Retroviral gene delivery is widely used in T cell therapies for hematological cancers. However, viral vectors are expensive to manufacture, they integrate genes in semi-random patterns, and their transduction efficiency is highly variable. In this study, several non-viral gene delivery vehicles, promoters, and additional variables were compared to optimize non-viral transgene delivery and expression in both Jurkat and primary T cells. Overall, transfecting Jurkat cells in X-VIVOTM 15 media with Lipofectamine LTX provided a high transfection efficiency (63.0±10.9% EGFP+). However, the same method yielded a much lower transfection efficiency in primary T cells (8.1±0.8% EGFP+). Subsequent confocal microscopy revealed that a majority of the lipoplexes did not enter the primary T cells, which might be due to relatively low expression levels of heparan sulfate proteoglycans (HSPGs) detected via mRNA-sequencing. PYHIN DNA sensors (e.g., AIM2, IFI16) were also expressed at high levels in Primary T cells, which can induce apoptosis when bound to cytoplasmic DNA. Therefore, transfection of primary T cells appears to be limited at the level of cellular uptake and/or DNA sensing in the cytoplasm, so both of these factors should be considered in the development of future viral and non-viral T cell gene delivery methods.
The mechanical properties of biofilms can be used to predict biofilm deformation, for example under fluid flow. We used magnetic tweezers to spatially map the compliance of Pseudomonas aeruginosa biofilms at the micron scale, then used modeling to assess its effects on biofilm deformation. Biofilms were grown in capillary flow cells with Reynolds numbers (Re) ranging 0.28 to 13.9, bulk dissolved oxygen (DO) concentrations from 1 mg/L to 8 mg/L, and bulk calcium ion (Ca2+) concentrations of 0 and 100 mg CaCl2/L. Higher Re numbers resulted in more uniform biofilm morphologies. The biofilm was stiffer at the center of the flow cell than near the walls. Lower bulk DO led to more stratified biofilms. Higher Ca2+ led to increased stiffness and more uniform mechanical properties. Using the experimental mechanical properties, fluid-structure interaction models predicted up to 64% greater deformations for heterogeneous biofilms, compared to a homogeneous biofilms with the same average properties. However, the error depended on the biofilm morphology and flow regime. Our results show significant spatial mechanical variability exists at the micron scale, and that this variability can potentially affect biofilm deformation. The average mechanical properties, provided in many studies, should be used with caution when predicting biofilm deformation.
The heavy metals pollution represents one of the important issues in the environmental field since they are involved in many pathologies from cancer, neurodegenerative and metabolic diseases. We propose an innovative portable biosensor for the determination of traces of trivalent Arsenic (AsIII) and bivalent mercury (HgII) in water. The system implements a strategy combining two advanced sensing modules consisting in (a) a whole cell based on engineered Escherichia coli as selective sensing element towards the metals and (b) an electrochemical miniaturised silicon device with three microelectrodes and a portable reading system. The sensing mechanism relies on the selective recognition from the bacterium of given metals producing the 4-aminophenol (PAP) redox active mediator detected through a cyclic voltammetry analysis. The miniaturized biosensor is able to operate a portable, robust and high-sensitivity detection of AsIII with a sensitivity of 0.122 µA ppb-1, LoD of 1.5 ppb and a LoQ of 5 ppb. The LoD value is one order of magnitude below of the value indicated to WHO to be dangerous (10 μg/L). The system was proved to be fully versatile being effective in the detection of Hg(II) as well. A first study on Hg(II) showed sensitivity value of 2.11 µA/ppb a LOD value of 0.1 ppb and LoQ value of 0.34 ppb. Also in this case, the detected LOD was ten time lower than that indicated by WHO (1 ppb). These results pave the way for advanced sensing strategies suitable for the environmental monitoring and the public safety.
