CS208147B2 - Method of making the stabile low antigennous or the antigene of the plain insuline preparation of the protracted effect - Google Patents

Abstract

A stable insulin preparation having a lasting action and having low antigenicity is prepared by reaction of acidic groups of the insulin with basic groups of organic compounds. The reaction is carried out in an aqueous buffer solution which contains a protein-dissociating or protein-depolymerising agent. Basic polypeptides, e.g. protamines, or cleavage products of basic polypeptides are preferred as organic compounds containing a basic group. The protein-dissociating or protein-depolymerising agent is preferably urea.

Description

(54) A method for producing a stable, low-antigen or antigen-free insulin preparation having a protracted activity

The invention relates to a process for the production of a stable, low-antigen or antigen-free insulin preparation having a protracted activity, by reacting insulin with an amino-containing organic base.

In the treatment of diabetes mellitus, preparations derived from pork or bovine pancreas are usually used. Approximately 30% of the world's insulin consumption is based on pig insulin and approximately 70% on bovine insulin. Insulin from other animals, such as sheep insulin, has been suggested, but has not yet gained much commercial significance.

Insulin therapy has previously included numerous disadvantages that have been manifested, among others, by allergy and lipodystrophy.

It has long been known that conventional insulin therapy induces the production of insulin antibodies in most patients, which may lead to increased insulin requirements, where unknown amounts of insulin may be bound by the antibodies, and by continuing this binding, insulin becomes ineffective as a blood sugar regulator.

It has long been believed that the main cause of antibody formation and the consequent high insulin consumption is the presence of various impurities in normal commercial insulin.

As examples of such impurities, insulin dimer, proinsulin, intermediate insulin (the stage between proinsulin and insulin), arginine insulin, insulin ethyl ester, mono-desamidoinsulin, didesamidoinsulin may be mentioned.

The first three of these compounds are known to be highly antigenic. It is also known that the fourth or fifth or both of these compounds are highly antigenic, whereas the sixth and seventh compounds and the insulin molecule alone are weakly antigenic or non-antigenic; see Schlichtcrull, Monocomponent Insulin and its Clinical Implications, 16th III. 1973.

It is known to remove said impurities by gel filtration and / or ion exchange, whereby it is possible to obtain a highly purified insulin comprising essentially a single component. In recent years, insulin preparations have been marketed which have been deprived of some of these impurities to some degree, and in many cases, diabetic patients have seen reduced antibody production.

It has been found that fast-acting preparations of both porcine and bovine insulin can be produced in such a purity that they do not induce or induce to a certain degree only an antibody formation; see Schlichtcrull, Monocomponent Insulin and its Clinical Implications, 16th III. 1973.

And the possibility of producing prolonged efficacy formulations with substantially reduced antigenicity has been tested if production is based on highly purified porcine insulin, see T. Deckert et al., Diabetologia, Vol. 10, pp. 703-708, 1974.

However, it has also been found that, despite the fact that bovine insulin is produced in such purity that it has to be considered as pure as highly purified pork insulin by today's analytical methods, which today can be so pure that, as mentioned above, it does not or only to a low degree when used in fast-acting preparations, low antigenicity and protracted formulations based on highly purified bovine insulin have not yet been on the market.

This is a serious drawback as beef insulin preparations are preferred internationally.

Antibody formation in a living organism against a chemical compound such as insulin can be caused by such chemical differences in the primary structure; see E. R. Arquilla et al., Immunochemistry of Insulin, Handbook of Physiology, Ch. 9, p. 160, 1972, wherein the chemical compound has a structure which is incompatible with and repelled by the organism; E. R. Arquilla et al .: Immunology Conformation and Biological Activity of Insulin, Diabetes, vol. 18, p. 194, 1969, or that the chemical compound has an aggregate that is incompatible with and repelled by the organism; E. R. Arquille et al., Immunochemistry of Insulin, Handbook of Physiology, Ch. 9, p. 60, 1972.

