Plastic article or earthenware shaping or treating: apparatus – Immersed shaping orifice discharging directly into liquid... – Shaping orifice and bath or shower means mounted for...
Reexamination Certificate
2002-02-07
2002-10-29
Saidha, Tekchand (Department: 1652)
Plastic article or earthenware shaping or treating: apparatus
Immersed shaping orifice discharging directly into liquid...
Shaping orifice and bath or shower means mounted for...
C435S252300, C435S320100, C435S325000, C435S404000, C435S069100, C514S002600, C514S012200, C514S814000, C536S023100
Reexamination Certificate
active
06471500
ABSTRACT:
The invention concerns a process for producing erythropoietin which is free of animal foreign proteins with the exception of proteins of the host cell.
Erythropoietin (EPO) is a human glycoprotein which stimulates the formation of erythrocytes. Its action and therapeutic application are described in detail for example in EP-B 0 148 605, Huang, S. L., Proc. Natl. Acad. Sci. USA (1984) 2708-2712, EP-B 0 205 564, EP-B 0 209 539 and EP-B 0 411 678 as well as Lai, P. H. et al., J. Biol. Chem. 261 (1986) 3116-3121 and Sasaki, H. et al., J. Biol. Chem. 262 (1987) 12059-12076. Erythropoietin for therapeutic use is produced by recombinant means (EP-B 0 148 605 and EP-B 0 209 539).
The recombinant production of erythropoietin is usually carried out in CHO cells with the addition of foetal calf serum and optionally bovine insulin in the culture medium. As a result an EPO preparation produced in this manner contains at least traces of substances which are derived from these additives even after purification. These may for example be bovine viruses and comparable agents, residual amounts of bovine proteins and/or bovine DNA. It is known that a serum-free fermentation of recombinant CHO cells which contain an EPO gene can be carried out using the methods of the state of the art.
Such methods are described for example in EP-A 0 513 738, EP-A 0 267 678 and in a general form by Kawamoto, T. et al., Analytical Biochem. 130 (1983) 445-453, EP-A 0 248 656, Kowar, J. and Franek, F., Methods in Enzymology 421 (1986) 277-292, Bavister, B., Expcology 271 (1981) 45-51, EP-A 0 481 791, EP-A 0 307 247, EP-A 0 343 635, WO 88/00967. It has turned out that in a serum-free culture of EPO-producing eukaryotic host cells the proportion of proteins of the host cell relative to the total protein amount is more than twice that of a culture in media containing serum. Such a protein preparation also contains nucleic acids from the host cells in a not inconsiderable amount.
In EP-A 0 267 678 an ion exchange chromatography on S-Sepharose, a preparative reverse phase HPLC on a C
8
column and a gel filtration chromatography are described for the purification of EPO produced in serum-free culture after dialysis. In this connection the gel filtration chromatography step can be replaced by ion exchange chromatography on S-Sepharose fast flow. It is also proposed that a dye chromatography on a Blue Trisacryl column be carried out before the ion exchange chromatography.
EPO purified in this manner has a purity of about 99% but still contains considerable amounts of protein and DNA from the host cell.
A process for the production of EPO in mammalian cells is described in EP-A 0 513 738 without elaborating on the purification.
A process for the purification of recombinant EPO is described by Nobuo, I. et al., J. Biochem. 107 (1990) 352-359. In this process EPO is treated however with a solution of Tween® 20, phenylmethylsulfonyl fluoride, ethylmaleimide, pepstatin A, copper sulfate and oxamic acid prior to the purification steps. Although the pharmaceutical preparation contains only traces of these additives they are critical from the therapeutic point of view.
Moreover, for a therapeutic use it is preferred that a therapeutically effective preparation should be completely free of proteins and nucleic acids from mammalian cells and largely free of proteins and nucleic acids from the host cell.
