KR860000894B1 - Producing method of human insulin - Google Patents

Abstract

Human insulin (I) was produced by transplanting a human cell capable of producing (I) into any warm-blooded animal other than a human or in a device, esp., a diffusion chamber, which supplies them with the nutrient body fluid directly from warm-blooded animals. The grown cells are then treated with an insulin inducer. Pref. cells which are recommended for use include human Langerhans β- cells, viral trasformed cells, human insuloma cells, lung tumor cells or cell lines established from the latter and lymphoblastoid leukemia hybridomas. Inducers are saccarides, amino acids, peptide hormones, and metal ions. The amt. of (I) prod. per cell under these conditions is 2 - 50 times greater than possible by in vitro tissue cultures.

Description

Method of manufacturing human insulin

The present invention relates to a method for preparing human insulin.

Human insulin contains A chains containing 21 amino acid residues and 30 amino acid residues, as described, for example, in Biochemistry Data Handbook I, pages 1354-1356, published by the Institute of Chemical Engineering (1979). It is a peptide holmone having a molecular weight of about 5800 consisting of the B chain.

As a method of preparing human insulin, chemical synthesis, in vitro tissue culture, and microorganism culture by genetic modification are known, but all of these methods have very low human insulin yield and very high production cost. Therefore, the reality is that insulin derived from cow or pig is inevitably used.

As a result of continuing research on the purpose of mass production of human insulin, the present inventors have surprisingly found that human-derived cells having human insulin-producing ability were terminated using warm-blooded animals other than humans. According to the present invention, the present inventors have found that human insulin production is significantly higher than cells obtained according to the present invention, which can achieve up to about 2 to 50 times per cell.

In more detail, the present invention is to implant the human-derived cells having human insulin production capacity in a warm-blooded animal other than the human body or to be placed in a chamber that can supply the nutrient fluid of the warm-blooded animal other than the human body to the human-derived cells The present invention relates to a method for producing human insulin, wherein the human-derived cells are proliferated and the human-derived cells proliferated according to one of the aforementioned proliferation methods are exposed to insulin-inducing agents to produce human insulin.

The method according to the present invention not only produces large amounts of human insulin compared with in vitro tissue culture, but also significantly reduces or eliminates the need for nutrient medium containing expensive serum required for cell proliferation. Maintenance of the medium is much easier.

In particular, all human-derived cells with human insulin-producing ability are transplanted into warm-blooded animals other than humans to use nutrient fluids supplied from these animals, or to suspend these cells in a diffusion chamber designed to supply nutrient fluids. It can be easily propagated by breeding as usual.

In addition, this method is characterized by more stable cell proliferation, higher proliferative cell volume, and greater human insulin production per cell.

As the cell which can be used in the present invention, any cell can be used as long as it has human insulin producing ability and easily proliferates in a warm-blooded animal other than human.

For example, human insulin is a woundless human pancreatic Langerhans island β-cells, cells transformed by EB virus or X-ray irradiation, or with tumor cells of pancreatic tumor patients (Human insuloma coll). Cells with the same intrinsic human insulin production capacity; Human lung cancer cells having ectopic human insulin production ability; And cells cultured with these cells can be conveniently used in the present invention.

Moreover, the means of cell fusion which uses the gene which has human insulin production capability of these cells, for example using polyethyleneglycol or Sendai virus; Or genetically modified technology means using DNA ligase, restriction enzyme (Nuclease) and DNA polymerase; As a result, when introduced into cultured human lymphocytes obtained by more easily maintaining cultures, the cell proliferation rate is not only increased when these cells are transplanted into non-human warm-blooded animals, but also by the human body per cell. Particularly preferred is an increase in insulin production of about 2-10 times.

In addition, transplanting the above-described cultured human lymphocytes into the animal forms a large amount of tumor, which is difficult to mix with the host animal cells and easily collapses, so that only the living human lymphocytes proliferated are easily Can be harvested.

As the animal which can be used in the present invention, any animal can be used according to the present invention as long as human cells proliferate in the body.

