Showing papers in "Biotechnology and Bioengineering in 2023"

Preparation of lignin nanoparticles via ultra‐fast microwave‐assisted fractionation of lignocellulose using ternary deep eutectic solvents

Abstract: Lignin separation from natural lignocellulose for the preparation of lignin nanoparticles (LNPs) is often challenging owing to the recalcitrant and complex structure of lignocellulose. This paper reports a strategy for the rapid synthesis of LNPs via microwave‐assisted lignocellulose fractionation using ternary deep eutectic solvents (DESs). A novel ternary DES with strong hydrogen bonding was prepared using choline chloride, oxalic acid, and lactic acid in a 1:0.5:1 ratio. Efficient fractionation of rice straw (0.5 × 2.0 cm) (RS) was realized by the ternary DES under microwave irradiation (680 W) within only 4 min, and 63.4% of lignin could be separated from the RS to prepare LNPs with a high lignin purity (86.8%), an average particle size of 48–95 nm, and a narrow size distribution. The mechanism of lignin conversion was also investigated, which revealed that dissolved lignin aggregated into LNPs via π–π stacking interactions.

Application of gas diffusion electrodes in bioeconomy: An update

Abstract: The transition of today's fossil fuel based chemical industry toward sustainable production requires improvement of established production processes as well as development of new sustainable and bio‐based synthesis routes within a circular economy. Thereby, the combination of electrochemical and biotechnological advantages in such routes represents one important keystone. For the electrochemical generation of reactants from gaseous substrates such as O2 or CO2, gas diffusion electrodes (GDE) represent the electrodes of choice since they overcome solubility‐based mass transport limitations. Within this article, we illustrate the architecture, function principle and fabrication of GDE. We highlight the application of GDE for conversion of CO2 using abiotic catalysts for subsequent biosynthesis as well as the application of microbial catalysts at GDE for CO2 conversion. The reduction of oxygen at GDE is summarized for the application of oxygen depolarized cathodes in microbial fuel cells and generation of H2O2 to drive enzymatic reactions. Finally, engineering aspects such as scale‐up and the modeling of GDE‐based processes are described. This review presents an update on the application of GDE in bio‐based production systems and emphasizes their large potential for sustainable development of new pathways in bioeconomy.

A CHO stable pool production platform for rapid clinical development of trimeric SARS‐CoV‐2 spike subunit vaccine antigens

Abstract: Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time‐consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non‐clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP‐toxicology and early‐phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS‐CoV‐2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost‐effective clinical evaluation of potentially life‐saving vaccines.

Exploring the potential of CRISPR/Cas genome editing for vegetable crop improvement: An overview of challenges and approaches

Abstract: Vegetables provide many nutrients in the form of fiber, vitamins, and minerals, which make them an important part of our diet. Numerous biotic and abiotic stresses can affect crop growth, quality, and yield. Traditional and modern breeding strategies to improve plant traits are slow and resource intensive. Therefore, it is necessary to find new approaches for crop improvement. Clustered regularly interspaced short palindromic repeats/CRISPR associated 9 (CRISPR/Cas9) is a genome editing tool that can be used to modify targeted genes for desirable traits with greater efficiency and accuracy. By using CRISPR/Cas9 editing to precisely mutate key genes, it is possible to rapidly generate new germplasm resources for the promotion of important agronomic traits. This is made possible by the availability of whole genome sequencing data and information on the function of genes responsible for important traits. In addition, CRISPR/Cas9 systems have revolutionized agriculture, making genome editing more versatile. Currently, genome editing of vegetable crops is limited to a few vegetable varieties (tomato, sweet potato, potato, carrot, squash, eggplant, etc.) due to lack of regeneration protocols and sufficient genome sequencing data. In this article, we summarize recent studies on the application of CRISPR/Cas9 in improving vegetable trait development and the potential for future improvement.

In vitro modeling of liver fibrosis with 3D co‐culture system using a novel human hepatic stellate cell line

Abstract: Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC‐1), and applied it to various types of three‐dimensional (3D) co‐culture systems with differentiated HepaRG cells. Furthermore, we compared LSC‐1 with a commercially available HSC line on conventional monolayer culture. LSC‐1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast‐like or transdifferentiated state. However, activated states reverted to a quiescent‐like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC‐1 exerted an overall positive effect on co‐cultured differentiated HepaRG, which significantly increased hepatic functionality upon long‐term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC‐1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix‐related protein expression mechanism. Therefore, the LSC‐1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies.

