Salt tolerant endonucleases for the removal of host cell DNA in Downstream Processing of enveloped viruses

Host cell DNA is a critical impurity in downstream processing of enveloped viruses. For vaccine applications, host cell DNA content should be below 10 ng per dose in the final product. Enveloped viruses exhibit an overall negative net charge on their surface and therefore have binding properties similar to DNA. Consequently, separation of virus from DNA can be cumbersome. In addition to DNA in its naked form, host cell DNA is present in virus preparations in form of chromatin. Chromatin (Figure 1) consists of complex and large structures which include DNA and highly positively charged histones. Therefore, different types of interactions of chromatin with chromatographic material and membranes can be observed, electrostatic interaction through negative charges of DNA and positive charges histones and hydrophobic interaction through hydrophobic patches of histones. Moreover, chromatin is often similar in size to viruses, further complicating their separation. We evaluated the performance of four different endonucleases, two salt tolerant endonucleases and two sensitive to salt, in the downstream processing of recombinant Measles virus. Endonuclease treatment was performed after clarification and followed by a purification step using flowthrough chromatography with Capto™ Core 700 resin. Nanoparticle tracking analysis (NTA) was used to determine size and particle concentration and TCID50 to determine the infectivity of the viruses. DNA and histones presence (in process and purified samples) were determined using PicoGreen™ assay and Western blot analysis using detecting anti-histone antibodies. The salt tolerant endonucleases are more efficient in the removal of chromatin and consequently in the removal of host cell DNA. A 97 % reduction of DNA could be observed. Please click Download on the upper right corner to see the full abstract

Virus-like particles adsorption in anion exchange chromatography media

Biotechnological and pharmaceutical industries have the development of modern vaccines and novel drug delivery systems as one of their main focus. At this point, Virus-Like Particles (VLPs) are key candidates once they have the ability to stimulate humoral and cellular immune responses combined with the inability to replicate or proliferate. VLPs are non-infectious self-assembled protein structures which mimic native viruses (lacking any viral genetic material). However great developments in VLPs manufacturing have already been achieved, their purification is still a complex process, usually slow and with low productivity. Accordingly, there is a demand for new purification strategies and unit operations. Anion exchange chromatography is well established and widely used in industry for the purification all sorts of biomolecules. It is already known that polymer-grafted media in form of charged hydrogels and/or chromatography beads have a very high protein binding capacity and they also bind large biomolecules such as plasmids and viruses. However, the separation mechanism of large biomolecules is still not well understood and this lack of knowledge hinders the development and optimization of the purification processes. To overcome this, our aim is to elucidate the adsorption mechanisms of VLPs, large proteins and protein superstructures into different types of anion exchange chromatography media including highly charged hydrogels and polymer-grafted media. The binding kinetics and equilibria of HIV-1 VLPs expressed in CHO cells and Influenza VLPs expressed in Baculovirus-Insect cell system have been measured for polymer grafted media to elucidate the effect of the charged polymer. Adsorption isotherms were measured in microtiter plates and kinetics in batch mode. Gerster, P., Kopecky, E.-M., Hammerschmidt, N., Klausberger, M., Krammer, F., Grabherr, R., Mersich, C., Urbas, L., Kramberger, P., Paril, T., Schreiner, M., Nöbauer, K., Razzazi-Fazeli, E., Jungbauer, A. Purification of infective baculoviruses by monoliths (2013) Journal of Chromatography A, 1290, 36-45. Jungbauer, A., Hahn, R. Polymethacrylate monoliths for preparative and industrial separation of biomolecular assemblies (2008) Journal of Chromatography A, 1184 (1-2), 62-79. Jungbauer, A. Chromatographic media for bioseparation (2005) Journal of Chromatography A, 1065 (1), 3-12

Polymer grafted chromatography media for direct capture and high-resolution purification of enveloped virus-like particles

