Showing papers in "Biotechnology and Bioengineering in 2021"

Scale-up economics for cultured meat

Abstract: This analysis examines the potential of "cultured meat" products made from edible animal cell culture to measurably displace the global consumption of conventional meat. Recognizing that the scalability of such products must in turn depend on the scale and process intensity of animal cell production, this study draws on technoeconomic analysis perspectives in industrial fermentation and upstream biopharmaceuticals to assess the extent to which animal cell culture could be scaled like a fermentation process. Low growth rate, metabolic inefficiency, catabolite inhibition, and shear-induced cell damage will all limit practical bioreactor volume and attainable cell density. Equipment and facilities with adequate microbial contamination safeguards have high capital costs. The projected costs of suitably pure amino acids and protein growth factors are also high. The replacement of amino-acid media with plant protein hydrolysates is discussed and requires further study. Capital- and operating-cost analyses of conceptual cell-mass production facilities indicate economics that would likely preclude the affordability of their products as food. The analysis concludes that metabolic efficiency enhancements and the development of low-cost media from plant hydrolysates are both necessary but insufficient conditions for displacement of conventional meat by cultured meat.

Metabolic engineering of Pichia pastoris for malic acid production from methanol.

Abstract: The application of rational design in reallocating metabolic flux to accumulate desired chemicals is always restricted by the native regulatory network. In this study, recombinant Pichia pastoris was constructed for malic acid production from sole methanol through rational redistribution of metabolic flux. Different malic acid accumulation modules were systematically evaluated and optimized in P. pastoris. The recombinant PP‐CM301 could produce 8.55 g/L malic acid from glucose, which showed 3.45‐fold increase compared to the parent strain. To improve the efficiency of site‐directed gene knockout, NHEJ‐related protein Ku70 was destroyed, whereas leading to the silencing of heterogenous genes. Hence, genes related to by‐product generation were deleted via a specially designed FRT/FLP system, which successfully reduced succinic acid and ethanol production. Furthermore, a key node in the methanol assimilation pathway, glucose‐6‐phosphate isomerase (g6pi) was knocked out to liberate metabolic fluxes trapped in the XuMP cycle, which finally enabled 2.79 g/L malic acid accumulation from sole methanol feeding with nitrogen source optimization. These results will provide guidance and reference for metabolic engineering of P. pastoris to produce value added chemicals from methanol. This article is protected by copyright. All rights reserved.

Advances in encapsulin nanocompartment biology and engineering.

Abstract: Compartmentalization is an essential feature of all cells. It allows cells to segregate and coordinate physiological functions in a controlled and ordered manner. Different mechanisms of compartmentalization exist, with the most relevant to prokaryotes being encapsulation via self-assembling protein-based compartments. One widespread example of such is that of encapsulins-cage-like protein nanocompartments able to compartmentalize specific reactions, pathways, and processes in bacteria and archaea. While still relatively nascent bioengineering tools, encapsulins exhibit many promising characteristics, including a number of defined compartment sizes ranging from 24 to 42 nm, straightforward expression, the ability to self-assemble via the Hong Kong 97-like fold, marked physical robustness, and internal and external handles primed for rational genetic and molecular manipulation. Moreover, encapsulins allow for facile and specific encapsulation of native or heterologous cargo proteins via naturally or rationally fused targeting peptide sequences. Taken together, the attributes of encapsulins promise substantial customizability and broad usability. This review discusses recent advances in employing engineered encapsulins across various fields, from their use as bionanoreactors to targeted delivery systems and beyond. A special focus will be provided on the rational engineering of encapsulin systems and their potential promise as biomolecular research tools.

A review of manufacturing capabilities of cell spheroid generation technologies and future development.

Abstract: Spheroid culture provides cells with a three-dimensional environment that can better mimic physiological conditions compared to monolayer culture. Technologies involved in the generation of cell spheroids are continuously being innovated to produce spheroids with enhanced properties. In this paper, we review the manufacturing capabilities of current cell spheroid generation technologies. We propose that spheroid generation technologies should enable tight and robust process controls to produce spheroids of consistent and repeatable quality. Future technology development for the generation of cell spheroids should look into improvement in process control, standardization, scalability and monitoring, in addition to advanced methods of spheroid transfer and characterization.

A CRISPR-Cas12a-derived biosensor enabling portable personal glucose meter readout for quantitative detection of SARS-CoV-2.