Aided‐efflux and high production of β‐amyrin realized by β‐cyclodextrin in situ synthesized on surface of Saccharomyces cerevisiae

Abstract: As a plant‐derived pentacyclic triterpenoid, β‐amyrin has been heterogeneously synthesized in Saccharomyces cerevisiae. However, β‐amyrin is intracellularly produced in a lower gram scale using recombinant S. cerevisiae, which limits the industrial applications. Although many strategies have been proven to be effective to improve the production of β‐amyrin, the intracellularly accumulation is still a challenge in reaching higher titer and simplifying the extraction process. To solve this problem, the amphiphilic β‐cyclodextrin (β‐CD) has been previously employed to aid the efflux of β‐amyrin out of the cells. Nevertheless, the supplemented β‐CD in the medium is not consistent with β‐amyrin synthesis and has the disadvantage of rather high cost. Therefore, an aided‐efflux system based on in situ synthesis of β‐CD was developed in this study to enhance the biosynthesis of β‐amyrin and its efflux. The in situ synthesis of β‐CD was started from starch by the surface displayed cyclodextrin glycosyltransferase (CGTase) on yeast cells. As a result, the synthesized β‐CD could capture 16% of the intracellular β‐amyrin and improve the total production by 77%. Furthermore, more strategies including inducing system remodeling, precursor supply enhancement, two‐phase fermentation and lipid synthesis regulation were employed. Finally, the production of β‐amyrin was increased to 73 mg/L in shake flask, 31 folds higher than the original strain, containing 31 mg/L of extracellular β‐amyrin. Overall, this work provides novel strategies for the aided‐efflux of natural products with high hydrophobicity in engineered S. cerevisiae.

Poly (beta‐amino ester) as an in vivo nanocarrier for therapeutic nucleic acids

Abstract: Therapeutic nucleic acids are an emerging class of therapy for treating various diseases through immunomodulation, protein replacement, gene editing, and genetic engineering. However, they need a vector to effectively and safely reach the target cells. Most gene and cell therapies rely on ex vivo gene delivery, which is laborious, time‐consuming, and costly; therefore, devising a systematic vector for effective and safe in vivo delivery of therapeutic nucleic acids is required to target the cells of interest in an efficient manner. Synthetic nanoparticle vector poly beta amino ester (PBAE), a class of degradable polymer, is a promising candidate for in vivo gene delivery. PBAE is considered the most potent in vivo vector due to its excellent transfection performance and biodegradability. PBAE nanoparticles showed tunable charge density, diverse structural characteristics, excellent encapsulation capacity, high stability, stimuli‐responsive release, site‐specific delivery, potent binding to nucleic acids, flexible binding ability to various conjugates, and effective endosomal escape. These unique properties of PBAE are an essential contribution to in vivo gene delivery. The current review discusses each of the components used for PBAE synthesis and the impact of various environmental and physicochemical factors of the body on PBAE nanocarrier.

In vitro glycation of a tissue‐engineered wound healing model to mimic diabetic ulcers

Abstract: Diabetic foot ulcers are a major complication of diabetes that occurs following minor trauma. Diabetes-induced hyperglycemia is a leading factor inducing ulcer formation and manifests notably through the accumulation of advanced glycation end-products (AGEs) such as N-carboxymethyl-lysin. AGEs have a negative impact on angiogenesis, innervation, and reepithelialization causing minor wounds to evolve into chronic ulcers which increases the risks of lower limb amputation. However, the impact of AGEs on wound healing is difficult to model (both in vitro on cells, and in vivo in animals) because it involves a long-term toxic effect. We have developed a tissue-engineered wound healing model made of human keratinocytes, fibroblasts, and endothelial cells cultured in a collagen sponge biomaterial. To mimic the deleterious effects induced by glycation on skin wound healing, the model was treated with 300 µM of glyoxal for 15 days to promote AGEs formation. Glyoxal treatment induced carboxymethyl-lysin accumulation and delayed wound closure in the skin mimicking diabetic ulcers. Moreover, this effect was reversed by the addition of aminoguanidine, an inhibitor of AGEs formation. This in vitro diabetic wound healing model could be a great tool for the screening of new molecules to improve the treatment of diabetic ulcers by preventing glycation.