For example, birds such as chickens and pigeons, as well as mammals such as dogs, cats, monkeys, goats, pigs, cattle, horses, rabbits, guinea pigs, mice, hamsters, mice and noumous mice, can be conveniently used in the present invention.

Since transplantation of human-derived cells into these animals may lead to an undesirable immune response, it is necessary to reduce the immune response as much as possible in newborn or childhood animals; Or it is preferred to use the youngest stage possible, namely egg, embryo, fetal animals.

In order to weaken the immune response, these animals are subjected to pre-treatment by X-ray or γ-ray irradiation of about 200 to 600 rem or by injection of antiserum or immunosuppressive agents prepared by conventional methods prior to cell transplantation. .

If the warm-blooded animal used is a growth mouse, the immune response is weak, so that all cultured human cells can be conveniently transplanted and rapidly proliferated in the body without the stated pretreatment.

In addition, cells derived from humans, for example, are first transplanted into hamsters to be expanded, and then transplanted using a combination of different non-human warm-blooded animals, such as when the cells are transplanted again to Nude mice to obtain a target product. This can further stabilize cell proliferation and increase the amount of human insulin produced from it. In this case, not only the same kind, the same speed, but also the same rape and alum can be transplanted.

As for the site for transplanting human-derived cells, human-derived cells can be transplanted to any site of the animal as long as the transplanted cells can proliferate.

For example, these cells can be implanted in urine, intravenous, intraperitoneal, subcutaneous, or the like.

It also prevents the incorporation of human-derived cells directly into the animal body, prevents the incorporation of host cells into the diffusion chamber, and allows porous blood membranes to supply the nutrient solution to culture-derived human-derived cells. Diffuse chambers of various shapes and sizes with porous membranes, ultrafiltration membranes or hollow fibers with 10 ^-7 to 10 ^-5 m are embedded in animals, for example intraperitoneally, and supplied from animals. Using the nutrient solution, the human-derived cells having the ability to produce human insulin produced in the preceding cultures can be expanded in the chamber.

In addition, if necessary, the diffusion chamber is attached to the surface of the host animal, for example, so that the body fluid in the animal can circulate into the chamber, and the proliferation state of the human-derived cells in the diffusion chamber is maintained through the projection side window provided on the wall of the chamber. The diffusion chamber can be designed to be able to see and to remove the replacement chamber and replace it with a new one, thereby increasing cell proliferation during animal life to higher levels without slaughtering the host animal. In addition, it may also increase cell production per animal. Moreover, when using such a diffusion chamber, since human-derived cells do not come in direct contact with host animal cells, only proliferated human-derived cells can be easily harvested, and undesirable immune responses do not occur. In the present invention without any pre-treatment to be able to use a variety of warm-blooded animals other than humans as a host animal freely.

Breeding of host animals transplanted with human-derived cells can proceed easily according to conventional methods even after transplantation of these cells and does not require any special handling.

Maximal cell proliferation is achieved about 1 to 20 weeks after cell transplantation. When the cultured coin cells transplanted into the animal are human tumor cells or human lymphocytes, their proliferation rate is so large that the maximum cell proliferation is usually achieved within 1 to 5 weeks after cell transplantation.

According to the invention, the number of human derived cells obtained per host ranges from about 10 ⁷ to 10 ¹² or more.

In other words, the number of human derived cells transplanted into an animal body is increased by about 10 ² to 10 ⁷ times or more, or about 10 to 10 ⁶ times or more of the number obtained by an in vitro culturing method using nutrient medium. Increasingly, these cells are conveniently used for human insulin production.

As an insulin induction method, any method may be used as long as the human-derived cells obtained by the above-described method can produce human insulin by acting on the insulin derivative. For example, proliferating human-derived cells obtained by proliferating in an intraperitoneal ascites and then collecting them from the ascites or extracting huge tumors formed subcutaneously and decomposing the tumors are maintained at a temperature of about 20 to 40 ° C Human nutrient medium may be induced by floating in a nutrient medium at a concentration of about 10 ⁴ to 10 ⁸ cells / ml and by inducing an insulin inducer for about 1 to 20 hours.

