Search

The hydromechanical stress is a relevant parameter for mammalian cell cultivations, especially regarding scale-up processes. It describes the mechanical forces exerted on cells in a bioreactor. The maximum local energy dissipation rate is a suitable parameter to characterize hydromechanical stress. In literature, different studies deal with the effects of hydromechanical stress on CHO cells in stirred tank reactors. However, they often focus on lethal effects. Furthermore, systematic examinations in smaller scales like shake flasks are missing. Thus, this study systematically considers the influence of hydromechanical stress on CHO DP12 cells in shake flask cultivations. By utilizing online monitoring of the oxygen transfer rate, the study simplifies and enhances the resolution of examinations. Results indicate that while lethal effects are absent, numerous sub-lethal effects emerge with increasing hydromechanical stress: The process time is prolonged. The time of glucose and glutamine depletion, and the lactate switch correlate positively linear with the logarithmic average energy dissipation rate while the maximum specific growth rate correlates negatively. Strikingly, the final antibody concentration only declines at the highest tested average energy dissipation rate of 3.84 W kg−1 (only tested condition with a turbulent flow regime and therefore a higher maximal local energy dissipation rate) from about 250 mg L−1 to about 180 mg L−1. This study presents a straightforward method to examine the impact of hydromechanical stress in shake flasks, easily applicable to any other suspension cell line. Additionally, it offers valuable insights for scale-up processes, for example into stirred tank reactors. • CHO cells are influenced by varying hydromechanical stress levels in shake flasks. • Respiration activity online monitoring is a valuable tool for studying the influence. • Nutrient depletion is influenced by varying levels of hydromechanical stress. • The antibody titer only declines at a turbulent flow regime. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has brought global attention towards the readiness of biotechnology platforms for rapid development of immunogens to be used as vaccine antigens, in diagnostics and in fundamental research. In the present study we provide a bioprocess that is both robust and industry-compatible for high level expression, efficient purification, and scale-up of IMT-CVAX, a prefusion-stabilized trimeric spike protein of SARS-CoV-2. The recombinant IMT-CVAX protein was produced using stable cell pool of Chinese Hamster Ovary (CHO-S) cells that were banked in compliance with cGMP (Current Good Manufacturing Practices) regulations. High quantity expression of IMT-CVAX was achieved through optimization of the shake flask-based process with regards to media, nutrient feeding regimen, incubation temperature, viable cell density and cell viability. We employed refined expression procedures to scale up IMT-CVAX using bioreactor, resulting in a significantly higher yield of around 500 mg/L. Subsequently, a straightforward and simple purification process was developed to produce high-quality IMT-CVAX protein. This procedure consisted of centrifugation, tangential flow filtration (TFF), and either one- or two-step liquid chromatography. A robust anti-spike IgG response was observed in mice after immunization with purified adjuvanted IMT-CVAX. In pseudoviral neutralization assay, mice-generated anti-IMT-CVAX sera could neutralize various SARS-CoV-2 variants. The human convalescent sera from COVID-19-recovered patients recognized IMT-CVAX effectively, which confirms the conformational integrity of IMT-CVAX epitopes. The bioprocess demonstrated here is able to produce sufficient quantities of biopharmaceutical-quality spike protein, which can be used as immunogen against emerging SARS-CoV-2 variants, and can aid in the rapid response to any other pandemic-potential pathogens. [Display omitted] • Bioprocess for high level expression of recombinant, prefusion-stabilized trimeric spike protein (named IMT-CVAX) of SARS-CoV-2 in CHO cells. • Scale-up of IMT-CVAX expression using bioreactor and simple purification process using tangential flow filtration and liquid chromatography. • In mice, IMT-CVAX was found to have a high immunogenic response, and antisera was able to effectively neutralize SARS-Cov-2 variants. • IMT-CVAX maintains its stability at a variety of temperatures and has also been recognized by human convalescent sera. • IMT-CVAX has potential of clinical development of protein-subunit based vaccine against SARS-CoV-2. [ABSTRACT FROM AUTHOR]

Biomarkers are valuable tools in clinical research where they allow to predict susceptibility to diseases, or response to specific treatments. Likewise, biomarkers can be extremely useful in the biomanufacturing of therapeutic proteins. Indeed, constraints such as short timelines and the need to find hyper-productive cells could benefit from a data-driven approach during cell line and process development. Many companies still rely on large screening capacities to develop productive cell lines, but as they reach a limit of production, there is a need to go from empirical to rationale procedures. Similarly, during bioprocessing runs, substrate consumption and metabolism wastes are commonly monitored. None of them possess the ability to predict the culture behavior in the bioreactor. Big data driven approaches are being adapted to the study of industrial mammalian cell lines, enabled by the publication of Chinese hamster and CHO genome assemblies which allowed the use of next-generation sequencing with these cells, as well as continuous proteome and metabolome annotation. However, if these different -omics technologies contributed to the characterization of CHO cells, there is a significant effort remaining to apply this knowledge to biomanufacturing methods. The correlation of a complex phenotype such as high productivity or rapid growth to the presence or expression level of a specific biomarker could save time and effort in the screening of manufacturing cell lines or culture conditions. In this review we will first discuss the different biological molecules that can be identified and quantified in cells, their detection techniques, and associated challenges. We will then review how these markers are used during the different steps of cell line and bioprocess development, and the inherent limitations of this strategy. • Biomarkers characterize the physiological state of cells and help predict their behavior. • A biomarker can consist of any macromolecule present in the cell that can be detected and quantified. • Discovering and identifying biomarkers requires specific and customized methods. • In an industrial context, biomarkers enable a more rational and efficient approach to bioprocessing. • Biomarkers are often specific to environmental conditions and, as such, have limitations. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells, widely acknowledged as the preferred host system for industrial recombinant protein manufacturing, play a crucial role in developing pharmaceuticals, including anticancer therapeutics. Nevertheless, mammalian cell-based biopharmaceutical production methods are still beset by cellular constraints such as limited growth and poor productivity. MicroRNA-21 (miR-21) has a major impact on a variety of malignancies, including glioblastoma multiforme (GBM). However, reduced productivity and growth rate have been linked to miR-21 overexpression in CHO cells. The current study aimed to engineer a recombinant CHO (rCHO) cell using the CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) system coupled with the Bxb1 recombinase-mediated cassette exchange (RMCE) to express a circular miR-21 decoy (CM21D) with five bulged binding sites for miR-21 sponging. Implementing the ribonucleoprotein (RNP) delivery method, a landing pad was inserted into the genome utilizing the CRIS-PITCh technique. Subsequently, the CM21D cassette flanked by Bxb1 attB was then retargeted into the integrated landing pad using the RMCE/Bxb1 system. This strategy raised the targeting efficiency by 1.7-fold, and off-target effects were decreased. The miR-21 target genes (Pdcd4 and Atp11b) noticed a significant increase in expression upon the miR-21 sponging through CM21D. Following the expression of CM21D, rCHO cells showed a substantial decrease in doubling time and a 1.3-fold increase in growth rate. Further analysis showed an increased yield of hrsACE2, a secretory recombinant protein, by 2.06-fold. Hence, we can conclude that sponging-induced inhibition of miR-21 may lead to a growth rate increase that could be linked to increased CHO cell productivity. For industrial cell lines, including CHO cells, an increase in productivity is crucial. The results of our research indicate that CM21D is an auspicious CHO engineering approach. Key points: • CHO is an ideal host cell line for producing industrial therapeutics manufacturing, and miR-21 is downregulated in CHO cells, which produce recombinant proteins. • The miR-21 target genes noticed a significant increase in expression upon the miR-21 sponging through CM21D. Additionally, sponging of miR-21 by CM21D enhanced the growth rate of CHO cells. • Productivity and growth rate were increased in CHO cells expressing recombinant hrs-ACE2 protein after CM21D knocking in. [ABSTRACT FROM AUTHOR]

