Monitoring Protein-Ligand Interactions in Human Cells by Real-Time Quantitative In-Cell NMR using a High Cell Density Bioreactor.

In-cell NMR is a unique approach to observe the structural and dynamic properties of biological macromolecules at atomic resolution directly in living cells. Protein folding, chemical modifications, and conformational changes induced by ligand binding can be observed. Therefore, this method has great potential in the context of drug development. However, the short lifetime of human cells confined in the NMR spectrometer limits the application range of in-cell NMR. To overcome this issue, NMR bioreactors are employed that can greatly improve the cell sample stability over time and, importantly, enable the real-time recording of in-cell NMR spectra. In this way, the evolution of processes such as ligand penetration and binding to the intracellular protein target can be monitored in real time. Bioreactors are often limited by low cell viability at high cell numbers, which results in a trade-off between the overall sensitivity of the experiment and cell viability. We recently reported an NMR bioreactor that maintains a high number of human cells metabolically active for extended periods of time, up to 72 h. This setup was applied to monitor protein-ligand interactions and protein chemical modification. We also introduced a workflow for quantitative analysis of the real-time NMR data, based on multivariate curve resolution. The method provides concentration profiles of the chemical species present in the cells as a function of time, which can be further analyzed to obtain relevant kinetic parameters. Here we provide a detailed description of the NMR bioreactor setup and its application to monitoring protein-ligand interactions in human cells.

Hydrofoam and oxygen headspace bioreactors improve cell-free therapeutic protein production yields through enhanced oxygen transport.

Protein therapeutics are powerful tools in the fight against diabetes, cancers, growth disorders, and many other debilitating diseases. However, availability is limited due to cost and complications of production from living organisms. To make life-saving protein therapeutics more available to the world, the possibility of magistral or point-of-care protein therapeutic production has gained focus. The recent invention and optimization of lyophilized "cell-free" protein synthesis reagents and its demonstrated ability to produce highly active versions of FDA-approved cancer therapeutics have increased its potential for low-cost, single-batch, magistral medicine. Here we present for the first time the concept of increased oxygen mass transfer in small-batch, cell-free protein synthesis (CFPS) reactions through air-water foams. These "hydrofoam" reactions increased CFPS yields by up to 100%. Contrary to traditional protein synthesis using living organisms, where foam bubbles cause cell-lysis and production losses, hydrofoam CFPS reactions are "cell-free" and better tolerate foaming. Simulation and experimental results suggest that oxygen transfer is limiting in even small volume batch CFPS reactors and that the hydrofoam format improved oxygen transfer. This is further supported by CFPS reactions achieving higher yields when oxygen gas replaces air in the headspace of batch reactions. Improving CFPS yields with hydrofoam reduces the overall cost of biotherapeutic production, increasing availability to the developing world. Beyond protein therapeutic production, hydrofoam CFPS could also be used to enhance other CFPS applications including biosensing, biomanufacturing, and biocatalysis.

Modeling tangential flow filtration using reverse asymmetric membranes for bioreactor harvesting.

Tangential flow filtration (TFF) has many advantages for bioreactor harvesting, as the permeate could be introduced directly to the subsequent capture step. However, membrane fouling has limited its widespread use. This is particularly problematic given the high cell densities encountered today. Here, a reverse asymmetric membrane, where the more open surface faces the feed stream and the tighter barrier layer faces the permeate stream, has been investigated. The open surface contains pores up to 40 µm in diameter while the tighter barrier layer has an average pore size of 0.4 µm. Filtration of yeast suspensions has been conducted under a range of conditions. The yeast cells are trapped in the open pore structure. The membrane stabilizes an internal porous cake that acts like a depth filter. This stabilized cake layer can remove particulate matter that would foul the barrier layer if it faced the feed stream. As filtration continues, a surface cake layer forms on the membrane surface. A resistance in series model has been developed to describe the permeate flux during TFF. The model contains three fitted parameters which can easily be determined from constant pressure normal flow filtration experiments and total recycle constant flux TFF experiments. The model can be used to estimate the capacity of the filter for a given feed stream. Our results suggest that using a reverse asymmetric membrane could avoid severe flux decline associated with fouling of the barrier layer during bioreactor harvesting.