Integrated continuous downstream processes with process analytical technology offer a promising opportunity to reduce production costs and increase process flexibility and adaptability. In this case study, an integrated continuous process was used to purify a recombinant protein on laboratory scale in a two-system setup that can be used as a general downstream setup offering multi-product and multi-purpose manufacturing capabilities. The process consisted of continuous solvent/detergent virus inactivation followed by periodic countercurrent chromatography in the capture step, and a final chromatographic polishing step. A real-time controller was implemented to ensure stable operation by adapting the downstream process to external changes. A concentration disturbance was introduced to test the controller. After the disturbance was applied, the product output recovered within 6 hours, showing the effectiveness of the controller. In a comparison of the process with and without the controller, the product output per cycle increased by 27%, the resin utilization increased from 71.4% to 87.9%, and the specific buffer consumption was decreased by 21% with the controller, while maintaining a similar yield and purity as in the process without the disturbance. In addition, the integrated continuous process outperformed the batch process, increasing the productivity by 95% and the yield by 28%.
Alcohol dehydrogenases (ADHs) play key roles in the production of various chemical precursors that are essential in pharmaceutical and fine chemical industries. To achieve practical application of ADHs in industrial processes, tailoring enzyme properties through rational design or directed evolution is often required. Here, we developed a secretion-based dual fluorescence assay (SDFA) for high-throughput screening of ADHs. In SDFA, an ADH of interest is fused to a mutated superfolder green fluorescence protein (MsfGFP), which could result in secretion of the fusion protein to culture broth. After a simple centrifugation step to remove the cells, the supernatant can be directly used to measure activity of the ADH based on a red fluorescence signal, whose increase is coupled to formation of NADH (a redox co-factor of ADHs) in the reaction. SDFA allows easy quantification of ADH concentration based on the green fluorescence signal of MsfGFP. This feature is useful in determining specific activity and may improve screening accuracy. Out of five ADHs we have tested with SDFA, four ADHs can be secreted and characterized. We successfully screened a combinatorial library of an ADH from Pichia finlandica and identified a variant with a 197-fold higher kcat/km value toward (S)-2-octanol compared to its wild-type.
Biofouling represents an important limitation in photobioreactor cultures. The biofouling propensity of different materials (polystyrene, borosilicate glass, polymethyl methacrylate and polyethylene terephthalate glycol-modified) and coatings (two spray-applied and nanoparticle-based superhydrophobic coatings and a hydrogel-based fouling release coating) was evaluated by means of a short-term protein test, using bovine serum albumin (BSA) as a model protein, and by the long-term culture of the marine microalga Nannochloropsis gaditana under practical conditions. The results from both methods were similar, confirming that the BSA test predicts microalgal biofouling on surfaces exposed to microalgae cultures; these secrete macromolecules, such as proteins, that have a high capacity for forming a conditioning film prior to cell adhesion. The hydrogel-based coating showed significantly reduced BSA and N. gaditana adhesion, whereas the other surfaces failed to control biofouling. Microalgal biofouling was associated with an increased concentration of sticky extracellular proteins at low N/P ratios (below 15).
Producing recombinant proteins in transgenic plant cell suspension cultures in bioreactors provides controllability, reproducibility, scalability, and low-cost production, although low yields remain the major challenge. The studies on scaling-up to pilot-scale bioreactors, especially in conventional stainless-steel stirred tank bioreactors (STB), to produce recombinant proteins in plant cell suspension cultures are very limited. In this study, we scaled-up the production of rice recombinant butyrylcholinesterase (rrBChE), a complex hydrolase enzyme that can be used to prophylactically and therapeutically treat against organophosphorus nerve agents and pesticide exposure, from metabolically-regulated transgenic rice cell suspension cultures in a 40-L pilot-scale STB. Employing cyclical operation together with a simplified-process operation (controlling gas sparging rate rather than dissolved oxygen and allowing natural sugar depletion) identified in lab-scale (5-L) bioreactor studies, we found consistent maximum total active rrBChE production level of 46-58 µg/g fresh weight in four cycles over 82 days of continuous operation. Additionally, maintaining the overall volumetric oxygen mass transfer coefficient (kLa) in the pilot-scale STB to be equivalent to the lab-scale STB improves the maximum total active rrBChE production level and the maximum volumetric productivity to 85 µg/g fresh weight and 387 µg L-1 day-1, respectively, which are comparable to the lab-scale culture. Here, we demonstrate pilot scale bioreactor performance using a metabolically-regulated transgenic rice cell culture for long-term, reproducible, and sustained production of rrBChE.
