NO854823L - PURE CHYMOPAPAIN B: INDUSTRIAL PROCESS AND THERAPEUTIC PREPARATION. - Google Patents

Description

The present invention relates to a method for isolating large amounts of chymopapain B and chymopapain C from either papaya latex or chymopapain raw preparations. The method according to the invention provides homogeneous concentrations of chymopapain B and chymopapain C, free from cross-contamination and free from other proteins. This process yields on an industrial/commercial scale large amounts of chymopapain B and large amounts of chymopapain C with low levels of pyrogens. The procedure is fast, simple and suitable for execution on an industrial scale without major adaptations or complications.

The present invention also includes therapeutic preparations which consist essentially of chymopapain B without contamination by chymopapain C. A new method for measuring the activity of the chymopapain is also provided.

Chymopapain generally refers to a group of proteolytic sulphide enzymes which are now recognized to be different from papain and form the main component of raw papaya latex derived from Carica papaya, Caricaceae. Chymopapain was first partially characterized in 1941 by Jansen and Balls. See: J. Biol. Chem. 137:459 (1941); US Patent 2,313,875 (1943). Chymopapain has been considered to consist of four components, two of which, chymopapain A and chymopapain B, have molecular weights of approx. 35,000 and has been isolated in the laboratory and studied. See: The Merck Index, 10th Edition (1983), Entry No. 2244, page 322.

Proteolytic enzymes derived from the papaya plant have long been used

in industry. See generally: US Patent 1,826,467, issued to Harteneck on October 6, 1931 (decoiling raw silk and coagulated rubber latex); US Patent 1,967,679, issued to Muench et al. 24 July 1934 (removal of hair from hides);

US Patent 2,095,300, issued to Wallerstein on October 12, 1937 (decoiling of silk); US Patent 2,219,209, issued to Neuf eld on October 22, 1940 (tendering of meat); US Patent 2,464,200, issued to Hall on March 15, 1949 (tendering of meat); US Patent 3,235,468, issued to Hogan on February 15, 1966 (tendering of meat); US Patent 3,296,094, issued to Cayle on January 3, 1967 (tendering of meat); US Patent 3,466,706, issued to Beuk on May 27, 1969 (tendering of meat); US patent 3,709,790, issued to Beuk on Jan. 9. 1973 (tendering of meat); US Patent 3,776,693, issued to Smith et al. December 4, 1973 (dry cleaning); US Patent 4,024,285, issued to Beuk et al. 17 May 1977 (tendering of meat); and US patent 4,118,515, issued to Rømmele et al. Oct. 3, 1978 (inhibition of cold-turbidity in beer).

Concurrent with such industrial applications, considerable efforts have been made to activate and stabilize these enzymes. See generally: US Patent 1,826,467, issued to Harteneck on October 6, 1931; US Patent 1,967,679, issued to Muench et al. 24 July 1934; US Patent 2,095,300, issued to Wallerstein on October 12, 1937; US Patent 2,130,137, issued to Klotz on September 13, 1938; US Patent 2,257,218, issued to Balls on September 30, 1941; US Patent 2,676,138, issued to Hinkel on April 20, 1954; US Patent 3,019,171, issued to Block et al. January 30, 1962; US Patent 3,284,316, issued to Cayle on November 8, 1966; US Patent 3,539,451, issued to Heinicke on November 10, 1970; US Patent 3,709,790, issued to Beuk et al. January 9, 1973; US Patent 4,011,169, issued to Diehl et al. March 8, 1977; US Patent 4,024,285, issued to Beuk et al. 17 May 1977; and US Patent 4,118,515, issued to Rommele et al.

3 October 1978.

Recently, medical applications for the proteolytic enzymes derived from papaya have also been investigated. See e.g. US Patent 3,019,171, issued to Block et al. 30 January 1962 (physiological debridement of burn scab, blood and pus); US Patent 3,072,532, issued to Innerfield on January 8, 1963 (chemotherapy); US Patent 3,320,131, issued to Smith on May 16, 1967 (treatment of herniated intervertebral discs); US Patent 3,558,433, issued to Stern on January 26, 1971 and assigned to Baxter Laboratories, Inc.

(treatment of herniated intervertebral discs and acceleration of the post-cryosurgical healing process); and US Patent 4,374,926, issued to Stern on February 22, 1983 and assigned to Smith Laboratories, Inc. (treatment of abnormal, damaged or herniated discs).

Medical applications of the proteolytic enzymes derived from the papaya plant have been delayed by toxicity and antigenicity problems. Unfortunate physiological effects were observed when such enzymes were injected into live cattle to effect ante-mortem tenderization of meat. See generally: US Patent 3,235,468, issued to Hogan on February 15, 1966; US Patent 3,446,706, issued to Beuk on May 27, 1969; and US Patent 3,707,790, issued to Beuk on June 9, 1973. US Patent 3,923,599, issued to Hess et al. 2 December 1975, reported a method for the preparation of plant enzyme preparations, e.g. papain, with low bacterial content. US Patent 4,024,285, issued to Beuk on May 17, 1977, reported a method of processing food grade papain or crude papain, whereby intravascular ante-morten injection reportedly did not result in adverse physiological reactions. Said US patent 4,024,285 also states that several proteolytic enzymes other than pure papain are present in both raw papain and food grade papain, and that chymopapain proteases can make up up to 90% of the total proteases in commercial powders of food grade papain and raw papain. US Patent 4,374,926, issued to Stern on February 22, 1983, discloses a method for the preparation of what is stated to be an improved chymopapain with lower toxicity risk and lower sensitive ringing propensity by intravenous and intradermal injection in laboratory animals.

Problems resulting from the toxicity and antigenicity of chymopapain preparations have so far done their part to thwart the development of chemo-nucleolytic therapies that represent great potential for alleviating the clinical manifestations resulting from herniation of intervertebral discs in general and of lumbar intervertebral discs in particular . The adult spinal column consists of a series of 24 bones that form the flexible, central axis of the skeleton. The spinal column encloses the spinal cord, supports the head, holds the ribs laterally, and is attached below to the pelvic ring. The bones that make up the spinal column are called vertebrae. Each vertebra is essentially a hollow cylinder of bone with flat upper and lower surfaces. The spinal cord and nerves run through the central cavity. The flat surfaces of adjacent vertebrae are connected by intervertebral cartilage discs. The discs are somewhat flexible, and allow bending of the spine. Each disc has rings of solid fibrocartilage, termed the annulus fibrosus, along its outer and inner circumference. These rings surround and hold back a soft, compressible center, called the nucleos pulposus, which acts as a shock-absorbing element. The nucleus pulposus is particularly well developed in the intervertebral discs between the five large lumbar vertebrae, located in the lower back, which supports the weight of the upper torso. These lower intervertebral discs are exposed to great forces, and are the ones most likely to herniate: damage or weakening of the annulus fibrosus, and surrounding ligaments causes the nucleus pulposus to be pressed into the neural canal and impinge on nerves leading from the spine to the legs and lower torso. Clinical manifestations of pain in the lower back, sciatica, and paralysis can be the result.

