An automatic system for the assessment of complex medium additives under cultivation conditions.

Complex medium additives such as yeast extract or peptone are often used in industrial cell culture processes to prolong cell growth and/or to improve product formation. The quality of those supplements is dependent on the preparation method and can differ from lot to lot. To guarantee consistent production these different lots have to be tested prior to use in fermentation processes. Because a detailed qualitative and quantitative analysis of all components of such a complex mixture is a very difficult task, another assessment method has to be chosen. The best way to evaluate the effect of such supplements is to monitor cell activity during real cultivation conditions with and without the added supplement lot. A bioreactor-based test system has been developed to determine the oxygen requirement of the cells as a response to the addition of a supplement to be tested under standardized conditions. Investigations were performed with a mouse-mouse hybridoma cell line and yeast extracts as an example for complex medium additives. The results showed differences in the impact between different extract lots and between different concentrations of an extract.

Endogenous retrovirus particles and their repercussion effects on the growth behaviour of continuous hybridoma cultivation processes.

Hybridoma cells used for the production of monoclonal antibodies are also known to form growth inhibitory substances. Growth inhibitors already described in the literature belong to the class of peptides and proteins likeTGF-ss (Transforming Growth Factor-ss). The endogenous retrovirus particles - a further potential substance producing this kind of effect - are described here. To examine whether the retrovirus particles participated in growth inhibitory effects hybridoma cells were cultivated in continuous perfusion mode by using a special reactor set-up. A rapid increase of the signal in the supernatant which coincided with a decrease of viability could be observed by monitoring the reverse transcriptase-activity during this type of fermentation process. The examination of concentrated and fractionated supernatant from this period showed growth inhibitory effects in the biological assay (MTT-assay). Investigations of respective fractions demonstrated retrovirus particles with reverse transcriptase-activity. Based on RT-PCR data it was shown that only inhibitory fractions contain retrovirus particles which were of E-MuLV and MCF origin.

Effects on growth behavior in continuous hybridoma cell cultures: The role of viral contamination.

This article describes the retrovirus expression with optimal nutrient supply and its potential growth inhibition effects in continuous hybridoma cell cultivation. A special reactor setup with total cell retention was developed to examine growth inhibition effects. Using this fermentation strategy we observed a decrease of viability cell rate which occurred at a defined state of the process despite sufficient nutrient supply. Therefore we assume that inhibitory substances are responsible for these effects. The molecular weight range of the inhibitory substances and the possible retrovirus cooperation of these growth inhibition effects were examined. To determine the molecular weight range we used the following methods: ultrafiltration, gelfiltration, ultracentrifugation and gel electrophoresis. Furthermore, RT-PCR and western-/immunoblot are used to detect retrovirus particles in the supernatant and to show a retrovirus participation on growth inhibition effects. The possible growth modulation was tested in a biological assay (MTT-assay).

Lysis-free separation of hybridoma cells by continuous disc stack centrifugation.

The non-destructive removal of hybridoma cells from fermentation broth with an improved disc stack centrifuge (CSA1, Westfalia Separator AG, Oelde, Germany) was investigated. The centrifuge was equipped with a hydrohermetic feed system, which allowed a gentle, shearless acceleration of the cells inside the bowl. No significant cell damage was observed during the separation of hybridoma cells from repeated batch fermentation in 100 liter scale. In the clarified liquid phase there was no increase in Lactate-Dehydrogenase (LDH) activity. Consequently, there was no increased exposure of the product to intracellular components.Due to continuous operation with a periodic and automatic discharge of sediment, a high throughput was achieved without any considerable loss of product. The clarification for mammalian cells was in the range of 99% to 99.9%, depending on the operating conditions. The content of cell debris and other small particles decreased about 30 to 50%, depending on the particle load in the feed stream. The centrifuge was fully contained; cleaning and sterilizing in place possible. Therefore, the decice could be integrated easily into the fermentation process.

Design of a bubble-swarm bioreactor for animal cell culture.

A stationary bubble-swarm has been used to aerate a mammalian cell culture bioreactor with an extremely low gas flow rate. Prolonging the residence time of the gas bubbles within the medium improved the efficiency of the gas transfer into the liquid phase and suppressed foam formation. An appropriate field of speed gradients prevented the bubbles from rising to the surface. This aeration method achieves an almost 90% transfer of oxygen supplied by the bubbles. Consequently, it is able to supply cells with oxygen even at high cell densities, while sparging with a gas flow of only 0.22 x 10(-3) -1.45 x 10(-3) vvm (30-200 ml/h). The reactor design, the oxygen transfer rates and the high efficiency of the system are presented. Two repeated batch cultures of a rat-mouse hybridoma cell line are compared with a surface-aerated spinner culture. The used cell culture medium was serum-free, either with or without BSA and did not contain surfactants or other cell protecting agents. One batch is discussed in detail for oxygen supply, amino acid consumption and specific antibody production.