It is also possible to induce antibody production by co-developing a chemical compound and a component capable of activating the immune mechanism.

Numerous assays tend to indicate that the physical state of the insulin molecule plays an important role in antibody production. / Kumar se sp.: Horn. Metab. Res. 6. (1974) 185-177 and Piers et al., Neth. J. Med. 17 (1974) 234-238].

The reason why the fast-acting highly purified insulin preparations do not induce antibody formation or only to some degree, based on the fact that insulin in thee HTO cases, except that they are highly purified, are present in monomeric form or ^J designation says only loose aggregates, whereas in previously manufactured and marketed highly purified insulin preparations, insulin appears to be in aggregated form and with properties that result in antibody formation.

This problem is greatly emphasized with respect to bovine insulin, which is probably due to the fact that bovine insulin has a greater tendency to aggregate than pig insulin.

bovine insulin without additives is known to have a wider isoelectric clotting zone or interval than porcine insulin. It is also known that this broad isoelectric clotting zone may be narrowed to the same range as is known for porcine insulin by the addition of certain compounds such as phenol or m-cresol; Briton. Fri. file No. 1 222 100.

Said wider isoelectric clotting zone of bovine insulin is due to the patina being caused by the fact that bovine insulin forms aggregations at pH values close to neutral.

The invention is based on the discovery that it is possible to stabilize highly purified insulin to the extent that it does not form aggregates that can induce antibody formation during manufacture or storage of sustained-release preparations or in use.

This object has been achieved by the invention, which is characterized in that the reaction is carried out with a highly purified insulin in a stabio-mono-mono or released aggregate form.

According to the process of the invention, highly purified insulin is reacted in the form of a stabilized monomer or as a released aggregate with an organic compound containing basic groups such as aninostopines or substituted amino stopins.

A preferred compound of the basic moiety is a polypeptide, for example polyarginine, ssmiaosal, trstaein or globin, or sell cleavage of such a basic polypeptide. JIRY příkldeem suitable basic compound is 1,3-bis / 4-Amii ^metih-2-y l-6-chinslylmoisviíl.

According to the invention, the reaction is conveniently carried out in the presence of urea as a stabilizer. Examples of other additional stabilizers are lower alpha-canols, for example, ethers, and ethanol, di-chloroformates, for example dimethylformamides, K, N-alkylacetamides, for example N-ethyl acetamide and 1Ciocirrile.

These stabilizers have an antisense or depolymerization effect on proteins and are therefore effective in stabilizing insulin in eonommerce or in freely aggregated form.

The invention may also be used in conjunction with insulin from various animal species such as pigs, cattle or sheep, but is particularly important in conjunction with bovine insulin.

A preferred embodiment of the invention is characterized in that the insulin solution containing the stabilizer is subjected to ion exchange by pumping with a wire containing a stabilizer containing insulin in the monomer. form or released aggregate form, after which the impurity-free insulin fraction is added to a solution containing the basic polypeptide or the base polypeptide cleavage product and the precipitated insulin complex is isolated.

This method combines effective protection against aggregation by isolating the highly purified insulin first as a result of the subsequent risk of aggregation. Furthermore, the method is simple in that the protracted insulin preparation precipitates directly in a stable form.

The invention will be explained in detail by the following examples.

PIÍIlI 1

250 mg of recrystallized bovine insulin was dissolved in 5.2 µl of a buffer solution containing 7 M (molar) deionized eivivine and 0.02 M tris (eldroxy-ethyl) iminomethane, pH 8.1.

The solution was mixed with 5.2 ml of 7M molar. The pH of the mixture was adjusted to 8.1. A 5 cm diameter column was filled with a 2.1 cm DEAE cellulose layer and supplemented with a buffer solution of the above composition. The insulin solution was loaded onto the column and eluted at 75 ml / h according to the following scheme:

2.5 hours with the buffer of the above composition, hours with the buffer of the above composition to which 0.0045 mol of sodium chloride in lithium was added, hours with the buffer of the above composition to which 0.011 mol of sodium chloride in liter was added.

