We have constructed a new bioreactor with reciprocal mixing that is better suited for the cultivation of delicate animal cells. In-silico simulation (computational fluid dynamics) suggested both maximum and average shear stresses in the bioreactor with reciprocal mixing to be remarkably lower than in conventional bioreactor with rotary mixing. Although we could not find any difference in growth speed and cell density between the bioreactors with reciprocal and rotary mixing, we did find cell viability in reciprocal-mixing bioreactor to be retained longer than in rotary-paddle bioreactor. This implied that cell culture in a bioreactor with reciprocal mixing could be prolonged for the production of target proteins. Leakage of lactate dehydrogenase activity into the culture medium was suppressed much more in the reciprocal-mixing bioreactor than in the rotary-paddle one. Production of human tissue plasminogen activator in the former was also observed to be much more than in the latter. Therefore, bioreactor with reciprocal mixing was concluded to be better suited for the cultivation of animal cells and efficient production of proteins, such as antibody drugs and various growth factors.
Harvesting of microalgae is essentially the most energy intensive process in commercial algal culture ventures. Developing innovative, cost effective harvesting systems is of paramount need for commercial algal culture ventures. The study thus aimed at investigating the use of eco-friendly Solar Powered Electroflocculation (SPEF) coupled with battery for harvesting marine microalgae using aluminium electrodes. Optimization of various operating parameters like initial algal density, time of operation (5, 10, 15 and 30 min), (initial biomass concentration 0.1, 0.5 and 1.0 gL-1) and electrode distance (35, 55, 75 and 95 cms) were done using direct current (D.C). Best flocculation efficiency (91.31 ± 2.91 %) was obtained using a current density of 37.2 Acm-2, a voltage of 24 V for 15 min at an electrode distance of 95 cm and pH 8.0. The quality of the harvested biomass was ascertained in-terms of biochemical components using Fourier Transform Infrared spectroscopy, total lipid and pigment profile. Due to the low resistance of seawater the energy required for electroflocculation was as low as 0.223 kWh/ton. The energy requirement for the electroflocculation system with or without solar power was also predicted. The results revealed SPEF can be developed as a potential alternative marine microalgal harvesting system for nutraceutical coupled biodiesel production.
A reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the quantitative determination of recombinant HIV-1 gp145 produced in CHO-K1 cells, as measured directly in culture supernatants. Samples were diluted in 50% D-PBS and 50% PowerCHO-2 (PC2) spent media, and resolved on a Zorbax 300SB-C8 Rapid Resolution (2.1 x 50 mm, 3.5 µm) column, fitted with a C8 guard column (Zorbax 300SB-C8, 2.1 x 12.5 mm, 5µm), using 0.1% TFA and 2% n-propanol as mobile phase A and 0.1% TFA, 70% isopropanol, and 20% acetonitrile as mobile phase B. The column temperature was 80ºC, the flow rate 1 ml/min and the absorbance monitored at 280 nm. The procedures and capabilities of the method were evaluated against the present criteria for linearity, limit of detection (LOD), accuracy, precision, and robustness of the International Conference on Harmonization (ICH) guidelines. Two different variants of the HIV-1 envelope protein (Env), CO6980v0c22 gp145 and SF162 gp140, were analyzed and their retention times were found to be different. The methods showed good linearity (R2 = 0.9996), a lower LOD of 2.4 µg/ml, and an average recovery of 101%. The analysis includes measurements of accuracy, inter-user precision, and robustness. Overall, we present a RP-HPLC method that could be applied for the quantitation of cell culture titers for this and other variants of HIV Env following ICH guidelines.
Surgery of the entire ear pinna even today presents a challenge to reconstructive surgeons, in the absence of a universally acceptable, quality construct for clinical use. In this article, the authors present a technique to generate a flexible, human-size ear with the aim to meet this limitation for ear reconstructive surgeries. The construct was engineered by using a decellularized goat ear cartilage. This was characterized by hematoxylin-eosin (H/E), diamidino-2-phenylindole (DAPI), Masson’s trichrome (MT), Alcian Blue (AB) staining and Scanning Electron Microscopy (SEM) for extracellular matrix (ECM) analysis. The decellularization protocol followed yielded complete removal of all cellular components without changing the properties of the ECM. In vivo biocompatibility of the ear, pinna showed demonstrable recellularization. Recellularization was tracked using HE, DAPI, MT, AB staining, toluidine staining, SEM, vascular-associated protein (VAP), and CD90+ expressing cells. VAP expression revealed specific vasculogenic pattern (angiogenesis). CD90+ expression reflected the presence of the stromal cell. The graft maintained the properties of ECM and displayed chondrocyte recruitment. In summary, the decellularized goat ear pinna (cartilage) exhibited xenograft biocompatibility, stable mechanical properties, and in vivo chondrocyte recruitment. Subsequently developed tissue-engineered ear pinna offer potential for cartilage flexibility and individualization of ear shape and size for clinical application.
