Fed-batch Research Articles

Enhancing monoclonal antibody production efficiency using CHO-MK cells and specific media in a conventional fed-batch culture

Enhancing monoclonal antibody production efficiency using CHO-MK cells and specific media in a conventional fed-batch culture

Production of a cellulose-aminating polysaccharide from a filamentous sulfur-oxidizing bacterium, Thiothrix nivea, grown lithotrophically or mixotrophically.

Glucosaminoglucan (β-1,4-linked glucose and glucosamine) produced by a mixotrophic sulfur-oxidizing bacterium, Thiothrix nivea, is a useful cellulose-aminating agent. Lithotrophic and mixotrophic glucosaminoglucan production were examined using fed-batch techniques. A jar fermenter was used for the fed-batch cultivation. Glucosaminoglucan was extracted from T. nivea using diluted HCl. Lithotrophic growth was detected by feeding with Na2S as the energy source, and 12mg L-culture-1 of glucosaminoglucan was obtained. In contrast, no growth was observed with Na2S2O3. Similarly, mixotrophic growth in the presence of acetic acid was promoted by Na2S, whereas Na2S2O3 had no effect. When acetic acid and Na2S were added, 470mg L-culture-1 of glucosaminoglucan was obtained. T. nivea was cultured and glucosaminoglucan was produced lithotrophically using Na2S for feeding. Na2S is also indispensable for mixotrophic growth and glucosaminoglucan production, indicating that sulfide oxidation pathways control the TCA cycle. The involvement of the SOX pathway (for thiosulfate oxidation) in the activation of energy metabolism is doubtful because neither lithotrophic nor mixotrophic growth was promoted by Na2S2O3. Based on these results, we assumed that T. nivea is facultatively mixotrophic (lithotrophic growth is possible in addition to organotrophic growth in the presence of sulfide (Na2S)), rather than obligately mixotrophic.

Repeated Fed-Batch Culture Strategy for the Synthesis of Polyhydroxybutyrate (PHB) Biopolymers from Sugar Cane Juice Using Azotobacter vinelandii

In this research work, a main biopolymer group of polyhydroxyalkanoates (PHAs) in the form of polyhydroxybutyrate (PHB) was synthesised by a pure bacterial strain of Azotobacter vinelandii via repeated fed-batch fermentation. An agricultural crop, sugar cane, was used as the sole carbon source. Firstly, batch fermentation was investigated considering variations in incubation times (24 h, 48 h, and 96 h). The highest dry cell weight (DCW) and PHAs of 5.15 ± 0.04 g/L and 4.00 ± 0.04 g/L were obtained after 48 h of incubation time. The optimum time obtained was further varied to investigate the effect of the sugar concentrations in the medium. It was found that bacteria could grow very well and produced the highest DCW and PHAs (11.17 ± 0.15 g/L and 8.77 ± 0.06 g/L) when the culture medium with a 100 g/L sugar concentration was added. Later, repeated fed-batch fermentation was carried out to improve productivity. The results obtained revealed that PHA production was increased in the next cycle of the process. Furthermore, the final productivity (0.104 g/L·h) was increased 1.65-fold compared to the first cycle (0.063 g/L·h). Moreover, the culture strategy showed remarkable results, with reductions in both fermentation time and preparation cost.

De Novo Production of the Bioactive Phenylpropanoid Artepillin C Using Membrane-Bound Prenyltransferase in Komagataella phaffii.

Artepillin C is a diprenylated phenylpropanoid with various pharmacological benefits for human health. Its natural occurrence is limited to a few Asteraceae plants, such as Baccharis species, necessitating a stable supply through synthetic biology. In Saccharomyces cerevisiae, the utilization of aromatic substrates within the cell was limited, resulting in very low production of artepillin C. In this study, we used AcPT1, a p-coumaric acid (p-CA)-specific diprenyltransferase, in Komagataella phaffii to produce artepillin C. Detailed studies revealed that the critical bottleneck in K. phaffii was the supply of prenyl diphosphates, not phenylpropanoid flux. By enhancing the prenyl substrate pathway through overexpression of isopentenyl diphosphate isomerase and a truncated HMG-CoA reductase, we achieved a strong increase in artepillin C production. A major part of artepillin C was accumulated in yeast cells. One of the advantages of K. phaffii is its superior growth and ability to achieve high cell density cultivation compared to that of S. cerevisiae. Therefore, fed-batch cultivation with glycerol was performed. As a result, the dry cell weight (DCW) reached 61.0 g/L, and the intracellular amount of de novo produced artepillin C reached 187.3 μg/DCW. Analysis of intermediates revealed that the supply of p-CA constituted a bottleneck in artepillin C production in the engineered strain. By enhancing the p-CA supply, the intracellular accumulation of artepillin C reached 1200 μg/DCW even in batch cultivation. Moreover, the total intra- and extracellular amounts of artepillin C reached 12.5 mg/L, marking the highest de novo synthesis amount of artepillin C reported thus far, even under batch cultivation conditions.