Abstract: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread rapidly throughout the whole world and caused significant difficulties in the prevention and control of the epidemic. In this case, several detection methods have been established based on nucleic acid diagnostic techniques and immunoassays to achieve sensitive and specific detection of SARS-CoV-2. However, most methods are still largely dependent on professional instruments, highly trained operators, and centralized laboratories. These limitations gravely diminish their practicality and portability. Herein, a clustered regularly interspaced short palindromic repeats (CRISPR) Cas12a based assay was developed for portable, rapid and sensitive of SARS-CoV-2. In this assay, samples were quickly pretreated and amplified by reverse transcription recombinase-aided amplification under mild conditions. Then, by combining the CRISPR Cas12a system and a glucose-producing reaction, the signal of the virus was converted to that of glucose, which can be quantitatively read by a personal glucose meter in a few seconds. Nucleocapsid protein gene was tested as a model target, and the sensitivity for quantitative detection was as low as 10 copies/μl, which basically meet the needs of clinical diagnosis. In addition, with the advantages of lower material cost, shorter detection time, and no requirement for professional instrument in comparison with quantitative reverse transcription-polymerase chain reaction, this assay is expected to provide a powerful technical support for the early diagnosis and intervention during epidemic prevention and control.

"High-risk" host cell proteins (HCPs): A multi-company collaborative view.

Abstract: Host cell proteins (HCPs) are process-related impurities that may copurify with biopharmaceutical drug products. Within this class of impurities there are some that are more problematic. These problematic HCPs can be considered high-risk and can include those that are immunogenic, biologically active, or enzymatically active with the potential to degrade either product molecules or excipients used in formulation. Some have been shown to be difficult to remove by purification. Why should the biopharmaceutical industry worry about these high-risk HCPs? What approach could be taken to understand the origin of its copurification and address these high-risk HCPs? To answer these questions, the BioPhorum Development Group HCP Workstream initiated a collaboration among its 26-company team with the goal of industry alignment around high-risk HCPs. The information gathered through literature searches, company experiences, and surveys were used to compile a list of frequently seen problematic/high-risk HCPs. These high-risk HCPs were further classified based on their potential impact into different risk categories. A step-by-step recommendation is provided for establishing a comprehensive control strategy based on risk assessments for monitoring and/or eliminating the known impurity from the process that would be beneficial to the biopharmaceutical industry.

The role of extracellular DNA in the formation, architecture, stability, and treatment of bacterial biofilms.

Abstract: Advances in biotechnology to treat and cure human disease have markedly improved human health and the development of modern societies. However, substantial challenges remain to overcome innate biological factors that thwart the activity and efficacy of pharmaceutical therapeutics. Until recently, the importance of extracellular DNA (eDNA) in biofilms was overlooked. New data reveal its extensive role in biofilm formation, adhesion, and structural integrity. Different approaches to target eDNA as anti-biofilm therapies have been proposed, but eDNA and the corresponding biofilm barriers are still difficult to disrupt. Therefore, more creative approaches to eradicate biofilms are needed. The production of eDNA often originates with the genetic material of bacterial cells through cell lysis. However, genomic DNA and eDNA are not necessarily structurally or compositionally identical. Variations are noteworthy because they dictate important interactions within the biofilm. Interactions between eDNA and biofilm components may as well be exploited as alternative anti-biofilm strategies. In this review, we discuss recent developments in eDNA research, emphasizing potential ways to disrupt biofilms. This review also highlights proteins, exopolysaccharides, and other molecules interacting with eDNA that can serve as anti-biofilm therapeutic targets. Overall, the array of diverse interactions with eDNA is important in biofilm structure, architecture, and stability.

Production of trimeric SARS-CoV-2 spike protein by CHO cells for serological COVID-19 testing.

Abstract: We describe scalable and cost‐efficient production of full length, His‐tagged severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) spike glycoprotein trimer by Chinese hamster ovary (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 human embryonic kidney (HEK) and CHO cells mediated by polyethyleneimine was increased significantly (up to 10.9‐fold) by a reduction in culture temperature to 32°C 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 enzyme‐linked immunosorbent assay format to detect immunoglobulin G antibodies against SARS‐CoV‐2 in sera from patient cohorts previously tested for viral infection by polymerase chain reaction, including those who had displayed coronavirus disease 2019 (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.

