Thomas Vogel

High cell density cultivations by alternating tangential flow (ATF) perfusion for influenza A virus production using suspension cells

High cell densities in animal cell culture can be obtained by continuous perfusion of fresh culture medium across hollow fiber membranes that retain the cells. Careful selection of the membrane type and cut-off allows to control accumulation of target molecules and removal of low molecular weight compounds. In this report, perfusion with the scalable ATF (alternating tangential filtration, Refine Technology) system was evaluated for two suspension cell lines, the avian cell line AGE1.CR and the human cell line CAP. Both were cultivated in chemically defined media optimized for batch cell growth in a 1L stirred tank bioreactor connected to the smallest ATF unit (ATF2) and infected with cell line-adapted human influenza A virus (A/PR/8/34 (H1N1), typical diameter: 80-100 nm). At concentrations of about 25 million cells/mL three different membrane cut-offs (50 kDa, 0.2 μm and 0.5 μm) were tested and compared to batch cultivations performed at 5 million cells/mL. For medium and large cut-offs no cell-density effect could be observed with cell-specific virus yields of 1428-1708 virions/AGE1.CR cell (infected with moi 0.001) and 1883-4086 virions/CAP cell (moi of 0.025) compared to 1292 virions/AGE1.CR cell and 3883 virions/CAP cell in batch cultures. Even at a concentration of 48 million AGE1.CR cells/mL (cut-off: 0.2 μm) a cell-specific yield of 1266 virions/cell was reached. Only for the small cut-off (50 kDa) used with AGE1.CR cells a decrease in cell-specific yield was measured with 518 virions/cell. Surprisingly, the ratio of infectious to total virions seemed to be increased in ATF compared to batch cultures. AGE1.CR cell-derived virus particles were present in the permeate (0.2 and 0.5 μm cut-off), whereas CAP cell-derived virions were not, suggesting possible differences in morphology, aggregation or membrane properties of the virions released by the two cell lines. To our knowledge, this is the first study that illustrates the potential of ATF-based perfusion of chemically defined media across cell-retaining membranes for production of an influenza A vaccine.

On the asymptotic linking number

We prove a theorem formulated by V. I. Arnold concerning a relation between the asymptotic linking number and the Hopf invariant of divergence-free vector fields. Using a modified definition for the system of short paths, we prove their existence in the general case.

Production of high-titer human influenza A virus with adherent and suspension MDCK cells cultured in a single-use hollow fiber bioreactor.

Hollow fiber bioreactors (HFBRs) have been widely described as capable of supporting the production of highly concentrated monoclonal antibodies and recombinant proteins. Only recently HFBRs have been proposed as new single-use platforms for production of high-titer influenza A virus. These bioreactors contain multiple hollow fiber capillary tubes that separate the bioreactor in an intra- and an extra-capillary space. Cells are usually cultured in the extra-capillary space and can grow to a very high cell concentration. This work describes the evaluation of the single-use hollow fiber bioreactor PRIMER HF (Biovest International Inc., USA) for production of influenza A virus. The process was setup, characterized and optimized by running a total of 15 cultivations. The HFBRs were seeded with either adherent or suspension MDCK cells, and infected with influenza virus A/PR/8/34 (H1N1), and the pandemic strain A/Mexico/4108/2009 (H1N1). High HA titers and TCID₅₀ of up to 3.87 log₁₀(HA units/100 μL) and 1.8 × 10(10)virions/mL, respectively, were obtained for A/PR/8/34 influenza strain. Influenza virus was collected by performing multiple harvests of the extra-capillary space during a virus production time of up to 12 days. Cell-specific virus yields between 2,000 and 8,000 virions/cell were estimated for adherent MDCK cells, and between 11,000 and 19,000 virions/cell for suspension MDCK.SUS2 cells. These results do not only coincide with the cell-specific virus yields obtained with cultivations in stirred tank bioreactors and other high cell density systems, but also demonstrate that HFBRs are promising and competitive single-use platforms that can be considered for commercial production of influenza virus.

Linking numbers of measured foliations

We generalize the average asymptotic linking number of a pair of divergence-free vector fields on homology three-spheres by considering the linking of a divergence-free vector field on a manifold of arbitrary dimension with a codimension-two foliation endowed with an invariant transverse measure. We prove that the average asymptotic linking number is given by an integral of Hopf type. Considering appropriate vector fields and measured foliations, we obtain an ergodic interpretation of the Godbillon–Vey invariant of a family of codimension-one foliations discussed by Kotschick ( Symplectic and Contact Topology: Interactions and Perspectives . Eds. Y. Eliashberg, B. Khesin and F. Lalonde. American Mathematical Society, Providence, RI).

On the uniqueness of the contact structure approximating a foliation

According to a theorem of Eliashberg and Thurston, a C-2-foliation on a closed 3-manifold can be C-0-approximated by contact structures unless all leaves of the foliation are spheres. Examples on the 3-torus show that every neighbourhood of a foliation can contain nondiffeomorphic contact structures. In this paper we show uniqueness up to isotopy of the contact structure in a small neighbourhood of the foliation when the foliation has no torus leaf and is not a foliation without holonomy on parabolic torus bundles over the circle. This allows us to associate invariants from contact topology to foliations. As an application we show that the space of taut foliations in a given homotopy class of plane fields is not connected in general.

Engel structures and weakly hyperbolic flows on four-manifolds

We study pairs of Engel structures on four-manifolds whose intersection has constant rank one and which define the same even contact structure, but induce different orientations on it. We establish a correspondence between such pairs of Engel structures and a class of weakly hyperbolic flows. This correspondence is analogous to the correspondence between bi-contact structures and projectively or conformally Anosov flows on three-manifolds found by Eliashberg--Thurston and by Mitsumatsu.

Synthetic Approach to Extended π-Systems

This paper describes a new method for the synthesis of ladder polymers which are potentially useful precursors for the synthesis of polymers with extended areas of π-conjugation.