GB2156821A - Process for purifying chymopapain - Google Patents

Abstract

A process which permits the commercial scale isolation of large amounts of homogeneous populations of Chymopapain B or Chymopapain C, free from cross-contamination and other proteins and with low levels of pyrogens, from either papaya latex or crude chymopapain preparations. This process comprises mixing the papaya latex or crude chymopapain with increments of cation exchange resin to selectively absorb some but not all of the chymopapain onto the resin until the point is reached at which protease activity in the supernatant has decreased to a predetermined minimum level. The chymopapain is then eluted from the cation exchange resin. Therapeutic chymopapain-containing compositions are also disclosed, as is a standardized, reproducible enzyme assay for Chymopapain B and C.

Description

SPECIFICATION Pure chymopapain B: Industrial process and therapeutic composition Field of the Invention This invention relates to a process for isolating large amounts of Chymopapain B and Chymopapain C from either papaya latex or crude chymopapain preparations. Said inventive process produces homogeneous populations of Chymopapain B and Chymopapain C, free from cross-contamination and free from other proteins. Said inventive process produces on an industrial/commercial scale large amounts of Chymopapain B and large amounts of Chymopapain C, with low levels of pyrogens. Said inventive process is rapid, simple, and suitable for industrial scale-up without major adaptations or complications.

This invention also encompasses therapeutic compositions which consist essentially of Chymopapain B without contamination by Chymopapain C.

Background of the Invention Chymopapain refers generally to a group of sulfhydryl proteolytic enzymes, now recognized to be distinct from papain and to constitute the major component of crude 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); U.S. Patent No. 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 about 35,000 and have been isolated in the laboratory and studied. See: The Merck Index, 1 0th Ed. (1983), Entry No. 2244, p. 322.

Proteolytic enzymes derived from the papaya plant have long been used in industry. See generally: U.S. Patent No. 1,826,467, issued to Harteneck on October 6, 1931 (degum raw silk and coagulate rubber latex); U.S. Patent No. 1,967,679, issued to Muench et al on July 24, 1934 (dehair hides); U.S. Patent No. 2,095,300, issued to Wallerstein on October 12, 1937 (degum silk); U.S. Patent No. 2,219,209. issued to Neufeld on October 1940 (tenderize meat); U.S. Patent No. 2,464,200, issued to Hall on March 15, 1 949 (tenderize meat); U.S. Patent No. 3,235,468, issued to Hogan on February 1 5, 1 966 (tenderize meat); U.S. Patent No. 3,296,094, issued to Cayle on January 3, 1 967 (tenderize meat); U.S.Patent No. 3,466,706, issued to Beuk on May 1969 (tenderize meat); U.S. Patent No.

3,709,790, issued to Beuk on January 9, 1973 (tenderize meat); U.S. Patent No. 3,776,693, issued to Smith et al on December 4, 1973 (dry cleaning); U.S. Patent No. 4,024,285, issued to Beuk et al on May 17, 1977 (tenderize meat); and U.S. Patent No. 4, 118,515, issued to Römmele et al on October 3, 1 978 (prevent cold turbidity in beer).

Concomitant with such industrial applications, considerable efforts have been made to activate and stabilize these enzymes. See generally: U.S. Patent No. 1,826,467, issued to Harteneck on October 6, 1931; U.S. Patent No. 1,967,679, issued to Muench et al on July 1934; U.S.

Patent No. 2,095,300, issued to Wallerstein on October 12, 1937; U.S. Patent No.

2,130,137, issued to Klotz on September 1 3, 1938; U.S. Patent No. 2,257,218, issued to Balls on September 30, 1941; U.S. Patent No. 2,676, 138, issued to Hinkel on April 20, 1954; U.S. Patent No. 3,019,71, issued to Block et al on January 30, 1962; U.S. Patent No.

3,284,316, issued to Cayle on November 8, 1966; U.S. Patent No. 3,539,451, issued to Heinicke on November 10, 1970; U.S. Patent-No. 3, 709, 790, issued to Beuk et al on January 9, 1973; U.S. Patent No. 4,011,169, issued to Diehl et al on March 8, 1977; U.S. Patent No.

4,024,285, issued to Beuk et al on May 17, 1977; and U.S. Patent No. 4, 118,515, issued to Römmele et al on October 3, 1 978.

Recently, medicinal uses for the proteolytic enzymes derived from papaya have also been sought. See, e.g., U.S. Patent No. 3,019,171, issued to Block et al on January 30, 1962 (physiological debridement of eschar, blood and pus); U.S. Patent No. 3,072,532, issued to Innerfield on January 8, 1963 (chemotherapy); U.S. Patent No. 3,320,131, issued to Smith on May 16,1967 (treatment of herniation of intervertebral discs); U.S. Patent No. 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 U.S. Patent No. 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 retarded by problems of toxicity and antigenicity. Adverse physiological effects were observed when such enzymes were injected into live cattie in order to effect ante-mortem meat tenderization. See generally: U.S. Patent No. 3,235,468, issued to Hogan on February 15, 1966; U.S. Patent No. 3,446, 706, issued to Beuk on May 27, 1969; and U.S. Patent No.

3,707,790, issued to Beuk on June 9, 1973. U.S. Patent No. 3,923,599, issued to Hess et al on December 2, 1 975 reported a process for preparing plant enzyme preparations, e.g., papa in, of low germ content. U.S. Patent No. 4,024,285, issued to Beuk on May 17, 1977, reported a method of treating food grade or crude papain whereby intravascular ante-mortem injection reportedly did not result in adverse physiological reactions. The '285 patent also noted that in both crude and food grade papains, there are present several proteolytic enzymes other than pure papain, and that chymopapain proteases may constitute up to 90% of the total proteases in food grade and crude papain commercial powders. U.S.Patent No. 4,374,926, issued to Stern on February 22, 1983, disclosed a method for producing what was reported to be an improved chymopapain with lower toxicity risk and lower sensitization liability when injected intravenously and intradermally into laboratory animals.