Optimising the biosynthesis of oxygenated and acetylated Taxol precursors in Saccharomyces cerevisiae using advanced bioprocessing strategies

Abstract: Taxadien‐5α‐hydroxylase and taxadien‐5α‐ol O‐acetyltransferase catalyse the oxidation of taxadiene to taxadien‐5α‐ol and subsequent acetylation to taxadien‐5α‐yl‐acetate in the biosynthesis of the blockbuster anti‐cancer drug, paclitaxel (Taxol®). Despite decades of research, the promiscuous and multispecific CYP725A4 enzyme remains a major bottleneck in microbial biosynthetic pathway development. In this study, an interdisciplinary approach was applied for the construction and optimisation of the early pathway in Saccharomyces cerevisiae, across a range of bioreactor scales. High‐throughput microscale optimisation enhanced total oxygenated taxane titre to 39.0±5.7 mg/L and total taxane product titres were comparable at micro and mini‐bioreactor scale at 95.4±18.0 and 98.9 mg/L, respectively. The introduction of pH control successfully mitigated a reduction of oxygenated taxane production, enhancing the potential taxadien‐5α‐ol isomer titre to 19.2 mg/L, comparable to the 23.8±3.7 mg/L achieved at microscale. A combination of bioprocess optimisation and increased GC‐MS resolution at 1L bioreactor scale facilitated taxadien‐5α‐yl‐acetate detection with a final titre of 3.7 mg/L. Total oxygenated taxane titres were improved 2.7‐fold at this scale to 78 mg/L, the highest reported titre in yeast. Critical parameters affecting the productivity of the engineered strain were identified across a range of scales, providing a foundation for the development of robust integrated bioprocess control systems. This article is protected by copyright. All rights reserved.

A genome-scale metabolic network model and machine learning predict amino acid concentrations in Chinese Hamster Ovary cell cultures.

Abstract: The control of nutrient availability is critical to large-scale manufacturing of biotherapeutics. However, the quantification of proteinogenic amino acids is time-consuming and thus is difficult to implement for real-time in situ bioprocess control. Genome-scale metabolic models describe the metabolic conversion from media nutrients to proliferation and recombinant protein production, and therefore are a promising platform for in silico monitoring and prediction of amino acid concentrations. This potential has not been realized due to unresolved challenges: (1) the models assume an optimal and highly efficient metabolism, and therefore tend to underestimate amino acid consumption, and (2) the models assume a steady state, and therefore have a short forecast range. We address these challenges by integrating machine learning with the metabolic models. Through this we demonstrate accurate and time-course dependent prediction of individual amino acid concentration in culture medium throughout the production process. Thus, these models can be deployed to control nutrient feeding to avoid premature nutrient depletion or provide early predictions of failed bioreactor runs.

Modeling of photosynthesis and respiration rate for microalgae‐bacteria consortia

Abstract: In this article, the influence of culture conditions (irradiance, temperature, pH, and dissolved oxygen) on the photosynthesis and the respiration rates of microalgae-bacteria consortia in wastewater treatment was analyzed. Specifically, some short photo-respirometric experiments, simulating outdoor raceway reactors, were performed to evaluate the response of microalgae, heterotrophic bacteria, and nitrifying bacteria to variations in environmental parameters. Results demonstrate that irradiance is the most dominant variable to determine microalgae photosynthesis rates. However, reduction in microalgae activity was not observed at higher irradiance, ruling out the existence of photoinhibition phenomena. Related to heterotrophic and nitrifying bacteria, their activities were strongly affected by the influence of temperature and pH. Moreover, the effect of dissolved oxygen concentrations on microalgae, and bacteria activities was studied, displaying a reduced photosynthetic rate at dissolved oxygen concentrations above 20 mg/L. Data have been used to develop an integrated model for each population (microalgae, heterotrophic bacteria, and nitrifying bacteria) based on considering the simultaneous influence of irradiance, temperature, pH, and dissolved oxygen. The models fit the experimental results in the range of culture conditions tested, and they were validated using data obtained by the simultaneous modifications of the variables. These individual models serve as a basis for developing a global biologic microalgae-bacteria model for wastewater treatment to improve the optimal design and management of microalgae-based processes, especially outdoors, where the cultures are subject to variable daily culture conditions.

Extracellular vesicles from organoids and 3D culture systems

Abstract: When discovered, extracellular vesicles (EVs) such as exosomes were thought of as junk carriers and a means by which the cell disposed of its waste material. Over the years, the role of EVs in cell communication has become apparent with the discovery that the nano-scale vesicles also transport RNA, DNA, and other bioactive components to and from the cells. These findings were originally made in EVs from body fluids of organisms and from in vitro two-dimensional (2D) cell culture models. Recently, organoids and other three-dimensional (3D) multicellular in vitro models are being used to study EVs in the context of both physiologic and pathological states. However, standard, reproducible methods are lacking for EV analysis using these models. As a step toward understanding the implications of these platforms, this review provides a comprehensive picture of the progress using 3D in vitro culture models for EV analysis. Translational efforts and regulatory considerations for EV therapeutics are also briefly overviewed to understand what is needed for scale-up and, ultimately, commercialization. This article is protected by copyright. All rights reserved.

