CS199576B2 - Method for the purification of insulin complex with alkaline metals or ammonium ions - Google Patents

Description

The present invention relates to a process for purifying an insulin complex with alkali metals or ammonium ions.

Accordingly, the present invention provides a method of purifying an insulin complex with an alkali metal or ammonium ion, comprising swelling a gel having a dry swelling of 4 to 98% by weight as well as a particle diameter of 20 to 80 microns, whereupon the column is filled with swollen gel, an aqueous solution of an alkali metal complex with ammonium ion of at least 80 percent purity is added to the packed column, the concentration of insulin being in the range of 1 to 8% w / v. % and the total amount of insulin is sufficient to provide a column load in the range of 0.8 to 100%

6.7 g per liter of bed volume and pH of the solution ranged from 2.0 to 3.5, after which the elution of insulin from the column was carried out at a temperature ranging from 5 to 30 ° C using an aqueous eluent containing an inorganic salt at a concentration of up to 0.02 M and a pH in the same range as it has. insulin solution.

The attached figure shows the elution diagram for Example I. The optical density at 280 πΐμ of some insulin solution fractions during gel filtration is applied.

Since the volume of insulin as a component of pancrea199576 su in 1921, it has a worldwide importance in the treatment of diabetes (Diabetes mellitus). Since substantial amounts of non-insulin proteins are also extracted from the pancreas tissue extraction procedures, considerable efforts have been made to identify insulin purification procedures, i.e. methods of separating insulin from noninsulin proteases. Some of the insulin purification procedures thus found are of commercial importance.

Of these applicable methods, one of the oldest is based on the general phenomenon that the protein is characterized by the lowest solubility at the isoelectric point under other constant conditions. US patent 1469 944 describes a method for purifying insulin, characterized in that noninsulin proteins are precipitated from the aqueous pancreas extract at the corresponding isoelectric points. Isoelectric precipitation of ihsulin from the aqueous extract of the pancreas at a pH in the range of about 4 to about 7 is described in U.S. Patent No. 1,520,673.

In another prior art, precipitation of ihsulin is accomplished by adding sufficient inorganic salt. Thus, U.S. Pat. No. 1,547,515 discloses a first method of salting out insulin from a solvent used in extraction by adding sodium chloride. A differential salting-out process is described in U.S. Pat. No. 2,448,076, which involves salting out the insulin from a neutralized extract by adding sodium chloride, filtering off the solid, redissolving the residue, and re-salting out insulin, this time at a lower salt concentration than it was used in the first salting out stage.

In a third operationally important method of purifying insulin, precipitation or crystallization of insulin from solution in the form of an insulin-zinc complex is used. Generally, this complex precipitates from a buffered aqueous solution containing zinc ions. U.S. Pat. No. 2,626,228 discloses, for example, the use of a citric acid buffer and a salt thereof from numerous possible other buffers.

Currently, the commercially available methods of purifying insulin utilize all 3 of the above methods, and since the zinc insulin complex is the most important commercial form of insulin, usually the final stage of insulin purification is the degree of crystallization of the zinc complex.

However, it has been found in recent years that noninsullin proteins, predominantly of proinsulin and proteins similar to it, constitute an increasingly small proportion of the commercial form of insulin, i.e., complex with zinc. Although these components typically represent less than about 8% by weight of the zinc insulin complex; it is believed that some of these components are antigenic or immunogenic in nature.

See, for example, Chance et al., Science 161, 165 (968); Steiner et al., Diabetes 18, 725 (1968); Bromer, BioScience 20, 701 (1970) and Rubinstein et al., Ann. Roar. Copper. 22, 1 (1971).

Thus, an ongoing problem is the removal of non-insulin proteins from insulin on an industrial scale.

The term "insulin" as used herein includes not only insulin as such, but also insulin-like proteins such as desamidoinsulin. Since insulin and insulin-like proteins also have similar hypoglycaemic properties, it is usually not necessary to separate insulin from other pancreatic proteins, i.e., it is usually not necessary to prepare homogeneous insulin.

Methods for separating biological substances on an analytical support, such as electrophoresis and ion exchange chromatography, are known, and thus the non-insulin components can be separated from the insulin component from commercial and crude insulin preparations. These methods can also separate insulin components into insulin per se and deamidoinsulins. However, these processes have problematic applicability in large-scale processes.

Gel filtration (equivalent to gel exclusion chromatography or * gel permeation chromatography) is a method that is easier to adjust for large scale use. In this procedure, proteins are separated on a gel-containing column cross-linked in such a way that pores are formed in each gel particle. They are characterized by a defined, measurable volume that is proportional to the degree of swelling of the gel and inversely proportional to the degree of crosslinking. Because smaller molecules have better access to these pores than larger molecules, the passage of smaller molecules through the column is hampered compared to larger molecules having only partial access to the pores, or no access at all.