Problems due to toxicity and antigenicity of chymopapain preparations have so far helped stymie the development of chemonucleolytic therapies, which hold great potential for alleviating the clinical manifestations resulting from herniation of intervertebral discs generally and of lumbar intervertebral discs in particular. The adult vetebral column is a series of 24 bones which form the flexible central axis of the skeleton. The vetebral column encloses the spinal cord, supports the head superiorly, suspends the ribs laterally, and attaches inferiorly to the pelvic girdle. The bones which compose the vertebral column are called vertebrae. Each vertebra is essentially a hollow cylinder of bone with flattened superior and inferior surfaces. The spinal cord and nerves run through the central cavity.The flattened surfaces of adjacent vertebrae are interconnected by cartilaginous intravertebral discs. The discs are somewhat flexible and allow the spine to bend. Each disc has rings of tough fibrocartilage, called the annulus fibrosus, along its outer and inner circumferences. These rings surround and restrain a soft compressible center, called the nucleus pulposus, which acts as a sock absorber. The nucleus pulposus is especially well developed in the intervertebral discs between the five large lumbar vertebrae, located in the lower back, which support the weight of the upper torso.These lower intervertebral discs are subject to great forces and are the most likely to herniate: injury or weakening of the annulus fibrosus and surrounding ligaments allows thenucleus pulposus to extrude into the neural canal and impinge upon nerves leading from the spinal cord to the legs and lower toroso. Clinical manifestations of lower back pain, sciatica, and paralysis may result.

Chemonucleolysis is a promising alternative to surgical methods, e.g., laminectomy and discecetomy, of repairing those lumbar disc herniations which fail to respond to conservative therapy. A hypodermic needle is carefully inserted and positioned into the herniated nucleus pulposus, and a small amount of proteolytic enzyme is injected therein in order to dissolve the nucleus pulposus and relieve the pressure on the spinal nerves. Chymopapain in 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., U.S. Patent No. 3,320, 131, issued to Smith on May 16, 1967 and assigned to Baxter Laboratories, Inc.However, the promise of this "chemical surgery" has been retarded by, inter alia, the marked toxicity and antigenicity of the chymopapain preparations heretofore available.

Mild to severe anaphylactoid reactions have been observed following the injection of even relatively refined chymopapain preparations in approximately 1% of all patients, and these reactions are life-threatening if not promptly and properly treated. An allergic reaction can occur immediately or up to an hour after injection and may last from minutes to several hours.

Moreover, reactions such as rash, urticaria, or itching may occur as late as two weeks after chemonucleolysis with the chymopapain compositions in the prior art. Due to the danger of anaphylaxis, chemonucleolysis is also presently contraindicated in patients who have previously been injected with any form of chymopapain.

The chymopapain compositions in the prior art were isolated by extraction protocols which typically represented refinement of early methods of isolating proteolytic enzymes from papaya plants generally. Such early methods generally involved various salting-out steps to precipitate out contaminants and to selectively recover papain fractions that exhibited enzymatic activity.

See generally the following United States Patents: No. 1,959,750, issued to Wada on 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 February 15, 1954 (salting out with ion exchange resins); No. 2,950,227, issued to Gibian on August 23, 1960 (thiocyanate precipitation); No. 2,958,632, issued to Schwartz et al on November 1, 1960 (sulfate salt precipitation); No. o.3,011,952, issued to Lesuk on December 5, 1961 (sequential lower alkanol precipitation); 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 respectively on July 1964, April 26, 1966, April 26, 1 966, September 20, 1 966 and September 20, 1 966 (acetone or ammonium sulfate precipitation); No. 3,210,257, issued to Cayle on October 5, 1965 (acid, ammonium sulfate, and solvent precipitations); and No. 3,694,315, issued to Boudart on September 26, 1 972.

Such salting-out steps have generally been supplemented by various chromatographic separation protocols. See: U.S. Patent No. 3,104,206, issued to Messing on September 17, 1963 (reported gel filtration of papain on dextran column); U.S. Patent No. 3,983,001, issued to Coupek et al on September 28, 1 976 (reported isolation of papain on a column of hydroxyethyl methacrylate gel with covalently bound p-aminophenyl mercury acetate); U.S.

Patent No. 4,020,268, issued to Nishikawa et al on April 26, 1 977 (reported isolation of papain on columns of affinity adsorbent, e.g., carboxymethyl cellulose, or cross-linked dextrans); and U.S. Patent No. 4,318,990, issued to Thompson et al on March 9, 1982 (reported separation of papain on column of titania particles).

Several other isolation protocols are known which either combine batch with chromatographic techniques or teach the need to achieve some sort of optimization of various isolation parameters.

U.S. Patent No. 3,011,952, issued to Lesuk on December 5, 1961, reported a process for purifying papain by dispersing the crude enzymes in water, adding a quantity of water-miscible lower alkanol to the incipient precipitation point of the proteolytic enzymes, thereby retaining the normal proteolytic activity in the solvent phase whole precipitating the major portion of the lower alkanol insoluble contaminants. It was stated that in order to precipitate the maximum proteolytic activity of the purified enzyme, it was advantageous to determine first the optimal concentration of lower alkanol to use. For example, to aliquot portions of a dispersion of 100 g of crude papain in 1 20 ml of water at 24"C were added varying amounts of methanol to yield various solvent concentrations to the range of 60-90% (v/v) in the solvent phase.After equilibration for one hour at 24to, the respective precipitates were isolated and the proteolytic activity of the precipitated enzymes were determined quantitatively for each.

U.S. Patent No. 3, 752, 741, issued to Courtois on August 14, 1973, reported a process for the extraction of proteases active in alkaline medium from fermentation broths by means of a non-ionic Amberlite resin. In general practice, 100 to 500 ml of the resin per liter of fermentation broth to be treated were reportedly added in one or several times. Generally, the resin was left in contact with the fermentation broth for 2 to 1 5 hours to insure good adsorption of the proteases on the resin. The resin was then isolated from the whole fermentation broth by, e.g., simple screening. The isolated resin was reportedly most advantageously placed in a column, and the proteases then eluted. The proteases were thereby reportedly recovered from the fermentation broth at yields of 70 to 90%.

U.S. Patent No. 3,947,324, issued to Lakshminaraya on March 30, 1976, reported a method for isolating peroxidase enzyme from plant tissue which involved treating an aqueous extract of said plant tissue with zinc ion. Following such treatment, the clear enzyme solution was preferentially adsorbed by the addition of hydrated carboxymethyl cellulose (Sephadex C50), which was reported to adsorb about 70% of the total peroxidase activity available, and the balance was recycled to the next batch. The carboxymethyl cellulose was then separated by filtration and the resulting cake was washed and eluted.

U.S. Patent No. 4,246,351, issued to Miyake et al on January 20, 1981, reported a copolymer adsorbent which was said to adsorb proteins generally and, inter alia, papain.

