NO146698B - PROCEDURE FOR MANUFACTURING A STABLE LONG-EFFECTIVE INSULIN PRODUCT - Google Patents

Description

This invention relates to a method for manufacturing

of a stable long-acting insulin product with low antigenicity by reacting highly purified insulin with an organic base containing amino groups, preferably the polypeptide protamine.

For the treatment of diabetes mellitus, it is common to use insulin products that have been extracted from the pancreas of pigs or bulls. Thus, approx. 30% of the world's insulin consumption is based on pig insulin and approx. 70% on beef insulin.

Insulin from other animal species has been suggested, e.g. sheep insulin,

but has so far not achieved great commercial importance.

The insulin therapy carried out so far has resulted in quite a lot

disadvantages such as has been shown in allergy and lipodystrophy.

It has long been known that the usual insulin treatment

for most patients has led to the formation of antibodies against insulin, which in some cases has led to an increased need for insulin, as unknown amounts of insulin can bind to the antibodies and will be ineffective for regulating blood sugar as long as this binding lasts .

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

Examples of such impurities are dimerized insulin, pro-

insulin, intermediate insulin (the stage between proinsulin and insulin), arginine insulin, ethyl ester insulin and mono-desamido-insulin and di-desamido-insulin. Of these, it is known that the first three are strongly antigenic, and furthermore it is known that the fourth or the fifth or both are strongly antigenic, while the sixth and the seventh compound as well as the insulin molecule itself are not or only to a small extent antigenic . Cf.: Schlichtkrull, Monocomponent Insulin and its Clinical Implications, 16 March 1973, as well as British patent 1 285 023, corresponding to German official document 1 940 130.

It is known to remove the aforementioned impurities by gel filtration and/or ion exchange, whereby highly purified insulin can be obtained which essentially only contains a single component. Thus it was already successful in or before 1960 for R. David Cole,

see "The Chromatography of Insulin in Urea-containing Buffer",

J. Biol. Chem. 235 (1960), pp. 2294-2299, to isolate highly purified insulin products by ion exchange chromatography using

of urea-containing buffer solutions as eluents.

Also J. Arthur Mirsky and Kazunori Kawamura, see “Heterogeneity

of Crystalline Insulin", Endocrinology 78, (1966), pp. 1115-1119, investigated the composition of insulin from many different animal species, including human insulin, and isolated highly purified insulin fractions by gel filtration and separation by polyacrylamide electrophoresis (DISC PAGE).

British patent no. 881 855 relates to a general method for purifying protein substances by a chromatographic process, whereby an alcohol-containing solution, such as 70% ethanol in an aqueous buffer, is preferably used as eluent. Depending on the conditions during the chromatography, this process can be used for the extraction of insulin in high yield or for the purification of insulin, since both cation exchange resins and anion exchange resins have been proposed as ion exchange resins.

In recent years, insulin products such as

is freed from more or fewer of the aforementioned impurities and which has also in many cases led to a reduced antibody formation in diabetics.

It has been shown that fast-acting products of both pig and beef insulin can be produced in such a pure state that they do not or only to a very small extent cause the formation of insulin antibodies. Cf. Schlichtkrull, Monocomponent Insulin and its Clinical Implications, 16 March k973. Furthermore, it has been shown that it is possible to produce long-acting products with significantly reduced antigenicity, when these products are formed on the basis of highly purified pig insulin, cf. T. Deckert et al. Diabetologia volume 10. pp. 703-8, 1974. On the other hand, it has been shown that even if bovine insulin is produced of such a high purity that with current analytical methods it must be considered as pure as the highly purified pig insulin that can be produced today, and as pure that, as mentioned above, it does not or only to a small extent causes the formation of antibodies when used in fast-acting products, long-acting low-antigenic products based on highly purified bovine insulin have not yet been marketed. This is a serious disadvantage, as long-acting products based on bovine insulin are the most widely used internationally.

Formation of antibodies in the living organism against a chemical compound, e.g. insulin, may be due to chemical primary structural differences, see Arquilla E.R. et al: Immunochemistry of Insulin, Handbook of Physiology, ch. 9,

p. 160, 1972, that the chemical compound has such a steric structure that it appears foreign and undesirable to the organism, see Arquilla E. R. et al: Immunology Conformation and Biological Activity of Insulin, Diabetes, vol. 18, p. 194, 1969, or that the chemical compound has such a total size in suspension (aggregate formation) that it appears foreign and undesirable to the organism, see Arquilla E.R. et al: Immunochemistry of Insulin, Handbook of Physiology, ch. 9, p. 160, 1972.