The eluate was separated into fractions. The highly purified fractions were collected and assayed for insulin content. Protamine was dissolved in the amount necessary to achieve an isophenic ratio of highly purified insulin to protamine in a 7 M molar solution of the same volume as the highly purified insulin fraction.

The insulin solution was added slowly and dropwise with stirring to the protamine solution and, if necessary diluted to 1 M molar concentration, the insulin complex precipitated in an amoophic form.

The precipitate was isolated by centrifugation and yielded virtually only one band when 300 µg was subjected to isoelectric focusing in a polyacrylamide gel and using 2% Anmhoin (pH 3-10) in 6 M deionized molar.

This precipitate can be used to prepare injectable insulin preparations by suspending in a known carrier medium.

Example 2

The stabilized buffer solution was prepared by mixing two solutions I and II in a ratio adjusting the pH to 6.0.

Solution I contains the mono-deionization of the molar and 0.13 ml of sodium dihydrogen phosphate.

Solution II contains the mono-monomeric molar and 0.13 molar dinatrimhydrogeenoethyl.

400 mg of recrystallized bovine insulin was dissolved in 3 ml of this buffer and the pH was then adjusted to 6.0 with a small amount of solution II and then the mixture obtained was then filtered.

The 2.5 cm diameter column was packed with a 27 cm layer of anmereit CG-50 type II resin equilibrated with a puff buffer of the above composition. The insulin solution was loaded onto a column, which was eluted at a rate of 40 ml of the solution of the above composition per hour.

The eluate was separated into a fraction. The purified insulin fractions were collected and determined for insulin content. The protamine was dissolved in an amount necessary to obtain an isophene ratio of purified insulin to protamine in a solution of the same volume as the mixture of purified fractions.

An aliquot of the insulin solution was added to the protemine solution with stirring, and upon dilution, if necessary, to the mono-mono-mono-terminal end, the protamine-insulin complex precipitated in the amorphous state.

The precipitate was isolated by centrifugation and had the same purity as the product described in Example 1.

. The precipitates can be used to prepare injectable insulin preparations by suspending them in known carriers.

Example 3

The procedure of Example 1 or 2 was repeated and zinc chloride was added to the protamine solution at a concentration of 0.5% zinc ions based on the amount of insulin and 0.3% m-cresol based on the volume of the mixes.

The pH of the smmsi was maintained in the range of 6 to 9, with the result that the new complex precipitated in the crystalline state. *

The precipitate was isolated by centrifugation and contained a single band when 300 µg was subjected to pulsacrylate gel iontophoresis using 2% Ammpoline (pH 3 to 10) in 6 M deionized monolysis.

The precipitates may be used for the preparation of injectable Ιο ^ Ι-πο ^^ Ο. by suspending them in known carriers.

Example 4

The procedure of Example 1 or 2 was repeated, but bovine insulin purified by gel filtration on Sephadex G-50 was used as the starting material.

The product was of the same purity as the product described in Example 1.

Example 5

Example 1 or 2 was repeated using pig insulin instead of bovine insulin.

The derived porcine insulin-propylmilea complex was as pure as the complex prepared in Example 1.

Example 6

Example 1 or 2 was repeated using porcine insulin as a starting material prepared by salting out the crude aqueous extract produced by adding NaCl to a concentration of 3.5 M solution at pH 8.5.

The salt cake obtained was desalted in the usual manner before ion exchange. The product was of the same purity as the product described in Example 1.

Pří] kaa7

The procedure of Example 6 was repeated and the obtained salt cake was gelled on Sephadex G-50.

Sell had the same purity as the product described in Example 1.

P Pikl a d 8

The procedure of Examples 1 or 2 was repeated, replacing the protamine with poly-L-arginine (degree of polymerization 296).

When 300 µg of the precipitate was subjected to polyacrylamide gel iontophoresis using 2% Ampholine (pH - 3 to 10) in 6 M deionized urea, said precipitate showed essentially a single band.

