“Magnetic Paper – Based ELISA” for IgM-dengue antibodies detection provide a system with improved analytical response.

BackgroundShake flasks are widely used during the development of bioprocesses for recombinant proteins. Cultures of recombinant Escherichia coli with orbital mixing (OM) have an oxygen limitation negatively affecting biomass growth and recombinant-protein production. With the aim to improve mixing and aeration in shake flask cultures, we analyzed cultures subjected to OM and the novel resonant acoustic mixing (RAM) by applying acoustic energy to E. coli BL21-Gold (DE3): a producer of recombinant phospholipase A2 (rPLA2) from Micrurus laticollaris snake venom.ResultsComparing OM with RAM (200 rpm vs. 7.5g) at the same initial volumetric oxygen transfer coefficient (kLa ≈ 80 h−1) ~69% less biomass was obtained with OM compared with RAM. We analyzed two more conditions increasing agitation until maximal speed (12.5 and 20g), and ~1.6- and ~1.4-fold greater biomass was obtained as compared with cultures at 7.5g. Moreover, the specific growth rate was statistically similar in all cultures carried out in RAM, but ~1.5-fold higher than that in cultures carried out under OM. Almost half of the glucose was consumed in OM, whereas between 80 and 100% of the glucose was consumed in RAM cultures, doubling biomass per glucose yields. Differential organic acid production was observed, but acetate production was prevented at the maximal RAM (20g). The amount of rPLA2 in both, OM and RAM cultures, represented 38 ± 5% of the insoluble protein. A smaller proportion of α-helices and β-sheet of purified inclusion bodies (IBs) were appreciated by ATR-FTIR from cultures carried out under OM, than those from RAM. At maximal agitation by RAM, internal E. coli localization patterns of protein aggregation changed, as well as, IBs proteolytic degradation, in conjunction with the formation of small external vesicles, although these changes did not significantly affect the cell survival response.ConclusionsIn moderate-cell-density recombinant E. coli BL21-Gold (DE3) cultures, the agitation increases in RAM (up to the maximum) was not enough to avoid the classical oxygen limitation that happens in OM shake flasks. However, RAM presents a decrease of oxygen limitation, resulting in a favorable effect on biomass growth and volumetric rPLA2 production. While under OM a higher recombinant protein yield was obtained.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0746-1) contains supplementary material, which is available to authorized users.

A careful selection of culture mediums and feeds has become necessary to maximize yields of recombinant proteins during bioprocesses of mammalian cells. Supplements contain a variety of concentrate nutrients, and their beneficial effects vary according to recombinant cell lines. In this study, the effects of PowerFeed A on growth kinetics, productivity and cellular metabolism were evaluated for two Chinese hamster ovary cell lines producing a monoclonal antibody in a batch culture. Supplemented cultures increased integral viable cell density of CRL-12444 and CRL-12445 cells by 2.4 and 1.6 times through extension of culture time at which viability was above 90% in 72 and 36 h, respectively, and increment of maximal cell concentration in 3.25 × 10 6 cells/ml (69%) for CRL-12445 cells. Product titer augmented 1.9 and 2.5 times for CRL-12444 and CRL-12445 cells, respectively, without changes in growth rate and specific productivity. Feed supplementation also stimulated full consumption of glucose and free glutamine and reduced 10 times lactate accumulation, while ammonium, sodium and potassium remained at similar concentrations at the end of the culture. About 44% of calcium, mainly provided by feed, was consumed by both cell lines. Maximization of cellular growth, viability and protein titer through feeding encourages extending its use to other cell lines and exploring novel combinations with other basal mediums or feeds. A thorough investigation of its impact on protein quality and the molecular mechanisms behind these effects will allow designing effective feeds and strategies to rationally optimize protein production in the biomanufacturing industry.

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