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

Alternative splicing of exon 10 in the tau gene as a target for treatment of tauopathies

Tau aggregation is one of the major features in Alzheimer's disease and in several other tauopathies, including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), and progressive supranuclear palsy (PSP). More than 35 mutations in the tau gene have been identified from FTDP-17 patients. A group of these mutations alters splicing of exon 10, resulting in an increase in exon 10 inclusion into tau mRNA. Abnormal splicing with inclusion of exon 10 into tau mRNA has also been observed in PSP and AD patients. These results indicate that abnormal splicing of exon 10, leading to the production of tau with exon 10, is probably one of the mechanisms by which tau accumulates and aggregates in tauopathic brains. Therefore, modulation of exon 10 splicing in the tau gene could potentially be targeted to prevent tauopathies. To identify small molecules or compounds that could potentially be developed into drugs to treat tauopathies, we established a cell-based high-throughput screening assay. In this review, we will discuss how realistic, specific biological molecules can be found to regulate exon 10 splicing in the tau gene for potential treatment of tauopathies

Preparative Size-Exclusion Chromatography

Size-exclusion chromatography in group separation mode is an efficient unit operation for the separation of influenza virions from host cell protein [1]. In order to avoid this operation becoming a bottleneck in vaccine production processes, however, the column load has to be optimised balancing productivity with purity. Design principles and the results of a loading study on Sepharose 4 FF are resented in this article

Monitoring Influenza Virus Content in Vaccine Production : Precise Assays for the Quantitation of Hemagglutination and Neuraminidase Activity

Robust and precise quantitation of influenza virus is a premise for the efficient development of vaccine production processes. In this article, revised assays for the determination of hemagglutination (NA)and neuraminidase (NA) activity are presented. Bias of traditional discontinuous HA assays and operator dependency was overcome by introduction of a regression procedure. At little effort, a continuous assay result is obtained with repeatability as good as +29% / -22% in the best case (95% confidence intervals reported). Similarly, neuraminidase activity determined in microtiter plates resulted in repeatability better than ±20%. NA activity decreased almost linearly for pH ranging from 5.8 to 7.8 and was enhanced by the addition of Ca2+. Non-linearity of the assay (due to unspecific adsorption) was overcome by addition of BSA. Using -methylumbelliferyl- -D-Nacetylneuraminic acid as substrate, Michaelis-Menten constants of 30 and 460 μM were determined for strains A/PR/8/34 (H1N1) and A/Equi 2/NM/1/93 (H3N8), respectively. The error introduced by approximation of Michaelis-Menten kinetics (zero and first order) was minimized by limiting substrate consumption to about 10%. Linearity of both assays was verified in dilution experiments. Applicability was demonstrated in three cases: virus propagation in mammalian cell culture, ultrafiltration and precipitation of nucleic acids. 056/08/$34.00 2007 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved. [accessed 2013 November 14th

Generic Process Scheme for Particle-Based Purification of Cell Culture Derived Influenza A Virus

With the ongoing shift from traditional egg-based production of viral vaccines to cell culture-based cultivation, new purification strategies need to be developed to account for the different starting conditions. In the case of viral vaccines high loads of cellular debris and impurities have to be faced - in contrast to recombinant proteins or viral vectors produced by packaging cell lines. Here we present a modular process scheme for particle-based purification of human and equine influenza A virus as a model system. Virus was replicated with adherent Madin-Darby canine kidney cells, either in parallelized roller bottle cultures or microcarrier fermentations using serum-containing (fetal bovine serum) and serum-free media. Individual tasks were defined (clarification, inactivation, concentration, separation of impurities) and then rendered by appropriate unit operations. A traditional „harvest - capture - purification“ scheme was followed with the additional inactivation step inserted between the harvest and capture steps. Clarification was achieved by sequential depth and static membrane filtration with an option for centrifugal techniques in the future. The main purpose of this step was the removal of cell debris and gel particles contained in microcarrier cultures. Virus inactivation was conducted chemically with the well-established reagents b-propiolactone or ethylenimine. For the capture step, dynamic ultrafiltration, affinity chromatography and adsorptive filters have been investigated aiming at a first purification and concentration of the product. The capture step was then followed by a combination of chromatographic steps and/or adsorptive filters that were selected for efficient stripping of colloidal impurities, in particular host cell protein and dsDNA. In order to characterize each unit operation, robust microtiter plate assays have been developed for the quantitation of viral activity (infectious, hemagglutinin, neuraminidase), protein and dsDNA content. An immunological assay was employed to quantitate the virions. Particle size distributions were determined where appropriate using dynamic light scattering techniques

Generic Process Scheme for Particle-Based Purification of Cell Culture Derived Influenza A Virus