Mixed-culture fermentation provides a means to recycle carbon from complex organic waste streams into valuable feedstock chemicals. Using complex microbial consortia, individual systems can be tuned to produce a range of biochemicals to meet market demand. However, the metabolic mechanisms and community interactions which drive product expression changes under differing conditions are currently poorly understood. Furthermore, predictable product transitions are currently limited to pH-driven changes between butyrate and ethanol, and chain-elongation (fed by CO2, acetate, and ethanol) to butyrate, valerate, and hexanoate. Lactate, a high-value biopolymer feedstock chemical, has been observed in transition states, but sustained production has not been described. In this work, a continuous stirred bioreactor was operated at low pH (5.5) with substrate concentration varied between limiting and non-limiting conditions. Using glucose as a model substrate, two sustained operational states were defined: butyrate production during substrate limitation, and lactate production in the non-limited state. Through SWATH-MS metaproteomics and 16S rDNA community profiling, the mechanism of change between butyrate and lactate was described primarily by redirected carbon flow through the methylglyoxal bypass by Megasphaera under substrate non-limiting concentrations. Crucially, butyrate production resumed upon return to substrate-limited conditions, demonstrating the reversibility of this transition.
Recent research has demonstrated that synthetic methanotroph-photoautotroph cocultures offer a highly promising route to convert biogas into value-added products. However, there is a lack of techniques for fast and accurate characterization of cocultures, such as determining the individual biomass concentration of each organism in real-time. To address this unsolved challenge, we propose an experimental-computational protocol for fast, easy and accurate quantitative characterization of the methanotroph-photoautotroph cocultures. Besides determining the individual biomass concentration of each organism in the coculture, the protocol can also obtain the individual consumption and production rates of O2 and CO2 for the methanotroph and photoautotroph, respectively. The accuracy and effectiveness of the proposed protocol was demonstrated using two model coculture pairs, Methylomicrobium alcaliphilum 20ZR - Synechococcus sp. PCC7002 that prefers high pH high salt condition, and Methylococcus capsulatus - Chlorella sorokiniana that prefers low salt and neutral pH medium. The performance of the proposed protocol was compared with a flow cytometry based cell counting approach. The experimental results show that the proposed protocol is much easier to carry out and delivers faster and more accurate results in measuring individual biomass concentration than the cell counting approach without requiring any special equipment.
We describe scalable and cost-efficient production of full length, His-tagged SARS-CoV-2 spike glycoprotein trimer by CHO cells that can be used to detect SARS-CoV-2 antibodies in patient sera at high specificity and sensitivity. Transient production of spike in both HEK and CHO cells mediated by PEI was increased significantly (up to 10.9-fold) by a reduction in culture temperature to 32ºC to permit extended duration cultures. Based on these data GS-CHO pools stably producing spike trimer under the control of a strong synthetic promoter were cultured in hypothermic conditions with combinations of bioactive small molecules to increase yield of purified spike product 4.9-fold to 53 mg/L. Purification of recombinant spike by Ni-chelate affinity chromatography initially yielded a variety of co-eluting protein impurities identified as host cell derived by mass spectrometry, which were separated from spike trimer using a modified imidazole gradient elution. Purified CHO spike trimer antigen was used in ELISA format to detect IgG antibodies against SARS-CoV-2 in sera from patient cohorts previously tested for viral infection by PCR, including those who had displayed COVID-19 symptoms. The antibody assay, validated to ISO 15189 Medical Laboratories standards, exhibited a specificity of 100% and sensitivity of 92.3%. Our data show that CHO cells are a suitable host for the production of larger quantities of recombinant SARS-CoV-2 trimer which can be used as antigen for mass serological testing.
In this study, the binding of multimodal chromatographic ligands to the IgG1 FC domain were studied using nuclear magnetic resonance and molecular dynamics simulations. Nuclear magnetic resonance experiments carried out with chromatographic ligands and a perdeuterated 15N-labeled FC domain indicated that while single mode ion exchange ligands interacted very weakly throughout the FC surface, multimodal ligands interacted with specific clusters of residues with relatively high affinity, forming distinct binding regions on the Fc. The multimodal ligand binding sites on the FC were concentrated in the hinge region and near the interface of the CH2 and CH3 domains. Further, the multimodal binding sites were primarily composed of positively charged, polar and aliphatic residues in these regions, with histidine residues exhibiting some of the strongest binding affinities with the multimodal ligand. Interestingly, comparison of protein surface property data with ligand interaction sites indicated that the patch analysis on FC corroborated molecular level binding information obtained from the nuclear magnetic resonance experiments. Finally, molecular dynamics simulation results were shown to be qualitatively consistent with the nuclear magnetic resonance results and to provide further insights into the binding mechanisms. An important contribution to multimodal ligand-FC binding in these preferred regions was shown to be electrostatic interactions and pi-pi stacking of surface exposed histidines with the ligands. This combined biophysical and simulation approach has provided a deeper molecular level understanding of multimodal ligand-FC interactions and sets the stage for future analyses of even more complex biotherapeutics.