Chemonucleolysis is a promising alternative to surgical methods, e.g. laminectomy and discectomy, for the repair of lumbar disc herniations that do not respond to conservative therapy. A needle is carefully inserted and placed into the herniated nucleus pulposus, and a small amount of proteolytic enzyme is injected into it to dissolve the nucleus pulposus and release pressure on the spinal nerves. Chymopapain is the proteolytic enzyme of choice for chemonucleolysis because it selectively hydrolyzes the chondromucoprotein structure of the nucleus pulposus but does not significantly attack the collagenous annulus fibrosus. The general chemonucleolysis technique has been known for over two decades, see e.g. US patent 3,320,131, issued to Smith on May 16, 1967 and assigned to Baxter Laboratories, Inc. The promise of this "chemical surgery" has been hindered by, among other things, of the marked toxicity and antigenicity of the chymopapain preparations hitherto available. Mild to severe anaphylactoid reactions have been observed after injection of even relatively refined chymopapain preparations in about 1% of all patients, and these reactions are life-threatening if not treated quickly and correctly. An allergic reaction can occur immediately or up to 1 hour after injection and can last from minutes to several hours. Moreover, reactions such as rash, urticaria or itching can occur as late as 2 weeks after chemonucleolysis with the previously known chymopapain preparations. Because of. Because of the risk of anaphylaxis, chemonucleolysis is now contraindicated in patients who have previously been injected with some form of chymopapain.

The chymopapain preparations in the prior art were isolated by extraction methods that typically represented an improvement on prior methods of isolating proteolytic enzymes from papaya plants in general. Such prior methods usually involved various salting-out steps to precipitate contaminants and to selectively recover papain fractions showing enzymatic activity. See generally the following US patents: No. 1,959,750, issued to Wada May 22, 1934 (acetone precipitation); no. 2,219,209, issued to Neufeld on October 22, 1944; No. 2,492,580, issued to Kauffman et al on December 27, 1949; No. 2,669,559, issued to Reid on Feb. 15, 1954 (salting out with ion exchange resins); No. 2,950,227, issued to Gibian on Aug. 23, 1960 (thiocyanate precipitation); No. 2,958,632, issued to Schwartz et al. 1 November 1960 (sulphate salt precipitation); No. 3,011,952, issued to Lesuk Dec. 5, 1961 (sequential precipitation with lower alkanol); No. 3,141,832, No. 3,248,300, No. 3,248,301, No. 3,274,072 and No. 3,274,073, all issued to Burdick on July 21, 1964, April 26, 1966, April 26, 1966, respectively. 20 September 1966 and 20 September 1966 (acetone or ammonium sulphate precipitation); No. 3,210,257, issued to Cayle on Oct. 5, 1965 (acid, ammonium sulfate, and solvent precipitations); and No. 3,694,315, issued to Boudart on September 26, 1972.

Such salting-out steps have generally been supplemented by various chromatographic separation methods. See: US Patent 3,104,206, issued to Messing September 17, 1963 (reporting gel filtration of papain and dextran column; US Patent 3,983,001, issued to Coupek et al. September 28, 1976 (reporting isolation of papain on a column of hydroxyethyl meth-actylate gel with covalently bound p-aminophenylmercuric acetate); US Patent 4,020,268, issued to Nishikawa et al. April 26, 1977 (reporting isolation of papain on affinity adsorbent columns); e.g., carboxymethyl cellulose, or cross-linked dextrans; and US patent 4,318,990, issued to Thompson et al March 9, 1982 (reporting separation of papain on column of titanium oxide particles).

Several other isolation methods are known and either combine batchwise with chromatographic techniques, or indicate the need to achieve a certain optimization of different isolation parameters.

US Patent 3,011,952, issued to Lesuk on December 5, 1961, reports a method for purifying papain by dispersing the crude enzymes in water, adding an amount of water-miscible lower alkanol to the initial precipitation point of the proteolytic enzymes, thereby retaining it normal proteolytic activity in the solvent phase while most of the contaminants insoluble in the lower alkanol are precipitated. It is stated that in order to precipitate the maximum proteolytic activity of the purified enzyme, it was advantageous to first determine the optimal concentration of lower alkanol to be used. For example, to aliquot portions of a dispersion of 100 g of crude papain in 120 ml of water at 24°C, varying amounts of methanol were added to achieve varying solvent concentrations in the range of 60-90% (v/v) in the solvent phase . After equilibration for 1 hour at 24°C, the respective precipitates were isolated, and the proteolytic activity of the precipitated enzymes was determined quantitatively for each of them.

US Patent 3,752,741, issued to Courtois on August 14, 1973, reports a method for the extraction of proteases active in alkaline medium from fermentation broths using a nonionic amberlite resin. In normal practice, 100-500 ml of the resin per liters of fermentation broth to be processed, allegedly added one or more times. In general, the resin was left in contact with the fermentation broth for 2-15 hours to ensure adsorption of the proteases on the resin. The resin was then isolated from the entire fermentation broth by e.g. easy aiming. The isolated resin was reportedly most advantageously in a column and the proteases then eluted. The proteases were thereby allegedly recovered from the fermentation broth at yields of 70-90%.

US Patent 3,947,324, issued to Lakshminaraya on March 30, 1976, reports a method for isolating peroxidase enzyme from plant tissue, which involved treating an aqueous extract of said plant tissue with zinc ions. After such treatment, the clear enzyme solution was selectively adsorbed by the addition of hydrated carboxymethylcellulose (Sephadex C50), which is stated to adsorb approx. 70% of the total available peroxidase activity and the rest was recycled to the next batch. The carboxymethyl cellulose was then separated by filtration and the resulting cake was washed and eluted.

US Patent 4,246,351, issued to Miyake et al. Jan 20 1981, reports a copolymer adsorbent which is stated to adsorb proteins in general and inter alia papain. Adsorption of a protein is generally reported as placing the adsorbent in contact with a solution or dispersion of the protein, either by adding the adsorbent to protein solutions (batch process) or by passing protein solutions through a column packed with the adsorbent (continuous process). It is reported that because the greater the amount of adsorbent, the greater the amount of adsorbed protein, and the faster the adsorption rate, the amount of adsorbent to be used should be determined depending on the amount of protein to be adsorbed, and the desired adsorption time: "Before practical adsorption operation, it is necessary to determine optimal conditions. This will be achieved by quantitatively determining the adsorption capacity of the adsorbent over time. The protein adsorbing capacity of the adsorbent was determined by the following methods. First, in the case that the adsorption was carried out by a batch process, the concentration of the protein was in the treated solution or dispersion determined. The amount of protein adsorbed by the adsorbent was calculated from the difference between the concentration of the protein in the original solution or dispersion and that in the treated solution or dispersion. Second, in the case that the adsorption was carried out by a continuous process, after the adsorption, the protein solution or dispersion present among the grains of the adsorbent was washed out using the same solvent as that of the protein solution or dispersion. The volume of the solvent to be used for washing was five times the apparent volume of the adsorbent. The amount of protein in the treated solution or dispersion including wash material was determined. The difference between the amount of protein in the original solution or dispersion and that in the treated solution or dispersion is the amount of protein adsorbed by the adsorbent. The protein adsorbing capacity of the adsorbent is calculated in mg of adsorbed protein per grams of dry adsorbent." (Translation) (see column 14, line 44, to column 15, line 2).

