, various feeding strategies were explored to increase culture performance. Dynamic feedings such as feeding based on current culture volume, viable cell density (VCD), oxygen uptake rate (OUR), and bio-capacitance signals were tested and compared to standard bolus addition. Increases in integral of viable cell concentration (IVCC) (1.25-fold) and protein yield (2.52-fold), as well as greater culture longevity (extension of 5 days) were observed in dynamic feeding strategies when compared to periodic bolus feeding. Our study emphasizes the benefits of designing feeding strategies around metabolically relevant signals such as OUR and bio-capacitance signals.

Continuous biomanufacturing for sustainable bioeconomy applications

The bioeconomy provides an alternative to non-renewable or fossil-based products. Biomanufacturing is emerging as a sustainable platform for the production of a wide range of value-added products, including chemicals and biodegradable materials. At present, the biotechnology industry is dominated by batch and fed-batch processes and there is increasing interest in continuous biomanufacturing (CBM) as an alternative to reduce capital and operating costs. However, CBM faces adoption challenges due to perceived operational complexities. This review critically evaluates the state-of-the-art in CBM, examining its benefits, limitations, and successful industrial applications across key sectors. Transitioning from batch to continuous processes can enhance productivity and reduce environmental impacts, making CBM a cornerstone technology for a sustainable bioeconomy.

Stepwise cell culture process intensification for high-productivity and cost-effective commercial manufacturing of a Mabcalin™ bispecifics

Process intensification and media optimization, as a crucial step for improving productivity and manufacturing cost of goods (COG), set the stage for commercialization readiness and redefine the landscape for patient access. This study described a stepwise approach to explore different intensified fed-batch processes along with optimized cell culture media for the production of a Mabcalin™ bispecifics. Initially, by leveraging perfusion expansion, intensified fed-batch (IFB) with an inoculation density of 10.3 × 106 cells/mL was developed to produce 6.1 g/L of products, compared to 3.9 g/L from the original traditional fed-batch (TFB). Following the IFB conversion, a high-performing production medium, MagniCHO™, was chosen to substitute the original one, which further boosted the titer to 9.1 g/L. The result underscored the significance of developing an optimized cell culture media for intensified cultivation. Furthermore, the approach of ultra-intensified intermittent-perfusion fed-batch was utilized, raising the seeding density to 73.6 × 106 cells/mL. A final harvest titer of 24.5 g/L was recorded. Additionally, manufacturing COG was calculated to evaluate how process intensification could lead to improved manufacturing cost-effectiveness, with up to 71 % COG reduction attainable with the UI-IPFB process. This study demonstrated that even for difficult-to-express modalities, applying a strategic development approach including process intensification and media optimization could effectively improve manufacturing efficiency and COG competitiveness.

PID Controller Design for an E. coli Fed-Batch Fermentation Process System Using Chaotic Electromagnetic Field Optimization

This paper presents an optimal tuning of a proportional integral differential (PID) controller used to maintain glucose concentration at a desired set point. The PID controller synthesizes an appropriate feed rate profile for an E. coli fed-batch cultivation process. Mathematical models are developed based on dynamic mass balance equations for biomass, substrate, and product concentration of the E. coli BL21(DE3)pPhyt109 fed-batch cultivation for bacterial phytase extracellular production. For model parameter identification and PID tuning, a hybrid metaheuristic technique—chaotic electromagnetic field optimization (CEFO)—is proposed. In the hybridization, a chaotic map is used for the generation of a new electromagnetic particle instead of the electromagnetic field optimization (EFO) search strategy. The CEFO combines the exploitation capability of the EFO algorithm and the exploration power of ten different chaotic maps. The comparison of the results with classical EFO shows the superior behaviour of the designed CEFO. An improvement of 30% of the objective function is achieved by applying CEFO. Based on the obtained mathematical models, 10 PID controllers are tuned. The simulation experiments show that the designed controllers are robust, resulting in a good control system performance. The closed-loop transient responses for the corresponding controllers are similar to the estimated models. The settling time of the control system based on the third PID controller for all estimated models is approximately 9 min and the overshoot is approximately 15%. The proposed CEFO algorithm can be considered an effective methodology for mathematical modelling and achievement of high quality and better performance of the designed closed-loop system for cultivation processes.

