GB2085891A

GB2085891A - Process for the production of human prolactin

Info

Publication number: GB2085891A
Application number: GB8125630A
Authority: GB
Original assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK; Hayashibara Biochemical Laboratories Co Ltd
Current assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK
Priority date: 1980-08-22
Filing date: 1981-08-21
Publication date: 1982-05-06
Also published as: CH649783A5; FR2488804B1; FR2488804A1; GB2085891B; JPS5756876B2; IT8149117A0; JPS5739795A; KR830005867A; IT1143224B; KR860000896B1

Abstract

A process for the mass production of human prolactin, comprising in vitro multiplication of human cells capable of producing human prolactin, or a hybrid human lymphoblastoid line introduced with human prolactin productibility by means of cell fusion, and exposure of the multiplied cells to a prolactin inducer to induce human prolactin.

Description

SPECIFICATION Process for the production of human prolactin The present invention relates to a process for the production of human prolactin.

Human prolactin is a hormone, secreted by human acidophile adenoma cells of the anterior pituitary, which stimulates the growth of mammary gland, prostate and seminal vesicle.

No process for the mass production of human prolactin has been established so far.

We have investigated processes for the mass production of low-cost human prolactin and have unexpectedly found that the yield of human prolactin obtained from human cells which are produced by multiplying human cells capable of producing human prolactin using a non-human warm-blooded animal body is much higher than that obtained by in vitro tissue culture; i.e. 2-50 times higher in terms of human prolactin production per cell.

According to the present invention there is provided a process for the production of human prolactin comprising multiplying human cells capable of producing human prolactin by transplanting said cells to a non-human warmblooded animal body, or alternatively multiply ing said cells by allowing said cells to multiply within a device in 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 prolactin inducer to induce human prolactin.

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

As to the cells which can be used in the present invention, any cells can be used as long as they produce human prolactin and multiply easily in a non-human warm-blooded animal body. Examples of suitable such cells are human cells which produce inherently human prolactin such as human acidophile cells from anterior pituitary, those transformed by EB virus or X-ray irradiation, and human acidophile adenoma cells from a patient suffering from acidophile adenoma of the pituitary gland; lung carcinoma cells which produce ectopic human prolactin; and established cell lines of the above cells.Also when the cells to be multiplied are transplanted to a non-human warm-blooded animal body the use of easily maintainable established human lymphoblastoid lines introduced with human prolactin production governing genes by means of genetic recombination techniques using enzymes such a DNA ligase, nuclease and DNA polymerase, or by cell fusion using agents such as polyethylene glycol or Sendai virus results in a remarkably increased cell multiplication, and in a 2 to 1 04old increase at least of human prolactin production per cell. Furthermore, since transplantation of the above mentioned human lymphoblastoid lined results in the formation of massive tumors, and said massive tumors are barely contaminated with the host animal cells and disaggregated easily, the multiplied live human lymphoblastoid cells can be harvested easily.

Any warm-blooded animal body can be used to perform the process of the present invention so long as the desired human cells multiply therein. Examples of suitable animals are poultry such as chickens or pigeons, and mammels such as dogs, cats, monkeys, goats, pigs, cows, horses, rabbits, guinea pigs, rats, hamsters, mice or nude mice. Since transplantation of human cells gives rise to undesirable immuno-reaction, the use of a newborn or infant animal, or an animal body in the youngest possible stage, for example in the form of an egg, embryo or foetus, is desirable. In order to reduce the incidence of immunoreactions, prior to the cell transplantation, the animal may be treated with X-ray or y-ray irradiation, at about 200-600 rem, or with an injection of antiserum or an immuno-suppressive agent prepared according to conventional methods.Nude mice, even in adult form, are found to exhibit weak immunoreactions; thus any established human cells can be transplanted in nude mice and multiplied therein rapidly without such pretreatment to suppress immunoreactions.

Stabilized cell multiplication and enhancement of human prolactin production can be both 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 human cells in hamsters and multiplying the cells therein, then by reimplanting the cells in nude mice. Further, the repeated transplantation may be carried out with animals of the same class or division as well as those of the same species or genus.