Comparison Between Simple Batch and Fed-Batch Bioreactor Cultivation of Recombinant BCG

Background/Objectives: Tuberculosis continues to be a significant global health concern, causing 1.3 million deaths in 2022, particularly affecting children under 5 years old. The Bacillus Calmette-Guérin (BCG) vaccine, developed in 1921, remains the primary defense against tuberculosis but requires modernized production methods. The recombinant BCG-pertussis strain shows potential in providing dual protection against tuberculosis and whooping cough, especially for vulnerable newborns, and enhanced efficacy against bladder cancer. Implementing submerged cultivation techniques for rBCG-pertussis production can offer increased productivity and standardization. Methods: This study explores a fed-batch cultivation strategy with pH-stat control to feed L-glutamic acid through the acid pump into 1 L bioreactor. Three pH values were evaluated for fed-batch and a simple batch without pH control was done for comparison. The viable cell concentration was compared before and after freeze-drying samples harvested during the cultures. Results: L-glutamic acid was identified as the preferred substrate for rBCG-pertussis. While the fed-batch strategy did not enhance the maximum specific growth rate compared to simple batch cultivation, it did improve the specific growth rate after day 4 in the pH 7.4-controlled fed-batch cultures, thereby reducing the cultivation time. Fed-batch cultures controlled at three pH levels exhibited lower optical density than the simple batch, although the viable cell counts were similar. Notably, samples harvested after day 8 from the simple batch cultures showed a reduction in CFU/mL after freeze-drying, whereas all fed-batch samples exhibited high recovery of viable cell counts post lyophilization. Conclusions: The additional glutamate supplied to the fed-batch cultures may have protected the cells during the lyophilization process.

Antibiotic-Free High-Level l-Methionine Production in Engineered Escherichia coli.

l-Methionine, a valuable sulfur-containing amino acid, holds great significance as a feed additive, nutraceutical, pharmaceutical, or even in the cosmetic industry. However, achieving efficient microbial production of l-methionine remains challenging due to its complex biosynthetic pathway and plasmid loss during fermentation. Herein, l-methionine biosynthesis was improved by enhancing succinyl-CoA supply, introducing a direct-sulfurylation pathway, and weakening the l-threonine branched pathway. The engineered strain produced 21.55 g/L l-methionine with a yield of 0.14 g/g glucose in a 5 L bioreactor. To eliminate the need for antibiotics and minimize plasmid loss, the hok/sok system was incorporated into the plasmid. The resulting plasmid pAm10 enabled strain M2 thrBA1G to produce 20.39 g/L of l-methionine without antibiotics in 5 L of fed-batch cultivation, a 42.58% increase compared to the control. This study highlights the potential of plasmid-based antibiotic-free fermentation for efficient and cost-effective production of l-methionine, as well as other amino acids or chemicals.

Experimental determination of maximum shear stress in Mobius® Breez perfusion microbioreactors and comparative analysis with stirred tank bioreactors

Perfusion processes have experienced increased popularity in recent years due to their ability to sustain high cell densities and productivities in biopharmaceutical production, offering advantages over traditional batch and fed-batch cultivation methods. The Mobius® Breez microbioreactor significantly reduces experimental effort by downsizing the classical volume of perfusion bioreactors to the mL range and thus represents a valuable tool for process development. However, miniaturization has raised questions regarding comparability with traditional bioreactors in terms of the physical environment, such as hydrodynamic shear stress. Therefore, the maximum hydrodynamic shear stress, cultivation performance, and membrane-wall contact were evaluated to elucidate the system's behavior. Findings reveal two distinct operational conditions, distinguished by the presence or absence of membrane-wall contact, resulting in varying levels of hydrodynamic stress. Conditions lacking membrane contact demonstrate stress levels within safe operating thresholds for CHO cells, while those involving membrane contact exceed these thresholds, potentially leading to cell damage. Through the identification of critical frequencies of membrane motion, this study offers insights for optimizing microbioreactor operation and enhancing overall bioprocess efficiency.

