Development of a lectin-affinity chromatography step for the downstream processing of influenza virus vaccines

Influenza remains due to its annual death rate and potential to cause pandemics a major public health concern. Efforts to control the annual spread of influenza have centered on prophylactic vaccinations. Human influenza vaccines are traditionally produced in embryonated hen s eggs. However, major constraints with this method, e.g. allergic reactions induced by egg proteins and lack of scalability have lead to the development of cell culture based production processes. In recent years, several continuous cell lines such as the Madin Darby canine kidney (MDCK) or the African green monkey kidney Vero cells have been successfully established for the production of influenza vaccines in cell culture. These processes require the modification of existing but also the development of new downstream strategies to account for the changed upstream technology. Downstream processing of biological products is conventionally subdivided into three steps: capture or concentration, separation or fractionation and polishing. The capture step is commonly the most expensive unit operation. Hence, the efficiency of this step has a large impact on the total process economics. The presented study focuses on the development of a proficient capture step based on lectin-affinity chromatography. Lectins are a class of carbohydrate specific proteins of non-immune origin that have a selective affinity for a carbohydrate or a group of carbohydrates. Immobilized lectins have been used successfully for many years to separate and isolate glycoconjugates, polysaccharides, soluble cell components, and cells containing glycoproteins with specific carbohydrate structures on its surface. The influenza A virus contains two spike glycoproteins on its surface: hemagglutinin (HA) and neuraminidase (NA). HA is the most abundant surface protein. It is a trimeric glycoprotein containing per subunit 3 to 9 N-linked glycosylation sites depending on the viral strain. Here the influenza A/PR/8/34 virus has been selected as a model. The HA molecule of this particular virus contains according to the NetNGlyc 1.0 Server prediction six glycosylation sites. Detailed analysis of these sites and their individual glycan structures are presently performed. Based on preliminary structural glycan analysis studies and literature data several HA-binding lectins are selected for a pre-screening via lectin-blots. The most promising lectinblot results are obtained from lectins specific for terminal galactose e.g. Erythrina cristagalli (ECL), Arachis hypogaea (PNA). Lectins, by which lectin-blot analysis suggests an interaction with viral membrane proteins, are currently screened for their suitability as an affinity matrix ligand. Therefore, centrifuged cultivation broths of influenza A/PR/8/34 virus infected MDCK cells are applied to various agaroseimmobilized lectins. Components interfering with the immobilized lectins are selectively adsorbed. Non or weak binding components are washed from the column. Subsequently, bound components are dissociated from the lectin by competitive elution with suitable hapten carbohydrates. This fraction contains the influenza virus particles and virally encoded membrane proteins, which have to be further processed for vaccine manufacturing. The extend of the subsequent purification depends on the specificity of the lectin binding to virally encoded surface proteins. Lectins with weak or no interaction with host cell proteins or medium components and strong interaction with viral membrane glycoproteins represent a powerful tool to concentrate and purify viral surface proteins from contaminating nucleic acids, medium components, and non-virally encoded host cell proteins

Superparamagnetic Nanoparticles as a Powerful Systems Biology. Characterization Tool in the Physiological Context

Recently, functionalized superparamagnetic iron oxide nanoparticles (SPIONs) have been utilized for protein separation and therapeutic delivery of DNA and drugs. The development of new methods and tools for the targeting and identification of specific biomolecular interactions within living systems is of great interest in the fields of systems biology, target and drug identification, drug delivery, and diagnostics. Magnetic separation of organelles and proteins from complex whole-cell lysates allows enrichment and elucidation of intracellular interaction partners for a specific immobilized protein or peptide on the surface of SPIONs

Lectin-affinity chromatography for downstream processing of MDCK cell culture derived human influenza A viruses

The presented study aims on the development of a capture step for the purification of cell culture derived influenza viruses using lectin affinity chromatography. Human influenza A/Puerto Rico/8/34 virus produced in Madin Darby canine kidney cells have been chosen as a model. The influenza A virus envelop possesses two viral glycoproteins: hemagglutinin and neuraminidase. Oligosaccharides of theses glycoproteins can be targeted as affinity ligands using specific lectins. First, lectins have been screened via lectin blots and spin columns. Adequate lectins have been chosen based on published glycan structures of hemagglutinin. The most specific binding was achieved via the galactose specific Erythrina cristagalli and Euonymus europaeus lectins. Second, the chromatographic separations characteristics of these lectins have been further determined via FPLC. These experiments revealed that the rate of hemagglutinin glycan binding to the ligands was higher with the Euonymus europaeus compared to the Erythrina cristagalli lectin. Third, viral recoveries in addition to the total protein and contaminating host cell DNA have been balanced in a series of Euonymus europaeus lectin chromatography runs. The total protein and dsDNA content in the product fraction of the affinity chromatography was reduced from the starting conditions to 21% and 0.1%, respectively. The average viral recovery in the product fraction was 97%. SDS-PAGE analysis indicated that the majority of the eluted proteins were of viral origin. The reproducibility and column stability was confirmed in more than 25 runs applying 6 different virus product batches. © 2006 Elsevier Ltd. All rights reserved [accessed 2013 November 14th

