CS208107B2 - Method of isolation of the glucagone - Google Patents

Description

The invention relates to a process for the isolation of glucagon from a supernatant from a process for the treatment of insulin by crystallization in an alkaline medium.

Soon after Banting and Best discovered insulin in 1921, there were several groups of workers, see, for example, Murlin et al., J. Biol. Chem. 56, 252 (1953) and Klmball and Murlin,

J. Biol. Chem. 58, 337 (1954), that several extracts of insulin from the pancreas had a hyperglycemic response. And the factor responsible for this hyperglycemic response was named glucagon.

Currently, the most important use of glucagon is the treatment of insulin-induced hypoglycaemia. And as the population affected by diabetes grows in the world, so must the demand and need for glucagon. In addition, the possibility of using glucagon in cardiac disorders is also being monitored. As a result, these two possibilities of use mean increased demand for glucagon production, and the demands can best be met by increasing the yield of glucagon from natural sources.

The first preparation of crystalline glucagon was described in 1953, see Staub et al.

Science 117, 628 (1953); also Staub et al., J. Biol. Chem. 214, 619 (1955). The starting material used was the amorphous fraction obtained during the commercial production of insulin, which involved extracting tissue from the pancreas with acidified alcohol, concentrating estract, sodium chloride precipitation, isoelectric precipitation, several fractions using alcohol, decolorizing, crystallizing in the presence of zinc salt from acetic acid buffer followed by washing to remove amorphous fractions and drying of the zinc insulin complex obtained by this procedure. The isolated amorphous fraction contains about 4% glucagon and about 7% insulin by weight. Generally, the yield of the amorphous fraction is in the range of about 5 to 10 mg per 0.45 kg of pancreas. Further purification of glucagon includes optionally fractionation at pH 6.7, fractionation using acetone, fractional precipitation at pH 4.3, two fractional precipitation at pH 2.5, and 2 crystallization from urea-glycine buffer at pH 8.6 . The yield of crystalline glucagon is about 20% by weight of the glucagon content of the dry amorphous material, which corresponds to a yield of about 0.04 to 0.08 mg of glucagon per 0.45 kg of pancreas.

The introduction of crystallization of an insulin-zinc complex from citric acid buffer as described in U.S. Pat. No. 2,626,228 means an overall reduction in the number of steps required for insulin production when bovine pancreas is used. Although approximately the same amount of amorphous fraction is obtained in the washing step as in the prior art, the glucagon content of the amorphous fraction decreases to about 0.2% by weight. It has been found that glucagon is retained in citric acid buffer during said crystallization. Isolation of glucagon from citric acid buffer means precipitation with excess zinc followed by salt precipitation and two precipitation! at pH values of 5.1 and 5.6 in each case, thereby removing zinc. The yield of crude glucagon from citric acid buffer corresponds to about 0.8 to 0.9 mg per 0.45 kg of pancreas, while the yield of purified glucagon from pancreas corresponds to about 0.1 to 0.3 mg per 0.45 kg of pancreas. The use of zinc for precipitation makes it difficult to completely remove the zinc while still increasing the amount of insulin in the final purified glucagon.

In studying pancreatic extracts, it has been found that the various acidified alcoholic extracts, as commonly used in insulin production, contain 4-6 mg glucagon per 0.45 kg pancreatic. After concentrating and defatting the extract, the glucagon content is reduced to 1.5-2.0 mg per 0.45 kg pancreas. However, as mentioned above, only 25 to 50 wt. this amount for further purification, depending on the starting material.

SUMMARY OF THE INVENTION The present invention provides a process for the isolation of glucagon, characterized in that glucagon-containing proteins are isolated from the supernatant after crystallization in an alkaline medium in the manufacture of insulin by isoelectric precipitation at pH values of 4.2 to 6.6 or ion exchange chromatography or followed by glucagon fractionation, whereupon the glucagon is separated from the other proteins by the formation of glucagon fibrils at a pH of 1.5 to 2.7 and the purification of the glucagon is carried out by crystallization at a pH of 4.5 to 8.5.

In the accompanying scheme, a preferred embodiment of the process according to the invention is also shown, which also shows the relationship between carrying out the process according to the invention and the degree of crystallization in an alkaline medium in the production of insulin.

