Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Patent
1995-08-14
1997-03-04
Fleisher, Mindy
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
435 702, 4351722, 4351738, 935 34, 935 89, 935 93, 935 95, 935106, 935109, C12P 2100, C12N 1502, C12N 1506, C12N 1567
Patent
active
056078455
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention relates to a method for obtaining a higher product expression in a producing mammalian cell line characterized by treating the original cell line with itself according to a fusion protocol, known in the art. The production of protein products is performed in mammalian tissue culture, and the product could be therapeutic proteins or other molecules intended for human use. In particular this invention relates to the use of cell fusion methods to obtain a stable high producing cell line.
DESCRIPTION OF THE RELATED ART
Expression of proteins in heterologous mammalian cells is usually performed in one of the following three ways, well known to the artisan in the field. proteins in mammalian cells may be introduced in an appropriate cell. continuous cell line. cell line and the resulting hybrid can be selected for expression of the wanted protein.
By further cultivation of cells derived from 1-3 above the protein of interest can be obtained from the culture medium or the cells themselves.
The latter technique 3 is the most common one and is used for production of monoclonal antibodies by hybridoma cells. Hybrid cell lines are usually characterized by presence of chromosomes or genes from the two different fusion parents and require some sort of selection procedure in order to eliminate the parental cells from the fusion mixture and allowing the emergence and outgrowth of the fused cell.
Cell fusion as a method of enhancing protein production has been described by Chenciner et al in Bio/Technology vol 8, 1990:858-862. The authors produced classical hybrids between a liver cell and a transfected Vero cell. The stable hybrids produced an increased amount of the wanted product.
Different cell fusion protocols are well known in the art. The use of polyethylene glycol (PEG), protoplasts or Sendaivirus can be mentioned as examples and reference is here given to Cell culture by William B. Jakoby and Ira H. Pastan, Methods in Enzymology Vol. LVIII, Academic Press, 1979. Electrofusion is another method that also can be used and reference is here given to Electromanipulation in hybridoma technology, Laboratory manual, by Carl A. K. Borrbaeck and Inger Hagen, Stockman Press, 1989 and Guide to Electroporation and Electrofusion, Chang et al, Academic Press, 1992. By variations of e.g. the used amount of cells, time, temperature etc., each laboratory often has its own protocol.
In summary the above described prior art discloses the use of classical cell fusion of two different cells, as means of obtaining and increasing production of a particular product in a particular cell which is a fusion cell, a combination of the two parent cells.
We have now found a method that can be used in the establishment of a protein producing cell line. The method will increase the production of the wanted product of the cell line.
Although the method resembles cell fusion it is not a classical cell fusion and the cell lines obtained are, to our great surprise, stable over a prolonged period.
SUMMARY OF THE INVENTION
The present invention relates to a method of obtaining a higher product expression in a producing cell line by fusing the original cell with itself according to a fusion protocol, known in the art.
Cell fusion protocols suitable for the present invention are methods utilizing polyethylene glycol (PEG) or electrofusion, protoplasts or Sendaivirus. The cell could be a Chinese Hamster Ovary (CHO) derived cell line, Baby Hamster
Kidney (BHK) or Cos (an African green monkey derived cell line) or any other commonly used mammalian cell line.
The product is preferably full length factor VIII, deletion derivatives of factor VIII or otherwise modified factor VIII but could also be another therapeutically active protein or molecule.
The resulting cell lines could have a chromosome and DNA content identical or very similar to the original cell line.
The cell fusion protocol is preferably performed without the presence of a selection protocol, but selection protocols could be used.
The fusion protoc
REFERENCES:
patent: 4659663 (1987-04-01), Wright
patent: 4822470 (1989-04-01), Chang
patent: 4832814 (1989-05-01), Root
patent: 4959321 (1990-09-01), Preece et al.
Hotta et al. "New Antibiotic-Producing Steptomyces, Selected By Antibiotic Resistance As a Marker" J. Antibiot. 38(1) 64-69 1985.
Chenciner et al. "Enhancement of Gene Expression By Somatic Hybridization w/Primary Cells" Bio/Technology 8 858-862 1990.
Roos et al. "Control of Virus-Induced Cell Fusion by Host Cell Lipid Composition" Virology 175 345-357 1990.
Adamsson Lars
Spira Jack
Degen Nancy J.
Fleisher Mindy
Pharmacia & UpJohn AB
LandOfFree
Method for obtaining an increased production of a producing cell does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Method for obtaining an increased production of a producing cell, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method for obtaining an increased production of a producing cell will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2145419