JPS5939422B2 - Insulin purification method - Google Patents

Description

[Detailed description of the invention] The present invention relates to a method for purifying insulin.

According to the method of the present invention, alkali metal or ammonium insulin can be purified by gel filtration under conditions of pH 2.0 to 3.5. The gist of the invention is that the dry state has a moisture content of at least about 4% (by weight) and about 1% (by weight).
A gel having a particle size not exceeding 0.00 μm is swollen, the swollen gel is packed into a column, and an aqueous alkali metal or ammonium insulin solution having a purity of at least about 80% and an insulin concentration in the aqueous solution of about 1 to 8 μm is prepared.
% (weight/volume), sufficient to load the column with a total amount of insulin in the range of about 0.8 to 6.7 y per 11 packed bed volumes, and the pH is about 2.
．． An aqueous solution in the range of 0 to 3.5 is added to the column, and insulin is removed from the column using an eluent having a pH in the same range as the pH of the insulin solution.
A process for the purification of alkali metal or ammonium insulin characterized by elution at a temperature in the range of °C. FIG. 1 is a chart showing the elution results of Example 1. This chart plots the optical density at 280 mμ of several fractions of insulin solution in the gel P filtration step. Since its discovery in 1921 as a component in the pancreas, insulin has gained worldwide importance in the treatment of diabetes.

Because the extraction methods used to isolate insulin from pancreatic tissue also result in the separation of substantial amounts of non-insulin proteins, methods for purifying insulin (i.e., separating insulin from non-insulin proteins) have been developed. A great deal of effort has been put into it. Several of the methods developed to purify insulin have achieved important economic results. One of the earliest discoveries of these commercially important methods is the general phenomenon that, other things being constant, a protein's solubility is at its minimum at its isoelectric point. For example, Banting et al.
No. 469,994 discloses a purification process consisting of precipitating non-insulin proteins contained in an aqueous pancreatic extract at their isoelectric point. Also, insulin has a pH of about 4~
7, it was reported by Walden in U.S. Pat. No. 152
It was disclosed in No. 0673.

In other conventional methods, precipitation of insulin was carried out by adding sufficient amounts of inorganic salts.

For example, Murlin, U.S. Patent No.
No. 7515, the first method of salting out insulin from the extraction solvent by adding sodium chloride was disclosed. The method of differential salting out is based on La
U.S. Patent No. 244 by Utenschlager et al.
No. 9076, the method involves salting out insulin from a neutral extract by adding sodium chloride, filtering, redissolving the residue, and salting out insulin again (first salting process). This method involves salting out at a salt concentration lower than the precipitation conditions. A third commercially important method of insulin purification in the prior art involves precipitating, or crystallizing, insulin from solution as a complex of zinc and insulin.

A common method is to precipitate zinc insulin from an aqueous buffer solution containing zinc ions. Various buffer solutions have been used; for example, Petersen et al.
No. 26228 disclosed the use of a citric acid-citrate buffer solution.

Current economic methods of insulin purification typically include all three of the above methods.

Since zinc insulin is the most important commercial form of insulin, the final step in insulin purification is usually a zinc crystallization step. However, non-insulin proteins (
Among these, proinsulin and proinsulin-like proteins are common.

) has recently been recognized as a minor component in commercially available zinc insulin. Although such ingredients generally represent less than about 8% by weight of commercially available zinc insulin, some of these ingredients are believed to be antigenic or immunogenic in nature (e.g., Chance
) et al. (Science Vol. 161, p. 165 (1968))
, Steiner et al. (Diabetes Vol. 18, p. 725 (1968)), Proma I (Br.
Omer) (Bioscience Vol. 20, p. 701 (19)
1970) and Rubenstein
) et al. (Ann-Rev-Med, Vol. 22, p. 1 (197
1 year))).

Therefore, a problem that requires continued research is how to remove non-insulin proteins from insulin on an economical scale.

The term insulin as used herein includes not only insulin itself, but also all insulin-like proteins such as desamide insulin.

Since insulin and insulin-like proteins have similar hypoglycemic effects, it is usually not necessary to separate insulin from all other pancreatic proteins. That is, an insulin-only homogenate is usually not required. Methods for separating biological substances on an analytical scale, such as electrophoresis and ion-exchange chromatography, are known as methods for separating non-insulin from insulin components in commercially available insulin preparations (or crude insulin). It is being

These methods even allow the insulin component to be divided into insulin itself and desamidated insulin. However, there are doubts about the large-scale use of such methods. A method that is more easily applicable to large-scale purification is gel filtration (synonymous with gel filtration chromatography and gel permeation chromatography).

