Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Rodent cell – per se
Patent
1996-04-15
1999-03-30
Prats, Francisco
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Rodent cell, per se
435358, 435243, 53612313, 5361231, C12N 100, C13K 500, C13K 700, C07H 100
Patent
active
058888152
DESCRIPTION:
BRIEF SUMMARY
BACKGROUND OF THE INVENTION
Factor VIII is a blood protein essential for the blood coagulation process. Lack of Factor VIII will reduce or prevent the coagulating ability of the blood, thereby causing a disease called hemophilia A. Patients suffering from this disease are treated with therapeutical preparations containing Factor VIII.
Factor VIII has conventionally been recovered from human blood plasma. Such Factor VIII concentrates contain several fragmented fully active Factor VIII forms (Andersson et al., Proc. Natl., Acad. Sci. U.S.A., Vol. 83, 2979-83, May 1986). The smallest active form has a molecular mass of 170 kDa and consists of two chains of 90 KDa and 80 KDa held together by a metal ion bridge, as disclosed in EP-A-197 901.
Factor VIII can, however, now also be produced as a recombinant protein expressed in mammalian cells, as described, for example, in J. Gitschier et al., Nature 312, 330-37, 1984 and EP-A-160 457. A truncated recombinant Factor VIII product which corresponds to the 170 kDa plasma Factor VIII is disclosed in WO 91/09122.
Media used for the cultivation of mammalian cells include a variety of nutrients and growth factors, many of which are traditionally supplied by serum. Serum is usually derived from either foetal calf, newborn calf or horse and added to the medium in concentrations from 0.5 to 20 % v/v. In addition to supplying growth enhancing components, serum also functions as a carrier/buffer/chelator for labile or water insoluble molecules, toxin neutraliser, protease inhibitor, cell attachment enhancer and as a protective agent in agitated suspension cultures.
The use of serum in cell culture media, however, has several disadvantages. It is comparatively expensive, it is not a defined component, and different lots of serum may vary in the concentration of compounds present and thus result in unpredictable culture growth and productivity. Serum may also be the source of contaminants such as mycoplasma, bacteriophages, virus and toxins. Additionally, the protein in serum may complicate the purification of cell products from cell culture media.
In efforts to overcome the disadvantages of serum containing medium, serum-free media have been developed in which serum is substituted with better defined or more characterised components. Due to the complexity of serum and the different growth requirements of cells, this has resulted in a variety of different media compositions. (For reviews it may be referred to Rizzino et al., Nutrition Reviews 37: 369-378 (1979); Barnes and Sato, Cell 22: 649-655 (1980); and Bodeker et al., Develop. Biol. Standard. 60: 93-100 (1985)). In most such serum-free media the serum is substituted by "cocktails" of trace elements, lipids, hormones, growth factors and purified proteins, for example serum albumin, and are therefore often only partially defined.
Media for the serum-free culture of Chinese hamster ovary (CHO) cells have been reported by Gasser et al., In-vitro Cellular Developmental Biology 21: 588-592 (1985), and Mendiaz et al., In-vitro Cellular Developmental Biology 22: 66-74 (1986). These media compositions, however, contain non-defined components derived from either human or animal source.
Albumin is considered a multifunctional transport protein for a broad spectrum of ligands including inorganic cations, organic anions, amino acids and hydrophobic molecules such as fatty acids (Meucci et al., Journal of Biological Chemistry 266: No. 8, 4692-4699 (1991). Due to the nature of human serum albumin (HSA), it is an important component in serum-free media used for the culture of cells expressing recombinant Factor VIII both in the context of cell growth and production. It is envisaged that it also acts as a stabiliser/protection factor for example against proteases and shear forces (van der Pol and Tramper, Effect of reducing the serum or albumin concentrations on the shear sensitivity of two hybridoma cell lines in sparged cultures. ESACT 11th Symposium, 1991).
The supplementation of cell culture media with HSA, however, potentially has
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Andersson et al., Isolation and characterization of human factor VIII: Molecular forms in commercial factor VIII concentrate, cryoprecipitate, and plasma, Medical Sciences, vol. 83 (1986), pp. 2979-2983.
Wood et al., Expression of active human factor VIII from recombinant DNA clones, Nature, vol. 312 (1984), pp. 330-337.
Rizzino et al., Defined Media and the Determination of Nutritional and Hormonal Requirements of Mammalian Cells in Culture, Nutrition Reviews, vol. 37, No. 12 (1979), pp. 369-378.
Barnes et al., Serum-free Cell Culture: a Unifying Approach, Cell, vol. 22 (1980), pp. 649-655.
Bodeker et al., A Screening Method to Develop Serum-Free Culture Media for Adherent Cell Lines, Develop. bio. Standard., vol. 60 (1985), pp. 93-100.
Gasser et al., Long-Term Multiplication of the Chinese Hamster Ovary (Cho) Cell Line in a Serum-Free Medium, In Vitro Cellular & Developmental Biology, vol. 21, No. 10 (1985), pp. 588-592.
Mendiaz et al., A Defined Medium for and the Effect of Insulin on the Growth, Amino Acid Transport, and Morphology of Chinese Hamster Ovary Cells . . . In Vitro Cellular & Developmental Biology, vol. 22, No. 2 (1986), pp. 66-74.
Meucci et al., Metal-catalyzed Oxidation of Human Serum Albumin: Conformational and Functional Changes, The Journal of Biological Chemistry, vol. 266, No. 8 (1991), pp. 4692-4699.
van der Pol et al., Effect of Reducing the Serum or Albumin.
Chenciner et al., Enhancement of Gene Expression by Somatic Hybridization with Primary Cells: High-Level Synthesis of the Hepatitis B Surface Antigen in Monkey Vero Cells by Fusion with Primary Hepatocytes, Bio/Technology, vol. 8 (1990), pp. 858-862.
Morrison & Boyd, "Organic Chemistry" 3rd ed. 1973, see p. 1126.
Boork Sarah
Nilsson Helena Brink
Pharmacia & Upjohn Aktiebolag
Prats Francisco
Ware Deborah K.
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