FI73130C - Process for the preparation of purified insulin suitable for the preparation of clinically useful injectable insulin preparations. - Google Patents

Description

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SUOMI FINLAND

(H) (21) Patent application - Patentansökning 793110 (22) Application date - Ansökningsdag 08.10.79

National Board of Patents and Registration (23) Start date-Giltighetsdag 31.07.69

Patent- och registarstyrelsen (41) Announced to the public - Biivit offentiig 08.10.79 (44) Date of publication and date of publication. - 29.05.87

Ansökan utlagd och utl.skriften publicerad (86) Kv. Application-int. ansökan (32) (33) (31) Privilege claimed - Begärd priority 09.08.68 United Kingdom-Great Britain (GB) 38081/68 (71) Novo Therapeutic Laboratory A / S, Fug 1 ebakkeve j 115, KeSbenhavn, Denmark-Denmark rk (DK) (72) Jörgen Sohi ichtkru 1 1, Br «5nsh ^ j, Denmark-Danmark (DK) (7 * 0 Oy Kolster Ab (5 * 0 Method for the preparation of purified insulin suitable for clinical use, for the preparation of injectable insulin preparations The present invention relates to a process for the preparation of injectables for clinical use, which are suitable for clinical use and to the manufacture of injectable injectables for clinical use. for the production of purified insulin starting from commercial or crude bovine and / or porcine insulin, which do not produce insulin antibodies or are highly present when insulin preparations are administered. and which insulin preparations are substantially one-component by discontinuous polyacrylamide gel electrophoresis analysis (DISC PAGE).

The method according to the invention is characterized in that commercial or crude: insulin obtained from porcine and / or bovine pancreatic tissue is purified by removing both pancreatic proteins having a molecular weight greater than 6000 and antigenic insulin-like substances having a molecular weight of about 2,73130, preferably gel filtrates. and column chromatography with an ion exchanger to collect those fractions that correspond to the major part of the insulin peak.

During the first years of insulin therapy, when injectable solutions of amorphous insulin were used, allergic skin reactions were common. With the introduction of crystalline insulin injectable solutions, the number and severity of skin reactions were reduced and virtually eliminated when patients switched to recrystallized insulin solutions. Today, all commercial insulin preparations for clinical use are made from crystalline insulin.

The aforementioned skin allergy was most likely caused by pancreatic protein material present as impurities in significant amounts in amorphous insulin, to a lesser extent in crystallized insulin, and only insignificant amounts, if any, in recrystallized insulin.

Side effects other than those explained due to the nature of the insulin molecule itself have not been attributed to modern insulin preparations containing recrystallized insulin. After a few months of treatment, the serum of a diabetic patient has been found to contain immunoglobulins that combine with insulin in vitro and in vivo. (Such sera from insulin-treated guinea pigs are now routinely used for the immunoassay of insulin.) This side effect is undesirable for several reasons: 1. The formation of insulin antibodies indicates a persistent antagonistic state of the body against the essential hormone, insulin.

2. This condition is likely to be detrimental to cells in the body, e.g., ^ '.' Cells that have been destroyed in rabbits when immunized with insulin and an excipient. It is hypothesized that subsequent diabetic complications such as hemorrhoids may become severe.

3. It has been found that high levels of insulin antibodies sometimes cause insulin resistance, i. high dose requirements and lack of metabolic control.

4. It has been estimated that the actual dosage requirement of the diabetic population far exceeds the need that would be necessary to avoid the formation of an antibody.

5. In some patients, antibodies bind bovine insulin much more strongly than porcine insulin and cases of hypoglycaemia have been reported following a change in the quality composition of insulin products.

6. When insulin antibodies begin to form in a patient, there is a concomitant increase in circulating exogenous serum insulin concentration. Some serum insulin binds to antibodies. Concentrations of total insulin and even free insulin can reach values that are 10 to 100 times higher than normal physiological concentrations. These high amounts are harmful to tissues. They can contribute to arterial sclerosis and can damage surviving fi cells. It has been shown that high insulin concentrations can have a inhibitory effect on / 4-cell secretion.

The mechanism behind the development of insulin antibodies and the properties of these agents have been studied with increasing interest over the past 5-10 years. It has been found that the type of insulin appears to be a determining factor in the development of antibodies. Bovine insulin, which differs (3 amino acid positions) from human insulin more than porcine insulin (1 amino acid position), is, as might be expected, more antigenic than porcine insulin. On the other hand, insulin from pigs also causes the formation of antibodies against porcine insulin in pigs. No explanation has been found for this latter consideration, although it has been hypothesized that abnormally high concentrations of porcine insulin in injection form may induce an immunological "apparatus" in porcine to produce antibodies against its own insulin.