With the ongoing shift from traditional egg-based production of viral vaccines to cell culture-based cultivation, new purification strategies need to be developed to account for the different starting conditions. In the case of viral vaccines high loads of cellular debris and impurities have to be faced - in contrast to recombinant proteins or viral vectors produced by packaging cell lines. Here we present a modular process scheme for particle-based purification of human and equine influenza A virus as a model system. Virus was replicated with adherent Madin-Darby canine kidney cells, either in parallelized roller bottle cultures or microcarrier fermentations using serum-containing (fetal bovine serum) and serum-free media. Individual tasks were defined (clarification, inactivation, concentration, separation of impurities) and then rendered by appropriate unit operations. A traditional „harvest - capture - purification“ scheme was followed with the additional inactivation step inserted between the harvest and capture steps. Clarification was achieved by sequential depth and static membrane filtration with an option for centrifugal techniques in the future. The main purpose of this step was the removal of cell debris and gel particles contained in microcarrier cultures. Virus inactivation was conducted chemically with the well-established reagents b-propiolactone or ethylenimine. For the capture step, dynamic ultrafiltration, affinity chromatography and adsorptive filters have been investigated aiming at a first purification and concentration of the product. The capture step was then followed by a combination of chromatographic steps and/or adsorptive filters that were selected for efficient stripping of colloidal impurities, in particular host cell protein and dsDNA. In order to characterize each unit operation, robust microtiter plate assays have been developed for the quantitation of viral activity (infectious, hemagglutinin, neuraminidase), protein and dsDNA content. An immunological assay was employed to quantitate the virions. Particle size distributions were determined where appropriate using dynamic light scattering techniques

Tangential Flow Filtration of Influenza Virus as a Model System

Background: Purification of virus particles for vaccines and viral vectors for gene therapy is a major large-scale separations challenge. Here we consider the purification of human influenza virus as a model system for enveloped viruses of about 100 nm in diameter. Methods: Virus produced in roller bottles (250 mL per bottle) and microcarrier cultures (2-3 L wv) was clarified by two static filtration steps and inactivated with ß-propiolactone. The use of tangential flow filtration and dialfiltration was investigated in order to concentrate the virus and to reduce the level of contaminating compounds. Small-scale hollow-fibre modules (40..200 cm2 filter area, Amersham Biosciences) with different molecular weight cut-offs and pore sizes (500 kDa, 750 kDa, 0.1 µm, 0.45 µm) were used in this study. Rejection of virus particles, permeation of protein and DNA, permeate flux and flux decline were examined for all the membranes. Results: No virus was found in the permeates of the 500 kDa and 750 kDa membranes whereas complete breakthrough was achieved with the 0.45 µm membrane. Virus particles seemed to enter the pores of the 0.1 µm membrane leading to very low recoveries. Most of the contaminating proteins could be removed by diafiltration using a 750 kDa membrane. Only partial removal of DNA could be achieved independent of the membrane type. Conclusion: 750 kDa hollow-fibre membranes seem to be an ideal choice for the concentration of influenza virus. They resulted in high recoveries of the product whereas most of the contaminating proteins could be removed. The partial removal of DNA suggests that genomic DNA fragments are either about the same size as or a

Maximizing productivity in size-exclusion chromatography of influenza virus : A modelling approach

Introduction. Influenza disease caused by influenza virus impacts every year up to 10% of the world’s population. Vaccination with attenuated or inactivated virus is the principal means of prophylaxis. One approach for the purification of influenza virus is separation based on size using size-exclusion chromatography (SEC). However, SEC often suffers from low productivity posing a bottleneck in downstream processes. Here we present an elegant approach for maximizing the productivity in SEC on the example of vaccine production from cell-culture derived influenza virus. Method. Based on experimental data, a simple method was developed for the prediction of purity, yield and dilution as a function of column load and fractionation. Virus-containing cell culture supernatant (concentrated by cross-flow ultrafiltration) was loaded onto an SEC column at various pulse lengths. Chromatograms were obtained by offline analysis of eluate fractions (HA activity, total protein). Numerical deconvolution of spline interpolated chromatograms followed by least-squares optimzation resulted in the Dirac response of the system. The response to arbitrary loading functions was predicted by convolution with this Dirac response. Predictions of the method were validated by comparison with a control group. Results and Conclusions. Applying the method to a previously established SEC operation, productivity was tuned to 0.15 cv h-1 (at a constant flow rate of 60 cm h-1) while dilution was limited to 0.5 (process requirements were the 95% recovery of eluting virus and a 33% reduction in total protein content). In combination with cross-flow ultrafiltration 20-fold reduction in total protein was achieved. Currently, the method is being extended to incorporate the effects of the flow rate and column length. The method is by no means limited to SEC of influenza virus but provides a general tool aiming at the reduction of experimental work. Due to the nature of the approach no a priori knowledge of the feed composition is required