FR2487851A1 - PROCESS FOR THE PRODUCTION OF HUMAN INSULIN - Google Patents

Abstract

METHOD FOR IMPROVING THE PRODUCTION OF HUMAN INSULIN. IT CONSISTS OF MULTIPLYING HUMAN CELLS CAPABLE OF PRODUCING HUMAN INSULIN BEFORE EXPOSING THEM TO THE ACTION OF AN INSULIN INDUCER, OR BY TRANSPLANT DIRECTLY INTO THE BODY OF A WARM BLOOD ANIMAL OR BY SUPPLYING THEM, USING A DEVICE, SUCH AS A DIFFUSION CHAMBER, THE BODY NUTRIENT FLUID OF AN ANIMAL WITH HOT BLOOD OTHER THAN MAN. THE PRODUCTION OF INSULIN ACHIEVED ACCORDING TO THE INVENTION IS 2 TO 50 TIMES GREATER, PER CELL, THAN OBTAINED WITH CONVENTIONAL METHODS OF IN VITRO TISSUE CULTURE.

Description

The present invention relates to an improved process

  for the production of human insulin.

  The conventional methods known for the production of human insulin, such as chemical synthesis, in vitro tissue culture, or recombinant microorganism culture

  genetically, only result in very low yields

  ments, with a high cost. That is why we use

  cement of insulin of bovine or porcine origin.

  The present invention avoids these inconveniences.

  disclaimers of the prior art. It is based on the

  tation that can unexpectedly be obtained from

  human cells, capable of producing human insulin in an amount of 2 to 50 times greater, per cell, than that obtained during tissue culture in vitro, thanks to the multiplication of human cells, capable of

  to produce human insulin, produced by means of ani-

hot-blooded ailments.

  The novel process according to the invention of

  human insulin consists in multiplying the human cells, capable of carrying out such a production, by transplanting them into the body of a warm-blooded animal, or by letting them multiply in a device, in which they are brought the nutrient body fluid of

  a warm-blooded animal; the multiplied cells are

  subsequently exhibited, by one of the preceding modes of operation

  multiplication, to the action of an insulin inducer.

  The method according to the invention, apart from the fact that it allows a higher production of human insulin, does not require or very little nutrient medium containing expensive serum, for the multiplication of the cells; it facilitates, much more than in the case of in vitro tissue cultures, the conservation of the culture medium during cell multiplication. In particular, all

  human cells, capable of producing human insulin

  can be easily multiplied by means of the nutrient body fluid provided by a warm-blooded animal, either by transplanting these cells to the animal or by suspending these cells in a blood chamber.

  diffusion designed to receive the body fluid nutrient

  tif; the animal is fed in the usual way.This process

  is also characterized by a multiplication of

  lules more stable andYeselected, and by more

  abundant human insulin, per cell.

  Cells that are suitable in accordance with the

  vention are human insulin-producing cells, which multiply easily in the body of warm-blooded animals. We can notably mention human cells

  which by nature produce insulin, such as cell

  the intact P-islands of Langerhans of the human pancreas,

  those transformed by the EB virus or irradiation

  X-ray and insuloma cells from patients with insuloma; carcinoma cells from

  human lung, which produce human insulin ecto-

  pike; also established cell lines, cells above. Similarly, the use of bloodlines

  established human lymphoblastoids, easily conserved

  introduced with the dominant genes for the production of human insulin, using genetic recombination techniques using enzymes such as DNA ligase,

  nuclease and DNA polymerase, or by cell fusion

  agents such as polyethylene glycol or Sendai virus, conveniently results in remarkably superior cell multiplication, when their cells are transplanted into the body of warm-blooded animals and to the production, per cell, of human insulin, 2 to 10 times higher. In addition, as the transplantation of established human lymphoblastoid lines, mentioned above,

  to the body of the animal, leads to the formation of massive tumors

  sive, and that these massive tumors are scarcely contaminated

  by the cells of the animal host, and are easily disaggregated

  the human lymphoblastoid cells

  folded, alive, can be easily harvested.

