Boris Hundt

Continuous purification of influenza virus using simulated moving bed chromatography

Continuous size exclusion chromatography for the separation of cell culture-derived influenza virus from contaminating proteins was established successfully. Therefore, an open loop simulated moving bed (SMB) setup with one column per zone was applied. Several operating conditions were tested and overall trends were found to be in agreement with expectations derived from theory. Furthermore, the separation performance was compared to an optimized conventional batch chromatography. The yield of influenza virus in the product fraction, based on a hemagglutination assay, was 70% (SMB) and 80% (batch), respectively. The amount of contaminating protein per product was 0.61μgkHAU(-1) (SMB) compared to 0.29μgkHAU(-1) (batch). This corresponds to a reduction of the respective amount in the feed solution by 60% and 80%, respectively. For both processes, the estimated amount of total protein per vaccine dose would meet the level required for manufacturing of human influenza vaccines prepared in cell cultures. Depending on the strategy chosen for sanitization and equilibration of columns the calculated overall productivity for the SMB process was up to 3.8 times higher compared to the batch mode. SMB, therefore, has the potential to replace single column discontinuous chromatography in order to design more efficient purification trains for production of cell culture-derived influenza vaccines.

Mono- and polyprotic buffer systems in anion exchange chromatography of influenza virus particles.

Different ions typically used in downstream processing of biologicals are evaluated for their potential in anion exchange chromatography of an industrially produced, pandemic influenza H1N1 virus. Capacity, selectivity and recovery are investigated based on single step elution parallel chromatography experiments. The inactivated H1N1 feedstream is produced in Madin-Darby Bovine Kidney cells. Interesting effects are found for sodium phosphate and sodium citrate. Both anions are triprotic kosmotropes. Anion exchange chromatography generally offers high scalability to satisfy sudden demands for vaccines, which may occur in case of an emerging influenza outbreak. Appropriate pH conditions for H1N1 adsorption are determined by Zeta potential measurements. The dynamic binding capacity of a salt tolerant polyamine-type resin is up to 6.4 times greater than the capacity of a grafted Q-type resin. Pseudo-affinity interactions of polyamines with the M2 protein of influenza may contribute to the obtained capacity increase. Both resins achieve greater capacity in sodium phosphate buffer compared to Tris/HCl. A recovery of 67% and DNA clearance close to 100% without DNAse treatment are achieved for the Q-type resin. Recovery of the virus from the salt tolerant resin requires the use of polyprotic acids in the elution buffer. 85% of the DNA and 60% of the proteins can be removed by the salt tolerant resin. The presence of sodium phosphate during anion exchange chromatography seems to support stability of the H1N1 particles in presence of hydrophobic cations.

Large pilot scale cultivation process study of adherent MDBK cells for porcine Influenza A virus propagation using a novel disposable stirred-tank bioreactor

Influenza is one of the major viral diseases and has tremendous importance for human and animal health worldwide. The etiologic agent, Influenza A virus belongs to the family Orthomyxoviridae and is a single stranded (-) enveloped RNA virus with round shape and a diameter of 90-130 nm. Its genome consists of 8 segments which code for up to 11 proteins. The major surface proteins and antigenic determinants are hemagglutinin (HA) and neuraminidase (NA). Pigs are of special importance for Influenza. They are susceptible for a number of Influenza A viruses from different species (human, avian, porcine); in the case of multiple infections of swine new and dangerous virus strains can be assembled by reassortment of genes (antigenic shift). The strain which was used in this study is part of a trivalent inactivated vaccine against porcine Influenza (IDT Biologika GmbH). The cultivation of Influenza virus for the production of vaccines is often still done in embryonated hens eggs, although there are a number of cell lines available which propagate the virus, e. g. VERO or MDCK cells. Egg production processes are limited in terms of capacity whereas cell culture based processes are more flexible and easy to scale up when facing pandemic situations (H5N1 in 2005 and H1N1pan in 2009/10). Currently, adherent MDBK cells are used for the production of porcine Influenza A viruses at commercial scale (IDT Biologika GmbH). This process is performed in roller bottles so far using a biphasic process scheme (cell growth and virus propagation phase) and is thus limited in capacity, yields and process control. The main goal of this study was to explore options for direct transfer of this process into a novel disposable stirred-tank bioreactor (BIOSTAT CultiBag STR, Sartorius Stedim Biotech GmbH) using microcarriers for the attachment of MDBK cells. The advantage of this system is its scalability up to 1000 l, better process control and presumably higher virus yields. The use of disposables has positive effects on costs and efforts but also for cleaning validation in a multi-purpose facility. Based on the results of this study further optimization steps can be done and the implementation of such systems in GMP routine vaccine production can be evaluated.

Size distribution analysis of influenza virus particles using size exclusion chromatography

Size exclusion chromatography is a standard method in quality control of biopharmaceutical proteins. In contrast, vaccine analysis is often based on activity assays. The hemagglutination assay is a widely accepted influenza quantification method, providing no insight in the size distribution of virus particles. Capabilities of size exclusion chromatography to complement the hemagglutination assay are investigated. The presented method is comparatively robust regarding different buffer systems, ionic strength and additive concentrations. Addition of 200mM arginine or sodium chloride is necessary to obtain complete virus particle recovery. 0.5 and 1.0M arginine increase the hydrodynamic radius of the whole virus particles by 5nm. Sodium citrate induces virus particle aggregation. Results are confirmed by dynamic light scattering. Retention of a H1N1v strain correlates with DNA contents between 5ng/mL and 670ng/mL. Quantitative elution of the virus preparations is verified on basis of hemagglutination activity. Elution of hemagglutination inducing compounds starts at a flow channel diameter of 7000nm. The universal applicability is demonstrated with three different influenza virus samples, including an industrially produced, pandemic vaccine strain. Size distribution of the pandemic H1N1v 5258, H1N1 PR/8/34, and H3N2 Aichi/2/68 preparations spreads across inter- and intra-particle volume and extends to the secondary interaction dominated range. Thus, virus particle debris seems to induce hemagglutination. Fragments generated by 0.5% Triton™ X-100 treatment increase overall hemagglutination activity.