Usually, the downstream processing of viruses and virus-like particles (VLP) does not include conventional chromatography media (beads) in the capture and/or purification steps. For large biomolecules, the binding capacity of conventional resins is limited to the outer surface of the beads. We developed a purification process based on polymer-grafted media, which allows a swift purification of HIV-1 VLP from CHO cell culture supernatant. The dynamic binding capacity is one order of magnitude lower than convective media but still in the range of 5-7x1011 particles/ml packed bed, which is unexpectedly high. For that reason, the binding mechanism was studied in detail. As expected, transmission electron micrographs showed that the VLPs only adsorb at the outer surface of the beads. This was corroborated by confocal microscopy using florescence labelled VLPs by incorporating cell membrane label. In batch update experiments, we observed a biphasic behavior with a fast uptake within minutes followed by a slow adsorption within hours. Desorption was also occurring very fast within minutes. Modeling linear gradient elutions with different gradient slopes showed that the number of effective charges involved in the adsorption is in the range of 30 and the adsorption is not really affected by salt. This explains why VLPs can be directly loaded from culture supernatant without further preconditioning. Scalability is not an issue, because these polymer grafted media can be packed in any scale from less than 0.5 ml to several hundred liters and in any column geometry

Haemoglobinopathies in Europe: health & migration policy perspectives

BACKGROUND: Major haemoglobinopathies (MH), such as thalassaemia syndromes (Thal) and sickle cell disorders (SCD), are genetic defects associated with chronic anaemia and other complications. In Europe, MH are rare diseases (RD) but their prevalence is significantly growing in many countries due to mobility and migration flows. This creates a growing health problem in the EU that has not yet been effectively addressed by Member States (MS) authorities. The present study has been conducted with the aim of: (i) providing an overview of policies for MH in 10 EU member states (MS) (ii) analysing the challenges linked to these RD due to growing requirements imposed by population, mobility and migration trends and (iii) identifying gaps, proposing improvements on existing policies, or developing new ones to fit the identified needs. METHODS: The study has been undertaken by a group of members of the European Network for Rare and Congenital Anaemias (ENERCA) and the Thalassaemia International Federation (TIF), in collaboration with the public affairs firm Burson-Marsteller Brussels. Data from 10 EU countries have been gathered using targeted desk research and one-to-one interviews with local stakeholders, including healthcare professionals, patients and public health officers/providers. RESULTS: 1. MH are the most common RD in all the 10 countries, 2. Data on prevalence, overall burden, trends, and clinical follow up costs are lacking in most countries. 3. Neonatal screening practices show a wide variation across and within countries. 4. Awareness on MH and their related complications is very low, exception made of Italy, Greece, Cyprus and UK, 5. No disaggregated data is available to understand the impact of mobility and migration on the prevalence of haemoglobinopathies, and how healthcare delivery systems should adapt to respond to this situation. 6. Targeted policy measures and/or actions are generally lacking and/or delayed. CONCLUSIONS: Ten policy recommendations have been drawn from this study, building on 2006 WHO recommendations for MH to include haemoglobinopathies in National Plans of Actions for Rare Diseases

Especies de Paracoccidioides circulantes en Paraguay y su relación con características epidemiológicas y clínicas de los pacientes.

El objetivo general del proyecto fue identificar especies de Paracoccidioides circulantes en Paraguay y relacionar con las características epidemiológicas y clínicas de los pacientes.CONACYT - Consejo Nacional de Ciencias y TecnologíaPROCIENCI

Observation of Higgs boson production in association with a top quark pair at the LHC with the ATLAS detector