In this method, a gel is used that has been cross-linked in such a way that pores are formed in the structure of the gel particles, and proteins are separated on a column containing this gel. These pores have a measurable finite volume that is directly proportional to the degree of swelling of the gel and inversely proportional to the degree of crosslinking. Small molecules fit more closely into these pores than larger molecules, so smaller molecules pass through the column compared to larger molecules that are only partially close to the pores or do not enter the pores at all. be prevented from doing so. Traditionally, a gel filtration method has been used to purify insulin.

Specifically, in the isolation of insulin taken from the pancreas of a cat, acid-alcohol extraction was followed by alkaline precipitation to remove inert substances, concentration, and isoelectric precipitation, followed by sodium chloride precipitation. Crude insulin is obtained by salting out precipitation. This crude insulin can then be loaded onto a cross-linked dextran gel column and eluted with 1.0M acetic acid (DavOre
n) Author (Biohimica Biophysica Acta No. 63)
Volume 150 (1962)) (see literature).

To isolate insulin from fish, the pancreatic glands are homogenized in water and the protein is precipitated with a trichloroacetic acid solution.

The precipitate is extracted with acid-ethanol and the extract is treated with methylene chloride to remove lipids. Isolate the remaining solid; 5.
Dissolved in 0M acetic acid and filtered through a cross-linked dextran gel column using 5.0M acetic acid as eluent (Humbel, Biochemical and Biophysical Research Communication Vol. 12, p. 333).
(1963))).

Yields of about 80% have been reported. It should be noted that Pampel states that in order to better separate insulin from other proteins, the elution conditions preferably dissociate the insulin molecule. He also referred to Yphantis and Waugh (Biohimica Biophysica Acta Vol. 26, 21).
8 (1957)) suggest that 1.0 M acetic acid is not a satisfactory solvent since insulin is not completely dissociated therein. A crude extract of bovine pancreas was described by Epstein et al. (see Biochemistry 1, Vol. 2, p. 461 (1963)) as being enriched with a cross-linked dextran gel.

The eluent was 0.2M ammonium bicarbonate, which is also a solvent that Pampel said to be poor in the literature. Insulin, which Epstein et al. reported in a yield of about 60%, has also been isolated from human pancreas. The pancreatic glands are first homogenized and extracted. Some inert material is removed by fractionation with ammonium sulfate.
Insulin is then precipitated with sodium chloride and isoelectrically focused. 0.5 of the insulin produced
Dissolve in N-acetic acid and filter over a cross-linked dextran gel. The excess insulin is converted into zinc insulin (see the literature by Jackson et al., Diaperties Vol. 18, p. 206 (1969)).
The insulin yield after gel filtration was about 60%.
Schllchtkrull et al. carried out purification by a combination of gel filtration and ion exchange chromatography (see South African Patent No. 69/5280 (corresponding to Perky Patent No. 737257)).

In accordance with the gel filtration only example (Example 1 in the above-mentioned Pelkey patent), zinc insulin was filtrated through a cross-linked dextran gel column with a yield of 80%. The eluent was 1.0M acetic acid. It should be noted that Schülichtkrul et al. recognized that after filtration, it was necessary to employ salting out, isoelectric precipitation and zinc crystallization methods to obtain zinc insulin. All of the above-mentioned prior art techniques in which gel filtration is applied to the purification of insulin are accompanied by significant losses of insulin.

Moreover, there is no certainty from the prior art that using gel filtration results in only insulin (including the insulin-like proteins mentioned above). As a result of various studies aimed at overcoming the above-mentioned drawbacks in the insulin purification method, the present inventor has found that an extremely good yield can be achieved by the method described below without repeating operations such as salting out and isoelectric precipitation. The present inventors have discovered that insulin can be purified at a relatively low yield, and have completed the present invention.

In general, any water-swellable gel suitable for use with protein solutes can be used in the method of the invention.

However, such gels, in their dry or unswollen state, have a water content of at least about 4% by weight (based on the weight of the dry gel).
or water content) and have a particle size not exceeding about 100 microns. Preferably, the water content of the gel is about 4-98%, most preferably about 5-20%. The particle size of the dry gel preferably does not exceed about 80 microns, with a particularly useful particle size range of about 20-80 microns. Examples of suitable gels include, among others, starches (including corn starch), cross-linked galactomannans, cross-linked dextran, agar or agarose, polyacrylamide, copolymers of acrylamide and methylene bis-acrylamide, methylene bis-acrylamide and There are copolymers of vinyl ethyl carbitol, copolymers of methylene bisacrylamide and vinylpyrrolidone, and similar products.