Generally, adsorption of a protein was reported by contacting the adsorbent with a solution or a 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 was reported that since the larger the amount of the adsorbent, the larger the amount of protein adsorbed and the faster the adsorption velocity, the amount of the adsorbent to be used should be determined depending on the amount of the protein to be adsorbed and the desired time of adsorption: "Before practical adsorption operation, setting of optimum conditions is required. This will be attained by determining quantitatively the adsorbing capacity of the asorbent with the lapse of time. The protein-adsorbing capacity of the adsorbent was determined by the following procedures. Firstly, in case the adsorption was carried out by a bath process, the concentration of the protein in the treated solution or dispersion was determined.The amount of the 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.

Secondly, in case 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 5 times the apparent volume of the adsorbent. The amount of the protein in the treated solution or dispersion including the washings was determined. The difference between the amount of the protein in the original solution or dispersion and that in the treated solution or dispersion is the amount of the protein adsorbed by the adsorbent.The protein-adsorbing capacity of the adsorbent is estimated in terms of mg adsorbed protein per gram of dry adsorbent." (See Col. 14, 1. 44 to Col. 15, 1. 2.) All of the working examples in the '351 patent involve the adsorption of predetermined amounts of a pure protein species by means of the general protein adsorbent; none taught monitoring the adsorption of specific enzyme from complex mixtures containing other proteins in addition to specific enzyme.

U.S. Patent No. 4,388,406 and 4,390,628, issued to Johnson on June 14 and 28, 1983, respectively, report a method for recovering Cu,Zn-superoxide dismutase on an industrial scale.

On Example 1, it was 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 column of 30 cm diameter, washed with the buffer, and then transferred to a column of 10 cm diameter for elution.

The following patents disclose various isolation protocols for chymopapain: U.S. Patent No. 2,313,875, issued to Jansen et al on March 16, 1943, reported the production of a new proteolytic enzyme, named chymopapain, from papaya latex. Contaminants were removed from a solution of undried latex by acid precipitation at pH 2 followed by salt precipitation at half saturation sodium chloride. Nearly pure chymopapain protein was then reportedly precipitated by arising the concentration of salt to full saturation at pH 2. Said protein was reportedly susceptible to further purification by reprecipitation and recrystallization.

U.S. Patent No. 3,235,468, issued to Hogan on February 15, 1966, reported the isolation of very highly purified materials, e.g., pure chymopapain, from crude papain. However, no disclosure of what was reported to be an involved separation system was made.

U.S. Patent No. 3,558,433, issued to Stern on January 26, 1971 and assigned to Baxter Laboratories, Inc., reported a process for purifying chymopapain by chromatography of an aqueous extract of crude chymopapain or papaya latex with carboxymethyl substituted crosslinked dextran copolymer, preferably Sephadex CM-50, equilibrated with aqueous buffer solution followed by eluting with aqueous buffer solution having about the same pH as, but greater ionic strength than, the buffer used for equilibrating the chromatographic column. Said crude chymopapain was defined therein as chymopapain separated from papaya by salt fractionation, and/or solvent fractionation, and/or pH adjustment and/or similar methods. Stern taught that the column form of the carboxymethyl substituted cross-linked dextran copolymer is essential, and that a batch technique does not result in satisfactory separation.Moreover, the '433 patent taught that the bed volume should be such that substantially all of the protein to be supplied will be retained on the column, with sufficient free carboxymethyl substituted crosslinked dextran copolymer remaining to effect good separation of components during passage through the column. Thus, in order to make substantially complete use of both the gel filtration and ion exchange properties of the copolymer, Stern taught that the chymopapain should be loaded on the column at far below the saturation concentration of the copolymer. In the sole working example, four grams 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 eluant at a flow rate of about 20 ml per hour.Chymopapains I and Il were reportedly desorbed at around 50% and 80% salt saturations, respectively. Four grams of crude chymopapain, which was said to constitute a typical processing, reportedly yielded 2.250 grams of chymopapain I and 0.430 grams of chymopapain II. Stern reported that essentially pure chymopapains I and II were isolated; yet his electrophoretic results indicate that there was cross-contamination. After stating that different proteins form different zones under electrophoresis, Stern reported the formation of single protein zones after electrophoresis on cellulose acetate. Moreover, the elution profile presented in the '433 patent's Fig. 1 tracks each fraction by only total protein content.

U.S. Patent No. 3,627,640, issued to Blumberg et al on December 14, 1971, reported a process of purifying and decolorizing a relatively crude enzyme material by forming a covalently bound polymer-enzyme product. Said process wherein the enzyme is chymopapain was the subject of that patent's claim 9.

U.S. Patent Nos. 4,212,945 and 4,212,946, issued to Nonaka et al on July 15, 1980, reported a process for recovering protease, e.g., chymopapain, from the reaction product of a peptide synthesis in the presence of protease.

U.S. Patent No. 4,374,926, issued to Stern on February 22, 1 983 and assigned to Smith Laboratories, Inc., reported a method for the production of what was called improved chymopapain. Said method bascially substituted a carboxymethyl agarose gel for the carboxymethyl dextran gel reported in Stern's '433 patent. Crude chymopapain (about 29 g) in aqueous solution was added to a column of carboxymethyl agarose gel resin and eluted with about 4.6 liters of eluant for about 40 hours. Elution profiles were presented in the '926 patent's Fig. 1 with regard to both total protein content and enzymatic activity. The reported yield, by protein content, of chymopapain I is about five times that of chymopapain 11. Moreover, the enzymatic activity curve is clearly displaced from the curve of total protein content; such an offset is recognized by enzymologists to indicate the presence of impurities. The eluant fractions collected containing chymopapain I and II were combined, dialyzed and lyophilized. It was reported that by purifying chymopapain to two active components a consistent product of lower toxicity was produced.

Various chymopapain compositions are disclosed in the prior art; see generally the claims in the following United States Patents: No. 1,967,679, issued to Muench et al on July 24, 1 934 (activated papain preparation); No. 2,676, 138, issued to Hinkel on April 20, 1954 (papain composition); No. 3,019, 171, issued to Bloch et al on January 30, 1962 (activated papain composition); No. 3,284,316, issued to Cayle on November 8, 1966 (storage stable papain derivative); and No. 4,011, 169, issued to Diehl et al on March 8, 1 977 (peptide peptidohydrolyse-containing compositions).Two chymopapain compositions are currently marketed, nationally and internationlly, for use in chemonucleolysis therapy. Discaseo, a product of Baxter-Travenol Laboratories, Inc., is described in that company's product insert of July 1 982 as a sterile preparation of the proteolytic enzyme, chymopapain, derived from papaya latex; it is reportedly essentially free of papain as determined by electrophoresis. Chymodiactino, a product of Smith Laboratories, Inc., is described in that company's product profile of January 1 983 as a refined proteolytic enzyme obtained from crude latex of the Carica papaya tree, containing two enzymatically active protein components.

