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Different cellular processes that contribute to protein production in Chinese hamster ovary (CHO) cells have been previously investigated by proteomics. However, although the classical secretory pathway (CSP) has been well documented as a bottleneck during recombinant protein (RP) production, it has not been well represented in previous proteomic studies. Hence, the significance of this pathway for production of RP was assessed by identifying its own proteins that were associated to changes in RP production, through subcellular fractionation coupled to shot-gun proteomics. Two CHO cell lines producing a monoclonal antibody with different specific productivities were used as cellular models, from which 4952 protein groups were identified, which represent a coverage of 59% of the Chinese hamster proteome. Data are available via ProteomeXchange with identifier PXD021014. By using SAM and ROTS algorithms, 493 proteins were classified as differentially expressed, of which about 80% was proposed as novel targets and one-third were assigned to the CSP. Endoplasmic reticulum (ER) stress, unfolded protein response, calcium homeostasis, vesicle traffic, glycosylation, autophagy, proteasomal activity, protein synthesis and translocation into ER lumen, and secretion of extracellular matrix components were some of the affected processes that occurred in the secretory pathway. Processes from other cellular compartments, such as DNA replication, transcription, cytoskeleton organization, signaling, and metabolism, were also modified. This study gives new insights into the molecular traits of higher producer cells and provides novel targets for development of new sub-lines with improved phenotypes for RP production.

Chinese hamster ovary cells have been the workhorse for the production of recombinant proteins in mammalian cells. Since biochemical, cellular and omics studies are usually affected by the lack of suitable fractionation procedures to isolate compartments from these cells, differential and isopycnic centrifugation based techniques were characterized and developed specially for them. Enriched fractions in intact nuclei, mitochondria, peroxisomes, cis-Golgi, trans-Golgi and endoplasmic reticulum (ER) were obtained in differential centrifugation steps and subsequently separated in discontinuous sucrose gradients. Nuclei, mitochondria, cis-Golgi, peroxisomes and smooth ER fractions were obtained as defined bands in 30–60% gradients. Despite the low percentage represented by the microsomes of the total cell homogenate (1.7%), their separation in a novel sucrose gradient (10–60%) showed enough resolution and efficiency to quantitatively separate their components into enriched fractions in trans-Golgi, cis-Golgi and ER. The identity of these organelles belonging to the classical secretion pathway that came from 10–60% gradients was confirmed by proteomics. Data are available via ProteomeXchange with identifier PXD019778. Components from ER and plasma membrane were the most frequent contaminants in almost all obtained fractions. The improved sucrose gradient for microsomal samples proved being successful in obtaining enriched fractions of low abundance organelles, such as Golgi apparatus and ER components, for biochemical and molecular studies, and suitable for proteomic research, which makes it a useful tool for future studies of this and other mammalian cell lines.

Alginate is a linear polymer composed of b-1,4 linked mannuronic acid and its epimer, a-L-guluronic acid, and frequently extracted from marine algae, as from bacteria such as Azotobacter and Pseudomonas. Here, we show the impact of conventional and unconventional carbon sources on A. vinelandii growth, alginate production, its mean molecular weight (MMW) and its viscosifying power. Starting with 20 g/L of sugars, the highest biomass concentration was obtained using deproteinized and hydrolyzed whey (6.67±0.72 g/L), and sugarcane juice (6.68±0.45 g/L). However, the maximum alginate production was achieved using sucrose (5.11±0.37 g/L), as well the highest alginate yield and specific productivity. Otherwise, the higher alginate MMW was obtained using sugarcane juice (1203±120 kDa), and the higher viscosifying power was obtained using deproteinized/ hydrolyzed whey (23.8±2.6 cps L/galg). This information suggests that it is possible to manipulate the productivity and molecular characteristics of alginates, as a function of the carbon source used. All this, together with the knowledge of the effects of environmental conditions will allow for high yields of high added value biopolymers.