The optimal growth, maturation and function of bioengineered tissues are mediated by both biochemical and physical cues. We here describe a 3D biomimetic environment directing stem cells towards a chondrogenic phenotype. This system comprises a collagen hydrogel and poly-lactic-co-glycolic acid microcarriers (PLGA-MCs) engineered to protect, carry and release a human Transforming Growth Factor b1 (hTFGb1) payload. PLGA-MCs were prepared using supercritical emulsion extraction technology and integrated into a collagen hydrogel co-seeded with human Bone Marrow Mesenchymal Stem Cells (hBM-MSCs). Testing different concentrations of hTFGb1 supplemented to cell monolayer cultures suggested 10 ng/mL as the most appropriate concentration to promote upregulation of SRY-Related HMG-BOXGene 9 (4-fold) and collagen type II (2-fold) specific markers, at Day 16. A similar growth factor concentration was delivered within the 3D bioengineered environment cultured in a dynamic via a custom perfusion bioreactor. A chondrogenic commitment was obtained as indicated by upregulation of collagen type II (5-fold) and downregulation of collagen types I and III (both 0.1-fold) at Day 16. Histological analysis confirmed the remodeling of the synthetic extracellular matrix in where an enhanced mass exchange was described by FEM analysis of fluid-dynamics and related nutrient mass transfer within the 3D construct. This study supports the use of 3D bioengineered scaffolds cultured in a dynamic environment as a suitable tissue engineered model to study chondrogenic differentiation in vitro and opens perspectives for an injectable collagen-based advanced therapy system.
This study explores the application of metabolic engineering in Aspergillus terreus to re-route the precursor flow towards the lovastatin biosynthetic pathway by simultaneously overexpressing the gene for acetyl-CoA carboxylase (acc) to increase the precursor and eliminating (+)-geodin biosynthesis (competing metabolite), by knocking out emodin anthrone polyketide synthase (gedC). Alterations to metabolic flux in the double mutant (gedCΔ*accox) strain and the effects of using two different substrate formulations were examined. Cultivation of gedCΔ*accox strain with a mixture of glycerol and lactose, had greatly increased levels of precursors malonyl-CoA (48%) and acetyl-CoA (420%), complete inhibition of (+)-geodin biosynthesis and a maximum production of lovastatin (152 mg/L), 143% more than the wild-type (WT) strain. This study demonstrates the manipulation of A. terreus metabolic pathways to increase the efficiency of carbon flux towards lovastatin, elevating its production. It provides a framework for new opportunities to synthesize valuable compounds using cheap and renewable carbon sources.
Deep learning has the potential to revolutionize process analytical technology in the pharmaceutical industry. Here, we used Raman spectroscopy-based deep learning strategies to develop a tool for detecting microbial contamination. We built a Raman dataset for microorganisms that are common contaminants in the pharmaceutical industry for Chinese Hamster Ovary (CHO) cells, which are often used in the production of biologics. Using a convolution neural network (CNN), we classified the different samples comprising individual microbes and microbes mixed with CHO cells with an accuracy of 95-100%. The set of 12 microbes spans across Gram-positive and Gram-negative bacteria as well as fungi. We also created an attention map for different microbes and CHO cells to highlight which segments of the Raman spectra contribute the most to help discriminate between different species. This dataset and algorithm provide a route for implementing Raman spectroscopy for detecting microbial contamination in the pharmaceutical industry.
The biological reduction of ferrous ethylenediaminetetraacetic acid (EDTA-FeII-NO and EDTA-FeIII) is an important process in integrated electrobiofilm reduction method, and this method has been regarded as a promising alternative for removing NOx from industrial boiler flue gas. EDTA-FeII-NO and EDTA-FeIII are crucial substrates that should be biologically reduced at a high rate. However, they would inhibit one another’s reduction processes when they are present together, which might limited the further promotion of this integrated method. In this study, an integrated electrobiofilm reduction system with high reduction rate of EDTA-FeII- NO and EDTA-FeIII was established. The microbial communities in electrobiofilms were mainly studied to analyze their changes during the reduction of these two substrates under different conditions. It presents a better performance of substrates- resistance shock loading and high microbial diversities compared with the conventional chemical absorption-biological reduction system. High-throughput sequencing analysis showed that the changes in concentrations of EDTA-FeII-NO and EDTA-FeIII significantly impacted the genera of the microbial community. Alicycliphilus, Enterobacteriaceae and Raoultella were found to be the dominant genera (>25%, respectively) involved in EDTA-FeII-NO reduction. As an EDTA-FeIII reducing bacteria, Chryseobacterium can endure shock loading of substrates. Ochrobactrum can reduce nitrate using electrons and exhibited better stability under shock loading. Furthermore, higher microbial diversity and stable reactor operation could be achieved when the concentrations of EDTA-FeII-NO and EDTA-FeIII approached the same value.