Engineering Escherichia coli for D-allulose biosynthesis from glycerol

D-allulose, a naturally occurring monosaccharide, is present in small quantities in nature. It is considered a valuable low-calorie sweetener due to its low absorption in the digestive tract and zero energy for growth. Most of the recent efforts to produce D-allulose have focused on in vitro enzyme catalysis. However, microbial fermentation is emerging as a promising alternative that offers the advantage of combining enzyme manufacturing and product synthesis within a single bioreactor. Here, a novel approach was proposed for the efficient biosynthesis of D-allulose from glycerol using metabolically engineered Escherichia coli. FbaA, Fbp, AlsE, and A6PP were used to construct the D-allulose synthesis pathway. Subsequently, PfkA, PfkB, and Pgi were disrupted to block the entry of the intermediate fructose-6-phosphate (F6P) into the Embden−Meyerhof−Parnas (EMP) and pentose phosphate (PP) pathways. Additionally, GalE and FryA were inactivated to reduce D-allulose consumption by the cells. Finally, a fed-batch fermentation process was implemented to optimize the performance of the cell factory. As a result, the titer of D-allulose reached 7.02 g/L with a maximum yield of 0.287 g/g.

Acclimation dynamics of Chlorella vulgaris to sudden light change

Chlorella vulgaris photoacclimation was monitored over eight instantaneous light intensity changes. The intensities ranged between 35μmol PhotonPAR.m−2.s−1 and 600μmolPhotonPAR.m−2.s−1. Cultures were grown in ultra-thin flat panel photobioreactors under continuous light and maintained in low cell density to ensure homogeneous light availability. Photoacclimation was evaluated through spectral quantification of pigments and fluorometric assays. The former gave access to a proxy of chlorophyll and carotenoid content, the latter to the Photosystem-II cross-section (σPSII) and qualification of the photosynthetic machinery (viaOJIP assays). Both the acclimated steady-state values of pigment content and the dynamic of their evolutions after sudden light intensity change were monitored. The characteristic times of the transitions were estimated based on a first-order assumption. Results consistently showed that antenna size adjustment of Chlorella vulgaris was primarily dictated by the light availability, both regarding the acclimated steady-state values and the acclimation dynamics. An energetic limitation was highlighted by the acclimation dynamics at low light. The characteristic time of transition was estimated to be 16.6±2.17h for the transition to the lowest light intensity (35μmol PhotonPAR.m−2.s−1) and 3.55±1.01h for intensities higher than the maximal intensity of photolimitation (120μmol PhotonPAR.m−2.s−1). No hysteresis effect was observed as light intensities were shifted once and reverted to their original values. These results extend the literature regarding photoacclimation dynamics of antenna size and photosynthetic apparatus. They are well-suited to calibrate photoacclimation models and can provide valuable insight into the strategies to implement for culture scale-up, fed-batch, and semi-continuous processes.

Comparative Approaches for Quantification of Product Yield in a Model Recombinant Green Fluorescent Protein Expressed in E. coli.

Process Analytical Technologies (PAT) used to monitor and control manufacturing processes are crucial for efficient and automated bioprocessing, which is in congruence with lights-off-manufacturing and Industry 4.0 initiatives. As biomanufacturing seeks to realize more high-throughput and automated operation, an increasing need for multimodal analysis of process metrics becomes essential. Herein, we detail a series of methods for analyzing product yield from a bioreactor and how to conduct cross-method comparisons. We employ a model system of Escherichia coli (E. coli) expression of green fluorescent protein (GFP), which is a simple, cost effective model for students and educators to replicate at different scales. GFP is an ideal analytical marker as it is easy to visualize due to its fluorescence which indicates cellular protein expression, cell localization and physiological changes of the cell population. In this study, samples from a 300 L bioreactor with GFP-expressing E. coli are analyzed to improve product yield and bioprocessing efficiency. Utilizing a fed-batch process for enhanced cell density and product titer, this bioreactor runs on a 24-hour schedule from inoculation to GFP induction and final harvest. To reliably quantify relative GFP expression and E. coli proliferation, we provide simple protocols and example results for comparing three different analytical methods: (1) in-line bioreactor measurements, (2) plate reader assays, and (3) microscopy. The GFP and cell density results follow similar trends based on the various inline and offline analytical methods and show a peak of GFP expression and cell density between 12.5 and 18 hours post inoculation.

Metabolomic characterization of monoclonal antibody-producing Chinese hamster lung (CHL)-YN cells in glucose-controlled serum-free fed-batch operation.

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.