  As animals, usable according to the

  all the animals in which the cells can multiply, and advantageously poultry such as chicken and pigeon and mammals such as dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster, mouse and nude mouse. As this cell transplantation causes an immunoreaction

  undesirable, it is desirable to use a new animal

  calf-born or young, or at the youngest stage possible,

  especially egg, embryo or fetus. In order to reduce the immunity

  tion, the animal can be treated before transplantation

  cell, by X-ray or Y-irradiation,

  from 200 to 600 rem, or receive an injection of

  tiserum or immunosuppressive agent, prepared according to

  ceded classic. Like the nude mouse, used as an animal

  warm-blooded, exhibits weaker immunoreaction,

  me in the adult state, it can be transplanted conveniently and

  let it multiply rapidly, without pre-treatment, always

  the established human cells.

  Stabilized cell multiplication and increased production of human insulin can be achieved by repeated transplants using a combination of different warm-blooded animals other than humans; for example, these goals can be achieved

  by first implantation of human cells into a hams-

  ter, where they multiply, then by reimplantation in the nude mouse. In addition, repeated transplantation can be performed with animals of the same class or division,

  as well as with those of the same species or genus.

  The place where human cells are implanted

  tables, can be any site of the animal where the cells multiply; for example the allantoic cavity, or the

  intravenous, intraperitoneal or subcutaneous routes.

  Apart from this direct transplantation of cells into the body of an animal, all established, classical human cells capable of producing 1 '

  insulin, can be multiplied thanks to the use of

  nutrient body fluid, provided by the body

  of an animal by inclusion, for example intraperitoneally

  tonic, in the body of the animal of a chamber of diffe-

  conventional fusion, of varied size and shape, provided with a porous filtering membrane, an ultra-filter, or hollow fiber whose pore size has a diameter of about 10a7 to 10-5 m, which pockets the contamination by penetration of host cells inside the

  diffusion chamber, and allows the delivery of body fluid

  Nutritive rel of the animal to the cells. In addition, since the diffusion chamber may have a suitable shape, it may be placed for example on the animal host, and let the body fluid of the body of the animal circulate to the chamber, allowing observation of the suspension of

  cells in the room by one or more windows la-

  transparent walls, provided in the wall of this chamber.

  as well as replacement by exchange with a chamber

  news; this way cell multiplication increases

  to an even higher level with respect to the life of the animal, and cell production per animal is increased without sacrificing the host. In addition, when

  such a diffusion chamber is used, such as cell

  multiplied humans can be harvested easily and no immunoreaction arises because of the lack of direct contact between human cells and

  those of the animal host, any warm-blooded animal may be

  be used as a host according to the invention, without

  previously to reduce immunoreaction.

  Feeding of the animal host implanted with human cells can be easily performed by a

  conventional process, even after transplantation of

  them, and does not require any particular care.

  The maximum multiplication of cells is reached after 1 to 20 weeks after transplantation. When established human cells, implanted in the animal, are human tumor cells, or human lymphoblastoid lines, maximal cell multiplication is reached in 1 to weeks after cell transplantation, due to

  their multiplication speeds are much higher.

  In accordance with the invention, the number of cells

  humans obtained per host ranges between 107 and 1012 or

  more. In other words, the number of human cells, trans-

  planted in the body of the animal, is multiplied by 102 to 107 or more, or is approximately equal to 10 to 106 times or more, that which is achieved by the in vitro tissue culture method, using a nutrient medium it well that these cells can be used for the production

of human insulin.

  With regard to the induction of insulin,

  can use any process in which the human cells

  obtained by the procedure mentioned above,

  release human insulin. For example, cell

  multiplied humans, obtained by multiplication in ascites, suspension and harvest, from this ascites, or by massive tumor extraction, formed under the skin

  and harvest after disintegration of said tumor, are half

  suspended in a concentration of the order of 1 to 108 cells / ml in a nutrient medium, maintained at a temperature in the region of 200 to 400C, and then subjected to this temperature by the action of an insulin inducer

  for 1 to 20 hours, to produce human insulin.

  Preferred insulin inducers are saccharides such as glucose, mannose, fructose, ribose and xylitol; of the

  amino acids such as arginine, lysine and leucine; hor-

  peptides such as glucagon and adrenocorticosteroid

  trope (ACTH); metallic cations like K and Ca ++.

  The human insulin thus obtained can be

  readily collected by purification and separation techniques using conventional procedures, such as salting, dialysis, filtration, centrifugation, concentration and lyophilization. When you want to have an even more purified human insulin preparation,

  it can be obtained by a combination of the techniques

  above, using conventional techniques such as adsorption and desorption with ion exchange, gel filtration, affinity chromatography, fractionation with

  isoelectric point and electrophoresis.