Moreover, it is possible to produce antibody formation if, together with the chemical compound, a component is administered which can activate the immune apparatus, such as the so-called adjuvant substances, e.g. "Freund's complete adjuvant" and "Freund's incomplete adjuvant".

Several studies indicate that the physical condition form

of insulin plays a significant role in antibody formation.

(Kumar et al: Horn. Metab. Res. 6 (1974) 175-177 and P.A. Piers et al: Neth J. Med. 17 (1974) pp. 234-238.

That the fast-acting, highly purified insulin products do not

or only to a small extent causes the formation of antibodies is probably due to the fact that in these cases, in addition to being highly purified, the insulins are also present in monomeric form or possibly only form loose aggregates, while in the marketed long-term-acting highly purified insulin products that have been manufactured to date, it is possible to get the insulin to appear in an aggregate-formed form and with such properties that it causes the formation of antibodies. This problem is particularly pronounced for bovine insulin, which is probably due to the fact that bovine insulin is more prone to form aggregates than porcine insulin is.

It is known that bovine insulin without additives has a wider isoelectric folding zone than is the case for pig insulin. It is further known that this wide isoelectric folding zone can be narrowed to what is known for pig insulin, by the addition of certain substances, such as e.g. phenol or m-cresol, see Norwegian patent no. 122 090. It must be assumed that this wider isoelectric folding zone for bovine insulin is due to the fact that bovine insulin aggregates at pH values close to the neutral point. The use of phenolic compounds, such as cresols, for the production of insulin products containing proteins is further known from Swedish patent 111 488 and US patent 3 509 120. However, these products are not antigen-free.

The invention is based on the realization that it is possible to stabilize highly purified insulin so that neither during production nor in long-acting products during storage or use does it form aggregates that cause anti-substance formation. Although highly purified insulin has previously been produced, it has not previously been noticed that the products produced from it form aggregates or polymers that can cause antigenicity. The method according to the invention is characterized by the fact that the reaction between the highly purified insulin and the organic base containing amino groups is carried out in an aqueous buffer solution, containing a stabilizer, which during the reaction maintains the insulin in dissolved and stabilized monomeric or loosely aggregated form, after which the formed insulin product is isolated.

Examples of suitable organic bases which can be used in the method according to the invention are those which are usually used to obtain long-acting insulin products, such as polypeptides, e.g. polyarginine, somatostatin, protamine or cleavage products thereof or globin. An example of a useful base that is not a polypeptide is surfene, i.e. 1,3-bis-(4-amino-2-methyl-6-quinolyl)-urea. The preferred base is protamine.

Stabilizers which can maintain insulin (and other proteins) in stabilized monomeric or loosely aggregated form are well-known from the literature, cf. e.g. the above DE-OS 1 940 130; Cole, J. Biol. Chem. 235 (1960) and Elbaum et al., Biochemistry, vol. 13, No. 6, 1974, pages 1268-1278. Such protein-dissociating stabilizers have particularly been used in the chromatographic purification of proteins to counteract the formation of hydrogen bonds and polymerization of the protein molecules. In those cases, cf. e.g. DE-OS 1 940 130, where the stabilizers have been used in connection with the purification of insulin, they have always been removed during the isolation of the purified insulin, and before this is possibly converted into long-acting products.

According to the invention, the conversion is suitably carried out in the presence of urea as a stabilizer. Examples of other suitable stabilizers are lower alkanols, such as methanol and ethanol, di-alkylformamide, e.g. dimethylformamide, acetamide, N-alkylacetamides such as N-methylacetamide, and acetonitrile. As mentioned, these stabilizers have a protein-dissociating or protein-depolymerizing effect and are therefore effective in stabilizing the insulin in monomeric or loosely aggregated form. By loosely aggregated form is meant a condition where the insulin can be reversibly converted into monomeric form, and the equilibrium is shifted towards monomeric form in the presence of the protein-dissociating or protein-depolymerizing stabilizer.

The invention can be used in connection with insulin from many different animal species, such as pigs, oxen or sheep, but the invention is particularly important in connection with bovine insulin.