Copyright of CBÜ Beden Eğitimi & Spor Bilimleri Dergisi is the property of CBU Beden Egitimi & Spor Bilimleri Dergisi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Foam is generated in mammalian cell cultures by excessive agitation or gas sparging. This occurs particularly in cultures that generate recombinant proteins at high cell concentrations. Three antifoam agents were tested for their compatibility with antibody‐producing Chinese hamster ovary (CHO) cells. One agent (antifoam 204) was completely inhibitory to growth at a concentration of 10 ppm, one agent (antifoam C) showed partial inhibition and a third (antifoam SE‐15) showed no inhibition at this concentration. A novel foam image analyzer (LabCam) was used to evaluate two antifoams (C and SE‐15) for their ability to dissipate foam generated in cell culture media by enhanced agitation. The presence of antifoam in the media reduced significantly the foam layer that was generated and this was shown to be rapidly dissipated in the presence of 10 ppm SE‐15. The antifoams were also tested for foam dissipation in cultures of CHO cells at >106 cells/mL. Supplementation of the cultures with SE‐15 resulted in dissipation of foam generated by excessive gas sparging within 2 min. Under equivalent conditions 75% of foam dissipated in the presence of antifoam C, within 2 min but there was a residual foam layer up to 25 min. This study showed the value of an optical monitoring system (LabCam) for measuring foam generation and dissipation in a bioreactor to assess the efficiency of antifoam agents to reduce foam in a bioreactor. This has the potential for use as a control system that could be designed for continuous monitoring and foam control in a mammalian cell bioprocess. [ABSTRACT FROM AUTHOR]

Cell line development (CLD) represents a complex but highly critical process during the development of a biological drug. To shed light on this crucial workflow, a team of BioPhorum members (authors) has developed and executed surveys focused on the activities and effort involved in a typical CLD campaign. An average of 27 members from different companies that participate in the BioPhorum CLD working group answered surveys covering three distinguishable stages of a standard CLD process: (1) Pre‐transfection, including vector design and construction; (2) Transfection, spanning the initial introduction of vector into cells and subsequent selection and analysis of the pools; and (3) Single Cell Cloning and Lead Clone Selection, comprising methods of isolating single cells and confirming clonal origin, subsequent expansion and screening processes, and methods for identifying and banking lead clones. The surveys were very extensive, including a total of 341 questions split between antibody and complex molecule CLD processes. In this survey review, the authors interpret and highlight responses for antibody development and, where relevant, contrast complex molecule development challenges to provide a comprehensive industry perspective on the typical time and effort required to develop a CHO production cell line. [ABSTRACT FROM AUTHOR]

Age-related macular degeneration (AMD) is a degenerative, progressive disease, which destroys the central retina. AMD has two main forms - „dry“ and „wet“ (exudative). In order to make the diagnosis of AMD with CNV, apart from ophthalmoscopy, specialized tests are needed - fluorescein angiography (FA), indocyanine angiography, OCT, OCT-angiography (OCT-A). OCTA is a new and non-invasive technology. Compared to the old, well-known techniques for retinal evaluation, it is very precious because of its high resolution and detailed images – which allow early diagnosis and objective monitoring of the lesions in the future. The aim of this review is to describe the principles of function, features of the device and its application in everyday clinical practice in diagnosing patients with macular pathology. A systematic search of the literature, published in the past decade was identified from PubMed and Ovid databases with reference to the Preferred Reporting items for Systematic Review. AMD is a disease that is increasingly socially important because of the aging population in developed countries. The increasing use of OCT-A nowadays suggests new classification systems. The non-invasiveness, the short examination time and the high informativeness that OCT-A brings in relation to AMD requires a deeper study of the methodology and the development of new classifications to facilitate clinical practice.

Abstract Engineered mammalian cells are key for biotechnology by enabling broad applications ranging from in vitro model systems to therapeutic biofactories. Engineered cell lines exist as a population containing sub-lineages of cell clones that exhibit substantial genetic and phenotypic heterogeneity. There is still a limited understanding of the source of this inter-clonal heterogeneity as well as its implications for biotechnological applications. Here, we developed a genomic barcoding strategy for a targeted integration (TI)-based CHO antibody producer cell line development process. This technology provided novel insights about clone diversity during stable cell line selection on pool level, enabled an imaging-independent monoclonality assessment after single cell cloning, and eventually improved hit-picking of antibody producer clones by monitoring of cellular lineages during the cell line development (CLD) process. Specifically, we observed that CHO producer pools generated by TI of two plasmids at a single genomic site displayed a low diversity (

Cell engineering strategies typically rely on energy‐consuming overexpression of genes or radical gene‐knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine‐tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters. [ABSTRACT FROM AUTHOR]

The search for promising substances aimed at creating new chemotherapeutic drugs based on them is relevant in modern oncology. In this regard, new azoloazine derivatives, which are analogs of the well-known anticancer drug temozolomide, may be promising. The goal was to study the antitumor activity of 11 new azoloazine derivatives in vitro and in vivo. The MTT assay was used to assess the cytotoxic effect of the azoloazines on MCF-7 and CHO cells. BALB/c mice were orthotopically injected with 106 MCF-7 cells to simulate a xenogenic model of breast cancer. The test substances were administered at doses 1/2 IC50 once intraperitoneally 72 hours after tumor transplantation. The degree of differentiation and tumor extent in the tissue and the nature of metastasis were studied by analyzing histological sections of animals removed from the experiment. The volume of the primary tumor was measured. An evaluation of the cytotoxic activity of the new azoloazine derivatives showed that compounds 4, 6, and 9 were the most toxic to MCF-7 cells. Therefore, they were selected for cytotoxicity studies on normal cells (cultured CHO cells). Selected compounds 6 and 9 had low cytotoxicity against CHO cells, which was regarded as a positive aspect for their use as anticancer drugs. An in vivo experiment showed that administration of selected compounds 4, 6, and 9 led to more pronounced inhibition of tumor growth than the use of epirubicin alone. Histological analysis also revealed that compounds 4, 6, and 9 exhibited antitumor activity. The antitumor effects of the new azoloazine derivatives obtained in in vitro and in vivo experiments proved their high activity as potential chemotherapeutic agents and their prospects for further studies aimed at creating new antitumor drugs based on them. [ABSTRACT FROM AUTHOR]