In the past, it was understood by one skilled in the art that insulin itself, when injected into the blood, caused the formation of insulin antibodies. However, it has now surprisingly been found that by very careful purification of insulin, insulin can be prepared which, when injected into the blood, does not cause any or at least no significant formation of insulin antibodies. Such pure porcine and bovine insulin (i.e., MC insulin) has not been previously prepared.

Thus, the insulin prepared according to the invention is pure, and it is surprising that it has very little or no antigenicity.

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From Biochem. 4 (1965) 1044 and Biochem. J. 106 (1968) 531 discloses fish insulin, which is a completely different substance from porcine or bovine insulin. Fish insulin has not been purified to result in a monocomponent product. It is not clear from the publication whether fish insulin is devoid of antigenicity.

In J. Biol. Chem. 235 (1960) 2294 describes a process for the preparation of insulin. This insulin is also not monocomponent.

The methods of GB Patent 871,541 and U.S. Patent 3,069,323 also do not allow the production of monocomponent insulin. The GB patent contains only one example of insulin (No. 14). The antigenicity of insulin has not been shown, nor has it been mentioned how to obtain monocomponent insulin.

The methods of GB patent 1,054,523 and FI patent 30,528 also do not produce monocomponent insulin.

The present invention provides a solution to the problem of the body's resistance to known insulin preparations. The insulin according to the invention does not cause an antagonistic reaction in the body, i.e. no or very little insulin antibodies are formed, because the new insulin preparation is free of certain substances which have always been present in known insulin preparations, higher molecular weight than insulin. In the past, it was assumed that insulin itself produced insulin antibodies, but it has now surprisingly been found that the cause is not the insulin molecule but the said impurities, which have higher molecular weights than insulin.

Insulin prepared according to the invention also does not contain insulin-like substances which have a molecular weight approximately equal to that of insulin but which differ slightly from insulin in terms of their chemical composition, for example insulin mono-desamido and a substance which appears to be! insulin arginine derivative, which substances have some degree of antigenicity. The pH of the new insulin preparation should preferably be about 7 to keep the formation of mono-desamido insulin to a minimum during storage.

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One known method for separating protein molecules of different sizes is to dissolve the protein mixture in a solvent in which the molecules dissociate and pass the solution through a molecular sieve, e.g. a column of organic matter cross-linked into a three-dimensional network of defined pore size. It has been shown that crystalline insulin dissolved in 1 m acetic acid can be fractionated on components a), b) and c) using a Sephadex ® G-50 column, respectively. These components can be separated from their solutions by conventional methods, e.g. by salting out of solution, precipitating with a neutral zinc salt, drying by cooling, etc. The amount of component a) can be 2-5% by weight of crystallized insulin and less than 1% by weight of recrystallized insulin. The amount of component b) can in each case be from 4 to 8%. Component b) can be further broken down into several proteins of approximately the same molecular size but different in charge ^ This has been demonstrated using the anion exchanger DEAE Sephadex k-'A 50 and a solution of component b) (bull) in a 7-m urea solution which is buffered with 0.04 m TRIS (tris (hydroxymethyl) aminomethane) to pH 7.7, NaCl gradient 0-0.2 m, cf. Figure 1, where peaks number 4, 6 and 7 correspond to known substances: proinsulin, intermediate and dimer, respectively. No other substances have been identified yet. These substances can all be separated from their solutions by known methods.

: Component c) can be decomposed by ion exchange chromatography. It contains the major fraction, which is pure insulin, mono-desamido insulin, and a fraction referred to herein as "arginine insulin" because it contains more arginine than the amount present in the insulin molecule. The fraction of mono-desamido insulin in component c) varies and depends on the "origin" of the insulin. Component c) is generally from 3 to 10% by weight. Arginine-insulin may be 2-3% by weight of component c).

* Figures 2a and 2b show the results of the fractions, where: Figure 2a shows graphically for recrystallized insulin; gel filtration performed: Sephadex® G 50, 1-m-acetic acid; the abscissa has volume, and the ordinate has absorption. Figure 2b shows: graphically the chromatography of fraction C of Figure 2a using an anion exchanger; the abscissa has volume and the ordinate has absorption.