γ-Aminobutyric acid (GABA) is a non-protein amino acid produced from the decarboxylation of glutamate by glutamate decarboxylase. Corynebacterium glutamicum is the most promising host of γ-aminobutyric acid production for its inherent glutamate precursor supply. However, the intracellularly expressed glutamate decarboxylase in C. glutamicum showed the weak catalysis capacity on the conversion of glutamate to γ-aminobutyric acid. Here we designed an different catalysis scenario by secretively overexpressing the glutamate decarboxylase in C. glutamicum and moving the decarboxylation reaction into the extracellular space for GABA synthesis. A signal peptide in the expression cassette directed the successful secretion of glutamate decarboxylase in C. glutamicum. The extracellular catalysis by secreted glutamate decarboxylase increased the γ-aminobutyric acid generation by three-folds, comparing with that by intracellular catalysis. Further efforts on enhancing the expression of glutamate decarboxylase and decreasing the degradation of γ-aminobutyric acid improved γ-aminobutyric acid generation by 39%. The fed-batch fermentation of the engineered C. glutamicum strain reached the record high titer (77.6g /L), overall yield (0.37 g/g glucose), and productivity (1.21 g/L/h) of γ-aminobutyric acid production. This study demonstrated a unique design of extracellular catalysis for efficient γ-aminobutyric acid production by C. glutamicum.
Therapeutic monoclonal antibodies and related products have steadily grown to become the dominant product class within the biopharmaceutical market. Production of antibodies requires special precautions to ensure safety and efficacy of the product. In particular, minimizing antibody product heterogeneity is crucial as drug substance variants may impair the activity, efficacy, safety and pharmacokinetic properties of an antibody, consequently resulting in the failure of a product in pre-clinical and clinical development. This review will cover the manufacturing and formulation challenges and advances of therapeutic monoclonal antibodies, with a focus on improved processes to minimize variants and ensure batch-to-batch consistency. Processes put in place by regulatory agencies such as Quality-by-Design (QbD) and current Good Manufacturing Practices (cGMP) will be reviewed, and how their implementation has aided drug development in pharmaceutical companies. Advances in formulation and considerations on the intended use of a therapeutic antibody, including route of administration and patient compliance, will be discussed.
The oleaginous yeast Yarrowia lipolytica is an important industrial microorganism used for the production of lipids, proteins and various chemicals. The design of effective biotechnological processes with this cell factory requires an in-depth knowledge of its metabolism. Here we present a transcriptomic study of Y. lipolytica grown in the presence of two important carbon sources, namely glycerol and glucose, as well as in a mixture of both at different carbon to nitrogen ratios. It emerged that the transcriptomic landscape of Y. lipolytica is more sensitive to the nitrogen availability than to the utilized carbon source, as evidenced by more genes being differentially expressed in lower carbon to nitrogen ratio. In particular, expression of hexokinase (HXK1) is significantly susceptible to changes in nitrogen concentrations. Moreover, high HXK1 expression in low nitrogen seems to impact the expression of other genes which are implicated in tricarboxylic acid cycle and erythritol biosynthesis. We further show that expression of HXK1 and two genes belonging to the sugar porter family might be controlled by GATA-like zinc-finger proteins.
With the pandemic emergence of SARS-CoV-2, the exposure of cell substrates used for manufacturing of medicines has become a possibility. Cell lines used in biomanufacturing were thus evaluated for their SARS-CoV-2 susceptibility, and the detection of SARS-CoV-2 in culture supernatants was tested by routine adventitious virus testing of fermenter harvest.
Current technological developments in the field of wireless communications has enabled the use of ultra-low power, lightweight, miniature types of smart health monitoring devices which are integrated into a Wireless Body Sensor Network (WBSN). Vital signs in humans such as temperature, heartbeat, and pulse can be tracked and detected from a remote location with help of biomedical wireless sensors. This article proposes a method to securely encrypt the patient’s data and transmit it to the authorized doctor using wireless devices with in the hospital premises. This article is aimed at generating electrocardiogram (ECG) based key agreement scheme for the sake of generating a common key in a body area network. This kind of smart health information communication method by means of sensors and internet connection to servers is more confidential and encrypted to protect the patient’s health data. Security and Privacy mechanisms use a significant part of the available energy and should therefore be energy efficient and light weight. The Proposed work in this article is implemented in Telosb running ContikiOS. The TelosB mote is designed for experimentation with low-power personal wireless area network (LoWPAN) with IEEE 802.15.4/ZigBee/Telosb compliant RF transceiver. By using iris or fingerprints, the security of this technique can be further improved.
