Process for the Production of Human Follicle-Stimulating Hormone

The present invention relates to a process for the production of human follicle-stimulating hormone (follitropin, abbreviated hFSH hereinafter).

The hFSH is a hormone which stimulates the growth of spermatogenesis in the male, and the growth of ovarian follicles and the secretion of estrogen in the female.

No process for the mass production of low-cost hFSH has been established so far.

The present inventor has investigated processes for the mass production of hFSH. These efforts have resulted in the unexpected finding that human cells capable of producing hFSH, multiplying rapidly using non-human warm-blooded animals, and have an extremely higher hFSH producibility per cell than that obtained by in vitro tissue culture; up to about 2-50-fold of the latter in terms of hFSH production per cell.

More precisely, the present invention relates to a process for the production of hFSH, characterized in multiplying human cells capable of producing hFSH by transplanting said cells to a non-human warm-blooded animal body, or alternatively multiplying said cells by allowing said cells to multiply with a device by which the nutrient body fluid of a non-human warm-blooded animal is supplied to said cells, and exposing the cells multiplied by either of the above multiplication procedures to a follicle-stimulating hormone inducer to induce hFSH.

The process according to the present invention, besides realizing a greater hFSH production, requires no, or much less, nutrient medium containing expensive serum for cell multiplication, and renders the maintenance of the culture medium during the cell multiplication much easier than in the case of in vitro tissue culture. Particularly, any human cells capable of producing hFSH can be multiplied easily while utilizing the nutrient body fluid supplied from non-human warm-blooded animal by transplanting said cells to the animal body, or suspending said cells in a diffusion chamber devised to receive nutrient body fluid from the animal body, and feeding the animal in the usual way. Also, the process is characterized by stabler and higher cell multiplication, and higher hFSH production per cell.

As to the human cells usable in the present invention, any human cells can be used so far as they produce hFSH, and multiply easily in the non-human warm-blooded animal body. For example, conveniently feasible for use in the present invention are human cells which inherently produce hFSH, such as human basophile adenoma cells from the anterior lobe of the pituitary gland, such basophile cells which have been transformed by EB virus or X-ray irradiation, or human basophile adenoma cells from a patient suffering from basophile adenoma of the pituitary gland; human lung carcinoma cells which produce ectopic hFSH; and established cell lines of the above cells. Furthermore, there may be used easily maintainable established human lymphoblastoid lines in which there has been introduced hFSH production governing genes by means of genetic recombination techniques using enzymes such as DNA ligase, nuclease and DNA polymerase, or by cell fusion using agents such as polyethylene glycol or Sendai virus. The use of such established lymphoblastoid lines in which have been introduced hFSH production genetic sites conveniently results in a remarkably higher cell multiplication when the cells are transplanted to a non-human warm-blooded animal body, i.e., about 2-10-fold or more higher hFSH production per cell. Furthermore, since transplantation of the above mentioned established human lymphoblastoid lines to the animal body results in the formation of a massive tumor, and said massive tumor is hardly contaminated with the host animal cells and is disaggregated easily, the multiplied live human lymphoblastoid cells can be harvested easily.

As to the animals usable in the present invention, any animals can be used so far as the cells multiply therein. For example, poultry, such as chicken or pigeon, or a mammalian, such as dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster, mouse or nude mouse, is advantageously usable in the invention. Since such cell transplantation elicits undesirable immunoreaction, the use of a newborn or infant animal, or those in the youngest possible stage, for example, egg, embryo or foetus, is desirable. In order to reduce the immunoreaction, prior to the cell transplantation, the animal may be treated with X-ray or .gamma.-ray irradiation, about 200-600 rem, or injection of antiserum or immunosuppressive agent prepared according to conventional methods. Since nude mice, used as the non-human warm-blooded animal, exhibit weaker immunoreaction even when in their adulthood, conveniently, any established human cell can be transplanted and multiplied rapidly therein without such pretreatment.