The subject matter of the invention is a therapeutically effective preparation of a protein with erythropoietin activity obtainable after culturing a host cell which produces erythropoietin characterized by:
a) a content of proteins which are derived from the host cell of not more than 100 ppm (w/w), preferably 40 ppm or less, and more preferably 20 ppm or less,
b) a content of DNA from the host cell of not more than 10 pg per 83 &mgr;g EPO, preferably 1 pg per 83 &mgr;g EPO or less, and in that
c) the preparation is completely free of natural mammalian proteins which are not derived from the host cell.
Such an EPO preparation which in addition is free of phenylmethylsulfonyl fluoride, pepstatin A and/or Cu ions is previously unknown and also cannot be produced by the methods of the state of the art especially in therapeutically effective amounts. In order to obtain and to isolate a homogeneous EPO preparation of this specification, it is necessary to combine the steps of the process according to the invention.
A protein with erythropoietin activity is understood as a protein which has the biological function of EPO. This biological function is to stimulate differentiation and division processes in erythroid precursor cells and thus to provide erythrocytes. The properties of this protein are preferably identical to or essentially identical to those of human erythropoietin and it is composed of 166 amino acids with a molecular weight of ca. 34-38 kD the percentage of glycosyl residues in the molecular weight being ca. 40%. Derivatives and fragments of EPO which have an analogous activity and are produced after culturing an EPO-producing host cell, can also be produced in a pure form by the processes according to the invention. The DNA and protein sequences of human EPO are described for example in EP-B 0 205 564 and EP-B 0 209 539.
“Completely free of natural mammalian proteins” means that due to the fact that no foreign proteins from natural sources such as bovine serum albumin or foetal calf serum are added during the culture of the host cell, no such foreign proteins are present to a detectable extent in the preparation. The preparation is thus completely free of such mammalian proteins added as planned that are not derived from the host cell and are usually added to the culture medium in a serum-free culture to maintain and improve cell growth and to optimize the yield. Natural mammalian proteins are understood as mammalian proteins from natural sources such as from human material or from animal material but not recombinant mammalian proteins which are for example produced in prokaryotes such as
E. coli.
Such mammalian proteins added during cell culture are for example bovine serum albumin, foetal calf serum, transferrin (human or bovine), insulin (porcine or bovine) or gelatin.
A “host cell” is understood as an animal or human cell whose genome contains an active EPO gene and this EPO gene is transcribed and translated during culture of the cell in a serum-free medium. The EPO gene can be introduced into this host cell as an exogenous gene, preferably with regulation elements (cf. e.g. EP-B 0 148 605, EP-B 0 209 539), already be present in the host cell as an active endogenous gene or become activated as an endogenous non-active gene. Such an activation of endogenous genes can for example be achieved by the specific introduction of regulation elements into the genome for example by homologous recombination. Such methods are known and are described for example in WO 91/09955.
Mammalian cells are usually used as host cells. If an exogenous human EPO gene is introduced, CHO or BHK cells can for example be used as host cells. If an endogenous EPO gene is used for the expression, it is expedient to use human cells such as for example kidney, liver or lymph cells.
“Proteins which are derived from the host cell” are understood as proteins which are formed during the culture of the host cells containing the active EPO gene and do not have the specifications of EPO described above. The protein content is stated in ppm relative to weight (w/w). The content of these proteins can for example be determined by an ELISA which is based on polyclonal antibodies that are directed against the proteins of the host cell. Such polyclonal antibodies are obtained by immunizing animals, preferably sheep, with an extract of the proteins of the host cell (extracellular and intracellular proteins). The test is preferably carried out as a sandwich test using an immobilized polyclonal antibody and a peroxidase-labelled second antibody. The total protein extract is used as a standard. The lower limit of detection of such a test is about 15 ng protein per ml.
Burg Josef
Fürst Werner
Schneider Walter
Sellinger Karl-Heinz
Wrba Alexander
Arent Fox Kintner Plotkin & Kahn
Saidha Tekchand
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