Production and monitoring of biomass and fucoxanthin with brown microalgae under outdoor conditions.

Abstract: The effect of light on biomass and fucoxanthin (Fx) productivities was studied in two microalgae, Tisochrysis lutea and Phaeodactylum tricornutum. High and low biomass concentrations (1.1 and 0.4 g L-1 ) were tested in outdoor pilot-scale flat-panel photobioreactors at semi-continuous cultivation mode. Fluorescence spectroscopy coupled with chemometric modeling was used to develop prediction models for Fx content and for biomass concentration to be applied for both microalgae species. Prediction models showed high R2 for cell concentration (.93) and Fx content (.77). Biomass productivity was lower for high biomass concentration than low biomass concentration, for both microalgae (1.1 g L-1 : 75.66 and 98.14 mg L-1 d-1 , for T. lutea and P. tricornutum, respectively; 0.4 g L-1 : 129.9 and 158.47 mg L-1 d-1 , T. lutea and P. tricornutum). The same trend was observed in Fx productivity (1.1 g L-1 : 1.14 and 1.41 mg L-1 d-1 , T. lutea and P. tricornutum; 0.4 g L-1 : 2.09 and 1.73 mg L-1 d-1 , T. lutea and P. tricornutum). These results show that biomass and Fx productivities can be set by controlling biomass concentration under outdoor conditions and can be predicted using fluorescence spectroscopy. This monitoring tool opens new possibilities for online process control and optimization.

Ultrahigh-cell-density heterotrophic cultivation of the unicellular green alga Chlorella sorokiniana for biomass production.

Abstract: Heterotrophic cultivation of Chlorella has achieved commercial success, but the application of Chlorella biomass is still limited due to the high cost of biomass production. In this study, an effective and industrially scalable heterotrphic cultivation technology has been developed for a production strain Chlorella sorokiniana GT-1. Under the optimized culturing conditions, the ultrahigh biomass concentration of 271 and 247 g L-1 was achieved in 7.5 L bench-scale and 1000 L pilot-scale fermenters, respectively. Technoeconomic (TE) analysis indicated that the production cost of C. sorokiniana GT-1 could be reduced to $1601.27 per ton of biomass if the biomass concentration reached 200 g L-1 , which is 24.2% lower than that of the reported highest Chlorella biomass production through fermentation with the same TE model. Under the same growth conditions, the maximum biomass concentration of a low-starch mutant SLM2 was reduced to 93 g L-1 , which was 54% lower than that of the wild type, indicating the capabilities of C. sorokiniana GT-1 cells in accumulating large amounts of starch are essential for achieving the ultrahigh-cell-density under the heterotrophic conditions. In addition, the ultrahigh-cell-density growth potential of C. sorokiniana GT-1 cells was inferred to be related to the intrinsic biological characteristics including the tolerance to low dissolved oxygen and a moderate doubling time under the heterotrophic conditions as well. The breakthrough in cultivation technology is promising for Chlorella industry and would expand its applications in food and feed.

Transforming data to information: A parallel hybrid model for real-time state estimation in lignocellulosic ethanol fermentation.

Abstract: Operating lignocellulosic fermentation processes to produce fuels and chemicals is challenging due to the inherent complexity and variability of the fermentation media. Real-time monitoring is necessary to compensate for these challenges, but the traditional process monitoring methods fail to deliver actionable information that can be used to implement advanced control strategies. In this study, a hybrid-modeling approach is presented to monitor cellulose-to-ethanol (EtOH) fermentations in real-time. The hybrid approach uses a continuous-discrete extended Kalman filter to reconciliate the predictions of a data-driven model and a kinetic model and to estimate the concentration of glucose (Glu), xylose (Xyl), and EtOH. The data-driven model is based on partial least squares (PLS) regression and predicts in real-time the concentration of Glu, Xyl, and EtOH from spectra collected with attenuated total reflectance mid-infrared spectroscopy. The estimations made by the hybrid approach, the data-driven models and the internal model were compared in two validation experiments showing that the hybrid model significantly outperformed the PLS and improved the predictions of the internal model. Furthermore, the hybrid model delivered consistent estimates even when disturbances in the measurements occurred, demonstrating the robustness of the method. The consistency of the proposed hybrid model opens the doors towards the implementation of advanced feedback control schemes.

Production of β-carotene in Saccharomyces cerevisiae through altering yeast lipid metabolism.