In all cases, all antigenic impurities are removed to the greatest extent possible or desirable, before the reaction with the protamine is carried out. This purification can take place in a manner known per se, such as by gel filtration and/or ion exchange chromatography. As mentioned, the thus highly purified insulin is maintained in stabilized monomeric or loosely aggregated form during the reaction. This avoids the polymerization or aggregate formation that could otherwise occur in the absence of the stabilizer, such as in the case of intermediate isolation and re-dissolution of the highly purified insulin. ■After the reaction with protamine, the insulin is fixed in a stable monomeric form and will not be able to form antigenic polymers or aggregates, even if the stabilizer is removed and the insulin-protamine complex is suspended in a normal carrier medium for injection.

A preferred embodiment of the invention is characterized in that an insulin-containing fraction, which has been obtained in a known manner by chromatographic purification of insulin using a buffered aqueous eluent containing a stabilizer, which maintains the insulin in stabilized monomer or loosely aggregated form, is reacted with a solution of the organic base containing amino groups, after which the formed insulin product which is an insulin-organic amine complex is precipitated and isolated. Thereby, a high degree of safety against aggregate formation is achieved, as the highly purified insulin is not first isolated as such with the resulting risk of aggregate formation. Furthermore, this embodiment is very simple, as the long-acting insulin product is directly precipitated in a stable form

According to another suitable embodiment of the invention, the reaction between highly purified insulin and the organic base containing amino groups is carried out in an aqueous buffer solution containing dissolved urea, after which the insulin product formed is precipitated by diluting the reaction mixture. A suitable stabilization can be achieved by using a buffer, which is approx. 7 molar with respect to urea.

After the elimination of the insulin product, e.g. an insulin-protamine complex, this can be suspended in a common carrier medium to obtain an injectable product, with long-term action.

The insulin-protamine product can also be isolated or recovered

in another way. If a protein-dissociating or protein-depolymerizing solvent is used as a stabilizer, this can be replaced with the desired carrier medium by decantation, filtration or centrifugation and resuspension of the product in the medium. If the stabilizer is a volatile solvent, such as methanol or ethanol, this can be removed by vacuum evaporation before or after addition of the carrier medium.

The invention is illustrated in more detail by means of the following examples.

Example 1

250 mg of recrystallized bovine insulin is dissolved in 5.2 ml of stabilized buffer solution consisting of 7 molar deionized urea and 0.02 molar tris-(hydroxymethyl)aminomethane with a pH value of 8.1. The solution is mixed with 5.2 ml of 7 molar urea solution. The pH value of the mixture is set to 8.1. A 5 cm diameter column is packed with a 2.1 cm high layer of DEAE cellulose ("Whatman DE 52") and equilibrated with a buffer solution of the above composition. The insulin solution is introduced into the column, and it is eluted at a rate of 75 ml per hours according to the following schedule:

2.5 hours with a buffer of the above composition,

3 hours with a buffer with the above composition, added 0.0045 mol of sodium chloride per litre, 12 hours with a buffer with the above composition, added 0.011 mol sodium chloride per litres.

The eluate is divided into fractions. The highly purified fractions are collected, and the insulin content is determined. In a 7 molar urea solution of the same volume as the mixture of the highly purified fractions, protamine is dissolved in the amount necessary to achieve the isophane ratio between the highly purified insulin and protamine.

The insulin solution is slowly dripped into the protamine solution while stirring, whereby, possibly after dilution to a urea concentration of 1 molar, a protamine-insulin complex is precipitated in an amorphous state.

The precipitate is isolated by centrifugation and shows essentially only one band when 300 µg is subjected to isoelectric focusing in polyacrylamide gel using 2% "Ampholine"

(system of amphoteric aliphatic polyamino-polycarboxylic acids)

(pH 3-10) in 6M deionized urea.

This precipitate can be used for the production of injectable insulin products by suspension in known carrier media.

Example 2

A stabilized buffer solution is prepared by mixing two solutions I and II in such a ratio that the pH value is 6.00.

Solution I consists of 7 molar deionized urea and

0.13 molar sodium dihydrogen phosphate

Solution II consists of 7 molar deionized urea and

0.13 molar disodium hydrogen phosphate.

400 mg of recrystallized bovine insulin is dissolved in 3 ml of this buffer, after which the pH value is readjusted to 6.00 with a little solution II, and the resulting mixture is filtered.

A 2.5 cm diameter column is packed with a 27 cm high layer of "Amberlite" ® resin CG-50 type II (weakly acidic cation exchange resin prepared on the basis of polyacrylic acid) and equilibrated with a buffer solution of the above composition. The insulin solution is introduced into the column, and it is eluted at a rate of 40 ml per hour with a buffer solution of the above composition.

