Bioreactor Process Research Articles

Critical Quality Predictive Control of Fed-Batch Mammalian Cell Bioreactors

Abstract A model predictive control (MPC) formulation for a mammalian cell fed-batch bioreactor processes is developed. A nonlinear fundamental model for the bioreactor is used to generate a database of historical runs comprising of the measurement variables and the manipulated input feed flow rate to the bioreactor. The database is used with subspace identification methods to develop a state-space model of the process. The identified model is used to design various MPC formulations with different objective criteria, including the conventional trajectory-tracking objective function and a novel terminal objective for maximizing the product yield at completion of a run. Case studies involving the simulated bioreactor process demonstrate the efficacy of the MPC algorithms subject to unknown disturbances, random variations in the inlet feed glucose and glutamine concentrations, and measurement noise. Compared to the traditional proportional-integral control algorithm, the trajectory-tracking predictive control algorithm is able to better track the reference glucose concentration set-point with an improvement of 5.1% in the tracking error. The critical quality attribute predictive control algorithm designed to maximize the product yield results in a 3.9% increase in the product concentration at the completion of the run.

An Investigation of the Membrane Fouling Behaviors in Constant Flux Mode

Studying the effect of different operation conditions (bovine serum albumin (BSA) concentration, flux and stirring speed) on the membrane fouling behaviors (the variation of transmembrane pressure (TMP), cake resistance (Rc), pore blocking resistance (Rc ) and available membrane area ratio (A/A0 ) is of paramount importance in the membrane bioreactor (MBR) process. The constant flux filtration experiment was conducted to investigate the fouling behaviors of BSA solution, which provides a guidance for controlling the membrane fouling. The intermediate blocking-cake combined model was established and it was described mathematically with good results. The results showed that the influence sequence of operating conditions on the TMP was obtained as flux > concentration > stirring speed. Meanwhile, Rc decreased with the increase of stirring speed, and the A/A0 decreased with the increase of BSA concentration and the flux.

Whey management based on bioreactor and membrane processes: clean technology gaining valuable components of whey

Whey management based on bioreactor and membrane processes: clean technology gaining valuable components of whey

Aerobic Utilization of Methanol for Microbial Growth and Production.

Methanol is a reduced one-carbon (C1) compound. It supports growth of aerobic methylotrophs that gain ATP from reduced redox equivalents by respiratory phosphorylation in their electron transport chains. Notably, linear oxidation of methanol to carbon dioxide may yield three reduced redox equivalents if methanol oxidation is NAD-dependent as, e.g., in Bacillus methanolicus. Methanol has a higher degree of reduction per carbon than glucose (6 vs. 4), and thus, lends itself as an ideal carbon source for microbial production of reduced target compounds. However, C-C bond formation in the RuMP or serine cycle, a prerequisite for production of larger molecules, requires ATP and/or reduced redox equivalents. Moreover, heat dissipation and a high demand for oxygen during catabolic oxidation of methanol may pose challenges for fermentation processes. In this chapter, we summarize metabolic pathways for aerobic methanol utilization, aerobic methylotrophs as industrial production hosts, strain engineering, and methanol bioreactor processes. In addition, we provide technological and market outlooks.

Global Sensitivity Analysis for a perfusion bioreactor system in tissue engineering

Abstract This work presents a global sensitivity analysis and simulations of a perfusion bioreactor process using the method of high-dimensional model representation (HDMR). This method was developed to express the input–output relationships of a complex model with a high dimensional input space. The comprehensive mathematical model of convection and diffusion in a perfusion bioreactor, combined with cell growth kinetics, is developed and implemented using Computational Fluid Dynamics (with the commercial software COMSOL Multiphysics v5.5). The model describes the spatio-temporal evolution of glucose concentration, oxygen concentration, lactate concentration and cell density within a 3D polymeric scaffold. A quantitative analysis of the complex kinetic mechanisms using recent development of advanced mathematical approaches to global sensitivity and uncertainty analysis through HDMR can be exploited to investigate the important features of the perfusion bioreactor process as well as possible factors underlying qualitative discrepancies.