Genome reduction improves octanoic acid production in scale down bioreactors.

Microorganisms in large-scale bioreactors are exposed to heterogeneous environmental conditions due to physical mixing constraints. Nutritional gradients can lead to transient expression of energetically wasteful stress responses and as a result, can reduce the titres, rates and yields of a bioprocess at larger scales. To what extent these process parameters are impacted is often unknown and therefore bioprocess scale-up comes with major risk. Designing platform strains to account for these intermittent stresses before introducing synthesis pathways is one strategy for de-risking bioprocess development. For example, Escherichia coli strain RM214 is a derivative of wild-type MG1655 that has had several genes and whole operons removed from its genome based on their metabolic cost. In this study, we engineered E. coli strain RM214 (referred to as WG02) to produce octanoic acid from glycerol in batch-flask and fed-batch bioreactor cultivations and compared it to an octanoic acid-producing E. coli MG1655 (WG01). In batch flask cultivations, the two strains performed similarly. However, in carbon limited fed-batch bioreactor cultivations, WG02 provided a greater than 22% boost to biomass compared to WG01 while maintaining similar titres of octanoic acid. Reducing the biomass accumulation of WG02 with nitrogen limited fed-batch cultivation resulted in a 16% improvement in octanoic acid titre over WG01. Finally, in a scale-down system consisting of a stirred tank reactor (representing a well-mixed zone) and plug flow reactor (representing an intermittent carbon starvation zone), WG02 again improved octanoic acid titre by almost 18% while maintaining similar biomass concentrations as WG01.

Continuous bioethanol production from glucose-rich hydrolysate derived from wheat straw using a unique fed-batch cultivation method in a bioreactor

The present study provides solutions to the bioethanol industry’s main issues of the absence of production of maximum fermentable sugars from lignocellulosic biomass, the unsuitability of a single microorganism to convert the fermentable sugars (xylose and glucose) together to bioethanol, and the unavailability of fermentation technique to run the process unlimitedly with consistent yield and productivity. A novel fractional acid hydrolysis technology was demonstrated in this study, which produced more than 90% (w/w) of available glucose in wheat straw as glucose-rich hydrolysate (GRH). This hydrolysate was then made acid-free using a membrane-based acid separation unit to reuse the separated acid to make the hydrolysis process environmentally safe and chemically inexpensive. Thereafter, this acid-free GRH was fermented to bioethanol using a unique constant volume fed-batch fermentation technique under an optimum glucose-feeding strategy. Remarkably, this fermentation technique yielded optimal average bioethanol productivity (g/Lh), yield (g/g), and titer (g/L) of 7.147±0.533, 0.508±0.002, and 58.835±0.766; 5.722±0.529, 0.501±0.006, and 33.748±0.322, respectively, with more than 99% glucose utilization during ten fed-batch cycles of synthetic and GRH glucose. Notably, the high production of GRH from lignocellulosic biomass, acid separation and reuse, and continuous and consistent bioethanol production sets this study apart as a pioneering endeavor. Furthermore, the bioethanol profit of USD 0.019/kg biomass from the second batch onwards at a bioethanol selling price of USD 0.745/L suggests the feasibility of the current study for industrial-scale bioethanol production from lignocellulosic biomass.

Highly-efficient co-production of microbial lipid and magnesium ammonium phosphate from N-acetyl-D-glucosamine

The valorization of chitin-rich wastes into chemicals and biofuels holds immense economic and environmental benefits. Here, N-acetyl-D-glucosamine (GlcNAc), the basic structural unit of chitin, was firstly described for co-producing microbial lipid and magnesium ammonium phosphate (MAP). Due to the strong substrate inhibition of GlcNAc, a fed-batch culture mode was successfully adopted to achieve high cell density by Cutaneotrichosporon oleaginosum. When a phosphate limitation strategy was applied, cell mass, lipid titer, content, yield, and productivity were 102.7 g/L, 74.2 g/L, 72.2 %, 21.4 g/100 g, and 0.69 g/L/h, respectively. The ammonium ion was efficiently precipitated by forming MAP with a removal rate around 95.4 %. The lipid samples showed high similarity to vegetable oil, which emerged as high-quality precursor for biodiesel production. This study offers a promising strategy for full conversion of GlcNAc into lipid and slow-release fertilizer, which provides an attractive technical route for turning the chitin-rich materials into valuable products.