The source of glucagon is a supernatant liquid from the crystallization stage of insulin in an alkaline medium as described in US Patent No. 3,719,655. The liquid is an aqueous solution having a pH of 7.2 to 10.0 with a cation concentration of 0.2 to 1. 0.1 M and dissolved proteins of which approximately 1 to 10% are insulin and 0.2 to 1.5% glucagon, depending on the pancreas used in insulin production. For example, when using bovine pancreas, the supernatant after crystallization in an alkaline medium contains a protein of which approximately 5 to 10% is insulin, 1.0 to 1.5% glucagon. When using porcine pancreas, the protein from the supernatant usually contains about 1-2% insulin and about 0.2-0.3% glucagon. It should be noted, however, that the actual yield of glucagon from batch to batch can vary considerably over a wide range due to the easy degradation of glucagon by enzymes.

Separation of the glucagon-containing protein from the supernatant after alkaline crystallization is the first step of the process. Generally speaking, separation can be achieved by any known method, e.g., the pH of the supernatant can be adjusted to about 3.0, followed by the addition of sodium chloride in an amount of 20% by weight. it precipitates glucagon and other proteins. Otherwise, precipitation can be achieved by adding an excess of zinc chloride at a pH of about 6.0. The third procedure involves the use of isoelectric precipitation. Yet another method involves the use of ion exchange chromatography. The second stage of the procedure is the separation of glucagon from other proteins. Basically, this separation can be carried out by any known method, such as gel filtration, ion exchange chromatography, isoelectric precipitation and glucagon fibril formation, of which the formation of glucagon fibrils is the most preferred.

The third and final stage of the process is purification of the glucagon obtained in the second stage. In general, the purification can be carried out by any known method known to those skilled in the art, such as crystallization, ion exchange chromatography, and the like. Crystallization is a preferred procedure.

The use of isoelectric precipitation or ion exchange chromatography, or a combination of the two, is a preferred method for separating glucagon-containing proteins from the supernatant after alkaline crystallization. Generally, isoelectric precipitation requires the ph value of the supernatant to be in the range of about 4.2 to about 6.6. A preferred pH range is from about 4.6 to about 5.2, with the most preferred range being from about 4.7 to about 5.0.

Since the supernatant after alkaline crystallization has a pH of from about 7.2 to about 10, the liquid should be acidified to the desired pH. Acidification is usually by adding a dilute solution of an inorganic or organic acid that does not break down glucagon without reacting with it. Examples of suitable acids include hydrochloric, phosphoric, formic, acetic, propionic and the like. Hydrochloric acid is preferred.

Optionally and preferably about 20% by volume of alcohol may be added, thereby reducing the solubility of the glucagon containing proteins in the supernatant after alkaline crystallization. The term alcohol means a saturated aliphatic monohydric alcohol having at most five carbon atoms, preferably at most three carbon atoms, such as methanol, ethyl alcohol, propanol and isopropyl alcohol, with ethyl alcohol being most preferred. If such an alcohol is used, it is usually added to the supernatant prior to acidification.

After acidification of the supernatant to the desired pH, the glucagon-containing protein precipitates usually within a few minutes. To achieve complete precipitation, the solution is allowed to stand, usually at a temperature of not more than 20 ° C. Preferably, the solution is cooled to a temperature of from about 3 to about 10 ° C. Although precipitation is generally complete within 24 hours, the mixture can be allowed to stand for as long as possible without detrimental effect.

After the precipitation is complete, the precipitate, hereinafter referred to as the isoelectric precipitate, is isolated by any known method, such as centrifugation or filtration; filtration is generally preferred. The isoelectric precipitate thus obtained does not need to be washed or dried, although these procedures can be used if desired.

Another method suitable for separating glucagon-containing proteins from the supernatant after crystallization in an alkaline medium is ion exchange chromatography.

Among the described processes of this kind, the process described in U.S. Pat. No. 3,715,345 is particularly effective and hence particularly preferred. In short, the ion exchange chromatography process is based on the flow of a glucagon-containing solution through a resin, a sulfonated macroreticular copolymer of styrene and divinylbenzene. in an alkali metal cycle at a pH of 7 to 8. Glucagon is adsorbed onto the resin and then eluted with a dilute base solution such as 0.1 N ammonia solution. The glucagon containing eluate is acidified to pH 2.5 and glucagon is precipitated using a conventional salting out procedure. The resulting glucagon-containing precipitate is thereby essentially separated from the isulin proteins, but the precipitate still contains other non-glucagon materials.