Especially for the production of artificial, difficult to express molecules a further development of the CHO production cell line is required to keep pace with the continuously increasing demands. However, the identification of novel targets for cell line engineering to improve CHO cells is a time and cost intensive process. Since plasma cells are evolutionary optimized for a high antibody expression in mammals, we performed a comprehensive multi-omics comparison between CHO and plasma cells to exploit optimized cellular production traits. Comparing the transcriptome, proteome, miRNome, surfaceome and secretome of both cell lines identified key differences including 392 potential overexpression targets for CHO cell engineering categorized in 15 functional classes like transcription factors, protein processing or secretory pathway. In addition, 3 protein classes including 209 potential knock-down/out targets for CHO engineering were determined likely to affect aggregation or proteolysis. For production phenotype engineering, several of these novel targets were successfully applied to transient and transposase mediated overexpression or knock-down strategies to efficiently improve productivity of CHO cells. Thus, substantial improvement of CHO productivity was achieved by taking nature as a blueprint for cell line engineering. • Multi-omic cross species comparison of plasma and CHO cells = naturally vs. forced antibody producers. • Next generation cell line engineering based on systemic big data analyses (omic technologies). • Target validation for CHO cell line engineering via transposase technology and siRNA knock down screening. [ABSTRACT FROM AUTHOR]

N‐linked glycosylation is one of the most important post‐translational modifications of monoclonal antibodies (mAbs) and is considered to be a critical quality attribute (CQA), as the glycan composition often has immunomodulatory effects. Since terminal galactose residues of mAbs can affect antibody‐dependent cellular cytotoxicity (ADCC), complement‐dependent cytolysis (CDC) activation, serum half‐life, and antiviral activity it has to be monitored, controlled and modulated to ensure therapeutic effects. The ability of small noncoding microRNAs (miRNAs) to modulate glycosylation in Chinese hamster ovary (CHO) production cells was recently reported establishing miRNAs as engineering tools for modulation of protein glycosylation. In this study, we report the characterization and validation of miRNAs as engineering tools for increased (mmu‐miR‐452‐5p, mmu‐miR‐193b‐3p) or decreased (mmu‐miR‐7646‐5p, mmu‐miR‐7243‐3p, mmu‐miR‐1668, mmu‐let‐7c‐1‐3p, mmu‐miR‐7665‐3p, mmu‐miR‐6403) degree of galactosylation. Furthermore, the biological mode of action regulating gene expression of the galactosylation pathway was characterized as well as their influence on bioprocess‐related parameters. Most important, stable plasmid‐based overexpression of these miRNAs represents a versatile tool for engineering N‐linked galactosylation to achieve favorable phenotypes in cell lines for biopharmaceutical production. [ABSTRACT FROM AUTHOR]

Monoclonal antibody drugs have grown into a drug category with a market size of over $100 billion since the first product was launched on the market, which naturally creates a large demand for production. At the same time, the $100 billion market is distributed among more than 200 listed drugs, which indicates that the production demand for monoclonal antibody drugs is diverse. To meet this demand, major suppliers offer single-use bioreactors of all sizes. These single-use bioreactors with different specifications, especially the inconsistency of aeration pore sizes, pose great challenges for technology transfer and scale-up production, and the conventional scale-up strategies of constant Power input/volume ratio (P/V) and constant vessel volume per minute (vvm) can no longer meet the needs. This study simplified the selection of technical parameters in bioreactors based on the differences in aeration pore size. Innovatively combined the aeration pore sizes with initial aeration vvm, and comprehensively investigated the relationship between P/V, vvm and aeration pore size by designing experiments (DoE) using the orthogonal test method. The results showed a quantitative relationship between the aeration pore size and the initial aeration vvm in the P/V range of 20 ± 5 W/m3. The appropriate initial aeration was between 0.01 and 0.005 m3/min for aeration pore size ranging from 1 to 0.3 mm, which was the optimal incubation condition in the bioreactors. The choice of initial ventilation was most related to the final expression. Follow-up studies validated these findings in a 15 L glass bioreactor and a 500 L single-use bioreactor, and the results were consistent with expectations.

Introduction: In the past decade, microRNAs (miRNAs), which are small, noncoding RNAs of ?22 nucleotides length, have gained attention as novel engineering tools for biopharmaceutical cell line development. As miRNAs provide the ability to fine-tune the regulation of their targets, they offer attractive options for the development of differentially glycosylated monoclonal antibodies (mAbs) in production hosts like Chinese hamster ovary (CHO) cells. However, as one miRNA can potentially interact with several hundred gene transcripts, targeted miRNA mediated glycosylation regulation is complex to implement. This circumstance raises the need for computational aid in the prediction of miRNA targets. Method: In our study, we present a workflow using the target prediction tool RNA22 in combination with a comprehensive dataset of sequences for CHO cells, followed by curation of prediction results. Results: We created a knowledge-based database consisting of biochemically relevant genes for mAb N-glycosylation, to rationally process relevant results. Comparison to experimental data of target regulation unraveled the potential of our method to correctly predict 55 of 69 (79%) regulations caused by 16 different miRNAs known to affect mAb glycosylation. Conclusion: This work could potentially serve as a starting point for the development of new bioinformatics-assisted workflows to select miRNAs serving the user's exact needs.

Biologics represent the fastest growing group of therapeutics, but many advanced recombinant protein moieties remain difficult to produce. Here, we identify metabolic engineering targets limiting expression of recombinant human proteins through a systems biology analysis of the transcriptomes of CHO and HEK293 during recombinant expression. In an expression comparison of 24 difficult to express proteins, one third of the challenging human proteins displayed improved secretion upon host cell swapping from CHO to HEK293. Guided by a comprehensive transcriptomics comparison between cell lines, especially highlighting differences in secretory pathway utilization, a co-expression screening of 21 secretory pathway components validated ATF4, SRP9, JUN, PDIA3 and HSPA8 as productivity boosters in CHO. Moreover, more heavily glycosylated products benefitted more from the elevated activities of the N- and O-glycosyltransferases found in HEK293. Collectively, our results demonstrate the utilization of HEK293 for expression rescue of human proteins and suggest a methodology for identification of secretory pathway components for metabolic engineering of HEK293 and CHO.