Abstract: Saccharomyces cerevisiae is a widely used cell factory for the production of fuels and chemicals. However, as a non-oleaginous yeast, S. cerevisiae has a limited production capacity for lipophilic compounds, such as β-carotene. To increase its accumulation of β-carotene, we engineered different lipid metabolic pathways in a β-carotene producing strain and investigated the relationship between lipid components and the accumulation of β-carotene. We found that overexpression of sterol ester synthesis genes ARE1 and ARE2 increased β-carotene yield by 1.5-fold. Deletion of phosphatidate phosphatase (PAP) genes (PAH1, DPP1, and LPP1) also increased β-carotene yield by twofold. Combining these two strategies resulted in a 2.4-fold improvement in β-carotene production compared with the starting strain. These results demonstrated that regulating lipid metabolism pathways is important for β-carotene accumulation in S. cerevisiae, and may also shed insights to the accumulation of other lipophilic compounds in yeast.

Manufacturing a chimpanzee adenovirus-vectored SARS-CoV-2 vaccine to meet global needs.

Abstract: Manufacturing has been the key factor limiting rollout of vaccination during the COVID-19 pandemic, requiring rapid development and large-scale implementation of novel manufacturing technologies. ChAdOx1 nCoV-19 (AZD1222, Vaxzevria) is an efficacious vaccine against SARS-CoV-2, based upon an adenovirus vector. We describe the development of a process for the production of this vaccine and others based upon the same platform, including novel features to facilitate very large-scale production. We discuss the process economics and the "distributed manufacturing" approach we have taken to provide the vaccine at globally-relevant scale and with international security of supply. Together, these approaches have enabled the largest viral vector manufacturing campaign to date, providing a substantial proportion of global COVID-19 vaccine supply at low cost.

Scaffolds obtained from decellularized human extrahepatic bile ducts support organoids to establish functional biliary tissue in a dish

Abstract: Biliary disorders can lead to life-threatening disease and are also a challenging complication of liver transplantation. As there are limited treatment options, tissue engineered bile ducts could be employed to replace or repair damaged bile ducts. We explored how these constructs can be created by seeding hepatobiliary LGR5+ organoids onto tissue-specific scaffold. For this, we decellularized discarded human extrahepatic bile ducts (EBD) that we recellularized with organoids of different origin, that is, liver biopsies, extrahepatic bile duct biopsies, and bile samples. Here, we demonstrate efficient decellularization of EBD tissue. Recellularization of the EBD extracellular matrix (ECM) with the organoids of extrahepatic origin (EBD tissue and bile derived organoids) showed more profound repopulation of the ductal ECM when compared with liver tissue (intrahepatic bile duct) derived organoids. The bile duct constructs that were repopulated with extrahepatic organoids expressed mature cholangiocyte-markers and had increased electrical resistance, indicating restoration of the barrier function. Therefore, the organoids of extrahepatic sources are identified to be the optimal candidate for the development of personalized tissue engineered EBD constructs.

Functional expression of eukaryotic cytochrome P450s in yeast.

Abstract: Cytochrome P450 enzymes (P450s) are a superfamily of heme-thiolate proteins widely existing in various organisms. Due to their key roles in secondary metabolism, degradation of xenobiotics, and carcinogenesis, there is a great demand to heterologously express and obtain a sufficient amount of active eukaryotic P450s. However, most eukaryotic P450s are endoplasmic reticulum-localized membrane proteins, which is the biggest challenge for functional expression to high levels. Furthermore, the functions of P450s require the cooperation of cytochrome P450 reductases for electron transfer. Great efforts have been devoted to the heterologous expression of eukaryotic P450s, and yeasts, particularly Saccharomyces cerevisiae are frequently considered as the first expression systems to be tested for this challenging purpose. This review discusses the strategies for improving the expression and activity of eukaryotic P450s in yeasts, followed by examples of P450s involved in biosynthetic pathway engineering.

Dynamics of microbial competition, commensalism and cooperation and its implications for coculture and microbiome engineering

Abstract: Microbial consortium is a complex adaptive system with higher-order dynamic characteristics that are not present by individual members. To accurately predict the social interactions, we formulate a set of unstructured kinetic models to quantitatively capture the dynamic interactions of multiple microbial species. By introducing an interaction coefficient, we analytically derived the steady-state solutions for the interacting species and the substrate-depleting profile in the chemostat. We analyzed the stability of the possible coexisting states defined by competition, parasitism, amensalism, commensalism, and cooperation. Our model predicts that only parasitism, commensalism, and cooperation could lead to stable coexisting states. We also determined the optimal social interaction criteria of microbial coculture when sequential metabolic reactions are compartmentalized into two distinct species. Coupled with Luedeking-Piret and Michaelis-Menten equations, accumulation of metabolic intermediates in one species and formation of end-product in another species could be derived and assessed. We discovered that parasitism consortia disfavor the bioconversion of intermediate to final product; and commensalism consortia could efficiently convert metabolic intermediates to final product and maintain metabolic homeostasis with a broad range of operational conditions (i.e., dilution rates); whereas cooperative consortia leads to highly nonlinear pattern of precursor accumulation and end-product formation. The underlying dynamics and emergent properties of microbial consortia may provide critical knowledge for us to understand ecological coexisting states, engineer efficient bioconversion process, deliver effective gut therapeutics as well as elucidate probiotic-pathogen or tumor-host interactions in general.

