GB2083824A - Process for the production of human growth hormone - Google Patents

Abstract

A process for the mass production of human growth hormone (hGH) comprising in vivo multiplication of human cells capable of producing hGH, or a hybrid human lymphoblastoid line introduced with hGH productibility by means of cell fusion technique, and exposure of the multiplied cells to a growth hormone inducer to induce hGH.

Description

SPECIFICATION Process for the production of human growth hormone The present invention relates to a process for the production of human growth hormone (hereinafter abbreviated hGH).

Although conventional processes for the production of hGH such as those by chemical synthesis, in vitro tissue culture, or cultivation of genetically recombinated microorganisms are known, the processes result in a very low hGH yield and high production cost.

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

According to the present invention there is provided a process for the production of hGH, comprising multiplying human cells capable of producing hGH by transplanting said cells to a non-human warm-blooded animal body, or alternatively multiplying said cells by allowing said cells to multiply within a device in which the nutrient body fluid of a non-human warmblooded animal is supplied to said cells, and exposing the cells multiplied by either of the above multiplication procedures to a growth hormone inducer to induce hGH.

The process according to the invention, besides realizing a greater hGH production, requires much less nutrient medium containing expensive serum for cell multiplication or no such medium, and renders much easier the maintainance of the culture medium during the cell multiplication than in in vitro tissue culture. Particularly, any human cells capable of producing hGH can be multiplied easily while utilizing the nutrient body fluid supplied from a hon-human warm-blooded animal 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 hGH 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 hGH and multiply easily in a non-human warm-blooded animal body.

Examples of suitable such cells are human cells which produce inherently hGH such as intact human acidophile cells from anterior pituitary, those transformed with EB virus or X-ray irradation and acidophile adenoma cells from a patient suffering from acidophile adenoma of the pituitary gland; human lung carcimona cells which produce ectopic hGH; and established cell lines of the above human 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 hGH production governing genes by means of gentic 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 conveniently results in a remarkably increased cell multiplication, and in a 2 to 10fold increase at least of hGH production per cell. Furthermore, since transplantation of the above mentioned established human lymphoblastoid lines to the animal body results in the formation of massive tumors, and said massive tumors are hardly contaminated with the host animal cells and are 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 as long as the desired human cells multiply therein. Examples of suitable animals are poultry such as chickens or pigeons, and mammals 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 immunoreactions, 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, the animal may be treated by X-ray or y-ray irradiation, at about 200-600 rem, or with an injection of antiserum or immunosuppressive agent prepared according to conventional methods.Nude mice, even in adult form, are found to exhibit weak immunoreactions; thus any established human cell lines can be transplanted and multiplied in nude mice rapidly without pre-treatment to suppress immunoreactions.

Stabilized cell multiplication and augmentation of hGH production can 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 the 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 of genus.

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

As well as direct cell transplantation of the cells to the animal body, it is also possible to multiply any of conventional established human cell lines capable of producing hGH by using nutrient body fluid supplied from an 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 10-7 to 10-5 m 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 window(s) equipped on the chamber wall(s), and so as to enable replacement and exchange with a fresh chamber. By such a method cell multiplication 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 arises owing to the absence of direct contact of human cells with the host animal cells, any non-human warmblooded animal can be used as the host without any pre-treatment to reduce immunoreactions.

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 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 1012 or more. In other words, the number of human cells transplanted in the animal body increases about 102-1 07-fold or more, or about 10 to 106 fold or more than that attained by in vitro tissue culture methods using nutrient medium; the cells are thus conveniently usable for hGH production.

Any suitable method can be employed for hGH induction as long as the human cells obtained by the above mentioned procedure release hGH. 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 to 108 cells per ml in a nutrient medium, kept at a temperature of about 20-40 . and then subjected to a growth hormone inducer at this temperature for about one to 20 hours to induce hGH. Preferable growth hormone inducers are amino acids such as lysine, arginine, triptophan, leucine, casamino acid and L-DOPA, amino acid metabolites such as a serotonin, and peptides.

The hGH thus obtained can be easily collected by purification and separation techniques using conventional procedures such as salting-out, dialysis, filtration, centrifugation, and lyophilization. If a more highly purified hGH preparation is desirable, a hGH 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 fractionaltion and electrophoresis.

Since the hGH preparation thus obtained is immunologically identical with standard hGH preparation, the preparation is advantageously usable alone or in combination with one or more agents for injection, or for external, internal, or diagnostical administration in the prevention and treatment of human diseases as well as in human growth stimulation.

The following Examples ilustrate the present invention.