When the supernatant is treated after the crystallization of insulin in an alkaline medium using the procedure of U.S. Pat. No. 3,715,345, an eluate containing insulin and a solid containing glucagon-containing salt is obtained. The insulin eluate may be recycled to the insulin production process if appropriate. The solid portion of the glucagon-containing salt is redissolved for further processing; for simplicity, the pH of the solution and the protein concentration are usually adjusted to about the same value as the supernatant after crystallization in an alkaline medium.

it may often be advantageous to carry out the first step of the process, i.e. separating the glucegon-containing proteins from the supernatant after alkaline crystallization by isoelectric precipitation followed by ion exchange chromatography one after the other. For example, the supernatant from alkaline crystallization may be treated by ion exchange chromatography. The solid portion of the glucagon-containing salt, as obtained, is redissolved and subjected to isoelectric precipitation as described above. For variation, the supernatant after crystallization in an alkaline medium is treated by isoelectric precipitation, the precipitate thus obtained may be dissolved in a weakly alkaline aqueous medium, and the solution is treated by ion exchange chromatography. In any case, the insulin-containing eluate can be recycled from the ion exchange chromatography, if necessary, to the insulin processing process.

As mentioned hereinbefore, the preferred method of separating glucagon from other proteins is the formation of glucagon fibrils. Generally, the formation of glucagon fibrils is carried out in an acidified aqueous solution. Typically, glucagon-containing proteins are dissolved in an acidified, aqueous medium of less than about 2.7. Although the pH can usually vary from about 1.5 to about 2.7, a preferred pH range is from about 2.0 to about 2.5. And although any of the acids that are suitable for use in acidifying the supernatant after crystallization in an alkaline medium prior to the use of isoelectric precipitation can be used, hydrochloric acid is again desirable. However, a preservative such as phenol, usually at a concentration of about 0.2%, calculated as weight / volume, should be used using hydrochloric acid. Typically, the glucagon-containing protein is dissolved in a 0.01 N hydrochloric acid solution containing 0.2% phenol, by weight / volume.

The concentration of glucagon-containing proteins in the solution can usually vary from about 2.5 to about 30 mg / ml. The preferred range is from about 5 to about 20 mg / ml, and the most preferred range is from about 5 to about 10 mg / ml.

Optionally, a water-soluble inorganic salt may be added to the acidified solution of the glucagon-containing protein to initiate fibril formation. The term soluble salt means an inorganic salt which is soluble in water at the concentration used as described above. Since it is believed that the inorganic salt has a primary salting effect on the initiation of fibril formation, the choice of inorganic salt is not critical. In practice, however, the use of radioactive, poisonous or colored salts, or salts which are characterized by oxidative effects, or which are reducing agents, or are strongly acidic or basic, is not desirable for obvious reasons. Thus, suitable inorganic salts generally include water-soluble ammonium salts, water-soluble alkali metal salts, and salts of elements up to the sixth group of the Periodic System, as well as water-soluble, alkaline earth metal salts containing elements of the sixth group periodic system. Examples of such salts include, but are not limited to, ammonium bromide, ammonium chloride, ammonium fluoride, ammonium iodide, magnesium ammonium sulfate, manganato ammonium sulfate, ammonium nitrate, ammonium sulfate, lithium chloride, lithium fluorosilicate, lithium fluorosulfonate, lithium iodide, lithium iodide, lithium iodide, lithium iodide. lithium nitrate, potassium-potassium sulfate, sodium-ammonium phosphate, sodium-ammonium sulphate, sodium bromide, sodium chloride, sodium iodide, sodium hexafluorophosphate, sodium fluorosulfonate, sodium magnesium sulphate, sodium nitrate, sodium hexametaphosphate, sodium phosphate orthophosphate, sodium phosphate orthophosphate sodium sulfate, potassium bromide, calcium chloride, potassium chloride, potassium fluoride, potassium iodide, magnesium magnesium sulfate, potassium magnesium sulfate, potassium magnesium chloride, potassium molybdate, potassium orthophosphate, sodium potassium sulfate, rubidium bromide, rubidium chloride, rubidium chloride ý, rubidium iodide, rubidium nitrate, cesium bromide, cesium chloride, cesium fluoride, cesium didium, cesium nitrate, cesium sulphate, beryllium bromide, beryllium chloride, beryllium nitrate, beryllium orthophosphate, magnesium iodide, magnesium bromide, magnesium nitrate, magnesium silicofluoride, calcium bromide, calcium chloride, calcium iodide, calcium nitrate, strontium bromide, strontium chloride, strontium iodide, barium bromide, barium iodide, and their corresponding water-soluble hydrates and the like. Preferred salts are ammonium salts, with ammonium sulfate being the most preferred salt. Suitable inorganic salts may generally be used at a concentration of up to about 0.5 M. Preferably, such salts may be used at a concentration of from about 0.01 to about 0.05 M.