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Figure 2c shows the components of the fractions as obtained by polyacrylamide gel electrophoresis (PAGE) performed according to the plate method described by Ornstein and Davis using 7.5% polyacrylamide and pH 8.9.

It has been found that new insulin preparations are surprisingly better tolerated by the body than known insulin preparations. The explanation for this is that components a) and b) present in known insulin preparations unexpectedly contain highly antigenic and apparently diabetogenic substances, while the new insulin preparations are surprisingly non-antigenic.

In the toxicity studies performed, rabbits were used as experimental animals because they show the same type of serum antibody response as humans when treated with known insulin preparations, i. similar antibody properties with respect to insulin binding capacity and insulin affinity. (Guinea pigs would produce antibodies with much higher activity and capacity. Insulin produced in rabbits differs from human insulin only at position B 30, which is serine in rabbits and threonine in humans. Guinea pig insulin, on the other hand, differs from human insulin at position 18).

Rabbits were injected three times a week with a constant dose of protein, 40 μq dissolved in 1 ml of 0.04 M sodium phosphate buffer solution at pH 7.4. In the case of insulin, this amount corresponds to about 0.5 international units per kilogram, which is approximately the average dose for the treatment of diabetes. Serum antibody levels were examined at regular intervals using standard methods. The amount is expressed as the% of insulin I (bovine) added that is bound to the antibodies in the sample. Some results are shown in Figure 3.

Abscissa number of days, ordinate% bound 125 I insulin (cattle). Curve 1: component a) (cattle), curve 2: fraction 3 of component b) (Figure 1, cattle), curve 3: dimer (cattle), curve 4: proinsulin (cattle), curve 5: recrystallized insulin (cattle) , curve 6: pure insulin (bovine).

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Two of the results are very surprising and unexpected: 1. An insulin preparation comprising a new pure insulin does not produce antibodies against insulin.

2. Component a), which is not insulin, causes the formation of an abundant amount of antibody against insulin. The most likely explanation for this is that it contains something that is chemically unrelated to insulin in a hitherto unknown way.

3. Ordinary very pure insulin, i.e. recrystallized insulin generates antibodies as it does in humans.

4. The substances belonging to component b), namely No. 3 (see Figure 1), dimer and proinsulin, are antigenic, causing the formation of antibodies against insulin.

The figure does not show the finding that the "arginine-insulin" of component c) produced a small but significant amount of antibody after 45 days.

In the course of the tolerance test, some rabbits showed a tendency to form elevated blood glucose levels, namely in those rabbits injected with component b), and in particular in those rabbits which had received one of the fractions of component b), namely dimer. The dimer does not produce the greatest amount of antibodies, but for unknown reasons, blood glucose levels were highest in these animals. This effect of component b) and the dimer can be called diabetogenic or anti-insulin-like, because in fact it seems that the animals have become resistant to insulin, as shown in Figure 4. Abscess minutes after insulin injection, ordinate serum glucose mg / 100 ml. Curve 1: dimer (bovine), curve 2: pure insulin (bovine).

After 14 weeks of dimer treatment (cattle, 40 μg in three weeks), the rabbits fasted for 16 hours and after; they were given intravenous insulin (0.5 international units per kilogram). The control group of rabbits had been treated with a preparation containing pure insulin. The serum-glucose curves shown in Figure 4 show that the control rabbits reacted to insulin as expected, while the rabbits treated with the dimer from component b) were resistant to insulin. Resistance may explain the rise in blood sugar levels of 8,731,130, but the phenomenon itself has not yet been elucidated. It appears to be similar to the resistance that develops in some patients during treatment when treated with known insulin preparations. It is believed that clinical resistance can be avoided by using insulin preparations that are free of component b) and dimer.

To take advantage of the above findings regarding the biological properties of impurities present in commercial insulin, insulin is purified in an amount not yet used on a commercial scale. The necessary purification can in principle be carried out by a wide variety of methods by which proteins can be purified; the preferred method is purification by gel filtration and column chromatography.