Process Analytical Technologies (PATs) are taking a key role in the run for automatization in the biopharmaceutical industry. Spectroscopic methods such as Raman spectroscopy or mid‐infrared (MIR) spectroscopy are getting more recognition in the recent years for inline monitoring of bioprocesses due to their ability to measure various molecules simultaneously. However, their dependency on laborious model calibration making them a challenge to implement. In this study, a novel one‐point calibration that requires a single reference point prior to the inline monitoring of glucose and lactate in bioprocesses with MIR spectroscopy is assessed with 22 mammalian cell perfusion (PER) processes in two different scales and four different products. Concentrations are predicted over all PERs runs with a root mean square error (RMSE) of 0.29 g/L for glucose and 0.24 g/L for lactate, respectively. For comparison conventional partial least square regression (PLSR) models were used and trained with spectroscopic data from six bioreactor runs in two different scales and three products. The general accuracy of those models (RMSE of 0.41 g/L for glucose and 0.16 g/L for lactate) are in the range of the accuracy of the one‐point calibration. This shows the potential of the one‐point calibration as an approach making spectroscopy more accessible for bioprocess development. [ABSTRACT FROM AUTHOR]

Inconsistent yield of subtropical table grape across seasons is often associated with low carbohydrate reserves during flowering. In an attempt to increase TNC and thus yield, sucrose was injected into treated trunks during periods of high carbohydrate demand (i.e., between budburst and flowering). Total non-structural carbohydrate (TNC) concentration dynamics were assessed in the grapevine root and trunk tissues of both control and treated vines. In the control (untreated) vines, the TNC concentration in root and trunk tissues was 13.5% and 7.5% w/dw at leaf fall and 7.2% and 3.7% w/dw at flowering, respectively. This decrease in carbohydrate reserve was estimated at ~500 g/vine and is associated with the re-establishment of the plant canopy in early spring. Carbohydrate reserves remained stable or rose slightly between flowering and harvest and recovered between crop harvest and leaf fall. In treated vines, a constantly pressurised low-pressure in situ trunk injection system (69 kPa) with 5% w/v sucrose solution over 45 days from budburst (to flowering), in each of the two seasons, delivered a widely variable amount of sucrose into each vine with variation ascribed to the amount of internal dead wood in the trunk. In the best circumstances, an average of 150 g sucrose/vine/season was injected, and sucrose-injected vines had higher trunk TNC reserve (4.1% compared to 3.6% w/dw in the control) at flowering. A δ13C (‰) analysis confirmed the presence of injected sucrose in the shoot at flowering. However, the correlation between the amount of loaded sucrose and δ13C in young shoot tissue was poor, indicative of variable partitioning patterns. Inflorescence number per vine and berry yield were markedly higher in sucrose-injected vines, but differences were not significant given the high variation between vines. The addition of KCl to the sucrose solution and use of the healthy vines are recommended to increase sucrose loading using the injection method to address inconsistent yielding of subtropical table grape. [ABSTRACT FROM AUTHOR]

This report expresses a sustainable methodology for the in situ synthesis of Ag nanoparticles (NPs) adorned carboxymethyl lignin (CML) functionalized Fe3O4 nanocomposite (Fe3O4@CML/Ag) and its subsequent chemical and biological applications. Structural and physicochemical features of such novel material was evaluated through a number of analytical techniques like FT-IR, FE-SEM, TEM, EDS, XRD, ICP and VSM. The nanomaterial was a competent catalyst towards the N-acylation of diverse amines using acetic anhydride at solvent-free conditions. The material was isolated by magnet and reused for 8 consecutive times. Furthermore, the material was employed in the biological evaluation of anti-oxidant properties by DPPH method as well as the anticancer studies of against HT-29 and Caco-2 colon cell lines following MTT method. The DPPH assay afforded a significant IC50 value that suggested the material to have good anticancer properties. Interestingly, % cell viability of the malignant cell lines was found to decrease dose-dependently over Fe3O4@NaLS/Ag nanocomposite. MTT assay afforded the corresponding IC50 values to be 395.4 μg/ml and 287.3 μg/ml respectively against the two cell lines. The absorbance rate was evaluated at 545 nm, which represented toxicity on normal cell line (CHO) even up to 2000 μg/mL. [ABSTRACT FROM AUTHOR]

The ring-opening polymerization (ROP) of epoxides, namely cyclohexene oxide (CHO), propylene oxide (PO), and tert-butyl glycidyl ether (tBuGE) catalyzed by Group 13 (Al, Ga, In) metal initiators is reported. These polymerization reactions were performed under solvent-free conditions and in the presence of chloroform (CHCl3) and toluene. InCl3 was the most efficient catalyst for the polymerization in the presence of CHCl3, and monomer loading up to 10,000 equivalents. It was observed that though the polymerization rate was slow in the presence of a solvent, the polymerization was much more controlled than in solvent-free conditions, producing exceptionally high molecular weights with narrow dispersities. The highest molecular weight of the polymer attained was 136.3 kg/mol, with CHO as the monomer. The order of reactivity for the epoxides was found to be CHO > tBuGE > PO. The polymerization reaction proceeds through an activated chain-end mechanism as established from the MALDI-TOF analysis of the polymer. These polymers were found to be atactic. [ABSTRACT FROM AUTHOR]

Rare diseases are, despite their name, collectively common and millions of people are affected daily of conditions where treatment often is unavailable. Sulfatases are a large family of activating enzymes related to several of these diseases. Heritable genetic variations in sulfatases may lead to impaired activity and a reduced macromolecular breakdown within the lysosome, with several severe and lethal conditions as a consequence. While therapeutic options are scarce, treatment for some sulfatase deficiencies by recombinant enzyme replacement are available. The recombinant production of such sulfatases suffers greatly from both low product activity and yield, further limiting accessibility for patient groups. To mitigate the low product activity, we have investigated cellular properties through computational evaluation of cultures with varying media conditions and comparison of two CHO clones with different levels of one active sulfatase variant. Transcriptome analysis identified 18 genes in secretory pathways correlating with increased sulfatase production. Experimental validation by upregulation of a set of three key genes improved the specific enzymatic activity at varying degree up to 150-fold in another sulfatase variant, broadcasting general production benefits. We also identified a correlation between product mRNA levels and sulfatase activity that generated an increase in sulfatase activity when expressed with a weaker promoter. Furthermore, we suggest that our proposed workflow for resolving bottlenecks in cellular machineries, to be useful for improvements of cell factories for other biologics as well. • Unique transcriptomics data set of cultures of Sulfatase producing CHO clones. • Systemsbiology analysis pinpoint limiting factors in CHO secretional machinery. • Systemsbiology and metabolic engineering improve secreted activity 150-fold. • Lowering of transgene transcription improve product quality. [ABSTRACT FROM AUTHOR]

In this study, we investigated the effect of adenosine and its derivative cordycepin on the production yield of a recombinant human monoclonal antibody (adalimumab) in two commonly used Chinese Hamster ovary (CHO) cell lines that have different gene amplification systems, namely CHO‐DHFR− and GS‐CHO knockout (GS‐KO CHO) cells and that were grown in batch culture, with and without glucose feeding. The results showed that adenosine suppressed the cell growth rate and increased the fraction of cells in S phase of the cell cycle for both CHO cell lines. Different concentrations and exposure times of adenosine feeding were tested. The optimal yield of adalimumab production was achieved with the addition of 1 mM adenosine on day 2 after start of the batch culture. Adenosine could significantly improve antibody titers and productivity in both CHO cell lines in cultures without glucose feeding. However, upon glucose feeding, adenosine did not improve antibody titers in CHO‐DHFR− cells but extended culture duration and significantly increased antibody titers in GS‐KO CHO cells. Therefore, adenosine supplementation might be useful for antibody production in GS‐KO CHO cells in medium‐ to large‐scale batches. In case of cordycepin, a derivative of adenosine, CHO‐DHFR− cells required higher concentration of cordycepin than GS‐KO CHO cells around 10 times to display the changes in cell growth and cell cycle. Moreover, cordycepin could significantly increase antibody titers only in CHO‐DHFR− cell cultures without glucose feeding. [ABSTRACT FROM AUTHOR]

