On-line immunoanalysis of monoclonal antibodies during a continuous culture of hybridoma cells |
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Authors: | Jens J van der Pol Marc Machnik Manfred Biselli Theresa Portela-Klein Cornelis D de Gooijer Johannes Tramper Christian Wandrey |
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Institution: | 1.Forschungszentrum Jülich GmbH, Institute of Biotechnology, D-52425 Jülich, Germany ;2.ABION GmbH, Technology Center Jülich, Karl-Heinz Beckurts straße 13, D-52428 Jülich, Germany ;3.Food and Bioprocess Engineering group, Wageningen Agricultural University, P.O. Box 8129, NL-6700 EV Wageningen, the Netherlands |
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Abstract: | The monoclonal-antibody production of an immobilized hybridoma cell line cultivated in a fluidized-bed reactor was monitored
on-line for nearly 900 h. The monoclonal antibody concentration was determined by an immuno affinity-chromatography method
(ABICAP). Antibodies directed against the product, e.g. IgG, were immobilized on a micro-porous gel and packed in small columns.
After all IgG present in the sample was bound to the immobilized antibodies, unbound proteins were removed by rinsing the
column. Elution of the bound antibodies followed and the antibodies were determined by fluorescence. The analytical procedure
was automated with a robotic device to enable on-line measurements. The correlation between the on-line determined data and
antibody concentrations measured by HPLC was linear.
A sampling system was constructed, which was based on a pneumatically actuated in-line membrane valve integrated into the
circulation loop of the reactor. Separation of the cells from the sample stream was achieved by a depth filter made of glass-fibre,
situated outside the reactor. Rapid obstruction of the filter by cells or cell debris and contamination of the sample system
was avoided by intermittent rinsing of the sample system with a chemical solution. The intermittent rinsing of the filter,
which had a surface of 4.8 cm2, resulted in an operational capacity of up to 40 samples (1.0 l total sample volume). Both the sampling system and the analytical
device functioned without failure during this long-term culture.
The culture temperature was varied between 34 and 40 °C. Raising the temperature from 34 up to 37 °C resulted in a simultaneous
increase of growth and specific antibody production rate. Specific metabolic rates of glucose, lactate, glutamine and ammonium
stayed constant in this temperature range. A further enhancement of temperature up to 40 °C had a negative effect on the growth
rate, whereas the specific monoclonal antibody production rate showed a small increase. The other specific metabolic rates
also increased in the temperature range between 38 to 40 °C.
This revised version was published online in July 2006 with corrections to the Cover Date. |
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Keywords: | fluidized-bed reactor monoclonal antibody on-line monitoring sample system temperature |
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