Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
  • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
  • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
  • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
  • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
  • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
  • G01N2333/96466—Cysteine endopeptidases (3.4.22)

Definitions

• 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.
• a new method of measuring the activity of the chymopapains is also provided.
• 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, 10th 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 22, 1940 (tenderize meat);
• 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 shock 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.
• 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., U.S. Patent No. 3,320,131, issued to Smith on May 16, 1967 and assigned to Baxter Laboratories, Inc.
• 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.
• isolation protocols which either combine batch with chromatographic techniques or teach the need to achieve some sort of optimization of various isolation parameters.
• 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:
• 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.
• 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.)
• 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 raising the concentration of salt to full saturation at pH 2. Said protein was reportedly susceptible to further purification by reprecipitation and recrystallization.
• chymopapain compositions are currently marketed, nationally and internationlly, for use in chemonucleolysis therapy.
• Disease ® a product of Baxter-Travenol Laboratories, Inc.
• Chymodiactin ® a product of Smith Laboratories, Inc.
• Chymodiactin ® is described in that company's product profile of January 1983 as a refined proteolytic enzyme obtained from crude latex of the Carica papaya tree, containing two enzymatically active protein components.
• 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.
• the invention also provides a new method of assaying, with substantially reproducibility, the activity of Chymopapain B and Chymopapain C.
• 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 1-10 of the following procedure can be omitted, and the manufacturing protocol can commence with the highly selective batch adsorption process of Steps 11 and 12.
• STEP 3 Bring the supernatant solution to 45% ammonium sulfate by the addition of small portions
• STEP 4 Centrifuge at 9,000xg for 30 minutes.
• 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.
• 1 N hydrochloric acid prepared by adding 85 ml concentrated hydrochloric acid to 915 ml distilled water
• 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 (5-10g) at a time. Stir the resulting slurry for one hour.
• STEP 8 Centrifuge the slurry for 30 minutes at
• 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 retentate 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
• STEP 12 Add the retentate from Step 10 to the swollen Carboxymethyl-Sephadex from Step 11 and stir briefly with a glass stirring rod.
• 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 containing 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 Lyophilize the resulting salt-free solution to obtain the fluffy, crystalline Chymopapain B. Separately lyophilize the desalted Chymopapain C.
• 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.
• the 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 cross-linked 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.
• LAL Limulus amoebocyte lysate
• 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.
• 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.
• the assay procedure with casein is tedious and time consuming, and so does not permit continuous monitoring of the reaction.
• this procedure does not allow for the expression of enzyme activity in standard units, e.g., micromoles of product formed per minute.
• BAPNA DL-benzoyl arginine- p-nitroanilide
• extinction coefficient was established as follows. When chymopapain hydrolyzes BAPNA, the product p-nitrophenol absorbs light in the visible region. It is necesssary 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 spectro-photometer. 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
• 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 establish 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.
• Fig. 7 presents this data in a double reciprocal
• the K m 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 K m would be used to assure zero order kinetics.
• BAPNA is not soluble in aqueous buffer at pH 6.4 at that concentration.
• a concentration of 5 mM should be used to assay the enzyme. This represents the highest concentration at which the substrate is still soluble.
• the reaction velocity approaches Vmax, so that slight variations in BAPNA concentration cause only small changes in the measured velocity.
• 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 ⁇ l of 0.1 M Na- citrate (pH 6.4 ) , 3.6 mg/ml cysteine and 0.5 mM EDTA, and 50 ⁇ l 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 spectro-photometer (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 nonenzymatic hydrolysis of BAPNA. 10 ⁇ l of Chymopapain B enzyme solution is then added to the cuvette, the contents mixed, and the enzymatic activity monitored and recorded at 410 nm.
• IEU International Enzyme Unit
• the present invention furnishes a solution to these problems by providing an assay method in which 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 at which different samples of chymopapain regain activity once resubjected to a sulfhydryl reagent before reacting with the enzyme substrate.