Alginate is a linear polymer composed of b-1,4 linked mannuronic acid and its epimer, a-L-guluronic acid, and frequently extracted from marine algae, as from bacteria such as Azotobacter and Pseudomonas. Here, we show the impact of conventional and unconventional carbon sources on A. vinelandii growth, alginate production, its mean molecular weight (MMW) and its viscosifying power. Starting with 20 g/L of sugars, the highest biomass concentration was obtained using deproteinized and hydrolyzed whey (6.67±0.72 g/L), and sugarcane juice (6.68±0.45 g/L). However, the maximum alginate production was achieved using sucrose (5.11±0.37 g/L), as well the highest alginate yield and specific productivity. Otherwise, the higher alginate MMW was obtained using sugarcane juice (1203±120 kDa), and the higher viscosifying power was obtained using deproteinized/ hydrolyzed whey (23.8±2.6 cps L/galg). This information suggests that it is possible to manipulate the productivity and molecular characteristics of alginates, as a function of the carbon source used. All this, together with the knowledge of the effects of environmental conditions will allow for high yields of high added value biopolymers.

The impact of flask geometry on Streptomyces lividans growth and morphology, production and Omannosylation of a recombinant O-glycoprotein (APA from Mycobacterium tuberculosis) was described and associated to the evolution of the volumetric power input (P/V) in three shake flask geometries -- During the exponential growth, the highest P/V was found in baffled flasks (BF) with 0.51 kW/m3, followed by coiled flasks (CF) with 0.44 kW/m3 and normal Erlenmeyer flasks (NF) with 0.20 kW/m3 (flasks volume of 250 mL, filling with 50 mL and agitated at 150 rpm) -- During the stationary phase, P/V decreased 20% in BF and CF, but increased two times in NF, surely due to changes in mycelial morphology and its effects on rheology -- Also, NF cultures were carried out at a filling volume and agitation of 15 mL, 150 rpm (15 mL-NF), and 25 mL, 168 rpm (25 mL-NF), in order to raise P/V closely to the values obtained in CF -- However, different growth, morphology and recombinant protein productivity were obtained -- These data indicate that P/V is not a definitive parameter that can determine bacteria growth and morphology, not even glycoprotein production -- But it can be proposed that the oxygen transfer in the center of the pellets and hydromechanical stress might be the more relevant parameters than P/V

Azospirillum brasilense has industrial significance as a growth promoter in plants of commercial interest -- However, there is no report in the literature disclosing a liquid product produced in pilot-scale bioreactors and is able to be stored at room temperature for more than 2 years -- The aim of this work was to scale up a process from a shake flask to a 10- L lab-scale and 1,000-L pilot-scale bioreactor for the production of plant growth-promoting bacterium A -- brasilense for a liquid inoculant formulation -- Furthermore, this work aimed to determine the shelf life of the liquid formulation stored at room temperature and to increase maize crops yield in greenhouses -- Under a constant oxygen mass transfer coefficient (KLa), a fermentation process was successfully scaled up from shake flasks to 10- and 1,000-L bioreactors -- A concentration ranging from 3.5 to 7.5×108 CFU/mL was obtained in shake flasks and bioreactors, and after 2 years stored at room temperature, the liquid formulation showed one order of magnitude decrease -- Applications of the cultured bacteria in maize yields resulted in increases of up to 95 % in corncobs and 70 % in aboveground biomass

En la industria biofarmacéutica actual, los requerimientos para el diseño de un biorreactor difieren sustancialmente de aquellos de hace 50 años. Hoy en día las nuevas estrategias de cultivo permiten obtener alta densidad celular con altos rendimientos de productos biotecnológicos que deben cumplir con elevados estándares de calidad y normatividad compleja en su producción. Lo anterior obliga el desarrollo de nuevas tecnologías para suplir estas necesidades, por lo que en los últimos diez años el diseño y uso de biorreactores desechables ha crecido de manera exponencial. Los beneficios de la tecnología desechable son principalmente la eliminación de largos procesos de limpieza, esterilización, calificación, validación, reducción de tiempo entre lotes, el procesamiento de diferentes productos en la misma área, entre otros, lo que conlleva la disminución de los costos que implican esta serie de procedimientos.