Characterization of ionic strength for X‐MuLV inactivation by low pH treatment for monoclonal antibody purification

Abstract: In the production of monoclonal antibodies (mAbs) intended for use in humans, it is a global regulatory requirement that the manufacturing process includes unit operations that are proven to inactivate or remove adventitious agents to ensure viral safety. Viral inactivation by low pH hold (LPH) is typically used to ensure this viral safety in the purification process of mAbs and other biotherapeutics derived from mammalian cell lines. To ascertain the effectiveness of the LPH step, viral clearance studies have evaluated LPH under worst‐case conditions of pH above the manufacturing set point and hold duration at or below the manufacturing minimum. Highly acidic conditions (i.e., pH < 3.60) provide robust and effective enveloped virus inactivation but may lead to reduced product quality of the therapeutic protein. However, when viral inactivation is operated above pH 3.60 to ensure product stability, effective (>4 log10 reduction factor) viral inactivation may not be observed under these worst‐case pH conditions in viral clearance studies. A multivariate design of experiments was conducted to further characterize the operating space for low pH viral inactivation of a model retrovirus, xenotropic murine leukemia virus (X‐MuLV). The statistically designed experiment evaluated the effect of mAb isotype, pH, temperature, acid titrant, sodium chloride (NaCl) concentration, virus spike timing, and post‐spike filtration on X‐MuLV inactivation. Data from the characterization study were used to generate predictive models to identify conditions that reliably achieve effective viral inactivation at pH ≥ 3.60. Results of the study demonstrated that NaCl concentration has the greatest effect on virus inactivation in the range studied, and pH has a large effect when the load material has no additional NaCl. Overall, robust and effective inactivation of X‐MuLV at pH 3.65–3.80 can be achieved by manipulating either the pH or the NaCl concentration of the load material. This study contributes to the understanding of ionic strength as an influential parameter in low pH viral inactivation studies.

A compendium of stable hotspots in the CHO genome.

Abstract: The use of targeted integration for industrial CHO cell line development currently requires significant upfront effort to identify genomic loci capable of supporting multigram per liter therapeutic protein production from a limited number of transgene copies. To address this barrier to widespread adoption, we characterized transgene expression from thousands of stable hotspots in the CHO genome using the Thousands of Reporters Integrated in Parallel high-throughput screening method. This genome-scale data set was used to define a limited set of epigenetic properties of hotspot regions with sizes on the order of 10 kb. Cell lines with landing pad integrations at eight retargeted hotspot candidates consistently exhibited higher transgene mRNA expression than a commercially viable hotspot in equivalent culture conditions. Initial benchmarking of NISTmAb and trastuzumab productivity from one of these hotspots yielded mAb productivities of approximately 0.7-2 g/L (qP range: 2.9-8.2 pg/cell/day) in small-scale fed-batches. These findings indicate the list of hotspot candidates identified here will be a valuable resource for targeted integration platform development within the CHO community.

In situ biological particle analyzer based on digital inline holography

Abstract: Obtaining in situ measurements of biological microparticles is crucial for both scientific research and numerous industrial applications (e.g., early detection of harmful algal blooms, monitoring yeast during fermentation). However, existing methods are limited to offer timely diagnostics of these particles with sufficient accuracy and information. Here, we introduce a novel method for real‐time, in situ analysis using machine learning (ML)‐assisted digital inline holography (DIH). Our ML model uses a customized YOLOv5 architecture specialized for the detection and classification of small biological particles. We demonstrate the effectiveness of our method in the analysis of 10 plankton species with equivalent high accuracy and significantly reduced processing time compared to previous methods. We also applied our method to differentiate yeast cells under four metabolic states and from two strains. Our results show that the proposed method can accurately detect and differentiate cellular and subcellular features related to metabolic states and strains. This study demonstrates the potential of ML‐driven DIH approach as a sensitive and versatile diagnostic tool for real‐time, in situ analysis of both biotic and abiotic particles. This method can be readily deployed in a distributive manner for scientific research and manufacturing on an industrial scale.

