The influence of gravity on a biotechnological process was the main research subject of our series of TEXUS experiments. These included a detailed investi-gation of the fusion process without disturbing convection and sedimentation forces, as well as the improvement of electrophoretic downstream processing techniques.
Analysis of the fusion process and cellular
reorganisation after fusion of cells under microgravity
Monoclonal antibodies are of great importance in
medicine and biotechnology. Myeloma cells (usually from mice) are
fused chemically or electrically to the spleen cells of immunised
animals or humans. ¹ Some of the hybrids (the so-called
hybridomas) make antibodies specific to the antigen used to
immunise the animal. These hybridoma cells can be used to produce
the antibody on even an industrial scale. However, the frequency
of hybrid formation using conventional fusion techniques is very
low (10-5). The new efficient electrofusion protocols,
developed mainly in our laboratory, result in hybrid yields of
the order of 10-³.
Fusion rates of 1-10% are required for production of human hybridomas because of the limited number of activated lymphocytes that can be drawn from a donor. Electrofusion of cells under µg offers several theoretical advantages. ² For example, cells under µg are free of sedimentation forces that may disturb the alignment phase. In addition, it was expected that the thermal convection mixing would be minimised. In ground-based fusions, these forces interfere with the establishment of close cell contact during electrically-mediated chain formation and require increased electrical field condition, which can be harmful to the cells. In a series of experiments on several TEXUS flights, we demonstrated that the fusion efficiency and the hybrid yield in different cell systems were significantly enhanced under g conditions.
In the light of the brief-exposure µg results obtained so far, it is also clear that fusion in space certainly affected the molecular and cellular steps involved in the fusion, and similarly the rounding up process of the hybrid ²,³,4 at the cellular level.
Our investigations of the mechanisms of reversible and irreversible electrical membrane breakdown have also led to a better understanding of the fusion process itself, as well as of the processes responsible for the electrically-induced uptake of macromolecules into living cells.5
One of the most promising applications of this research area is the production of human antibody-secreting hybridoma cells obtained by electrofusion of heteromyeloma cells and human lymphocytes (either from biopsy-material or from peripheral blood). Highly efficient electrofusion protocols5 allowed us to produce hybridoma-clones secreting functional human monoclonal antibodies directed against Hepatitis C and stomach cancer antigens.6 The purification procedure for the antibodies has been optimised, allowing manufacture on a semi- industrial scale at this point. The first clinical studies using these human monoclonal antibodies have already been performed.
A powerful method for downstream processing of
biotechnological products under
microgravity
Electrophoresis is the migration of ions
under the influence of an applied electric field. The process
depends primarily on the charge density of the molecule or
particle and is one of the most important and gentle techniques
for analytical and preparative separation of biotechnological
products such as proteins (enzymes, hormones, etc.) or cells and
organelles. There are many effective instrumental methods of
electrophoresis, varying widely in sensitivity and specificity.
Among these, free-flow electrophoresis offers the distinct
advantage of continuous preparative and analytical separation of
charged molecules and cells.7 It has therefore had a
major impact on both clinical laboratories and industries
exploiting advances in biotechnology.
Briefly, the basic principle of free-flow electrophoresis is as follows. A laminar stream of buffer flows vertically from top to bottom of a flat separation chamber, where a perpendicular electric field is applied. A buffer of low ionic strength is used to minimise heat production. When the cells or protein molecules, introduced into the medium near the top, are exposed to the electric field they migrate laterally towards the positively- charged electrode. The migration velocity depends on the density of the cells negative surface charge. Thus particles with significant differences in surface charge densities migrate at different speeds and arrive at different points along the bottom line of the separation chamber, where they can be collected for preparative isolation.
Problems arise under terrestrial conditions from thermal convection, sedimentation and the velocity profile of the flowing film, which considerably affects the resolution. In an experiment on TEXUS 24 we demonstrated beyond doubt the significantly enhanced resolution and throughput of this technique under g conditions. In this experiment, a mixture of red blood cells from different species was separated in a specially designed electrophoresis module. Owing to energy input and the associated thermal convection, the resolution under terrestrial conditions was poor.7 Clear separation was obtained aboard the rocket in µg.
Our results unambiguously demonstrate the potential of free- flow electrophoresis for downstream processing under µg.
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