All the embodiments in US patent 4,246,351 involve the adsorption of specific amounts of a pure protein type by means of the general protein adsorbent; nothing teaches monitoring the adsorption of specific enzyme from complex mixtures containing other proteins in addition to specific enzyme.

US patents 4,388,406 and 4,390,628, issued to Johansen on June 14 and 28, 1983, respectively, report a method for the recovery of Cu,Zn superoxide dismutase on an industrial scale. In connection with Example 1, it is reported that microgranular carboxymethyl cellulose which had been equilibrated with buffer was added to a concentrated yeast crude extract, and the mixture was stirred for 1 hour. The carboxymethyl cellulose was then collected on a 30 cm diameter column, washed with the buffer and then transferred to a 10 cm diameter column for elution.

The following patents describe various isolation methods for chymopapain: US Patent 2,313,875, issued to Jansen et al. March 16, 1943, reports the preparation of a new proteolytic enzyme, designated chymopapain, from papaya latex. Contaminants were removed from a solution of undried latex by acid precipitation at pH 2, followed by salt precipitation by half-saturation with sodium chloride. Almost pure chymopapain protein was then reportedly precipitated by raising the concentration of the salt to full saturation at pH 2. Said protein was reportedly amenable to further purification by reprecipitation and recrystallization.

US patent 3,235,468, issued to Hogan on 15 April 1966, reports the isolation of highly purified materials, e.g. pure chymopapain, from crude papain. However, there is no description of what is reported to be a separation system involved.

US Patent 3,558,433, issued to Stern on January 26, 1971 and assigned to Baxter Laboratories, Inc., reports a method for the purification of chymopapain by chromatography of an aqueous extract of crude chymopapain or papaya latex with carboxymethyl substituted cross-linked dextran copolymer, preferably Sephadex CM-50, equilibrated with aqueous buffer solution followed by elution with aqueous buffer solution of approximately the same pH as, but greater ionic strength than, the buffer used to equilibrate the chromatographic column. Said crude chymopapain is defined therein as chymopapain separated from papaya by salt fractionation, and/or solvent fractionation, and/or pH adjustment and/or similar methods. Stern states that the column shape of the carboxymethyl substituted cross-linked dextran copolymer is important and that a batch technique does not result in satisfactory separation. Also, US patent 3,558,433 states that the bed volume should be such that substantially all protein to be fed will be retained on the column, with sufficient free carboxymethyl substituted, cross-linked dextran copolymer remaining to effect good separation of components during passage through the column. Thus, to make substantially full use of the gel filtration and ion exchange properties of the copolymer, Stern teaches that the chymopapain should be applied to the column at well below the saturation concentration of the copolymer. In the only embodiment, 4 g of crude chymopapain in aqueous solution was added directly to the top of the column and the column was eluted at 25°C with 400 ml of eluent at a flow rate of approx. 20 ml per hour. The chymopapain I and II were reportedly desorbed at approx. 50% and 80% salt saturations, respectively. 4 g of crude chymopapain, which is said to constitute a typical treatment result, reportedly yielded 2.250 g of chymopapain I and 0.430 g of chymopapain II. Stern reports that substantially pure chymopapains I and II were isolated; nevertheless, his electrophoretic results indicate that there was cross-contamination. After stating that different proteins form different zones during electrophoresis, Stern reports the formation of single protein zones after electrophoresis on cellulose acetate. Moreover, the elution profile presented in Fig. 1 in US patent 3,558,433, each fraction by total protein content only.

US Patent 3,627,640, issued to Blumberg et al. December 14, 1971, reports a method for purifying and decolorizing a relatively impure enzyme material by forming a covalently bound polymer-enzyme product. Said method in which the enzyme is chymopapain is defined in claim 9 of the patent.

US Patents 4,212,945 and 4,212,946, issued to Nonaka et al.15. July 1980, reports a method for the recovery of protease, e.g. chymopapain, from the reaction product of a peptide synthesis in the presence of protease.

US Patent 4,374,926, issued to Stern on February 22, 1983 and assigned to Smith Laboratories, Inc., reports a process for making what is termed improved chymopapain. Said method essentially replaces a carboxymethyl agarose gel with the carboxymethyl dextran gel reported in Stern's US patent 3,558,433. Crude chymopapain (about 29 g) in aqueous solution was added to a column. of carboxymethylagarose gel - resin and eluted with approx. 4.6 1 eluent in approx. 40 hours. Elution profiles are given in fig. 1 in US patent 4,374,926 with regard to both the total protein content and enzymatic activity. The reported yield, calculated for protein content, of chymopapain I is about five times that of chymopapain II. Moreover, the enzymatic activity curve is clearly shifted from the curve for total protein content; the enzymologists recognize that such a shift indicates the presence of impurities. The collected eluent fractions containing chymopapain I and II were combined, dialyzed and lyophilized. It is reported that by purifying chymopapain into two active components, a uniform product with lower toxicity was obtained.

Various chymopapain preparations are described in the prior art; see generally the claims of the following US patents: No. 1,967,679, issued to Muench et al. July 24, 1934 (activated papain preparation); No. 2,676,138, issued to Hink et al on April 20, 1954 (papain preparation); No. 3,019,171, issued to Bloch et al. January 30, 1962, (activated papain preparation); No. 3,284,316, issued to Cayle Nov. 8, 1966 (storable papain derivative); and No. 4,011,169, issued to Diehl et al. March 8, 1977 (peptide-peptidohydrolysis-containing preparations). Two chymopapain preparations are currently marketed, nationally and internationally, for use in chemonucleolysis therapy. "Discase", a product of Baxter-Travenol Laboratories, Inc., is described in said company's July 1982 product insert as a sterile preparation of the proteolytic enzyme chymopapain, derived from papaya latex; it is reportedly substantially free of papain as determined by electrophoresis. "Chymodiactin", a product of Smith Laboratories, Inc., is described in that company's January 1983 product profile as a refined proteolytic enzyme obtained from the crude latex of the Carica papaya tree, containing two enzymatically active protein components.

The present invention relates to a method for isolating large amounts of chymopapain B and chymopapain C from either papaya latex or raw chymopapain preparations. Homogeneous populations of chymopapain B and chymopapain C can now be extracted separately in commercial quantities without cross-contamination.

The present invention also relates to therapeutic preparations which consist essentially of pure chymopapain B without any contamination by other proteases, e.g. chymopapain C, or other proteins. Such therapeutic preparations according to the present invention are particularly useful for chemonucleolysis of herniated invertebral disc tissue, and their exceptional purity is an improvement that significantly reduces the risk of severe anaphylactoid and other allergic reactions. The invention also provides a new method for determining, with substantial reproducibility, the activity of chymopapain B and chymopapain C.

It has now been discovered that there are at least six proteases in papaya latex: papain, chymopapain A, chymopapain B1-B3, and chymopapain C. The purification process according to the present invention results in the isolation of chymopapain B and chymopapain C in homogeneous forms. It has now been discovered that chymopapain B exists in three subforms, which are termed chymopapains B1-B3, and that the three subforms of chymopapain B (B1-B3) are due to different oxidation states of the SH groups on this dithiol protease.