Removal of pancreatic proteins with a molecular weight greater than about 6000 contained in the starting insulin can be accomplished by subjecting the insulin to gel filtration. It is preferred to perform gel filtration in an aqueous medium which may contain water-miscible non-aqueous liquids, but if desired, it is also possible to perform gel filtration in organic solvents containing some water. When using an aqueous medium, a pH range of 2-4 is recommended. The stability of insulin below pH 2 and above pH 9 is poor, so insulin degradation products are formed during the process in these pH ranges. Very low or very high pH is therefore less suitable. The pH range of 4-9 can be used if the solubility of insulin is improved, e.g. by adding a urea which also has the ability to degrade the association products that may form between insulin molecules and between insulin molecules and impurities during the neutral reaction steps. When working at low pH, the use of an organic acid / such as acetic acid is recommended to adjust the pH, but concentrated mineral acids may also be used, with a concentration of 0.5 to 4 m acetic acid being preferred.

9,73130 Temperature is not critical to gel filtration and is primarily limited by insulin stability and solubility. In this case, it is advantageous to work at approximately room temperature, e.g. between 20 and 25 ° C. Fractions eluting before insulin-containing component c) are discarded. Fractions of component c) which are free of a substance with a molecular weight greater than 6000 are combined and insulin can be recovered, e.g. by evaporation, salting out of solution or precipitation with zinc ions. Insulin can be used as such or after crystallization for the preparation of insulin pharmaceutical preparations.

According to the invention, insulin-like substances which have almost the same molecular weight as insulin but which differ slightly from insulin in terms of their chemical composition, for example mono-desamido insulin, and a substance which appears to be an arginine derivative of insulin are also removed. The preferred method for removing the arginine derivative and deamidated insulin is column chromatography using an ion exchanger. Almost all commercially available ion exchangers that allow the penetration of an insulin molecule can be used, provided that the method is performed in a medium in which the insulin molecules do not form aggregates with each other or with impurities.

The following table provides examples of suitable agents for purifying insulin.

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Column packings for chromatographic purification of insulin

Substances Binders Synthetic Derived from natural compounds

Polyac- Poly- Poly- Dexst- Cellulose- Algine- ryl- sty-acrylic, loose acid amide rhenium meat press-type

Gel filtration- Bio-Gel Sephadex substances P-10 G 50

Bio-Gel Sephadex P-30 G 75

Anion exchanger species Dowex 1 QAE

Sephadex

Strongly basic groups

Weakly DEAE

basic Bio-Gel DEAE pulp groups DM Sephadex loosa

Ratio- Butter- Dowex SE- Phosphorin Dew 50 W Sephadex Found Exchangeable Acid Pulp Groups Loose

Weakly Bio-Gel Amber- CM- CM-sel- Alginic acid CM lite Sephadex Lulose acid groups CG 50

The ion exchange process can be performed with an aqueous organic solvent eluent, and it is preferred that the organic solvent be miscible with water. Examples of such solvents are tetramethyl urea, dimethylformamide, dioxane, water-miscible ketones such as acetone, and lower alcohols such as methanol, ethanol and propanols. Preferred are alcohols which can be used in a concentration of 30-80% (v / v), preferably in concentrations of 50-70% (v / v).

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When the eluent contains less than 50% (v / v) of a water-miscible organic solvent, it may be referred to as an aqueous medium, in which case the organic solvent may be replaced by urea used in an amount to give 4 to 8 molar, preferably about 7 -molar solutions. When a urea solution is used, it must be free of ammonium cyanate because the cyanate ion reacts with the amino group in the insulin molecule.

The eluent always contains a buffering agent to adjust the pH of the eluent. It is preferable to work at a constant pH. When a cation exchanger is used, the pH of the eluent must be between 3 and 6.5, preferably between 4 and 6, and when an anion exchanger is used, the pH of the eluent must be between 5.5 and 10, preferably between 6 and 9.

Said pH values are measured with a glass electrode adjusted in the usual way relative to the standard buffer solution.

Suitable buffers can be found in the available literature.

It is preferred that the temperature be relatively constant during the ion exchange operation. The temperature may be selected from -10 to 40 ° C, preferably from 0 to 30 ° C, except when the eluent is an aqueous solution of the urea, in which case it is preferable to keep the temperature below 5 ° C.

In the purification process, commercial insulin is generally used as the insulin starting material, i.e. amorphous insulin or crystalline insulin that may have been crystallized several times. Crude insulin can also be used, such as insulin-containing salt cake, which is formed upon recovery of insulin from the pancreas and which generally contains 10-30% by weight of insulin, but high impurity concentrations make this starting material less suitable for purification according to the invention. A better starting material can be obtained by subjecting the salt cake solution to isoelectric precipitation by adjusting the pH of the solution to 5.5 and separating the precipitate by centrifugation.

