HU196624B - Process for producing chymopapain with high specific activity - Google Patents

Abstract

The present invention relates to a process for preparing kimopain from crude kimopain by dissolving the crude chimera in the presence of a reducing agent in water or buffer, the clear supernatant being gel chromatographed, and the fractions containing the active enzyme are collected and freeze-dried. -1-

Description

The present invention relates to a process for the preparation of high purity, high specific activity chymopamine from crude chymopapine by gel chromatography.

Chimeropamine is known to be a thiol protease, which, together with papain and papaya peptidase, forms the proteolytic protein-degrading component of papaya latex. Papaya latex can be obtained from the unripe fruit of the tropical tree, Carica papaya, by scratching the fruit in several places and catching the milky juice that is spilled and then drying it.

Papain is the best known and best studied enzyme in latex. It was first crystallized by Balls et al. (Balls, A. K. Lineweaver, H. and Thompson, R. R. / 1937 / Science 86, 3794). Jansen and Balls (Jansen, E.P. and Balls, J. K. (1941) .1, Bioi. This relatively soluble enzyme, which is stable at pH 2, has been termed chymopamine. By increasing the concentration of ammonium sulphate, this enzyme can also be precipitated. This so-called "crude chymopamine" is not homogeneous material, as revealed by gel-chromatography and ion-exchange chromatography (Kálin, I. U. and Polgár,

L. Biochim. Biophys. Acta 760, 350-356 (1983); European Patent Specification 0 065 395).

Chymopapain is widely used in clinical practice for the treatment of spinal disc hernia because, when used in an appropriate amount, it selectively solubilizes the portion of the intervertebral disc nucleus (U.S. Patent No. 3,320,131). However, as a result of the treatment - approx. With a 50% incidence of mild side effects, approximately 1% of patients develop dangerous anaphylactic symptoms (U.S. Patent No. 4,039,682; Robinson, CP Drugsof Today Vol. 19, No. 8, 1983 / 454,460). .

According to the aforementioned European Patent No. 0.065.395, one of the superficial components of the heterogeneous chymopamine preparation, which gives off a precipitate with barium chloride, is responsible for the anaphylactic side reaction and can be removed by ion exchange chromatography. This proteolytically inactive substance is also characterized by a colorless, odorless substance.

It is an object of the present invention to overcome the disadvantages of the prior art by providing a solution which, in a simpler and less expensive manner, produces higher specificity chymopapine.

The present invention is based on the discovery that, apart from chymopamine, the deleterious barium chloride precipitating component can be separated by gel chromatography, which is technologically simpler, less expensive and at least ten times faster than conventional ion-exchange chromatography.

The present invention is further based on the discovery that partial inactivation of chymopamine by gel chromatography can be prevented by the addition of reducing agents.

The present invention relates to a process for the preparation of chymopamine by crude chymopamine gel chromatography. Accordingly, the crude chymopapine is dissolved in water or buffer in the presence of a reducing agent, the pure supernatant is subjected to gel chromatography, and the fractions containing the active enzyme are collected and freeze-dried. The crude chymopapine is conveniently dissolved in a phosphate or acetate buffer of pH 5 to 7, containing cysteine, mercaptoethanol or sodium sulfite as reducing agent. Preferably, the chromatographic column is a Sephadex G-50 or Sephadex G-25 column previously equilibrated with a buffer or saline solution, preferably a cysteine-containing pH 6.5 phosphate buffer. For effective chromatography, the column is chosen to have a length to diameter ratio of 5 to 50: 1, preferably 20: 1.

Freezing is carried out between -20 ° C and -70 ° C, preferably 40 ° C, and the ice is removed in vacuo at a rate of 1.333 Pa and 13.33 Pa, preferably 6.665 l'a.

The odorous material which precipitates with barium chloride is found in the chromatographic peak following the active enzyme.

The main advantages of the process of the invention may be summarized as follows.

Removal of the barium chloride protein component responsible for adverse reactions by gel chromatography is cheaper, approx. ten times faster and more gentle, so much more suitable for industrial scale implementation than ion exchange chromatography. The latter approx. 40 hours, during which time enzyme damage is inevitable. Gel chromatography, on the other hand, is not feasible in less than 4 hours and also provides the ability to chemically protect the enzyme with physiologically non-harmful ionic reducing agents. Gel chromato- graphy gives a cleaner product with higher separation efficiency, which is expected to result in a significant reduction in side effects.