Shortening the biologics clinical timeline with a novel method for generating stable, high‐producing cell pools and clones

Abstract: Reducing drug development timelines is an industry-wide goal to bring medicines to patients in need more quickly. This was exemplified in the coronavirus disease 2019 pandemic where reducing development timelines had a direct impact on the number of lives lost to the disease. The use of drug substances produced using cell pools, as opposed to clones, has the potential to shorten development timelines. Toward this goal, we have developed a novel technology, GPEx® Lightning, that allows for rapid, reproducible, targeted recombination of transgenes into more than 200 Dock sites in the Chinese hamster ovary cell line genome. This allows for rapid production of high-expressing stable cell pools and clones that reach titers of 4-12 g/l in generic fed-batch production. These pools and clones are highly stable in both titer and glycosylation, showing strong similarities in glycosylation profiles.

Critical challenges and advances in recombinant adeno-associated virus (rAAV) biomanufacturing.

Abstract: Gene therapy is a promising therapeutic approach for genetic and acquired diseases nowadays. Among DNA delivery vectors, recombinant adeno-associated virus (rAAV) is one of the most effective and safest vectors used in commercial drugs and clinical trials. However, the current yield of rAAV biomanufacturing lags behind the necessary dosages for clinical and commercial use, which embodies a concentrated reflection of low productivity of rAAV from host cells, difficult scalability of the rAAV-producing bioprocess, and high levels of impurities materialized during production. Those issues directly impact the price of gene therapy medicine in the market, limiting most patients' access to gene therapy. In this context, the current practices and several critical challenges associated with rAAV gene therapy bioprocesses are reviewed, followed by a discussion of recent advances in rAAV-mediated gene therapy and other therapeutic biological fields that could improve biomanufacturing if these advances are integrated effectively into the current systems. This review aims to provide the current state-of-the-art technology and perspectives to enhance the productivity of rAAV while reducing impurities during production of rAAV.

Overview of the advances in plant microbial fuel cell technology for sustainable energy recovery from rhizodeposition

Abstract: In plant microbial fuel cells (p‐MFCs) electrochemically active microbes present around the plant root convert rhizodeposits or the organic matter into electrons, protons, and CO2. This work covers the increasing trend in research with p‐MFCs with their mechanism of operation. Different plant species and their selection criteria are also covered. Furthermore, the long‐term evaluation of such systems with its cost effectiveness and commercial and environmental perspectives are also presented. A critical aspect for bioelectricity production is the photosynthetic pathway of the plant. Additionally, the microbial communities and reactor configurations employed across different capacities are also reviewed. The challenges with bioelectricity production and the opportunity for developing p‐MFCs in conjunction with traditional MFCs are also covered. These electrogenic reactor systems harness bioelectricity without harvesting the plant and has the capacity to utilize this energy for remote power applications.

Experimental and theoretical investigations of rotating algae biofilm reactors (RABRs): Areal productivity, nutrient recovery, and energy efficiency.

Abstract: Microalgae biofilms have been demonstrated to recover nutrients from wastewater and serve as biomass feedstock for bioproducts. However, there is a need to develop a platform to quantitatively describe microalgae biofilm production, which can provide guidance and insights for improving biomass areal productivity and nutrient uptake efficiency. This paper proposes a unified experimental and theoretical framework to investigate algae biofilm growth on a rotating algae biofilm reactor (RABR). Experimental laboratory setups are used to conduct controlled experiments on testing environmental and operational factors for RABRs. We propose a differential-integral equation-based mathematical model for microalgae biofilm cultivation guided by laboratory experimental findings. The predictive mathematical model development is coordinated with laboratory experiments of biofilm areal productivity associated with ammonia and inorganic phosphorus uptake by RABRs. The unified experimental and theoretical tool is used to investigate the effects of RABR rotating velocity, duty cycle (DC), and light intensity on algae biofilm growth, areal productivity, nutrient uptake efficiency, and energy efficiency in wastewater treatment. Our framework indicates that maintaining a reasonable light intensity range improves biomass areal productivity and nutrient uptake efficiency. Our framework also indicates that faster RABR rotation benefits biomass areal productivity. However, maximizing the nutrient uptake efficiency requires a reasonably low RABR rotating speed. Energy efficiency is strongly correlated with RABR rotating speed and DC.