Co-treatment of mixed municipal sewage and landfill leachates via the hydrolytic acidification–sequencing batch reactors–membrane bioreactor process

Co-treatment of mixed municipal sewage and landfill leachates via the hydrolytic acidification–sequencing batch reactors–membrane bioreactor process

Model Reference Adaptive Control for Milk Fermentation in Batch Bioreactors

This paper presents the advanced control theory’s original utilisation to realise a system that controls the fermentation process in batch bioreactors. Proper fermentation control is essential for quality fermentation products and the economical operation of bioreactors. Batch bioreactors are very popular due to their simple construction. However, this simplicity presents limitations in implementing control systems that would ensure a controlled fermentation process. Batch bioreactors do not allow the inflow/outflow of substances during operation. Therefore, we have developed a control system based on a stirrer drive instead of material flow. The newly developed control system ensures tracking of the fermentation product time course to the reference trajectory by changing the stirrer’s speed. Firstly, the paper presents the derivation of the enhanced mathematical model suitable for developing a control system. A linearisation and eigenvalue analysis of this model were made. Due to the time-consuming determination of the fermentation model and the variation of the controlled plant during operation, the use of adaptive control is advantageous. Secondly, a comparison of different adaptive approaches was made. The model reference adaptive control was selected on this basis. The control theory is presented, and the control realisation described. Experimental results obtained with the laboratory batch bioreactor confirm the advantages of the proposed adaptive approach compared to the conventional PI-control.

Modeling of heat exchange processes in the Metanetka bioenergy plant for individual use

A bioenergy plant with a solar heating system for individual use has been developed and built, intended for the processing and disposal of agricultural waste of organic origin to obtain biogas and liquid high-quality organic fertilizers under conditions of anaerobic fermentation. The main features of heat transfer processes in bioreactors are considered. A mathematical model is proposed that describes these processes. The use of the resulting model will improve the control processes of reactors and comply with technological regimes

Limits to biomass productivity during fed-batch cultivation of Laminaria saccharina female gametophyte cells in a stirred-tank photobioreactor

This study compared the growth of Laminaria saccharina female gametophyte filamentous cell suspension cultures in a stirred-tank photobioreactor under batch and fed-batch nutrient addition modes over a 48-day cultivation period. Cultures were grown on GP2 artificial seawater medium (0.75 mM nitrate, N:P = 16:1) at pH 8.3 without iron or copper. Total equivalent nutrient loadings ranged from 0.5X to 9.1X GP2 for batch cultivation and 1.3X to 10.4X GP2 for fed-batch cultivation at delivery rates of 0.0067–0.16 mmol N L−1 day−1 based on nitrate. The multicellular, L. saccharina filamentous clumps were dispersed to nominal size of 100 μm by mechanical blending (~ 16,000 rpm, 5 s) prior to inoculation. Fed-batch addition of all nutrients enhanced biomass productivity by a factor of two over a batch cultivation process at equivalent total nutrient loadings in a stirred-tank photobioreactor. Peak productivity through fed-batch cultivation was 57 mg DCW L−1 day−1, and average final biomass densities exceeded 1800 mg DCW L−1, vs. 30 mg DCW L−1 day−1 and 800–900 mg DCW L−1 for batch cultivation. However, there was a limit to biomass productivity enhancement at cumulative nutrient loadings greater than 3X GP2 that was not the result of insufficient CO2 or light delivery. It is suggested that the formation of large, multicellular clumps approaching 1-mm diameter during stirred-tank cultivation may have ultimately reduced biomass productivity during fed-batch cultivation under nutrient-replete conditions. Therefore, future bioreactor processing strategies might consider mechanical blending to disperse the filament clumps during the cultivation process.

Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18–59 years: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial

Safety, tolerability, and immunogenicity of an inactivated SARS-CoV-2 vaccine in healthy adults aged 18–59 years: a randomised, double-blind, placebo-controlled, phase 1/2 clinical trial

Steady-State Methodology for Activated Sludge Model 1 (ASM1) State Variable Calculation in MBR

The complexity of Activated Sludge Model No. 1 (ASM1) is one of the main obstacles slowing its widespread use, particularly among wastewater treatment plant (WWTP) professionals. In this paper, a simplification procedure based on steady-state mass balances is proposed for the conventional activated sludge process (ASP) configuration, consisting of an aerated bioreactor and a perfect settler (without particular compounds in the outlet). The results do, in fact, show perfect suitability to a membrane bioreactor process (MBR). Both organic carbon and nitrogen removal were investigated. The proposed approach was applied to ASM1, and simple analytical expressions of the state variables were obtained. These analytical expressions were then validated by comparison to simulations given by the original ASM1 (implemented in GPS-X software). A strong match (less than 4% of error overall) was obtained between both results in the steady-state; consequently, these analytical expressions may be useful as tools for quickly estimating the main state variables, feeding the filtration models, or identifying the interaction between operating parameters. Moreover, this enables a sensitivity analysis, covering relevant factors such as kinetics or operating parameters. For instance, the sludge retention time (SRT) effect is lower on XBH and XS at high SRT (˃20 days), while it is more pronounced on XP and XI as their variations with SRT are linear.