A large number of companies observe polysorbate (PS) degradation and associated (sub‐)visible particle formation in biological drug formulations, which compromise the stability of the drug product, ultimately posing a risk toward delivering innovative medicines to patients. The main culprits of PS degradation are hydrolytic host cell proteins (HCPs) originating from the production cell lines, which are mostly Chinese hamster ovary (CHO) cell derived. Here, a small portion of particularly difficult‐to‐remove HCPs—mainly lipases—cause hydrolytic cleavage of PS resulting in the accumulation of free fatty acid aggregates/particles. One possible mitigation strategy is the removal of such critical HCPs in the production cell line. Multigene regulation can be achieved via microRNAs (miRNAs) thereby serving as a smart tool to reduce the expression of different target genes using a single miRNA. To enable a tailored gene regulation of multiple specific target lipases self‐designed and non‐naturally occurring artificial miRNAs (amiRNA) can be designed. Based on micro‐conserved regions in the mRNA sequence of two sets of target HCPs, we provide a proof‐of‐concept for a simultaneous multi‐lipase knockdown in CHO cells using single amiRNAs. By this, we were not only able to reduce PS degradation but laid the foundation to expand this tool to other areas of cell line phenotype engineering. [ABSTRACT FROM AUTHOR]

Abstract Cell engineering strategies typically rely on energy‐consuming overexpression of genes or radical gene‐knock out. Both strategies are not particularly convenient for the generation of slightly modulated phenotypes, as needed in biosimilar development of for example differentially fucosylated monoclonal antibodies (mAbs). Recently, transiently transfected small noncoding microRNAs (miRNAs), known to be regulators of entire gene networks, have emerged as potent fucosylation modulators in Chinese hamster ovary (CHO) production cells. Here, we demonstrate the applicability of stable miRNA overexpression in CHO production cells to adjust the fucosylation pattern of mAbs as a model phenotype. For this purpose, we applied a miRNA chaining strategy to achieve adjustability of fucosylation in stable cell pools. In addition, we were able to implement recently developed artificial miRNAs (amiRNAs) based on native miRNA sequences into a stable CHO expression system to even further fine‐tune fucosylation regulation. Our results demonstrate the potential of miRNAs as a versatile tool to control mAb fucosylation in CHO production cells without adverse side effects on important process parameters.

This article discusses the interpretation of “yomachi” in the English translation of The Tale of Genji. “Yomachi” describes the size of the house Rokujōin, one of the houses in the story. The construction of Rokujō-in is completed in “the 21st chapter, Otome” volume. To make it easier to visualize the house’s vastness, English translations of books are heavily annotated, but the English translation and the notes show a transition and are not consistent. Heian-kyō was laid out in a grid pattern based on what was called the Jōbō City System. In the Jōbō City System, “yomachi” is a block unit. The “yomachi” that Arthur Waley used in his English-language edition was based on the Japanese Weights and Measurement Law at the time he translated it, likely because the status of Japanese units, which had changed over time, had not been communicated. This story, established in the Heian period, is written in hiragana, and the word is read as “yomachi.” It is important to clearly understand the size of the house Rokujō-in, built by the protagonist, Genji, because the story tells of the actual rituals and events that took place in the aristocratic society at that time.

Chinese hamster ovary (CHO) cells are the primary host for manufacturing of therapeutic proteins. However, productivity loss is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein expression. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide variants in DNA repair genes. Comparison with primary Chinese hamster cells confirmed DNA repair to be compromised in CHO. Correction of key DNA repair genes by single-nucleotide variant reversal or expression of intact complementary DNAs successfully improved DNA repair and mitigated karyotypic instability. Moreover, overexpression of intact copies of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show that correction of DNA repair genes yields improvements in genome stability in CHO, and provide new opportunities for cell line development for sustainable protein expression.

Abstract Process Analytical Technologies (PATs) are taking a key role in the run for automatization in the biopharmaceutical industry. Spectroscopic methods such as Raman spectroscopy or mid‐infrared (MIR) spectroscopy are getting more recognition in the recent years for inline monitoring of bioprocesses due to their ability to measure various molecules simultaneously. However, their dependency on laborious model calibration making them a challenge to implement. In this study, a novel one‐point calibration that requires a single reference point prior to the inline monitoring of glucose and lactate in bioprocesses with MIR spectroscopy is assessed with 22 mammalian cell perfusion (PER) processes in two different scales and four different products. Concentrations are predicted over all PERs runs with a root mean square error (RMSE) of 0.29 g/L for glucose and 0.24 g/L for lactate, respectively. For comparison conventional partial least square regression (PLSR) models were used and trained with spectroscopic data from six bioreactor runs in two different scales and three products. The general accuracy of those models (RMSE of 0.41 g/L for glucose and 0.16 g/L for lactate) are in the range of the accuracy of the one‐point calibration. This shows the potential of the one‐point calibration as an approach making spectroscopy more accessible for bioprocess development.

Chinese hamster ovary (CHO) cells have a long history in the biopharmaceutical industry and currently produce the vast majority of recombinant therapeutic proteins. A key step in controlling the process and product consistency is the development of a producer cell line derived from a single cell clone. However, it is recognized that genetic and phenotypic heterogeneity between individual cells in a clonal CHO population tends to arise over time. Previous bulk analysis of CHO cell populations revealed considerable variation within the mtDNA sequence (heteroplasmy), which could have implications for the performance of the cell line. By analyzing the heteroplasmy of single cells within the same population, this heterogeneity can be characterized with greater resolution. Such analysis may identify heterogeneity in the mitochondrial genome, which impacts the overall phenotypic performance of a producer cell population, and potentially reveal routes for genetic engineering. A critical first step is the development of robust experimental and computational methods to enable single cell mtDNA sequencing (termed scmtDNAseq). Here, we present a protocol from cell culture to bioinformatic analysis and provide preliminary evidence of significant mtDNA heteroplasmy across a small panel of single CHO cells.