The purification steps always result in a purified insulin solution fraction from which the insulin can be recovered in the usual way, e.g. by evaporation, salting out of solution or precipitation in the presence of zinc ions. If desired, insulin can be crystallized and prepared into injectable insulin preparations as is known in the art.

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The purification of insulin according to the invention is described by means of the following examples.

Example 1a 5 kg of fine quality Sephadex® G 50 (manufactured by PHARMACIA, Uppsala, Sweden) are swollen in 1 m acetic acid and the finest particles are removed by decantation. The substance is used for packing a column with a diameter of 15 cm and a height of 130 cm. The column is equilibrated with 1 M acetic acid.

10 g of porcine insulin, crystallized once from the citrate-drop buffer solution, are dissolved in a mixture of 390 ml of 1-acetic acid and 8 ml of 1-n hydrochloride. This solution is poured onto a column. Elution is performed with 1 M acetic acid at a rate of 1 liter / hour.

Collect 200 ml fractions.

Extinctions at 276 nm are measured and plotted with fraction numbers on the abscissa. The fractions corresponding to the major part of the insulin peak (highest peak) are combined and the insulin is precipitated by adding 200 g of sodium chloride per liter of liquid. The precipitate is separated by centrifugation and subjected to isoelectric precipitation and crystallization from a citrate buffer solution containing acetate in a known manner. The yield is 8 g of crystalline insulin, which is free of proteins of pancreatic origin with a molecular weight of more than about 6000.

Example 1b

The crystalline purified insulin obtained as described in Example 1a is further purified by ion exchange chromatography using Amberlite ion exchange resin. 7-m solutions of urea are prepared and deionized by passing them through columns containing a mixed layer of ion exchange resin (Amberlite MB-1). This deionized urea is then used to prepare a buffer solution: 0.13-m sodium phosphate - 7-m urea - 1% n-butanol, pH 6.00 at 25 ° C. The buffer solution is stored and used at 4.0 ° C, 1.5 kg of Amberlite CG 50 Type II is treated with the following substances: 1) water; 2) acetone; 3) water + sodium hydroxide; 4) water; 5) water + hydrochloride and 6) water. The washed resin is suspended in 5 liters of buffer solution. The pH of the suspension decreases immediately and after every few minutes about 30 ml portions of 40% (w / v) hydrochloride are added to maintain the pH at about 6.0.

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When the pH of the suspension remains at 6.00 after stirring for 15 minutes, the mixture is stirred overnight at 4 ° C. The resin is filtered and washed with 30 liters of buffer solution for 8 hours. The pH of the final filtrate should be the same as that of the inflowing buffer solution. This material is used to fill a column with a diameter of 7.5 cm and a height of 80 cm. The column is equilibrated with buffer solution.

15 g of insulin prepared by the method described in Example 1a are applied to the column as a 10% (w / v) solution in a buffer solution. Elution is performed with a buffer solution at a temperature of 4 ° C and a rate of 200 ml / hour. Collect 100 ml fractions.

Extinctions at 276 nm are measured and plotted with fraction numbers on the abscissa. The fractions corresponding to the major part of the insulin peak (highest peak) are combined and the insulin is precipitated by adding 2 liters of water, 15 ml of 4-n hydrochloride and 600 g of sodium chloride per liter of liquid. The precipitate is separated by centrifugation and subjected in a known manner to isoelectric precipitation and crystallization from a citrate buffer solution containing acetone.

The yield is 10 g of crystalline insulin containing only one component when subjected to polyacrylamide gel electrophoresis. In other words, crystalline insulin must be considered as pure insulin.

Claims (2)

14,731 30 A process for the preparation of purified insulin suitable for the manufacture of injectable insulin preparations for clinical use, starting from commercial or crude bovine and / or porcine insulin, which, when administered, do not produce insulin antibodies or have very low levels of insulin antibodies, and which insulin preparations discontinuous polyacrylamide gel electrophoresis (DISC PAGE) are essentially one-component, characterized in that commercial or crude insulin from porcine and / or bovine pancreatic tissue is purified by removing both pancreatic proteins with a molecular weight greater than 6000 and antigen the molecular weight is about 6000, preferably by gel filtration and column chromatography with an ion exchanger, collecting those fractions which correspond to the major part of the insulin peak. Process according to Claim 1, characterized in that the insulin is bovine insulin crystallized as sharp-edged rhombohedrons with planar crystal surfaces.