The following examples illustrate the process of the present invention.

Example 7

2.5 g of crude chymopapain are dissolved in 135 ml of 3 mM phosphate buffer pH 6.5 containing 10 mM cysteine.

If turbid, centrifuge. After incubation for 15-30 minutes, 135 ml of the solution is gel chromatographed on a Sephadex G-25 column (4X X100 cm) in 3 mM phosphate, pH 6.5 1 mM cysteine. The first peak, indicated by A in Figure 1, contains chymopapain, ca. 1.3 g.

Peak B contains a barium chloride inactive material which gives off a precipitate. The enzyme at Peak A is sterile filtered and lyophilized.

In all cases, the activity of chymopamine was measured with a substrate saturation of a benzyloxycarbonylglycine p-nitrophenyl ester (ZGN) (Asbótli, B., Polgár, L. / 1977 / Acta Biochim. Biophys. Acad. Sci. Hung. 12, 223-230). ), in the following reaction mixture:

100 µl of 1 mg / ml ZGN in acetonitrile.

2.8 mL of 0.1 M pH 5.5 acetate in 0.5 mM ITA EDTA.

100 µl Kimopain.

196,624

To characterize the composition, the change in extinction at 340 nm at 25 ° C for 1 minute caused by 1 mg of chymopamine / ml reaction mixture was reported. Specific activity of the enzyme: 62.

Example 10

Example 1 except that 5 mM cysteine was used instead of 1 mM cysteine. Specific activity: 64.

Example 2

The procedure of Example 1, except that a larger diameter column (5 x 100 cm) was used. Specific activity: 70.

Example 3

The procedure of Example 1, except that a short and fast column (10 x 16 cm) was used. Here, the contaminating protein precipitating with barium chloride is not separated from the chymopapine. Specific activity: 42.

Example 4

The procedure of Example 1 except that I mM cysteine was omitted from the gel chromatography buffer. Specific activity: 51.

Example 5

The procedure of Example 1, with the exception that the crude chymopapine is more concentrated, is such that the solution has a protein content of 5% (w / v). Specific activity: 55.

Example 6

The procedure of Example 1, except that the isolation is carried out at 2-10 ° C instead of room temperature. Specific activity: 65.

Example 7

The procedure of Example 1, except that Sephadex G-25 is chromatographed on a Sephadex G-50 column. Specific, activity: 53.

Example 8

The procedure of Example 1, except that 10 mM phosphate buffer was used instead of 3 mM phosphate. Specific activity: 63.

Example 9

The procedure of Example 1, with the exception that a pH 5.5 steel buffer is used in place of the pH 6.5 phosphate buffer. Specific activity: 67.

Example 11

The procedure of Example 1, except that the fraction containing the active substance (Peak A) is added in the amount of cis / lculus to 10% (% lcg) of protein c present. Specific activity: 63.

Example 12

The procedure of Example 1 except that sodium sulfite reducing agent was used instead of cysteine. Specific activity: 60.

Example 13

The procedure of Example I, except that 1 mL of each of the chromatographic peaks is taken and 0.2 mL of 1N hydrochloric acid is added, followed by 0.2 mL of 12% barium chloride. Precipitation is obtained only at peak B.

Example 14

Reference Measurement:

The specific activity of "Chimodiactin R" (Smilh Laboratories, Inc. USA) as measured by the method of Example 1 is 28.

Claims (6)

Claims CLAIMS 1. A process for the preparation of chymopamine from crude chymopamine, comprising dissolving the crude chymopapain in water or buffer in the presence of a reducing agent, gel chromatography and collecting the fractions containing the active enzyme and freeze-drying. The process according to claim 1, wherein the reducing agent is cysteine, mercaptoethanol or sodium sulfite. 3. The process of claim 1 or 2, wherein the buffer is a phosphate or acetate buffer having a pH of from 5 to 7. 4. A process according to any one of claims 1 to 4, wherein the chromatographic column is preferably a Sephadex G-50 or Sephadex G-25 column. 5. A process according to any one of claims 1 to 4, characterized in that the column used has a length to diameter ratio of 5 to 50: 1, preferably 20: 1. 6. Process according to any one of claims 1 to 3, characterized in that freeze-drying is preferably carried out at 40 ° C at 6,665 µ a.