Evaluation of Aerobic Propagation of Yeasts as Additional Step in Production Process of Corn Ethanol

Yeast is one of the co-products of ethanol plants, which can be used as a nutritional supplement in animal feed due to its high protein content. Given the importance of yeast contribution to the nutritional properties of DDG (dried distillers’ grains), the aim of this study was to assess how different levels of aeration affect the biomass production and the quality of yeast providing new insights into yeast production, offering an alternative source of income for the corn ethanol industry. For this purpose, yeasts were grown in a fed-batch process, and different concentrations of aeration in the medium were tested, namely 0.5, 1.0, and 1.5 volume of air per volume of wort per minute (v v−1 min−1). At the end of the cellular biomass production process, yeasts grown with 0.5 (v v−1 min−1) aeration in the reactor showed higher biomass formation (19.86 g L−1), cellular yield (g g−1), and a lower formation of succinic acid (0.70 g L−1) and acetic acid (0.11 g L−1). Aeration influenced an increase of 1.0% in the protein content in yeast. In conclusion, lower levels of aeration in the yeast production process enables more efficient sugar utilization for biomass formation and is a potential strategy to increase the protein content and the commercial value of DDG.

Soft sensor for viable cell counting by measuring dynamic oxygen uptake rate

Regulatory authorities in biopharmaceutical industry emphasize process design by process understanding but applicable tools that are easy to implement are still missing. Soft sensors are a promising tool for the implementation of the Quality by Design (QbD) approach and Process Analytical Technology (PAT). In particular, the correlation between viable cell counting and oxygen consumption was investigated, but problems remained: Either the process had to be modified for excluding CO2 in pH control, or complex kLa models had to be set up for specific processes. In this work, a non-invasive soft sensor for simplified on-line cell counting based on dynamic oxygen uptake rate was developed with no need of special equipment. The dynamic oxygen uptake rates were determined by automated and periodic interruptions of gas supply in DASGIP® bioreactor systems, realized by a programmed Visual Basic script in the DASware® control software. With off-line cell counting, the two parameters were correlated based on linear regression and led to a robust model with a correlation coefficient of 0.92. Avoidance of oxygen starvation was achieved by gas flow reactivation at a certain minimum dissolved oxygen concentration. The soft sensor model was established in the exponential growth phase of a Chinese Hamster Ovary fed-batch process. Control studies showed no impact on cell growth by the discontinuous gas supply. This soft sensor is the first to be presented that does not require any specialized additional equipment as the methodology relies solely on the direct measurement of oxygen consumed by the cells in the bioreactor.

Simultaneous Formate and Syngas Conversion Boosts Growth and Product Formation by Clostridium ragsdalei.

Electrocatalytic CO2 reduction to CO and formate can be coupled to gas fermentation with anaerobic microorganisms. In combination with a competing hydrogen evolution reaction in the cathode in aqueous medium, the in situ, electrocatalytic produced syngas components can be converted by an acetogenic bacterium, such as Clostridium ragsdalei, into acetate, ethanol, and 2,3-butanediol. In order to study the simultaneous conversion of CO, CO2, and formate together with H2 with C. ragsdalei, fed-batch processes were conducted with continuous gassing using a fully controlled stirred tank bioreactor. Formate was added continuously, and various initial CO partial pressures (pCO0) were applied. C. ragsdalei utilized CO as the favored substrate for growth and product formation, but below a partial pressure of 30 mbar CO in the bioreactor, a simultaneous CO2/H2 conversion was observed. Formate supplementation enabled 20-50% higher growth rates independent of the partial pressure of CO and improved the acetate and 2,3-butanediol production. Finally, the reaction conditions were identified, allowing the parallel CO, CO2, formate, and H2 consumption with C. ragsdalei at a limiting CO partial pressure below 30 mbar, pH 5.5, n = 1200 min-1, and T = 32 °C. Thus, improved carbon and electron conversion is possible to establish efficient and sustainable processes with acetogenic bacteria, as shown in the example of C. ragsdalei.

A high-density microfluidic bioreactor for the automated manufacturing of CAR T cells.

The manufacturing of autologous chimaeric antigen receptor (CAR) T cells largely relies either on fed-batch and manual processes that often lack environmental monitoring and control or on bioreactors that cannot be easily scaled out to meet patient demands. Here we show that human primary T cells can be activated, transduced and expanded to high densities in a 2 ml automated closed-system microfluidic bioreactor to produce viable anti-CD19 CAR T cells (specifically, more than 60 million CAR T cells from donor cells derived from patients with lymphoma and more than 200 million CAR T cells from healthy donors). The in vitro secretion of cytokines, the short-term cytotoxic activity and the long-term persistence and proliferation of the cell products, as well as their in vivo anti-leukaemic activity, were comparable to those of T cells produced in a gas-permeable well. The manufacturing-process intensification enabled by the miniaturized perfusable bioreactor may facilitate the analysis of the growth and metabolic states of CAR T cells during ex vivo culture, the high-throughput optimization of cell-manufacturing processes and the scale out of cell-therapy manufacturing.