Brief Description of the Invention The present invention relates to a process for isolating large amounts of Chymopapain B and Chymopapain C from either papaya latex or crude chymopapain preparations. Homogeneous populations of Chymopapain B and Chymopapain C can now be recovered separately, in commercial quantities, without cross-contamination.

This invention also relates to therapeutic compositions which consist essentially of pure Chymopapain B without any contamination by other proteases, e.g., Chymopapain C, or other proteins. Such therapeutic compositions of the present invention are particularly useful for the chemonucleolysis of herniated intervertebral disc tissue, and their exceptional purity is an improvement which significantly reduces the risk of severe anaphylactoid and other allergic reactions.

Detailed Description of the Invention It has now been discovered that there are at least six proteases in papya latex: Papain, Chymopapain A, Chymopapain B,-B3, and Chymopapain C. The purification process of the present invention results in the isolation of Chymopapain B and Chymopapain C in homoge neons forms. It has now been discovered that Chymopapain B exists in three subforms, which we call Chymopapain B,-B3, and that the three subforms of Chymopapain B (B,-B3) are due to different states of oxidation of the SH groups on this dithiol protease.

The present invention is directed to the development of a process for large-scale isolation of Chymopapain B and Chymopapain C as separate homogeneous enzymes. The most preferred method of isolating pure fractions of Chymopapain B and Chymopapain C will now be described in detail, starting with the raw material papaya latex. Note, also, that if partially purified chymopapain is used as the starting material, then Steps 1-1 0 of the following procedure can be omitted, and the manufacturing protocol can commence with the highly selective batch adsorption process of Steps 11 and 1 2.

All steps, unless otherwise stated, are carried out at approximately 4"C in order to minimize any possible microbial contamination and to preserve enzyme activity. Pyrogen-free distilled water is used throughout.

STEP 1: Weigh 2 kg of papaya latex in a 6 liter container. Add 3.2 liters of distilled water. Stir this mixture at room temperature (22-25"C) for one hour.

STEP 2: Rapidly centrifuge the mixture (9,000xg for 30 minutes). Discard the pellet, which contains papain. Save the supernatant for the next step.

STEP 3: Bring the supernatant solution to 45% ammonium sulfate by the addition of small portions (5-109) at a time. Stir the resulting slurry for 30 minutes.

STEP 4: Centrifuge at 9,000xg for 30 minutes. Discard the pellet. save the supernatant for the next step.

STEP 5: Slowly add 1 N hydrochloric acid (prepared by adding 85 ml concentrated hydrochloric acid to 915 ml distilled water) until the pH is brought to 2.0 upon which a turbid solution results.

STEP 6: Centrifuge the above solution at 9,000xg for 30 minutes. Discard the pellet. Save the supernatant for the next step.

STEP 7: Bring the supernatant to 75% ammonium sulfate by the addition of small portions (5-109) at a time. Stir the resulting slurry for one hour.

STEP 8: Centrifuge the slurry for 30 minutes at 9,000xg. Decant the supernatant and discard.

Save 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 0.2 M sodium acetate buffer (pH 5.0).

STEP 10: Transfer the resulting solution into dialysis tubes (prewashed with distilled water) and dialyze against 0.2 M sodium acetate buffer (pH 5.0). Several changes (3-5) of buffer solution should be used. The resulting retenate is partially purified but still contains several contaminating proteins. This crude chymopapain is purified as follows.

STEP 11: Weigh 100 grams of Carboxymethyl-Sephadex into a 4 liter beaker. Add 3.0 liters of 0.2 M sodium acetate buffer (pH 5.0) and stir. Leave this mixture overnight at 4"C.

The swollen Carboxymethyl-Sephadex (approximately 3 liters) is ready for the next step.

STEP 12: Add the retentate from Step 10 to the swollen Carboxymethyl-Sephadex from Step 11 and stir briefly with a glass stirring rod. Allow the Carboxymethyl-Sephadex to settle.

Assay an aliquot (50 microliters) of the supernatant for chymopapain activity using the standardized 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 remains in the solution.

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

STEP 14: Elute the enzymes off 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 presented in Fig. 1. The large protein peak emerging in the early fractions is devoid of chymopapain activity, while the two later eluting fractions containing activity represent Chymopapain B and Chymopapain C.

STEP 15: Carry out acid polyacrylamide gel electrophoresis on representative fractions of the eluant. Determine the fractions containing pure Chymopapain B and the fractions containing pure Chymopapain C. A typical electrophoretic profile is presented in Fig.

2.

STEP 16: Pool the fractions containing pure Chymopapain B, and reduce the volume either by lyophilization or ultrafiltration. Separately pool and concentrate those fractions con taining pure Chymopapain C.

STEP 17: Either dialyze the concentrated Chymopapain B 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 presented in Fig. 3.

Separately desalt the concentrated Chymopapain C in like manner.

STEP 18: Lyphilize the resulting salt-free solution to obtain the fluffy, crystalline Chymopapain B. Separately lyophilize the desalted Chymopapain C.

While this procedure has been described in terms of an initial starting material of 2 kg of papaya latex, it is understood that it can be further scaled up, e.g., 1 04old, without excessive delay or changes in the procedure. For example, an industrial-scale purification has been performed using 60 g of crude chymopapain as the starting material in Step 1 2.

While this invention has been described in terms of the preferred method of salting-out contaminants at 45% ammonium sulfate and at pH 2.0, followed by salting-out crude chymopapain at 75% ammonium sulfate, it is understood that crude chymopapain produced by other protocols can be employed advantageously and without undue experimentation in the novel selective batch adsorption process in Step 1 2. See Example 2, wherein a commercial source of partially purified chymopapain is purified by means of the aforementioned Steps 11-18. Similarly, it is contemplated that satisfactory elution can be achieved using linar gradients from 0.2 to 1.0 M (pH 5.0) of salts other than the preferred sodium acetate.

A critical element of the present process invention is the highly selective batch adsorption step (the aforementioned Step 12). A solution of crude chymopapain is mixed with a batch 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 assayed to determine the residual chymopapain activity in solution.