The present invention is directed towards the development of a method for the isolation on a large scale of chymopapain B and chymopapain C as separate homogeneous enzymes. The most preferred method for isolating pure fractions of chymopapain B and chymopapain C will now be described in detail starting from the raw material papaya latex. It should also be pointed out that if partially purified chymopapain is used as starting material, then steps 1-10 in the following procedure can be omitted, and the production method can begin with the highly selective batch adsorption process in steps 11 and 12.

Unless otherwise stated, all steps are performed at about 4°C to minimize any microbial contamination and to preserve enzyme activity. Pyrogen-free distilled water is used in all contexts.

Step 1: Weigh 2 kg of papaya latex into a 6 liter container. Add 3.2 1 distilled water. Stir this mixture at room temperature (22-25°C) for 1 hour.

Step 2: Quickly centrifuge the mixture (9000 x g for 30 min). Discard the pellet containing papain. Save the supernatant for the next step.

Step 3: Bring the supernatant solution to 45% ammonium sulfate by adding small amounts (5-10 g) at a time. Stir the resulting slurry for 30 min.

Step 4: Centrifuge at 9000 x g for 30 min. Discard the pellet. Toe

save the supernatant for the next step.

Step 5: Slowly add IN hydrochloric acid (made by adding 85

ml of concentrated hydrochloric acid to 915 ml of distilled water) until the pH value is brought to 2.0 whereby a cloudy solution results.

Step 6: Centrifuge the above solution at 9000 x g for 30 min.

Discard the pellet. Save the supernatant for the next step.

Step 7: Bring the supernatant to 75% ammonium sulfate by adding small amounts (5-10 g) at a time. Stir the resulting slurry for 1 hour.

Step 8: Centrifuge the slurry for 30 min at 9000 x g.

Decant the supernatant and discard. Keep the pellet, which contains chymopapain B and chymopapain C, for the next step.

Step 9: Dissolve the pellet from step 8 in a minimum volume (100-200

ml) of 0.2M sodium acetate buffer (pH 5.0).

Step 10: Transfer the resulting solution to dialysis tubes (pre-washed with distilled water) and dialyze against 0.2M sodium acetate buffer (pH 5.5). Several changes (3-5) of buffer solutions should be used. The resulting retentate is partially purified, but still contains several contaminating proteins. This crude chymopapain is purified as follows.

Step 11: Weigh 100 g of carboxymethyl Sephadex into a 4 liter beaker.

Add 3.0 liters of 0.2M sodium acetate buffer (pH 5.0) and mix. Store this mixture overnight at 4°C. The swollen carboxymethyl-Sephadex (about 3 liters) is ready for the next step.

Step 12: Add the retentate from step 190 to the swollen carboxymethyl Sephadex from step 11 and stir briefly with a glass stirring rod. Allow the carboxymethyl Sephadex to settle. Analyze an aliquot (50 µl) of the supernatant for chymopapain activity using the standard assay described further below. Add additional carboxymethyl-Sephadex, and repeat the process until the chymopapains are selectively bound to the matrix and less than 5% of the original activity is left in the solution.

Step 13: Pack the chymopapain-bound carboxymethyl-Sephadex onto a suitable column. Wash the column with 0.2 M sodium acetate until the absorbance (280 nm) of the effluent reaches the baseline level.

Step 14: Elute the enzymes from the column with a linear gradient of sodium acetate from 0.2 to 1.0 M (pH 5.0). Monitor the fractions for protein content and enzymatic activity. A typical elution profile is shown in fig. 1. The large protein peak appearing in the early fractions lacks chymopapain activity while the two later elution fractions containing activity represent chymopapain B chymopapain C.

Step 15: Perform acid polyacrylamide gel electrophoresis on representative fractions of the eluent. Determine the fractions containing pure chymopapain B and the fractions containing pure chymopapain C. A typical electrophoretic profile is shown in fig. 2.

Step 16: Collect the fractions containing pure chymopapain B and reduce the volume either by lyophilization or ultrafiltration. Separately collect and concentrate fractions containing pure chymopapain C.

Step 17: Either dialyze the concentrated chymopapain C solution from the previous step against distilled water with at least three changes of water, or pass the concentrated enzyme solution through a Sephadex G-25 gel filtration column to eliminate salts. A typical elution profile for Sephadex G-25 gel filtration is shown in fig. 3. Carry out separate desalting of the concentrated chymopapain C in a similar manner.

Step 18: Lyophilize the resulting salt-free solution to obtain the fluffy, crystalline chymopapain B. Perform separate lyophilization of the desalted chymopapain C.

While this method is described on the basis of an initial starting material of 2 kg of papaya latex, it should be understood that it can be further scaled up, e.g. 10 times, without too much delay or changes in procedure. A purification on an industrial scale has e.g. has been carried out using 60 g of crude chymopapain as starting material in step 12.

While the present description has been described on the basis of the preferred method for salting out contaminants at 45% ammonium sulfate and at pH 2.0, followed by salting out crude chymopapain at 75% ammonium sulfate, it should be understood that crude chymopapain prepared by other methods with advantage can be used, and without excessive experimentation in the new, selective batchwise adsorption process in step 12. See Example 2, where a commercial source of partially purified chymopapain is purified using the above-mentioned steps 11-18. Likewise, it is understood that satisfactory elution can be achieved using linear gradients from 0.2 to 1.0 M (pH 5.0) of salts other than the preferred sodium acetate.

A critical element in the present process is the highly selective batch adsorption step (the previously mentioned step 12). A solution of crude chymopapain is mixed with a portion of cation exchange resin, preferably a carboxymethyl substituted, cross-linked dextran copolymer, to specifically adsorb the chymopapain enzymes onto the resin, leaving contaminants in solution. The supernatant is then analyzed to determine the remaining chymopapain activity in solution. Additional cation exchange resin is added to specifically adsorb additional chymopapain enzymes from solution, and the supernatant is then reanalyzed. This titration of the solution with cation exchange resin is continued until a certain residual activity of chymopapain is back in solution - until the chymopapain enzymes have almost saturated the available binding sites on the cation exchange resin.

It has been found advantageous to add the resin slowly until the remaining activity in the supernatant is less than approx. 10% and preferably from approx. 5 to approx. 2%, of the initial total activity in the solution of crude chymopapain. Ideally, at least approx. 2% of the initial chymopapain activity remains in the solution. The aforementioned cation exchange resin is preferably a carboxymethyl substituted, crosslinked dextran copolymer. Carboxymethyl-Sephadex (Pharmacia) type C-50 is particularly preferred, but type C-25 is also acceptable. It is understood that carboxymethyl agarose gels will also be acceptable.