  The human insulin preparation, thus obtained,

  can be used alone or in combination with one or more

  agents, for injection, internal or external administration, or diagnostic, for the prevention and

treatment of human diseases.

  Human insulin is assayed in the culture medium by the enzyme immunoassay method described by K.Kato et al., J. Biochem. Flight. 78, pp. 235-237 (1975) and its quantity is expressed in International Units of

  Human insulin (IU), with 1 IU of human insulin being

  like the amount of insulin that decreases the level of

  rabbit blood at 64 mg / dl in 1 hour or at 45 mg / dl,

  2 hours after subcutaneous injection.

  The following examples are illustrative

  non-limitative embodiments of the method se-

the invention.

EXAMPLE 1

  Disaggregated cells of human insuloma, obtained

  extracted from an insuloma patient, and ha-

  chage, are implanted subcutaneously in mice

  adults, who are then fed in the usual way

  on, for 3 weeks. The massive, resulting tumors that form under the skin and weigh about 10 g each,

  are disaggregated by extraction, hashing and

  pension in a saline solution, physiological, containing collagenase. After washing with Earle's medium 199

  (pH 7.2), supplemented with 10% v / v foe serum

  bovine, the cells are resuspended,

  to obtain a concentration of about

  per ml of fresh preparation of the same medium containing mM of Dglucose as an insulin inducer; they are then incubated at 37 ° C for 4 hours, for

  give human insulin. Then the cells are

  ultrasound, and the amount of human insulin

  in the supernatant is dosed. The production

  is close to 1000 / IU / cell. The control cells, ob-

  by in vitro culture of human insuloma cells in Earle 199 medium (pH 7.2), supplemented with 10%

  volume / volume of fetal bovine serum, and incubation

  at 37 ° C, are treated as before with the inductive

  insulin. The production of human insulin is

than 200 / LUI / cell.

EXAMPLE 2

  Disaggregated cells of human insuloma, ob-

as

  in Example 1, and a lymphocyte cell line

  Blastoides, human leukemics, from Namalwa, are put

  together in suspension in a saline solution container

  containing 140 M NaCl, 54 mM KCl, 1 mM NaH2PO4

  and 2 mM CaCl 2, to give respective concentrations of

  are close to 103 cells / ml. The cell suspension, cooled with ice, is mixed with a preparation of

  the same saline solution containing Sendai virus pre-

  activated, transferred approximately 5 minutes after mixing in a 370C incubator, and stirred therein for 30 minutes

  approximately to achieve the fusion of the cells, introduced

  the ability to produce human insulin

  human insuloma cells in the lymphoblastoma line

  human leukemia. After cloning according to a conventional method of the hybridoma cell strain capable of producing human insulin, this is intraperitoneally implanted in adult nude mice, which

  are then fed habitually for 5

  Maines. The resulting massive tumors, 15 g each, are extracted and treated as in Example 1 to induce human insulin, but replacing 20 mM.

  of D-glucose per 30 mM L-arginine.-The production of

  human sulium is of the order of 3200.O-IU / cell.

  as the control experiment is carried out / in Example 1, by

  in vitro culture of the Namalwa line of lymphoblastoid fungi

  humans, and exposure of the multiplied cells to the insulin inducer. The production of human insulin does not

  is only about 100 O / IU / cell.

EXAMPLE 3

  After injection of antiserum, prepared from

  rabbits by a conventional method, to new hamsters

  born in order to reduce their possible possible immunoreaction

  transplantation of cells, these animals are implanted

  subcutaneously a line of human leukemic lymphoblastoid JBL, in which the insulin production ability of human insuloma cells, is

  introduced as shown in Example 2, and then fed

  laughs in the usual way for 3 weeks. Tumors

  resulting mass, formed under the skin and weighing approximately

  10 g each are extracted and treated as in

  Example 1 to produce human insulin. This pro-

  duction is of the order of 2300y-IU / cells.

  The control experiment is performed as in the example

  ple 1, by in vitro culture of lymphoblastotides

  fused human leukemia JBL, and the exposure of

  lules multiplied to an insulin inducer. The production

  human insulin is only of the order of 200 μl / cell.

