J. Schulze-Horsel

Infection dynamics and virus-induced apoptosis in cell culture-based influenza vaccine production – flow cytometry and mathematical modeling

Cell culture-based influenza vaccine manufacturing is of growing importance. Depending on virus strains, differences in infection dynamics, virus-induced apoptosis, cell lysis and virus yields are observed. Comparatively little is known concerning details of virus-host cell interaction on a cellular level and virus spreading in a population of cells in bioreactors. In this study, the infection of MDCK cells with different influenza A virus strains in lab-scale microcarrier culture was investigated by flow cytometry. Together with the infection status of cells, virus-induced apoptosis was monitored. A mathematical model has been formulated to describe changes in the concentration of uninfected and infected adherent cells, dynamics of virus particle release (infectious virions, hemagglutinin content), and the time course of the percentage composition of the cell population.

Flow cytometric monitoring of influenza A virus infection in MDCK cells during vaccine production

BackgroundIn cell culture-based influenza vaccine production the monitoring of virus titres and cell physiology during infection is of great importance for process characterisation and optimisation. While conventional virus quantification methods give only virus titres in the culture broth, data obtained by fluorescence labelling of intracellular virus proteins provide additional information on infection dynamics. Flow cytometry represents a valuable tool to investigate the influences of cultivation conditions and process variations on virus replication and virus yields.ResultsIn this study, fluorescein-labelled monoclonal antibodies against influenza A virus matrix protein 1 and nucleoprotein were used for monitoring the infection status of adherent Madin-Darby canine kidney cells from bioreactor samples. Monoclonal antibody binding was shown for influenza A virus strains of different subtypes (H1N1, H1N2, H3N8) and host specificity (human, equine, swine). At high multiplicity of infection in a bioreactor, the onset of viral protein accumulation in adherent cells on microcarriers was detected at about 2 to 4 h post infection by flow cytometry. In contrast, a significant increase in titre by hemagglutination assay was detected at the earliest 4 to 6 h post infection.ConclusionIt is shown that flow cytometry is a sensitive and robust method for the monitoring of viral infection in fixed cells from bioreactor samples. Therefore, it is a valuable addition to other detection methods of influenza virus infection such as immunotitration and RNA hybridisation. Thousands of individual cells are measured per sample. Thus, the presented method is believed to be quite independent of the concentration of infected cells (multiplicity of infection and total cell concentration) in bioreactors. This allows to perform detailed studies on factors relevant for optimization of virus yields in cell cultures. The method could also be used for process characterisation and investigations concerning reproducibility in vaccine manufacturing.

High-density microcarrier cell cultures for influenza virus production

Influenza virus A/PR/8/34 virus propagation in adherent Madin–Darby canine kidney cells in high‐density microcarrier cultures is described. To improve virus yields, perfusion and repeated fed‐batch modes were applied using cell‐specific feed rates. Cell densities up to 1.1 × 107 cells/mL were achieved. Cell‐specific virus yields in high‐density cultures were at similar levels compared with standard, low‐density cultivations. In the average 2,400 and 3,300 virions per cell were obtained for two variants of the virus strain A/PR/8/34, PR8‐National Institute for Biological Standards and Control (NIBSC) and PR8‐Robert Koch Institute, respectively. Maximum virus titer (HA activity = 1,778 HAU/100 μL) for virus variant PR8‐NIBSC was obtained for a cultivation infected before maximum cell concentration was reached.

Growth Behavior of Number Distributed Adherent MDCK Cells for Optimization in Microcarrier Cultures

An assay for measuring the number of adherent cells on microcarriers that is independent from dilution errors in sample preparation was used to investigate attachment dynamics and cell growth. It could be shown that the recovery of seeded cells is a function of the specific rates of cell attachment and cell death, and finally a function of the initial cell‐to‐bead ratio. An unstructured, segregated population balance model was developed that considers individual classes of microcarriers covered by 1–220 cells/bead. The model describes the distribution of initially attached cells and their growth in a microcarrier system. The model distinguishes between subpopulations of dividing and nondividing cells and describes in a detailed way cell attachment, cell growth, density‐dependent growth inhibition, and basic metabolism of Madin‐Darby canine kidney cells used in influenza vaccine manufacturing. To obtain a model approach that is suitable for process control applications, a reduced growth model without cell subpopulations, but with a formulation of the specific cell growth rate as a function of the initial cell distribution on microcarriers after seeding was developed. With both model approaches, the fraction of growth‐inhibited cells could be predicted. Simulation results of two cultivations with a different number of initially seeded cells showed that the growth kinetics of adherent cells at the given cultivation conditions is mainly determined by the range of disparity in the initial distribution of cells on microcarriers after attachment.

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.

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.

Influenza Vaccines : Challenges in Mammalian Cell Culture Technology

Experimental data as well as simulation results obtained by mathematical models for an influenza vaccine process with adherent MDCK cells clearly show that total cell number, specific virus replication rate and cell death due to virus infection (apoptosis) are the main factors to be taken into account for achieving high virus yields. In contrast, supply of cellular precursors and ATP for the synthesis of viral genome and virus specific proteins as well as multiplicity of infection seems not to limit virion formation.

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.

Single-cell approach in influenza vaccine production : apoptosis and virus protein production

The induction of apoptosis by influenza virus infection has been shown in vitro and in vivo. Here, we present a quantitative investigation of apoptosis occurring during influenza A vaccine production in Madin-Darby canine kidney microcarrier culture. Flow cytometry was employed for single-cell based analysis of infection, apoptosis and intracellular accumulation of viral nucleoprotein. Apoptotic DNA strand breaks were observed especially during late influenza A virus infection phase. Apoptosis was mainly detected in infected cells, only a small fraction of uninfected cells was apoptotic.