The new selective batch adsorption process in conjunction with the conventional column chromatography step has been found to offer several advantages over simple column chromatography methods alone, in which all the contaminants must pass completely through the column. The selective adsorption process according to the present invention substantially avoids the passage of polluting materials through the column. The polluting materials never pass through the column because they are never adsorbed on the cation exchange resin. The chymopapain enzymes are specifically and selectively adsorbed onto the chosen resin and the above titration step ensures that insufficient resin is added to the system for the contaminants to also be adsorbed. The titration of the resin in the impure enzyme solution is stopped near * the resin's saturation point for the enzymes, but before the saturation point is exceeded, at which point the contaminants would be adsorbed on the then available surplus of binding sites. Thus, recovery of the desired product can be maximized with minimal contamination.

Other advantages of this preload include much faster processing times and much greater escalation potential for industrial extractions, isolations and purifications. In the above dehydration, only sufficient carboxymethyl dextran is added to almost completely bind the chymopapain enzymes. This results in maximizing the efficiency of the ion exchange and, because the contaminants do not need to be laboriously pumped through the subsequently formed column, a much faster treatment is possible. It should be understood that treatment time is a factor affecting the pyrogen content of the final product, and that this parameter becomes particularly important where, as here, the addition of bacteriostatic agents would inactivate the enzyme. Table 1 summarizes representative samples of chymopapain B according to the invention with regard to endotoxin levels.

Limulus amoebocyte lysate (LAL) test (Associates of Cape Cod, Inc. Woods Wole, Massachusetts).

Because the batch titration process of the invention eliminates the need to pass contaminating materials through the column prior to elution, the scale of production can be easily increased. Chymopapain enzymes can now be recovered separately and rapidly in commercial quantities, and the homogeneous purity of the resulting enzymes is significantly better than that obtained by any existing commercial method.

An essential component of the above purification method is the use of a standardized enzymatic activity assay which is necessary to effect optimal loading (step 12) and recovery (step 14) of chymopapain B and chymopapain C.

It was necessary to provide a standardized reproducible enzyme assay for chymopapain B. The method traditionally used using casein as a substrate was unsatisfactory for several reasons. First, casein is a heterogeneous substrate comprising a mixture of peptides; furthermore, each hydrolytic cleavage provides a new, different substrate with different affinity and catalytic parameters. Thus, non-linear kinetics occur resulting in a lack of reproducible rates and non-linearity. Secondly, the method of analysis with casein is trivial and time-consuming and therefore does not allow continuous monitoring of the reaction. Third, this method does not allow expression of enzyme activity in standard units, e.g. micromole product formed per minute. An alternative method uses the synthetic substrate DL-benzoylarginine-p-nitroanilide (BAPNA). This substrate is homogeneous and provides a single bond type for hydrolysis. This substrate also allows a more appropriate and faster analysis because the reaction can be monitored photometrically. However, previous analyzes using BAPNA were not performed under substrate-saturation conditions, i.e. at zero-order kinetics. Under non-saturating conditions, small changes in substrate concentration result in large changes in reaction rate, and thus a non-standardized BAPNA assay lacks reproducibility. Previous BAPNA assays have also used the antiquarian nomenclature of ktal or pktal instead of expressing activity in international enzyme units (micromoles per min.) See e.g. US Patent 4,374,926.

Thus, in order to optimize the selective batch loading of chymopapain B onto the carboxymethyl-dextran and to improve and monitor its elution from the subsequently formed column, it was necessary to establish a standard assay for chymopapain B using BAPNA at substrate-saturation conditions under optimal conditions for enzyme activity. Those skilled in the field of enzymology will appreciate that before such a standardized method of analysis could be provided, the following parameters had to be investigated and established: the molar extinction coefficient of the hydrolytic products in BAPNA under optimal assay conditions, the enzyme's optimal pH, the optimal conditions for enzyme activation, and the substrate-saturation curve .

Said extinction coefficient was determined as follows: When chymopapain hydrolyzes BAPNA, the product p-nitrophenol absorbs light in the visible region. It is necessary to determine the molar extinction coefficient for this product under the conditions of analysis. BAPNA was completely hydrolyzed with chymopapain B and the visible absorbance spectrum determined using a Gilford model 2600 spectrophotometer. Fig. 4 shows absorbance spectra for both BAPNA and the hydrolysis products of BAPNA. 410 nm was chosen as the wavelength for this analysis, because at this wavelength BAPNA does not absorb. A wavelength of 410 nm was used for all future studies. A 0.10 M BAPNA standard solution was prepared by dissolving 130.47 mg of BAPNA (Sigma Chemical Co., product no. B-4875, molecular weight 434.9) in 3.0 ml of dimethyl sulfoxide (DMSO). This solution was diluted in DMSO to obtain stock solutions of 10 mM and 1 mM. It is necessary to dissolve BAPNA in DMSO because it does not dissolve easily in water. Aliquots of the 1 mM solution were transferred to envelopes containing assay buffer (0.1 mM sodium citrate, 0.5 mM EDTA, 3.6 mg/ml cysteine, pH 6.4), and the spectrophotometric zero baseline was established at 410 nm. The substrate was then hydrolyzed enzymatically using an aliquot (30 L) of either trypsin or chymopapain. The reaction was continuously monitored until all substrate was hydrolysed, and no further increase in absorbance was recorded. Completion of the reaction was confirmed by additional aliquots of either enzyme or substrate. The results given in Table 2 establish an average mM extinction coefficient for the hydrolytic products of BAPNA at 410 nm equal to 4.86 mM-<1>cm-<1>. ,

The pH optimum for chymopapain B was determined as follows: Chymopapain B activity was determined as a function of pH using 5 mM BAPNA. All incubations were in the 0.1 M sodium citrate assay buffer described above. Fig. 5 shows the results from these studies. All subsequent studies used pH 6.4 as the optimal pH value for the analysis.

Optimal enzyme assay conditions: Activator cysterin is included to ensure that the essential SH groups in the enzyme remain reduced. EDTA is added to prevent inactivation of heavy metals; citrate buffer also helps in this regard because its chelating properties. All the analyzes are carried out at 37°C since this is closest to human body temperature, whereby the enzyme is to be used for chemonucleolysis. Although the enzyme can be assayed at different temperatures, correction for the significant temperature effect on enzyme rate would otherwise be necessary.

Substrate saturation was determined as follows: Pure chymopapain B was used to establish a saturation curve. A constant amount of enzyme was assayed, in the above assay buffer as a function of the BAPNA concentration phase. The resulting data indicating the effect of BAPNA concentration on chymopapain B activity are reported in Table 3 and given as a saturation curve in Fig. 6.

Fig. 7 presents these data in a double reciprocal Lineweaver-Burk plotting, from which the following is calculated:

Correlation = 0.99956

Intercept = 77.948

Slope = 179.38

Km = 2.3 mM

The Km value of 2.3 mM indicates that at this concentration for BAPNA the reaction rate will be 1/2 the maximum. Ideally, a substrate concentration of 10 x Km would be used to ensure zero-order kinetics. However, BAPNA is not soluble in aqueous buffer at pH 6.4 at this concentration. As the best compromise, a concentration of 5 mM should be used to assay the enzyme. This represents the highest concentration at which the substrate is still soluble. At 5 mM, the reaction rate also approaches at max. so that small variations in the BAPNA concentration cause only small changes in the measured rate.

