Scale-Down Characterisation of Post-Centrifuge Flocculation Processes and the Study of its Impact upon Downstream Processing during Mammalian Cell Antibody Production

The high demand for biopharmaceuticals has been met thanks to improved upstream productivity. Downstream, which has not advanced at the same pace, calls for new clarification strategies to cope with the increased process-related impurities associated with upstream improvements. This thesis has focused on the implementation of polyelectrolyte flocculation between centrifugation and depth filtration during the primary recovery of a monoclonal antibody Chinese hamster ovary (CHO) cell process. A high-throughput, automated ultra scale-down (USD) flocculation methodology was developed with industrial relevance in mind for process development activities. The impact flocculation had on depth filtration and protein A chromatography performance was subsequently investigated and supported by analytical assays and techniques to assess impurity removal. Cell densities at time of harvest > 20x10^6 cells/mL were critical for flocculation to occur on centrifuged CHO cell cultures. Flocculant addition time limited the flocculation scale-up success between two vessels with different geometrical ratios and working volumes (1.5 L and 800 µL). The influence of mixing time scales (macromixing, mesomixing and micromixing) on this variable defined the USD flocculation scale-up basis, which was: predominance of micromixing in the vessels (flocculant addition time scale-up) and constant power input per unit volume (impeller speed scale-up). The implementation of constant pressure, single-layer USD filtration methods with flocculated feeds was precluded due to the low filter capacity values obtained ( 490 L/m^2). Flocculation achieved larger depth filtration capacities and lower process-related impurities before protein A chromatography when compared to the current manufacturing processing option. Post-protein A chromatography, the clear eluate obtained with flocculation contrasted with the turbid eluate of the current processing option. Mass spectrometry data confirmed the turbid eluate was mainly constituted of negatively charged CHO cell protein precipitates

Analysis of serum proteome after treatment of osteoporosis with anabolic or antiresorptive drugs

The aim of the study was to explore new markers in serum proteome associated with the response to antiosteoporosis drugs, namely teriparatide and denosumab. We obtained serum samples from 14 patients with osteoporosis, both at baseline and after 6 months of treatment with teriparatide (n = 10) or denosumab (n = 4). Samples were analyzed by nanoliquid chromatography coupled to high-resolution mass spectrometry on a QTOF 5600 (SCIEX) apparatus. The spectrometry data were analyzed with Mascot against the UniProtKB base and then several quality-control filters were applied for the identification of peptides (false discovery rate, FDR q < 0.02) and their quantification (FDR q < 0.05). In the group treated with teriparatide, 28 proteins were identified with significant differences before and after treatment. A pathway analysis by using the Reactome database revealed significant enrichment in the Insulin Like Growth Factor 1 (IGF-I) (FDR q 4 × 10-²) and innate immune system (FDR q 2 × 10-³) pathways. Among patients treated with denosumab, we observed significant differences in the levels of 10 proteins, which were also enriched in the pathways related to the innate immune system (FDR q 3 × 10-²). These results suggest that the innate immune system may be involved in the response to antiosteoporosis drugs.Funding: Supported by grants from FEIOMM (Grant No.17/0025) and ProteoRed-ISCIII. Acknowledgments: Alvaro del Real received support by the postdoctoral grant Augusto Gonzalez de Linares of the University of Cantabria