The temperature range for the formation of glucagon fibrils is usually in the range of about 20 ° C to about 30 ° C. A preferred temperature range is from about 24 ° C to about 26 ° C, and most preferably the temperature is 25 ° C.

Generally, fibril formation, which is aided by agitation, usually begins within about 3 to 4 hours from the time of preparation of the acidic aqueous glucagon solution and is completed in about 48 hours. Times above about 48 hours are usually not necessary.

Once fibril formation is complete, the fibrils formed can be isolated by any of the known methods, such as filtration or centrifugation, and the latter can be described as preferred. If desired, the glucagon fibrils may be washed by suspending the fibrils in an acidified aqueous medium, which may optionally contain an inorganic salt. The temperature of the wash medium can range from about 20 to about 30 ° C, with 25 ° C being preferred. The fibrils are then collected as before and stored refrigerated, usually at a temperature below about 10 ° C, unless the next step is performed immediately.

If desired, a chelating agent such as ethylenediaminetetraacetic acid may be added to the fibril forming environment. The concentration of the chelating agent is usually up to about 0.01 M, with a preferred concentration being 0.004 M. However, the use of a chelating agent is not preferred unless zinc ions or other divalent metal ions are present in solution simultaneously.

As mentioned hereinbefore, crystallization is a preferred method of purifying glucagon as obtained by separation from other proteins. Generally, crystallization of glucagon is accomplished by dissolving glucagon in an alkalized aqueous medium, acidifying the medium to a slightly alkaline or acidic region, thereby initiating crystallization, meaning that the pH should be in the range of about 4.5 to about 8, 5.

Dissolution of glucagon can be done in two ways; firstly, glucagon may be dissolved directly in an alkaline aqueous medium or the glucagon may be suspended in distilled water and the pH adjusted by the addition of an aqueous base solution. Suitable bases are generally alkali metal hydroxides and ammonia, with potassium hydroxide being the preferred compound.

Generally, the glucagon solution thus prepared may have a pH in the range of about 9.0 to about 11.5, with a range of about 9.5 to about 10.5 being preferred.

The concentration of glucagon in the alkaline solution should be in the range of about 2 to about 10 mg / ml. A preferred concentration is from about 4 to about 8 mg / ml, with a concentration of about 5 mg / ml being most preferred.

Since acidification often results in the immediate precipitation of a small amount of non-glucagon proteins, the acidification procedure below, although not essential, is preferred.

The alkaline glucagon solution is heated to a temperature of from about 55 to about 65 ° C, then the solution is acidified to a pH of from about 4 to about 6 by the addition of 10% phosphoric acid. Although any of the acids useful in the preceding step can be used, it is preferred to use phosphoric acid. The resulting solution is then filtered while maintaining the higher temperature. Generally speaking, this degree of filtration is merely optional and is often neglected if the amount of precipitate resulting from acidification is minimal. Furthermore, other methods may be used instead of filtration, for example centrifugation, if desired.

If the glucagon solution is decolorized, the activated carbon is added either before or after acidification, preferably before.

After acidification and filtration if necessary, leave the glucagon. the solution stands at a temperature in the range of about 2 to about 10 ° C and crystallizes for about 24 to about 120 hours. Preferred temperatures are 4 ° C, and preferably the crystallization time is from about 72 to about 120 hours, most preferably the crystallization time is 72 hours.

The bulk of the supernatant is decanted from the precipitated glucagon crystals, usually through a filter. The remaining portion of the supernatant is separated from the glucagon crystals by centrifugation. The purified glucagon is then washed sequentially with dilute sodium chloride solution (usually 0.001%) and water, then lyophilized and refrigerated.

Depending on the purity of the glucagon from the separation stage from the other proteins, i.e., the second stage (B), and depending on the desired purity of the final product, one or more additional crystallizations may be used. However, it has been found that a total of 2 crystallizations are usually sufficient to obtain glucagon with a purity of at least 80% based on the bioassays in cats.