Engineering Halomonas bluephagenesis for synthesis of polyhydroxybutyrate (PHB) in the presence of high nitrogen containing media

The trade-offs exist between microbial growth and bioproduct synthesis including intracellular polyester polyhydroxybutyrate (PHB). Under nitrogen limitation, more carbon flux is directed to PHB synthesis while growth is inhibited with diminishing overall carbon utilization, similar to the suboptimal carbon utilization during glycolysis-derived pyruvate decarboxylation. This study reconfigured the central carbon network of Halomonas bluephagenesis to improve PHB yield theoretically and practically. It was found that the downregulation of glutamine synthetase (GS) activity led to a synchronous improvement on PHB accumulation and cell growth under nitrogen non-limitation condition, increasing the PHB yield from glucose (g/g) to 85% of theoretical yield, PHB titer from 7.6 g/L to 12.9 g/L, and from 51 g/L to 65 g/L when grown in shake flasks containing a rich N-source, and grown in a fed-batch cultivation conducted in a 7-L bioreactor also containing a rich N-source, respectively. Results offer better metabolic balance between glucose conversion efficiency and microbial growth for economic PHB production.

Integration of mathematical and experimental modeling for sustainable phycobiliprotein production via fed-batch cultures

The production of phycobiliproteins, such as cyanobacterial phycocyanins, is a growing interest due to their diverse industrial and biotechnological applications. This study focuses on optimizing phycocyanin production using the strain Potamosiphon sp. through experimental techniques and mathematical modeling in fed-batch cultures. The methodology applied includes determining the kinetic constants by linearizing the Monod equation evaluating the concentrations of biomass, C-phycocyanin (C-PC), nitrates (NO3), and phosphates (PO4). A mathematical model of periodic fed-batch feeding was subsequently established, applying mass conservation principles and evaluating the accuracy of the Monod, Contois, Moser, and Tessier models. The results indicate that phycocyanin production is highly dependent on phosphorus and nitrogen concentrations, with optimal conversion observed at specific levels of these elements (0.832 for phosphorus and 0.805 for nitrogen in terms of C-PC and biomass, respectively). The Tessier model demonstrated the highest accuracy in predicting production and optimizing operational conditions, with a Mean Squared Error (MSE) of 0.005000 for biomass production, 0.200000 for C-PC production, and 0.000010 for substrate consumption. It also achieved high R² values of 0.980 for biomass, 0.999 for C-PC production, and 0.997 for substrate consumption. It presented the lowest Akaike Information Criterion (AIC) scores, indicating its robustness and reliability in modeling these processes and manipulating cultivation conditions and providing adequate nutrition allowed for achieving growth rates of 1.23 g/L and a C-PC concentration of 37 mg/L, which are essential for industrial applications such as natural colorants and antioxidants, among others.

Recent Trends in the Production and Recovery of Bioplastics Using Polyhydroxyalkanoates Copolymers

Polyhydroxyalkanoates (PHAs) are polyesters synthesized as a carbon and energy reserve material by a wide number of bacteria. These polymers are characterized by their thermoplastic properties similar to those of plastics derived from the petrochemical industry, such as polyethylene and polypropylene. PHAs are widely used in the medical field and have the potential to be used in other applications due to their biocompatibility and biodegradability. Among PHAs, P(3HB-co-3HV) copolymers are thermo-elastomeric polyesters that are typically soft and flexible with low to no crystallinity, which can expand the range of applications of these bioplastics. Several bacterial species, such as Cupriavidus necator, Azotobacter vinelandii, Halomonas sp. and Bacillus megaterium, have been successfully used for P(3HB-co-3HV) production, both in batch and fed-batch cultures using different low-cost substrates, such as vegetable and fruit waste. Nevertheless, in recent years, several fermentation strategies using other microbial models, such as methanotrophic bacterial strains as well as halophilic bacteria, have been developed in order to improve PHA production in cultivation conditions that are easily implemented on a large scale. This review aims to summarize the recent trends in the production and recovery of PHA copolymers by fermentation, including different cultivation modalities, low-cost raw materials, as well as downstream strategies that have recently been developed with the purpose of producing copolymers, such as P(3HB-co-3HV), with suitable mechanical properties for applications in the biomedical field.

Overexpression and characterization of recombinant hCGβ-LTB vaccine in fed-batch culture of yeast Pichia pastoris

Overexpression and characterization of recombinant hCGβ-LTB vaccine in fed-batch culture of yeast Pichia pastoris

Automated yeast cultivation control using a biosensor and flow cytometry.