Stabilized cell multiplication and enhancement of hFSH production can both be carried out by repeated transplantation using combination(s) of different non-human warm-blooded animals; for example, the objectives are attainable first by implanting said cells in hamster and multiplying therein, then by reimplanting in nude mouse. Further, the repeated transplantation may be carried out with animals of the same class or division as well as those in the same species or genus.

As to where the human cells are implantable, the human cells can be implanted in any site of the animal so far as the cells multiply therein; for example, in allantoic cavity, or intravenously, intraperitoneally, or subcutaneously.

Besides direct cell transplantation of the cells to the animal body, any conventional established human cell line capable of producing hFSH can be multiplied while utilizing the nutrient body fluid supplied from the animal body by embedding, for example, intraperitoneally, in the animal body a conventional diffusion chamber, of any of various shapes and sizes, and equipped with a porous membrane filter, ultra filter or hollow fiber having port sizes of about 10.sup.-7 to 10.sup.-5 m in diameter which prevents contamination with the host cells into the diffusion chamber and allows the animal to supply the cells with its nutrient body fluid. Additionally, the diffusion chamber can be designed, if necessary, so it could be placed, for example, on the host animal, and the body fluid allowed to circulate from the animal body into the chamber, to enable observation of the cell suspension in the chamber through transparent side window(s), equipped on the chamber wall(s), and to enable replacement and exchange with a fresh chamber; cell multiplication thereby increases to a further higher level over the period of the animal life, and the cell production per animal is further augmented without any sacrifice of the host animal. Furthermore, when such a diffusion chamber is used, since the multiplied human cells can be harvested easily and no immunoreaction is elicited due to the absence of direct contact of human cells with the host animal cells, any non-human warm-blooded animal can be used as the host in the present invention without any pretreatment to reduce the immunoreaction.

Feeding of the host animal implanted with the human cells can be carried out easily by conventional methods even after the cell transplantation, and no special care is required.

Maximum cell multiplication is attained about 1-20 weeks after the cell transplantation. When the established human cell line implanted in the animal is human tumor cell or human lymphoblastoid line, maximum cell multiplication is attained within one to five weeks after the cell transplantation due to their extremely higher cell multiplication rates.

According to the present invention, the number of human cells obtained per host ranges from about 10.sup.7 to 10.sup.12 or more. In other words, the number of human cells implanted in the animal body increases about 10.sup.2 -10.sup.7 -fold or more, or about 10.sup.1 -10.sup.6 -fold or more than that attained by in vitro tissue culture method: using nutrient medium; thus, the cells are conveniently usable for hFSH production.

As to the method for hFSH induction, any method can be employed so far as the human cells obtained by the above mentioned procedure release hFSH thereby. For example, the multiplied human cells, obtained by multiplying in ascite in suspension and harvesting from said ascite, or by extracting the massive tumor formed subcutaneously and harvesting after the disaggregation of the massive tumor, are suspended to give a cell concentration of about 10.sup.4 to 10.sup.8 cells per ml in a nutrient medium, kept at a temperature of about 20.degree.-40.degree. C., and then subjected to a follicle-stimulating hormone inducer at this temperature for about 1-50 hours to induce hFSH. Preferable follicle-stimulating hormone inducers are amino acids such as lysine, arginine, tryptophan, leucine or casamino acid; inorganic salts such as sodium chloride, potassium chloride, calcium chloride or magnesium sulfate; and hormones such as luteinizing hormone releasing hormone.

Simultaneous production of other human hormone such as human luteinizing hormone (abbreviated hLH hereinafter) can also be carried out according to the present invention.

The hFSH thus obtained can be collected easily by purification and separation techniques using conventional procedures such as salting-out, dialysis, filtration, centrifugation, concentration and lyophilization. If a further purified hFSH preparation is desirable, a preparation of the highest purity can be obtained by the above mentioned techniques in combination with other conventional procedures such as adsorption and desorption with ion exchange, gel filtration, isolectric point fractionation and electrophoresis.