Additional cation exchange resin is added in order to specifically adsorb additional chymopapain enzymes from solution, and the supernatant is then reassayed. This titration of the solution with cation exchange resin is continued until a predetermined residual activity of chymopapain remains in solution-until the chymopapain enzymes have nearly saturated the available binding sites on the cation exchange resin.

It has been found advantageous to add the resin slowly until the residual activity of the supernatant is less than about 10%, and preferably about 5% to about 2%, of the initial total activity of the crude chymopapain solution. Optimally, at least about 2% of the initial chymopapain activity should remain in solution.

The aforementioned cation exchange resin is preferably a carboxymethyl substituted crosslinked dextran copolymer. Carboxymethyl Sephadex (Pharmacia) type C-50 is especially preferred, but type C-25 is also acceptable. It is contemplated that carboxymethyl agarose gels will also prove acceptable.

The novel selective batch adsorption process in conjunction with the conventional column chromatography step has been found to provide several advantages over simple column chromatographic procedures alone, wherein all contaminants must pass completely through the column. The selective adsorption process of the present invention substantially avoids the passing of contaminating materials through the column. The contaminating materials never pass through the column because they are never adsorbed onto the cation exchange resin.The chymopapain enzymes specifically and preferentially adsorb onto the selected resin, and the aforementioned titration step insures that insufficient resin is added into the system to allow the contaminants to also be adsorbed The titration of resin into the crude enzyme solution is stopped near the saturation point of the resin for the enzymes, but before that saturation point is exceeded, at which point contaminants would adsorb onto the then available excess binding sites. Thus, recovery of the desired product can be maximized with minimal contamination.

Other advantages of this selective preloading include much more rapid processing times and much greater scale-up potential for industrial extractions, isolations, and purifications. In the aforementioned embodiment, only sufficient carboxymethyl dextran is added to almost fully bind the chymopapain enzymes. This results in maximizing the efficiency of the ion exchange and, because contaminants need not be laboriously pumped through the subsequently formed column, much more rapid processing is possible. It is understood that processing time is a factor that affects the pyrogen content of the ultimate product, and that this parameter becomes particularly important where, as here, the addition of bacteriostatic agents would inactivate the enzyme. Table I summarizes representative samples of the Chymopapain B of this invention in terms of endotoxin levels.

Table 1.

Pyrogen/5 mg Enzyme Lot Number Lot Size (Endotoxin Units) 0308-307 7.4 grams 3 E.U.

0308-202 2.4 grams 30 E.U.

0307-100 4.0 grams 3 E.U.

0308-408 8.2 grams 3 E.U.

0308-503 3.6 grams 30 E.U.

Limulus amoebocyte lysate (LAL) test (Associates of Cape Cod, Inc., Woods Hole, Massachusetts.) Moreover, because the inventive batch titration process eliminates the need to pass contaminating materials through the column prior to elution, the scale of the production can be readily enhanced. Chymopapain enzymes can now be separately and rapidly recovered in commercial quantities, and the homogeneous purity of the resulting enzymes is significantly better than that produced by any existing commercial method.

An integral component of the aforementioned purification procedure is employment 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.

A standardized reproducible enzyme assay for Chymopapain B needed to be established. The method traditionally employed using casein as a substrate was unsatisfactory for several reasons. First, casein is a heterogeneous substrate which comprises a mixture of peptides; furthermore, each hydrolytic cleavage produces a new, different substrate with different affinity and catalytic parameters. Thus, nonlinear kinetics occurs resulting in lack of reproducible rates and nonlinearity. Second, the assay procedure with casein is tedious and time consuming, and so does not permit continuous monitoring of the reaction. Third, this procedure does not allow for the expression of enzyme activity in standard units, e.g., micromoles of product formed per minute. An alternate procedure employed the synthetic substrate DL-benzoyl arginine-p-nitro anilide (BAPNA).This substrate is homogeneous and provides a single type of bond for hydrolysis. This substrate also permits a more convenient and faster assay because the reaction can be monitored photometrically. However, previous assays using BAPNA were not carried out under substrate saturating conditions, i.e., at zero order kinetics. Under nonsaturating conditions, slight changes in substrate concentration result in large changes in the reaction velocity, and hence a non-standardized BAPNA assay lacks reproducibility. Moreover, previous BAPNA assays have used the antiquated nomenclature of ktal or pktal rather than express activity in International Enzyme Units (micromoles per minute). See, e.g., U.S. Patent No.

4,374,926.

Thus, in order to optimize the selective batchwise loading of Chymopapain B onto the carboxymethyl dextran gel, and to better 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 versed in the art of enzymology will understand that before such a standardized assay protocol could be devised, the following parameters had to be investigated and established: the molar extinction coefficient for the hydrolytic products of BAPNA under optimal assay conditions, the pH optimum of the enzyme, the optimal conditions for enzyme activation, and the substrate saturation curve.

Said extinction coefficient was established as follows. When chymopapain hydrolyzes BAPNA, the product p-nitrophenol absorbs light in the visible region. It is necessary to establish the molar extinction coefficient for this product under conditions of the assay. BAPNA was hydrolyzed to completion with Chymopapain B and the visible absorbance spectrum determined using a Gilford Model 2600 spectrophotometer. Fig. 4 shows the absorbance spectra of both BAPNA and the hydrolysis products of BAPNA. 410 nm was chosen as the wavelength for the assay since at this wavelength BAPNA does not absorb. A wavelength of 410 nm was employed for all future studies. A 0.10 M BAPNA standard solution was prepared by dissolving 130.47 mg of BAPNA (Sigma Chemical Co., Product Number B-4875, Molecular Weight 434.9) in 3.0 ml of dimethylsulfoxide (DMSO).This solution was diluted in DMSO to give stock solutions of 10 mM and 1 mM. It is necessary to dissolve the BAPNA in DMSO since it does not readily dissolve in water. Aliquots of the 1 mM solution were transferred to cuvettes containing assay buffer (0.1 M sodium citrate, 0.5 M EDTA, 3.6 mg/ml cysteine, pH 6.4), and the spectrophotometric zero baseline was established at 410 nm. The substrate was then enzymatically hydrolyzed using an aliquot (30 yI) of either trypsin or chymopapain. The reaction was continuously monitored until all the substrate was hydrolyzed and no further increase in absorbance was noted. The completion of the reaction was verified by additional aliquots of either enzyme or substrate.The results, which are presented in Table 2, establish an average mM extinction coefficient for the hydrolytic products of BAPNA at 410 nm equal to 4.86 mM~'cm~'.