If 2 crystallizations are used, the following procedure is preferred: The first crystallization is carried out as described hereinabove and the crystals are dissolved in an alkaline solution. In the second crystallization, acidification is performed to a pH of from about 7.0 to about 8.5, preferably from about 7.3 to about 7.5, and most preferably to about 7.4. The dissolution and filtration process, if performed, is preferably carried out at a temperature of about 35 to about 45 ° C, most preferably at about 40 ° C. The glucagon concentration is most preferably about 10 mg / ml.

If desired, the supernatant may be recycled from any or all of the crystallizations. E.g. any portion of the protein optionally present may be precipitated from said supernatant by adjusting the pH using dilute hydrochloric acid to a range of 2.5 to 1.0 followed by addition of 20% sodium chloride, by weight / volume. The precipitate obtained can be used in the first step of the process of the invention either separately or after dissolution in an alkaline liquid above the sediment after crystallization.

As mentioned hereinbefore, usually the alkaline supernatant after crystallization in solution contains substantially more insulin than glucagon. Said proportion of insulin is a component of the glucanone-containing proteins as obtained by isoelectric precipitation. Although in the second stage of the process, i.e., when separating glucagon from other proteins, glucagon is effectively separated from insulin effectively, the separation process may appear to be very detrimental to the insulin component of the protein mixture. As a result, it is often desirable to carry out the first step of the process using ion exchange chromatography, which also permits the separation of insulin from glucagon.

It should be pointed out, however, that ion exchange chromatography is not the only way of separating insulin from glucagon before separating glucagon from other proteins. E.g. after isoelectric precipitation, the precipitate may be treated by a process known as hyperglycemic factor fractionation. In short, this procedure depends on salting out glucagon from a mildly alkaline, phenol-aqueous medium. The fractionation of the hyperglycemic factor is described by Staub and coworkers, supra. The supernatant containing insulin is recycled to the insulin purification process, while the precipitated crude glucagon is used in the second step of the process.

Although not essential to the process of the present invention, it is preferred that the supernatant from the fibril formation step be recycled to the insulin process, usually to the beginning of the alkaline crystallization step as described in U.S. Patent 3,719,655. recycling is necessary if insulin has been separated from glucagon as described herein before fibril formation. However, in such a case, the separated insulin is recycled to the insulin process.

The process of the invention and its relationship to the insulin process may be better understood with reference to the illustration, which is a schematic of one embodiment of the process of the invention. For the sake of simplicity, the liquids obtained as a fraction of the sediment from the first and third stages of the process are plotted as if they were not needed, and it is understood that these liquids can be recycled as described herein above.

The alkaline crystallization process of U.S. Pat. No. 3,715,655 is shown at the top of Scheme J. In this process, an alkaline or ammonium salt of insulin 6 and supernatant 2 are obtained. Parts of the insulin process are indicated by dashed line 2. outside the dashed line region 2 forms a glucagon process.

The supernatant 2 is recovered from which an isoelectric precipitate is obtained. The remaining supernatant 2 is discharged to waste 10. Further, glucagon fibrils J and crystalline glucagon 4 are obtained. The supernatant 11 of the crystallization of glucagon is discharged to waste 12.

Temperature values are given in degrees Celsius.

Example 1

The supernatant after alkaline crystallization from a treatment of 5 750 kg of 14.75 liter cattle and swine pancreas with a solids content of 40.7 mg / l was diluted by adding 1.45 liters of absolute ethanol. The pH of the resulting solution was adjusted to 5.2 by addition of 3 N hydrochloric acid and the solution was cooled to 5 ° C overnight. The precipitate formed is filtered off, dissolved in 11.28 liters of a 0.01 N hydrochloric acid solution containing 0.2% phenol (w / v, hereinafter referred to as a mixture of acid, phenol and water), and testing the obtained The following values were found in the solution:

total solids content: 512.6 g (45.4 mg / ml) insulin: 87.43 units / ml (1.93 units / mg solids) glucagon: 463.7 µg / ml (1.02% of shares).

For testing, a total of 870 ml was removed from the solution leaving 10.4 liters of a solution containing about 473 g of solids.