The human cells to be multiplied can be implanted in any sites of the animal as long as the cells will multiply at that site. For example, in the allantoic cavity, or intrave nously, intraperitoneally, or subcutaneously.

As well as direct transplantation of the cells to the animal body, it is also possible to multiply any conventional established human cell lines capable of producing human prolactin by using the nutrient body fluid supplied from the animal body by embedding, for example, intraperitoneally, in said animal body a conventional diffusion chamber, of various shapes and sizes, and equipped with porous membrane filter, ultra filter or hollow fiber with pore sizes of about 10-, to 10-5m in diameter which prevents contamination with host animal 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 so that it can be placed, for example, on the host animal in such a manner as to enable observation of the cell suspension in the chamber through transparent side windows(s), equipped on the chamber wall(s), and so as to enable replacement and exchange with a fresh chamber. By such a method cell mutliplication 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. Furthernore, when such a diffusion chamber is used, since the multiplied human cells can be harvested easily and no immunoreaction arises owing 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 without any pretreatment to reduce immunoreactions.

Feeding of the animal implanted with the human cells can be carried out easily by conventional methods even after the cell tranplantation, 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 the extremely high cell multiplication rates of these cells.

According to the invention, the number of the human cells obtained per host ranges from about 107 to 1 012 or more. In other words, the number of the human cells transplanted in the animal body increases about 102-107-fold or more, or about 101-106-fold or more than that attained by in vitro tissue culture methods using nutrient medium; the cells are thus conveniently usable for human prolactin production.

Any suitable method can be employed for human prolactin induction as long as the human cells obtained by the above mentioned procedure release human prolactin. 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 said massive tumor, are suspended in a concentration of about 104 toll8 cells per ml in a nutrient medium, kept at a temperature of about 20-40"C, and then subjected to a prolactin inducer at this temperature for about 1-50 hours to induce human prolactin.

Preferable prolactin inducers are organic compounds such as reserpin, phenothiazine and chlorobromadine; hormones such as thyrotropin releasing hormone and estrogen; amino acids such as lysine, arginine and cysteine; and inorganic salts such as sodium chloride, potassium chloride, calcium chloride and magnesium suifate. In this case, other hormones such as human growth hormone (hGH) may be produced simultaneously according to the invention.

The human prolactin thus obtained can be easily collected by purification and separation techniques using conventional procedures such as salting-out, dialysis, filtration, centrifugation, concentration and lyophilization. If a more highly purified human prolactin preparation is desirable, a human prolactin 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, affinity chromatography, isoelectric point fractionation and electrophoresis.

The human prolactin preparation thus obtained is immunologically identical with the NIH standard human prolactin preparation, and it is advantageously usable alone or in combination with one or more agents as an injection, or for external, internal, or diagnostical administration for prevention and treatment of human diseases such as hypogalactia.

The following Examples illustrate the present invention.

In this specification, human prolactin production was deternined by radio-immunoassay method as described in P. Hang et al ., Proc.

Nat. Acad. Sci. USA, Vol. 68, pp.

1902-1906 (1971), and expressed by weight per ml cell suspension in terms of the NIH standard human prolactin preparation.

The simultaneous hGH production was determined by radio-immunoassay method as described in S.M. Glick et aL, Nature, Vol. 1 99, page 784 (1963), and expressed by weight in terms of the NIH standard human growth hormone preparation.

EXAMPLE 1 Disaggregated human acidophile adenoma cells, obtained by extracting from a patient suffering from acidophile adenoma of the pituitary gland and mincing, were implanted subcutaneously in adult nude mice which were then fed in usual way for three 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 glucose-free Earle's 1 99 medium (pH 7.2), supplemented with 10 v/v% foetal bovine serum, the cells were resuspended to give a cell concentration of about 105 cells per ml in a fresh preparation of the same medium which contained 30 mM L-arginine as the prolactin inducer, and then incubated at 37"C for six hours to induce human prolactin.Thereafter, the cells were ultra-sonicated, and the human prolactin in the supernatant was determined.

