Compartment-specific metabolome analysis reveals the tight link between IgG1 formation and necessarily high mitochondrial shuttle activities in Chinese Hamster Ovary cells

Chinese hamster ovary (CHO) cells are the dominating host for the production of pharmaceutical proteins, in particular monoclonal antibodies (mABs). Although production titers improved more than 100 fold during the last 2 decades, similar enhancements of cell specific productivities are less pronounced. They demand for detailed subcellular studies to identify promising metabolic engineering targets. In this context, our study focused on compartment specific metabolome analysis to measure metabolic patterns in the cytosol and in the mitochondrion during cell cultivation. Thereof, in vivo shuttle activities were calculated and correlated with cell specific IgG1 formation rates. The compartment-specific metabolome and labelling analysis (13C) distinguishes between cytosol and mitochondrion. Metabolomics and instationary 13C metabolic flux analysis build on preliminary own studies of 13C analytics (Teleki et al., Anal Biochem 2015; Teleki et al. Metab Eng 2017) and compartment-specific metabolomics (Matuszczyk et al., Biotechnol J 2015; Pfitzenmaier et al., Biotechnol J 2016). Further development and optimization has been performed finally reaching the current status that allows monitoring compartment-specific flux distributions and shuttle activities during the course of cell cultivation. Studying multiple periods of an IgG1 production process the crucial role of the mitochondrion not only as a provider of ATP but also as an essential part of metabolism was unraveled. 13C flux analysis disclosed the time-variant activities of the mitochondrial shuttles that are tightly linked to mitochondrial and cytosolic metabolism. Clear evidence was found that mAB production strongly depends on sufficient NADPH supply provided by cytosolic malic enzyme activity and malate export from the mitochondrio

The Less the Better: How Suppressed Base Addition Boosts Production of Monoclonal Antibodies With Chinese Hamster Ovary Cells

Biopharmaceutical production processes strive for the optimization of economic efficiency. Among others, the maximization of volumetric productivity is a key criterion. Typical parameters such as partial pressure of CO2 (pCO2) and pH are known to influence the performance although reasons are not yet fully elucidated. In this study the effects of pCO2 and pH shifts on the phenotypic performance were linked to metabolic and energetic changes. Short peak performance of qmAb (23 pg/cell/day) was achieved by early pCO2 shifts up to 200 mbar but followed by declining intracellular ATP levels to 2.5 fmol/cell and 80% increase of qLac. On the contrary, steadily rising qmAb could be installed by slight pH down-shifts ensuring constant cell specific ATP production (qATP) of 27 pmol/cell/day and high intracellular ATP levels of about 4 fmol/cell. As a result, maximum productivity was achieved combining highest qmAb (20 pg/cell/day) with maximum cell density and no lactate formation. Our results indicate that the energy availability in form of intracellular ATP is crucial for maintaining antibody synthesis and reacts sensitive to pCO2 and pH-process parameters typically responsible for inhomogeneities after scaling up

The new Felsenkeller 5 MV underground accelerator

The field of nuclear astrophysics is devoted to the study of the creation of the chemical elements. By nature, it is deeply intertwined with the physics of the Sun. The nuclear reactions of the proton-proton cycle of hydrogen burning, including the 3He({\alpha},{\gamma})7Be reaction, provide the necessary nuclear energy to prevent the gravitational collapse of the Sun and give rise to the by now well-studied pp, 7Be, and 8B solar neutrinos. The not yet measured flux of 13N, 15O, and 17F neutrinos from the carbon-nitrogen-oxygen cycle is affected in rate by the 14N(p,{\gamma})15O reaction and in emission profile by the 12C(p,{\gamma})13N reaction. The nucleosynthetic output of the subsequent phase in stellar evolution, helium burning, is controlled by the 12C({\alpha},{\gamma})16O reaction. In order to properly interpret the existing and upcoming solar neutrino data, precise nuclear physics information is needed. For nuclear reactions between light, stable nuclei, the best available technique are experiments with small ion accelerators in underground, low-background settings. The pioneering work in this regard has been done by the LUNA collaboration at Gran Sasso/Italy, using a 0.4 MV accelerator. The present contribution reports on a higher-energy, 5.0 MV, underground accelerator in the Felsenkeller underground site in Dresden/Germany. Results from {\gamma}-ray, neutron, and muon background measurements in the Felsenkeller underground site in Dresden, Germany, show that the background conditions are satisfactory for nuclear astrophysics purposes. The accelerator is in the commissioning phase and will provide intense, up to 50{\mu}A, beams of 1H+, 4He+ , and 12C+ ions, enabling research on astrophysically relevant nuclear reactions with unprecedented sensitivity.Comment: Submitted to the Proceedings of the 5th International Solar Neutrino Conference, Dresden/Germany, 11-14 June 2018, to appear on World Scientific -- updated version (Figure 2 and relevant discussion updated, co-author A. Domula added

Implementation of a formula for the correction of NT-proBNP for impaired renal function