For hyperglycemic factor fractionation, the above solution is diluted by adding 111.8 liters of an acid / phenol / water mixture to a total volume of 122.2 liters (the solids content is then 0.387). 245.8 ml of liquefied phenol, 941 g of sodium chloride and a sufficient quantity of 40% aqueous sodium hydroxide solution are added to the dilute solution to adjust the pH to 9.0 to dissolve all solids. The pH is then adjusted to 7.5 by addition of 3 N hydrochloric acid and cooled to 5 ° for 1-2 days. During this time a precipitate formed, the supernatant was decanted and the residue was suctioned off. In addition, the precipitate is centrifuged, the solids from filtration and centrifugation are combined and dissolved in 10 liters of acidified phenolic water. Testing the solution gives the following values:

total solids content: 75.0 g (7.5 mg / ml) insulin: 6.33 units per ml glucagon: 275 µg / ml (5.01% of total solids)

The solution is diluted to a solids concentration of 5.0 mg / ml by adding an additional 5 liters of a mixture of acid, phenol and water, and 5.0 liters of the solution thus prepared are collected to give 10.0 liters of a solution containing about 50 g of solids. .

To the remaining solution was added, with stirring, 8.0 ml of a 0.5 M aqueous solution of ethylenediaminetetraacetic acid as the tetrasodium salt and 60 ml of a 50% aqueous solution of ammonium sulfate. Stirring is continued for 16 hours at room temperature, the glucagon fibrils which are formed are washed twice after centrifugation with a mixture of acid, phenol and water containing ethylenediaminetetraacetic acid and ammonium sulphate, as hereinbefore described.

Glucagon fibrils are suspended in 10 liters of water containing, by weight, 0.2% phenol per volume of water. The mixture is heated to 40 °, the pH is adjusted to 10.5 by addition of 150 ml of 10% aqueous potassium hydroxide solution, the solution is heated to 60 ° and the pH is adjusted to 7.8 by adding 68 ml of 0% phosphoric acid solution. . When the temperature reached 60 ° C, the pH was further lowered to 5.0 by addition of an additional 68 ml of 10% phosphoric acid solution, whereupon the solution was filtered while hot without suction and the filtrate was cooled with stirring. Cooling to 5 ° is carried out for 72 hours and the precipitated glucagon is filtered off. The solid thus obtained was dissolved as described above for glucagon fibrils, but after adjusting the pH to 10.5 the total volume was 870 ml. The solution is heated to 60 °, the pH is adjusted to 5.0 by the addition of a 10% phosphoric acid solution, and the resulting solution is filtered while hot without suction. The filtrate is cooled with stirring as described above. The precipitated glucagon is centrifuged and lyifilized, yielding 1.84 g of glucagon. This corresponds to 74% calculated on glucagon contained in the mixture prior to fibril formation and 39% glucagon contained in the mixture after precipitation of proteins from the supernatant after crystallization in an alkaline medium. (for these findings samples were taken).

Example 2

Alkaline mother liquors from 3 crystallizations of insulin using 28,365.5 kg, 36,902.7 kg and 48,778.6 kg of a 2: 1 mixture of bovine and porcine pancreas are centrifuged from the insulin crystals. The volume of said mother liquors is 460, 515 and 680 liters in each case, and the proportions are diluted by adding 51, 57.2 and 75.5 liters of absolute ethanol in each case, in each case acidifying to pH 5.0 by adding 3 N hydrochloric acid solution, and after stirring for 15 minutes, the reaction mixtures are cooled for at least 24 hours. Tests of mother liquors before precipitation and precipitate solutions:

1. 1,150 µg of glucagon and 228 units of insulin per 0.45 kg of the original pancreas in the mother liquor; 839 µg of glucagon and 291 units / 0.45 kg of the original pancreas, 59.7 mg solids per 0.45 of the original pancreas in clot solution, volume 158 liters;

2. 736 µg of glucagon and 139 units of insulin per 0.45 of the original pancreas in the mother liquor; 345, µg of glucagon and 123 units of insulin per 0.45 kg of the original pancreas, as well as 27.8 mg solids per 0.45 kg of the original pancreas in the clot solution, volume 1.50 liters;

3. 1 214, µg of glucagon and 218 units of insulin per 0.45 kg of the original pancreas in the mother liquor and 946 µg of glucagon and 191 units of insulin per 0.45 kg of the original pancreas, as well as 59.0 mg solids per 0.45 kg of the original pancreas in a precipitate solution with a flow rate of 156 liters. The precipitates were filtered off and dissolved in a mixture of acid, phenol and water, prepared as described in Example 1.