ALLCAR19: Updated Data Using AUTO1, a Novel Fast-Off Rate CD19 CAR in Relapsed/Refractory B-Cell Acute Lymphoblastic Leukaemia and Other B-Cell Malignancies

Introduction: Prognosis for adult B-cell Acute Lymphoblastic Leukaemia (B-ALL) is poor and there is currently no licensed CD19 Chimeric Antigen Receptor (CAR) therapeutic. We developed a novel CD19 CAR (CAT-41BBz CAR) with a fast off-rate, designed to result in more physiological T-cell activation, reduce toxicity and improve engraftment. We describe updated data from the Phase I ALLCAR19 (NCT02935257) study of AUTO1 in relapsed/refractory adult B-ALL. Methods: Manufacturing: AUTO1 utilises non-mobilised autologous leukapheresate. The first 6 products were generated using a standard dynabead/WAVE bioreactor process and subsequent products using a semi-automated closed process. Study design: Patients aged >16y underwent lymphodepletion with fludarabine (30mg/m2 x3) and cyclophosphamide (60mg/kg x1) followed by split dose CAR T-cell infusion (Day 0: if ≥20% Bone Marrow (BM) blasts, infuse 10 x 106 CAR T-cells; if <20% BM blasts, 100 x 106 CAR T-cells. At Day+9: if no grade 3-5 Cytokine Release Syndrome (CRS)/immune effector cell associated neurotoxicity syndrome (ICANS), infuse Dose 2, to a total dose of 410 x106 CAR T-cells). Study endpoints include feasibility of manufacture, grade 3-5 toxicity and remission rates at 1 and 3 months. Results: As of 13 May 2020, 24 patients have been leukapheresed, 23 products manufactured and 19 patients received at least 1 dose of AUTO1. The median age was 43y (range 18-62), 26% had prior blinatumomab, 47% had prior inotuzumab ozogamicin and 63% had prior hematopoietic stem cell transplantation (HSCT). At the time of pre-conditioning, 42% had ≥50% BM blasts. No patients experienced ≥Grade 3 CRS (Lee criteria), 3/19 (16%) experienced Grade 3 ICANS that swiftly resolved with steroids. Of 19 infused patients, 16/19 (84%) achieved Minimal Residual Disease (MRD) negative complete response (CR). Currently 6 patients have died, none related to AUTO1. 11/19 (58%) patients remain on study and continue in MRD negative remission at a median follow up of 12.2 months (range 0.6-24.4m). To date, only 2 patients underwent HSCT whilst in remission. For all treated patients, the event-free survival (EFS) at 6 months was 62% and 76% for those whose products were manufactured using the closed process. Patients exhibited robust CAR expansion (mean peak CAR T levels 716,769 copies/µg DNA). Conclusions: AUTO1 has a tolerable safety profile in adult patients with r/r B-ALL despite high disease burden. Early data shows high remission rates with 84% achieving MRD negative CR. This preliminary data supports the further development of AUTO1 as a standalone treatment in patients with r/r B-ALL. Data from additional patients and longer follow up will be presented. Furthermore, data from extension cohorts of patients with low- and high- grade B-cell Non-Hodgkin's lymphoma and chronic lymphocytic leukaemia will be presented. Figure Disclosures Roddie: Celgene: Honoraria; Gilead: Honoraria; Novartis: Honoraria. O'Reilly:Gilead: Honoraria; Novartis: Honoraria, Other: Travel support. Hartley:ADC Therapeutics: Consultancy, Current equity holder in publicly-traded company, Research Funding. Linch:Autolus: Consultancy. Pule:UCLB: Patents & Royalties; Mana Therapeutics: Other: entitled to share of revenue from patents filed by UCL; Autolus: Current Employment, Other: owns stock in and receives royalties, Patents & Royalties. Peggs:Autolus: Consultancy.

