GB2083826A

GB2083826A - Process for the production of human insulin

Info

Publication number: GB2083826A
Application number: GB8122892A
Authority: GB
Original assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK; Hayashibara Biochemical Laboratories Co Ltd
Current assignee: Hayashibara Seibutsu Kagaku Kenkyujo KK
Priority date: 1980-07-30
Filing date: 1981-07-24
Publication date: 1982-03-31
Also published as: IT8148915A0; JPS5756878B2; KR830005866A; GB2083826B; FR2487851B1; IT1196534B; JPS5729294A; CH649782A5; FR2487851A1; KR860000894B1

Abstract

A process for the mass production of human insulin, comprising multiplying human cells capable of producing human insulin, or hybrid cells from said cells and an established human lymphoblastoid line, obtained by transplanting said cells or the hybrid cells to a non-human warm-blooded animal body, or alternatively multiplying said cells or the hybrid cells by allowing the cells to multiply within a device in which the nutrient body fluid of a non-human warm-blooded animal is supplied to the cells, and exposing the cells multiplied by either of the above multiplication procedures to an insulin inducer to induce human insulin.

Description

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

Although conventional processes for the production of human insulin 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 human insulin yield and high production cost. Therefore, inevitably, insulin from bovine or porcine has been utilized.

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

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

The process according to the invention, besides realizing a greater human insulin 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 cell multiplication than in in vitro tissue culture. Particularly, any human cells capable of producing human insulin can be multiplied easily while utilizing the nutrient body fluid supplied from a non-human warmblooded animal by transplanting the cells to the animal, or suspending the cells in a diffusion chamber devised to receive the nutrient body fluid, and feeding the animal in the usual way. Also, in the present process one obtains more stable and increased cell multiplication, and higher human insulin production per cell.

As to the cells which can be used in the present invention, any cells can be used so long as they produce human insulin and multiply easily in a non-human warm-blooded animal body. Examples of suitable such cells are human cells which produce inherently human insulin such as intact human pancreas Langerhans island cells, those transformed by EB virus or X-ray irradiation, and insuloma cells from insuloma patient; human lung carcinoma cells which produce ectopic human insulin; 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 insulin 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 results conveniently in a remarkably higher cell multiplication and in a 2-10 fold increase of human insulin 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 cell 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 as long as the desired human cells multiply therein. Examples of suitable animals are poultry such as chickens and pigeons, and mammals such as dogs, cats, monkeys, goats, pigs, cows, horses, rabbits, guinea pigs, rats, hamsters, mice and 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, 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 of an immunosuppressive 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 and multiplied in nude mice rapidly without pretreatment to suppress immunoreactions.

Stablized cell multiplication and augmentation of human insulin production can be both carried out by repeated transplantation using combination(s) of different non-human warmblooded 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 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, the cells are implantable in the allan toic 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 cells capable of producing human insulin by using nutrient body fluid supplied from an animal body by embedding, for example, intraperitoneally, in the 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 10-5m in diameter which prevents contamination with 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 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 wal l(s), and so as to enable replacement and exchange with a fresh chamber. By such 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 warm-blooded animal can be used as the host without any pretreatment 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 re quires.

Maximum cell multiplication is attained about 1-20 weeks after the cell transplantation. When the established human cells implanted in the animal are human tumor cells or human lymphoblastoid lines; 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 the human cells transplanted in the animal body increases about 102-107-fold or more, or about 10 to 1 06-fold or more than that attained by in vitro tissue culture methods using nutrient medium; the cells are thus conveniently usable for human insulin production.

Any suitable method can be employed for insulin induction as long as the human cells obtained by the above mentioned procedure release human insulin. 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"C, and then subjected to an insulin inducer at this temperature for about one to 20 hours to induce human insulin.Preferable insulin inducers are saccharides such as glucose, mannose, fructose, ribose and xylitol; amino acids such as arginine, lysine and leucine; peptide hormones such as glucagon and adrenocorticotropic hormone (ACTH); and metal cations such as K+ and Ca++.

Human insulin thus obtained can be collected easily purification and separation techniques using conventional procedures such as salting-out, dialysis, filtration, centrifugation, concentration and lyophilization. If a more highly purified human insulin preparation is desirable, a human insulin preparation of the highest purity can be obtained by the above mentioned techniques in combination with conventional techniques such as adsorption and desorption with ion exchange, gel filtration, affinity chromatography, isoelectric point fractionation and electrophoresis.

The human insulin preparation thus obtained is advantageosuly 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.

The following Examples illustrate the present invention.

In the Examples, the human insulin in the culture medium was determined by enzymeimmunoassay method as described in K. Kato et al., J. Biochem, Vol. 78, pp. 235-237 (1975), and expressed by International Human Insulin Unit (IU) wherein 1 1U of human insulin is defined as the amount of insulin that decreases the rabbit blood sugar level to 64 mg/dl within one hour, or 45 mg/dl two hours after subcutaneous injection of the insulin preparation.

