Method of preparation of stable crystalline analogue of zinc - insulin

 

(57) Abstract:

Describes a new method of obtaining crystalline LizB28AboutB29human insulin, which includes crystallization LizB28AboutB29human insulin from a solution containing LizB28AboutB29human insulin, zinc at a concentration of from about 400 mg to 1 g LizB28AboutB29human insulin, at least 0,3 N organic acid selected from the group consisting of acetic acid, citric acid or glycine, and phenolic compounds in a concentration of from 0.15 to 0.25% vol. at pH from about 5.5 to 6.5. The proposed method allows to obtain insulin analogs with a faster Board activity while maintaining the biological activity of native human insulin. 3 C.p. f-crystals.

The present invention relates to Monomeric analogue of human insulin. More specifically, the present invention relates to a method for producing crystalline insulin analogue. This method is used in the purification and production of LizB28AboutB29-human insulin. LizB28AboutB29-human insulin used to treat diabetes.

Since the discovery of insulin in 1920 the harsh conditions and the availability of insulin. Were also developed a variety of drugs of different duration. Despite these improvements, therapy with subcutaneous injection has not yet provided to the patient should the regulation and control of blood sugar levels.

During the life of the patient frequent deviations from normal levels of sugar in the blood lead to Hyper - or hypoglycemic conditions and complications, including retinopathy, nephropathy, neuropathy and micro, macroangioapthy.

To avoid extreme levels of blood glucose, diabetics often practice multiple injections, when insulin is injected during each meal. This therapy, however, is far from optimized.

The high-speed commercial insulin too late peaks after the injection, and it continues too long that you can't effectively control glucose levels. Recently taken serious attempt to create preparations of insulin and its analogues, which accelerate the process of absorption of the drug percutaneously.

Since all commercial pharmaceutical preparations contain insulin insulin in samosatene condition and, mainly, in the form of zinc-hexane asociacia samosatenus insulin hexamer. To speed up the process of absorption, were developed Monomeric insulin analogs.

These Monomeric analogues have faster compared to insulin activity, at the same time preserving the biological activity of native human insulin. They provide rapid absorption from the injection site and reach the peak of his actions close in time to increase the level of glucose in the blood, associated with food intake.

In the present invention proposes a new method of preparation of crystals of one of these Monomeric analogues, LizB28AboutB29-human insulin (LizB28AboutB29-Chi). LizB28AboutB29Chi disclosed in EPO publication N 383472. However, this publication does not disclose commercially available method of preparation of crystalline LizB28AboutB29Chi.

Crystallization of insulin is well known to specialists. First discoveries date back to the year 1926, when Abel got crystalline insulin in the isoelectric point of the solution with a buffer in the form of brucine, pyridine and ammonium acetate (Abel J. J. Proc. Natl. Acad. Sci. U. S. 12: 132, (1926). Peterson and others in the U.S. patent 2920104 describes the crystals of insulin, drugs and methods for their preparation.

B28AboutB29Chi is placed in conditions that allow human insulin to form the zinc crystals, such crystallization is not observed.

The present invention, a method of crystallization LizB28AboutB29Chi where human insulin does not crystallize. This method provides high-quality zinc-crystals at a high yield. These crystals are stable solid form of the substance.

Crystalline solid is especially convenient in practice, since they are easy to characterize, purify, they are more elegant from a pharmaceutical point of view, than amorphous solids. In addition, this process is convenient for production.

The present invention provides a method of obtaining a crystalline LizB28AboutB29-human insulin, including: crystallization LizB28AboutB29-human insulin from a solution containing LizB28AboutB29-human insulin, zinc, at least 0,3 N organic acid, the higher the present invention provides a method of obtaining a crystalline human insulin LizB28AboutB29. The term LizB28AboutB29-insulin person" or "LizB28AboutB29Chi" is a fast-acting insulin analog that is less prone to dimerization or self. LizB28AboutB29Chi is human insulin, in which the Proline at position B28 B-chain is substituted by lysine and the lysine in position B29 of the B-chain is substituted with Proline as described in EPO publication N 383472, incorporated herein by reference.

All abbreviations of the amino acids used in the present description, are abbreviations accepted by the patent office of the USA and is provided in 37 C. F. R. 1. 822 (C) (2).

The term "phenol" or "derived phenol" means a collective designation of phenol, m-cresol, resorcinol, methyl-p-hydroxybenzoate or mixtures thereof.