Spectroscopy integration to miniature bioreactors and large scale production bioreactors-Increasing current capabilities and model transfer.

Spectroscopy techniques are being implemented within the biopharmaceutical industry due to their non-destructive ability to measure multiple analytes simultaneously, however, minimal work has been applied focussing on their application at small scale. Miniature bioreactor systems are being applied across the industry for cell line development as they offer a high-throughput solution for screening and process optimization. The application of small volume, high-throughput, automated analyses to miniature bioreactors has the potential to significantly augment the type and quality of data from these systems and enhance alignment with large-scale bioreactors. Here, we present an evaluation of 1. a prototype that fully integrates spectroscopy to a miniature bioreactor system (ambr®15, Sartorius Stedim Biotech) enabling automated Raman spectra acquisition, 2. In 50 L single-use bioreactor bag (SUB) prototype with an integrated spectral window. OPLS models were developed demonstrating good accuracy for multiple analytes at both scales. Furthermore, the 50 L SUB prototype enabled on-line monitoring without the need for sterilization of the probe prior to use and minimal light interference was observed. We also demonstrate the ability to build robust models due to induced changes that are hard and costly to perform at large scale and the potential of transferring these models across the scales. The implementation of this technology enables integration of spectroscopy at the small scale for better process understanding and generation of robust models over a large design space while facilitating model transfer throughout the scales enabling continuity throughout process development and utilization and transfer of ever-increasing data generation from development to manufacturing.

Suspension Culture of Human Induced Pluripotent Stem Cells in Single-Use Vertical-Wheel™ Bioreactors Using Aggregate and Microcarrier Culture Systems.

Human induced pluripotent stem cells (hiPSCs) have the potential to be used in a variety of biomedical applications, including drug discovery and Regenerative Medicine. The success of these approaches is, however, limited by the difficulty of generating the large quantities of cells required in a reproducible and controlled system. Bioreactors, widely used for industrial manufacture of biological products, constitute a viable strategy for large-scale production of stem cell derivatives. In this chapter, we describe the expansion of hiPSCs using the Vertical-Wheel™ bioreactor, a novel bioreactor configuration specifically designed for the culture of shear-sensitive cells. We provide protocols for the expansion of hiPSCs in suspension, both as floating aggregates and using microcarriers for cell adhesion. These methods may be important for the establishment of a scalable culture of hiPSCs, allowing the manufacturing of industrial- or clinical-scale cell numbers.

Model-Based Optimization of a Fed-Batch Bioreactor for mAb Production Using a Hybridoma Cell Culture.

Production of monoclonal antibodies (mAbs) is a well-known method used to synthesize a large number of identical antibodies, which are molecules of huge importance in medicine. Due to such reasons, intense efforts have been invested to maximize the mAbs production in bioreactors with hybridoma cell cultures. However, the optimal control of such sensitive bioreactors is an engineering problem difficult to solve due to the large number of state-variables with highly nonlinear dynamics, which often translates into a non-convex optimization problem that involves a significant number of decision (control) variables. Based on an adequate kinetic model adopted from the literature, this paper focuses on developing an in-silico (model-based, offline) numerical analysis of a fed-batch bioreactor (FBR) with an immobilized hybridoma culture to determine its optimal feeding policy by considering a small number of control variables, thus ensuring maximization of mAbs production. The obtained time stepwise optimal feeding policies of FBR were proven to obtain better performances than those of simple batch operation (BR) for all the verified alternatives in terms of raw material consumption and mAbs productivity. Several elements of novelty (i-iv) are pointed out in the "conclusions" section (e.g., considering the continuously added biomass as a control variable during FBR).

Bioprocess development for scalable production of cultivated meat.