The observation of Higgs boson production in association with a top quark pair ( tt¯H ), based on the analysis of proton–proton collision data at a centre-of-mass energy of 13 TeV recorded with the ATLAS detector at the Large Hadron Collider, is presented. Using data corresponding to integrated luminosities of up to 79.8 fb −1 , and considering Higgs boson decays into bb¯ , WW⁎ , τ+τ− , γγ , and ZZ⁎ , the observed significance is 5.8 standard deviations, compared to an expectation of 4.9 standard deviations. Combined with the tt¯H searches using a dataset corresponding to integrated luminosities of 4.5 fb −1 at 7 TeV and 20.3 fb −1 at 8 TeV, the observed (expected) significance is 6.3 (5.1) standard deviations. Assuming Standard Model branching fractions, the total tt¯H production cross section at 13 TeV is measured to be 670 ± 90 (stat.) −100+110 (syst.) fb, in agreement with the Standard Model prediction.Peer Reviewe

Measurement of photon–jet transverse momentum correlations in 5.02 TeV Pb + Pb and pppp collisions with ATLAS

Jets created in association with a photon can be used as a calibrated probe to study energy loss in the medium created in nuclear collisions. Measurements of the transverse momentum balance between isolated photons and inclusive jets are presented using integrated luminosities of 0.49 nb$^{-1}$ of Pb+Pb collision data at $\sqrt{s_\mathrm{NN}}=5.02$ TeV and 25 pb$^{-1}$ of $pp$ collision data at $\sqrt{s}=5.02$ TeV recorded with the ATLAS detector at the LHC. Photons with transverse momentum $63.1 31.6$ GeV and pseudorapidity $\left|\eta^\mathrm{jet}\right| 7\pi/8$. Distributions of the per-photon jet yield as a function of $x_\mathrm{J\gamma}$, $(1/N_\gamma)(\mathrm{d}N/\mathrm{d}x_\mathrm{J\gamma})$, are corrected for detector effects via a two-dimensional unfolding procedure and reported at the particle level. In $pp$ collisions, the distributions are well described by Monte Carlo event generators. In Pb+Pb collisions, the $x_\mathrm{J\gamma}$ distribution is modified from that observed in $pp$ collisions with increasing centrality, consistent with the picture of parton energy loss in the hot nuclear medium. The data are compared with a suite of energy-loss models and calculations.Peer Reviewe

A large scale hearing loss screen reveals an extensive unexplored genetic landscape for auditory dysfunction

The developmental and physiological complexity of the auditory system is likely reflected in the underlying set of genes involved in auditory function. In humans, over 150 non-syndromic loci have been identified, and there are more than 400 human genetic syndromes with a hearing loss component. Over 100 non-syndromic hearing loss genes have been identified in mouse and human, but we remain ignorant of the full extent of the genetic landscape involved in auditory dysfunction. As part of the International Mouse Phenotyping Consortium, we undertook a hearing loss screen in a cohort of 3006 mouse knockout strains. In total, we identify 67 candidate hearing loss genes. We detect known hearing loss genes, but the vast majority, 52, of the candidate genes were novel. Our analysis reveals a large and unexplored genetic landscape involved with auditory function

Immune and spermatogenesis-related loci are involved in the development of extreme patterns of male infertility

We conducted a genome-wide association study in a large population of infertile men due to unexplained spermatogenic failure (SPGF). More than seven million genetic variants were analysed in 1,274 SPGF cases and 1,951 unaffected controls from two independent European cohorts. Two genomic regions were associated with the most severe histological pattern of SPGF, defined by Sertoli cell-only (SCO) phenotype, namely the MHC class II gene HLA-DRB1 (rs1136759, P = 1.32E-08, OR = 1.80) and an upstream locus of VRK1 (rs115054029, P = 4.24E-08, OR = 3.14), which encodes a protein kinase involved in the regulation of spermatogenesis. The SCO-associated rs1136759 allele (G) determines a serine in the position 13 of the HLA-DR beta 1 molecule located in the antigen-binding pocket. Overall, our data support the notion of unexplained SPGF as a complex trait influenced by common variation in the genome, with the SCO phenotype likely representing an immune-mediated condition. A GWAS in a large case-control cohort of European ancestry identifies two genomic regions, the MHC class II gene HLA-DRB1 and an upstream locus of VRK1, that are associated with the most severe phenotype of spermatogenic failure