The term "crystallization" refers to the formation of crystals LizB28AboutB29-human insulin.

The term "physiologically acceptable base" is well known to specialists. Physiologically acceptable bases include sodium hydroxide, potassium hydroxide and ammonium hydroxide. Predpochtitel the nom production of human insulin crystallizes in the presence of 0.25 N acetic acid, the 1.6-2.1 g/l of insulin and 2% zinc at pH 5,95-6,05. Crystallization occurs when approaching the point of crystallization with acidic side by adding the base, usually sodium hydroxide.

It was unexpectedly found that soluble LizB28AboutB29Chi does not crystallize under normal conditions of crystallization of insulin. Self-Association and aggregation at LizB28AboutB29Chi is minimized. The fact that LizB28AboutB29Chi is not aggregatum, was first noted Brems and other Protein Engineering, 5:6, 527-533 (1992).

Consider that the minimum self-Association and aggregation, which causes monomeres analogue, is the absence of crystallization LizB28AboutB29Chi under conditions designed to insulin.

The present invention describes the conditions under which LizB28AboutB29Chi crystallizes with zinc and phenolic compound and forms a stable solid crystalline substance. The preferred phenols are selected from the group consisting of phenol, resorcinol and mixtures thereof. Zinc, and phenol are the essential factors to achieve crystallization.

The solution LizB28AboutB29Chi is prepared by dissolving analogue Ann is. repectfully concentration equivalent to about 2 g/L. Dissolution may contribute to the so-called acid dissolution, i.e. reduce pH to approximately 3.0 to 3.5 with a physiologically tolerated acid, preferably hydrochloric. Other physiologically acceptable acids include acetic acid, citric acid and phosphoric acid.

The concentration of organic acid selected from the group consisting of acetic acid, citric acid or glycine, is at least 0,3 N; at lower concentrations obtained largely amorphous product. Preferably, LizB28AboutB29Chi dissolved in 0.8 and 1.2 N acetic acid solution; more preferably in 1 N acetic acid solution.

The concentration of added zinc is such that the final concentration is approximately from 40 to 400 mg per gram equivalent. It is preferable to add the zinc in the form of a salt. Examples of zinc salts can serve as zinc acetate, zinc bromide, zinc chloride, zinc fluoride, zinc iodide and zinc sulfate.

Experienced professionals know that there are many other zinc salts, which can also be used in the method of the present invention. Preferably the Lenna developed methods. The optimal concentration of zinc for crystallization is from about 100 to 300 mg per gram LizB28AboutB29Chi.

Conditions of crystallization are sensitive to the presence of phenol, the base and the zinc concentration; therefore, the specialist performing the crystallization process will choose the optimal parameters, mentioned above, to produce a well-designed crystals. Optimal conditions for each combination of phenol-based range set forth within. Preferably as phenolic compounds to use phenol in a concentration of from about 0.15 to 0.25% (vol./about., the final concentration). It is more preferable to use a phenol at a concentration of 0.2%.

The way is the dissolution LizB28AboutB29Chi, or the order in which the phenol, zinc and LizB28AboutB29Chi added to the solution, are not fundamental to the present method. However, the principal is the fact that phenol interacts with LizB28AboutB29Chi below the isoelectric point LizB28AboutB29Chi.

Accordingly, the crystallization may be initiated by either the addition of zinc, and transfer the solution acidity from acidic pH d the include sodium hydroxide, the potassium hydroxide and ammonium hydroxide. It is preferable to use ammonium hydroxide.

The crystallization can be performed by transferring the acidity of a solution containing LizB28AboutB29Chi, zinc, at least 0,3 N organic acid selected from the group of acetic acid, citric acid or glycine, and phenol to about pH 5.5 to 6.5 with physiologically acceptable bases. Preferably the pH is brought to values approximately 5.9 to 6.2, then formed crystals.

More preferably, LizB28AboutB29Chi dissolve approximately 1 N acetic acid solution (if you want to contribute to the dissolution, the pH can be increased up to 3.0-3.5). Then to the solution was added the phenol, and the solution is balanced. the pH of the solution was adjusted to about 5.5 to 6.5 with physiologically acceptable bases. It is preferable to use ammonium hydroxide, and the pH adjusted to approximately 5.9 to 6.2. Then, crystallization is initiated by the addition of zinc.

Under different methods of crystallization, the crystals are formed by the mixing or no mixing and can be collected and washed. It is preferable to perform the crystallization with premesis the crystals can be collected and dried using standard techniques.