Table 2.

CONTENTS APNA AA410 emM-1cm-1 100IL1 of 1 mM BAPNA 0.1 mM 0.492 4.92 870 ILl of Assay Buffer 30 111 of Enzyme 100 yl of 1 mM BAPNA 0.1 mM 0.487 4.87 870 ,ul of Assay Buffer 30 yl of Enzyme 50 yl of 1 mM BAPNA 0.05 mM 0.240 4.80 870 j of Assay Buffer 30 yl of Enzyme The pH optimum of Chymopapain B was established as follows. Chymopapain B activity was assayed 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 of these studies. All future studies utilized pH 6.4 as the optimal pH for the assay.

Optimal enzyme assay conditions: The activator cysteine is included to insure that the essential SH groups of the enzyme remain reduced. EDTA is added to prevent inactivation by heavy metals; citrate buffer also assists in this respect because of its chelating properties. All assays are run at 37 since this approximates most closely the human body temperature, at which the enzyme is to be utilized for chemonucleolysis. Although the enzyme can be assayed at different temperatures, correction for the marked effect of temperature on enzyme velocity would be necessary otherwise.

Substrate saturation was established as follows: Pure Chymopapain B was utilized to established a saturation curve. A constant amount of enzyme was assayed in the aforementioned assay buffer as a function of BAPNA concentration. The resulting data, which indicate the effect of BAPNA concentration on Chymopapain B activity, are reported in Table 3 and presented as a saturation curve in Fig. 6.

Table 3.

[BAPNA 1 mM hOD/min l.U. 1/[BAPNA] 1/lU.

0.1 0.0026 0.000535 10 1869.23 0.2 0.0050 0.0010288 5 972.01 0.5 0.0110 0.0022634 2 441.82 1.0 0.0163 0.0033539 1 298.16 2.0 0.0300 0.0061728 0.5 162.00 3.0 0.0358 0.0073663 0.33 135.75 4.0 0.0482 0.0099177 0.25 100.83 5.0 0.0475 0.0097737 0.2 102.32 Fig. 7 presents this data in a double reciprocal Line-weaver-Burk Plot, from which the following are 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 of BAPNA, the velocity of the reaction will be 1/2 maximal. Ideally a substrate concentration of 10 X Km would be used to assure zero order kinetics. However, BAPNA is not soluble in aqueous buffer at pH 6.4 at that 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.

Moreover, at 5 mM the reaction velocity approches Vmax, so that slight variations in BAPNA concentration cause only small changes in the measured velocity.

Once the above-mentioned optimal conditions were established, the standardized, reproducible enzyme assay for Chymopapain B was devised. The following solutions are employed: assay buffer, BAPNA stock solution, and Chymopapain B solution. The assay buffer is 0.1 M Nacitrate, 3.6 mg/ml cysteine, 0.5 mM EDTA, pH 6.4. Dissolve 29.41 g of sodium citrate (Na3C6HsO7 2H2O; M.W. = 294.10; Fisher Scientific Co.) in 1 1 of distilled water. Add 3.6 g cysteine and 168.1 mg EDTA, disodium salt (M.W. = 336.2; Sigma Chemical Co., Product Number ED255), and stir until dissolved. Adjust pH to 6.4 with 0.1 M citric acid, prepared by dissolving 1 9.2 g of citric acid (M.W. = 1 92.1 3; MCB Manufacturing Chemists) per liter. The BAPNA stock solution is 100 mM BAPNA in DMSO.Dissolve 130.47 mg BAPNA (M.W. = 434.9; Sigma Chemical Co., Product Number 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 procedure is as follows: For the standard assay procedure, the level of BAPNA is 5 mM. The recording spectrophotometer is set at 410 nm (visible light source); temperature is set at 37"C; 940 yl of 0.1 M Nacitrate (pH 6.4), 3.6 mg/ml cysteine and 0.5 mM EDTA, and 50 its of 100 mM BAPNA in DMSO are added to a 1.0 ml cuvette (1 cm pathlength). The contents of the cuvette are mixed, and the cuvette is inserted into the spectrophotometer (at 37"C). All the BAPNA, if not already in solution, will go into solution when 37"C is reached.The recorder is turned on, a zero baseline is established to confirm that there is no nonenymatic hydrolysis of BAPNA. 10 pl of Chymopapain B enzyme solution is then added to the cuvette, the contents mixed and the enzymatic activity monitored and recorded at 410 nm.

One International Enzyme Unit (IEU) of Chymopapain B is that amount of enzyme which produces one micromole of p-nitroaniline per minute from a 5 mM solution of BAPNA substrate at 37 and pH 6.4.

Brief Description of the Drawings Reference is now made to the illustrations accompanying this application wherein: Figure 1 is a graph which plots both total protein content in units of Absorbance at 280 nm

and enzymatic activity in International Enzyme units per ml

versus molar concentration and fraction number of eluant, as described in Example 4; Figure 2 is the gel plate which resulted from electrophoretic analysis on acid polyacrylamide gel of every tenth fraction of eluant, as described in Example 2; Figure 3 is a graph which depicts a typical elution profile after desalting (see Step 1 7 in the Detailed Description), wherein both enzymatic activity in International Enzyme Units per ml

and salt concentration as a function of conductivity

are plotted against eluant traction number;; Figure 4 is a graph, wherein Absorbance is plotted versus wavelength, which shows the absorbance spectra of both BAPNA and the hydrolysis products of BAPNA, as described in the Detailed Description; Figure 5 is a graph, wherein enzymatic activity in thousands of International Enzyme Units is plotted versus pH, which establishes the pH optimum of Chymopapain B, as described in the Detailed Description; Figure 6 is a graph, wherein enzymatic activity in thousands of International Enzyme Units is plotted against BAPNA concentration (in mM), as reported in Table 3; Figure 7 is a double reciprocal Lineweaver-Burk Plot of the data in Table 3; and Figure 8 is a gel plane which resulted from electrophoretic analysis on acid polyacrylamide gel of four chymopapain preparations: DiscaseS (far left), Chemolase (middle left), Chymodiactins (middle right), and the purified Chymopapain B of this invention (far right), as described in Example 6.

In order that those skilled in the art can more fully understand the present invention, the following examples are set forth. These examples are given solely for purposes of illustration, and they should not be considered as expressing limitations unless so set forth in the appended claims.

