Britta Isken

Distributed modeling of human influenza a virus-host cell interactions during vaccine production

This contribution is concerned with population balance modeling of virus–host cell interactions during vaccine production. Replication of human influenza A virus in cultures of adherent Madin–Darby canine kidney (MDCK) cells is considered as a model system. The progress of infection can be characterized by the intracellular amount of viral nucleoprotein (NP) which is measured via flow cytometry. This allows the differentiation of the host cell population and gives rise to a distributed modeling approach. For this purpose a degree of fluorescence is introduced as an internal coordinate which is linearly linked to the intracellular amount of NP. Experimental results for different human influenza A subtypes reveal characteristic dynamic phenomena of the cell distribution like transient multimodality and reversal of propagation direction. The presented population balance model provides a reasonable explanation for these dynamic phenomena by the explicit consideration of different states of infection of individual cells. Kinetic parameters are determined from experimental data. To translate the emerging infinite dimensional parameter estimation problem to a finite dimension the parameters are assumed to depend linearly on the internal coordinate. As a result, the model is able to reproduce all characteristic dynamic phenomena of the considered process for the two examined virus strains and allows deeper insight into the underlying kinetic processes. Thus, the model is an important contribution to the understanding of the intracellular virus replication and virus spreading in cell cultures and can serve as a stepping stone for optimization in vaccine production. Biotechnol. Bioeng. 2013; 110: 2252–2266.

Productivity, apoptosis, and infection dynamics of influenza A/PR/8 strains and A/PR/8-based reassortants.

In cell culture-based influenza vaccine production significant efforts are directed towards virus seed optimization for maximum yields. Typically, high growth reassortants (HGR) containing backbones of six gene segments of e.g. influenza A/PR/8, are generated from wild type strains. Often, however, HA and TCID₅₀ titres obtained do not meet expectations and further optimization measures are required. Flow cytometry is an invaluable tool to improve our understanding of mechanism related to progress of infection, virus-induced apoptosis, and cell-specific productivity. In this study, we performed infections with two influenza A/PR/8 variants (from NIBSC and RKI) and two A/PR/8-based HGRs (Wisconsin-like and Uruguay-like) to investigate virus replication, apoptosis and virus titres at different multiplicities of infection (MOI 0.0001, 0.1, 3). Flow cytometric analyses showed similar dynamics in the time course of infected and apoptotic cell populations for all four tested strains at MOI 0.0001. Interestingly, higher MOI resulted in an earlier increase of the populations of infected and apoptotic cells and showed strain-specific differences. Infections with A/PR/8 NIBSC resulted in an earlier increase in both cell populations compared to A/PR/8 RKI. The Uruguay-like reassortant showed the earliest increase in the concentration of infected cells and a late induction of apoptosis at all tested MOIs. In contrast, the Wisconsin-like reassortant showed strong apoptosis induction at high MOIs resulting in reduced titres compared to lower MOI. Maximum HA titres were unaffected by changes in the MOI for the two A/PR/8 and the Uruguay-like reassortant. Maximum TCID₅₀ titres, however, decreased with increasing MOI for all strains. Overall, infections at very low MOI (0.0001) resulted not only in similar dynamics concerning progress of infection and induction of apoptosis but also in maximum virus yields. Highest HA titres were obtained for virus seed strains combining a fast progress in infection with a late onset of apoptosis. Therefore, both factors should be considered for the establishment of robust influenza vaccine production processes.

Distributed Modeling and Parameter Estimation of Influenza Virus Replication During Vaccine Production

Abstract This contribution is concerned with population balance modeling of influenza virus replication in mammalian cell cultures. The cells are heterogeneous with respect to intracellular compounds like viral proteins. The amount of viral NP protein can be measured directly by means of flow cytometry. The corresponding degree of fluorescence is introduced as internal coordinate for a distributed deterministic modeling approach. The resulting model includes kinetic processes like infection, virus replication and release, apoptosis and cell death. It consists of three partial differential equations describing the distribution dynamics which are coupled to two differential equations that characterize the concentration of active and inactive virions in the medium. Kinetic parameters are determined from experimental data. The parameters are assumed to depend on the internal coordinate. The emerging infinite dimensional parameter estimation problem is translated to a finite dimension using a hermite spline representation of the distributed parameters. Hence the resulting inverse problem can be solved in a weighted nonlinear least squares framework. Spline approaches of different complexity are discussed and the estimation results are compared.

Population balance modelling of influenza virus replication during vaccine prodcution - Influence of apoptosis

Abstract This contribution is concerned with population balance modelling of virus infection in cell cultures during vaccine production. As a model system infection of human influenza A virus in microcarrier cell cultures of Madin-Darby canine kidney (MDCK) cells is considered. Differentiation on the population level is described by a degree of fluorescence, which is proportional to the amount of intracellular viral proteins and can be measured directly using flow cytometry. Additional measurement information allows distinguishing between nonapoptotic and apoptotic cells. Apoptosis or programmed cell death is a natural process during cell development and can be activated by a large variety of external and internal stimuli particularly by viral infection. It invariably leads to cell lysis and has major influence on the process productivity.

Monitoring of Host-Cell Infection and Virus-Induced Apoptosis in Influenza Vaccine Production

Detailed knowledge of status and progress of host-cell infection in cell culture-derived vaccine manufacturing can be of great value for process design and optimization. Therefore, cell populations in a 5 L microcarrier system using adherent MDCK cells were monitored by flow cytometry for degree of infection and induction of apoptosis. Cells attached to microcarriers as well as detached cells were analyzed. About 8 h post infection the concentration of cells in the supernatant increased, followed by an increase in HA titers 2 h later. About 30 h post infection most cells had detached from the microcarriers and were apoptotic, while the virus particle concentration (HA) did not increase further. Virus yields mainly depended on the total number of adherent cells, and a high concentration of detached cells clearly indicated the end of the productive phase of cultivations. Therefore, measures to delay cell detachment, i.e. virus-induced apoptosis, could lead to higher virus titers in influenza vaccine production processes.