Recombinant measles viruses are currently tested in clinical trials as oncolytic agent to be applied in cancer therapy. Contrary to their use as vaccine where 10 3 infectious virus particles per dose are needed, for cancer therapy 10 9 virus particles should be provided per dose. This leads to other challenges for the production process when compared to vaccine production. This study presents measles virus stability with regard to conditions during production and storage of the virus. Relevant process parameters such as temperature (4-37°C), pH (pH 4-11), conductivity (1.5 to 137.5 mS cm ) and oxygen partial pressure were analyzed. The infectivity of measles virus particles decreased highly at 37 and 32°C, while at 22 and 4°C it remained stable for several hours or even days, respectively. The thermal inactivation reactions followed first order kinetics and the thermodynamic parameters enthalpy and entropy were estimated. Towards changes in pH measles virus particles were very sensitive, while no inactivation could be observed with varying conductivity. Measles virus incubation at an oxygen partial pressure of 100% did not lead to any loss of infectivity. The results show which parameters should be considered and controlled strongly in the production process to further raise measles virus yields for the high amount needed in cancer therapy approaches.

Human herpesvirus type 6 (HHV-6) causes roseola infantum (exanthema subitum) upon primary infection in young children. Thereafter it persists lifelong in the organism. Like other herpesviruses, HHV-6 can be reactivated in periods of immunosuppression - e.g., after organ transplantation. In order to study the incidence and the time to reactivation after orthotopic liver transplantation (OLT) we tested buffy coat lysates before and up to 10 weeks after transplantation for the presence of HHV-6 DNA by polymerase chain reaction (PCR). Forty-six patients (male n=27, female n=19) with a median age of 48 years (range 20-66) were studied. Altogether, 30 of 287 (10.5%) buffy coat samples were PCR-positive. Before OLT 2 of 21 (9.5%) patients were positive. This ratio is not different from healthy blood donor controls. After OLT 13 of 46 (23.8%) patients were positive on one or more occasions. However, there was no statistically significant difference before and after OLT. Ten patients were analyzed for HHV-6 variants by restriction enzyme digestion of PCR products. One patient carried variant A and 9 variant B. In conclusion, HHV-6 can be detected in buffy coat cells after OLT. Our observations do not argue in favor of a reactivation.

Measles virus (MV) with attenuated pathogenicity has potential as oncolytic agent. However, the clinical translation of this therapy concept has one major hurdle: the production of sufficient amounts of infectious oncolytic MV particles. The current study describes oncolytic MV production in Vero cells grown on microcarrier using serum-free medium. The impact of the number of harvests, cell concentration at infection (CCI), multiplicity of infection (MOI), and temperature on MV production was determined in different production scales/systems (static T-flasks, dynamic spinner, and bioreactor system) and modes (batch, repeated-batch, and perfusion). Cell growth, metabolic, and production kinetics were analyzed. It was found that the number of harvests had the strongest positive impact on MV yield in each production scale, and that high temperatures affected MV yield adversely. Moderate MV titers were produced in T- and spinner flasks at 37°C (∼107 TCID50 mL−1, where TCID50 is tissue culture infective doses 50%), but stirred tank reactor (STR) MV production at 37°C yielded up to 10 000-fold lower MV titers. In contrast, at lower temperatures (32°C, 27°C), 1.4 × 107 TCID50 mL−1 were achieved in the STR. Variations in MOI and CCI had almost no influence on MV production yield. The current study improves oncolytic MV production process understanding and identifies process bottlenecks for large-scale production

Modern bioprocesses demand for a careful definition of the critical process parameters (CPPs) already during the early stages of process development in order to ensure high-quality products and satisfactory yields. In this context, online monitoring tools can be applied to recognize unfavorable changes of CPPs during the production processes and to allow for early interventions in order to prevent losses of production batches due to quality issues. Process analytical technologies such as the dielectric spectroscopy or focused beam reflectance measurement (FBRM) are possible online monitoring tools, which can be applied to monitor cell growth as well as morphological changes. Since the dielectric spectroscopy only captures cells with intact cell membranes, even information about dead cells with ruptured or leaking cell membranes can be derived. The following chapter describes the application of dielectric spectroscopy on various virus-infected and non-infected cell lines with respect to adherent as well as suspension cultures in common stirred tank reactors. The adherent mammalian cell lines Vero (African green monkey kidney cells) and hMSC-TERT (telomerase-immortalized human mesenchymal stem cells) are thereby cultured on microcarrier, which provide the required growth surface and allow the cultivation of these cells even in dynamic culture systems. In turn, the insect-derived cell lines S2 and Sf21 are used as examples for cells typically cultured in suspension. Moreover, the FBRM technology as a further monitoring tool for cell culture applications has been included in this chapter using the example of Drosophila S2 insect cells.

Attenuated measles virus has revealed selective tumor cell killing and is currently tested in clinical trials for the therapy of cancer patients. The amount of infectious particles per dose needed for oncolytic therapy can be more than a million times higher compared to vaccination. This requires highly effective production processes which guarantee the measles virus quantities needed for its use in cancer therapy. Referring to measles virus production itself, several factors are influencing process parameters and subsequent virus yields. These factors are medium optimization, feeding of nutrients, an optimal multiplicity of infection, localization of produced virus particles, temperature sensitivity and time of harvest. This review summarizes the available data concerning measles virus production in cell culture and factors influencing the virus yield.