CS271346B2 - Method of high-purity chymopapain production - Google Patents

Abstract

The solution applies to the production of highly pure chymopapain from raw chymopapain. According to the solution, raw chymopapain dissolves in the presence of a reductant in the water of a buffer. The pure upper part of the dissolution undergoes gel-permeation chromatography and fractions containing active enzyme are picked and dried by freezing.

Description

(57) The solution concerns a process for the production of high or hundredth chymopapain from crude chymopapain. According to the solution, crude chymopapain is dissolved in the presence of a reducing agent in aqueous buffer. The clear upper part of the solution is subjected to gel chromatography and the fractions containing the active enzyme are collected and freeze-dried.

271 346 (11) (13) 82 (52) Int. Cl?

OJ C 12 N 9/50

The invention relates to a process for producing high purity chymopapine with high specific activity from crude chymopapain by gel chromatography.

Chymopapain is a thiol protease that, together with papain and papalyapeptidase, forms the proteolytic (protein degrading) components of papaya latex. Papaya latex is obtained from the still immature fruits of the carica papaya (melon tree), which grows in the tropics, by cutting the fruit in several places, and the leaking milk juice is collected and finally dried. F

The best known and best studied latex enzyme is papain. This was first obtained by Balls et. al. crystallization (Balis, A.K., Lineweaver, H. and Thompson, R.R., Science, 86, 3794, 1937). Jensen and Balls have shown that - if papain is separated from the papaya-derived latex extract by ammonium sulfate precipitation - another thiol protease remains in solution. This relatively soluble and stable pH 2 enzyme was called chymopapain. By increasing the concentration of ammonium sulfate, this enzyme can also be precipitated. The crude chymopapain obtained in this way is not a homogeneous substance, as evidenced by gel electrophoresis and ion exchange chromatography. (Kahn, I.U. and Polgar, L., Biochem. 8iophys.Acta, 760, 350-356, 1983; European Patent No. 0 065, 395.

Chymopapain is used in clinical practice, on a large scale, for the treatment of intervertebral disc fractures, since when used in an appropriate amount it selectively dissolves the pulp nucleus (USP 3,320,131). In addition to the mild side effects occurring in about 50% of the treated patients, about 1% of severe anaphylactic disorders (hypersensitivity to foreign proteins) can be observed. (USP 4,039,682; Robinson, C.P., Drugs of Today, Vol. 19, No. 8, 1983, 454-460). These can be derived from one of the protein components of heterogeneous chymopapain according to the aforementioned European Patent No. 0 065 395. This component gives a precipitate with barium chloride and can be removed by ion exchange chromatography. According to said patent, the proteolytically inactive substance is colored and shows an unpleasant odor.

It is an object of the present invention to provide a process which does not exhibit the drawbacks of the known processes and makes it possible to easily and cheaply produce chymopapain with a high specific activity.

The invention is based on the discovery that the harmful components precipitated by barium chloride can be removed. from chymopapain by gel chromatography. This is technologically simpler, cheaper and at least ten times faster than conventional ion exchange chromatography. The invention is further based on the finding that partial inactivation of chymopapine during gel chromatography can be prevented by the addition of reducing agents.

Accordingly, the present invention provides a process for producing high purity chymopapain from crude chymopapain. The process is characterized in that crude chymopapain is dissolved in the presence of a reducing agent in water or in a buffer. The clear upper part of the solution is subjected to gel chromatography and the fractions containing the active enzyme are collected and freeze-dried. The crude chymopapain is preferably dissolved in a phosphate or acetate buffer having a pH of 5 to 7, which contains mercaptoethanol or sodium sulfite as reducing agent. A column of Sephadex G-50 or Sephadex G-25 which has been previously equilibrated with a buffer or saline solution, preferably a cysteine-containing phosphate buffer, is preferably used as the chromatography column. In the interest of efficient chromatography, the column is chosen such that its length to the diameter is in the ratio of 5 to 50: 1, preferably 20: 1.

The freezing is carried out at -30 to -70 ° C, preferably at -40 ° C; the ice is removed in a manner known per se under a vacuum of between 1.333 Pa and 13.33 Pa, preferably 6.665 Pa.

The unpleasant odorous substance precipitated by barium chloride is found in the chromatographic peak following the active enzyme.

2.

The method according to the invention has the following advantages:

Separation of the protein component responsible for the harmful side effects and providing a barium chloride precipitate by means of cannon chromatography is cheaper, more gentle and ten times faster than known methods. Therefore, the process is considerably more suitable for large scale operation than ion exchange chromatography, which lasts about 40 hours. In this long time, the enzyme will inevitably be damaged. In contrast, gel chromatography can be performed in less than 4 hours and provides the ability to chemically protect the enzyme with physiologically acceptable, ionic reducing agents. Since gel chromatography shows a higher effective degree of separation, the product obtained is much purer, which, as expected, leads to a significant attenuation of the side effects.