Culture pH is a critical process parameter during CHO cell bioreactor operations that is key for proper cell growth, protein production, and maintaining the critical quality attributes of a monoclonal antibody drug substance. The traditional means of measuring pH in bioreactors is with an electrochemical probe that can withstand and maintain accuracy through repeated sterilization cycles. An alternative technique for measuring pH is an optical sensor composed of a fluorescent dye that is sensitive to the hydrogen ion concentration. In this work we explore single-use electrochemical and single-use optical pH sensors in stirred-tank and rocking bioreactors, respectively, to understand how their overall performance compares to traditional electrochemical probes in benchtop glass stirred tank bioreactors. We found that the single-use optical pH sensors were generally less accurate than the electrochemical probes, especially in detecting large pH drifts from the setpoint. The single-use electrochemical probes were increasingly accurate as pH was increased from

Chinese hamster ovary (CHO) cells are a popular choice in biopharmaceuticals because of their beneficial traits, including high-density suspension culture, safety, and exogenously produced proteins that closely resemble natural proteins. Nevertheless, a decline in the expression of exogenous proteins is noted as culture time progresses. This is a consequence of foreign gene recombination into chromosomes by random integration. The current investigation employs CRISPR-Cas9 technology to integrate foreign genes into a particular chromosomal location for sustained expression. Results demonstrate the successful integration of enhanced green fluorescent protein (EGFP) and human serum albumin (HSA) near base 434814407 on chromosome NC_048595.1 of CHO-K1 cells. Over 60 successive passages, monoclonal cell lines were produced that consistently expressed all relevant external proteins without discernible variation in expression levels. In conclusion, the CHO-K1 cell locus, NC_048595.1, proves an advantageous locus for stable exogenous protein expression. This study provides a viable approach to establishing a CHO cell line capable of enduring reliable exogenous protein expression. [ABSTRACT FROM AUTHOR]

Background: Prior implementations of the channelized Hotelling observer (CHO) model have succeeded in assessing the performance of X‐ray angiography systems under a variety of imaging conditions. However, often times these conditions do not resemble those present in routine clinical imaging scenarios, such as having complex anthropomorphic backgrounds in conjunction with moving test objects. Purpose: This work builds up on prior established CHO methods and introduces a new approach to switch from the already established "multiple‐sample" CHO implementation to a "single‐sample" technique. The proposed implementation enables the inclusion of moving test objects upon nonuniform backgrounds by allowing only a single sample to represent the test object present condition that is to be used within the statistical test to estimate the detectability index. Methods: To assess the proposed method, two image data sets were acquired with a clinical X‐ray angiography system. The first set consisted of a uniform background in combination with static test objects while the second consisted of an anthropomorphic chest phantom in conjunction with moving test objects. The first set was used to validate the proposed approach against the multiple‐sample method while the second was used to assess the feasibility of the proposed method under a variety of imaging conditions, including seven object sizes and seven detector target dose (DTD) levels. Results: For the uniform background data set, considering all detectability indices greater or equal than 1, the ratio between the detectability indices of the proposed single‐sample approach versus the multiple‐sample method was 0.997 ± 0.056 (range 0.884–1.159). The average single‐direction width of the 95% confidence intervals (CIs) of the detectability index estimates for the multiple‐sample method was 0.38 ± 0.43 (range 0.03–2.20). For the single‐sample approach, the average width was 2.52 ± 0.63 (range 1.11–5.44). For the anthropomorphic background image set, the results were consistent with classical quantum‐limited signal‐to‐noise ratio (SNR) theory. The magnitude of the detectability indices varied predictably with changes in both object size and DTD, with the highest value associated with the highest dose and the largest object size. Additionally, the proposed method was able to capture differences in the imaging performance for a given test object across the field of view, which was associated with the attenuation levels provided by different features of the anthropomorphic background. Conclusions: A new single‐sample variant of the CHO model to assess the performance of X‐ray angiography imaging systems is proposed. The new approach is consistent with quantum‐limited image quality theory and with a standard implementation of the CHO model. The proposed method enables the assessment of moving test objects in combination with complex, nonuniform image backgrounds, thereby opening the possibility to assess imaging conditions which more closely resemble those used in clinical care. [ABSTRACT FROM AUTHOR]

Stable, highly productive mammalian cells are critical for manufacturing affordable and effective biological medicines. Establishing a rational design of optimal biotherapeutic expression systems requires understanding how cells support the high demand for efficient biologics production. To that end, we performed transcriptomics and high‐throughput imaging studies to identify putative genes and morphological features that underpin differences in antibody productivity among clones from a Chinese hamster ovary cell line. During log phase growth, we found that the expression of genes involved in biological processes related to cellular morphology varied significantly between clones with high specific productivity (qP > 35 pg/cell/day) and low specific productivity (qP < 20 pg/cell/day). At Day 10 of a fed‐batch production run, near peak viable cell density, differences in gene expression related to metabolism, epigenetic regulation, and proliferation became prominent. Furthermore, we identified a subset of genes whose expression predicted overall productivity, including glutathione synthetase (Gss) and lactate dehydrogenase A (LDHA). Finally, we demonstrated the feasibility of cell painting coupled with high‐throughput imaging to assess the morphological properties of intracellular organelles in relation to growth and productivity in fed‐batch production. Our efforts lay the groundwork for systematic elucidation of clone performance using a multiomics approach that can guide future process design strategies. [ABSTRACT FROM AUTHOR]

Media and feed optimization have fueled many-fold improvements in mammalian biopharmaceutical production, but genome editing offers an emerging avenue for further enhancing cell metabolism and bioproduction. However, the complexity of metabolism, involving thousands of genes, makes it unclear which engineering strategies will result in desired traits. Here we present a comprehensive pooled CRISPR screen for CHO cell metabolism, including ~16,000 gRNAs against ~2500 metabolic enzymes and regulators. Using this screen, we identified a glutamine response network in CHO cells. Glutamine is particularly important since it is often over-fed to drive increased TCA cycle flux, but toxic ammonia may accumulate. With the screen we found one orphan glutamine-responsive gene with no clear connection to our network. Knockout of this novel and poorly characterized lipase, Abhd11, substantially increased growth in glutamine-free media by altering the regulation of the TCA cycle. Thus, the screen provides an invaluable targeted platform to comprehensively study genes involved in any metabolic trait, and elucidate novel regulators of metabolism.

During the development of traditional Chinese hamster ovary (CHO) cell lines, target genes randomly integrate into the genome upon entering the nucleus, resulting in unpredictable productivity of cell clones. The characterization and screening of high-yielding cell lines is a time-consuming and expensive process. Site-specific integration is recognized as an effective approach for overcoming random integration and improving production stability. We have designed a multifunctional expression cassette, called CDbox, which can be manipulated by the site-specific recombination systems Cre/lox and Dre/rox. The CDbox expression cassette was inserted at the Hipp11(H11) locus hotspot in the CHO-K1 genome using CRISPR/Cas9 technology, and a compliant CHO-CDbox cell platform was screened and obtained. The CHO-CDbox cell platform was transformed into a pool of EGFP-expressing cells using Cre/lox recombinase-mediated cassette exchange (RMCE) in only 2 weeks, and this expression remained stable for at least 75 generations without the need for drug stress. Subsequently, we used the Dre/rox system to directly eliminate the EGFP gene. In addition, two practical applications of the CHO-CDbox cell platform were presented. The first was the quick construction of the Pembrolizumab antibody stable expression strain, while the second was a protocol for the integration of surface-displayed and secreted antibodies on CHO cells. The previous research on site-specific integration of CHO cells has always focused on the single functionality of insertion of target genes. This newly developed CHO cell platform is expected to offer expanded applicability for protein production and gene function studies.