Once the above optimal conditions were established, the standardized, reproducible enzyme assay for chymopapain B was provided. The following solutions are used: Analysis buffer, BAPNA stock solution and chymopapain B solution. The analysis buffer is 0.1 M Na citrate, 3.6 mg/ml cysteine, 0.5 mM EDTA, pH 6.4. Dissolve 29.41 g of sodium citrate (Na3C6H507-2H20; molecular weight = 294.10; Fisher Scientific Co.) in 1 L of distilled water. Add 3.6 g cysteine and 168.1 mg EDTA, disodium salt (molecular weight = 336.2; Sigma Chemical Co., Product No. ED255), and stir until dissolved. Adjust the pH to 6.4 with 0.1 M citric acid, prepared by dissolving 19.2 g of citric acid (molecular weight = 292.13; MCB Manufacturing Chemists) per litres. The BAPNA stock solution is 100 mM BAPNA in DMSO. Dissolve 130.47 mg of BAPNA (molecular weight = 434.9; Sigma Chemical Co., Product No. B-4875) in 3.0 mL of DMSO at 37 °C. The enzyme solution is prepared by dissolving 8 mg of chymopapain B in 1.0 ml of distilled water.

The standard assay method is as follows: For the standard assay method, the level of BAPNA is 5 mM. The recording spectrophotometer is set at 410 nm (visible light source); the temperature is set at 37°C; 940 µl of 0.1 M Na citrate, (pH 6.4), 3.6 mg/ml cysteine, and 0.5 mM EDTA, and 50 µl of 100 mM BAPNA in DMSO are added to a 1.0 ml cuvette (1 cm road length). The contents of the cuvette are mixed, and the cuvette is introduced into the spectrophotometer (at 37°C). All BAPNA, if not already in solution, will dissolve when 37°C is reached. The recorder is turned on, a zero baseline is established to confirm that there is no non-enzymatic hydrolysis of BAPNA. 10 µl of chymopapain B enzyme solution is then added to the cuvette, the contents are mixed, and the enzymatic activity is monitored and recorded at 410 nm.

An international enzyme unit (IEU) of chymopapain B is the amount of enzyme that produces one micromole of p-nitroaniline per min. from a 5 mM solution of BAPNA substrate at 37°C and pH 6.4.

A significant feature of decisive importance in the standardized analysis, and obvious variations thereof, is the use of continuous monitoring. Problems with inactivity, instability and reproducibility in previous enzyme assays for chymopapain have been recognised. See e.g. US Patent 2,676,138. The present invention provides a solution to these problems by providing an analysis method whereby the increase in absorbance from the products of the enzyme-substrate reaction is continuously monitored. Such monitoring avoids, for example, the problem of variability rates whereby different samples of chymopapain regain activity when first re-exposed to a sulfhydryl reagent prior to reaction with the enzyme substrate.

While the standard analysis method as described above is the preferred embodiment of the present invention, it will be understood that the analysis method can be modified in a number of different ways in accordance with the principles of the technical field. By way of illustration, the use of EDTA or a comparable chelating agent is not strictly necessary because metal ions are not usually contaminants in the assay buffer or mixtures thereof. The addition of a chelating agent such as EDTA will, however, reduce the effect of metal ions, if such are present.

Other sulfhydryl reagents can be used instead of, or in combination with, cysteine. Included among such sulfhydryl reagents are dithiothreitol, β-mercaptoethanol, and glutathione. Commonly used buffers, e.g. phosphate or acetate buffers can be used instead of sodium citrate, and buffer concentrations varying from approx. 0.25 M to approx. 0.025 M can be used.

A number of different substrates for chymopapain B or chymopapain C can be useful as homogeneous non-proteinaceous photometric substrates for the photometric measurement of enzymatic activity. Besides BAPNA, such substrates include, but are not limited to, N-cx-benzoyl-L-arginine ethyl ester (BAEE); N-α-benzoyl-L-arginine methyl ester; N-oc-benzoyl-D,L-arginine-p-naphthylamide; N-α-benzoyl-D,L-arginine-para-nitroamide. BAPNA itself has been reported by others to be useful in measuring chymopapain. See e.g. US Patent 4,439,423.

While the concentration of BAPNA or another substrate is preferably approx. 5 mM, and in all cases substantially below 10 x Km, it will be understood that concentrations lower or higher than approx. 5 mM in principle is useful when performing the present standardized method of analysis provided that sufficient reproducibility continues to be achieved. It has been discovered that the reproducibility of the present standardized analysis is greater than it would have been expected to be for analyzes carried out at approx. 2 x Km, or 5 mM in the case of BAPNA.

The standard analysis method for chymopapain described above can also be used for measuring the C form of chymopapain, also known as chymopapain C. See e.g. fig. 1. These two forms of chymopapain have previously been designated by other nomenclature.

According to the general principles for enzyme analyses, the above-described standard analysis methods for chymopapain B can also be modified by changing the temperature, volume, pressure and concentration of reactants. It is understood that variations f of these parameters as well as others are covered by the present invention.

In the following, reference must be made to the accompanying illustrations, where:

Fig. 1 is a graphical presentation which plots both total protein content in units for absorbance at 280 nM (<*>—<*>) and enzymatic activity in international enzyme units per ml (□—□) versus molar concentration and fraction number for eluent, as described in example 4: fig. 2 is the gel plate resulting from electrophoretic analysis on acid-polyacrylamide gel of every 10th fraction of eluent, as described in Example 2;

fig. 3 is a graphical representation showing a typical elution profile after desalting (see step 17 in the description given above), where both enzymatic activity in international enzyme units per ml —Q) and the salt concentration as a function of conductivity (<*>—<*>) are plotted against eluent - fraction number;

fig. 4 is a graphical representation where absorbance is plotted against wavelength, which shows the absorbance spectrum for both BAPNA and the hydrolysis products of BAPNA as described above;

fig. 5 is a graphical representation where enzymatic activity in 1000 of international enzyme units is plotted against pH value, which establishes the pH optimum for chymopapain B, as described above;

fig. 6 is a graphical representation where enzymatic activity in thousands of international enzyme units is plotted against BAPNA concentration (in mM) as reported in Table 3;

fig. 7 is a double reciprocal Lineweaver-Burk plot of the data in Table 3; and

fig. 8 is a gel plate resulting from acid-polyacrylamide gel electrophoretic analysis of four chymopapain preparations: Discase® (far left), Chemolase® (middle left), Chymodiactin® (middle right), and the purified chymopapain B according to the invention (far right), as described in example 6.

In order for the expert in the field to understand the present invention more fully, the following examples are given. These examples are provided for illustrative purposes only and shall not be deemed to express limitations unless stated in the accompanying claims.

Example 1

A typical calculation of the enzymatic activity for chymopapain B, determined by the standardized enzyme assay according to the invention and expressed in international enzyme units (herein referred to as "units") per mg, was carried out as follows. 8 mg of Chemolase<®> (Pharmotex, Inc., lot no. 01153-38) was dissolved in 1.0 ml of distilled water, 10 liters of this enzyme solution was analyzed.

A buffer solution (0.1 M sodium citrate, 3.6 mg/ml cysteine, 0.5 mM EDTA, pH 6.4) was prepared as previously described. 940 µl of said buffer solution was used in this analysis. A BAPNA stock solution (100 mM BAPNA in DMSO was prepared as described previously. 50 µl of said BAPNA stock solution was used in this analysis.