Systematic identification of safe harbor regions in the CHO genome through a comprehensive epigenome analysis.

Abstract: The Chinese hamster ovary (CHO) cell lines that are used to produce commercial quantities of therapeutic proteins commonly exhibit a decrease in productivity over time in culture, a phenomenon termed production instability. Random integration of the transgenes encoding the protein of interest into locations in the CHO genome that are vulnerable to genetic and epigenetic instability often causes production instability through copy number loss and silencing of expression. Several recent publications have shown that these cell line development challenges can be overcome by using site-specific integration (SSI) technology to insert the transgenes at genomic loci, often called "hotspots," that are transcriptionally permissive and have enhanced stability relative to the rest of the genome. However, extensive characterization of the CHO epigenome is needed to identify hotspots that maintain their desirable epigenetic properties in an industrial bioprocess environment and maximize transcription from a single integrated transgene copy. To this end, the epigenomes and transcriptomes of two distantly related cell lines, an industrially relevant monoclonal antibody-producing cell line and its parental CHO-K1 host, were characterized using high throughput chromosome conformation capture and RNAseq to analyze changes in the epigenome that occur during cell line development and associated changes in system-wide gene expression. In total, 10.9% of the CHO genome contained transcriptionally permissive three-dimensional chromatin structures with enhanced genetic and epigenetic stability relative to the rest of the genome. These safe harbor regions also showed good agreement with published CHO epigenome data, demonstrating that this method was suitable for finding genomic regions with epigenetic markers of active and stable gene expression. These regions significantly reduce the genomic search space when looking for CHO hotspots with widespread applicability and can guide future studies with the goal of maximizing the potential of SSI technology in industrial production CHO cell lines.

Production of high-quality SARS-CoV-2 antigens: Impact of bioprocess and storage on glycosylation, biophysical attributes, and ELISA serologic tests performance.

Abstract: Serological assays are valuable tools to study SARS-CoV-2 spread and, importantly, to identify individuals that were already infected and would be potentially immune to a virus reinfection. SARS-CoV-2 Spike protein and its receptor binding domain (RBD) are the antigens with higher potential to develop SARS-CoV-2 serological assays. Moreover, structural studies of these antigens are key to understand the molecular basis for Spike interaction with angiotensin converting enzyme 2 receptor, hopefully enabling the development of COVID-19 therapeutics. Thus, it is urgent that significant amounts of this protein became available at the highest quality. In this study, we produced Spike and RBD in two human derived cell hosts: HEK293-E6 and Expi293F™. We evaluated the impact of different and scalable bioprocessing approaches on Spike and RBD production yields and, more importantly, on these antigens' quality attributes. Using negative and positive sera collected from human donors, we show an excellent performance of the produced antigens, assessed in serologic enzyme-linked immunosorbent assay (ELISA) tests, as denoted by the high specificity and sensitivity of the test. We show robust Spike productions with final yields of approx. 2 mg/L of culture that were maintained independently of the production scale or cell culture strategy. To the best of our knowledge, the final yield of 90 mg/L of culture obtained for RBD production, was the highest reported to date. An in-depth characterization of SARS-CoV-2 Spike and RBD proteins was performed, namely the antigen's oligomeric state, glycosylation profiles, and thermal stability during storage. The correlation of these quality attributes with ELISA performance show equivalent reactivity to SARS-CoV-2 positive serum, for all Spike and RBD produced, and for all storage conditions tested. Overall, we provide straightforward protocols to produce high-quality SARS-CoV-2 Spike and RBD antigens, that can be easily adapted to both academic and industrial settings; and integrate, for the first time, studies on the impact of bioprocess with an in-depth characterization of these proteins, correlating antigen's glycosylation and biophysical attributes to performance of COVID-19 serologic tests.

End-to-end continuous bioprocessing: impact on facility design, cost of goods and cost of development for monoclonal antibodies.