A preferred gel is a cross-linked dextran such as Sephadex, manufactured and sold by Pharmacia Fine Chemicals, Inc., Bisgataway, New Chassis, USA. The type of column used in the method of the invention is not critical.

The choice of column height, diameter, or configuration may be determined by the desired operating conditions. The flow rate decreases as the column height increases. In other words, the back pressure at the bottom of the column is directly proportional to the height of the column. For this reason, stacked column configurations such as Pharmacia Sectional Column KS37O type are preferred. For normal manufacturing purposes, a six-layer stack is very useful. According to the method of the invention, the packed bed capacity is 11
Satisfactory insulin purification can be achieved by loading the column with about 4,57 parts of alkali metal insulin or ammonium insulin per liter, and such loadings are most preferred.

The range of load amount is packed bed capacity 1f! Approximately 0.8 to 6.7 per
y, preferably about 3.5 to 5.07 per 11 packed bed volumes
The range is fine. Optimal conditions can be easily determined for any particular column. The gel can be swollen and the column filled with the swollen gel by any of a variety of methods known to those skilled in the art.

Generally, the gel is swollen in the elution medium. Alternatively, the gel can be swollen in 30% aqueous ethanol, the supernatant decanted, and the swelling medium replaced with the elution medium. Alkali metal insulin or ammonium insulin solutions can be prepared by conventional means. For example, insulin may be dissolved with an appropriate amount of eluent. Alternatively, insulin may be dissolved in an aqueous solvent that is somewhat more acidic than the eluent (but within the PH range of the eluent). For example, if the eluent is 0.5N acetic acid with a pH of 2.5, insulin is an aqueous solvent with a pH of about 2.3, i.e. 1.0
It can be dissolved in specified acetic acid. Alkali metal insulin or ammonium insulin is usually alkaline.

As a result, when this insulin is dissolved in a neutral or acidic aqueous solvent, the pH of the resulting solution increases. In this way, if insulin is dissolved in 1.0N acetic acid, the pH of the resulting solution will rise to about 2.7. However, since the pH depends on the concentration of insulin used, the insulin solution may have a pH of about 3.2.
An increase in pH is normal and does not cause problems as long as the pH of the insulin solution does not exceed about 3.5.
Alternatively, insulin can be dissolved in 0.5N acetic acid and
The pH may be adjusted to about 3.2 or less with dilute hydrochloric acid. Generally, alkali metal insulin or ammonium insulin can be prepared by any known method.

However, it is preferred that the alkali metal insulin or ammonium insulin be prepared by the method of US Pat. No. 3,719,655. However, as previously indicated, such insulin must have a purity of at least about 80%. That is, the content of insulin, including insulin-like proteins that have hypoglycemic effects, should be at least about 80% of the total protein present. As mentioned above, the concentration of alkali metal insulin or ammonium insulin in the insulin solution to be passed through the column is about 1 to 8% (weight/volume).

The preferred concentration is about 4-6%. To obtain satisfactory results, the alkali metal insulin or ammonium insulin should be dissolved in a volume of solvent less than the separation volume, as described below. In chromatography, the partition coefficient Kd is defined as the ratio of the solute concentration in the mobile phase to the solute concentration in the stationary phase.

In gel filtration, the mobile phase is the solvent that moves through the gaps between the gel particles, and the stationary phase is the solvent absorbed by the gel particles,
That is, the solvent is trapped within the pores of each gel particle. Therefore, Kd represents the fraction of absorbed solvent that can be penetrated by solute. Using Kd, the solute elution volume Ve can be expressed by the following formula: [where VO is the interstitial volume in the column,
Vl is the volume of solvent absorbed into the gel.

]0Kd, the equation becomes as follows.

If the density of water is uniform, the relationship Vi-a-Wr holds true (where a is the weight of the dry gel and Wr is the water content).

]o Therefore, Kd can be expressed by the following formula: This Kd value can be determined experimentally by those skilled in the art.

If the solution contains two solutes, the elution volume of each solute can be expressed by the following equation: Since the separation volume Vs is the difference between the elution volumes of the two solutes, the following equation holds: From the above, It is clear that the degree of separation of two (or more) solutes depends in part on the column loading.