The regulation of cell density is an important segment in microfluidic cell culture, particularly in the repeated assays. Traditionally, the consistent cell density is difficult to achieve owing to the inaccurate regulation of cell density with manual feedback. A novel microfluidic culture method with automatic feedback is proposed for real-time regulation of cell density in this paper. Here, an integrated microfluidic system combining cell culture, density detection and control of proliferation rate was developed. Interdigital electrode structures (IDES) are sputtered on the microchannel for automatically providing the real-time feedback information of impedance. The most sensitive frequency is studied to improve the detection resolution of the sensing chip. Cells were cultured on the chip surface and the cell density was detected by monitoring the alternation of the impedance. The feedback controller is established by least squares support vector machines (LS-SVM). Then, the cell proliferation rate was automatically controlled using the feedback controller to achieve the desired cell density in the repeated assays. The results show that the standard error of this method is 2.8% indicating that the method can keep consistency of cell density in the repeated assays. This study provides a basis for improving the accuracy and repeatability in the further assays of finding the optimal drug concentration.
Algae are promising feedstock of biofuel. The screening of competent species and proper fertilizer supply are of the most important tasks. To accelerate this rather slow and laborious step, we developed an integrated high-throughput digital microfluidic (DMF) system that uses discrete droplet to serve as micro-bioreactor, encapsulating microalgal cells. Based on the fundamental understanding of various droplet hydrodynamics induced by the existence of different sorts of ions and biological species, an incorporation of capacitance-based position estimator, electrode-saving-based compensation and deterministic splitting approach was performed to optimize the DMF bioreactor. Thus, it enables all processes (e.g. nutrient gradient generation, algae culturing and analyzing of growth and lipid accumulation) occurring automatically on-chip especially in a high-fidelity way. The ability of the system to compare different micro algal strains on chip was investigated. Also, the Chlorella sp. were stressed by various conditions and then growth and oil accumulation were analyzed and compared, which demonstrated its potential as a powerful tool to investigate microalgal lipid accumulation at significantly lower laborites and reduced time.
ABSTRACT: AcCHMO, a cyclohexanone monooxygenase from Acinetobacter calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase. AcCHMOM6 is a mutant of AcCHMO we obtained previously that could oxidase the omeprazole sulfide to chiral sulfoxide drug esomeprazole. Based on the structural characteristics of AcCHMO, focused mutagenesis strategy was adopted at the intersections of FAD binding domain, NADPH binding domain and α-helical domain. By the focused mutagenesis and subsequent global evolution, two key residues (55-Leu and 497-Pro) at the intersection of subdomains were identified, of which the L55Y mutagenesis accelerated the H- transfer from NADPH to FAD, while the P497S mutagenesis widened the bottleneck radius of the substrate tunnel and alleviated the substrate inhibition remarkably. By combination of the two mutagenesis, AcCHMOM7 (L55Y/P497S) increased its specific activity from 18.5 U/g to 108 U/g, and its Ki of the substrate sulfide was increased from 34 μM to 265 μM. These results indicated that the catalytic performance can be elevated by modification of the sensitive sites in the intersection of subdomains of AcCHMO, which also provided some insights for the engineering of other type I BVMOs or other multi-subdomain proteins.