The hFSH preparation can be used advantageously alone or in combination with one or more agents for injection, external, internal, or diagnostical administration in the prevention and treatment of human diseases.

The hFSH in the culture medium was determined by radioimmunoassay method as described in C. Faiman, R. J. Ryan, J. Clin. Endocrinol. Metab., Vol. 27, pp. 444-447 (1967), and expressed by the International Unit (IU). Also, the simultaneous hLH production was determined by radio-immunoassay method as described in A. R. Midgley, Jr., Endocrinology, Vol. 79, pp. 10-18 (1966), and expressed by the International Unit (IU).

Several embodiments of the present invention are disclosed hereinafter.

Disaggregated human basophile adenoma cells, obtained by extracting from a patient suffering from basophile adenoma of the pituitary gland and mincing, were implanted subcutaneously in adult nude mice which were then fed in the usual way for four weeks. The resulting massive tumors formed subcutaneously and about 10 g each were extracted and disaggregated by mincing and suspending in a physiological saline solution containing trypsin. After washing the cells with Earle's 199 medium (pH 7.2), supplemented with 10 v/v % foetal calf serum, the cells were resuspended to give a cell conentration of about 10.sup.5 cells per ml in a fresh preparation of the same medium which contained 30 mM L-arginine as the follicle-stimulating hormone inducer, and incubated at 37.degree. C. for 15 hours to induce hFSH. Thereafter, the cells were ultra-sonicated, and the hFSH in the supernatant was determined. The hFSH production was about 500 mIU per ml cell suspension.

The simultaneous hLH production in the supernatant was about 400 mIU per ml cell suspension.

Control cells, obtained by cultivating in vitro the human basophile adenoma cells in Earle's 199 medium (pH 7.2), supplemented with 10 v/v % foetal calf serum, and incubating at 37.degree. C., were treated similarly as above to induce hFSH. The hFSH production was only about 80 mIU per ml cell suspension.

Disaggregated human basophile adenoma cells, obtained by extracting from a patient suffering from basophile adenoma of the pituitary gland and mincing, and a human leukemic lymphoblastoid line Namalwa, were suspended together in a vessel to give a respective cell concentration of about 10.sup.3 cells per ml with a salt solution containing 140 mM NaCl, 54 mM KCl, 1 mM NaH.sub.2 PO.sub.4 and 2 mM CaCl.sub.2. The ice-chilled cell suspension was mixed with a fresh preparation of the same salt solution containing UV-irradiation preinactivated Sendai virus, transferred into a 37.degree. C. incubator five minutes after the mixing, and stirred therein for 30 minutes to effect cell fusion, introducing the hFSH producibility of the human basophile adenoma cells into the human leukemic lymphoblastoid line. After cloning according to conventional methods, the hybridoma cell strain capable of producing hFSH was implanted intraperitoneally in adult nude mice which were then fed in the usual way for five weeks. The resulting massive tumors, about 15 g each, were extracted and treated similarly as in EXAMPLE 1 to induce hFSH except that 30 mM L-arginine was replaced with about 10 ng luteinizing hormone. The hFSH production was about 1,600 mIU per ml cell suspension.

The simultaneous hLH production in the supernatant was about 1,400 mIU per ml cell suspension.

A control experiment was carried out similarly as in EXAMPLE 1 by cultivating in vitro the hybridoma cell strain, and exposing the multiplied human cells to the follicle-stimulating hormone inducer. The hFSH production was only about 80 mIU per ml cell suspension.