The eluate is divided into fractions. The purified insulin fractions are collected and the insulin content determined. In a 7 molar urea solution with the same volume as the mixture of the highly purified fractions, protamine is dissolved in the amount necessary to achieve the isophane ratio between the highly purified insulin and protamine.

The insulin solution is slowly added to the protamine solution while stirring, whereby, possibly after dilution to a urea concentration of 1 molar, a protamine-insulin complex is precipitated in an amorphous state.

The precipitate is isolated by centrifugation and exhibits the same purity as the product described in example 1.

This precipitate can be used for the production of injectable insulin products by suspension in known carrier media.

Example 3

The procedure according to example 1 or 2 is repeated, with zinc chloride corresponding to 0.5% Zn ions in relation to the amount of insulin and 0.3% m-cresol based on the volume of the mixture being introduced into the protamine solution. The mixture's pH value is maintained in the range 6-9, whereby the protamine-insulin complex is precipitated in a crystalline state.

The precipitate is isolated by centrifugation and shows essentially only one band when 300 µg is subjected to isoelectric focusing in polyacrylamide gel using 2% "Ampholine"

(pH 3-10) in 6M deionized urea.

This precipitate can be used for the production of injectable insulin products by suspension in known carrier media.

Example 4

The procedure according to example 1 or 2 is repeated, that

as starting material, recrystallized bovine insulin is used which has been purified by gel filtration on "Sephadex<®> G-50" (dextran cross-linked with epichlorohydrin).

The manufactured product has the same purity as the product described in example 1.

Example 5

The procedures according to example 1 or 2 are repeated, using pig insulin instead of ox insulin. The protamine-porcine insulin complex formed is as pure as the complex prepared in Example 1.

Example 6

The procedures from example 1 or 2 are repeated, using as starting material pig insulin produced by salting out an aqueous crude extract formed during insulin production by adding NaCl to 3.5M at pH 8.5. The obtained salt cake is desalted in a known manner before the ion exchange.

The product obtained has the same purity as the product described in example 1.

Example 7

The procedure according to example 6 is repeated, the obtained salt cake being subjected to gel filtration on "Sephadex G-50".

The product has the same purity as the product described in example 1.

Example 8

The procedure according to example 1 or 2 is repeated, using poly-L-arginine with a degree of polymerization of 296 instead of protamine.

By isoelectric focusing in polyacrylamide using 2% "Ampholine" (pH 3-10) in 6M deionized urea, 300 µg of precipitate shows essentially only one band.

Example 9

The procedure according to example 1 or 2 is repeated, however, a 2% aqueous solution of bis-(4-amino-2-methyl-6-quinolyl)-urea hydrochloride is added to the total highly purified insulin fractions in an amount corresponding to 10% by weight of the amount of insulin present. This precipitates, possibly after dilution to a urea concentration of 1 molar with 0.025M Na-phosphate buffer pH 7.3, an insulin complex which can be isolated by centrifugation after a period of time. The manufactured product has the same purity as the product described in example 1.

Example 10

The procedure according to example 9 is repeated, using as starting material recrystallized bovine insulin which has been purified by gel filtration on "Sephadex G-50". The manufactured product has the same purity as the product described in example 1.

Example 11

The procedure according to example 9 is repeated, using pig insulin instead of ox insulin.

The porcine insulin complex formed is as pure as the complex prepared according to Example 1.

Example 12

The procedure according to example 9 is repeated, using as starting material pig insulin produced by salting out an aqueous crude extract formed during insulin production by adding NaCl to 3.5M at pH 8.5. The obtained salt cake is desalted in a known manner before the ion exchange. The product obtained has the same purity as the product described in example 1.

Example 13

The procedure according to example 12 is repeated, the obtained salt cake being subjected to gel filtration on "Sephadex G-50".

The product has the same purity as the product described in example 1.

In order to illustrate the improved immunogenic properties of the protamine-insulin complex isolated according to the invention, comparative experiments have been carried out on rabbits, as these animals are usually used when investigating the antigenic properties of insulin and insulin-like components.

The following products have been used in the comparative studies: 1. Conventional NPH bovine insulin produced in the usual way from recrystallized bovine insulin containing the impurities mentioned in the description. 2. Purified NPH bovine insulin prepared in the usual way from purified crystalline bovine insulin, but freed from the impurities mentioned in the description by column chromatography. 3. Purified NPH bovine insulin produced according to the invention as described in example 3, New NPH bovine insulin.