If the crystals are collected by filtration, to the filtrate, you can add an additional amount of zinc or stock solution for further recovery LizB28AboutB29Chi. Crystals prepared according to the present invention, are high quality and give a great yield on an industrial scale.

The crystals provide a stable solid form of mass drug substances suitable for storage, and to perform the subsequent process operations. The crystallization procedure does not change the purity and aggregation characteristics of the material.

The crystallization temperature is not crucial. Acceptable temperature is approximately from 4 to 26oC. it is Preferable to maintain the temperature approximately from 22 to 24oC.

LizB28AboutB29Chi can be obtained using any of the methods of peptide synthesis, including classical (solution), the method of solid phase synthetic methods and the most modern methods with recombinant DNA. For example, Chance, etc., EPO publication N 383472, disclose the preparation of human insulin.

The following examples are given for purposes alluaudia the scope of the present invention.

Example 1

846,5 g LizB28AboutB29Chi 116 l acetic acid buffer was diluted to absorption of 2.25 at 276 nm of purified water reaches 260.6 l) and glacial acetic acid (19,44 l) drum of stainless steel to a final concentration of acetic acid 1 n

To the solution was added a liquid phenol concentrations up to 2 ml/l (the total number of 792 ml) and the pH of the resulting solution was below 3. Next, the pH of the solution was brought to 6,01 by adding 24 l of ammonium hydroxide and warmed up to the 22oC.

After this was added 5,35 l of a solution of zinc chloride (2% wt./about. in water). Then we were convinced that the pH of the solution is within the desired range (5,9-6,1), and the solution was stirred for 12 hours. Then the solution was cooled to 8oC, stop stirring and the crystals were allowed to settle for 18 hours at 2-8oC.

After sedimentation of the crystals 370 l of the supernatant solution decantation, and the remaining adosados and the crystals were transferred into the drum, smaller incremental subsidence using desantiruemogo nadeshiko for washing the first reel. After 14 hours of sedimentation 61 l nadeshiko decantation, leaving approximately 9 l of a crude crystals and nadeshiko.

Crystals suspended antifuse DPR6000 within 45 minutes. Centrifugation of the whole crystalline sludge took two loading centrifuge. Centrifugal decantation.

Crystals (approximately 250 ml per flask) suspended in 500 ml of purified water to the centrifuge flask and again centrifuged at 4000 rpm./min for 45 minutes. The crystals are then re-suspended in centrifuge tubes in approximately 500 ml of chilled (2-8oC) absolute alcohol SD N 3A on the flask and was recentrifuged at 4000 rpm./min for 15 minutes.

Alcohol centrifugal decanted and such alcohol laundering was repeated two more times. The crystals are washed with alcohol (1.9 kg wet weight), and dried under vacuum.

Example 2

The solution LizB28AboutB29Chi approximately 2 g/l were prepared to a final concentration of 1 N acetic acid (determined by absorption at 280 nm). After adding liquid phenol (3,3 ml/l) the pH of the solution was brought to 5.9 and 6.2, concentrated ammonium hydroxide was added zinc chloride in water 2% (weight. /about. ) or 20% (wt./about.) to a final concentration of 40-160 mg of zinc chloride per gram LizB28AboutB29Chi.

The obtained crystals were settled, and the mother liquor was removed by decantation after centrifugation. Crystals otbyvali in a vacuum.

The second collection of crystals can be obtained by adding zinc to the stock solution up to a limit of 160 mg/g LizB28AboutB29Chi.

Example 3

LizB28AboutB29-human insulin (222 mg) was dissolved in 100 ml of water Milli-Q. In the solution was determined by the content of 2.0 mg per ml of solution LizB28AboutB29Chi using liquid chromatography high resolution. The solution was osvetleni by bringing approximately pH to 3.0 with 10% HCl. Selected four 5 ml aliquot was added glacial acetic acid to a concentration of 1 N, and pH was below a 3.5.

To each sample was added in ten Microlitre liquid phenol and then six microlitres solution of zinc chloride (20% wt./about. in water). pH is brought to 6.0 by addition of concentrated ammonium hydroxide, sodium hydroxide (10% wt./about.) or potassium hydroxide (10% wt./vol.).

The solutions were stirred for about 15 minutes and then left to stand under the lid at room temperature. After approximately 2 hours, in all three solutions was well defined rhombohedral crystals, and the fastest crystals formed in the solution, which was added potassium hydroxide.