Traditional farm-based products based on livestock are one of the main contributors to greenhouse gas emissions. Cultivated meat is an alternative that mimics animal meat, being produced in a bioreactor under controlled conditions rather than through the slaughtering of animals. The first step in the production of cultivated meat is the generation of sufficient reserves of starting cells. In this study, bovine adipose-derived stem cells (bASCs) were used as starting cells due to their ability to differentiate towards both fat and muscle, two cell types found in meat. A bioprocess for the expansion of these cells on microcarriers in spinner flasks was developed. Different cell seeding densities (1,500, 3,000, and 6,000 cells/cm2 ) and feeding strategies (80%, 65%, 50%, and combined 80%/50% medium exchanges) were investigated. Cell characterization was assessed pre- and postbioprocessing to ensure that bioprocessing did not negatively affect bASC quality. The best growth was obtained with the lowest cell seeding density (1,500 cells/cm2 ) with an 80% medium exchange performed (p < .0001) which yielded a 28-fold expansion. The ability to differentiate towards adipogenic, osteogenic, and chondrogenic lineages was retained postbioprocessing and no significant difference (p > .5) was found in clonogenicity pre- or postbioprocessing in any of the feeding regimes tested.

Bioprocess considerations for T-cell therapy: Investigating the impact of agitation, dissolved oxygen, and pH on T-cell expansion and differentiation.

Adoptive T-cell therapy (ACT) has emerged as a promising new way to treat systemic cancers such as acute lymphoblastic leukemia. However, the robustness and reproducibility of the manufacturing process remains a challenge. Here, a single-use 24-well microbioreactor (micro-Matrix) was assessed for its use as a high-throughput screening tool to investigate the effect and the interaction of different shaking speeds, dissolved oxygen (DO), and pH levels on the growth and differentiation of primary T cells in a perfusion-mimic process. The full factorial design allowed for the generation of predictive models, which were used to find optimal culture conditions. Agitation was shown to play a fundamental role in the proliferation of T cells. A shaking speed of 200 rpm drastically improved the final viable cell concentration (VCC), while the viability was maintained above 90% throughout the cultivation. VCCs reached a maximum of 9.22 × 106 cells/ml. The distribution of CD8+ central memory T cells (TCM ), was found to be largely unaffected by the shaking speed. A clear interaction between pH and DO (p < .001) was established for the cell growth and the optimal culture conditions were identified for a combination of 200 rpm, 25% DO, and pH of 7.4. The combination of microbioreactor technology and Design of Experiment methodology provides a powerful tool to rapidly gain an understanding of the design space of the T-cell manufacturing process.

[Development and optimization of perfusion process for mammalian cell culture].

In recent years, the demand of biologics has increased rapidly. Cell culture process with perfusion mode has become more and more popular due to its high productivity, good quality and high efficiency. In this paper, the unique operation and the details of process optimization for perfusion culture mode are discussed by comparing with traditional batch culture process. Meanwhile, the progress and strategies in the development and optimization of perfusion culture process in recent years are summarized to provide reference for the future development of mammalian cell perfusion culture technology.

[Numerical simulation and optimization of impeller combination used in stirred bioreactor].

Bioreactors have been central in monoclonal antibodies and vaccines manufacturing by mammalian cells in suspension culture. Numerical simulation of five impeller combinations in a stirred bioreactor was conducted, and characteristics of velocity vectors, distributions of gas hold-up, distributions of shear rate in the bioreactor using 5 impeller combinations were numerically elucidated. In addition, genetically engineered CHO cells were cultivated in bioreactor installed with 5 different impeller combinations in fed-batch culture mode. The cell growth and antibody level were directly related to the maximum shear rate in the bioreactor, and the highest viable cell density and the peak antibody level were achieved in FBMI3 impeller combination, indicating that CHO cells are sensitive to shear force produced by impeller movement when cells were cultivated in bioreactor at large scale, and the maximum shear rate would play key roles in scaling-up of bioreactor at industrial scale.

Technoeconomic analysis of semicontinuous bioreactor production of biopharmaceuticals in transgenic rice cell suspension cultures.