The standard analysis method was followed as described above. The 1.0 ml cuvette containing the aforementioned aliquots of buffer and BAPNA solutions was equilibrated at 37°C in a spectrophotometer, and a zero baseline at 410 nm was established. Then the aforementioned aliquot of enzyme solution was added, stirred and the change in absorbance at 410 nm at 37°C was monitored. Said observed change in absorbance (A) was 0.044/min.

The relevant formula is:

A = cbc

where

A absorbance = 0.044/min.

c = extinction coefficient = 4.86 mM~<l>cm~<l>b = path length = 1 cm

c = concentration

Rearranging the formula:

Since the final volume in the cuvette is 1 ml and unit =<i>^-y (0 , 009053 5 ^~--t- )<1 ml) = 0 , 009 05 35 = 9^535 x 10~<3>units. ml min min

Since 10 µl of chemolase is used for the analysis, one obtains (\f X0.01 ml) = 0.08 mg of chemolase used for the analysis. Therefore, 9.0535 x 10^ units/0.08 mg = 0.113 units)/mg weighed chemolase.

Example 2

The purification process according to the present invention is illustrated by the following production method which used a partially purified chymopapain as starting material. 100 g of carboxymethyl-dextran (CM-Sephadex) beads were suspended in 3 liters of 0.2 M sodium acetate buffer, pH 5.0, and left overnight at 4°C. The next morning the swollen CM-Sephadex was ready for use. 30 g of partially purified chymopapain (Sigma, lot no. 13F-8155) was dissolved in 1.5 liters of 0.2 M sodium acetate buffer, pH 5.5, in a 2 liter beaker using a magnetic stir bar. An aliquot of the resulting crude enzyme solution was assayed for activity using the method described in Example 1, and the total enzymatic activity for the 1,500 mL was calculated to be 5,245 international enzyme units, or 3,497 units/mL.

About. 1.5 L of the settled CM-Sephadex beads and 750 ml of the crude enzyme solution were mixed and stirred with a glass rod. The beads were allowed to settle, and the supernatant was analyzed for enzymatic activity. No activity was observed.

A further 250 ml of impure enzyme solution was added and stirred. After the CM-Sephadex beads had settled, an aliquot of the supernatant was analyzed and a weak enzymatic activity was observed.

About. 650 ml additional CM-Sephadex and the remaining 500 ml of the crude enzyme solution were added and stirred. The supernatant was again analyzed, and this time a weak enzymatic activity - less than 5% of the calculated total activity - was observed.

The resulting chymopapain-bound CM-Sephadex was packed into a 5 x 100 cm column and washed overnight with 0.2 M sodium acetate buffer, pH 5.0, at a flow rate of 1.5 ml/min. 4 liter volumes of 0.2 M and 1 M sodium acetate solutions, pH 5.0, were applied to the column to obtain a linear ionic strength gradient of eluent from 0.2 to 1.0 M, at pH 5.0. Said gradient took about 4 days through the column. 2.5 ml sequential fractions of eluent were collected as they exited the column.

Every 10th fraction was analyzed for chymopapain activity and also subjected to acid-polyacrylamide gel electrophoretic analysis. The gel plate from the thus tested fractions in this experiment is reproduced in fig. 2. Note that chymopapain B occupies the upper band, and that chymopapain C occupies the lower band that only appears in the fractions that eluted at high molar concentrations.

The fractions which only contained chymopapain B, and especially those represented by fractions 150-220 in fig. 8, was collected and lyophilized to a smaller volume. This concentrated combined material was then dialyzed against distilled water with 4 water changes. The resulting retentate was then lyophilized to obtain the fluffy, crystalline chymopapain B.

Pyrogen content was analyzed using the Limulus amoebocyte lysate (LAL) test (Associates of Cape Cod, Inc., PO Box 244, Woods Hole, Massachusetts 02543). The results showed the presence of only three pyrogenic endotoxin units (5 mg of this crystalline chymopapain

B).

Example 3

7.4 g of purified chymopapain B, prepared by several executions of the process described above, was determined to have a specific activity of 0.15 units/mg. This 7.4 g of purified chymopapain B or 1,110 international enzyme units was sent to Connaught Laboratories, Inc. which lot no.

0308-307, for mixing with conventional excipients to form a Chemolase® preparation for use in chemonucleolysis therapy.

Chemolase® is a sterile, lyophilized, proteolytic enzyme powder for injection between slices. Chemolase® contains 6 international enzyme units Chymopapain B, 3.5 mg L-cysteine hydrochloride monohydrate and 0.37 mg disodium edetate; with 1 mg of sodium disulfide added by mixing. Said therapeutic preparation essentially consists of chymopapain Buten mixture with chymopapain C or other contaminating proteins.

Example 4

Fig. 1 represents an elution profile which is representative of the total recoveries effected by the present method. Enzymatic activity (international enzyme units/ml) and total protein content (absorbance at 280 nm) are plotted on the abscissa, molar salt concentration on the diagonal and fraction number on the ordinate. Note that the large protein peak that appeared first lacked protease activity, while the two fractions that subsequently eluted from the column did show such activity. Chymopapain B, the second fraction, exited the column at an approximate salt concentration from ca. 0.65 to approx. 0.75 M (fractions 165-220 here). Chymopapain C eluted as a third fraction at a salt concentration from approx. 0.80 to 0.90 M (fractions 230-280 here).

Example 5

The physiochemical properties of the pure chymopapain B isolated by the representative method of Example 2 were investigated. Said homogeneous chymopapain B showed three closely related bands on polyacrylamide gel electrophoresis. These three bands were found to represent three subforms of chymopapain B, designated B1-B3, which are due to different oxidation states of the SH groups on this dithiol protease. Chymopapains BJ-B3 are convertible into each other by oxidation or reduction. For example, fig. 8 shows an electrophoretic gel of pure chymopapain B in both partially reduced (middle left, with cysteine) and oxidized

(far right, without cysteine) condition. These bands often merge into a single zone, which is generally referred to in the prior art as chymopapain B (or chymopapain I).

From SDS-polyacrylamide gels, it appears that the molecular weight for chymopapain B according to the present invention is 34-35 K. The best estimate in the present context is 35,200 daltons. This enzyme is a monomer.

The isoelectric point of chymopapain B according to the invention is very basic. Electrophoretic and isoelectric directed studies indicate that chymopapain B (BJ-B3) has an apparent pl = 10.3-10.5.

N-terminal analysis of chymopapain B according to the invention showed primarily tyrosine. This is in agreement with findings in the literature, which also indicate that chymopapain A has an N-terminal glutamic acid and chymopapain C an N-terminal leucine.

The amino acid compositions of two representative samples of chymopapain B according to the invention were determined using conventional techniques. The results are shown in table 4.