The solutions were combined to give 464 liters of 12 570 g solids; Dilution with 630 L of an acid / phenol / water mixture gave a solution containing 2% solids and then adjusted to pH 2.1 by further addition of 3 N hydrochloric acid. To the solution was added 0.3% ammonium sulfate as a 50% solution (6 mg per liter, a total of 3,780 mL) and stirred for 24 hours at 25 ° while forming fibrils, then allowed to stand for 48 hours at room temperature. 15 ° · Glucagon fibrils are centrifuged from the remaining solution and the supernatant is tested (containing 222 µg of glucagon and 114 units of insulin per 0.45 kg of the original pancreas) before being recycled to insulin processing. The glucagon fibrils are suspended in 450 liters of cold water containing 0.2 Ϊ phenol, 2700 ml of 50% ammonium sulfate solution are added to the suspension, and the reaction mixture is stirred slowly for 30 minutes to wash the fibrils, which are centrifuged again. to the need). The glucagon fibrils are suspended in 275 liters of water containing 0.2% phenol and the pH is adjusted to 3.85 using 10% phosphoric acid. Testing revealed: solids content of 5.48 mg per 0.45 kg of the original pancreas (0.52%), i.e. 1,404 g of the original 115,047 kg of the original pancreas. Glucagon content: 212 ^ / 0.45 kg of original pancreas, total 54.2 g.

275 liters of glucagon fibril suspension are divided into 2 parts, 1 135 liters and 2 liters.

140 liters for the first crystallization. The first fraction is diluted by adding 140 liters of 0.2% phenol water to give a 0.5% solution. The other fraction is left at a solids content of 0.52%. Each of the fibril suspensions is heated to 60 ° and the pH is adjusted to 9.0 to 11.0, namely in the case of 1. to 9.9 and in the case of 2. to 9.7 using 10% aqueous potassium hydroxide solution. . The result is a clear solution. 281 g of activated carbon (Norit A, 0.4 g per g of solids) are added and after stirring for 10 minutes, the pH of the solution is again adjusted to 5.0 by addition of a 10% phosphoric acid solution. The reaction mixture is filtered while still hot under suction with ED No. 613 filter paper. The filtrate (crystallization solution) is further slowly stirred for 16-20 hours under cooling to 4 °, promoting uniform crystallization of glucagon. The precipitate, which was collected at pH 5.0 at 60 °, was further processed to obtain an additional portion of crystalline glucagon by suspending the precipitate in 120 liters of 0.2% phenol water at a solids concentration of 0.5% (solid test: 8.0 g) heating the suspension to 60 °, adjusting the pH to 10.0, dissolving the solids, stirring for 10 minutes and adjusting the pH to 5.0 again by adding · 10% phosphoric acid solution ; while still hot, the reaction mixture was filtered without suction. The filtrate was slowly stirred for 16-20 hours under cooling to 4 °, whereupon crystallization was complete in 72-120 hours.

A second precipitate at pH 5.0 is not needed. The mother liquor proper after crystallization from the first and second crystallization mixtures is filtered off with suction after decantation, to the first mother liquor 20% sodium chloride solution is added in such a quantity as to precipitate any part of the glucagon that has not crystallized. These precipitates are mixed with other similar portions and reprocessed by precipitation at pH 4.6 with further crystallization to give an additional portion of glucagon crystals. The glucagon crystals from the first two fractions representing the first yield and the crystals from the subsequent treatment of the combined precipitates after the first crystallization represent the second yield; The batches are combined, centrifuged for 3 minutes, separating the mother liquors and then transporting the solid to the lyophilization containers using water as the suspension medium and lyophilizing. Glucagon crystals after intermediate crystallization are weighed and samples are tested. Glucagon Crystals from Crystallization Environment: Yield 1: 1. 41.0 g (91.7% purity) and 2. 44.0 g (86.8% purity), equivalent to 85.0 g of 89% pure glucagon 1%, i.e., 75.8 g of pure glucagon, i.e. 292 µg per 0.45 kg of the original pancreas, or 332 µg / 0.45 kg of the original pancreas in the glucagon state as isolated.

First processing of the crystals (second yield): 19.0 g (100% purity), 75 µg per 0.45 kg of the original pancreas, calculated on both the pure product and the product obtained. Second processing of crystals (first mother liquor), yield 22.0 g (purity 46%) or

9.9 g of pure substance, i.e. 39 µg ^per 0.45 kg of original pancreas (calculated as pure) or 85 pigs per 0.45 kg of original pancreas, calculated on the substance as isolated. Total yield 126.0 g. Solids content: 126.0 g (476 fig for 0.45 kg of original pancreas), purity 83.1% or 104.7 g of pure glucagon (409 fig for 0.45 kg of original pancreas) .