Design, characterization and in vitro evaluation of antifungal polymeric films

The objective of the present paper was the development and the full characterization of antifungal films. Econazole nitrate (ECN) was loaded in a polymeric matrix formed by chitosan (CH) and carbopol 971NF (CB). Polyethylene glycol 400 and sorbitol were used as plasticizing agents. The mechanical properties of films were poorer when the drug was loaded, probably because crystals of ENC produces network outages and therefore reduces the polymeric interactions between the polymers. Polymers–ECN and CH–CB interactions were analyzed by Fourier-transform infrared spectroscopy (FTIR), thermal gravimetry analysis, and differential thermal analysis (DTA-TGA). ECN did not show structure alterations when loaded into the films. In scanning electron microphotographs and atomic force microscopy analysis, films prepared with CB showed an evident wrinkle pattern probably due to the strong interactions between the polymers, which were observed by FTIR and DTA-TGA. The in vitro activity of the formulations against Candida krusei and Candida parapsilosis was twice as greater as the commercial cream, probably as a result of the antifungal combination of the drug with the CH activity. All these results suggest that these polymeric films containing ECN are potential candidates in view of alternatives dosages forms for the treatment of the yeast assayed.Fil: Real, Daniel Andres. Universidad Nacional de Rosario; ArgentinaFil: Martinez, Maria V.. Universidad Nacional de Rosario; ArgentinaFil: Frattini, Agustin Lujan. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas; ArgentinaFil: Soazo, Marina del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; ArgentinaFil: Luque, Alicia G.. Universidad Nacional de Rosario; ArgentinaFil: Biasoli, Marisa S.. Universidad Nacional de Rosario; ArgentinaFil: Salomon, Claudio Javier. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; ArgentinaFil: Olivieri, Alejandro Cesar. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; ArgentinaFil: Leonardi, Darío. Universidad Nacional de Rosario; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Química Rosario; Argentin

Differential analysis of genome-wide methylation and gene expression in mesenchymal stemcells of patients with fractures and osteoarthritis

Insufficient activity of the bone-forming osteoblasts leads to low bone mass and predisposes to fragility fractures. The functional capacity of human mesenchymal stem cells (hMSCs), the precursors of osteoblasts, may be compromised in elderly individuals, in relation with the epigenetic changes associated with aging. However, the role of hMSCs in the pathogenesis of osteoporosis is still unclear. Therefore, we aimed to characterize the genome-wide methylation and gene expression signatures and the differentiation capacity of hMSCs from patients with hip fractures. We obtained hMSCs from the femoral heads of women undergoing hip replacement due to hip fractures and controls with hip osteoarthritis. DNA methylation was explored with the Infinium 450K bead array. Transcriptome analysis was done by RNA sequencing. The genomic analyses revealed that most differentially methylated loci were situated in genomic regions with enhancer activity, distant from gene bodies and promoters. These regions were associated with differentially expressed genes enriched in pathways related to hMSC growth and osteoblast differentiation. hMSCs from patients with fractures showed enhanced proliferation and upregulation of the osteogenic drivers RUNX2/OSX. Also, they showed some signs of accelerated methylation aging. When cultured in osteogenic medium, hMSCs from patients with fractures showed an impaired differentiation capacity, with reduced alkaline phosphatase activity and poor accumulation of a mineralized matrix. Our results point to 2 areas of potential interest for discovering new therapeutic targets for low bone mass disorders and bone regeneration: the mechanisms stimulating MSCs proliferation after fracture and those impairing their terminal differentiation

Evaluation of seminal characteristics of Pelibuey and East Friesian rams at two different times of the year

Objective: To determine the changes in seminal quality of Pelibuey and East Friesian rams during the non-breeding (long days; March-June) and breeding seasons (short days; September-December) at 19° north latitude. Design/methodology/approach: To determine changes in seminal quality over time, seminal parameters of rams, collected with an artificial vagina were evaluated over 32 weeks. An analysis of variance was performed with a completely randomized design in a 2 x 2 factorial arrangement (breed and season). Results: No differences were found within breeds or between breeds in the same season in the evaluated seminal parameters; however, differences were reported between seasons in the live weight parameters, lower in the non-reproductive season, in addition to an increased scrotal circumference and mass motility during the reproductive season. Study limitations/implications: Semen parameters estimation, in field trials, is subjective compared to computerized semen evaluation systems, it is therefore desirable to have extensive experience in semen evaluation at the field level before starting the study. To confirm the results obtained in this study, a new experiment with a larger number of experimental units is suggested. Findings/conclusions: It is concluded that in the environmental and management conditions, where the seminal evaluation took place, no differences were found between breeds, suggesting that the Pelibuey and East Friesian breeds at 19° north latitude do not decrease their seminal parameters during the non-breeding season compared to the breeding season. This suggest that these two sheep breeds are able to reproduce, in such conditions, all year round