Cell-to-cell variability in cell populations arises from a combination of intrinsic factors and extrinsic factors related to the milieu. However, the heterogeneity of high cell density suspension cultures for therapeutic protein production remains unknown. Here, we illustrate the increasing heterogeneity in the cellular transcriptome of serum-free adapted CHO K1 cells during high cell density suspension culture over time without concomitant changes in the genomic sequence. Cell cycle--dependent subpopulations and cell clusters, which typically appear in other single-cell transcriptome analyses of adherent CHO K1 cultures, were not found in these suspension cultures. Our results indicate that cell division changes the intracellular microenvironment and leads to cell cycle--dependent heterogeneity. Whole mitochondrial single-cell genome sequencing showed cell-to-cell mitochondrial genome variation and heteroplasmy within cells. Indeed, the mitochondrial genome sequencing method developed here enables the validation of cell clonality. The culture time-dependent increase in cellular heterogeneity observed in this study did not show any attenuation in this increasing heterogeneity. Future advances in bioengineering such as culture upscaling, prolonged culturing, and complex culture systems will be confronted with the need to assess and control cellular heterogeneity, and the method described here may prove useful for this purpose.
Process analytical technology (PAT) has been defined by the Food and Drug Administration (FDA) as a system for designing, analyzing, and controlling manufacturing through timely measurements to ensure final product quality. Based on quality-by-design (QbD) principles, real-time or near-real-time data monitoring is essential for timely control of critical quality attributes (CQAs) to keep the process in a state of control. To facilitate next-generation continuous bioprocessing, deployment of PAT tools for real-time monitoring is integral for process understanding and control. Real-time monitoring and control of CQAs is essential to keep the process within the design space and align with the guiding principles of QbD. The contents of this manuscript are pertinent to the online/at-line monitoring of upstream titer and downstream product quality with timely process control. We demonstrated that a UPLC system interfaced with a process sample manager (UPLC-PSM) can be utilized to measure titer and CQAs directly from bioreactors and downstream unit operations, respectively. We established online titer measurements from fed-batch and perfusion-based alternating tangential flow (ATF) bioreactors as well as product quality assessments of downstream operations for real-time peak collection. This integrated, fully automated system for online data monitoring with feedback control is designed to achieve desired product quality.
Yeast has been engineered for cost-effective organic acid production through metabolic engineering and synthetic biology techniques. However, cell growth assays in these processes were performed in bulk at the population level, thus obscuring the dynamics of rare single cells exhibiting beneficial traits. Here, we introduce the use of monodisperse picolitre droplets as bioreactors to cultivate yeast at the single-cell level. We investigated the effect of acid stress on growth and the effect of potassium ions on propionic acid tolerance for single yeast cells of different species, genotypes and phenotypes. The results showed that the average growth of single yeast cells in microdroplets was identical to those of yeast populations grown in bulk, and microdroplet compartments do not significantly affect cell viability. This approach offers the prospect of detecting cell-to-cell variations in growth and physiology and is expected to be applied for the engineering of yeast to produce value-added bioproducts.
E. coli BL21 (DE3) is an excellent and widely used host for recombinant protein production. Many variant hosts were developed from from BL21 (DE3), but improving the expression of specific proteins remains a major challenge in biotechnology. In this study, we found that when BL21 (DE3) overexpressed glucose dehydrogenase (GDH), a significant industrial enzyme, serious autolysis was induced. Subsequently, we observed this phenomenon in the expression of 10 other recombinant proteins. This precludes a further increase of the produced enzyme activity by extending the fermentation time, which is not conducive to the reduction of industrial enzyme production costs. The membrane structure and mRNA expression analysis showed that cells suffered programmed cell death (PCD) during autolysis period. However, blocking three known PCD pathway in BL21 (DE3) cannot alleviate autolysis completely. Furthermore, we attempted to develop a strong expression host resistant to autolysis by controlling the speed of recombinant protein expression. To find a more suitable protein expression rate, the high- and low-strength promoter lacUV5 and lac were shuffled and recombined to yield the promoter variants lacUV5-1A and lac-1G. The results showed that only one base in lac promoter needs to be changed, and the A at the +1 position was changed to a G, resulting in a host of BL21 (DE3-lac1G), which successfully withstand the PCD of the host. The GDH activity at 43h was greatly increased from 37.5 U/mL to 452.0 U/mL. In scale-up fermentation, the new host was able to produce the model enzyme with a high rate of 89.55 U/mL/h at 43h, compared to the 3 U/mL/h of BL21 (DE3). Importantly, BL21 (DE3-lac1G) also successfully improved the production of other 10 enzymes. The engineered E. coli strain in the study conveniently optimizes recombinant protein overexpression by suppressing cell autolysis, and shows potential industrial applications.