Chymopapain B produced by the present method can be clearly distinguished from chymopapain C (also called chymopapain II), which is eliminated by the present method, and which does not contaminate the present therapeutic preparation. Preliminary investigations of chymopapain C isolated by the present method indicate that this monothiol enzyme appears to be monomeric and has the approximate size of the other thiol proteases from papaya. This chymopapain C is more basic than chymopapain B and has an apparent pl value of about 10.8. This chymopapain C exhibits positive BAPNA hydrolytic activity and, on a specific activity basis, has a higher BAPNA "turnover" than chymopapain B.

Example 6

Four chymopapain preparations were analyzed by gel electrophoresis under acidic conditions by the following method: J. V. Maizel, Methods in Virology, vol. 5, Chapter 5, Pages 179-246 (1971).

The results are shown in fig. 8. On the far left is Discase® (Baxter-Travenol Laboratories, lot no. AV15F5M). To the left of the center is Chemolase® (lot no. 021031-86), which is representative of purified chymopapain B prepared by a more detailed method than the method in example 2, and here mixed with L-cysteine hydrochloride monohydrate and disodium edate, with added sodium disulfite. To the right of center is chymodiactin® (Smith Laboratories, lot no. BM201). On the far right is a representative sample of chymopapain B purified by the method in example 2.

It should be noted that both Discase and Chymodiactin are mixtures of chymopapain B (upper band) and C (lower band), while the preparation according to the present invention (far right) contains chymopapain B without any contaminating chymopapain C.

Example 7

The toxicity and antigenicity of two different chymopapain preparations were compared in a double-blind study. A Chemolase® preparation which had been prepared here by preparing the purified chymopapain B according to the invention as described in example 3, was compared with Chymodiactin® (Smith Laboratories), which contained both chymopapain B and C. A control study with a double blind reference without placebo; was conducted at Yale University under FDA protection (IND 21087). including chemonucleolysis with human patients. Neither the doctors involved nor the patients knew which of the two above-mentioned chymopapain preparations were used.

Out of 43 injections to date, an adverse reaction which could possibly be an allergic type of reaction has been reported, and which occurred after injection of the Chymodiactin® compound. In this particular case, the physician reported that a full-body skin rash occurred 24-48 hours after injection of a preparation that was later determined to be the Chymodiactin® compound. The records show that the affected patient had previous episodes of this type of reaction. Although the physician is not convinced that this was actually a drug reaction, similar serum-type illness reactions following chymopapain injection have previously been reported in the medical literature, and this clinical observation was reported to the FDA. These preliminary results indicate that the new therapeutic preparation according to the invention, which consists essentially of chymopapain B without mixing with chymopapain C, can reduce the risk of adverse allergic reactions vis a vis injection with preparations containing several chymopapain enzymes. This suggestion follows the general principle that to minimize the risk of any type of allergic reaction, it is advisable to incorporate the fewest number of different types of proteins into the inoculum.

While the invention has been described in connection with particular embodiments thereof, it will be understood that it can be further modified, and that the present application shall cover any variation, use or adaptation of the invention which generally follows the principles of the invention and includes such deviations from what is here is described which is covered by the relevant technology to which the invention belongs, and which can be used on the previously indicated essential features, within the idea of the invention and the framework for the accompanying claims.

Claims (26)

1. Method for purifying chymopapain protease from a complex in solution, characterized in that one . (a) mixing said complex solution with increments of cation exchange resin to selectively adsorb some but . not all protease on the resin until the point is reached at which protease activity in the supernatant has decreased to a certain minimal level; and (b) eluting the protease from the resin. 2. Method according to claim 1, characterized in that the complex solution is an aqueous solution of crude chymopapain. 3. Method according to claim 2, characterized in that the crude chymopapain is isolated from papaya latex by (a) forming an aqueous dispersion containing papaya latex, (b) centrifuging said dispersion to obtain a supernatant and a precipitate, (c) bringing the supernatant from step (b) to 45% ammonium sulfate to form a resulting supernatant and a precipitate, (d) acidify the resulting supernatant from step (c) to approx. pH 2.0 to form a resulting solution and a precipitate, (e) bringing the resulting solution from step (d) to 75% ammonium sulfate to precipitate crude chymopapain; and (f) recovering the precipitated crude chymopapain. 4. Method according to claim 1, characterized in that the cation exchange resin is a carboxymethyl derivative of a polymeric carbohydrate such as carboxymethyl dextran, carboxymethyl agarose etc. 5. Method according to claim 4, characterized in that the cation exchange resin is a CM-Sephadex. 6. Method according to claim 1, characterized in that the determined minimum protease activity is less than approx. 5%, but greater than approx. 2% of the total protease activity initially present in the complex solution. 7. Method according to claim 1, characterized in that the elution is carried out by column chromatography. 8. Method according to claim 1, characterized in that the elution is carried out with a linear ionic strength gradient of eluent. 9. Method according to claim 8, characterized in that the elution is dried with a linear gradient of sodium acetate from 0.2 to 1.0 M (pH 5.0). 10. Method according to claim 1, characterized in that chymopapain B is extracted separately from chymopapain C. 11. Preparation, characterized in that it essentially consists of pure chymopapain B produced by the method according to claim 1. 12. Preparation, characterized in that it essentially consists of pure chymopapain C produced by the method according to claim 1. 13. Therapeutic preparation, characterized in that it essentially consists of chymopapain B without chymopapain C. 14. Therapeutic preparation according to claim 13, characterized in that it is mixed with suitable pharmaceutical excipients. 15. Therapeutic preparation according to claim 14, characterized in that it is mixed with cysteine and disodium edetate. 16. Therapeutic preparation according to claim 15, characterized in that it is mixed with sodium disulfate. 17. Enzyme assay for chymopapain, characterized by the following steps: (a) providing a suitable amount of buffer containing (1) a sulfhydryl reagent and (2) a homogeneous photometric substrate, (b) contacting said buffer with sufficient chymopapain to provide for the occurrence of detectable photometric changes, and (c) continuously monitoring said photometric changes as they occur. 18. Enzyme analysis according to claim 17, characterized in that said chymopapain is chymopapain B. 19. Enzyme analysis according to claim 18, characterized in that the substrate is BAPNA. 20. Enzyme analysis according to claim 18, characterized in that the buffer is Na citrate with a molarity from approx. 0.025 M to approx. 0.25M. 21. Enzyme analysis according to claim 20, characterized in that the Na citrate buffer has a molarity of approx. 0.1M. 22. Enzyme analysis according to claim 18, characterized in that the buffer contains a chelating agent. 23. Enzyme analysis according to claim 18, characterized in that the sulfhydryl reagent is cysteine, p-mercaptoethanol and glutathione. 24. Enzyme analysis according to claim 23, characterized in that the sulfhydryl reagent is cysteine. 25. Enzyme analysis according to claim 19, characterized in that the photometric changes are monitored at approx. 410 nm. 26. Enzyme assay for chymopapain B, characterized by the following steps: (a) providing a suitable amount of approx. 0.1 M Na-citrate buffer at approx. pH 6.4, containing approx. 3.6 mg/ml cysteine and approx. 0.5 mM EDTA and approx. 5 mM BAPNA, equilibrating the buffer at 37°C, (b) contacting the buffer with sufficient chymopapain B to allow detectable increases in absorbance at ca. 410 nm, and (c) continuously monitoring said increases in absorbance at 410 nm as they occur.