miR-2861 as novel HDAC5 inhibitor in CHO cells enhances productivity while maintaining product quality.

Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells.

Hippocampal subfield plasticity is associated with improved spatial memory.

Physical exercise studies are generally underrepresented in young adulthood. Seventeen subjects were randomized into an intervention group (24.2 ± 3.9 years; 3 trainings/week) and 10 subjects into a passive control group (23.7 ± 4.2 years), over a duration of 6 months. Every two months, performance diagnostics, computerized spatial memory tests, and 3 Tesla magnetic resonance imaging were conducted. Here we find that the intervention group, compared to controls, showed increased cardiorespiratory fitness, spatial memory performance and subregional hippocampal volumes over time. Time-by-condition interactions occurred in right cornu ammonis 4 body and (trend only) dentate gyrus, left hippocampal tail and left subiculum. Increases in spatial memory performance correlated with hippocampal body volume changes and, subregionally, with left subicular volume changes. In conclusion, findings support earlier reports of exercise-induced subregional hippocampal volume changes. Such exercise-related plasticity may not only be of interest for young adults with clinical disorders of hippocampal function, but also for sedentary normal cohorts.

The impact of endotoxin masking on the removal of endotoxin during manufacturing of a biopharmaceutical drug product.

Endotoxins are a highly pyrogenic and immunogenic contaminant of bacterial origin that must be avoided during the manufacturing of biopharmaceutical products to ensure safety and efficacy. Low endotoxin recovery, also known as a masking effect, is defined as the ability to detect <50% [21] of the expected endotoxin in an endotoxin assay. Masking can be caused by the ability of endotoxins to build aggregates, bind to the protein or organise in micelles or vesicles that in turn inhibit detection of the endotoxin in the solution being tested. Therefore, a masking effect can result from physical parameters of the molecule being tested or from the buffer/environmental conditions of the solution the molecule is in. This can subsequently lead to the underestimation of endotoxin contaminations and lead to a potential false negative test. Tight control over the effectiveness of the downstream process and the use of well-characterised endotoxin testing assays are needed to ensure optimal endotoxin removal. This manuscript demonstrates the capacity to remove the endotoxins within a proven acceptable range by also controlling and evaluating the potential masking effects during downstream process at ambient temperature and also during sample storage condition until the analyse was performed. The endotoxin removal study (ERS) is divided in the initial part to evaluate the process buffers and the conditions of the molecule to avoid the underestimation of endotoxins in process samples in advance. This pre-study is a necessary prerequisite to evaluate the results after the endotoxin spiked downstream unit operations. With those aspects, the removal capacity can be demonstrated. A study was carried out to characterise the endotoxin removal capability of the purification process including controlling of masking effects. The endotoxin removal capacity on ion exchange chromatography and during ultrafiltration/diafiltration unit operations of the downstream processing of an immunoglobulin G1 antibody was conducted using various process parameters to understand their impact on endotoxin removal. In the small-scale study, the processing steps from each tested unit operation were spiked with Escherichia coli endotoxins. The potential masking effect during purification was addressed by controlling the hold time by spiking studies of the different generated pools at ambient temperature. By conducting a masking study, all generated protein pools (flow-through/wash, eluate and regeneration pools) had no masking effect caused through sample handling prior to analysis. Overall, this study showed that endotoxins could be successfully removed by anion exchange chromatography. A partial removal could be achieved by cation exchange chromatography and endotoxins could not be removed with ultrafiltration/diafiltration.