Gel filtration has recently been used to purify insulin. Isolation of insulin from cat pancreas yields crude insulin by extraction with acidified alcohol followed by precipitation in an alkaline medium to remove inactive materials, followed by isoelectric precipitation after concentration, followed by precipitation with saline. The crude insulin is then loaded onto a cross-linked dextran gel column, eluting with a 0.1 M acetic acid solution, see Davoren, Biochem. Biophys. Acta 63: 150 (1962).

To isolate insulin from fish, the gland is homogenized in water and proteins are precipitated with a trichloroacetic acid solution. The precipitate was extracted with acidified ethanol, and the lipid fractions were removed from the extract with methylene chloride. The remaining solids were isolated, dissolved in 5.0 M acetic acid solution, and the solution was filtered through a gel column from cross-linked dextran using 5.0 M acetic acid solution to elute, see Hurabel, Biochem. Biophys. Res. Commun. 12, 333 (1963). Yields of about 80% are reported. In addition, Humbel states that for good separation of insulin from other proteins, elution conditions should promote the dissociation of insulin molecules. It further states that a 1.0 M acetic acid solution is not a suitable solvent because, according to Yphantis and Waugh, Biochem. Biophys. Acta 26, 218 (1957) fails to fully dissociate insulin.

The crude bovine pancreas extract is filtered on a cross-linked dextran gel according to Epstein et al., Biochemlstry 2, 461 (1963). A 0.2 M ammonium bicarbonate solution is used as the solvent for elution, and this reagent is also supported by Humbel (cited above). Epstein and coworkers mention a yield of about 60 percent.

Insulin has also been isolated from human pancreas. The gland is first homogenized and extracted, further inactive materials are removed by ammonium sulfate fractionation, insulin is precipitated first with sodium chloride, then isoelectrically. The thus prepared insulin was dissolved in a 0.5 N acetic acid solution, and the solution was filtered on a cross-linked dextran gel. The filtered insulin solution is then converted to a zinc-insulin complex, see Jackson et al., Diabetes 18, 206 (1969). The yield of insulin after gel filtration is about 60%.

Subsequently, according to South African Patent 69/5280 (corresponding to Belgian Patent 737 257), a combination of ge199578 ion filtration and ion exchange chromatography is used. In a single example of gel filtration (Example 1J, the zinc insulin complex was filtered through a cross-linked dextran gel column in a 80% yield. A 1.0 M acetic acid solution was used as the elution solvent. After filtration, desalting, isoelectric precipitation, as well as a process for crystallizing the zinc complex to obtain a zinc insulin complex, must be carried out.

In each of the aforementioned uses of gel filtration for purification of insulin, substantial losses of insulin occur. Furthermore, it is not clear from the prior art that it can be relied on that only insulin, including insulin-like proteins, as mentioned above, can be obtained by gel filtration.

In general, any gel swellable by water and useful in working with protein solutions can be used in the process of the invention. Such a gel should be characterized by a dry or swollen ability to absorb water of at least 4% by weight, calculated on the weight of the dry gel, as well as a particle diameter below about 100 microns. Preferably, the water uptake capacity is from about 4 to about 98%, most preferably from about 5 to about 20%. Preferably, the dry gel particle diameter is also below about 80 microns, and a particularly suitable particle diameter range is from about 20 to about 80 microns.

Examples of suitable gels include, but are not limited to: starches, including corn starch, cross-linked galactomannan, cross-linked dextran, agar or agarose, polyacrylamides, copolymers of acrylamide and methylene-bis-acrylamide, copolymers of methylene-bis-acrylamide with vinylethylcarbinol and yinylpyrrolidine . Crosslinked dextrans such as sephadex series are preferred gels.

The type of column used in the process of this invention is not critical. The choice of column height, diameter, or arrangement depends on the operating parameters. It is clear that as the column height increases, the flow rate decreases; in other words, the back pressure in the column is proportional to the column height. For this reason, a tray column is preferred. A six-stage tray column is sufficient to use conventional production.

Satisfactory insulin purification can be performed under a column load of about 4.5 g of the insulin-alkali metal or ammonium salt complex per bed volume bed, which is the most advantageous load. However, the column load can vary from 0.8 to 6.7 g per liter bed volume, with the most preferred range being 3.5 to 5.0 g per liter bed volume. Of course, the best conditions are readily determined for any given column.