Example 9

The procedure of Examples 1 or 2 was repeated, but to the combined insulin fractions was added a 0.2% aqueous solution of bis- (4-amino-2-methyl-6-quinolyl) urea hydrochloride in an amount equivalent to 10% by weight, the amount of insulin present in the fractions.

The insulin complex was then precipitated after a possible dilution of the urea to a concentration of 1 M with 0.025 M sodium phosphate buffer, which was allowed to stand for a period of time and then isolated by centrifugation.

The product was of the same purity as the product described in Example 1.

Example 10

The procedure of Example 9 was repeated using recrystallized bovine insulin, purified by gel filtration on Sephadex G-50 as the starting material. The product was of the same purity as the product described in Example 1.

Example 11

The procedures of Example 9 were repeated using pig insulin instead of bovine insulin.

The derived porcine insulin complex was as pure as the complex prepared in Example 1.

Example 12

The procedure of Example 9 was repeated using porcine insulin as a starting material prepared by salting out the aqueous crude extract obtained in the manufacture of insulin by adding NaCl to obtain a 3.5 M solution at pH 8.5. The salt cake obtained was desalted in the usual manner before ion exchange.

The product was of the same purity as the product described in Example 1.

Example 13

The procedure of Example 12 was repeated and the obtained salt cake was subjected to gel filtration on Sephadex G-50.

The product was of the same purity as the product described in Example 1.

Example 14

The 2.6 x 80 cm column was packed with DEAE-Sephadex, previously swollen and equilibrated in a buffer of 0.15 M ammonium hydroxide in 7 M dimethylformamide adjusted to pH 9.0 with 5 N hydrochloric acid. · 300 mg. bovine insulin Cross-linked to gel filtration on Sephadex G-50 was dissolved in 40 ml of the above buffer and loaded onto the column, eluting for 2 hours with the above buffer at a rate of 200 ml per hour.

Elution was then continued at the same rate on a 1% gradient, the gradient being formed from 2700 ml of the above buffer and 2700 ml of the 0.25 M ammonium hydroxide buffer in 7. M dimethylformamide, pH 9, adjusted with hydrochloric acid.

The eluate was separated into fractions. The highly purified fractions were pooled and the insulin content was evaluated. The pH was adjusted to 7.9 with 5 N hydrochloric acid and m-taresol was added until the solution contained 0.2% of said m-thesol.

When the total zinc content of the solution was adjusted to 0.5% insulin content, the protamine sulphate solution needed to obtain the isethane ratio as a 1% aqueous solution was added and the mixture was mixed thoroughly.

After standing for 15 at 20 ° C, 6 volumes of 1/75 sodium phosphate buffer, pH = 7.3, containing 0.2% m-toesol, were added and the reaction mixture was allowed to stand after stirring briefly. at 20 ° C for 16 hours.

In this way, the amorphous precipitated complex of prsnarin-iozuli crystallized.

By replacing the above liquid with a medium of a known carrier, the precipitate was converted into an injectable insulin preparation.

When 300 yug of precipitate was subjected? eleknřsfsřéze v. polyacrylnide gel using 2% AmrPoSiou (pH - 3 to 10) in 6 M deionized molar, said precipitate showed essentially only a single band. ’

Example 15

The column, 2.6 cm in diameter and 4.4 cm in height, was packed with QE-Sephadex, pre-swollen and equilibrated in a buffer of 0.1 M ngSs- (hydooyme-thylarmOnomenhan in 60 vol% ethanol, adjusted to pH = 7.35 with 5 N hydrochloric acid.

250 mg of bovine insulin purified by gel filtration on Sephadex G-50 was dissolved in 10 ml of 0.1 M tris buffer in 60% ethanol adjusted to pH = 8.5 with hydrochloric acid and after filtration the solution was applied to the columns.

Then the column was eluted with equilibration buffer at a rate of 10 ml per hour. The eluate was separated into fractions, the highly purified fractions were pooled and the insulin content determined. Then m-cesol was added until the solution contained 0.2% of said m-cresol, after which the total zinc content was adjusted to 0.5% by weight, insulin content.