After injection of antiserum, prepared with rabbit according to conventional methods, into newborn hamsters, the animals were implanted subcutaneously with a human leukemic lymphoblastoid line JBL wherein the hFSH producibility of the human basophile adenoma cells was introduced similarly as in EXAMPLE 2, and then fed in usual way for three weeks. The resulting massive tumors formed subcutaneously and about 10 g each were extracted and treated similarly as in EXAMPLE 1 to induce hFSH. The hFSH production was about 1,500 mIU per ml cell suspension.

The simultaneous hLH production was about 1,300 mIU per ml cell suspension.

A control experiment was carried out similarly as in EXAMPLE 1 by cultivating in vitro the fused human leukemic lymphoblastoid line JBL, and exposing the multiplied cells to the follicle-stimulating hormone inducer. The hFSH production was only about 110 mIU per ml cell suspension.

Newborn rats were implanted intravenously with a human leukemic lymphoblastoid line Namalwa wherein the hFSH producibility of the human basophile adenoma cells was introduced similarly as in EXAMPLE 2, and then fed in the usual way for four weeks. The resulting massive tumors, about 40 g each, were extracted and treated similarly as in EXAMPLE 2 to induce hFSH. The hFSH production was about 1,200 mIU per ml cell suspension.

A control experiment was carried out similarly as in EXAMPLE 1 by cultivating in vitro the fused human leukemic lymphoblastoid line Namalwa, and exposing the multiplied human cells to the follicle-stimulating hormone inducer. The hFSH production was only about 80 mIU per ml cell suspension.

After about 400 rem X-ray irradiation of adult mice to reduce their immunoreaction, the animals were implanted subcutaneously with human basophile adenoma cells, obtained similarly as in EXAMPLE 1, and then fed in the usual way for four weeks. The resulting massive tumors formed subcutaneously and about 15 g each were extracted and treated similarly as in EXAMPLE 3 to induce hFSH. The hFSH production was about 500 mIU per ml cell suspension.

A control experiment was carried out similarly as in EXAMPLE 1 by cultivating in vitro the human basophile adenoma cells, and exposing the multiplied human cells to the follicle-stimulating hormone inducer. The hFSH production was only about 80 mIU per ml cell suspension.

A human leukemic lymphoblastoid line JBL wherein the hFSH producibility of the human basophile adenoma cells was introduced similarly as in EXAMPLE 3 was suspended in physiological saline solution, and transferred into a plastic cylindrical diffusion chamber, inner volume about 10 ml, and equipped with a membrane filter having a pore size of about 0.5.mu. in diameter. After intraperitoneal embedding of the chamber into an adult rat, the animal was fed in the usual way for four weeks, the chamber was removed. The human cell density in the chamber attained by the above operation was about 2.times.10.sup.9 cells per ml which was about 10.sup.3 -fold or more higher than that attained by in vitro cultivation using a CO.sub.2 incubator. The human cells thus obtained were treated similarly as in EXAMPLE 2 to induce hFSH. The hFSH production was about 1,300 mIU per ml cell suspension.

The simultaneous hFSH production was about 1,600 mIU per ml cell suspension.

A control experiment was carried out similarly as in EXAMPLE 1 by cultivating in vitro the fused human leukemic lymphoblastoid line, and exposing the multiplied human cells to the follicle-stimulating hormone inducer. The hFSH production was only about 110 mIU per ml cell suspension.

A human leukemic lymphoblastoid line JBL wherein the hFSH producibility of the human basophile adenoma cells was introduced similarly as in EXAMPLE 3 was implanted in allantoic cavities of embryonated eggs which had been preincubated at 37.degree. C. for five days, and the eggs were incubated at this temperature for an additional one week. The multiplied human cells were harvested and treated similarly as in EXAMPLE 1 to induce hFSH. The hFSH production was about 1,300 mIU per ml cell suspension.

A control experiment was carried out similarly as in EXAMPLE 1 by cultivating in vitro the fused human leukemic lymphoblastoid line JBL, and exposing the multiplied human cells to the follicle-stimulating hormone inducer. The hFSH production was only about 110 mIU per ml cell suspension.