EXAMPLE 1 Disaggregated human insuloma cells, obtained by extracting from an insuloma patient 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 disaggregated by extracting, mincing and suspending in a physiological saline solution containing collagenase. After washing the cells with Earle's 1 99 medium (pH 7.2), supplemented with 10 v/v % foetal bovine serum, the celts were resuspended to give a cell concentration of about 105 cells per ml in a fresh preparation of the same medium which contained 20 mM D glucose as the insulin inducer, and then incubated at 37"C for four hours to induce human insulin.Thereafter, the cells were ultra-sonicated, and then the human insulin in the supernatant was determined. The human insulin production was about 1 ,000 sulU per cell.

The control cells, obtained by cultivating in vitro the human insuloma cells in Earle's 1 99 medium (pH 7.2), supplemented with 10 v/v % foetal bovine serum, and incubating at 37"C, were treated similarly as above with the insulin inducer. The human insulin production was only about 200 alU per cell.

EXAMPLE 2 Disaggregated human insuloma cells, obtained by extracting from an insuloma patient and mincing, and a human leukemic lymphoblastoid cell line Namalwa were suspended together in a vessel with a salt solution, containing 140 mM NaCI, 54 mM KC1, 1 mM NaH2PO4 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 insulin productibility of the human insuloma cells into the human leukemic lymphoblastoid line.

After cloning according to conventional method the hybridoma cell strain capable of producing human insulin, 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 insulin except that 20 mM D-glucose was replaced with 30 mM L-arginine. The human insulin production was about 3,200 jul U per cell.

A control experiment was carried out in a manner similar to that described in EXAMPLE 1 by cultivating in vitro the fused human lymphoblastoid line Namalwa, and exposing the multiplied cells to the insulin inducer. The human insulin production was only about 100 ,ul U per cell.

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 human insulin productibility of the human insuloma cells was introduced similarly as 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 in EXAMPLE 1 to induce human insulin. The human insulin production was about 2,300 IlIU per cell.

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 insulin inducer.

The human insulin production was only about 200 ,ulU per cell.

EXAMPLE 4 Newborn rats were implanted intravenously with a human leukemic lymphoblastoid line Namalwa wherein the human insulin productibility of the human insuloma 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 human insulin. The human insulin production was about 2,600 pIU per cell.

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

EXAMPLE 5 Adult mice were irradiated with about 400 rem X-ray to reduce their immunoreaction, implanted subcutaneously with human insuloma cells obtained as described in EXAMPLE 2, 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 1 to induce human insulin. The human insulin production was about 1 ,000 iLlU per cell.

A control experiment was carried out similarly as described in EXAMPLE 1 by cultivating in vitro the human insuloma cells, and exposing the multiplied cells to the insulin inducer. The human insulin production was only about 200 yIU per cell.

EXAMPLE 6 A human leukemic lymphoblastoid line JBL wherein the human insulin productibility of the human insuloma 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 0.5y in diameter, and the chamber was embedded intraperitoneally in an adult rat. After feeding the rat for four weeks in usual way, the chamber was removed. The human ceil density in the chamber attained by the above operation was about 5 x 109 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 human insu lin.The human insulin production was about 2,500 ,ulU per cell.

A control experiment was carried out simi larly as described in EXAMPLE 1 by cultivat ing in vitro the fused human leukemic lym phoblastoid line JBL, and exposing the multi plied cells to the insulin inducer. The human insulin production was only about 200 yIU per cell.

EXAMPLE 7 A human leukemic lymphoblastoid line JBL wherein the human insulin productibility of the human insuloma cells was introduced sim ilarly as described in EXAMPLE 3 was im planted 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 human insulin. The human insulin production was about 2,000 ,ulU per cell.

A control experiment was carried out similarly as described in EXAMPLE 1 by cultivating in vitro the fused human leukemic lym phoblastoid line JBL, and exposing the multiplied cells to the insulin inducer. The human insulin production was only about 200 ,ulU per cell.

Claims

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

2. A process as claimed in Claim 1, wherein said human cells capable of producing human insulin are intact human pancrease Langerhans island cells, human cells transformed by EB virus or X-ray irradiation, human insuloma cells, human lung carcinoma cells, or established cell lines of the above mentioned cells.

3. A process as claimed in Claim 1, wherein said human cells capable of producing human insulin are hybridoma cells obtained by cell fusion of a human leukemic lymphoblastoid line with cells of the type set forth in Claim 2.

4. A process as claimed in Claim 1, 2 or 3, wherein said insulin inducer is one or more materials selected from saccharides such as glucose, mannose, fructose, ribose and xylitol, amino acids such as arginine, lysine and leucine, peptide hormones such as glucagon and adrenocorticotropic hormone (ACTH), and metal cations such as K+ and Ca+ +.

5. A process as claimed in any one of the preceding claims, wherein said non-human warm-blooded animal 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 insulin whenever prepared by a process as claimed in any one of the preceding claims.