In this specification, the hGH production in the culture medium was determined by the radio-immunoassay method as described in S.M. Glick et al., Nature, Vol. 199, page 784 (1963), and expressed by weight in terms of hGH standard preparation distributed by the National Institute of health.

EXAMPLE 1 Dissagregated 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 10g each, were disaggregated by extracting, 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 bovine serum, the cells were resuspended in a fresh preparation of-the same medium which contained 30 mM Larginine as the growth hormone inducer, and then incubated at 37 for six hours to induce hGH. Thereafter, the cells were ultra-sonicated, and the hGH in the supernatant was determined. The 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 % foetal bovine serum, and incubating at 37 , were treated similarly as above with the growth hormone inducer to induce hGH. The hGH 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, 54 mM KCI, 1 mM NaH2PO4 and 2mM 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 incubator about five minutes after the mixing, and stirred therein for about 30 minutes to effect cell fusion, introducing the hGH productibility of the human acidophile adenoma cells into the human leukemic lymphoblastoid line.

After cloning according to conventional method the hybridoma cells capable of producing hGH, the hybridoma cells were 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 hGH except that 30 mM L-arginine was replaced with L DOPA. The hGH production was about 1,88 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 hybridoma cells, and exposing the multiplied cells to the growth hormone inducer. The hGH production was only about 100 ng per ml cell suspension.

EXAMPLE 3 Newborn hamsters were injected with an antiserum, prepared from rabbit 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 hGH 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 hGH. The hGH production was about 2,000 ng per ml suspansion.

A control experiment was carried out in a manner similar to that described in EXAMPLE 1 by cultivating in vitro the fused human leukemic lymphoblastoid line JBL, and exposing the multiplied cells to the growth hormone inducer. The hGH 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 hGH 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 1 to induce hGH. The hGH production was about 1,500ng per ml cell suspension.

A control experiment was carried out similarly as described in EXAMPLE 1 by cultivating in vitro the fused human lymphoblastoid line Namalwa, and exposing the multiplied cells to the growth hormone inducer. The hGH 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 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 hGH. The hGH production was about 600 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 growth hormone inducer. The hGH production was only about 200 ng per ml cell suspension.

EXAMPLE 6 A human leukemic lymphoblastoid line JBL wherein the hGH productibility of the human acidophile adenoma cells was introduced similarly as described in EXAMPLE 3 was suspended in physiological saline solution, and transferred into a diffusion chamber having an inner volume of about 10 ml and a membrane filter of pore sizes of about 0.5 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 operation was about 5 > < x 109 10 cells per ml which was about 103-fold 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 hGH.The hGH production was about 2,200 ng per ml cell suspension.

A control experiment was carried out similarly as described in EXAMPLE 1 by cultivating in vitro the fused human leukemic lymphoblastoid line JBL, and exposing the multipled cells to the growth hormone inducer. The hGH production was only about 200 ng per ml cel suspension.

EXAMPLE 7 A human leukemic lymphoblastoid line JBL wherein the hGH productibility of the human acidophile adenoma cells was introduced similarly as described in EXAMPLE 3 was implanted in 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 1 to induce hGH. The hGH production was about 1,300 ng per ml cell suspension.

A control experiment was carried out similarly as described in EXAMPLE 1 by cultivating in vitro the fused human leukemic lymphoblastoid line JBL, and exposing the multiplied cells to the growth hormone inducer.

The hGH production was only about 200 ng per ml cell suspension.

Claims (8)

1. A process for the production of hGH, which process comprises (1) multiplying human cells capable of producing hGH by transplanting said cells to a non-human warm-blooded animal body, and exposing the multiplied cells to a growth hormone inducer to induce hGH, or (2) multiplying human cells capable of producing hGH 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 growth inducer to induce hGH.

2. A process as claimed in Claim 1, wherein said human cells capable of producing hGH are intact human acidophile cells from the anterior pituitary, those transformed by EB virus or X-ray irradiation, or human acidophile adenoma cells.

3. A process as claimed in Claim 1, wherein said human cells capable of producing hGH are established cells of the type set forth in Claim 2.

4. A process as claimed in Claim 1, wherein said human cells capable of producing hGH are hybridoma cells obtained by cell fusion of a human leukemic lymphoblastoid line with one of the cells described in Claim 2 or Claim 3.

5. A process as claimed in any one of the preceding claims, wherein said growth hormone inducer is one or more materials selected from amino acids such as lysine, arginine, triptophan, leucine, casamino acid and L DOPE, amino acid metabolites such as serotonin, and peptides.

6. A process as claimed in any 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, cow, horse, rabbit, guinea pig, rat, hamster, mouse or nude mouse.

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

8. Human growth hormone whenever prepared by a process as claimed in any one of the preceding claims.