Effect of protein fouling on filtrate flux and virus breakthrough behaviors during virus filtration process

Abstract: Virus filtration process is used to ensure viral safety in the biopharmaceutical downstream processes with high virus removal capacity (i.e., >4 log10). However, it is still constrained by protein fouling, which results in reduced filtration capacity and possible virus breakthrough. This study investigated the effects of protein fouling on filtrate flux and virus breakthrough using commercial membranes that had different symmetricity, nominal pore size, and pore size gradients. Flux decay tendency due to protein fouling was influenced by hydrodynamic drag force and protein concentration. As the results of prediction with the classical fouling model, standard blocking was suitable for most virus filters. Undesired virus breakthrough was observed in the membranes having relatively a large pore diameter of the retentive region. The study found that elevated levels of protein solution reduced virus removal performance. However, the impact of prefouled membranes was minimal. These findings shed light on the factors that influence protein fouling during the virus filtration process of biopharmaceutical production.

An enhanced electron transport chain improved astaxanthin production in Phaffia rhodozyma

Abstract: Astaxanthin (AX) is a carotenoid pigment with antioxidant properties widely used as a feed supplement. Wild‐type strains of Phaffia rhodozyma naturally produce low AX yields, but we increased AX yields 50‐fold in previous research using random mutagenesis of P. rhodozyma CBS6938 and fermentation optimization. On that study, genome changes were linked with phenotype, but relevant metabolic changes were not resolved. In this study, the wild‐type and the superior P. rhodozyma mutant strains were grown in chemically defined media and instrumented fermenters. Differential kinetic, metabolomics, and transcriptomics data were collected. Our results suggest that carotenoid production was mainly associated with cell growth and had a positive regulation of central carbon metabolism metabolites, amino acids, and fatty acids. In the stationary phase, amino acids associated with the TCA cycle increased, but most of the fatty acids and central carbon metabolism metabolites decreased. TCA cycle metabolites were in abundance and media supplementation of citrate, malate, α‐ketoglutarate, succinate, or fumarate increased AX production in the mutant strain. Transcriptomic data correlated with the metabolic and genomic data and found a positive regulation of genes associated with the electron transport chain suggesting this to be the main driver for improved AX production in the mutant strain.

Engineering of xylanases for the development of biotechnologically important characteristics

Abstract: Xylanases are the main biocatalysts used for the reduction of the xylan backbone from hemicellulose, randomly splitting off β‐1,4‐glycosidic linkages between xylopyranosyl residues. Xylanase market has been annually estimated at 500 million US Dollars and they are potentially used in broad industrial process ranges such as paper pulp biobleaching, xylo‐oligosaccharide production, and biofuel manufacture from lignocellulose. The highly stable xylanases are preferred in the downstream procedure of industrial processes because they can tolerate severe conditions. Almost all native xylanases can not endure adverse conditions thus they are industrially not proper to be utilized. Protein engineering is a powerful technology for developing xylanases, which can effectively work in adverse conditions and can meet requirements for industrial processes. This study considered state‐of‐the‐art strategies of protein engineering for creating the xylanase gene diversity, high‐throughput screening systems toward upgraded traits of the xylanases, and the prediction and comprehensive analysis of the target mutations in xylanases by in silico methods. Also, key molecular factors have been elucidated for industrial characteristics (alkaliphilic enhancement, thermal stability, and catalytic performance) of GH11 family xylanases. The present review explores industrial characteristics improved by directed evolution, rational design, and semi‐rational design as protein engineering approaches for pulp bleaching process, xylooligosaccharides production, and biorefinery & bioenergy production.

Lactic acid bacteria as a tool for biovanillin production: A review

Abstract: Vanilla is the most commonly used natural flavoring agent in industries like food, flavoring, medicine, and fragrance. Vanillin can be obtained naturally, chemically, or through a biotechnological process. However, the yield from vanilla pods is low and does not meet market demand, and the use of vanillin produced by chemical synthesis is restricted in the food and pharmaceutical industries. As a result, the biotechnological process is the most efficient and cost‐effective method for producing vanillin with consumer‐demanding properties while also supporting industrial applications. Toxin‐free biovanillin production, based on renewable sources such as industrial wastes or by‐products, is a promising approach. In addition, only natural‐labeled vanillin is approved for use in the food industry. Accordingly, this review focuses on biovanillin production from lactic acid bacteria (LAB), which is generally recognized as safe (GRAS), and the cost‐cutting efforts that are utilized to improve the efficiency of biotransformation of inexpensive and readily available sources. LABs can utilize agro‐wastes rich in ferulic acid to produce ferulic acid, which is then employed in vanillin production via fermentation, and various efforts have been applied to enhance the vanillin titer. However, different designs, such as response surface methods, using immobilized cells or pure enzymes for the spontaneous release of vanillin, are strongly advised.