In the current pharmaceutical industry the requirements for the design of a bioreactor are far different from those necessary 50 years ago. Nowadays, new strategies for growing cells produce high cell densities and high yields of biotechnology products with high standards regulations. That requires the development of new technologies to meet these needs, consequently in the past ten years, the design and use of disposable bioreactors has grown exponentially. The benefits of disposable technology, ranging from the elimination of lengthy and costly processes as cleaning, sterilization and validation, reduction in the time between production batches, production of various products in the same area, to diminish the costs because of this set of procedures., En la industria biofarmacéutica actual, los requerimientos para el diseño de un biorreactor difieren sustancialmente de aquellos de hace 50 años. Hoy en día las nuevas estrategias de cultivo permiten obtener alta densidad celular con altos rendimientos de productos biotecnológicos que deben cumplir con elevados estándares de calidad y normatividad compleja en su producción. Lo anterior obliga el desarrollo de nuevas tecnologías para suplir estas necesidades, por lo que en los últimos diez años el diseño y uso de biorreactores desechables ha crecido de manera exponencial. Los beneficios de la tecnología desechable son principalmente la eliminación de largos procesos de limpieza, esterilización, calificación, validación, reducción de tiempo entre lotes, el procesamiento de diferentes productos en la misma área, entre otros, lo que conlleva la disminución de los costos que implican esta serie de procedimientos.

En la industria biofarmacéutica actual, los requerimientos para el diseño de un biorreactor difieren sustancialmente de aquellos de hace 50 años. Hoy en día las nuevas estrategias de cultivo permiten obtener alta densidad celular con altos rendimientos de productos biotecnológicos que deben cumplir con elevados estándares de calidad y normatividad compleja en su producción. Lo anterior obliga el desarrollo de nuevas tecnologías para suplir estas necesidades, por lo que en los últimos diez años el diseño y uso de biorreactores desechables ha crecido de manera exponencial. Los beneficios de la tecnología desechable son principalmente la eliminación de largos procesos de limpieza, esterilización, calificación, validación, reducción de tiempo entre lotes, el procesamiento de diferentes productos en la misma área, entre otros, lo que conlleva la disminución de los costos que implican esta serie de procedimientos.

In the current pharmaceutical industry the requirements for the design of a bioreactor are far different from those necessary 50 years ago. Nowadays, new strategies for growing cells produce high cell densities and high yields of biotechnology products with high standards regulations. That requires the development of new technologies to meet these needs, consequently in the past ten years, the design and use of disposable bioreactors has grown exponentially. The benefits of disposable technology, ranging from the elimination of lengthy and costly processes as cleaning, sterilization and validation, reduction in the time between production batches, production of various products in the same area, to diminish the costs because of this set of procedures., En la industria biofarmacéutica actual, los requerimientos para el diseño de un biorreactor difieren sustancialmente de aquellos de hace 50 años. Hoy en día las nuevas estrategias de cultivo permiten obtener alta densidad celular con altos rendimientos de productos biotecnológicos que deben cumplir con elevados estándares de calidad y normatividad compleja en su producción. Lo anterior obliga el desarrollo de nuevas tecnologías para suplir estas necesidades, por lo que en los últimos diez años el diseño y uso de biorreactores desechables ha crecido de manera exponencial. Los beneficios de la tecnología desechable son principalmente la eliminación de largos procesos de limpieza, esterilización, calificación, validación, reducción de tiempo entre lotes, el procesamiento de diferentes productos en la misma área, entre otros, lo que conlleva la disminución de los costos que implican esta serie de procedimientos.

In the current pharmaceutical industry the requirements for the design of a bioreactor are far different from those necessary 50 years ago. Nowadays, new strategies for growing cells produce high cell densities and high yields of biotechnology products with high standards regulations. That requires the development of new technologies to meet these needs, consequently in the past ten years, the design and use of disposable bioreactors has grown exponentially. The benefits of disposable technology, ranging from the elimination of lengthy and costly processes as cleaning, sterilization and validation, reduction in the time between production batches, production of various products in the same area, to diminish the costs because of this set of procedures., En la industria biofarmacéutica actual, los requerimientos para el diseño de un biorreactor difieren sustancialmente de aquellos de hace 50 años. Hoy en día las nuevas estrategias de cultivo permiten obtener alta densidad celular con altos rendimientos de productos biotecnológicos que deben cumplir con elevados estándares de calidad y normatividad compleja en su producción. Lo anterior obliga el desarrollo de nuevas tecnologías para suplir estas necesidades, por lo que en los últimos diez años el diseño y uso de biorreactores desechables ha crecido de manera exponencial. Los beneficios de la tecnología desechable son principalmente la eliminación de largos procesos de limpieza, esterilización, calificación, validación, reducción de tiempo entre lotes, el procesamiento de diferentes productos en la misma área, entre otros, lo que conlleva la disminución de los costos que implican esta serie de procedimientos.