Effect of cell media on polymer coated superparamagnetic iron oxide nanoparticles (SPIONs): Colloidal stability, cytotoxicity, and cellular uptake studies

The influence of the composition of the polymer coated polyvinyl alcohol (PVA), vinyl alcohol/vinyl amine copolymer (A-PVA) and polyethylenimine (PEI) coated superparamagnetic iron oxide nanoparticles (SPIONs) on the colloidal stability, cytotoxicity and cellular uptake of these particles in different cell media is reported in this paper. Although all examined polymer coated SPIONs were stable in water and PBS buffer these colloidal systems had different stabilities in DMEM or RPMI media without and supplemented with fetal calf serum (FCS). We found that A-PVA coating onto the surface of the SPIONs decreased the cytotoxicity of the polymer compared to the same concentration of A-PVA alone. As well, polyplexes of PEI-SPIONs with DNA in concentration used for transfection experiments showed no cytotoxicity compared to PEI and PEI-SPIONs. Our data show that the choice of medium largely influences the uptake of these particles by HeLa cells. The optimal medium is different for the different examined polymer coated SPIONs and it should be determined in each case, individually

Lectin-affinity purification of cell culture derived human Influenza A viruses

The presented study aims on the development of a capture step for the purification of human Influenza viruses using lectin-affinity chromatography. This enables a high product concentration factor and a separation of the target biomolecule from the major bulk of contaminants with a single step. In the following studies a Madin Darby canine kidney (MDCK) cell produced human Influenza A/PR/8/34 virus has been chosen as a model strain. The Influenza A virus surface possesses two viral spike glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). The HA is the most abundant and immunogenic surface glycoprotein. Based on HA glycoanalysis some adequate lectins have been chosen for lectin binding screening via lectin blots. Therefore, the proteins of the concentrated cultivation broth were separated by SDS-PAGE analysis and transferred onto PVDF-membranes. Treatment of the membrane with biotinylated lectins and detection by chemiluminescence indicated the lectin affinity to viral and host cell glycoproteins. The most specific binding to the viral glycoprotein HA was achieved via the galactose specific lectins Erythrina cristagalli lectin (ECL, gal(betta1,4)glcNAc) and Euonymous Europaeus Lectin (EEL, gal(alpha1,3)gal). These lectins have been selected for further investigations of chromatographic separations. The virus and the viral membrane glycoproteins from concentrated cell culture broth adsorbed to the lectin-polymer-matrix specifically. The majority of host cell proteins did not bind to the lectins. These proteins were washed out from the column. Viral proteins were desorbed from the column by competitive elution with the appropriate carbohydrate. HA recoveries of up to 80% based on activity measurements have been achieved with this procedure with a high degree of removal of host cell proteins and nucleic acids. On that account, lectin affinity chromatography represents a promissing tool for an effective capture step for the downstream processing of MDCK-cell derived Influenza vaccines

Improved dynamic response assessment for intra-articular injected iron oxide nanoparticles

The emerging importance of nanoparticle technology, including iron oxide nanoparticles for monitoring development, progression, and treatment of inflammatory diseases such as arthritis, drives development of imaging techniques. Studies require an imaging protocol that is sensitive and quantifiable for the detection of iron oxide over a wide range of concentrations. Conventional signal loss measurements of iron oxide nanoparticle containing tissues saturate at medium concentrations and show a nonlinear/nonproportional intensity to concentration profile due to the competing effects of T1 and T2 relaxation. A concentration calibration phantom and an in vivo study of intra-articular injection in a rat knee of known concentrations of iron oxide were assessed using the difference-ultrashort echo time sequence giving a positive, quantifiable, unambiguous iron signal and monotonic, increasing concentration response over a wide concentration range in the phantom with limited susceptibility artifacts and high contrast in vivo to all other tissues. This improved dynamic response to concentration opens possibilities for quantification due to its linear nature at physiologically relevant concentrations