The human prolactin production was about 600 ng per ml cell suspension. The simultaneous hGH production was about 500 ng per ml cell suspension.

The control cells, obtained by cultivating in vitro the human acidophile adenoma cells in Earle's 199 medium (pH 7.2), supplemented with 10 v/v /0 foetal bovine serum, and incubating at 37"C, were treated similarly as above with the prolactin inducer. The human prolactin production was only about 100 ng per ml cell suspension.

EXAMPLE 2 Disaggregated human acidophile adenoma cells, obtained by extracting from a patient suffering from acidophile adenoma of the pituitary gland and mincing, and a human leukemic lymphoblastoid line Namalwa were suspended together in a vessel with a salt solution, containing 140 mM NaCI, 1 mM KCI, 1 mM NaH2P04 and 2 mM CaCI2, to give respective cell concentration of about 103 cells per ml. The ice-chilled cell suspension was mixed with a preparation of the same salt solution containing UV-irradiation preinactivated Sendai virus, transferred into a 37"C incubator about five minutes after the mixing, and stirred therein for about 30 minutes to effect cell fusion, introducing the human prolactin productability of the human acidophile adenoma cells into the human leukemic lymphoblastoid line.After cloning according to conventional method the hybridoma cell strain capable of producing human prolactin, the hybridoma cell strain was implanted intraperitoneally in adult nude mice which were then fed in the usual way for five weeks. The resulting massive tumors, about 1 5 g each, were extracted and treated similarly as in EXAMPLE 1 to induce human prolactin except that 30 mM L-arginine was replaced with about 10 ng thyrotropin releasing hormone and the incubation was carried out at 35"C.

The human prolactin production was about 3,300 ng per ml cell suspension.

A control experiment was carried out in a manner similarly to that described in EXAM PLE 1 by cultivating in vitro the hybrid human leukemic lymphoblastoid line Namalwa, and exposing the multiplied cells to the prolactin inducer. The human prolacting production was only about 200 mg per ml cell suspension.

EXAMPLE 3 Newborn hamsters were injected with an antiserum, prepared from rabbits according to conventional methods in order to reduce immunoreaction of the hamsters resulting from cell transplantation. The hamsters were then implanted subcutaneously with a human leukemic lymphoblastoid line JBL wherein the human prolactin productibility of the human acidophile adenoma cells was introduced similarly as described in EXAMPLE 2. The hamsters were then fed in the usual way for three weeks. The resulting massive tumors, formed subcutaneously and about 10 g each were extracted and treated similarly as described in EXAMPLE 1 to induce human prolactin. The human prolactin production was about 1,800 ng per ml cell suspension. The simultaneous hGH production was about 2,000 ng per ml cell suspension.

A control experiment was carried out in a manner similar to that described in EXAMPLE 1 by cultivating in vitro the hybrid human leukemic lymphoblastoid line JBL, and exposing the multiplied cells to the prolactin inducer. The human prolactin production was only about 200 ng per ml cell suspension.

EXAMPLE 4 Newborn rats were implanted intravenously with a human leukemic lymphoblastoid line Namalwa wherein the human prolactin productibility of the human acidophile adenoma cells was introduced similarly as described 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 described in EXAMPLE 2 to induce human prolactin. The human prolactin production was about 2, 700 ng per ml cell suspension.

A control experiment was carried out similarly as described in EXAMPLE 1 by cultivating in vitro the hybrid human leukemic lymphoblastoid line Namalwa, and exposing the multiplied cells to the prolactin inducer. The human prolactin production was only about 100 ng per ml cell suspension.

EXAMPLE 5 Adult mice were irradiated with about 400 rem X-ray to reduce their immunoreaction, implanted subcutaneously with human acidophile adenoma cells obtained similarly as described in EXAMPLE 1, and fed in the usual way for three weeks. The resulting massive tumors, formed subcutaneously and about 1 5 g each, were extracted and treated similarly as described in EXAMPLE 2 to induce human prolactin. The human prolactin production was about 800 ng per ml cell suspension.