ABSTRACTIntrons are included in genes encoding therapeutic proteins for their well-documented function of boosting expression. However, mis-splicing of introns in recombinant immunoglobulin (IgG) heavy chain (HC) transcripts can produce amino acid sequence product variants. These variants can affect product quality; therefore, purification process optimization may be needed to remove them, or if they cannot be removed, then in-depth characterization must be carried out to understand their effects on biological activity. In this study, HC transgene engineering approaches were investigated and were successful in significantly reducing the previously identified IgG HC splice variants to

Sphingosine 1-phosphate (S1P) is one of the lipid mediators involved in diverse physiological functions. S1P circulates in blood and lymph bound to carrier proteins. Three S1P carrier proteins have been reported, albumin, apolipoprotein M (ApoM) and apolipoprotein A4 (ApoA4). The carrier-bound S1P exerts its functions via specific S1P receptors (S1PR1-5) on target cells. Previous studies showed several differences in physiological functions between albumin-bound S1P and ApoM-bound S1P. However, molecular mechanisms underlying the carrier-dependent differences have not been clarified. In addition, ApoA4 is a recently identified S1P carrier protein, and its functional differences from albumin and ApoM have not been addressed. Here, we compared the three carrier proteins in the processes of S1P degradation, release from S1P-producing cells and receptor activation. ApoM retained S1P more stable than albumin and ApoA4 in the cell culture medium when compared in the equimolar amounts. ApoM facilitated theS1P release from endothelial cells most efficiently. Furthermore, ApoM-bound S1P showed a tendency to induce prolonged activation of Akt via S1PR1 and S1PR3. These results suggest that the carrier-dependent functional differences of S1P are partly ascribed to the differences in the S1P stability, S1P-releasing efficiency and signaling duration. [ABSTRACT FROM AUTHOR]

The use of transposase in cell line development (CLD) programs has experienced increased popularity over the past decade. However, few studies have described the mechanism of action and the genomic and phenotypic characteristics of clones derived from transposase. Additionally, how these traits impact long-term bioproduction is unknown. Here, we use chromosome painting, deep sequencing, and ddPCR to characterize the unique fingerprints associated with transposase-derived clones. Transposase reduces the cellular pool of transient vector as early as three days post transfection following transfection and expedites stable pool establishment by up to two weeks. Furthermore, recombinant DNA expression is significantly improved up to ∼3 fold along with a greater balance of antibody heavy and light chain transcripts, resulting in higher titers in transposase generated pools. Transposase derived pools contained an often innumerable number of integration sites, representing a vast increase in integration site diversity over randomly generated pools, which were bottlenecked at 1–3 integration sites per pool. These transposase mediated integrations typically occurred in clean singlets, free of genomic scars such as deletions, inversions, and other modifications associated with legacy transfection methods which exhibited higher copy numbers per integration site. Relative declines in gene expression occur with copy number increase in the randomly generated, but not the transposase derived clones. Furthermore, transposase-derived clones were more likely to exhibit enhanced a long term stability profile, including product quality attributes such as mannose-5. This improved stability may result from circumventing mechanisms associated with the silencing of tandem repeats. Thus, transposase-mediated approaches can provide multifaceted molecular and phenotypic advantages in cell line development when compared to legacy random-integration methods. • Improved productivity and stability of product quality during cell line development using transposase. • Interrogation of integration sites and their effect on the genome in recombinant bioproduction cell lines. • Chromosome painting and transgene locus amplification (TLA) for genetic characterization of bioproduction cell lines. • Mechanisms and strategies to prevent transgenic silencing during cell line development. [ABSTRACT FROM AUTHOR]

The accumulation of metabolic wastes in cell cultures can diminish product quality, reduce productivity, and trigger apoptosis. The limitation or removal of unintended waste products from Chinese hamster ovary (CHO) cell cultures has been attempted through multiple process and genetic engineering avenues with varied levels of success. One study demonstrated a simple method to reduce lactate and ammonia production in CHO cells with adaptation to extracellular lactate; however, the mechanism behind adaptation was not certain. To address this profound gap, this study characterizes the phenotype of a recombinant CHO K-1 cell line that was gradually adapted to moderate and high levels of extracellular lactate and examines the genomic content and role of extrachromosomal circular DNA (eccDNA) and gene expression on the adaptation process. More than 500 genes were observed on eccDNAs. Notably, more than 1000 genes were observed to be differentially expressed at different levels of lactate adaptation, while only 137 genes were found to be differentially expressed between unadapted cells and cells adapted to grow in high levels of lactate; this suggests stochastic switching as a potential stress adaptation mechanism in CHO cells. Further, these data suggest alanine biosynthesis as a potential stress-mitigation mechanism for excess lactate in CHO cells. [ABSTRACT FROM AUTHOR]

Despite advances in genetic characterization of Chinese hamster ovary (CHO) cell lines regarding identification of integration sites using next generation sequencing, e.g. targeted locus amplification sequencing (TLA-seq), the concatemer structure of the integrated vectors remains elusive. Here, the entire integration locus of two CHO manufacturing cell lines was reconstructed combining CRISPR/Cas9 target enrichment, nanopore sequencing and the Canu de novo assembly pipeline. An IgG producing CHO cell line integrated 3 vector copies, which were near full-length and contained all relevant vector elements such as transgenes and their promoters on each of the vector copies. In contrast, a second CHO cell line producing a bivalent bispecific antibody integrated 7 highly fragmented vector copies in different orientations leading to head-to-head and tail-to-tail fusions. The size of the vector fragments ranged from 3.0 to 11.4 kbp each carrying 1–3 transgenes. The breakpoints of the genome-vector and vector-vector junctions were validated using Sanger sequencing and Southern blotting. A comparison to TLA-seq data confirmed the genomic breakpoints, but most of the breakpoints of the vector-vector fusions were missed by TLA-seq. For the first time, the complete transgene locus of CHO manufacturing cell lines could be deciphered. Strikingly, the application of the nanopore long-read sequencing technology led to novel insights into the complexity of genomic transgene integrations of CHO manufacturing cell lines generated via random integration. • Nanopore sequencing resolves complete concatemer structure of randomly integrated vectors. • Assembly of concatemer structure reveals highly fragmented and depleted genetic architecture. • Cas9-based enrichment leads to 37-fold increase of expression vector coverage. • Complete concatemer structure allows for a thorough interpretation of complex Southern blot data. [ABSTRACT FROM AUTHOR]

Chinese hamster ovary (CHO) cells are the preferred workhorse for the biopharmaceutical industry, and CRISPR/Cas9 has proven powerful for generating targeted gene perturbations in CHO cells. Here, we expand the CRISPR engineering toolbox with CRISPR activation (CRISPRa) to increase transcription of endogenous genes. We successfully increased transcription of Mgat3 and St6gal1, and verified their activity on a functional level by subsequently detecting that the appropriate glycan structures were produced. This study demonstrates that CRISPRa can make targeted alterations of CHO cells for desired phenotypes.