Expanding the CRISPR toolbox for Chinese hamster ovary cells with comprehensive tools for Mad7 genome editing

Abstract: The production of high-value biopharmaceuticals is dominated by mammalian production cells, particularly Chinese hamster ovary (CHO) cells, which have been widely used and preferred in manufacturing processes. The discovery of CRISPR-Cas9 significantly accelerated cell line engineering advances, allowing for production yield and quality improvements. Since then, several other CRISPR systems have become appealing genome editing tools, such as the Cas12a nucleases, which provide broad editing capabilities while utilizing short guide RNAs (gRNAs) that reduce the complexity of the editing systems. One of these is the Mad7 nuclease, which has been shown to efficiently convey targeted gene disruption and insertions in several different organisms. In this study, we demonstrate that Mad7 can generate indels for gene knockout of host cell proteins in CHO cells. We found that the efficiency of Mad7 depends on the addition of protein nuclear localization signals and the gRNAs employed for genome targeting. Moreover, we provide computational tools to design Mad7 gRNAs against any genome of choice and for automated indel detection analysis from next-generation sequencing data. In summary, this paper establishes the application of Mad7 in CHO cells, thereby improving the CRISPR toolbox versatility for research and cell line engineering.

Electrospinning of honey and propolis for wound care

Abstract: Phytochemicals and naturally derived compounds, such as plant extracts and bee products, are regarded as complementary and alternative medicines for the treatment of skin wounds, due to their antibacterial, anti‐inflammatory, and antioxidant properties. In recent years, it has been shown that dressings impregnated with honey (particularly Manuka honey) are effective for the topical treatment of wounds and burns, and some of them are currently used in clinics. This has stimulated the development of more advanced dressings based on polymeric nanofibres that can release honey and other bee products (like propolis) to promote wound healing. In this review, the current literature on the electrospinning of honey and propolis is analyzed and the effectiveness of the resulting dressings to inhibit bacterial growth and stimulate cellular proliferation and tissue repair is discussed.

Enhanced bioprocess control to advance the manufacture of mesenchymal stromal cell-derived extracellular vesicles in stirred-tank bioreactors.

Abstract: Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) act as signalling mediators of cellular responses. However, despite representing a promising alternative to cell-based therapies, clinical translation of EVs is currently limited by their lack of scalability and standardized bioprocessing. Herein, we integrated scalable downstream processing protocols with standardized expansion of large numbers of viable cells in stirred-tank bioreactors to improve EV production. Higher EV yields were linked to EV isolation by tangential flow filtration followed by size exclusion chromatography, rendering 5 times higher number of EVs comparatively to density gradient ultracentrifugation protocols. Additionally, when compared to static culture, EV manufacture in bioreactors resulted in 2.2 higher yields. Highlighting the role of operating under optimal cell culture conditions to maximize the number of EVs secreted per cell, MSCs cultured at lower glucose concentration favored EV secretion. While offline measurements of metabolites concentration can be performed, in this work, Raman spectroscopy was also applied to continuously track glucose levels in stirred-tank bioreactors, contributing to streamline the selection of optimal EV collection timepoints. Importantly, MSC-derived EVs retained their quality attributes and were able to stimulate angiogenesis in vitro, therefore highlighting their promising therapeutic potential. This article is protected by copyright. All rights reserved.

An adaptive soft‐sensor for advanced real‐time monitoring of an antibody‐drug conjugation reaction