Biopharmaceutical protein production using transgenic plant cell bioreactor processes offers advantages over microbial and mammalian cell culture platforms in its ability to produce complex biologics with simple chemically defined media and reduced biosafety concerns. A disadvantage of plant cells from a traditional batch bioprocessing perspective is their slow growth rate which has motivated us to develop semicontinuous and/or perfusion processes. Although the economic benefits of plant cell culture bioprocesses are often mentioned in the literature, to our knowledge no rigorous technoeconomic models or analyses have been published. Here we present technoeconomic models in SuperPro Designer® for the large-scale production of recombinant butyrylcholinesterase (BChE), a prophylactic/therapeutic bioscavenger against organophosphate nerve agent poisoning, in inducible transgenic rice cell suspension cultures. The base facility designed to produce 25 kg BChE per year utilizing two-stage semicontinuous bioreactor operation manufactures a single 400 mg dose of BChE for $263. Semicontinuous operation scenarios result in 4-11% reduction over traditional two-stage batch operation scenarios. In addition to providing a simulation tool that will be useful to the plant-made pharmaceutical community, the model also provides a computational framework that can be used for other semicontinuous or batch bioreactor-based processes.

Process Optimization using High Throughput Automated Micro-Bioreactors in Chinese Hamster Ovary Cell Cultivation.

Optimization of bioprocesses to increase the yield of desired products is of importance in the biopharmaceutical industry. This can be achieved by strain selection and by developing bioprocess parameters. Shake flasks have been used for this purpose. They, however, lack the capability to control the process parameters such as pH and dissolved oxygen (DO). This limitation can be overcome with the help of an automated micro-bioreactor. These bioreactors mimic cultivation at a larger scale. One of the major advantages of this system is the integration of the Design of Experiment (DOE) in the software. This integration enables establishing a design where multiple process parameters can be varied simultaneously. The critical process parameters and optimum bioprocess conditions can be analyzed within the software. The focus of the work presented here is to introduce the user to the steps involved in process design in the software and incorporation of the DOE within the cultivation run.

Suitability of SBR for Wastewater Treatment and Reuse: Pilot-Scale Reactor Operated in Different Anoxic Conditions.

The present study investigates the performance of a pilot-scale Sequencing Batch Reactor (SBR) process for the treatment of wastewater quality parameters, including turbidity, total suspended solids (TSS), total solids (TS), nitrogen (ammonia (NH3-N), nitrite (NO2-), and nitrate (NO3-), phosphate (PO43-), the chemical oxygen demand (COD), and the 5-day biological oxygen demand (BOD5), from municipal wastewater. Two scenarios, namely, pre-anoxic denitrification and post-anoxic denitrification, were investigated to examine the performance of a pilot-scale SBR on the wastewater quality parameters, particularly the nitrogen removal. The correlation statistic was applied to explain the effects of operational parameters on the performance of the SBR system. The results revealed that the post-anoxic denitrification scenario was more efficient for higher qualify effluent than the first scenario. The effluent concentrations of the targeted wastewater quality parameters obtained for the proposed SBR system were below those of the local standards, while its performance was better than that of the North Sewage Treatment Plant, Dharan, Eastern province, Kingdom of Saudi Arabia (KSA), in terms of the BOD5, COD, TN, and PO43- treatment efficiencies. These results indicated the suitability of SBR technology for wastewater treatment in remote areas in the KSA, with a high potential of reusability for sustainable wastewater management.

Improved purified terephthalic acid wastewater treatment using combined UAFB-SBR system: At mesophilic and ambient temperature.

In this study, a combined UAFB-SBR process was introduced to improve the treatment efficiency of PTA wastewater. The techno-economic feasibility of the process was evaluated in terms of organic removal efficiencies under mesophilic (37 °C) and ambient temperature (15-25 °C) during the long-term run. The lab-scale study revealed that all organic compounds present in the PTA wastewater could be efficiently removed under both mesophilic and ambient temperature, and p-toluic acid is probably the critical pollutant regulating the overall process performance in anaerobic stage, which should be seriously considered. The Miseq Sequencing results suggested that, along with the system temperature variation from mesophilic to ambient temperature, greater effects on bacterial community than archaeal community were detected in the UAFB reactor, while only slight variations were observed in the SBR reactor. Further taxonomy analysis demonstrated that within the UAFB reactor, the syntrophic partnership of Syntrophorhabdus, Syntrophus and Desulfovibrio with hydrogenotrophic methanogens were the main impetus for aromatic organics reduction. In the meanwhile, the intensively identified Thauera and Azoarcus groups were speculated of important roles in the aerobic degradation of aromatic compounds.