Example 4

Priblizitel the Oh of the acid. In the first sample were added forty-four microliters of liquid phenol, and the second 34 microliters of liquid phenol. In both cases, the pH is brought to 6.0 by addition of concentrated ammonium hydroxide and then added forty five microlitres solution of zinc chloride (20% wt./about. in water).

The solution was stirred for approximately 5 minutes, after which they were left to stand under the lid at room temperature. After approximately 24 hours watched well-decorated rhombohedral crystals.

Example 5

LizB28AboutB29-human insulin (222 mg) was dissolved in 100 ml of water Milli-Q. In the solution was determined by the content of 2.0 mg LizB28AboutB29Chi in a milliliter of the solution by liquid chromatography high resolution. The solution was osvetleni bringing the pH to about 3.0 by the addition of 10% HCl. Selected four 5 ml aliquot was added glacial acetic acid to a concentration of 1 N, and pH was determined below a 3.5.

In the aliquots was added or m-cresol (12 μl), or phenol (10 μl), or resorcinol (2,1 ál of 100 mg/ml solution in water), or methylparaben (1.6 ml 10 mg/ml solution in water), which resulted in the aliquot of the same molar ratio of peptide and phenolic compounds.

using concentrated ammonium hydroxide. The solutions were stirred for about 15 minutes and then left to stand under the lid at room temperature.

After approximately 24 hours in solutions containing phenols, watched a well-decorated rhombohedral crystals. In the solution containing methylparaben, formed a small number of poorly defined flat crystals. Solutions containing m-cresol and resorcinol, under these conditions, the crystals are not formed.

A similar procedure was undertaken with one tenth the number of resorcinol and bringing the pH with sodium hydroxide (10% wt./about. solution); this worked well decorated rhombohedral crystals. This example shows that phenolic compounds can form well-shaped crystals in routine optimization of conditions.

Example 6

LizB28AboutB29Chi (222 mg) was dissolved with 100 ml of water Milli-Q. In the solution was determined by the content of 2.0 mg LizB28AboutB29Chi in a milliliter of the solution by liquid chromatography high resolution.

The solution was osvetleni bringing approximately pH to 3.0 with 10% HCl. Selected one aliquot of 5 ml was added glacial acetic acid to a concentration of about 0.25. about. in water). the pH of the solution was brought to 6.0 by the addition of concentrated ammonium hydroxide. The solution was stirred for approximately 15 minutes, after which it was left to stand under the lid at room temperature. After approximately 24 hours the solution was not observed the formation of crystals, but on the bottom of the vessel fell amorphous precipitate.

As a control took another aliquot of 5 ml and treated her with ammonium hydroxide according to the procedure described in example 1. Formed well-decorated rhombohedral crystals LizB28AboutB29. When human insulin (2,3 mg/ml in 1 N acetic acid) was subjected to the procedure described in example 1, using ammonium hydroxide, within 7 days, the crystals were not formed.

This experiment showed that the conditions usually used for the crystallization of biosynthetic human insulin, are not suitable for crystallization LizB28AboutB29-human insulin, and the conditions of crystallization described for crystallization LizB28AboutB29Chi will not produce crystals of human insulin.

Example 7

The method described in example 1 was performed crystallization, replacing 1 N acetic acid 1 N citric kilocalorie crystalline LizB28AboutWhuman insulin, characterized in that it includes crystallization LizB28AboutWhuman insulin from a solution containing LizB28AboutWhuman insulin, zinc at a concentration of about 40 to 400 mg per 1 g LizB28AboutWhuman insulin, at least 0,3 N organic acid selected from the group consisting of acetic acid, citric acid or glycine, and phenolic compounds at a concentration of 0.15 to 0.25% vol. at a pH of approximately 5.5 to 6.5.

2. The method according to p. 1, characterized in that the concentration of LizB28AboutWhuman insulin is approximately 1.8 - 2.5 g/l; phenol compound is phenol and the concentration of zinc is approximately 40 - 400 mg/l LizB28AboutWhuman insulin.

3. The method according to p. 1, characterized in that the pH is approximately 5.9 to 6.2.

4. The method according to p. 1, characterized in that it includes the addition of phenol in the amount of 0,15 - 0,25% of the total volume of the solution LizB28AboutWhuman insulin at a concentration of approximately 1.8 - 2.5 g/l in approximately 1N acetic acid, bringing the pH to values of about 5.9 to 6.2 ammonium hydroxide and to W human insulin.

 

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