As the amount of low molecular weight solute approaches the limit of the pore that can accommodate it, the degree of separation between the two solutes necessarily decreases. The degree of separation can be varied by varying the loading within the range of loadings specified as part of this invention. In some cases, the column loading can be increased due to trade-offs between productivity and resolution. As mentioned above, the eluent has a pH in the range of about 2.0 to 3.5. Generally, the eluent is an aqueous solution of an inorganic or organic acid to which the protein is stable. Examples of such acids include hydrochloric acid, phosphoric acid, formic acid, acetic acid, propionic acid, butyric acid,
There are isobutyric acid, valeric acid and similar acids. Acetic acid is preferred. If hydrochloric acid is used, a preservative such as phenol must be added to the eluent.
Acetic acid also acts as a preservative. The preferred pH is about 2
．． It is 5. Therefore, the most preferable eluent is a 0.5N acetic acid solution. Sodium chloride in the eluent if desired,
It may contain an amount of inorganic salts such as potassium chloride, ammonium chloride, and the like, not exceeding about 0.02 mole/l.

The preferred concentration is 0.01M and the preferred salt is sodium chloride. It is preferred to use such salts to improve solubility and to prevent basic substances from being absorbed by the gel. Column elution is approximately 5
It can be carried out at temperatures ranging from -30°C.

Preferably, the processing temperature is room temperature or lower. Elution may be carried out in any convenient manner.

However, it is particularly useful to monitor each fraction by performing UV absorption measurements at 280 mμ. Fractions representing the purified desired insulin component are combined and typically converted to zinc insulin. As aforementioned,
Zinc insulin is the most important form of insulin.

Therefore, the high purity alkali metal insulin or ammonium insulin obtained by the treatment method of the present invention can usually be converted into zinc insulin. Preferably, the conversion to zinc insulin is carried out in the presence of ammonium acetate buffer at pH 6. Do it with O. It is not necessary to isolate the alkali metal insulin or ammonium insulin from the eluate or to concentrate the purified insulin solution before crystallizing with zinc. An advantage of the method of the present invention is that the insulin in the eluted solution is sufficiently concentrated that no salting out or isoelectric precipitation is necessary, although such methods may be used if desired. The purity of the zinc insulin obtained by the method of the invention is improved as a result of the treatment. This increased purity is demonstrated by several methods. Zinc insulin can be directly assayed for non-insulin protein components such as proinsulin and glucagon. proteolytic enzyme,
The presence of high molecular weight proteins, which typically contain protaminase, can be determined by measuring the stability of protamine insulin suspensions at heating temperatures. If protaminase is present, due to the degradation of protamine,
The protamine insulin complex dissociates. Physical properties such as the solubility of zinc insulin at neutral PH and the color of the resulting solution can be determined. In addition, the specific activity of insulin can be measured by biological and immunological test methods. Since the zinc insulin obtained by the above process has a high purity, it can be used in various preparations of insulin, such as ordinary insulin, isophane insulin, protamine zinc insulin, zinc insulin suspension, glopine insulin, and the like. It is suitable for preparing similar products.

Furthermore, neutral insulin, which is more stable than acidic insulin, can be made from higher purity zinc insulin. Conventionally, impurities present in zinc insulin often precipitate in part at neutral pH values. Such impurities often included enzymes that degrade insulin, such as trypsin and chymotrypsin, which reduced the efficacy of insulin preparations. Purified zinc insulin also allows for the preparation of extended potency porcine and bovine isosulin formulations without the additional purification required in the past. Finally, in the absence of acidic protein impurities, it is possible to precipitate wood nut insulin by using the small amount of protamine needed to precipitate insulin, making it possible to precipitate even more pure zinc insulin. Isophane insulin can be produced using small amounts of protamine. The method of the present invention can be combined with other purification methods as desired.

For example, an alkali metal insulin or ammonium insulin solution may be subjected to one or more sifting operations prior to gel filtration. Additionally, the eluted insulin can be subjected to one or more filtration operations prior to being converted to zinc insulin. Next, specific embodiments of the present invention will be described with reference to Examples.

Example 1 A cross-linked dextran gel with a moisture content of 5% and a particle size of 20-80μ is equilibrated with 0.5N acetic acid.