Then, the amount of protamine sulfate needed to obtain the isphosphate ratio as a 1% solution was added, and the mixture was thoroughly mixed. After standing for 15 minutes. 9 volumes of 1/75 sodium phosphate buffer, pH = 7.3 containing 0.2% m-cresol were added at 20 ° C, and after brief stirring the reaction mixture was allowed to stand at 20 ° C. for 16 hours.

Thereby, the amorphous precipitated protamine-insulin complex crystallized.

By replacing the above liquid with a medium of a known carrier, the precipitate was converted into an injectable insulin preparation.

When 300 µg of the precipitate was subjected to polyacryammonium gel electrophoresis using 2% Ampholine (pH = 3-10) in 6 M deionized urea, said precipitate showed only one band. .

Example 16

Cm ^ diC ^ tepLH box at 5 DEG C. ^b YLA ^P flax Olona ^to 2.6 cm diameter and ¹¹ cm height ^Y SehOαdexep QAE-A-25 that was previously in swelling and ebrillboován pafru obsahouícím 0.5M tris (hydroxymethylaminomethyl) in 8 M acetamide adjusted to pH = 8.0 with glacial acetic acid.

300 mg of bovine insulin, which was purified by gel filtration on Se ^p hadeex G-50, was dissolved in 30 ml of the above buffer and loaded onto the column, eluting for 1 hour at a rate of 80 ml per hour.

The elution was continued at the same linear gradient, the gradient being prepared from 2000 ml of the above buffer and 2000 ml of the same buffer containing 0.15 M sodium chloride.

The u-uét was divided into factions. The highly purified fractions were pooled and the insulin content determined. After addition of m-cresol to a content of 0.2% by weight, the total zinc content was adjusted to 0.5% Oppo, with a 0.15 liter zinc chloride solution, and then the amount of sulphate required to obtain the isoholone was added. in a 1% aqueous solution.

The mixture was mixed thoroughly and after standing for 15 minutes, seven volumes of 1/75 M sodium phosphate buffer, pH 7.3, added 0.2% H 2 O were added. m-cresol, after which the reaction mixture was allowed to stand for an additional 16 hours at 20 ° C.

The ultrafined insulin-insulin complex was then recovered.

By replacing the above liquid with a known carrier medium, the precipitate was converted into an injectable insulin preparation.

When 300 µg of the precipitate was subjected to holyacrylpmil gel electrophoresis using 2%. Amm @ 2 Hnu (pH = 3-10) in 6 M deionized molar, said precipitate showed only a single band.

Example 17

In a freezer at 5 ° C, a 2.6 cm diameter and 12 cm high column was packed with QAS-Sehexadex A-25, which was previously swelled and equilibrated in a buffer consisting of 0.05 M tungsten (hydropolymeric maleate). in 7M acetone, adjusted to pH = 8.2 with 5 N hydrochloric acid.

300 mg of bovine insulin, which was purified by gel filtration on Sephadex G-50, was dissolved in 30 µl of the above buffer, adjusted to pH 9.3 with 2 N sodium hydroxide and then with 8.2 2 N hydrochloric acid and The column was run on the column for one hour and eluted with the above buffer at a rate of 75 ml per hour.

Elution was then continued at the same rate with a linear gradient, producing a gradient of 1800 mL of the above pxUfru and 1800 mL of the same buffer containing an additional 0.15 M sodium chloride.

The eluate was separated into fractions. The highly purified fractions were pooled and the insulin content determined. After addition to a content of 0.2% by weight, the total zinc content of the solution was adjusted to 0.5 percent by weight. The insulin content of 0.15 M insulin-zinc chloride was then added and then added. The amount of protamine sulphate required to obtain the isopropin ratio in the form of a 1% aqueous solution.