The goal of this research is to leverage computational molecular biophysics to guide process development, reduce experimental burden and focus purification activities on feasible targets. Here, we distill a complex separation problem (e.g. chromatographic retention of monoclonal antibodies) into a tangible model (ligand/protein complex), which is computationally feasible while preserving enough detail (atomistic level for interaction site) to support industrially relevant separation challenges. Computational docking, coupled with molecular dynamics simulation, produces results that are directionally consistent with chromatography for proteins (mAb). This approach is generalizable and can be applied to a range of ligands (AEX, CEX, and Mixed Mode). A detailed model of the chromatography base matrix (agarose) was constructed to obtain a biophysical understanding of potential protein/base matrix interactions. The base matrix was then modified in silico with ligands over a range of ligand densities representative of commercial chromatography resins to generate an agarose/ligand complex. A generic approach was developed to model the impact of avidity and ligand density on mAb/ligand interaction. The results revealed that increasing ligand density mask contributions of base matrix binding. Increasing the number of ligands that can interact with mAb results in more favorable free energy of binding or ΔG (more negative) with a limited incremental increase in ΔG by increasing N (number of ligands per agarose cluster) above three. Additionally, for protein/ligand interactions at each binding site, not all ligands contribute equally to the binding affinities or interaction energies and a redistribution of binding interactions/energies occur as N increases. These observations yield insights into the impact of avidity on retention (macroscopic affinity measurement via k’). The generic approach described in this manuscript can be leveraged to inform resin selection and design as well as targeted ligand selection/purification development in a rational manner.
Glutarate is an attractive C5 platform chemical having wide application in nylon and plasticizer. However, microbial glutarate is mainly accumulated in the degradation of lysine and other methods were rarely explored for producing the glutarate from glucose directly. Here, we utilized a reversed adipic acid degradation pathway (RADP) and improved the glutarate production by increasing the precursors (malonyl-CoA and acetyl-CoA) based on the previously studied strain Bgl4146. Specifically, the conversion system of intracellular acetyl-CoA to malonyl-CoA was firstly constructed and optimized to balance acetyl-CoA synthase and acetyl-CoA carboxylase under different dissolved oxygen, enhancing the glutarate production from 0.09 g/L to 0.49 g/L. Then, modulating CoA balance by monitoring the expression of acetate kinase and pyruvate dehydrogenase resulting in a rise in glutarate titer up to 0.70 g/L. Finally, the optimized strain Bgl51464 was able to produce 7.97 g/L glutarate in a 5-L bioreactor. This strategy was described here, which could lay a certain foundation for the development of effective CoA balance to produce industrially high value-added chemicals.
Bacillus subtilis is an attractive host for directed evolution of the enzymes whose substrates cannot be transported across the cell membrane. However, generation of mutant library in B. subtilis still suffers problems of small library size, plasmid instability and heterozygosity. Here, large library of random mutant was created through inserting error-prone PCR (epPCR) product to the chromosome of B. subtilis. Specifically, epPCR product was fused with flanking regions and antibiotic resistant marker using a PCR-based multimerization method, generating insertion construct. epPCR product was integrated into chromosome via homologous recombination after insertion construct was transformed into the supercompetent cells of B. subtilis strain SCK6. The transformation efficiency of insertion construct was improved though increasing the number of competent cell and the length of flanking regions. A library containing 3.5×105 random mutant was construction using per μg insertion construct, which is sufficient for directed evolution. Moreover, the library generation process could be accomplished within one day. The effectiveness of this method was confirmed by improving the activity of Methyl Parathion Hydrolase (MPH) toward chlorpyrifos and to enhance the secretion level of MPH in B. subtilis. Taken together, present work provides a fast and efficient method to integrate epPCR product into the chromosome of B. subtilis, facilitating directed evolution and expression optimization of target protein.
Avian transgenesis has served as a suitable approach to generate bioreactors for the manufacturing of recombinant proteins. Production in chicken cells comes with significant advantages over other systems including providing the human-like glycosylation on target proteins. In this regard, the oviduct-specific ovalbumin promoter has been one of the ideal candidates to drive the expression of transgenes. Previous plasmid-based studies on the regulatory sequences of ovalbumin promoter have led researchers to exploit ovalbumin regulatory elements out of their native genomic context (ex situ) to direct transgene expression in the transgenic chicken bioreactors. Although the inherent limitations on the ex situ use of ovalbumin promoter have promoted the use of native ovalbumin promoter for the expression of a transgene, generation of transgenic chicken is relatively difficult, inefficient, and time-consuming. To overcome these obstacles, in this study we show that CRISPR-mediated deletion of some distal ovalbumin promoter sequences in a non-oviduct cell can lead to the significant expression of the ovalbumin gene, and also a knocked-in reporter, in an estrogen-independent manner. These findings overcome the limitation of cloned promoters, where the promoter regulatory sequences have to be taken out of their cis context and also their native spatial nuclear organization into a plasmid.