Abstract: In the production of antibody‐drug conjugates (ADCs), the conjugation reaction is a central step defining the final product composition and, hence, directly affecting product safety and efficacy. To enable real‐time monitoring, spectroscopic sensors in combination with multivariate regression models have gained popularity in recent years. The extended Kalman filter (EKF) can be used as so‐called soft‐sensor to fuse sensor predictions with long‐horizon forecasts by process models. This enables the dynamic update of the current state and provides increased robustness against experimental noise or model errors. Due to the uncertainty associated with sensor and process models in biopharmaceutical applications, the deployment of such soft‐sensors is challenging. In this study, we demonstrate the combination of an uncertainty‐aware sensor model with a kinetic reaction model using an EKF to monitor a site‐directed ADC conjugation reaction. As the sensor model, a Gaussian process regression model is presented to realize a time‐variant determination of the sensor uncertainty. The EKF fuses the time‐discrete predictions of the amount of conjugated drug from the sensor model with the time‐continuous predictions from the kinetic model. While the ADC species are not distinguishable by on‐line recorded UV/Vis spectra, the developed soft‐sensor is able to dynamically update all relevant reaction species. It could be shown that the use of time‐variant process and sensor noise computation approaches improved the performance of the EKF and achieved a reduction of the prediction error of up to 23% compared with the kinetic model. The developed framework proved to enhance robustness against noisy sensor measurements or wrong model initialization and was successfully transferred from batch to fed‐batch mode. In future, this framework could be implemented for model‐based process control and be adopted for other ADC conjugation reaction types.

Correction to “Recent advances in yeast genome evolution with stress tolerance for green biological manufacturing”

Abstract: Xu, K., Zhang, Y.‐f., Guo, D.‐y., Qin, L., Ashraf, M., & Ahmad, N. (2022). Recent advances in yeast genome evolution with stress tolerance for green biological manufacturing. Biotechnology and Bioengineering, 119, 2689–2697. https://doi.org/10.1002/ bit.28183 In the original article, Xiao‐peng Gao, Jin‐wei Zhang, and Chun Li were inadvertently left off the author list. The correct author list is shown below: Jin‐wei Zhang, Ke Xu, Xiao‐peng Gao, Yun‐feng Zhang, Dong‐yu Guo, Lei Qin, Munaza Ashraf, Nadeem Ahmad and Chun Li Key Laboratory of Medical Molecule Science and Pharmaceutics Engineering, Ministry of Industry and Information Technology, Institute of Biochemical Engineering, School of Chemistry and Chemical Engineering, Beijing Institute of Technology, Beijing, People's Republic of China Key Laboratory for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing, People's Republic of China Key Laboratory of Agricultural Pathogenic Fungi and Toxins, Department of Life Science, Tangshan Normal University, Tangshan, People's Republic of China School of Life Science, Yan′an University, Shanxi, People's Republic of China Department of Zoology, University of Sargodha, Sargodha, Pakistan Department of Pharmacy, COMSATS University Islamabad, Abbottabad Campus, Abbottabad, Pakistan Note: Jin‐wei Zhang, Ke Xu, and Xiao‐peng Gao contributed equally to this study. Nadeem Ahmad (nadeemahmad@cuilahore.edu.pk) and Chun Li (lichun@tsinghua.edu.cn) are the corresponding authors. We apologize for this error.

The microRNomes of Chinese hamster ovary (CHO) cells and their extracellular vesicles, and how they respond to osmotic and ammonia stress

Abstract: A new area of focus in Chinese Hamster Ovary (CHO) biotechnology is the role of small (exosomes) and large (microvesicles or microparticles) extracellular vesicles (EVs). CHO cells in culture exchange large quantities of proteins and RNA through these EVs, yet the content and role of these EVs remain elusive. MicroRNAs (miRs) are central to adaptive responses to stress and more broadly to changes in culture conditions. Given that EVs are highly enriched in miRs, and that EVs release large quantities of miRs both in vivo and in vitro, EVs and their miR content likely play an important role in adaptive responses. Here we report the miRNA landscape of CHO cells and their EVs under normal culture conditions and under ammonia and osmotic stress. We show that both cells and EVs are highly enriched in five miRs (among over 600 miRs) that make up about half of their total miR content, and that these highly enriched miRs differ significantly between normal and stress culture conditions. Notable is the high enrichment in miR-92a and miR-23a under normal culture conditions, in contrast to the high enrichment in let-7 family miRs (let-7c, let-7b and let-7a) under both stress conditions. The latter suggests a preserved stress-responsive function of the let-7 miR family, one of the most highly preserved miR families across species, where among other functions, let-7 miRs regulate core oncogenes, which, depending on the biological context, may tip the balance between cell cycle arrest and apoptosis. While the expected –based on their profound enrichment – important role of these highly enriched miRs remains to be dissected, our data and analysis constitute an important resource for exploring the role of miRs in cell adaptation as well as for synthetic applications.This article is protected by copyright. All rights reserved.

