Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Using tissue cell culture to make a protein or polypeptide
Patent
1993-12-14
1995-06-20
Beisner, William H.
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Using tissue cell culture to make a protein or polypeptide
43524022, 43524025, 43524027, 435286, 435296, 435312, 435315, 435316, C12M 300, C12M 128, C12N 508, C12P 2108
Patent
active
054260370
DESCRIPTION:
BRIEF SUMMARY
FIELD OF THE INVENTION
This invention relates to a method of and apparatus for the production of monoclonal antibodies (MAbs).
BACKGROUND TO THE INVENTION
MAbs are typically prepared by in vivo cultivation of hybridomas in the peritoneal cavity of mice as ascitic rumours (Goding, 1980), which produces antibody concentrations of the order of 20 mg/ml. The simplicity, efficiency, and high concentration of the final product make ascitic fluid the preferred choice for laboratories requiring relatively small amounts of a large number of MAbs. There are ethical reasons to look for alternatives, but it would in any case be attractive to develop a simpler, and possibly cheaper, alternative tissue culture method capable of substituting ascitic fluid.
One known tissue culture method for MAb production involves growing hybridoma cultures to large volumes in roller bottles, and then concentrating the antibodies produced by use of ammonium sulphate precipitation. With this method, the cells can only be maintained healthily below 10.sup.6 /ml. Another method involves concentrating the cells to 2.times.10.sup.6 cells/ml or more in serumless medium for 3-4 days (Galfre and Milstein, 1981). This can improve the yield, and has added advantages in that the volume of the product is lower, and there are no foreign proteins present (eg. Foetal bovine proteins) to complicate purification. Moreover, these methods are cumbersome, take up valuable space in the incubator, and necessitate the use of large quantities of media, foetal calf serum (FCS), and ammonium sulphate.
Hollow fibre systems allow cells to be grown between narrow diameter hollow fibres, within a cartridge into which fresh medium is continuously pumped. Hybridomas can reach high densities, and production of MAbs is efficient (Schonherr and Gelder, 1988) However, the cost of such systems with associated software is high, and only justified when relatively large amounts of individual MAbs are required. This is also the case with fermenters and bioreactors. Growth of cells in dialysis bags within small culture bottles (Sjorgren-Jansson and Jeanson, 1990), although appropriate for laboratory use, is restricted to 25 ml per bottle.
THE INVENTION
According to one aspect of the present invention, there is provided a method of producing monoclonal antibodies according to which a dialysis tube immersed in a growth medium is filled with a culture of hybridomas to the extent necessary to leave a bubble in the tube and is sealed, and the bubble is caused to oscillate back and forth along the sealed dialysis tube in order to keep the culture of hybridomas in suspension.
In a preferred method, oscillation of the bubble is achieved by an oscillatory or rotary motion of the dialysis tube taking said tube continuously through two orientations, in one of which the bubble lies at one end the tube and in the second of which the bubble lies at the other end of the tube.
The required oscillatory or rotary motion of the dialysis tube can be achieved by fixing the tube within a container for the growth medium, and rocking or rotating the container. A preferred container is a roller bottle mounted with its longitudinal axis horizontal for oscillatory or preferably continuous rotation about a horizontal axis, the sealed dialysis tube being fixed within and between the respective ends of the bottle at positions angularly spaced around said longitudinal bottle axis, ie. so that the tube is skewed relative to said axis. The longitudinal axis of the bottle is preferably coincident with the axis of rotation.
Thus, according to another aspect of the invention, there is provided apparatus for the production of monoclonal antibodies, comprising a container for a growth medium, a dialysis tube fixed to and within the container so as to be immersed in the growth medium, and means for imparting such a motion to the container that, when the dialysis tube is filled with a culture of hybridomas to the extent necessary to leave a bubble in the tube and is sealed, the bubble will be caused to oscilla
Milstein Cesar
Pannell Richard
Beisner William H.
Degen Nancy J.
Medical Research Council
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