The Experts below are selected from a list of 15717 Experts worldwide ranked by ideXlab platform
Jérôme F. L. Duval - One of the best experts on this subject based on the ideXlab platform.
-
impact of the Virus Purification protocol on aggregation and electrokinetics of ms2 phages and corresponding Virus like particles
Physical Chemistry Chemical Physics, 2013Co-Authors: Jérôme F. L. Duval, Cheryl Dika, Christophe Gantzer, A PerrinAbstract:Previous experimental and theoretical studies have established that electrokinetic and aggregation properties of soft MS2 phages are not only governed by the physico-chemical features of their proteinaceous outer surface but are also significantly impacted by those of their inner RNA component (Dika et al. Appl. Environ. Microbiol., 2011, 14, 4939–4948). These conclusions contradict the recent findings of Nguyen et al. (Soft Matter, 2011, 7, 10449–10456) who reported identical electrokinetic and aggregation characteristics for MS2 and corresponding Virus like particles (VLPs) that lack the internal RNA component. We demonstrate here that this contradiction originates from the different Purification methods adopted prior to measurements. More generally, we show that stability and electrohydrodynamics of Viruses differ according to Purification by (i) dialysis, (ii) isopycnic centrifugation in the cesium chloride gradient, and (iii) precipitation using polyethylene glycol (PEG). Methods (i) and (iii) lead to aggregation of MS2 phages at pH ≤ 4 and pH ≤ 6 in 1–100 mM NaNO3 solutions, respectively, while under such conditions aggregation is not observed for MS2 and VLP suspensions prepared according to (ii). In addition, VLPs prepared following methods (i) and (iii) aggregate only at the isoelectric point (pH ∼ 3–4) in 1 mM NaNO3 solution. Electrophoretic mobility data of stable MS2 and VLP particles were further examined using a recent formalism for electrokinetics of soft multilayered colloids. The analysis qualitatively shows how the Purification protocol may affect either the outer particle surface properties and/or the inner particle content. Finally, the non-DLVO aggregation behavior of MS2 and VLPs purified via the above protocols is discussed in terms of the possible change in corresponding interparticular interactions.
Udo Reichl - One of the best experts on this subject based on the ideXlab platform.
-
hydrophobic interaction chromatography for Purification of influenza a and b Virus
Journal of Chromatography B, 2019Co-Authors: Thomas Weigel, Remon Soliman, Michael W Wolff, Udo ReichlAbstract:Abstract Options for hydrophobic-interaction chromatography (HIC) for Purification of cell culture-derived influenza A and B Virus have been assessed using a 96-well plate format using a semi-high-throughput approach. Follow-up experiments at preparative scale were used to characterize dynamic binding capacity, viral hemagglutinin protein (HA protein) recovery, and the influence of influenza Virus (IV) strains on yield and contamination levels. Virus recoveries of up to 96% with a residual DNA level of about 1.3% were achieved. To achieve DNA contamination levels required for manufacturing of influenza vaccines for human use, a Purification train comprising clarification, inactivation, concentration, column-based anion-exchange chromatography (AEC), and HIC was used in a final set-up. AEC using strong quaternary ammonium ligands was applied as an orthogonal method for DNA depletion by adsorption. Subsequently, HIC (with polypropylene glycol as functional group) was used to reversibly bind Virus particles for capture and to remove residual contaminating DNA and proteins (flow-through). This two-step chromatographic process, which requires neither a buffer exchange step nor nuclease treatment had a total Virus particle yield for IV A/PR/8/34 (H1N1) of 92%. The protein and the DNA contamination level could be reduced to 42% and at least 1.0%, respectively. With 17.2 μg total protein and 2.0 ng DNA per monovalent dose, this purity level complies with the limits of the European Pharmacopeia for cell culture-derived human vaccines. Overall, the presented downstream process represents a valuable alternative to existing Virus Purification schemes. Furthermore, it utilizes only off-the-shelf materials and is a simple as well as an economic process for production of cell culture-derived Viruses and viral vectors.