A control experiment was carried out similarly as described in EXAMPLE 1 by cultivating in vitro the human acidophile adenoma cells, and exposing the multiplied cells to the prolactin inducer. The human prolactin production was only about 200 ng per ml cell suspension.

EXAMPLE 6 A human leukemic lymphoblastaid line JBL wherein the human prolactin productibility of the human acidophile adenoma cells was introduced similarly as described in EXAMPLE 3 was suspended in physiological saline solution, transferred into a plastic cylindrical diffusion chamber, with an inner volume of about 10 ml, and a membrane filter having pore sizes of about 0.5p in diameter, and the chamber was embedded intraperitoneally in an adult rat. After feeding the rat for four weeks in the usual way, the chamber was removed. The human cell density in the chamber attained by the above operations was about 2 x 109 cells per ml which was about 1034old higher or more than that attained by in vitro cultivation using a CO2 incubator. The cells thus obtained were treated similarly as described in EXAMPLE 1 to induce human prolactin.The human prolactin production was about 1,900 ng per ml cell suspension.

The simultaneous hGH production was about 2,200 ng per ml cell suspension.

A control experiment was carried out similarly as described in EXAMPLE I by cu[tivating in vitro the hybrid human leukemic lymphoblastoid line JBL, and exposing the multiplied cells to the prolactin inducer. The human prolactin production was only about 200 ng per ml cell suspension EXAMPLE 7 A human leukemic lymphoblastoid line JBL wherein the human prolactin productibility of the human acidophile adenoma cells was introduced similarly as described in EXAMPLE 3 was implanted in the allantoic cavities of embryonated eggs which had been preincubated at 37"C for five days. After incubation of the eggs at this temperature for an additional one week, the multiplied human cells were harvested. The cells were treated similarly as described in EXAMPLE 2 to induce human prolactin. The human prolactin production was about 1,700 ng per ml cell suspension.

A control experiment was carried out similarly as described in EXAMPLE 1 by cultivating in vitro the hybrid human leukemia lym.- phoblastoid line JBL, and exposing the multiplied cells to the prolactin inducer. The human prolactin production was only about 200 ng per ml cell suspension.

Claims

1. A process for the production of human prolactin, which process comprises (1) multiplying human cells capable of producing human prolactin by transplanting said cells to a non-human warm-blooded animal body, and exposing the multiplied cells to a prolactin inducer to induce human prolactin; or (2) multiplying human cells capable of producing human prolactin by allowing said cells to multiply within a device in which the nutrient body fluid of a non-human warm-blooded animal is supplied to said cells, and exposing the multiplied cells to a prolactin inducer to induce human prolactin.

2. A process as claimed in Claim 1, wherein said human cells are intact human acidophile cells of the anterior pituitary, those transformed by EB virus or X-ray irradiation, human acidophile adenoma cells of the pitui- ' tary gland, lung carcinoma cells, or established cell lines of the above described cells.

3. A process as claimed in Claim 1, wherein said human cells are hybrid cells obtained by cell fusion of a human lymphoblastoid with one of the cells of the type set forth in Claim 2.

4. A process as claimed in any one of the preceding claims, wherein said prolactin inducer is one or more materials selected from organic compounds such as reserpin, phenothiazine and chlorobromadine; hormones such as thyrotropin releasing hormone and estrogen; amino acids such as lysine, arginine, and cysteine; and inorganic salts such as sodium chloride, potassium chloride, calcium chloride and magnesium sulfate.

5. A process as claimed in an one of the preceding claims, wherein said non-human warm-blooded animal body is poultry such as chickens or pigeons, or a mammal such as a dog, cat, monkey, goat, pig, cow, horse, rabbit guinea pig, rat, hamster, mouse or nude mouse.

6. A process according to Claim 1 substantially as hereinbefore described in any one of Examples 1 to 7.

7. Human prolactin whenever prepared by a process as claimed in any one of the preceding claims.