The invention is further illustrated by the following examples.

Example 1

Dissolve 2.5 g of crude chymopapain in 135 ml of phosphate buffer containing 10 mmol of cysteine (3 mmol, pH 6.5). If the solution is cloudy, it is filtered. After 15 to 30 minutes, 135 ml of the solution is chromatographed on a Sephadex G-25 column (4 x 100 cm) with a buffer containing 3 mmol of phosphorus and 1 mmol of cysteine and exhibiting a pH of 6.5. The first peak (designated A in Fig. 1) contains chymopapain of about 1.3 g. Peak 0 contains an unpleasant odor, which forms a precipitate with barium chloride. The enzyme present in peak A is sterilized and lyophilized. ,

In each case, chymopapain activity was determined by saturating the substrate (Asboth, d., Polgar, L., Acta Biochim. Acad. Sci., Hung., 12, 223-230, 1977) on benzyloxycarbonylglycine p-nitrophenyl ester (ZGN) as a substrate in the following reaction mixtures:

100 µl ZGN solution prepared with 1 mg / ml acetonitrile, 2.0 ml 0.1 M acetate buffer pH 5.5 containing 1 mmol EOTA, 100 µl chymopapine.

To characterize the preparation, the change in optical density (extinction) measurable at 25 ° C over 1 minute at 340 nm induced by 1 mg chymopapain per ml of reaction mixture is further reported.

The enzyme produced has an activity of 62.

Example 2

It is operated as described in Example 1, except that the column has a larger diameter (5 x 100 cm), specific activity: 70.

Example 3.

The procedure is as described in Example 1 except that a short and fast column (10 x 16 cm) is used. There is no separation of the substance giving the barium chloride precipitate from chymopapain. Specific activity: 42.

Example 4

The procedure is as described in Example 1 except that the buffer used for gel chromatography contains no cysteine. Specific activity: 51.

Example 5

The procedure is as described in Example 1, except that a concentrated solution is prepared from chymopapain (protein content 5 4 - w / v - 4). Specific activity:. .

CS 271546 82

Example 6

The procedure is as described in Example 1 except that the isolation is carried out not at room temperature but at 2 to 10 ° C. Specific activity: 65.

Example 7 ‘

The procedure is as described in Example 1, except that Sephadex G-50 is used instead of Sephadex G-25. Specific activity:.

Example 8

The procedure is as described in Example 1 except that the phosphate buffer concentration is not 3 mmol but 10 mmol. Specific activity:.

Example 9

The procedure described in the example of the new buffer at pH 6.5 uses acetate buffer

Example 10.

The procedure is as described in the example of cysteine, but 5 mmol of cysteine. Specific

Example 11

The procedure is as described in the example of the active substance (peak A), based on the specific activity present: 63.

except that instead of phosphorus, a pH of 5.5. Specific activity:.

with the difference that the buffer does not contain 1 mmol activity: 64.

1, except that 10 tons of cysteine is added to the protein-containing fraction.

Example 12 _%

The procedure is as described in Example 1, except that sodium sulfite is used as the reducing agent instead of cysteine. Specific activity; 60.

Example 13

The procedure is as described in Example 1, except that 1 ml of each of the three fractions represented by the chromatographic peaks is taken and 0.2 ml of n hydrochloric acid and 0.2 ml of 12¾ barium chloride solution are added to each sample. . A precipitate formed only in fraction 8.

Example 14 o

The activity of the Chimodiactin preparation used as the reference preparation is measured as described in Example 1. The activity is 28.

SUBJECT OF THE INVENTION

Claims (6)

A process for the production of chymopapain from crude chymopapain, characterized in that crude chymopapain is dissolved in the presence of a reducing agent in water or buffer, the clear upper part of the solution is subjected to gel chromatography and the fractions containing the active enzyme are collected and freeze-dried. 2. A process according to claim 1, wherein the reducing agent is cysteine, mercaptoethanol or sodium sulfite. 3. A process according to claim 1 or 2, wherein the buffer used is a phosphate or acetate buffer having a pH of between 5 and 7. CS 271346 02 Method according to one of Claims 1 to 3, characterized in that a column of Sephadex G-50 or Sephadex G-25 is used as the chromatography column. Method according to one of Claims 1 to 4, characterized in that the ratio between the length and diameter of the column used is between 5: 1 and 50: 1, preferably 20: 1. Method according to one of Claims 1 to 5, characterized in that the freeze-drying is carried out in a manner known per se, preferably at -40 ° C and a pressure of 6.665 Pa.