Use of sulfated-cellulose membrane adsorbers to intensify purification of cell culturederived influenza A and B viruses

New generation of cell culture-based vaccines enables faster response to pandemic outbreaks and helps coping with the increasing demand for seasonal vaccines. Besides efficient upstream processing technologies, appropriate economic and robust downstream processing is key to consistently achieve high yields. Chromatography membranes have been extensively evaluated to capture viruses at laboratory scale, for influenza, adenovirus or virus like particles. They have shown great potential to intensify processes thanks to a high binding capacity, high flow-rate and ease of use and are now commonly used in new generation of vaccine processes. We present here Sartobind SC, a novel sulfated cellulose membrane adsorber, for the capture of Influenza. In a recent study, the binding capacity and the purification performance of two bead-based resins and Sartobind SC was compared for three influenza virus strains (H1N1, H3N2 and B) produced in MDCK suspension cells in a chemically defined medium. The dynamic binding capacity for the sulfated cellulose membrane adsorbers was consistently higher than for the resins (8 to 22-fold). Overall, recovery of virus varied between 66% and 81%. Total protein and DNA removal were\u3e74% and\u3e96%, respectively. Due to the higher operating flow rate and binding capacity, the productivity with the membrane adsorbers was on average 25-times higher than with the resins. This purification platform based on sulfated cellulose can intensify processes and therefore reduce the cost of influenza purification. This ligand is also a promising candidate for other viruses such as vaccinia, RSV, HSV or measles

Continuous purification of cell culture-derived influenza A virus particles through pseudo- affinity membrane chromatography

Continuous manufacturing is a relevant trend in biopharmaceutical production to reduce the process footprint and to improve the process economy. Vaccines against world-spread diseases, such as influenza, should benefit in particular from such an approach, given the increasing demand for seasonal vaccines and the need for a fast response in case of a pandemic outbreak. Upstream processing of viral vaccines has seen important progress in continuous production of viral vaccines [1], which further supports the development of hybrid or fully continuous flow-schemes for downstream processing. Please click Additional Files below to see the full abstract

Purifying viruses with a sheet of paper: Single-use steric exclusion chromatography as a capture platform for vaccine candidates

Steric exclusion chromatography (SXC) is a method in which a crude sample is mixed with polyethylene glycol (PEG) and fed to a hydrophilic stationary phase. Selectivity in SXC is strongly influenced by the target species’ size, so it is particularly well suited for purification of large biomolecules such as viruses and virus-like-particles. The product is captured without a direct chemical interaction thanks to the mutual steric exclusion of PEG between the product and the stationary phase (cellulose membranes with micron-sized pores). Product elution is achieved by removing the PEG from solution, and can theoretically be made in any buffer system. The low cost of the cellulose membranes allows this operation to be single-use. Using SXC, we have achieved virtually full recovery of several viruses produced in serum-free mammalian cell culture: influenza virus, yellow fever virus, and Modified Vaccinia Ankara (MVA) virus. For influenza virus, four different strains were produced separately in MDCK cell suspension cultures using either chemically defined medium or serum-free medium. Full recovery of all strains was observed using identical SXC conditions (loading with 8% PEG-6000) for both infectious and chemically inactivated virus particles. Coupling a nuclease treatment for DNA digestion prior to SXC, dsDNA was depleted \u3e99.98%. The column capacity in terms of the viral hemagglutinin antigen was at least 50 mg m−2. In the case of yellow fever virus, two attenuated strains used for commercial manufacture were produced separately in adherent Vero cells grown in serum-free medium. Full recovery of infective virus titer for both strains was attained using 10% PEG-6000 for sample load. The elution fraction was concentrated \u3e100-fold compared to the feed with the very high titer of 6×109 plaque forming units, equivalent to ≈100 000 doses. Total recovery was also observed for MVA virus loaded at 4% PEG-6000; produced in an avian cell line in chemically defined medium, the SXC elution pools contained ≈3.7×109 virions as estimated by TCID50 assay. In conclusion, SXC can drastically reduce process development in terms of time and equipment requirements. The convenience of purifying different virus strains using similar chromatography conditions is almost impossible to match by other methods, as are the high product recoveries typically achieved with SXC. The latter gives space to include additional polishing operations without risking low overall process yields. We deem membrane-based SXC as a promising platform technology for capturing viruses and virus-like particles in vaccine manufacturing

Optimization of sulfated cellulose membrane adsorbers for the purification of influenza virus