AKlOO/1 with diameter 10CTI 1 length 100Cr 1L
The above-mentioned swollen gel was packed into a 00 laboratory column (manufactured by Pharmacia Fine Chemicals Co., Ltd.) so that the packed bed volume was 71 (packing depth 89 CTIL). Prepared by the method of U.S. Pat. No. 3,719,655, activity 23.8 Un
Sodium insulin (extracted from bovine pancreas) 17,5y having a proinsulin concentration of 4.21% (weight) is dissolved in 400 ml of 0.5N acetic acid. This insulin solution was supplied to the above column, and 0,5
Elute with normal acetic acid under 5 psig pressure. The flow rate is 9
00 m1/Hr (linear flow rate 0.19 m1/Cd/min). After eluting the interstitial volume (1725 ml), fractions of approximately 23.5 ml each are taken. Protein concentration is 280mμ
Determined by measuring the ultraviolet absorption of Solid concentration? /Mj is also determined. Take 240 fractions.

These fractions are grouped into four groups as shown below. Add 3.0ml of 20% zinc chloride solution to group C;
The obtained solution was diluted with 3025 ml of 0.5N ammonia water.
Dilute to give

Adjust the pH of the final solution to 6.0 with dilute aqueous ammonia. After the precipitated zinc insulin was isolated by centrifugation and thoroughly washed with water, absolute alcohol and ether,
Dry in vacuum. The yield of zinc insulin is 13.9y.
, insulin activity is 25,6 Unit/Rflf7 and proinsulin concentration is 0.44%. The solids content of Group C is 86.4% of the total eluted solids, and the maximum yield of solids obtained from Group C is 86.4% of the total eluted solids.
6.4% or 15.17 insulin units (U
This is approximately 360,000 Units.

The yield of zinc insulin from group C corresponds to a recovery of 91.9% by weight and 98.9% of units of insulin. Weight recovery rate is 79.4 when converted to starting material.
%, the recovery rate in terms of insulin units was 85.4%.
Group B-2 is precipitated with excess zinc ion as described above for group C, dissolved in 0.5N acetic acid, and refiltered through a small column.

The insulin peak is recrystallized as described above for group C to yield 1.0y of zinc insulin with an activity of 26.7Unit/ヮ. As a result, the yield of zinc insulin was 85.1% by weight based on the starting material, and 91.8% in terms of insulin units. The method described in Example 1 may be better understood by referring to FIG. FIG. 1 is an elution diagram of Example 1. i.e. 280 mμ of each fraction for fraction number
This is a plot of the optical density of . This diagram shows the protein concentration for each fraction collected during the elution step.
A number of fractions are grouped into several groups as described in Example 1 and are indicated by vertical dotted lines. It is clear from FIG. 1 that the starting material consists essentially of insulin and that the insulin component is effectively separated from proinsulin and its analogous proteins. Example 2 Porcine sodium insulin 307 was used instead of bovine sodium insulin, and 600Tf1 of 0.5N acetic acid was added to dissolve the insulin.
The procedure is carried out in the same manner as in Example 1 except that the amount is increased to 1.

Porcine sodium insulin has an activity of 23.3 Units/wa and a proinsulin content of 2.42% (by weight). The following results are obtained. 25.3U from Group C
Zinc insulin 26.57 with nit/immediate activity and a proinsulin content of 0.57% is obtained.

The yield of zinc insulin was 98.1% by weight, corresponding to a regeneration of 95.9% in terms of insulin units.
Zinc insulin prepared from the same batch of pancreas yields the same yield per pound of pancreas when traditional isoelectric precipitation from water or hydroalcoholic solvents is used instead of the alkaline crystallization and gel filtration methods. It can be obtained with but,
The activity is 24.3 Unit/TrL9 and the proinsulin concentration is 3.18%.

[Brief explanation of drawings]

FIG. 1 is a chart plotting the optical density of the protein eluted in Example 1 for each fraction.

Claims (1)

[Claims] 1. Swelling a gel having a water content of at least about 4% (by weight) in the dry state and a particle size not exceeding about 100 microns and packing the swollen gel into a column of alkali metal or ammonium insulin having a purity of at least about 80%. It is an aqueous solution and the insulin concentration in the aqueous solution is about 1 to
8% (weight/volume), sufficient to load the column with a total amount of insulin in the range of about 0.8 to 6.7 g per liter of packed bed volume, and a pH of about 2. ．．
Adding an aqueous solution in the range of 0 to 3.5 to the column,
Insulin is removed from the column at about 5-30°C using an eluent having a pH in the same range as the pH of the insulin solution.
A process for the purification of alkali metal or ammonium insulin characterized by elution at a temperature in the range of .