Examples of preferred insulin inducers include sugars such as glucose, mannose, fructose ribose and xylitol; Amino acids such as arginine, lysine and leucine; Metal cations such as peptide hormones K ⁺ and Ca ⁺⁺ such as glucagon and corticosteroids (ACTH).

The human insulin thus obtained can be easily collected by a purification separation method using conventional methods such as salting out, dialysis, filtration, centrifugation, concentration and freeze drying.

In addition, when a high degree of purification is required, the above known methods may be combined with conventional techniques such as adsorption and desorption gel filtration and affinity chromatography, isoelectric point fractionation, and electrophoresis on ion exchangers. The highest purity human insulin preparation can be obtained.

The human insulin thus obtained is advantageously used for the prevention and treatment of human disease as an injection, external medicine, oral medicine, or diagnostic administration alone or in combination with one or more other medicines.

Production of human insulin was measured by the enzyme-immunoassay method described by K. Kato's male person in J. Biochem, Vol. 78, pp. 235-237 (1975), and the blood glucose level of rabbits after subcutaneous injection of insulin preparation 1 The amount of insulin that reduced to 64 mg / dL within 2 hours and to 45 mg / dL within 2 hours was expressed as the International Human Insulin Unit (IU), defined as 1 IU.

Some embodiments of the invention are described below.

Example 1

Human pancreatic tumor cells (Humaninsuloma coll) obtained by subcutaneously dissecting and dissecting from pancreatic tumor patients subcutaneously from grown Nude mice were transplanted for 3 weeks.

About 10 g of a massive subcutaneous tumor was extracted, divided, and suspended in physiological saline containing collagenase to disperse the cells. After washing these cells with Earle medium 199 (pH 7.2) supplemented with 10v / v% fetal bovine serum, these cells were concentrated at about 10 ⁵ cells / ml in a new identical medium containing 20 mM D-glucose as an insulin inducer. After resuspending to 4 hours incubation at 37 ℃ to induce human insulin. Thereafter, the cell bag was sonicated and the amount of human insulin in the obtained supernatant was measured. Human insulin production was about 1000 μIU per cell.

Control cells obtained by growing human tumor cells in vitro in Earle medium 199 (pH 7.2) supplemented with 10v / v% of bovine fetal serum and incubated at 37 ° C were treated with an insulin inducer in a similar manner as described above.

Production of human insulin was only about 200 μIU per cell.

Example 2

Human pancreatic tumor cells (Human insuloma coll) and human leukemia lymphocytes (Namalwa) cells obtained from the patients with pancreatic tumors, cut and dispersed, contained 140 mM NaCl, 54 mM MKCl, 1 mM NaH ₂ PO ₄ and 2 mM CaCl ₂ . Along with the saline solution was suspended in the vessel to the corresponding cell concentration of about 10 ³ cells / ml.

The ice-cold cell suspension was mixed with the same salt solution as described above containing Sendai virus, which had been previously irradiated with ultraviolet light, and transferred to a 37 ° C. constant temperature water bath after about 5 minutes of mixing, followed by stirring for about 30 minutes, followed by cell fusion. Human insulin production of human pancreatic tumor cells was introduced into lymphocytes of the leukemia lymphocytes. After the hybrid tumor cell line having human insulin-producing ability was isolated by a conventional method, the hybrid tumor cell line was transplanted intraperitoneally of the grown Nude mouse and reared for 5 weeks by a conventional method.

About 15 g of each of the obtained huge tumors was extracted and human insulin was induced by treatment similarly to Example 1 except that 20 mM of D-glucose was changed to 30 mM of L-arginine. Human insulin production was about 3200 μIU per cell.

Human lymphocyte namalba cells fused with the control test were cultured in vitro and the proliferating cells were exposed to insulin inducers to proceed similarly to Example 1. Human insulin production was only about 100 μIU per cell.