Double‐reporter‐guided targeted activation of the oxytetracycline silent gene cluster in Streptomyces rimosus M527

Abstract: In Streptomyces rimosus M527, the oxytetracycline (OTC) biosynthetic gene cluster is not expressed under laboratory conditions. In this study a reported‐guided mutant selection (RGMS) procedure was used to activate the cluster. The double‐reporter plasmid pAGT was constructed in which gusA encoding a β‐glucuronidase and tsr encoding a thiostrepton resistance methyltransferase were placed under the control of the native promoter of oxyA gene (PoxyA). Plasmid pAGT was introduced and integrated into the chromosome of S. rimosus M527 by conjugation, yielding initial strain M527‐pAGT. Subsequently, mutants of M527‐pAGT were generated by using ribosome engineering technology. The mutants harboring activated OTC gene cluster were selected based on visual observation of GUS activity and thiostrepton resistance. Finally, mutant M527‐pAGT‐R7 was selected producing OTC in a concentration of 235.2 mg/L. In this mutant transcriptional levels of oxysr genes especial oxyAsr gene were increased compared to wild‐type strain S. rimosus M527. The mutant M527‐pAGT‐R7 showed antagonistic activities against Gram‐negative and Gram‐positive strains. All data indicate that the OTC gene cluster was successfully activated using the RGMS method.

Cover Image, Volume 120, Number 7, July 2023

Abstract: The cover image is based on the Research Article Three-dimensional co-culture model employing silica nonwoven fabrics to enhance cell-to-cell communication of paracrine signaling between hepatocytes and fibroblasts by Hidenori Otsuka et al., https://doi.org/10.1002/bit.28425.

Real-time monitoring of biomass during Escherichia coli high-cell-density cultivations by in-line photon density wave spectroscopy.

Abstract: An efficient monitoring and control strategy is the basis for a reliable production process. Conventional optical density (OD) measurements involve superpositions of light absorption and scattering, and the results are only given in arbitrary units. In contrast, photon density wave (PDW) spectroscopy is a dilution-free method that allows independent quantification of both effects with defined units. For the first time, PDW spectroscopy was evaluated as a novel optical process analytical technology tool for real-time monitoring of biomass formation in Escherichia coli high-cell-density fed-batch cultivations. Inline PDW measurements were compared to a commercially available inline turbidity probe and with offline measurements of OD and cell dry weight (CDW). An accurate correlation of the reduced PDW scattering coefficient µs ' with CDW was observed in the range of 5-69 g L-1 (R2 = 0.98). The growth rates calculated based on µs ' were comparable to the rates determined with all reference methods. Furthermore, quantification of the reduced PDW scattering coefficient µs ' as a function of the absorption coefficient µa allowed direct detection of unintended process trends caused by overfeeding and subsequent acetate accumulation. Inline PDW spectroscopy can contribute to more robust bioprocess monitoring and consequently improved process performance.

Back Cover Image, Volume 120, Number 4, April 2023

Abstract: The cover image is based on the Article Rapid prototyping enzyme homologs to improve titer of nicotinamide mononucleotide using a strategy combining cell-free protein synthesis with split GFP by Qingyan Yuan et al., https://doi.org/10.1002/bit.28326.

Protection of biomanufacturing processes from virus contamination through upstream virus filtration of cell culture media.

Abstract: Cell-based manufacturing processes have occasionally been exposed to adventitious viruses, leading to manufacturing interruptions and unstable supply situations. The rapid progress of advanced therapy medicinal products needs innovative approaches to avoid any unwelcome reminder of the universal presence of viruses. Here, we investigated upstream virus filtration as a clearance step for any product too complex for downstream interventions. Culture media virus filtration was investigated with respect to virus clearance capacities under extreme conditions such as high process feed loading (up to ~19,000 L/m²), long duration (up to 34 days), and multiple process interruptions (up to 21 h). The small nonenveloped Minute virus of mice was used as relevant target virus, and as worse-case challenge for the investigated virus filters with a stipulated pore-size of about 20 nm. Certain filters-especially of the newer second generation-were capable of effective virus clearance despite the harsh regimen they were subjected to. The biochemical parameters for un-spiked control runs showed the filters to have no measurable impact on the composition of the culture media. Based on these findings, this technology seems to be quite feasible for large volume premanufacturing process culture media preparations.