Real municipal wastewater treatment using simultaneous pre and post-ozonation combined biological attached growth reactor: Energy consumption assessment

In this study, simultaneous pre and post-ozonation processes combined biological attached growth reactor were used in order to investigate the removal of COD, ammonium and total coliforms from real municipal wastewater. The biological process was carried out in a fixed bed aerobic bioreactor with an aeration rate of 6 L/min and hydraulic retention time of 5 h. The ozonation process was performed simultaneously in pre-ozonation and post-ozonation reactors, with 1 g/h ozone mass flow within 30 min every 8 h. The results showed that complete ammonium removal and 94 % COD removal were obtained. Also, total coliforms in the final effluent were measured to be about 0.51 MPN/mL. The analysis of energy consumption showed that the ozonation process was the dominant energy consumption source. The combination of fixed bed bioreactor and ozonation process showed a promising technology for the treatment of real municipal wastewater.

Metabolic understanding of disulfide reduction during monoclonal antibody production.

The disulfide reduction of intact monoclonal antibodies (mAbs) and subsequent formation of low molecular weight (LMW) species pose a direct risk to product stability, potency, and patient safety. Although enzymatic mechanisms of reduction are well established, an understanding of the cellular mechanisms during the bioreactor process leading to increased risk of disulfide reduction after harvest remains elusive. In this study, we examined bench, pilot, and manufacturing-scale batches of two mAbs expressed in Chinese hamster ovary (CHO) cells, where harvested cell culture fluid (HCCF) occasionally demonstrated disulfide reduction. Comparative proteomics highlighted a significant elevation in glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels in a highly reducing batch of HCCF, compared to a non-reducing batch. Analysis during production cell culture showed that increased GAPDH gene and protein expression correlated to disulfide reduction risk in HCCF in every case examined. Additionally, glucose 6-phosphate dehydrogenase (G6PD) activity and an increased (≥ 300%) lactate/pyruvate molar ratio (lac/pyr) during production cell culture correlated to disulfide reduction risk, suggesting a metabolic shift to the pentose phosphate pathway (PPP). In all, these results suggest that metabolic alterations during cell culture lead to changes in protein expression and enzyme activity that in turn increase the risk of disulfide reduction in HCCF. KEY POINTS: • Bioreactor conditions resulted in reduction susceptible harvest material. • GAPDH expression, G6PD activity, and lac/pyr ratio correlated with mAb reduction. • Demonstrated role for cell metabolic changes in post-harvest mAb reduction. Graphical abstract.

Performance evaluation of conventional membrane bioreactor and moving bed membrane bioreactor for synthetic textile wastewater treatment

In this study, conventional membrane bioreactor (MBR) and moving bed-membrane bioreactor (MB-MBR) processes were compared in synthetic textile wastewater treatment. For this purpose, the bioreactors were operated as a conventional MBR, an MB-MBR with a biocarrier filling ratio of 20 % and an MB-MBR with a biocarrier filling ratio of 10 %, respectively. In the conventional MBR operation, 93.1 % chemical oxygen demand (COD) and 87.1 % color (Reactive Red 390) removal efficiencies were obtained. In both MB-MBR operations, almost equal COD and color removal efficiencies were found as 98.5 % and 89.5 %, respectively. Moreover, offline physical and chemical membrane cleaning processes were applied every other day and every 15 days throughout the conventional MBR operation, respectively, while no physical or chemical membrane cleaning was required during both MB-MBR operations. Furthermore, lower polysaccharide concentrations of extracellular polymeric substances (EPS) and floc sizes of sludge and higher zeta potential of sludge were determined in MB-MBR. Considering the obtained results, it may be stated that the MB-MBR process is an attractive treatment technology for reducing membrane fouling propensity for treatment of textile wastewater.

Glutathione-S-transferase Fusion Protein Nanosensor.