Example 1 A typical calculation of the enzymatic activity of Chymopapain B, determined by the standardized enzyme assay of this invention and expressed in International Enzyme Units (herein referred to as "units") per mg, was made as follows.

8 mg of Chemolases (Pharmotex, Inc., Lot 4#01 1 53-38) was dissolved in 1.0 ml of distilled water; 10 ILl iof this enzyme solution was assayed.

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 itl of said buffer solution was used in this assay.

A BAPNA stock solution (100 mM BAPNA in DMSO) was prepared as previously described.

50 l of said BAPNA stock solution was used in this assay.

The standard assay procedure was followed as described above. A 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 = #bc where A = Absorbance = 0.044/min.

E= Extinction Coefficient = 4.86 mM-1cm-1 b = Pathlength = 1 cm c = Concentration.

Rearranging the formula, A 0.044/min mM c=-=----------------= 0.0090535 --- Eb (4.86 mM-1cm-1) (1 cm) min and poles moles mM = , so c = 0.0090535 ml ml min Since the final volume in the cuvette is 1 ml and unit = ymoles moles ymoles , (0.0090535 ) (1 ml) = 0.0090535 = = 9.0535 X min ml min min 10 - units. Since 10 its of Chemolase is used for the assay, 8 mg ( ----) (0.010 ml) = 0.08 mg of Chemolase used for the assay.

ml Therefore, 9.0535 X 103 units/0.08 mg = 0.11 3 units/mg of Chemolse weighed.

Example 2 The purification process of the present invention is illustrated by the following production run, which used a partially purified chymopapain as the 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 to use.

30 g of partially purified chymopapain (Sigma, Lot 13F-8155) was dissolved in 1.5 liters of 0.2 M sodium acetate buffer, pH 5.0, in a 2 liter beaker using a magnetic stirring bar. An aliquot of the resulting crude enzyme solution was assayed for activity, using the procedure described in Example 1, and the total enzymatic activity of the 1 500 ml was calculated to be 5,245 International Enzyme Units, or 3.497 units/ml.

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

Another 250 ml of crude enzyme solution was added and stirred. After the CM-Sephadex beads had settled, an aliquot of the supernatant was assayed, and slight enzymatic activity was observed.

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

The resulting chymopapain-bound CM-Sephadex was packed into a 5 = 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.

Four-liter volumes of 0.2 M and 1 M sodium acetate solutions, pH 5.0, were loaded onto the column to produce a linear ionic strength gradient of eluant from 0.1o1.0 M, at pH 5.0. Said gradient took approximately 4 days to run through the column. Sequential 2.5 ml fractions of eluant were collected as they came off the column.

Every tenth fraction was assayed for chymopapain activity and also subjected to electrophoretic analysis on acid polyacrylamide gel. The gel plate from the fractions so tested in this run is reproduced in Fig. 2. Note that Chymopapain B occupies the upper band, and that Chymopapain C occupies the lower band which only appears in the fractions which eluted at high molar concentrations.

The fractions which contained only Chymopapain B, and specifically those represented by Fractions 150-220 in Fig. 8, were pooled and lyophilized to a smaller volume. This concentrated pool was then dialyzed against distilled water, with four changes of water. The resulting retentate was then lyophilized to obtain the fluffy crystalline Chymopapain B.

Pyrogen content was assayed using the Limulus amoebocyte lysate (LAL) test (Associates of Cape Cod, Inc., Post Office Box 244, Woods Hole, Masachusetts 02543). The results showed the presence of only 3 pyrogen endotoxin units/5 mg of this crystalline Chymopapain B.

Example 3 7.4 9 of purified Chymopapain B, produced by several runs of the above-described process, was determined to have a specific activity of 0.15 units/mg. This 7.4 9 of purified Chymopapain B, or 1110 International Enzyme Units, was sent to Connaught Laboratories, Inc., as Lot #0308-307, for admixture with conventional excipients to produce a Chemolaseo composition for use in chemonucleolysis therapy.

Chemolaseo is a sterile, lyophilized proteolytic enzyme powder for interdiscal injection.

Chemolasea contains 6 International Enzyme Units of Chymopapain B, 3.5 mg of L-cysteine hydrochloride monohydrate, and 0.37 mg of disodium edetate; with 1 mg of sodium disulfite added at compounding. Said therapeutic composition consists essentially of Chymopapain B without admixture with Chymopapain C or other contaminating proteins.

Example 4 Fig. 1 represents an elution profile which is representative of the overall recoveries effected by the present inventive process. Enzymatic activity (International Enzyme Units/ml) and total protein content (Absorbance at 280 nm) are plotted on the abscissas, molar salt concentration on the diagonal, and fraction number on the ordinate. Note that the large protein peak which first merged was devoid of protease activity, while the two fractions which next eluted off the column did exhibit such activity. Chymopapain B, the second fraction, emerged from the column at an approximate salt concentration of about 0.65 to about 0.75 M (Fractions 165-220 here).

The Chymopapain C eluted as a third fraction at a salt concentration of approximately 0.80 to 0.90 M (Fractions 230-280 here).

Example 5 The physicochemical properties of the pure Chymopapain B isolated by the represented procedure in Example 2 was investigated. Said homogeneous Chymopapain B exhibited three closely related bands on polyacrylamide gel electrophoresis. These three bands were found to represent three subforms of Chymopapain B, which we call B1-B3, which are due to different states of oxidation of the SH groups on this dithiol protease. Chymopapains B1-B3 are interconvertible by oxidation or reduction. For example, Fig. 8 shows an electrophoretic gel of pure Chymopapain B in both partially reduced (left center, with cysteine) and oxidized (far right, without cysteine) states. 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, the molecular weight of the Chymopapain B of the present invention appears to be 34-35 K. Our best estimate is 35,200 daltons. This enzyme is a monomer.

The isoelectric point of the Chymopapain B of the present invention is very basic. Electrophoretic and isoelectric focusing studies indicate that Chymopapain B (B1-B3) has an apparent pl = 10.3-10.5.

N-terminal analysis of the Chymopapain B of the present inventional revealed primarily tyrosine. This is consistent 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 the Chymopapain B of the present inventions were determined by conventional techniques. The results are shown in Table 4.

Table 4.

