Proteomic Landscape of Adeno-Associated Virus (AAV)-Producing HEK293 Cells

Adeno-associated viral (AAV) vectors are widely used for gene therapy, providing treatment for diseases caused by absent or defective genes. Despite the success of gene therapy, AAV manufacturing is still challenging, with production yields being limited. With increased patient demand, improvements in host cell productivity through various engineering strategies will be necessary. Here, we study the host cell proteome of AAV5-producing HEK293 cells using reversed phase nano-liquid chromatography and tandem mass spectrometry (RPLC-MS/MS). Relative label-free quantitation (LFQ) was performed, allowing a comparison of transfected vs. untransfected cells. Gene ontology enrichment and pathway analysis revealed differential expression of proteins involved in fundamental cellular processes such as metabolism, proliferation, and cell death. Furthermore, changes in expression of proteins involved in endocytosis and lysosomal degradation were observed. Our data provides highly valuable insights into cellular mechanisms involved during recombinant AAV production by HEK293 cells, thus potentially enabling further improvements of gene therapy product manufacturing.

High Throughput miRNA Screening Identifies miR-574-3p Hyperproductive Effect in CHO Cells

CHO is the cell line of choice for the manufacturing of many complex biotherapeutics. The constant upgrading of cell productivity is needed to meet the growing demand for these life-saving drugs. Manipulation of small non-coding RNAs—miRNAs—is a good alternative to a single gene knockdown approach due to their post-transcriptional regulation of entire cellular pathways without posing translational burden to the production cell. In this study, we performed a high-throughput screening of 2042-human miRNAs and identified several candidates able to increase cell-specific and overall production of Erythropoietin and Etanercept in CHO cells. Some of these human miRNAs have not been found in Chinese hamster cells and yet were still effective in them. We identified miR-574-3p as being able, when overexpressed in CHO cells, to improve overall productivity of Erythropoietin and Etanercept titers from 1.3 to up to 2-fold. In addition, we validated several targets of miR-574-3p and identified p300 as a main target of miR-574-3p in CHO cells. Furthermore, we demonstrated that stable CHO cell overexpressing miRNAs from endogenous CHO pri-miRNA sequences outperform the cells with human pri-miRNA sequences. Our findings highlight the importance of flanking genomic sequences, and their secondary structure features, on pri-miRNA processing offering a novel, cost-effective and fast strategy as a valuable tool for efficient miRNAs engineering in CHO cells.

The Effect of Tin Compounds on the Lipid Peroxidation Level of Russian Sturgeon Fresh and Cryopreserved Sperm

The effect of organotin compounds (OTs) on the accumulation of the lipid peroxidation (LPO) carbonyl by-products, which react with thiobarbituric acid (TBARS) in fresh and cryopreserved sperm of Russian sturgeon, was studied. It was found that incubation (1 hour) of Russian sturgeon sperm with OTs (CH3SnCl3, (CH3)2SnCl2, (CH3)3SnCl, (n-C4H9)2SnCl2, (n-C4H9)3SnCl, (C6H5)2SnCl2, (C6H5)3SnCl) in concentration 0.1 mM led to the promotion of the accumulation of TBARS in native semen. Dimethyl- (DMT) and diphenyltin dichlorides (DPT) exhibited the greatest promoting activity, and the LPO level of both native and cryopreserved sperm of Russian sturgeon, including those in modified Stein's cryomedium, increased in the presence of these compounds. It was found that Russian sturgeon's cryopreserved sperm had lower sensitivity to the promotion of sperm LPO by DMT and DPT compared with the native sperm. The protective effect of Stein’s cryomedium decreased in the presence of the studied OTs. The results suggest that accumulation of OTs by gonad of fish is another stress factor affecting the cell productivity in the cryopreservation process.

PROCESS PARAMETERS OF THE CALCINED ANODE PRODUCTION PROCESS AS A FACTOR AFFECTING THE CELL PRODUCTIVITY

The main technological stages of production of the annealed anode are analyzed, the technological operation providing obtaining of the set complex of properties is revealed.

Optimization of Light Intensity and NaNO3 Concentration in Amazon Cyanobacteria Cultivation to Produce Biodiesel

The objective of this study, for the first time, was to optimize Amazonian cyanobacterial culture conditions for improving cell productivity and lipid content, by analyzing the effect of light intensity and nitrogen concentration, for empirically evaluating biodiesel quality parameters. The strains Synechocystis sp. CACIAM05, Microcystis aeruginosa CACIAM08, Pantanalinema rosaneae CACIAM18, and Limnothrix sp. CACIAM25, were previously identified by morphological and molecular analysis (16S rRNA) and were selected based on their production of chlorophyll a and dry cell weight. Then, factorial planning (22) with central points was applied, with light intensity and NaNO3 concentration as independent variables. As response variables, cell productivity and lipid content were determined. Statistical analysis indicated that for all strains, the independent variables were statistically significant for cell productivity. Analysis of the fatty acid composition demonstrated diversity in the composition of the fatty acid profile from the experimental planning assays of each strain. The Biodiesel Analyzer software predicted the biodiesel quality parameters. CACIAM05 and CACIAM25 obtained better parameters with low levels of light intensity and NaNO3 concentration, whereas CACIAM08 and CACIAM18 obtained better parameters with low NaNO3 concentrations and high luminous intensity.

CULTIVATION OF THE MICROALGAE CHLORELLA PYRENOIDOSA USING THE PROCESSES OF BIOTECHNOLOGY

The results obtained here, show that the use of Chlorella pyrenoidosa microalgae and biotechnology, using the discontinuous process, presented satisfactory results. With this, the study of the microalga Chlorella sp. has proved to be important because it has a wealth of proteins, carbohydrates, amino acids, fatty acids, carotenoids, vitamins and minerals in its constitution, which may represent commercial importance. This research revealed the best results for obtaining a lipoprotein-rich biomass, taking into account three different culture media, calculations of cell concentration, cell productivity, to the content (%) of protein, lipid, carbohydrate and ash present in the microalgal biomass.

Intensive Production of Carboxylic Acids Using C. butyricum in a Membrane Bioreactor (MBR)

This work reports on the use of a bench-scale chemostat (CSTR) in continuous mode and of a pilot-scale membrane bioreactor (MBR) in fed-batch mode to intensively produce acetic and butyric acids using C. butyricum grown on synthetic media. These studies were then used to perform a cost estimation study of the MBR system to assess the potential economic impact of this proposed methodology, regarding the production of carboxylic acids. The MBR system was found to be highly productive, reaching 37.88 g L−1 h−1 of acetic and 14.44 g L−1 h−1 of volumetric cell productivity, favoring acetic acid production over butyric acid at a ratio of 3 moles to 1. The cost of preparation and production of carboxylic acid using this system was found to be 0.0062 £PS/kg with up to 99% carbon recovery.

Intensive Production of Carboxylic Acids Using Cl. butyricum in a Membrane Bioreactor (MBR)

This work reports of the use of a bench scale chemostat (CSTR) in continuous mode and of a pilot scale membrane bioreactor (MBR) in fed-batch mode to intensively produce acetic and butyric acids using Cl. butyricum grown on synthetic media. These studies were then used to perform a cost estimation study of the MBR system to assess the potential economic impact of this proposed methodology. The MBR system was found highly productive reaching 37.88 g L-1 h-1 of acetic and 14.44 g L-1 h-1 of volumetric cell productivity, favoring acetic acid production to butyric at a ratio 3:1. The cost of preparation and production of carboxylic acids using this system was found as 0.0062 £PS/kg respectively and an up to 99% carbon recovery.