Fusion protein tags are widely used to capture and track proteins in research and industrial bioreactor processes. Quantifying fusion-tagged proteins normally requires several purification steps coupled with classical protein assays. Here, we developed a broadly applicable nanosensor platform that quantifies glutathione-S-transferase (GST) fusion proteins in real-time. We synthesized a glutathione-DNA-carbon nanotube system to investigate glutathione-GST interactions via semiconducting single-walled carbon nanotube (SWCNT) photoluminescence. We found that SWCNT fluorescence wavelength and intensity modulation occurred specifically in response to GST and GST-fusions. The sensor response was dependent on SWCNT structure, wherein mod(n - m, 3) = 1 nanotube wavelength and intensity responses correlated with nanotube diameter distinctly from mod(n - m, 3) = 2 SWCNT responses. We also found broad functionality of this sensor to diverse GST-tagged proteins. This work comprises the first label-free optical sensor for GST and has implications for the assessment of protein expression in situ, including in imaging and industrial bioreactor settings.

Potentiality of municipal sludge for biological gas production at Soba Station South of Khartoum (Sudan)

Potentiality of municipal sludge for biological gas production at Soba Station South of Khartoum (Sudan)

Simplified bioreactor processes for recombinant butyrylcholinesterase production in transgenic rice cell suspension cultures

Metabolically regulated transgenic rice cell suspension culture has been developed aiming to be an efficient and cost-effective platform for production of rice-recombinant butyrylcholinesterase (rrBChE) that could be used as a bioscavenger enzyme against organophosphate nerve agents. In two-stage batch cultures, switching between growth and induction phases by exchanging media, the maximum active rrBChE levels were comparable among the bioreactor runs with controlled dissolved oxygen (DO) concentrations from 10–40% DO and without controlled DO at constant aeration rate. For a single-stage batch operation with no media exchange at 40% DO, production of rrBChE is triggered simply through sugar depletion due to the uptake of sugar by the cells, showing similar maximum active cell-associated rrBChE level compared to two-stage batch operations. However, the bioreactor process using uncontrolled DO, no media exchange, and half-concentration of sucrose resulted in a 1.4-fold increase in the maximum total active rrBChE (77 μg/g fresh weight) and 1.6-fold increase of total active rrBChE specific productivity (86 μg (g dry weight)−1 day−1) compared to the two-stage batch cultures. We found that monitoring the culture pH is a simple non-invasive technique that can help identify the start and progress of the induction phase in a single-stage culture. Altogether, we demonstrate a simplified, efficient, economical, and scalable bioreactor process for rrBChE production in metabolically regulated transgenic rice cell cultures.

Detachment mechanism and energy consumption model for the ex-situ rinsing process in membrane bioreactors

In this paper, underlying mechanism of the detachment of foulant particles from the planar surface was analyzed. Meanwhile a new mathematical model was proposed, from an energy viewpoint, to study the variation of energy consumption in the rinsing process. The surface properties of the effluent suspensions at various rinsing time were systematically measured and the interaction energy between different foulant layers were analyzed in detail. The results showed that the model simulation could predict reasonably the energy consumption in the rinsing process under different conditions (R2 > 0.97). Moreover, the reliability of the model was evaluated by model parameters and model constraints. Additionally, the wide adaptability of the proposed model was verified by considering the planar surface was random rough (R2 > 0.97) and the situation of real wastewater as feed suspension (R2 > 0.98). Furthermore, the actual energy consumption for foulant particles breaking away from the planar surface was also analyzed. This paper would provide a novel approach for comprehensive understanding the ex-situ rinsing process in membrane bioreactors.

Development of an integrated aerobic granular sludge MBR and reverse osmosis process for municipal wastewater reclamation

The reclamation of municipal wastewater to obtain high-grade product water is a growing need due to the pressing global water shortage. However, the existing municipal wastewater treatment plants (WWTPs) with the conventional activated sludge process as a core is not a sustainable engineering solution towards future water sustainability. To tackle such an emerging water-wastewater nexus, a ferrous-assisted aerobic granular sludge membrane bioreactor and reverse osmosis (AGSMBR-RO) process was developed for municipal wastewater reclamation. Results show that about 99.9%, 99.7% and nearly 100% of dissolved organic carbon (DOC), ammonium-N and total phosphorus (TP), respectively, could be removed in the ferrous-assisted AGSMBR-RO process, while the product water could meet the typical NEWater quality of Singapore with respect to the parameters analysed in this study. Moreover, it was found that an addition of 6 mg/L of ferrous could improve the stability of aerobic granular sludge (AGS) through the coagulation and flocculation of suspended flocs as well as phosphorus removal. These in turn led to reduced membrane fouling in both AGSMBR and RO units. Consequently, the proposed process is a promising alternative for municipal wastewater reclamation.