-
a flow through chromatography process for influenza a and b Virus Purification
Journal of Virological Methods, 2014Co-Authors: Thomas Weigel, Michael W Wolff, Udo Reichl, Thomas Solomaier, Alessa Peuker, Trinath PathapatiAbstract:Vaccination is still the most efficient measure to protect against influenza Virus infections. Besides the seasonal wave of influenza, pandemic outbreaks of bird or swine flu represent a high threat to human population. With the establishment of cell culture-based processes, there is a growing demand for robust, economic and efficient downstream processes for influenza Virus Purification. This study focused on the development of an economic flow-through chromatographic process avoiding Virus strain sensitive capture steps. Therefore, a three-step process consisting of anion exchange chromatography (AEC), Benzonase(®) treatment, and size exclusion chromatography with a ligand-activated core (LCC) was established, and tested for Purification of two influenza A Virus strains and one influenza B Virus strain. The process resulted in high Virus yields (≥68%) with protein contamination levels fulfilling requirements of the European Pharmacopeia for production of influenza vaccines for human use. DNA was depleted by ≥98.7% for all strains. The measured DNA concentrations per dose were close to the required limits of 10ng DNA per dose set by the European Pharmacopeia. In addition, the added Benzonase(®) could be successfully removed from the product fraction. Overall, the presented downstream process could potentially represent a simple, robust and economic platform technology for production of cell culture-derived influenza vaccines.
-
n glycan analysis by cge lif profiling influenza a Virus hemagglutinin n glycosylation during vaccine production
Electrophoresis, 2008Co-Authors: J Schwarzer, Udo Reichl, Erdmann RappAbstract:Glycoproteins, such as monoclonal antibodies as well as recombinant and viral proteins produced in mammalian cell culture play an important role in manufacturing of many biopharmaceuticals. To ensure consisting quality of the corresponding products, glycosylation profiles have to be tightly controlled, as glycosylation affects important properties of the corresponding proteins, including bioactivity and antigenicity. This study describes the establishment of a method for analyzing N-glycosylation patterns of mammalian cell culture-derived influenza A Virus glycoproteins used in vaccine manufacturing. It comprises Virus Purification directly from cell culture supernatant, protein isolation, deglycosylation, and clean-up steps as well as "fingerprint" analysis of N-glycan pools by CGE-LIF, using a capillary DNA-sequencer. Reproducibility studies of CGE-LIF, Virus Purification, and sample preparation have been performed. For demonstrating its applicability, the method was exemplarily used for monitoring batch-to-batch reproducibility in vaccine production, with respect to the glycosylation pattern of the membrane protein hemagglutinin of influenza A/PR/8/34 (H1N1) Virus. This method allows characterization of variations in protein glycosylation patterns, directly by N-glycan "fingerprint" alignment.
Nicholas Muzyczka - One of the best experts on this subject based on the ideXlab platform.
-
recombinant adeno associated Virus Purification using novel methods improves infectious titer and yield
Gene Therapy, 1999Co-Authors: Sergei Zolotukhin, Barry J Byrne, E Mason, Irene Zolotukhin, Mark R Potter, Kye Chesnut, C Summerford, Richard J Samulski, Nicholas MuzyczkaAbstract:Conventional methods for rAAV Purification that are based on cesium chloride ultracentrifugation have often produced vector preparations of variable quality and resulted in significant loss of particle infectivity. We report here several novel Purification strategies that involve the use of non-ionic iodixanol gradients followed by ion exchange or heparin affinity chromatography by either conventional or HPLC columns. These methods result in more than 50% recovery of rAAV from a crude lysate and routinely produce vector that is more than 99% pure. More importantly, the new Purification procedures consistently produce rAAV stocks with particle-to-infectivity ratios of less than 100, which is significantly better than conventional methods. The new protocol increases the overall yield of infectious rAAV by at least 10-fold and allows for the complete Purification of rAAV in 1 working day. Several of these methods should also be useful for large-scale production.