Abstract: This article presents a systematic approach to evaluate the business case for continuous processing that captures trade-offs between manufacturing and development costs for monoclonal antibodies (mAbs). A decisional tool was built that integrated cost of goods (COG) with cost of development models and new equipment sizing equations tailored to batch, hybrid and end-to-end continuous processes. The COG analysis predicted that single-use continuous facilities (sized using a dedicated DSP train per bioreactor) offer more significant commercial COG savings over stainless steel batch facilities at annual demands of 100-500 kg (~35%), compared to tonnage demands of 1-3 tons (~±10%) that required multiple parallel continuous trains. Single-use batch facilities were found to compete with continuous options on COG only at 100 kg/year. For the scenarios where batch and continuous facilities offered similar COG, the analysis identified the windows of operation required to reach different COG savings with thresholds for the perfusion rate, volumetric productivity and media cost. When considering the project lifecycle cost, the analysis indicated that while end-to-end continuous facilities may struggle to compete on development costs, they become more cost-effective than stainless steel batch facilities when considering the total out-of-pocket cost across both drug development and commercial activities. This article is protected by copyright. All rights reserved.

Pilot-scale demonstration of an end-to-end integrated and continuous biomanufacturing process.

Abstract: There has been increasing momentum recently in the biopharmaceutical industry to transition from traditional batch processes to next-generation integrated and continuous biomanufacturing. This transition from batch to continuous is expected to offer several advantages which, taken together, could significantly improve access to biologics drugs for patients. Despite this recent momentum, there has not been a commercial implementation of a continuous bioprocess reported in the literature. In this study, we describe a successful pilot-scale proof-of-concept demonstration of an end-to-end integrated and continuous bioprocess for the production of a monoclonal antibody (mAb). This process incorporated all of the key unit operations found in a typical mAb production process, including the final steps of virus removal filtration, ultrafiltration, diafiltration, and formulation. The end-to-end integrated process was operated for a total of 25 days and produced a total of 4.9 kg (200 g/day or 2 g/L BRX/day) of the drug substance from a 100-L perfusion bioreactor (BRX) with acceptable product quality and minimal operator intervention. This successful proof-of-concept demonstrates that end-to-end integrated continuous bioprocessing is achievable with current technologies and represents an important step toward the realization of a commercial integrated and continuous bioprocessing process.

Effect of alginate matrix engineered to mimic the pancreatic microenvironment on encapsulated islet function

Abstract: Islet transplantation is emerging as a therapeutic option for type 1 diabetes, albeit, only a small number of patients meeting very stringent criteria are eligible for the treatment because of the side effects of the necessary immunosuppressive therapy and the relatively short time frame of normoglycemia that most patients achieve. The challenge of the immune-suppressive regimen can be overcome through microencapsulation of the islets in a perm-selective coating of alginate microbeads with poly-l-lysine or poly- l-ornithine. In addition to other issues including the nutrient supply challenge of encapsulated islets a critical requirement for these cells has emerged as the need to engineer the microenvironment of the encapsulation matrix to mimic that of the native pancreatic scaffold that houses islet cells. That microenvironment includes biological and mechanical cues that support the viability and function of the cells. In this study, the alginate hydrogel was modified to mimic the pancreatic microenvironment by incorporation of extracellular matrix (ECM). Mechanical and biological changes in the encapsulating alginate matrix were made through stiffness modulation and incorporation of decellularized ECM, respectively. Islets were then encapsulated in this new biomimetic hydrogel and their insulin production was measured after 7 days in vitro. We found that manipulation of the alginate hydrogel matrix to simulate both physical and biological cues for the encapsulated islets enhances the mechanical strength of the encapsulated islet constructs as well as their function. Our data suggest that these modifications have the potential to improve the success rate of encapsulated islet transplantation.

Overview on immobilization of enzymes on synthetic polymeric nanofibers fabricated by electrospinning

Abstract: The arrangement of supports has a significant impact on the efficiency of immobilized enzymes. Fibrous materials can be one of the most desirable supports for enzyme immobilization. This is due to their high surface area to volume ratio, internal porosity, ease of handling, and high mechanical stability, all of which allow a higher enzyme loading, release and finally leads to better catalytic efficiency. Fortunately, the enzymes can reside deep inside individual nanofibers to remain encapsulated and retain their three-dimensional structure. These properties can protect the enzyme's tolerance against harsh conditions such as pH variations and high temperature, probably enhancing the enzyme's stability. This review article will discuss the immobilization of enzymes on synthetic polymers, which are fabricated into nanofibers by electrospinning. Electrospinning is rapidly gaining popularity as one of the most practical ways to make polymer, metal oxide, and composite fibers. As a result, there is interest in using electrospun nanofibers to immobilize enzymes. However, only a few methods can be used to examine the impact of nanofiber surfaces on the immobilized enzymes' behaviors. Furthermore, present research on electrospun nanofibers for enzyme immobilization is primarily limited to the lab scale and is still pending. The primary future research objectives in electrospun nanofibers for enzyme immobilization include increasing yield to transfer biological products into commercial applications. This article is protected by copyright. All rights reserved.