(NPH = Neutral Protamine Hagedorn insulin)

The rabbits were injected subcutaneously every 10 days with a constant dose of 20 i.e. of the insulin product to be investigated. As it was not possible to detect any significant antibody formation after injection of conventional NPH bovine insulin without adjuvant, it was necessary for the sake of comparison to use an immunization procedure that is normally used in the production of antibodies, i.e. inject the insulin products at the 1st injection emulsified in Freund's Complete Adjuvant (FCA) and in subsequent injections emulsified in Freund's Incomplete Adjuvant (FIA) which differs from FCA by not containing killed bacteria. Both products also contain mineral oils and emulsifiers. The times of the injections are shown with arrows in Figures 1, 2 and 3.

Insulin antibody formation was examined at intervals of 20 days using the method described by Ortved Andersen et al.

method (Acta Endocr. (Kbh.) 69, 195-208, 1972). The results obtained are shown in fig. 1, 2 and 3, where the dots indicate the insulin-binding capacity in yU/ml. High binding capacity indicates a high amount of antibodies.

As can be seen from the results, it was possible to demonstrate antibody formation against conventional NPH bovine insulin and a reduced antibody formation against purified NPH bovine insulin, while it was practically not possible to demonstrate any antibody formation against the new NPH bovine insulin produced according to the invention. It is noted that products 2 and 3 were purified by the same chromatographic purification, whereby the purified insulin meets the purity criteria stated in German publication 1 940 130, and the only difference was that product 2 was prepared by first isolating the insulin and then reacting it with protamine in a known manner, while on the other hand product 3 was prepared by carrying out the reaction in the urea-containing eluate without prior isolation of the insulin.

Another series of experiments has also been carried out using a milder immunization procedure, where the initial stimulation of the immune system with killed bacteria is omitted, i.e. the products are in all cases injected emulsified in Freund's Incomplete Adjuvant. The rabbits were injected every 10 days with

a constant dose of 20 i.e. (see arrows on fig. 4-6).

The insulin antibody formation was examined at intervals

in 10 days using a newly developed method which is based on precipitation with polyethylene glycol (PEG) of insulin-antiinsulin-125 complexes using radioactively labeled insulin, I insulin as a tracer.

PEG method for antibody determination

100 ul rabbit serum, 100 ul 12 5I-insulin, approx. 2 yU/ml, 100 ul insulin solution, 250 yU/ml and 700 ul phosphate buffer, 0.04M pH 7.4 with 0.15M NaCl and 0.5% human albumin are incubated for 2 days at 4°C. 500 µl of PEG 6000 (polyethylene glycol with an average molecular weight of 6000) is added, 360 g/l is mixed on a rotamixer, and the sample is allowed to stand for 1 hour at 20°C.

Centrifuge for 10 minutes at 3000 rpm, the overlying 125

liquid is decanted from and discarded, the precipitate is counted for I.

In each set-up, a sample with an excess of guinea pig antioxinsulin, max. binding, and a sample with rabbit serum from unimmunized rabbits, 0 sample. Results are calculated as measured binding (%B) as a percentage of maximum binding.

where T = counting number for unknown

TQ = " " O sample

TM = " " max. binding.

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

As can be seen from the results, it was not possible to detect antibody formation against the new NPH bovine insulin produced according to the invention (fig. 6), while the other insulins caused a significant antibody formation (fig. 4 and 5). Since the sample values are determined with reference to a standard, some of the values below the abscissa are due to uncertainties in the measurement.

Claims (5)

1. Process for producing a stable long-acting insulin product with low antigenicity, by reacting highly purified insulin with an organic base containing amino groups, preferably the polypeptide protamine, characterized in that the reaction is carried out in an aqueous buffer solution containing a stabilizer which during the reaction maintains the insulin in dissolved and stabilized monomeric or loosely aggregated form, after which the insulin product formed is isolated. 2. Method according to claim 1, characterized in that bovine insulin is used. 3. Method according to claim 1 or 2, characterized in that an insulin-containing fraction obtained in a known manner by chromatographic purification of insulin using a buffered, aqueous eluent containing a stabilizer which maintains the insulin in solution in stabilized monomeric or loosely aggregated form, is reacted with an aqueous solution of the organic base containing amino groups, after which the insulin product formed is precipitated and isolated. 4. Method according to one of claims 1-3, characterized in that urea is used as stabilizer. 5. Method according to claim 4, characterized in that the reaction between the highly purified insulin and the organic base containing amino groups is carried out in an aqueous buffer solution containing dissolved urea in a concentration of approx. 7 molar, after which the insulin product formed is precipitated by dilution of the reaction mixture and isolated.