The mixture was thoroughly mixed and after standing for 15 minutes was added 6 volumes of 1/75 pxrfru frsfoteδilnu solution, pH = 7.3, 0.2% obsaahjícího hmoo, ml ^] es ⁿ (^ ru, then the reaction mixture allowed to stand for a further 16 hours at 20 ° C.

Thus, the amorphous precipitated prrtamine-iizuliii complex crystallized.

By replacing the above liquid with a medium of a known carrier, the product was converted into an injectable insulin preparation.

When the precipitate was 300 electrophoresed in pol ^ j ^ aki ylmiio ^ ^{(^,} ^ (m-gel using 2% NNU Amm ^ (pH = 3-10) in deionized 6 M mooooině, said precipitate showed only a single band,

Example 18

Example 16 was repeated, but the buffer now contained 0.05 M trSs (hhidoox; mt uyl) aminomethane in 5 M aqueous N-methylacetamide adjusted to pH = 8.2 with 5 N hydrochloric acid, and only 4 volumes of phosphate buffer were added to the inzuino solution. during crystallization.

When 300 µg of the precipitate was subjected to polyacrylamide gel electrolysis using 2% Amphurine (pH = 3 to 10) in 6 M deoxyphenol, said precipitate showed only a single band.

Example 19

The procedure of Example 17 was repeated, combining the highly purified III fractions. were run on a column packed with SepUldex G-25, which was swelled in a medium containing 1/60 M in / triphosphate doreenorphate in 1 M acetamide at a pH of 7.65, so that said volume did not exceed 50 ml. 20% of the total column volume.

The column was then ace of the above medium at a rate of 20 ml per hour per cm and the insulin-recovering fractions were pooled. After determination of the insulin content, the total zinc content of the solution was adjusted to 0.5 percent by weight of the insulin content of a 0.15 M zinc chloride solution in 1 M acetamide, and a 1/4 volume of 1/100 M hydrochloric acid containing 1.5% was added. mass, m-cresol and multiply! protamine sulphate, necessary to obtain the isophan ratio.

^p o ^p o ^d stall OBU ¹⁶ din ^ ^p s at 20 ° C by precipitation of the complex protunin ^insulin-y stllrolll Abbr. The product thus obtained was of the same purity as the product described in Example 17.

By replacing the aforementioned copal with a medium of a known carrier, the precipitates can be slaughtered for the preparation of injectable insulin preparations.

Immunological tests

In order to illustrate the altered imwiogenic properties of the protamine-insulin complex isolated according to the invention, comparative tests were performed on rabbits, animals commonly used to study the antigenic properties of insulin and insulin-like substances.

The following preparations were used in the comparative tests:

1. Conventional NPH bovine insulin prepared in conventional manner from recrystallized bovine insulin containing the impurities listed in the application.

2. A purified NPH bovine insulin prepared in the conventional manner from purified crystalline bovine insulin but free of impurities reported in the application by column chromatography.

3. Purified NPH bovine insulin prepared according to the invention as described in Example 3, new NPH bovine insulin.

Subcutaneous injections were administered every 10 days in the form of a constant dose of 20 l U. of the insulin preparation to be tested.

It was not possible to detect any essential antibody after injection of conventional NPH bovine insulin without adjuvant, and for comparison it was necessary to adopt the immunization procedure usually used in antibody preparation, ie injectable insulin preparations containing Dreund's adjuvant emulsified in the first injection and Freund's emulsified in subsequent injections. did not take tni adjuvans. Injection times are indicated by the arrows in the attached Figures 1, 2 and 3.

The production of insulin antibody was monitored at 20-day intervals using the method described by Ortved-Andersen sp. (Acta Endoor. / Copenhagen / 69, 195, already 2018, 1972).

The results obtained are shown in Figures 1, 2 and 3, where the demonstration points of insulin binding capacity in vU / ml. (10 meet insulin units per ml.) High binding ability suggests high antibodies.

The results show that antibody formation to conventional NPH bovine insulin and reduced antibody production to purified NPH bovine insulin could be demonstrated, while it was virtually impossible to show any antibody production to the new NPH bovine insulin prepared according to the invention.