EXAMPLE 4

  Newborn rats are implanted indiscriminately

  traveineuse, with a line of lymphoblastoid leukemia

  Namalwa human probes, in which the ability to produce insulin human insulin cells, is introduced as in Example 2, and then fed in the usual manner for 4 weeks. The resulting massive tumors, approximately 40 g each, are extracted and treated as in Example 1, to produce human insulin. This production is

the order of 2600 A & IU / cell.

  The control experiment is performed as in the example

  3. The production of human insulin is only 100%.

EXAMPLE 5

  Adult mice are irradiated with 400 rem of X-ray to reduce their immunoreaction, implanted subcutaneously with human insuloma cells, obtained as in Example 2, and fed

  usual for 3 weeks. Massive tumors result

  The aunts, formed under the skin and weighing 15 g each, are extracted and treated as in Example 1 to produce human insulin. The production of this insulin is

of the order of 1000 xLUI / cell.

  The control experiment is carried out as in Example 1, by in vitro culture of human insuloma cells, and

  exposure of the multiplied cells to the insulin inducer

  line. The production of human insulin is only 200 IU / cell.

EXAMPLE 6

  A line of human leukemic lymphoblastoid

  JBL hands, in which the ability to produce insul-

  no human, human insuloma cells is introduced

  as in Example 3, is suspended in

  physiological rumor, and everything is transferred to a room

  diffusion cell having an internal volume of the order of 10 ml, and whose filter membrane has a pore size of about 0.5 dia diameter; and this chamber is incorporated intraperitoneally into an adult rat. After 4 weeks during which the rat is fed in the usual manner, this chamber is removed. The density in human cells reached in the chamber by the above operation is of the order of 5 × 10 9 cells / ml, which is 103 times greater than that obtained during in vitro culture. way of a

  CO2 incubator * The cells thus obtained are treated

  as in Example 1 to induce human insulin. The production of the latter is close to

2500 / -UI / cell.

  The control experiment is performed as in the example

  3. The production of human insulin is only about

200, / LUI / cell.

EXAMPLE 7

  A line of human leukemic lymphoblastoid

  JBL hands, in which the ability to produce insu-

  human line of human insuloma cells is introduced

  as in Example 3, is implanted in the cavity

  total of embryonated eggs, previously incubated at 370C for 5 days. After incubation of these

  eggs at this temperature for an additional week

  the multiplied human cells are harvested. El-

  are then treated as in Example 1 to give human insulin. The production of this insulin is

of the order of 2,000 / LUI / cell.

  The control experiment is carried out as in Example 3. The production of human insulin is only of the order of

200 / LUI / cell.

Claims (9)

claims   A method for the production of human insulin by multiplication of human cells capable of producing the same, characterized in that, before exposing them to the action of an insulin inducer, said cells are subjected to in vivo of a nutrient body fluid of a warm-blooded animal.   2. Method according to claim 1, characterized in that said cells are transplanted directly   in the body of the warm-blooded animal.   3. Method according to claim 1, characterized in that said cells multiply in a device,   which brings them the body's nutritive fluid of the animal.   4. Method according to claim 3, characterized in   what the device is a broadcast chamber.   5. Method according to one of claims 1 to 4,   terized in that human cells capable of producing   insulin, are intact cells-A of the islands of Lan-   gerans of the human pancreas, those transformed by EBV or X-ray irradiation, insulin cells of human patients, human lung carcinoma cells, or cells of established cells above.   6. Method according to one of claims 1 to 4, characterized   rised in that the cells, capable of producing insulin   are hybridoma cells obtained by cell fusion.   of a human leukemia lymphoblastoid line   with one of the cells described in claim 5.   7. Method according to one of claims 1 to 6, characterized   in that the insulin inducer is a saccharide   me glucose, mannose, fructose, ribose, xylitol, an amino-a-   such as arginine, lysine, leucine; a peptide hormone   as glucagon, adrenocorticotropic hormone (ACTH); a metal cation such as K +, Ca + t or the mixture of several of them.   8. Method according to one of claims 1 to 7,   characterized in that the warm-blooded animal is a fowl such as chicken or pigeon, a mammal such as dog, cat, monkey, goat, pig, cow, horse, rabbit, guinea pig, rat, hamster, mouse or nude mouse. 9. Human insulin obtained by the process according to Claims 1 to 8.