Lektin-Affinitätschromatographie zur Aufreinigung humaner Influenza Viren

Humane Influenza Impfstoffe werden traditionell aus beimpften Hühnereiern hergestellt. Ein neuer Weg stellt die Impfstoffherstellung mit Zellkulturen dar, welcher eine Anpassung der Virusaufarbeitung erfordert. Ziel dieser Studie ist es, die Aufreinigung von humanen Influenzaviren aus der Zellkulturbrühe über eine Lektin-Affinitätschromatographie zu entwickeln. Lektine sind Proteine, welche spezifisch an Kohlenhydrate bzw. Kohlenhydratgruppen binden. Als Modellvirus wurde Influenza A/PuertoRico/8/34 in Madin Darby canine kidney (MDCK) Zellen produziert. Die Oberfläche des Influenza A Virus enthält zwei Spikeglykoproteine: das Hämagglutinin (HA) und die Neuraminidase (NA). Das HA ist das am häufigsten vorkommende Oberflächenprotein. Basierend auf dessen Glykostruktur wurden geeignete Lektine für Lektin-Virus Bindungsstudien ausgewählt. Diese Studien umfassten Lektin-Blot-Analysen und Small-scale-Affinitätschromatographie-Experimente. Unter den getesteten Liganden wurde die höchste spezifische Bindung an viralen Proteinen bei den Galactose spezifischen Lektinen Erythrina cristagalli Lectin (ECL) und Euonymous Europaeus Lectin (EEL) beobachtet. Beide Lektine wurden zur Aufreinigung der Influenzaviren über die Affinitätschromatographie eingesetzt. Dabei erreichte man HA-Wiederfindungsraten von bis zu 80% bei gleichzeitiger starker Verringerung der Zellprotein- und Nukleinsäurekontaminationen. Aus diesem Grund repräsentiert die Lectin-Affinitätschromatographie einen vielversprechenden Weg zur Aufarbeitung von Influenzaviren aus Zellkulturen

Lectin-affinity purification of cell culture derived human Influenza A viruses

Influenza remains due to its annual death rate and potential to cause pandemics a major public health concern. Strategies to control influenza outbreaks are mainly focused on prophylactic vaccinations in conjuction with antiviral medications. Human Influenza vaccines are conventionally produced in embryonated hen's eggs. However, major constraints with this method, e.g. allergic reactions induced by egg proteins and lack of scalability have lead to the development of cell culture based production processes. These upstream modifications demand the development of appropriate downstream procedures. Purification of biological products is generally divided into three parts: capture or concentration, separation or fractionation and polishing. The capture step is the most important unit operation concerning the overall process economics. The product selectivity and the concentration factor of the capture step define the efforts required for all further purification steps. The presented study aims on the development of a capture step for the purification of human Influenza viruses using lectin-affinity chromatography. Lectins are proteins, which specifically bind to a carbohydrate or a group of carbohydrates. Introduction of an affinity chromatography step at the beginning of the downstream process enables a high product concentration factor. Furthermore, it separates the target biomolecule from the major bulk of contaminants with a single step. In the following studies a Madin Darby canine kidney (MDCK) cell produced human Influenza A/PR/8/34 virus has been chosen as a model strain. The Influenza A virus surface possesses two viral spike glycoproteins: the hemagglutinin (HA) and the neuraminidase (NA). The HA is the most abundant and immunogenic surface glycoprotein. Based on HA glycoanalysis some adequate lectins have been chosen for lectin binding screening via lectins blots. Therefore, the proteins of the concentrated fermentation broth were separated by SDS-PAGE analysis and transferred onto PVDF-membranes. Treatment of the membrane with biotinylated lectins and detection by chemiluminescence indicated the lectin affinity to viral and host cell glycoproteins. The most specific binding to the viral glycoprotein HA was achieved via the galactose specific lectins Erythrina cristagalli lectin (ECL, gal(beta1,4)glcNAc) and Euonymous Europaeus Lectin (EEL, gal(alpha1,3)gal). These lectins have been selected for further investigations of chromatographic separations. The virus and the viral membrane glycoproteins from concentrated cell culture broth adsorbed to the lectin-polymer-matrix specifically. The majority of host cell proteins did not bind to the lectins. These proteins were washed out from the columns. Viral proteins were desorbted from the column by competitive elution with appropriate carbohydrate. HA recoveries of up to 80% based on activity measurements have been achieved with this procedure with a high degree of removal of host cell proteins and nucleic acids. On that account, lectin affinity chromatography represents a promissing tool for an effective capture step for the downstream processing of MDCK-cell derived Influenza vaccine