Microaerated UASB reactor treating textile wastewater: The core microbiome and removal of azo dye Direct Black 22.

Sequential anaerobic and aerobic processes have been recommended to treat textile wastewater reliably. In this work, the focus was on finding an energetically more competitive system to remove tetra-azo dye Direct Black 22 (DB22). We operated two upflow anaerobic sludge blanket (UASB) reactors (R1 and R2) in three phases (PI, PII, and PIII). R1 was operated as a conventional UASB, while R2 was microaerated in the upper part (0.18 ±â€¯0.05 mg O2. L-1), aiming to remove DB22 simultaneously with the aromatic amine byproducts. PI consisted of feeding reactors with synthetic textile wastewater (STW), PII had higher salinity in the STW, and PIII was the same as PII, plus sulfate. The results showed that color and COD removal efficiencies were similar for both reactors (67-72% for R1 and 59-78% for R2) without a substantial influence of oxygen in R2. However, microaeration played a crucial role in R2 by removing the anaerobically formed aromatic amines; during PIII, the effluent was 16 times less toxic than that of R1. The microbial community that developed in the sludge bed of both reactors was quite similar, with the core microbiome represented by Trichococcus, Syntrophus and Methanosaeta genera. The increase in salinity in PII and PIII promoted a shift in the microbial community, excluding salty-sensitive genera from the core microbiome. The putative genera Brevundimonas and Ornatilinea were associated to aromatic amine microaerobic removal. Therefore, there is a potential application of a compact microaerated anaerobic system for textile wastewater treatment.

CAR-T Cell Expansion in a Xuri Cell Expansion System W25.

Cell expansion is typically a long and labor-intensive step in CAR-T cell manufacture. The Xuri Cell Expansion System (CES) W25 semiautomates this step while functionally closing the process. Cells for autologous or allogeneic cell therapies are cultured inside a single-use Xuri Cellbag™ bioreactor. Wave-induced agitation, performed by a rocking Base Unit, transfers gas and mixes the culture. The integral UNICORN™ software allows customization of culture conditions and media perfusion schedules. Culture volumes can range from 300 mL to 25 L, making the Xuri CES W25 system suitable for both scale-up and scale-out manufacturing processes. CAR-T cell therapies have been successfully generated using the Xuri CES W25 system, which reduces manual labor compared with static culturing methods. This chapter details how to initiate a culture, install the Xuri CES W25, and install a 2 L Cellbag bioreactor. Protocols on inoculation, monitoring, and sampling are also outlined in this chapter.

Ammonia, thiocyanate, and cyanate removal in an aerobic up-flow submerged attached growth reactor treating gold mine wastewater.

The objective of the study was to investigate the nitrification process, as well as the bio-chemical removal of cyanate and thiocyanate, while treating gold mining wastewater using an aerobic up-flow SAGR. A total of six SAGRs, each packed with locally sourced pea gravel (estimated specific surface area of 297 m-2 m-3), were operated at various HRTs and tested on both low- and high-strength gold mining wastewaters. The two sets of three SAGRs were operated at HRTs of 0.45 days, 1.20 days, and 2.40 days. Nitrification was successfully achieved in all six SAGRs regardless of the wastewater strength or HRT examined. The steady-state, 20 °C surface area loading rate was determined to be 1.2 g-TAN m-2 d-1 in order to comply with an effluent discharge limit at 10 mg-TAN L-1 (i.e., with the wastewater sources examined). At all ammonia loading rates, thiocyanate was successfully removed, and residual concentrations were below 2 mg-SCN-N L-1. Cyanate appeared to be hydrolyzed and subsequently nitrified. Acute toxicity tests conducted on both daphnia and trout revealed the effluent to be safe for direct discharge.

Salinity and low temperature effects on the performance of column biochemical reactors for the treatment of acidic and neutral mine drainage.