Statins Disrupt CCR5 and RANTES Expression Levels in CD4(+) T Lymphocytes In Vitro and Preferentially Decrease Infection of R5 Versus X4 HIV-1

BACKGROUND: Statins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1 (HIV-1) through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1 incorporation into viral particles. PRINCIPLE FINDINGS: Here we demonstrate that treatment of an enriched CD4(+) lymphocyte population with lovastatin (Lov), mevastatin (Mev) and simvastatin (activated and non-activated, Sim(A) and Sim(N), respectively) can reduce the cell surface expression of the CC-chemokine receptor CCR5 (P<0.01 for Sim(A) and Lov). The lowered CCR5 expression was associated with down-regulation of CCR5 mRNA expression. The CC-chemokine RANTES protein and mRNA expression levels were slightly increased in CD4(+) enriched lymphocytes treated with statins. Both R5 and X4 HIV-1 were reduced for their infection of statin-treated cells; however, in cultures where statins were removed and where a decrease in CCR5 expression was observed, there was a preferential inhibition of infection with an R5 versus X4 virus. CONCLUSIONS: The results indicate that the modulation of CC-chemokine receptor (CCR5) and CC-chemokine (RANTES) expression levels should be considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1 activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment could be considered as a way to modulate immune induction such as during vaccination protocols

Brain structural correlates of sensory phenomena in patients with obsessive-compulsive disorder

Background: sensory phenomena (SP) are uncomfortable feelings, including bodily sensations, sense of inner tension, 'just-right' perceptions, feelings of incompleteness, or 'urge-only' phenomena, which have been described to precede, trigger or accompany repetitive behaviours in individuals with obsessive-compulsive disorder (OCD). Sensory phenomena are also observed in individuals with tic disorders, and previous research suggests that sensorimotor cortex abnormalities underpin the presence of SP in such patients. However, to our knowledge, no studies have assessed the neural correlates of SP in patients with OCD. Methods: we assessed the presence of SP using the University of São Paulo Sensory Phenomena Scale in patients with OCD and healthy controls from specialized units in São Paulo, Brazil, and Barcelona. All participants underwent a structural magnetic resonance examination, and brain images were examined using DARTEL voxel-based morphometry. We evaluated grey matter volume differences between patients with and without SP and healthy controls within the sensorimotor and premotor cortices. Results: we included 106 patients with OCD and 87 controls in our study. Patients with SP (67% of the sample) showed grey matter volume increases in the left sensorimotor cortex in comparison to patients without SP and bilateral sensorimotor cortex grey matter volume increases in comparison to controls. No differences were observed between patients without SP and controls. Limitations: most patients were medicated. Participant recruitment and image acquisition were performed in 2 different centres. Conclusion: we have identified a structural correlate of SP in patients with OCD involving grey matter volume increases within the sensorimotor cortex; this finding is in agreement with those of tic disorder studies showing that abnormal activity and volume increases within this region are associated with the urges preceding tic onset

Performance of prototypes for the ALICE electromagnetic calorimeter

The performance of prototypes for the ALICE electromagnetic sampling calorimeter has been studied in test beam measurements at FNAL and CERN. A $4\times4$ array of final design modules showed an energy resolution of about 11% /$\sqrt{E(\mathrm{GeV})}$ $\oplus$ 1.7 % with a uniformity of the response to electrons of 1% and a good linearity in the energy range from 10 to 100 GeV. The electromagnetic shower position resolution was found to be described by 1.5 mm $\oplus$ 5.3 mm /$\sqrt{E \mathrm{(GeV)}}$. For an electron identification efficiency of 90% a hadron rejection factor of $>600$ was obtained.Comment: 10 pages, 10 figure