The impact of influenza virus worldwide drives significant efforts and resources into R&D of vaccine manufacturing processes. A major challenge is to improve the flexibility of these processes, without substantially compromising productivity. As in other biotechnological processes, implementation of a chromatographic capturing step is favored since it removes the majority of the impurities (host cell proteins and DNA) and concentrates the product before polishing. Over the last years, significant efforts towards the development of sulfated membrane adsorbers were made. An improved membrane structure and the direct sulfation of a cellulose matrix resulted in a membrane adsorber (SCMA) with pseudo-affinity characteristics which can be used in the main chromatographic separation step for influenza virus. Pore size and ligand density are structural characteristics critical for the performance of the SCMA. For the best combination tested, the dynamic binding capacity (DBC) of the SCMA was shown to be 5.6×106 HAU/mlmembrane, which corresponds to an approximately 5.5 times higher capacity than bead-based media. However, both SCMA and bead-based media show a similar performance in terms of product recovery (86%-96%) and contaminant removal. Experimental results revealed the importance of operational parameters like virus concentration, flow rate, conductivity and elution salt concentration. Therefore, a DoE was used to determine the optimal process conditions in terms of product losses in the flow through as well as overall product yield and purity for an optimized SCMA using an influenza H1N1 virus strain. Currently undergoing experiments aim the identification of the best operating conditions, reevaluation of the DBC and purification performance of the optimized SCMA for the same virus strain. The robustness of the process for other virus (e.g. influenza H3N2 and B) is also being considered. Combining the advantages of a membrane chromatographic support with a chemical modification that is shown to be specific for the purification of influenza virus is a significant technological advance. After optimization of the SCMA and the respective operating conditions significant improvements in the influenza vaccine production process are to be expected

Addressing the challenges of influenza virus-like particles purification

Virus-like particles (VLPs) have been widely used in vaccine development over the last decades [1]. In fact, there are already several approved human vaccines against viruses that use recombinant VLPs as antigen, e.g. for hepatitis B virus and human papillomavirus [2]. Vaccination remains the most effective way to prevent infection with influenza viruses. However, their constant antigenic drift requires an annual update of the seasonal vaccine to prevent influenza epidemics [3-4]. To use the full potential of VLPs as vaccines efficient upstream processing as well as downstream processing (DSP) trains need to be established. The latter is of particular importance as it often accounts for the major biomanufacturing costs. Here we describe the establishment of an improved DSP unit train platform, adapted from virus particles to influenza VLPs, using pseudo-affinity sulfated cellulose membrane adsorbers (SCMA) [5]. An initial clarification step prepares the bulk for the subsequent purification steps. SCMA performance was optimized using a design of experiments (DoE) approach. More than 80% of the product was recovered with removal of host cell protein and DNA above 89% and 80%, respectively. This represents a significant improvement in performance compared to the traditional use of ion exchangers commercially available. Using this SCMA platform for influenza virus particles purification we were able to speed up the process by decreasing the number of DSP steps, to improve the scale-up and to reduce costs due to the removal of other chromatographic steps. References [1] L. Lua, et al., Biotechnology and Bioengineering, 111(3): p. 425-440 (2014). [2] Q. Zhao, et al., Trends in Biotechnology, 31(11): p. 654-663 (2013). [3] D. Smith, et al., Science, 305(5682): p. 371-376 (2004). [4] C. Thompson, et al., Virology Journal, 10 (2013). [5]M. Wolff, and U. Reichl, Expert Review of Vaccines, 10(10): p. 1451-1475 (2011)

Influenza virus capture using membrane chromatography: Improving selectivity by matrix design and pseudo-affinity ligand interactions

Because next generation cell-based influenza vaccines have to be produced faster and in greater quantities than traditional vaccines, future purification processes will require more efficient unit operations for their isolation and purification. Membrane chromatography has already demonstrated a number of positive characteristics for the bind&elute purification of viral particles like e.g. adenoviruses or influenza viruses. The technology not only addresses the diffusion limitations of porous particle media but also offers dramatic advantages in binding capacity in a disposable format. Therefore, the last remaining challenge for the easy adoption of this technology in the vaccine industry represents selectivity and recovery. We present here a novel cellulose based stationary phase whose active specific surface area is designed for maximum virus accessibility. The resulting gain in selectivity and recovery but also in binding capacity is further maximized by using highly selective pseudo affinity ligands for influenza viruses

Tuberculosis control: decentralization, local planning and management specificities