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.

Clavulanic Acid Overproduction: A Review of Environmental Conditions, Metabolic Fluxes, and Strain Engineering in Streptomyces clavuligerus

Clavulanic acid is a potent β-lactamase inhibitor produced by Streptomyces clavuligerus, widely used in combination with β-lactam antibiotics to combat antimicrobial resistance. This systematic review analyzes the most successful methodologies for clavulanic acid overproduction, focusing on the highest yields reported in bench-scale and bioreactor-scale fermentations. Studies have demonstrated that glycerol is the preferred carbon source for clavulanic acid production over other sources like starch and dextrins. The optimization of feeding strategies, especially in fed-batch operations, has improved glycerol utilization and extended the clavulanic acid production phase. Organic nitrogen sources, particularly soybean protein isolates and amino acid supplements such as L-arginine, L-threonine, and L-glutamate, have been proven effective at increasing CA yields both in batch and fed-batch cultures, especially when balanced with appropriate carbon sources. Strain engineering approaches, including mutagenesis and targeted genetic modifications, have allowed for the obtainment of overproducer S. clavuligerus strains. Specifically, engineering efforts that overexpress key regulatory genes such as ccaR and claR, or that disrupt competing pathways, redirect the metabolic flux towards CA biosynthesis, leading to high clavulanic acid titers. The fed-batch operation at the bioreactor scale emerges as the most feasible alternative for prolonged clavulanic acid production with both wild-type and mutant strains, allowing for the attainment of high titers during cultivations.

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.

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii

BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Continuous Process Verification 4.0 application in upstream: adaptiveness implementation managed by AI in the hypoxic bioprocess of the Pichia pastoris cell factory.

The experimental approach developed in this research demonstrated how the cloud, the Internet of Things (IoT), edge computing, and Artificial Intelligence (AI), considered key technologies in Industry 4.0, provide the expected horizon for adaptive vision in Continued Process Verification (CPV), the final stage of Process Validation (PV). Pichia pastoris producing Candida rugosa lipase 1 under the regulation of the constitutive GAP promoter was selected as an experimental bioprocess. The bioprocess worked under hypoxic conditions in carbon-limited fed-batch cultures through a physiological control based on the respiratory quotient (RQ). In this novel bioprocess, a digital twin (DT) was built and successfully tested. The implementation of online sensors worked as a bridge between the microorganism and AI models, to provide predictions from the edge and the cloud. AI models emulated the metabolism of Pichia based on critical process parameters and actionable factors to achieve the expected quality attributes. This innovative AI-aided Adaptive-Proportional Control strategy (AI-APC) improved the reproducibility comparing to a Manual-Heuristic Control strategy (MHC), showing better performance than the Boolean-Logic-Controller (BLC) tested. The accuracy, indicated by the Mean Relative Error (MRE), was for the AI-APC lower than 4%, better than the obtained for MHC (10%) and BLC (5%). Moreover, in terms of precision, the same trend was observed when comparing the Root Mean Square Deviation (RMSD) values, becoming lower as the complexity of the controller increases. The successful automatic real time control of the bioprocess orchestrated by AI models proved the 4.0 capabilities brought by the adaptive concept and its validity in biopharmaceutical upstream operations.

Application of a Novel Disposable Flow Cell for Spectroscopic Bioprocess Monitoring

The evaluation of the analytical capabilities of a novel disposable flow cell for spectroscopic bioprocess monitoring is presented. The flow cell is presterilized and can be connected to any kind of bioreactor by weldable tube connections. It is clamped into a reusable holder, which is equipped with SMA-terminated optical fibers or an integrated light source and detection unit. This modular construction enables spectroscopic techniques like UV-Vis spectroscopy or turbidity measurements by scattered light for modern disposable bioreactors. A NIR scattering module was used for biomass monitoring in different cultivations. A high-cell-density fed-batch cultivation with Komagataella phaffii and a continuous perfusion cultivation with a CHO DG44 cell line were conducted. A high correlation between the sensor signal and biomass or viable cell count was observed. Furthermore, the sensor shows high sensitivity during low turbidity states, as well as a high dynamic range to monitor high turbidity values without saturation effects. In addition to upstream processing, the sensor system was used to monitor the purification process of a monoclonal antibody. The absorption module enables simple and cost-efficient monitoring of downstream processing and quality control measurements. Recorded absorption spectra can be used for antibody aggregate detection, due to an increase in overall optical density.