It should be noted that preparations 2 and 3 were purified by sterile chromatographic purification, one difference being that preparation 2 was prepared first by isolating insulin and then reacting it in a known manner with lrotaminep, while preparation 3 was prepared by reacting with protamine in the eluate containing urea without prior isolation of insulin.

The second series of tests was carried out using a milder immunization procedure, which omitted the initial stimulation of the reaction of the immunological system with the Freum's commlet adjuvant containing killed bacteria, i.e. the injectable preparations were in all cases emulsified in Freund's noncopper adjuvant.

The rabbits were injected every 10 days with a constant dose of 20 I.U. (see arrows in Figures 4-6).

Insulin antibody production was monitored at 10 day intervals using a new developed method based on the slaughtering of polyethylene glycoprotein (PEG) gland-ainsin insulin commpexes, using radiolabeled insulin, ¹²⁵ J insulin as a tracer:

PEG evaluation method of antibody, ¹⁰⁰ μΐ králičtí of sera ^A, I, ⁰⁰ μ ^{1 '25} J-insulin (a ^and ^ 2 U / ml) I ⁰⁰ μ! insulin solution (250 μυ / ml) and 700 μΐ phosphate buffer, 0.04 M, pH 7.4 and 0.15 M NaCl and 0.5% human albumin were incubated for 48 hours at 4 ° C. .

Was added 500 μΐ PEG 6000, 360 g / 1 mixed into the blender and the sample stand byl.ponechán ^p o ^{d b} on one of the ^{d h} in ^y when t EPTO ²⁰ ° C. ^p o spin-alpha ^p o ^d OBU 3 for 10 minutes at 00 ° Ho ^"1 was discarded and the supernatant removed and the occiput determined nooství ¹²¹ J ^{e s e} Nina rye.

In each assay, a sample containing an excess of anti-bovine anti-bovine insulin too much bound and another rabbit serum sample from unindicated rabbits unbound ”is included as standard.

The results, expressed as measured binding, as a percentage of the binding (% B) were calculated as follows:

TT _of% B = T -T ----

M ¹ o

100 where

T = the number of imjuls in the К К Кх vz vzok vzokokokokokokokokokokokokokokokokokokok

Τθ = number of pulses at tAT

Ty = number of pulses of mf-acimally bound substance ”.

The results obtained are shown in Figures 4, 5 and 6.

From the results it is evident that it was not possible to detect any significant antibody production to the new NPH bovine insulin prepared according to the invention (Fig. 6), while other insulins significantly increased antibody production (Figs. 4 and 5).

Because the test values are set to · standard, some of the values appear below the abscissa as a result of the measurement accuracy.

Claims (2)

OBJECT OF THE INVENTION CLAIMS 1. A process for the production of a stable insulinotropic or aototin-free insulin preparation having a protracted activity by reacting insulin with an organic base comprising aeinostajpioy, characterized in that a solution of insulin, preferably bovine insulin, free of amtiger impurities, in aqueous buffer at pH 9 and which comprises a dissociating or depollecting protein stabilizer, such as monoovine, C 1 -C 4 -alkMool, in particular ethano or methaool, di-C 1 -C 1 -alkyl acetamide or aceto-oxil, keeping insulin in dissolved mono- or freely aggregated form, preferably urea. aS in a seven-molar concentration, is reacted with an organic base containing an amino group, preferably a polypeptide such as protamine, poly-L-argioin, soeetostat: Lnee or globin, or 1,3-bis- (4-aior-2-ethyl-6-). quinolyl) urea, with the amount of insulin and the organic base approaching the isophaone ratio, while stabilizing the insulin the preparation isolates. 2. A method according to claim 1, wherein a solution of insulin in an aqueous buffer containing a stabilizer, preferably a mono-α in a seven-mol concentration, is subjected to ion exchange chromium ^and aii on milder azoic or cationic, followed by a substantially deionized fraction. impurities, added to the solution of an inorganic base with amino groups.