-
recombinant adeno associated Virus Purification using novel methods improves infectious titer and yield
Gene Therapy, 1999Co-Authors: Sergei Zolotukhin, Barry J Byrne, E Mason, Irene Zolotukhin, Mark R Potter, Kye Chesnut, C Summerford, Richard J Samulski, Nicholas MuzyczkaAbstract:Recombinant adeno-associated Virus Purification using novel methods improves infectious titer and yield
Cheryl Dika - One of the best experts on this subject based on the ideXlab platform.
-
impact of the Virus Purification protocol on aggregation and electrokinetics of ms2 phages and corresponding Virus like particles
Physical Chemistry Chemical Physics, 2013Co-Authors: Jérôme F. L. Duval, Cheryl Dika, Christophe Gantzer, A PerrinAbstract:Previous experimental and theoretical studies have established that electrokinetic and aggregation properties of soft MS2 phages are not only governed by the physico-chemical features of their proteinaceous outer surface but are also significantly impacted by those of their inner RNA component (Dika et al. Appl. Environ. Microbiol., 2011, 14, 4939–4948). These conclusions contradict the recent findings of Nguyen et al. (Soft Matter, 2011, 7, 10449–10456) who reported identical electrokinetic and aggregation characteristics for MS2 and corresponding Virus like particles (VLPs) that lack the internal RNA component. We demonstrate here that this contradiction originates from the different Purification methods adopted prior to measurements. More generally, we show that stability and electrohydrodynamics of Viruses differ according to Purification by (i) dialysis, (ii) isopycnic centrifugation in the cesium chloride gradient, and (iii) precipitation using polyethylene glycol (PEG). Methods (i) and (iii) lead to aggregation of MS2 phages at pH ≤ 4 and pH ≤ 6 in 1–100 mM NaNO3 solutions, respectively, while under such conditions aggregation is not observed for MS2 and VLP suspensions prepared according to (ii). In addition, VLPs prepared following methods (i) and (iii) aggregate only at the isoelectric point (pH ∼ 3–4) in 1 mM NaNO3 solution. Electrophoretic mobility data of stable MS2 and VLP particles were further examined using a recent formalism for electrokinetics of soft multilayered colloids. The analysis qualitatively shows how the Purification protocol may affect either the outer particle surface properties and/or the inner particle content. Finally, the non-DLVO aggregation behavior of MS2 and VLPs purified via the above protocols is discussed in terms of the possible change in corresponding interparticular interactions.
Tânia P Pato - One of the best experts on this subject based on the ideXlab platform.
-
Purification of yellow fever Virus produced in vero cells for inactivated vaccine manufacture
Vaccine, 2019Co-Authors: Tânia P Pato, Marta Cristina O Souza, Diogo A Mattos, Elena Caride, Davis Fernandes Ferreira, Luciane Pinto Gaspar, Marcos Da Silva Freire, Leda R CastilhoAbstract:Abstract Yellow fever (YF) is a high-lethality viral disease, endemic in tropical regions of South America and Africa, with a population of over 900 million people under risk. A highly effective attenuated vaccine, produced in embryonated eggs, has been used for about 80 years. However, egg-based production limits manufacturing capacity, and vaccine shortage led to the emergency use of a fractional dose (1/5) by the WHO in an outbreak in Africa in 2016 and by Brazilian authorities during an outbreak in 2018. In addition, rare but fatal adverse events of this vaccine have been reported since 2001. These two aspects make clear the need for the development of a new vaccine. In an effort to develop an inactivated YF vaccine, Bio-Manguinhos/FIOCRUZ started developing a new vaccine based on the production of the attenuated 17DD Virus in serum-free conditions in Vero cells propagated in bioreactors, followed by chromatography-based Purification and β-propiolactone inactivation. Virus Purification was studied in this work. Capture was performed using an anion-exchange membrane adsorber (Sartobind® Q), resulting in a Virus recovery of 80.2 ± 4.8% and a residual DNA level of 1.3 ± 1.6 ng/dose, thus in accordance with the recommendations of the WHO (