% Amino Acid Composition Amino Acid Sample #1 Sample #2 Average Aspartic Acid 23.7 23.3 23 Threonine 19.8 20.1 20 Serine 23.4 23.4 23 Glutamic Acid 26.3 25.7 26 Glycine 39.2 38.9 39 Alanine 14.1 12.5 13 Valine 21.7 20.7 21 Methionine 1.0 1.0 1 Isoleucine 7.6 7.5 8 Leucine 11.7 11.3 11 Tyrosine 28.2 27.4 27 Phenylalamine 9.4 9.3 9 Lysine 26.1 26.6 27 Arginine 9.1 8.9 9 Histidine 6.5 4.5 5 Proline 12.9 13.2 13 Cysteine Tryptophan "These data do not include values for cysteine and tryptophan which are the subject of current studies and some controversy. The SH content of Chymopapain B as reported in the literature has ranged from 2-11 residues/mole. Since tryptophan in the past was destroyed by acid hydrolysis, accurate determinations present problems and usually required independent spectral analysis. We are currently undertaking a complete tryptophan analysis by several independent methods.

The Chymopapain B produced by the process of the present invention is clearly distinguishable from the Chymopapain C (also called Chymopapain II) which is eliminated by our process and which does not contaminate our therapeutic composition. Preliminary investigations of the Chymopapain C isolated by the process of the present invention indicates that this monothiol enzyme seems to be monomeric and of the approximate size of the other thiol proteases from papaya. This Chymopapain C is more basic than the Chymopapain B and has an apparent pl around 10.8. This Chymopapain C exhibits positive BAPNA hydrolytic activity, and on a specific activity basis it has a higher BAPNA turnover than the Chymopapain B.

Example 6 Four chymopapain compositions were analyzed by gel electrophoresis under acidic conditions by the following procedure: J. V. Maizel, Methods in Virology, Vol. 5, Ch. 5, pp. 179-246 (1971).

The results are presented in Fig. 8. At the far left is Discuses (Baxter-Travenol Laboratories, Lot #AV15F5M). At left center is Chemolaseo (Lot #021031-86), which is representative of purified Chymopapain B prepared by a more elaborate protocol than the method of Example 2, and here compounded with L-cysteine hydrochloride monohydrate and disodium edate, with sodium disulfite added. At right center is Chymodiactini (Smith Laboratories, Lot #BM201).

And at the far right is a representative sample of the Chymopapain B as purified by the process in Example 2.

Note that both the Discase and the Chymodiactin are mixture of Chymopapains B (upper bands) and C (lower band), while the composition of the present invention (far right) contains Chymopapain B without any contaminating Chymopapain C.

Example 7 The toxicity and antigenicity of two different chymopapain compositions were compared in a double-blind study. A Chymolaseo composition, which had been here prepared by compounding the purified Chymopapain B of this invention as described in Example 3, was compared with Chymodiactino (Smith Laboratories), which contained both Chymopapains B and C. A doubleblind reference control study, without placebo, was conducted at Yale University under FDA auspices (IND 21087) involving chemonucleolysis with human patients. Neither the attending physicians nor the patients knew which of the two abovementioned chymopapain compositions was employed.

Out of 43 injections to date, one adverse reaction that could possibly be an allergic-type reaction has been reported, and that occurred following injection of the Chymodiactino compound. In that particular case, the attending physician reported a total body skin rash occurred 24-48 hours post-injection of a preparation which was later determined to be the Chymodiactino compound. The records indicate that the afflicted patient had had a history of prior episodes of this type of reaction. Although the attending physician is not convinced that this was in fact a drug reaction, similar serum sickness type reactions following chymopapain injection have been previously reported in the medical literature, and this clinical observation was reported to the FDA.These preliminary results suggest that the novel therapeutic composition of the present invention, which consists essentially of chymopapain B without admixture with Chymopapain C, may reduce the risk of adverse allergic reactions vis-a-vis injection with compositions which contain a plurality of chymopapain enzymes. This suggestion follows the general proposition that, in order to minimize the risk of any type of allergic reaction, it is advisable to incorporate the fewest different kinds of proteins into the innoculum.

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modification and that this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within the pertinent art to which the invention pertains, and as may be applied to the essential features hereinbefore set forth, within the spirit of the invention and the scope of the appended claims.

Claims (16)

1. A process of purifying a chymopapain protease from a complex solution, comprising: (a) mixing said complex solution with increments of cation exchange resin to selectively adsorb some but not all of the protease onto the resin until the point is reached at which protease activity in the supernatant has decreased to a predetermined minimal level; and (b) eluting the protease from the resin.

2. A process as in claim 1 wherein the complex solution is an aqueous solution of crude chymopapain.

3. A process as in claim 2 wherein 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 of step (b) to 45% ammonium sulfate to form a resulting supernatant and a precipitate; (d) acidifying the resulting supernatant of step (c) to about pH 2.0 to form a resulting solution and a precipitate; (e) bringing the resulting solution of step (d) to 75% ammonium sulfate to precipitate out crude chymopapain; and (f) recovering the precipitated crude chymopapain.

4. A process as in claim 1 wherein the cation exchange resin is a carboxymethyl derivative of a polymeric carbohydrate such as carboxymethyl dextran, carboxymethyl agarose, and the like.

5. A process as in claim 4 wherein the cation exchange resin is a CM-Sephadex.

6. A process as in claim 1 wherein the predetermined minimal protease activity is less than about 5% but greater than about 2% of the total protease activity initially present in the complex solution.

7. A process as in claim 1 wherein the eluting is carried out by column chromatography.

8. A process as in claim 1 wherein the eluting is effected by a linear ionic strength gradient of eluant.

9. A process as in claim 8 wherein the eluting is effected by a linear gradient of sodium acetate from 0.2 to 1.0 M (pH 5.0).

10. A process as in claim 1 wherein Chymopapain B is recovered separately from Chymopapain C.

11. A composition consisting essentially of pure Chymopapain B produced by the process of claim 1.

1 2. A composition consisting essentially of pure Chymopapain C produced by the process of claim 1.

1 3. A therapeutic composition consisting essentially of Chymopapain B without Chymopapain C.

14. The therapeutic composition as in claim 13, admixed with suitable pharmaceutical excipients.

1 5. The therapeutic composition as in claim 14, admixed with cysteine and disodium edetate.

16. The therapeutic composition as in claim 15, admixed with sodium disulfate.

1 7. A process as claimed in claim 1 substantially as hereinbefore described in Example 2.

1 8. A composition as claimed in claim 1 3 substantially as hereinbefore described in any of the Examples.