The usage of composite nanomaterials in biomedical engineering applications.

Abstract: Nanotechnology is still developing over the decades and it is commonly used in biomedical applications with the design of nanomaterials due to the several purposes. With the investigation of materials on the molecular level has increased the develop composite nanomaterials with exceptional properties using in different applications and industries. The application of these composite nanomaterials is widely used in the fields of textile, chemical, energy, defense industry, electronics, and biomedical engineering which is growing and developing on human health. Development of biosensors for the diagnosis of diseases, drug targeting and controlled release applications, medical implants and imaging techniques are the research topics of nanobiotechnology. In this review, overview of the development of nanotechnology and applications which is use of composite nanomaterials in biomedical engineering is provided.

Autofluorescence of blood and its application in biomedical and clinical research.

Abstract: Autofluorescence of blood has been explored as a label free approach for detection of cell types, as well as for diagnosis and detection of infection, cancer, and other diseases. Although blood autofluorescence is used to indicate the presence of several physiological abnormalities with high sensitivity, it often lacks disease specificity due to use of a limited number of fluorophores in the detection of several abnormal conditions. In addition, the measurement of autofluorescence is sensitive to the type of sample, sample preparation, and spectroscopy method used for the measurement. Therefore, while current blood autofluorescence detection approaches may not be suitable for primary clinical diagnosis, it certainly has tremendous potential in developing methods for large scale screening that can identify high risk groups for further diagnosis using highly specific diagnostic tests. This review discusses the source of blood autofluorescence, the role of spectroscopy methods, and various applications that have used autofluorescence of blood, to explore the potential of blood autofluorescence in biomedical research and clinical applications.

Plasticized poly(vinylalcohol) and poly(vinylpyrrolidone) based patches with tunable mechanical properties for cardiac tissue engineering applications.

Abstract: Polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) are the two most investigated biopolymers for various tissue engineering applications. However, their poor tensile strength renders them unsuitable for cardiac tissue engineering (CTE). In this study, we developed and evaluated PVA-PVP-based patches, plasticized with glycerol or propylene glycol (0.1 - 0.4%; v-v), for their application in CTE. The cardiac patches were evaluated for their physico-chemical (weight, thickness, folding endurance, FT-IR, swelling behaviour) and mechanical properties. The optimized patches were characterized for their ability to support in vitro attachment, viability, proliferation, and beating behaviour of neonatal mouse cardiomyocytes (CMs). In vivo evaluation of the cardiac patches was done under the sub-cutaneous skin pouch and heart of rat models. Results showed that the optimized molar ratio of PVA: PVP with plasticizers (0.3%; v-v) resulted in cardiac patches, which were dry at room temperature and had desirable folding endurance of at least 300, a tensile strength of 6-23 MPa and, percentage elongation at break of more than 250%. Upon contact with phosphate-buffered saline (PBS), these PVA-PVP patches formed hydrogel patches having the tensile strength of 1.3 to 3.0 MPa. The patches supported the attachment, viability, and proliferation of primary neonatal mouse CMs and were non-irritant and non-corrosive to cardiac cells. In vivo transplantation of cardiac patches into a subcutaneous pouch and on the heart of rat models revealed them to be biodegradable, biocompatible, and safe for use in CTE applications. This article is protected by copyright. All rights reserved.

Evaluating the effects of microparticle addition on mycelial morphology, natural yellow pigments productivity, and key genes regulation in submerged fermentation of Monascus purpureus.

Abstract: Morphology plays an important role in fungal fermentation and secondary metabolites biosynthesis. One novel technique, microparticle-enhanced cultivation was successfully utilized to control the morphology of Monascus purpureus precisely and enhance the yield of yellow pigments. The production of yellow pigments increased to 554.2 U/ml when 4 g/L 5000 mesh talc added at 24 h. Field emission scanning electron microscope observation indicated that the actual effect depends on the properties of microparticle. Sharp-edged microparticles showed better stimulatory effects than smooth, round-shaped ones. Particle size analysis, scanning electron microscope, and cell integrity evaluation proved obvious morphological changes were induced by talc addition, including smaller mycelial size, rougher hyphae, and decreased cell integrity. Furthermore, the expression levels of MrpigG, MrpigD, MrpigE, and MrpigH were significantly upregulated by the addition of talc. It indicated that the microparticle could not only affect the mycelial morphology, but also influence the expression levels of key genes in biosynthetic pathway of Monascus yellow pigments.