The gel can be swelled as well as the column can be loaded with swollen gel using any of a number of procedures known to those skilled in the art. Generally, the gel swells by the elution medium; otherwise, the gel can be swollen with 30% aqueous ethanol to decant off the fines, then replace the swelling medium with the elution medium.

The alkali metal insulin salt solution or the ammonium salt solution can be prepared by any suitable method. For example, insulin may dissolve in an appropriate amount of elution medium, or insulin may dissolve in an aqueous medium that is somewhat acidic but still within the pH range of the elution medium compared to the elution medium. For example, if a 0.5 M acetic acid solution having a pH of 2.5 is used as the eluent, Insulin can be dissolved in an aqueous medium having a pH of about 2.3, which corresponds to a 1.0 N acetic acid solution.

The solution of the insulin-alkali metal salt or the solution of the ammonium salt of insulin is usually alkaline in nature.

As a result, upon dissolution of the insulin in a neutral or acidic aqueous medium, the pH of the obtained solution is increased. Thus, when insulin is dissolved in a 1.0 N acetic acid solution, the pH of the resulting solution is raised to about 2.7. However, depending on the concentration of insulin used, the insulin solution may have a pH of up to about 3.2. This increase in pH is normal and does not cause problems, provided that the pH of the insulin solution does not exceed about 3.5. Alternatively, the insulin may be dissolved in a 0.5 N acetic acid solution and the pH adjusted to below about 3.2 by the addition of dilute hydrochloric acid.

Generally, the alkali metal salt of insulin or the ammonium salt of insulin can be prepared by any of the known methods. However, it is preferred to prepare the alkali metal salt of insulin or the ammonium salt of insulin according to the process of U.S. Patent 3,719,655. As mentioned hereinbefore, said insulin must have a purity of at least about 80%. That is, the insulin content, including insulin-like proteins, characterized by hypoglycaemic activity, must be at least about 80% of all proteins present.

The concentration of the alkali metal or insulin ammonium salt in the insulin solution as applied to the column is, as mentioned herein, from about 1 to about 8% (w / v). A preferred concentration is 4 to about 6%.

For satisfactory results, the alkali metal salt of insulin and the ammonium salt of insulin should be dissolved in a solvent volume that is less than the partition volume, as discussed herein below.

In chromatography, the distribution coefficient K _{d is} defined as the ratio of the concentration of the dissolved component in the mobile phase to the concentration of the dissolved component in the stationary phase. In gel filtration, the mobile phase is roz188578

The T solvent moving in the free space between the gel particles and the stationary phase is a solvent soaked into the gel particles, i.e. enclosed in the pores within each gel particle. Thus, K _d denotes the fraction of the soaked solvent into which the dissolved component has formed.

The elution volume V _{e of the} dissolved component can be expressed using K _d by the equation:

'Ve = V ₀ + K _d . VI where V _o is _the void volume of the column, and Vi is the volume vsáknutého solvent. For K _d , the equation can be adjusted to a relation

If the water density is considered to be 1,

Vj = aW _r , where a is the weight of the dry gel and W _r is the increment of water. Thus, for K _{d, one} can deduce and this can be determined experimentally by a skilled person.

Assuming a solution of two solutes, the elution volume for each solute can be expressed

V _s = V _e ”-Ve '

V ₀ ”== V ₀ + K _d '' V _i

The partition volume V _s is the difference between the elution volumes of the two dissolved components:

V _s = V _e '-V ₆ '

V _s = (K _d ”—KďJVj

It is clear from the foregoing that the degree of separation of two or more dissolved components is in part dependent on column load. As the proportion of the lower molecular weight component approaches the limit of available pores, the separation of the two dissolved components must necessarily decrease. Within the range of possible loads that form part of the invention, the degree of separation may vary. In some cases, a higher column load can be selected to balance the degree of separation against productivity.

As mentioned hereinabove, the elution medium may have a pH in the range of between about 2.0 to about 3.5. Generally speaking, an aqueous solution of any inorganic or organic acid may be used as the eluent, as long as the proteins are stable in such an environment. Such acids include, among numerous others, hydrochloric, phosphoric, _t formic, acetic, propionic, n-butyric, isobutyric, pentanoic acid and the like. Acetic acid is preferred. If hydrochloric acid is used, a preservative such as phenol must be added to the eluent. It should be noted that acetic acid is itself a preservative. A preferred pH range is about 2.5. Therefore, the most preferred elution medium corresponds to a 0.5 N acetic acid solution.

If desired, the elution medium may contain up to about 0.02 moles of inorganic salt per liter, and the useful salt is sodium, potassium, ammonium chloride and the like. A preferred concentration is 0.01 M, and a preferred salt is sodium chloride. The use of such a salt is preferred; this improves the separation and avoids possible adsorption of the basic substances to the gel.