Passive biochemical reactors (PBRs) represent a promising option for the treatment of mine drainage. In this study, the influence of temperature (22 and 5 °C), salinity (0 and 20 g/L) and hydraulic retention time (HRT) on the efficiency of PBRs for the treatment of acidic and neutral mine drainage (AMD and NMD) was evaluated. To do so, eight 11 L PBRs were set-up and operated with vertically upward flow. Synthetic AMD and NMD, with two salinities (0 and 20 g/L), were tested at ambient temperature (22 ±â€¯0.5 °C) during the first 3 months, then at low temperature (5 ±â€¯1 °C), for 5 additional months. The HRT tested was 0.5 and 1 day, for NMD, and 2.5 and 5 days, for AMD. Results showed a consistent efficiency, above 65%, with higher HRTs (1 vs. 0.5 day for NMD and 5 vs. 2.5 for AMD). At room temperature, metals and sulfate removal was better for non-saline synthetic effluents (>99% vs 95% for Cu, 99% vs >74% for Ni, 90% vs 75% for Fe, and <99% vs <96% for SO42-), after 3 months. At 5 °C, removal efficiency decreased especially for Ni, from 99% to 74%, for both mine drainage qualities. However, sulfate removal was found to be better in saline AMD (<40% vs <10%). The simultaneous effect of low temperature and high salinity further decreased PBR performance. Although higher HRTs entailed better removal efficiency, hydraulic problems such as decreases in permeability of the reactive mixture may still lead to inhibition of long-term PBR efficiency.

Performance enhancement by adding ferrous to a combined modified University of Cape Town and post-anoxic/aerobic-membrane bioreactor.

The removal of nutrients in a combined modified University of Cape Town and post-anoxic/aerobic-membrane bioreactor (UCT-A/MBR) was investigated. Denitrifying phosphorus removal (DPR) and nitrate-dependent anaerobic ferrous oxidation (NAFO) were applied to enhance the nutrient removal performances. The results showed that NAFO with the addition of Fe(II) and DPR could promote nitrogen and phosphorus removal. The total nitrogen removal efficiency gradually increased from 71.05 ±â€¯2.00% to 73.84 ±â€¯1.74% and 75.70 ±â€¯1.47% with no Fe(II) addition, addition to the post-anoxic tank, and addition to the anoxic tank, and the total phosphorus removal efficiency increased from 89.37 ±â€¯1.91% to 95.21 ±â€¯0.85% and 96.01 ±â€¯1.10%, respectively. Gene sequencing was conducted, and Saprospiraceae was determined to be the dominant DPR-related bacteria, with its abundance increasing from 16.31% to 22.45% after Fe(II) addition. Additionally, the proportion of the NAFO-related bacteria Azospira increased from 0.58% to 1.91% after Fe(II) addition. The microbial succession caused by the addition of Fe(II) may have resulted in the enhanced removal performance.

Streamlined production of genetically modified T cells with activation, transduction and expansion in closed-system G-Rex bioreactors.

BACKGROUND: Gas Permeable Rapid Expansion (G-Rex) bioreactors have been shown to efficiently expand immune cells intended for therapeutic use, but do not address the complexity of the viral transduction step required for many engineered T-cell products. Here we demonstrate a novel method for transduction of activated T cells with Vectofusin-1 reagent. Transduction is accomplished in suspension, in G-Rex bioreactors. The simplified transduction step is integrated into a streamlined process that uses a single bioreactor with limited operator intervention. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were thawed, washed and activated with soluble anti-CD3 and anti-CD28 antibodies either in cell culture bags or in G-Rex bioreactors. Cells were cultured in TexMACS GMP medium with interleukin (IL)-7 and IL-15 and transduced with RetroNectin in bags or Vectorfusin-1 in the G-Rex. Total viable cell number, fold expansion, viability, transduction efficiency, phenotype and function were compared between the two processes. RESULTS: The simplified process uses a single vessel from activation through harvest and achieves 56% transduction with 29-fold expansion in 11 days. The cells generated in the simplified process do not differ from cells produced in the conventional bag-based process functionally or phenotypically. DISCUSSION: This study demonstrates that T cells can be transduced in suspension. Further, the conventional method of generating engineered T cells in bags for clinical use can be streamlined to a much simpler, less-expensive process without compromising the quality or function of the cell product.