Recrystallization was performed in conjunction with other intermediate crystallization crystals as these fractions accumulated. The final crystals of glucagon are 85% by weight of the crystals used. Recrystallization was carried out at pH 7.5 after dissolution of the obtained crystals at pH 8.0-10.0 in 10% aqueous potassium hydroxide solution at 40 °, with a solids content of 1.0%, followed by pH adjustment to 7, 5 by adding a 10% phosphoric acid solution followed by filtration and cooling. The final crystals were isolated by centrifugation after decanting the major portion of the mother liquor (which was recycled until further crystallization), washing twice with 0.001% sodium chloride solution, followed by cold water washing and lyophilization. The calculated yield is 89.0 g or 348 µg per 0.45 kg of the original pancreas and the recovery yield from the alkaline mother liquors after crystallization is 33.2%.

Example 3

The mother liquor after crystallization in an alkaline medium of 425 liters pH 7.5 containing 65 µg / ml glucagon is passed through an ion exchange resin consisting of the alkali metal or alkaline earth metal salt of a sulfonated macromolecular copolymer of styrene and divinylbenzene (Amberlite 200). .

The 630 liter fraction containing glucagon contains 33.6 g of glucagon in 4047 g of solids. An aliquot of this 32 L fraction was concentrated to a volume of 4.38 L at pH 3. Potassium hydroxide was added to this concentrate until a pH of 9.5 was reached. The resulting solution contained 1.3 g of glucagon in 82.5 g of solid.

Half of the concentrate, containing 0.65 g of glucagon, was adjusted to pH 2.1 by addition of dilute hydrochloric acid followed by addition of phenol at 2.2 ml / l, sodium salt of ethylenediaminetetraacetic acid at a concentration of 0.1 M at 4 ml / l, and 50% ammonium sulphate in an amount of 0 ml / l. The mixture was then stirred at room temperature for two days.

The resulting glucagon fibrils are separated from the mother liquor by centrifugation. The mother liquor contained 0.42 g of glucagon. The fibril fractions were dissolved in dilute potassium hydroxide solution to give 212 mL of a solution containing 1.89 g of solid and 0.307 g of glucagon.

The solution was heated to 60 ° and the pH was adjusted to 5.0 by addition of hydrochloric acid. The solution was then cooled to 5 ° for 72 hours. The precipitate was collected by filtration. The mother liquor contains 0.004 g of glucagon. The precipitate consists of 1.35 g solids containing 0.157 g glucagon. Yield 24%.

Claims (10)

OF THE INVENTION Glucagon isolation method, characterized in that glucagon-containing proteins are isolated from the supernatant after crystallization in an alkaline medium in the production of insulin by isoelectric precipitation at a pH of from 4.2 to 6.6 or by ion exchange chromatography or isoelectric precipitation followed by subsequent precipitation. hyperglycemic factor fractionation,. thereafter, glucagon is separated from other proteins by the formation of glucagon fibrils at a pH in the range of from 1.5 to 2.7 and purification of the glucagon is accomplished by crystallization at a pH in the range of from 4.5 to 8.5. 2. The process according to claim 1, wherein the isoelectric precipitation is carried out in the presence of up to 20% by volume of a saturated aliphatic monovalent alcohol having 1 to 4 carbon atoms. 3. The process according to claim 2, wherein the aliphatic monovalent alcohol is ethyl alcohol. 4. The process of claim 1 wherein said fibril formation is carried out in the presence of an inorganic salt. 5. The process of claim 4 wherein said salt is ammonium sulfate. 6. The process of claim 5, wherein the concentration of ammonium sulfate is from 0.01 to 0.05 M. 7. The process of claim 1 wherein the crystallization is carried out at a glucagon concentration of from 2 to 10 mg glucagon per ml of solution. 8. The process of claim 1, wherein two successive crystallizations are carried out. 9. The process of claim 8 wherein the first crystallization is carried out at a pH in the range of 4.5 to 5.5 and the second crystallization is carried out at a pH in the range of 7.0 to 8.5. 10. The process of claim 1, wherein the glucagon-containing protein is isolated from the supernatant by the crystallization process in an alkaline medium by isoelectric precipitation at pH 5.2, followed by hyperglycemic factor fractionation at pH 7.5. whereupon glucagon is separated from other proteins by the formation of glucagon fibrils in phenolic water, acidified with hydrochloric acid in the presence of ethylenediaminetetraacetic acid and ammonium sulfate, and the glucagon thus obtained is purified by crystallization at pH 5.0.