Das NT-proBNP ist ein Herzmarker, der eine Herzinsuffizienz frühzeitig erkennt bzw. ausschließen kann. Allerdings steigt dieser auch im Falle einer eingeschränkten Nierenfunktion an. Um herauszufinden, welchen Einfluss eine Niereninsuffizienz auf das NT-proBNP hat, wurden mit Hilfe einer Formel zur Korrektur des NT-proBNP bei eingeschränkter Nierenfunktion für ein Patientenkollektiv von 5716 Personen die angepassten Konzentrationen berechnet. Es zeigte sich eine umgekehrte Korrelation zwischen dem NT-proBNP und der glomerulären Filtrationsrate (GFR). Je niedriger die GFR wurde, desto mehr stieg das NT-proBNP an. Des Weiteren zeigte sich, dass das Delta (Differenz zwischen dem NT-proBNP-Wert vor der Korrektur und dem NT-proBNP-Wert nach der Korrektur) geringer wurde, je mehr die GFR ansteigt. Das bedeutet, die Werte konnten mit Zunahme der GFR weniger stark korrigiert werden, da mit zunehmender GFR das NT-proBNP vermehrt renal ausgeschieden werden kann und sich nicht im Blutplasma ansammelt. Jedoch wurde deutlich, dass eine Korrektur bis zu einer GFR von 72 ml/min/1,73 m2 notwendig ist, damit das NT-proBNP zuverlässig als Herzmarker eingesetzt werden kann. Beim Vergleich der Kreatinin-GFR und der Cystatin C-GFR wurde deutlich, dass das Kreatinin im GFR-Bereich von 40 ml/min/1,73 m2 und 80 ml/min/1,73 m2 kein zuverlässiger Parameter zur Ermittlung der GFR ist, da die GFR vermindert ist, aber das Kreatinin im Normalbereich liegt. Cystatin C ist hier der bessere Marker. Dieser steigt bereits bei kleinen Funktionseinschränkungen der Niere an

Epstein-Barr Virus-Induced Genes and Endogenous Retroviruses in Immortalized B Cells from Patients with Multiple Sclerosis

The immune pathogenesis of multiple sclerosis (MS) is thought to be triggered by environmental factors in individuals with an unfavorable genetic predisposition. Epstein–Barr virus (EBV) infection is a major risk factor for subsequent development of MS. Human endogenous retroviruses (HERVs) can be activated by EBV, and might be a missing link between an initial EBV infection and the later onset of MS. In this study, we investigated differential gene expression patterns in EBV-immortalized lymphoblastoid B cell lines (LCL) from MS-affected individuals (MSLCL) and controls by using RNAseq and qRT-PCR. RNAseq data from LCL mapped to the human genome and a virtual virus metagenome were used to identify possible biomarkers for MS or disease-relevant risk factors, e.g., the relapse rate. We observed that lytic EBNA-1 transcripts seemed to be negatively correlated with age leading to an increased expression in LCL from younger PBMC donors. Further, HERV-K (HML-2) GAG was increased upon EBV-triggered immortalization. Besides the well-known transactivation of HERV-K18, our results suggest that another six HERV loci are up-regulated upon stimulation with EBV. We identified differentially expressed genes in MSLCL, e.g., several HERV-K loci, ERVMER61-1 and ERV3-1, as well as genes associated with relapses. In summary, EBV induces genes and HERV in LCL that might be suitable as biomarkers for MS or the relapse risk

Screening Clinical Cell Products for Replication Competent Retrovirus: The National Gene Vector Biorepository Experience

Replication-competent retrovirus (RCR) is a safety concern for individuals treated with retroviral gene therapy. RCR detection assays are used to detect RCR in manufactured vector, transduced cell products infused into research subjects, and in the research subjects after treatment. In this study, we reviewed 286 control (n = 4) and transduced cell products (n = 282) screened for RCR in the National Gene Vector Biorepository. The transduced cell samples were submitted from 14 clinical trials. All vector products were previously shown to be negative for RCR prior to use in cell transduction. After transduction, all 282 transduced cell products were negative for RCR. In addition, 241 of the clinical trial participants were also screened for RCR by analyzing peripheral blood at least 1 month after infusion, all of which were also negative for evidence of RCR infection. The majority of vector products used in the clinical trials were generated in the PG13 packaging cell line. The findings suggest that screening of the retroviral vector product generated in PG13 cell line may be sufficient and that further screening of transduced cells does not provide added value. Keywords: retrovirus, safety testing, replicating virus, lentiviru

Management of anaphylaxis in schools: Evaluation of an epinephrine auto-injector (EpiPen®) use by school personnel and comparison of two approaches of soliciting participation

<p>Abstract</p> <p>Background</p> <p>There has been no large study characterizing selection bias in allergy and evaluating school personnel’s ability to use an epinephrine auto-injector (EpiPen®). Our objective was to determine if the consent process introduces selection bias by comparing 2 methods of soliciting participation of school personnel in a study evaluating their ability to demonstrate the EpiPen®.</p> <p>Methods</p> <p>School personnel from randomly selected schools in Quebec were approached using a 1) <it>partial</it> or 2) <it>full disclosure</it> approach and were assessed on their ability to use the EpiPen® and identify anaphylaxis.</p> <p>Results</p> <p>343 school personnel participated. In the <it>full disclosure</it> group, the participation rate was lower: 21.9% (95%CI, 19.0%-25.2%) versus 40.7% (95%CI, 36.1%-45.3%), but more participants achieved a perfect score: 26.3% (95%CI, 19.6%-33.9%) versus 15.8% (95%CI, 10.8%-21.8%), and identified 3 signs of anaphylaxis: 71.8% (95%CI, 64.0%-78.7%) versus 55.6% (95%CI, 48.2%-62.9%).</p> <p>Conclusions</p> <p>Selection bias is suspected as school personnel who were fully informed of the purpose of the assessment were less likely to participate; those who participated among the fully informed were more likely to earn perfect scores and identify anaphylaxis. As the process of consent can influence participation and bias outcomes, researchers and Ethics Boards need to consider conditions under which studies can proceed without full consent. Despite training, school personnel perform poorly when asked to demonstrate the EpiPen®.</p