The elution of the column can be carried out at temperatures ranging from about 5 to about 30 ° C. Preferably, it is operated at or below room temperature.

The elution pattern is monitored by any suitable method, and a particularly useful procedure is the measurement of ultraviolet adsorption at 280 nm of each fraction. Those fractions containing the expected purified insulin are pooled together and then converted to a zinc complex.

As mentioned above, the zinc-insulin complex is the most important form of insulin. Therefore, usually the alkali metal salt of insulin or the high purity ammonium salt of insulin obtained by the process of the present invention is converted to a zinc-insulin complex. This is done at pH 6.0 in the presence of ammonium acetate buffer. There is no need to isolate the alkali metal salt of insulin or the ammonium salt of the insulin from the eluent, or concentrate the purified insulin solution before crystallizing the zinc complex. An advantage of the process of the invention is that the insulin in solution as recovered is sufficiently concentrated after elution that there is no need for salt precipitation or isoelectric precipitation, although these procedures may be used if desired.

As a result of the process according to the invention, an insulin-zinc complex of increased purity is obtained. This can be done in several ways. On the one hand, it is possible to analyze the insulin-zinc complex for the content of noninsulin protein components such as proinsulin or glucogone. The presence of high molecular weight proteins, such as protaminase, a proteolytic enzyme, can be determined by monitoring the stability of the protamine-insulin suspension at elevated temperatures; in the presence of protaminase, the protamine-insulin complex dissociates due to protamine degradation. Physical properties such as the solubility of the zinc insulin complex at neutral pH as well as the color of the resulting solution can be measured. Subsequently, specific insulin activity can be monitored by biological and immunological tests.

Due to the higher purity, the insulin-zinc complex obtained after the process of the present invention is a desirable material for use in various pharmaceutical forms of insulin, such as insulin per se, isophan-insulin, protamine zinc insulin complex, complex suspension zinc insulin, globininsulin and the like. Furthermore, the purer insulin-zinc complex allows the preparation of a neutral insulin that is more stable than acidic insulin; impurities that have hitherto been present in the insulin-zinc complex precipitate partially to neutral pH. These impurities often contain enzymes that break down insulin, such as trypsin and chymotrypsin, reducing the effectiveness of insulin preparations. The purer insulin-zinc complex also permits the preparation of protruding preparations containing insulin from pigs and cattle, without further purification as needed in previous years. Finally, the preparation of isophane-insulin from a purer insulin-zinc complex requires less protamine, since the absence of acidic protein impurities allows only the amount of protamine required to precipitate insulin.

The process may be combined, as appropriate, with other purification processes. For example, the alkali metal insulin complex solution or the insulin ammonium salt solution may be filtered one or more times prior to the treatment.

AND 1405 10—69 B-l 1193 70—119 B-2 978 120—160. C 1520 161—225 and) % of total solid elution-

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To Group C, 3.0 mL of a 20% zinc chloride solution was added, and the resulting solution was diluted to 3025 mL with 0.5 N ammonia solution. The pH of the final solution is adjusted to 6.0 by addition of dilute ammonia solution, after which the precipitated zinc insulin complex is isolated by centrifugation and washed successively with water, absolute alcohol and ether, and then dried in vacuo. The yield of the zinc-insulin complex was 13.9 g; the insulin activity was 25.6 units / mg, the proinsulin content was 0.44%.

Since the solids content of Group C is 86.4% of all eluted solids, the maximum yield of solids from Group C is 86.4% of 17.5 g, or 15.1 g; on the basis of the units is 10.

gel filtration. It is also possible to filter the eluted insulin one or more times before the conversion to the zinc insulin complex.

Example 1

The crosslinked dextran gel with a water increment of 5% and a particle size of 20 to 80 μ is equilibrated in a 0.5 N acetic acid solution. The K100 / 100 laboratory column with a cross-sectional diameter of 10 cm and a length of 100 cm is packed with swollen gel to a bed volume of 7 liters (bed depth 89 cm). Next, 17.5 g of bovine pancreatic insulin sodium, prepared according to U.S. Pat. No. 3,719,655 and having an efficiency of 23.8 units / mg, were dissolved with a proinsulin content of 4.21% by weight in 400 ml of a 0.5 N acetic acid solution. The insulin solution was loaded onto the column followed by elution with 0.5 N acetic acid solution at 0.035 kPa to give a flow rate of 900 mL per hour (linear flow rate of 0.19 mL / cm ² / min).