The goal was to analyze, according to the perception of health managers, the practices that guide tuberculosis control actions in cities in the metropolitan region of João Pessoa - PB, Brazil. This qualitative study involved eight professionals in management functions. Testimonies were collected through semi-structured interviews between May and June 2009 and organized through content analysis. Despite the acknowledged benefits of tuberculosis control action decentralization, local planning indicates the predominance of a bureaucratic model that is restricted to negotiation and supplies. Local programming is centered on the coordinator, which shows a command line and vertical management that lead to the fragmentation of the work process. Management action should follow an innovative and transformative route that surpasses bureaucratic barriers and faces the biggest challenge it is proposed: to balance professional interrelations with a view to improving health work performance.Se buscó analizar, según la percepción de los gestores de salud, las prácticas que orientan las acciones de control de la tuberculosis en municipios de la región metropolitana de Joao Pesoa, estado de Paraíba. Se trata de un estudio cualitativo que envolvió ocho profesionales que ejercían cargos de gestión. Las declaraciones fueron recolectadas por medio de entrevistas semiestructuradas, entre mayo y julio de 2009, y organizadas mediante análisis de contenido. A pesar de que se reconozcan los beneficios de la descentralización de las acciones de control de la tuberculosis, la planificación local señala la predominancia del modelo burocrático restricto a negociación y suministro de insumos. La programación local se centra en la figura del coordinador, retratando una línea de comando y gestión vertical que induce a la fragmentación del proceso de trabajo. La tarea de administrar debe explorar un camino innovador y transformador, que ultrapase las barreras burocráticas y alcance el mayor desafío que le es impuesto: equilibrar las interrelaciones profesionales con la finalidad de perfeccionar el desempeño del trabajo en salud.Buscou-se analisar, segundo a percepção dos gestores de saúde, as práticas que norteiam as ações de controle da tuberculose, em municípios da região metropolitana de João Pessoa, PB. Trata-se de estudo qualitativo que envolveu oito profissionais que exerciam cargos de gestão. Os depoimentos foram coletados por meio de entrevista semiestruturada, entre maio e julho de 2009, e organizados mediante análise de conteúdo. Embora se reconheça os benefícios da descentralização das ações de controle da tuberculose, o planejamento local sinaliza a predominância de modelo burocrático restrito à negociação e provisão de insumos. A programação local centra-se na figura do coordenador, retratando uma linha de comando e gestão vertical que induzem à fragmentação do processo de trabalho. A tarefa de gerenciar deve trilhar caminho inovador e transformador que ultrapasse as barreiras burocráticas e alcance o maior desafio que lhe é imposto: equilibrar as inter-relações profissionais no intuito de aperfeiçoar o desempenho do trabalho em saúde

Global disparities in surgeons’ workloads, academic engagement and rest periods: the on-calL shIft fOr geNEral SurgeonS (LIONESS) study

: The workload of general surgeons is multifaceted, encompassing not only surgical procedures but also a myriad of other responsibilities. From April to May 2023, we conducted a CHERRIES-compliant internet-based survey analyzing clinical practice, academic engagement, and post-on-call rest. The questionnaire featured six sections with 35 questions. Statistical analysis used Chi-square tests, ANOVA, and logistic regression (SPSS® v. 28). The survey received a total of 1.046 responses (65.4%). Over 78.0% of responders came from Europe, 65.1% came from a general surgery unit; 92.8% of European and 87.5% of North American respondents were involved in research, compared to 71.7% in Africa. Europe led in publishing research studies (6.6 ± 8.6 yearly). Teaching involvement was high in North America (100%) and Africa (91.7%). Surgeons reported an average of 6.7 ± 4.9 on-call shifts per month, with European and North American surgeons experiencing 6.5 ± 4.9 and 7.8 ± 4.1 on-calls monthly, respectively. African surgeons had the highest on-call frequency (8.7 ± 6.1). Post-on-call, only 35.1% of respondents received a day off. Europeans were most likely (40%) to have a day off, while African surgeons were least likely (6.7%). On the adjusted multivariable analysis HDI (Human Development Index) (aOR 1.993) hospital capacity > 400 beds (aOR 2.423), working in a specialty surgery unit (aOR 2.087), and making the on-call in-house (aOR 5.446), significantly predicted the likelihood of having a day off after an on-call shift. Our study revealed critical insights into the disparities in workload, access to research, and professional opportunities for surgeons across different continents, underscored by the HDI

Educomunicação e suas áreas de intervenção: Novos paradigmas para o diálogo intercultural

oai:omp.abpeducom.org.br:publicationFormat/1O material aqui divulgado representa, em essência, a contribuição do VII Encontro Brasileiro de Educomunicação ao V Global MIL Week, da UNESCO, ocorrido na ECA/USP, entre 3 e 5 de novembro de 2016. Estamos diante de um conjunto de 104 papers executivos, com uma média de entre 7 e 10 páginas, cada um. Com este rico e abundante material, chegamos ao sétimo e-book publicado pela ABPEducom, em seus seis primeiros anos de existência. A especificidade desta obra é a de trazer as “Áreas de Intervenção” do campo da Educomunicação, colocando-as a serviço de uma meta essencial ao agir educomunicativo: o diálogo intercultural, trabalhado na linha do tema geral do evento internacional: Media and Information Literacy: New Paradigms for Intercultural Dialogue