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

Continuous-flow liquid-phase dehydrogenation of 1,4-cyclohexanedione in a structured multichannel reactor

A highly selective, scalable and continuous-flow process is developed for the liquid-phase dehydrogenation of 1,4-cyclohexanedione to hydroquinone in a millimetre-scale structured multichannel reactor. The square shaped channels (3 mm × 3 mm) were filled with 10 wt% Pd/C catalyst particles and utilized for the dehydrogenation reaction in single-pass and recycle modes. For the purpose to enhance process understanding and to maximize conversion and selectivity by process optimization, Design of Experiment (DoE) methodology was utilized by studying the effect of operating parameters on the catalytic performance in kinetic regime. The results demostrated the strong influence of temperature and liquid feed flow on the conversion and selectivity, with liquid feed and N₂ flows influencing pressure drop significantly. A multi-objective optimization methodology was used to identify the optimum process window with the aid of sweet spot plots, with design space plots developed to establish acceptable boundaries for process parameters. In single-pass mode, complete conversion per pass per channel was not achievable whereas conversion increased from 59.8% in one-channel to 78.3% for two-channel-in-series while maintaining selectivity (> 99%) with intermediate hydrogen removal. However, for without intermediate H₂ removal step, selectivity was declined from > 99% in one-channel to 82.3% at the outlet of second-channel. In recycle mode, dehydrogenation reaction was resulted in almost complete conversion (> 99%) with very high selectivity (> 99%) and yield (> 98%). This combination of mm-scale multichannel reactor and DoE methodology opens the way to developing highly selective and scalable dehydrogenation proocesses in the fine chemical and pharmaceutical industries

Advanced surface derivatization of superparamagnetic iron oxide nanoparticles in a fixed bed magnetic reactor for bio-application

There is an enormous interest in exploiting nanoparticles in various biomedical applications since their size scale is similar to that of biological molecules (e.g., proteins, DNA) and structures (e.g., viruses and bacteria). As the field continues to develop, quantitative and qualitative studies on particle-cell interaction, with respect to their size and surface, are required in order to advance nanotechnology for biomedical applications. This will be important for assessing nanoparticle toxicity (i.e. translocation into cells and interference with viability and cellular function), for advancing nanoparticles for imaging, drug delivery, and therapeutic applications (i.e. targeting specific cells, organs, or tumors), and for designing multifunctional nanoparticles. Due to the lack of systematic study to date, data are difficult to compare since the parameters and particles in each of the published studies differ substantially. However, the scientific community, in general, agrees that the size and colloidal behavior play a crucial role in cellular interaction/uptake, biodistribution, clearance and cytotoxicity. Surface functionalization of nanoparticles still remains a difficult task and represents not only a chemical challenge but constitutes a basic requirement for future scientific investigations. Alteration of surface charges and/or stabilization by the addition of bi- / multi-functional molecules, such as differently charged proteins or plasmids, frequently leads to particle flocculation and rapid sedimentation. The biological functionality, in such cases, is achieved by covalent binding of bio-active molecules on a preexisting single surface coating. A fixed bed magnetic reactor has been developed with a quadrupole repulsive arrangement of permanent magnets which allows for surface derivatization by magnetically immobilizing superparamagnetic iron oxide nanoparticles (SPIONs). The yield and quality of the resulting functionalized SPIONs was significantly improved with reduction in reaction times using solid phase synthesis strategy. In this way, pH changes across the isoelectric point, washing steps or even solvent exchanges could be easily tolerated thereby avoiding the problems of colloidal instability during the derivatization steps. It was shown that the surface functionalization of SPIONs using a magnetic fixed bed reactor was superior to the liquid phase reaction in terms of reaction yield, particle size distribution, colloidal stability and scalability. In particular, cell organelle targeting peptide derivatized on SPIONs surface was obtained from the reactor. The combination of functionalized SPIONs and their ability to be recovered using a magnetic column coupled with biomolecular mass spectrometry has allowed to explore a complex intracellular pathway using a peptide that is known to target HeLa cell organelle. Here the concept of biomolecular interaction network elucidation with an organelle-targeting peptide was demonstrated. Besides that, the colloidal stability and cellular uptake of polymer coated SPIONs were also studied. Preliminary results showed that minor modifications of the nanoparticle surface lead to an altered behavior in stability, uptake, and toxicity. Also, different charges on the particle surfaces were found responsible for differential uptake of particles in cell media. Colloidal stability and its influence on biological properties will provide a profound base for future discussions on toxicity and potential application of nanoparticles in the field of biomedicine

Influence of serum supplemented cell culture medium on colloidal stability of polymer coated iron oxide and polystyrene nanoparticles with impact on cell interactions in vitro

When nanoparticles interact with cells, the possible cellular responses to the particles depend on an array of parameters, in both particle and biological aspects. On the one hand, the physicochemical properties of the particles (e.g., material, size, shape, and surface charge) are known to play a key role in particle-cell interactions. On the other hand, it has been shown that prior to coming into contact with cells, nanoparticle interaction with the surrounding biological fluid may lead to a change of the initial particle properties. For example, the colloidal behavior of nanoparticles is strongly influenced by the density and viscosity of the surrounding media in both in vitro and in vivo systems

Protein Corona Composition of Superparamagnetic Iron Oxide Nanoparticles with Various Physico-Chemical Properties and Coatings

International audienc

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