Following elution of the void space (1725 mL), fractions of about 23.5 mL were collected and the protein content was determined by measuring ultraviolet absorption at 280 nm. The concentration of solids in mg / ml was also determined and a total of 240 fractions were collected. These fractions are combined into 4 groups as outlined below

Percentage of solids ³ ' Identification ί 1.4 High-molecular proteins and enzymes 2.4 mainly proinsulin , 9,7, proinsulin and insulin intermediates 86.4 insulin

higher possible yield of about 360,000 units. Thus, the yield of the Group Z zinc insulin complex corresponds to 91.9% of the yield, based on the weight of the support, and 98.9%, based on the units. Based on the starting material, the weight and unit yields are 79.4% and 85.4% respectively.

Group B-2 is precipitated with an excess of zinc ions as described above for Group C, whereupon the precipitate is dissolved in a 0.5 N acetic acid solution, and the solution is filtered on a smaller column. Insulin was recrystallized as described above for Group C to give an additional portion of 1.0 g of zinc insulin complex with an efficiency of 26.7 units per mg. Thus, the total yield of the zinc-insulin complex calculated on the starting material is 85.1% on a weight basis and

91.8% based on units.

For a better understanding of the procedure described in Example 1, reference is now made to the 1995 drawing. This is an elution diagram for Example 1, i.e., deposition of the optical density at 280 nm of different fractions versus number of fractions. Thus, the diagram shows the protein content of the collected fractions against the dilution process. Collection of fractions into several groups as described in Example 1 is represented by jerky vertical lines. It is clear from the diagram that the starting material is essentially insulin and that the insulin component is thereby effectively separated from proinsulin and similar proteins.

Example 1 a d 2

The procedure of Example 1 is repeated except that the bovine insulin insulin 2 is replaced by 30 g of the porcine insulin insulin and the amount of 0.5 N acetic acid solution in which the insulin is dissolved is increased to 600 ml. Pig sodium insulin having an efficiency of 23.3 units per mg and a proinsulin content of 2.42% by weight. The following results were achieved:

Group volume, ml Fractions % Solids ¹ AND 1410 10— 69 1.3 B — 1 1200 70—119 2.7 B — 2 820 120—154 6.0 C 1920's 155—235 ‘ 90.0

^al percentage of all eluted solids

From group C, 26.5 g of a zinc-insulin complex of 25.3 units per mg and a proinsulin content of 0.57% are obtained. Thus, the yield of the zinc insulin complex corresponds to a yield of 98.1% on a weight basis or 95.9% on a weight basis.

The zinc insulin complex prepared from the same batch of pancreas using a procedure whereby the alkaline crystallization and the gel filtration process is replaced by conventional isoelectric precipitation with an aqueous and aqueous-alcoholic medium is obtained in the same yield after recalculation to the same weight of the pancreas. However, the efficiency is only

24.3 units per mg, and the proinsulin content is 3.18%.

Claims (12)

SUBJECT A process for purifying an alkali metal complex with ammonium ions, comprising swelling a gel having a dry swelling of 4 to 98% by weight and a particle diameter of 20 to 80 microns, after which the column is swelled gel, an aqueous solution of an alkali metal complex with an ammonium ion of at least 80% purity is added to the packed column, with an insulin concentration ranging from 1 to 8% (w / w), and the total amount of insulin is sufficient to provide a column load in the range of 0.8 to 6.7 g per liter of bed volume and a pH of the solution ranging from 2.0 to 3.5, then eluting the insulin from the column at a temperature in the range of 5 to 30 ° C using an aqueous eluent containing up to 0.02 M inorganic salt and pH in the same range as the insulin solution. 2. A method according to claim 1, wherein a gel capable of receiving water in the range of 5 to 20% is used. 3. The process of claim 2 wherein the gel is cross-linked dextran. 4. A method according to claim 3, characterized in that a gel with a water absorption capacity of 5% is used. 5. A method according to claim 1, characterized in that the column is operated at a loading of from 3.5 to 5.0 g per liter of bed volume. 6. The method of claim 5, wherein the column is operated at a loading of 4.5 g per liter of bed volume. 7. The process of claim 1 wherein the insulin is dissolved in a 1.0 N acetic acid solution. 8. The process of claim 1 wherein the insulin is dissolved in a 0.5 N acetic acid solution and the pH is adjusted by adding dilute hydrochloric acid. 9. The process of claim 1 wherein the eluent is a 0.5 N acetic acid solution. 10. The process of claim 1, wherein the inorganic salt is used at a concentration of 0.01 M. 11. A process according to claim 10, wherein the inorganic salt is sodium chloride. 12. The method of claim 1, wherein the elution is carried out at room temperature.