• sulfhydryl reagents may be used instead of, or in combination with, cysteine. Included among such sulfhydryl reagents are dithiothreitol, beta-mercaptoethanol, and glutathione. Commonly used buffers, eg., phosphate or acetate buffers, may be substituted for Na-citrate, and buffer concentrations ranging from about 0.25 M to about 0.025 M may be employed.
• a variety of substrates for Chymopapain B or Chymopapain C may be useful as homeogeneous, non-proteinoceous photometric substrates for the photometric measurement of enzymatic activity.
• such substrates include, but are not limited to, N-alpha-benzoyl-L-arginine ethyl ester (BAEE) ; N-alpha-benzoyl-L-arginine methyl ester; N-alphabenzoyl-D,L-arginine-beta-naphthylamide; N-alphabenzoyl-D,L-arginine-para-nitroamide.
• BAEE N-alpha-benzoyl-L-arginine ethyl ester
• BAPNA itself has been reported by others as being useful in the measurement of chymopapain. See, e.g., U.S. Patent 4,439,423.
• concentration of BAPNA or other substrate is preferably about 5 mM, and in any case substantially below 10 x K m , it will be understood that concentrations lower or higher than about 5 mM are in principle useful in carrying out the standarized assay procedure of this invention, provided that adequate reproducibility continues to be achieved.
• the reproducibility of the standarized assay of this invention has been discovered to be greater than would have been expected for assays run at about 2 x K m , or 5 mM in the case of BAPNA.
• the standard assay procedure as described here-inabove for chymopapain may also be used for measur ing the C form of chymopapain, also known as Chymopapain C. See, e.g., Fig. 1. These two forms of chymopapain have been designated in the past by other nomenclatures.
• the standard assay procedures as described hereinabove for Chymopapain B can also be modified by changing temperature, volume, pressure, and concentration of reactants. It is contemplated that variations of these parameters as well as others are within the scope of the present invention. Brief Description of the Drawings Reference is now made to the illustrations accompanying this application wherein:
• Fig. 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;
• Fig. 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;
• Fig. 3 is a graph which depicts a typical elution profile after desalting (see Step 17 in the Detailed Description), wherein both enzymatic activity in
• Fig. 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;
• Fig. 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;
• Fig. 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;
• Fig. 7 is a double reciprocal Lineweaver-Burk Plot of the data in Table 3 ;
• Fig. 8 is a gel plate which resulted from electrophoretic analysis on acid polyacrylamide gel of four chymopapain preparations: Disease ® (far left), Chemolase ® (middle left), Chymodiactin ® (middle right), and the purified Chymopapain B of this invention (far right), as described in Example 6.
• Chemolase ® (Pharmotex, Inc., Lot #01153-38) was dissolved in 1.0 ml of distilled water; 10 1 of 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 ⁇ l 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.
• 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.
• CM-Sephadex carboxymethyl-dextran
• CM-Sephadex beads 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.
• 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.
• LAL Limulus amoebocyte lysate
• Example 3 7.4 g 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 g 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 Chemolase ® composition for use in chemonucleolysis therapy.
• Chemolase ® is a sterile, lyophilized proteolytic enzyme powder for interdiscal injection.
• Chemolase ® 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.
• 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
• total protein content Absorbance at 280 nm
• Chymopapain B the second fraction
• Chymopapain C 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 representative 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 B 1 -B 3 , which are due to different states of oxidation of the SH groups on this dithiol protease. Chymopapains B 1 -B 3 are interconvertible by oxidation or reduction. For example. Fig.
• Chymopapain B 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.
• 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 pI 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.
• 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.
• Disease ® Boxter-Travenol Laboratories, Lot #AV15F5M.
• Chemolase ® (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.
• Chymodiactin ® Smith Laboratories, Lot #BM201).
• composition of the present invention contains Chymopapain B without any contaminating Chymopapain C.
• 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.

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• 1984
  • 1984-07-03 GB GB08416926A patent/GB2156821A/en not_active Withdrawn
  • 1984-08-09 ES ES535035A patent/ES8604303A1/es not_active Expired
• 1985
  • 1985-04-02 JP JP60501768A patent/JPS61502444A/ja active Pending
  • 1985-04-02 EP EP85902217A patent/EP0175786A1/en not_active Withdrawn
  • 1985-04-02 AU AU42178/85A patent/AU4217885A/en not_active Abandoned
  • 1985-04-02 WO PCT/US1985/000559 patent/WO1985004417A1/en not_active Application Discontinuation
  • 1985-08-23 ES ES546394A patent/ES8700319A1/es not_active Expired
  • 1985-11-29 NO NO854823A patent/NO854823L/no unknown
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