Abstract Background The global extant literature acknowledge that housing serves as a key social determinant of health. Housing interventions that involve group homes have been found to support the recovery of persons with mental illness and those with addiction issues. The current study explored the views of homeowners in relation to a supportive housing program called Community Homes for Opportunity (CHO) that modernised a provincial group home program (Homes for Special Care [HSC]) and provided recommendations for improving the program implementation in other geographical areas of Ontario. Methods We applied ethnographic qualitative techniques to purposefully recruit 36 homeowner participants from 28 group homes in Southwest Ontario, Ontario Canada. Focus group discussions were conducted at two time points, during CHO program implementation (Fall 2018, and post implementation phases (Winter 2019) respectively. Results Data analysis yielded 5 major themes. These include: (1) general impressions about the modernization process, (2) perceived social, economic and health outcomes, (3) enablers of the modernization program, (4) challenges to implementation of the modernization program, and (5) suggestions for implementation of the CHO in future. Conclusions A more effective and expanded CHO program will need the effective collaboration of all stakeholders including homeowners for successful implementation.

Abstract Background Recovery-oriented programs provide individuals with opportunities for well-being through community integration processes that enhance the degree to which individuals could live, work, and recreate in their community. The current evaluation assessed how tenants experience their home environment after the modernization of Homes for Special Care (HSC) to Community Homes for Opportunity (CHO) in Southwest Ontario, Canada. Our study identifies existing policies and practices that could interfere with or promote the modernization process. Methods We applied ethnographic qualitative techniques to purposefully recruit 188 participants with severe mental illness from 28 group homes. Focus groups were conducted at three time points, i.e., at pre-implementation/Baseline/Time I – spring 2018; Transition/Time II – fall 2018, and Final/Time III – winter 2019. Results Study findings suggest that the transition of HSC to CHO supports activities that empower tenants towards personal growth and development. Participants were largely satisfied with the support they were getting in relation to the program-related services. Tenants disclosed that their quality of life and well-being had been enhanced through participating in the program, and that their social interaction and support for each other had also improved. Most tenants demonstrated autonomy in terms of personal and financial independence. The enhanced financial support for tenants did not only improve their quality of life, but also helped to raise their purchasing power, decision making, sense of responsibility and accountability towards healthy spending of their resources. Despite tenants’ good impression about the CHO, some still encountered problems and provided suggestions to further improve the program. Conclusion It is expected that a more effective and expanded CHO will lead to tenant empowerment and successful social integration.

Monoclonal antibodies are increasingly dominating the market for human therapeutic and diagnostic agents. For this reason, continuous methods—such as perfusion processes—are being explored and optimized in an ongoing effort to increase product yields. Unfortunately, many established cell retention devices—such as tangential flow filtration—rely on membranes that are prone to clogging, fouling, and undesirable product retention at high cell densities. To circumvent these problems, in this work, we have developed a 3D-printed microfluidic spiral separator for cell retention, which can readily be adapted and replaced according to process conditions (i.e., a plug-and-play system) due to the fast and flexible 3D printing technique. In addition, this system was also expanded to include automatic flushing, web-based control, and notification via a cellphone application. This set-up constitutes a proof of concept that was successful at inducing a stable process operation at a viable cell concentration of 10–17 × 106 cells/mL in a hybrid mode (with alternating cell retention and cell bleed phases) while significantly reducing both shear stress and channel blockage. In addition to increasing efficiency to nearly 100%, this microfluidic device also improved production conditions by successfully separating dead cells and cell debris and increasing cell viability within the bioreactor. [ABSTRACT FROM AUTHOR]

Calcium (Ca) and carbohydrate (CHO) supply in sweet cherry have been associated with fruit quality at harvest and during storage. There is little published information integrating CHO and Ca availability and distribution in sweet cherry and their effects on fruit quality. Accordingly, in the 2019–20 season, vascular restrictions were imposed on the phloem (girdling, G, stopping phloem flow) and xylem (transverse incision, S, cutting 50% of xylem cross–section area) of individual vertical branches of the sweet cherry combination 'Lapins'/Colt trained as Kym Green Bush system to modify mineral and CHO composition in fruit and associate such changes with quality at harvest and storage. The girdling to the phloem was used to induce changes in CHO distribution. The transverse incision to the xylem was a tool to modify Ca distribution. Five treatments (TR) were implemented: TR1–CTL = Control (without vascular restriction), TR2–G, at its base, TR3–G + G: at its base, and G further up at the change of year between the second and the third years of growth TR4––S and TR5–S + G. The vegetative (i.e., shoot and leaf growth), reproductive (i.e., fruit set and yield) development and stomatal conductance were monitored. Each branch was divided into the upper (1–and 2–year–old wood) and the lower (3–and 4–year–old wood) segments of the restriction applied. The quality and mineral composition (Ca, Mg, K, and N) of fruit borne on each segment were measured at harvest. The upper segment of TR3–G + G branches were harvested 10 d before the lower segment. The fruit from the upper segment of TR3–G + G was the largest, the sweetest, and had the higher titratable acidity concentration. However, fruits of this segment were the softest, had the lowest Ca concentrations, and had the highest ratios of N:Ca and K:Ca, compared with the other TRs. TR3–G + G branches developed the highest number of lateral current season shoots including shoots below the second girdling in the lower segment of the branch. This vegetative flow of growth would explain the mineral unbalance produced in the fruit from the upper segment of the branch. TR2–G did not register changes in fruit quality and mineral concentration compared with TR1–CTL. Surprisingly, the fruit from the branches with xylem restriction did not show changes in Ca concentration, suggesting that the xylem stream was enough to supply the fruit in branches without lateral shoot development. Fruit firmness was positively related to fruit Ca concentration and negatively related to the ratios of K:Ca and N:Ca. [ABSTRACT FROM AUTHOR]

N-linked glycosylation is a crucial post-translational modification of many biopharmaceuticals, including monoclonal antibodies (mAbs), capable of modifying their biological effect in patients and thus considered as a critical quality attribute (CQA). However, expression of desired and consistent glycosylation patterns remains a constant challenge for the biopharmaceutical industry and constitutes the need for tools to engineer glycosylation. Small non-coding microRNAs (miRNAs) are known regulators of entire gene networks and have therefore the potential of being used as tools for modulation of glycosylation pathways and for glycoengineering. Here, we demonstrate that novel identified natural miRNAs are capable of altering N-linked glycosylation patterns on mAbs expressed in Chinese hamster ovary (CHO) cells. We established a workflow for a functional high-throughput screening of a complete miRNA mimic library and identified 82 miRNA sequences affecting various moieties including galactosylation, sialylation, and α-1,6 linked core-fucosylation, an important glycan feature influencing antibody-dependent cytotoxicity (ADCC). Subsequent validation shed light on the intra-cellular mode of action and the impact on the cellular fucosylation pathway of miRNAs reducing core-fucosylation. While multiplex approaches increased phenotypic effects on the glycan structure, a synthetic biology approach utilizing rational design of artificial miRNAs further enhanced the potential of miRNAs as novel, versatile and tune-able tools for engineering of N-linked glycosylation pathways and expressed glycosylation patterns towards favourable phenotypes. • Engineering glycosylation is essential for production of qualitative biopharmaceuticals. • 82 microRNAs were identified to regulate glycosylation in Chinese hamster ovary cells. • Natural and artificial miRNAs were used to engineer cellular glycosylation pathways. • miRNAs were developed as a novel system to engineer and fine-tune fucosylation of monoclonal antibodies. [ABSTRACT FROM AUTHOR]