Example 3

In order to suppress the immune response that may be caused by cell transplantation, the anti-serum prepared by a known method from rabbits was previously injected into rabbit hamsters, and the human insulin production capacity of human pancreatic tumor cells was subcutaneously similar to that of Example 2. Human leukemia lymphocytes JBL, which had been briefly introduced, was transplanted and bred for three weeks in a conventional manner.

Thus, 10 g of each obtained subcutaneous tumor formed subcutaneously was removed and treated similarly to Example 1 to induce human insulin. The production of human insulin was about 2300 μIU per cell.

The control test proceeded similarly to Example 1 by culturing fused human leukemia lymphocytes JBL cells in vitro and exposing these proliferating cells to insulin inducers. Human insulin production was only about 200 μIU per cell.

Example 4

Similar to Example 2, human leukemia lymphocytes, which were introduced with human insulin-producing ability of human pancreatic tumor cells, were transplanted into a vein of neonatal rats, and then reared in a conventional manner for 4 weeks.

About 40 g of each of the giant tumors thus obtained were extracted and treated similarly to Example 1 to induce human insulin. Human insulin production was about 2600 μIU per cell.

The control test proceeded similarly as in Example 1 by culturing similarly fused human leukemia lymphocytes Namalba cells in vitro and acting these proliferating cells on insulin inducers. Human insulin production was only about 100 μIU per cell.

Example 5

After irradiating about 400 rem of X-rays to the grown ordinary mice, the immune response of the mice was attenuated, and human pancreatic tumor cells obtained in the same manner as in Example 2 were implanted subcutaneously, and grown in a conventional manner for 3 weeks. . Thus, about 15 g of each of the giant obtained tumors formed subcutaneously were extracted and treated similarly to Example 1 to induce human insulin.

Human insulin production was about 1000 μIU per cell.

The control test proceeded similarly as in Example 1 by culturing human pancreatic tumor cells in vitro and exposing the insulin inducing agent to the proliferative cells. Human insulin production was only about 200 μIU per cell.

Example 6

In a diffusion chamber with a volume of about 0.5 μm with a pore diameter of about 0.5 μm, similarly to Example 3, human leukemia lymphocytes JBL cells were introduced into physiological saline solution. The cells were suspended and buried in the abdominal cavity of the grown mice.

The chambers were removed after breeding the mice for 4 weeks in the usual manner.

The human-derived cell density in the chamber obtained as described above was about 5 × 10 ⁹ per ml, which was about 10 ³ times or more than that obtained by in vitro culture using a CO ₂ incubator.

Cells thus obtained were treated similarly to Example 1 to induce human insulin. Human insulin production was about 2500 μIU per cell.

Human leukemia lymphoblastic JBL cells fused to the control test were cultured in vitro and progressed similarly to Example 1 by exposing the proliferative cells to insulin inducers.

Human insulin production was only about 200 μIU per cell.

Example 7

Similarly to Example 3, human leukemia lymphocytes JBL cells into which human pancreatic tumor cells introduced human insulin-producing ability were implanted into fertilized urine cavity of fertilized eggs previously incubated at 37 ° C. for 5 days.

After further fermenting these fertilized eggs for one week at the same temperature, proliferating human-derived cells were harvested. The cells were treated similarly to Example 1 to induce human insulin. Human insulin production was about 2000 μIU per cell.

The control test proceeded similarly to Example 1 by culturing human leukemia lymphocytes JBL cells in vitro and exposing proliferating cells to insulin inducers. Human insulin production was only about 200 μIU per cell.

Claims (1)

Implanting human insuloma cells, human, non-JBL cells or Namalva cells with human insulin production into the body of a nude mouse, mouse, hamster, rat or chicken, or The animals were terminated using a chamber with a volume of about 10 mL equipped with a membrane diameter of about 0.5 micron in which the body fluids can be supplied, and D-glucose or L-arginine was applied to the macrophages to obtain molecular weight. Method for producing human insulin, characterized in that the production of human insulin of 5800.