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|Title:||Method for the production of improved chymopapain|
|Abstract:||A method of preparing a purified proteolytically active chymopapain from crude chymopapain extracts by the use of exchange resin absorption purification techniques is provided. The purified chymopapain is characterized by relative freedom from proteolytically inactive, colored and/or toxic components and by its failure to form a precipitate with barium chloride test solution (U.S.P.) under acid conditions.|
|Inventor(s):||Stern; Ivan J. (Chicago, IL)|
|Assignee:||Smith Laboratories, Inc. (Northbrook, IL)|
|Filing Date:||May 13, 1981|
|Claims:||1. A process for the purification of crude chymopapain to produce a purified chymopapain of reduced toxicity comprising: |
(a) contacting an aqueous buffered solution of crude chymopapain with a weakly acidic cationic exchanger comprising a column of carboxymethyl substituted cross-linked agarose gel, said exchanger having been previously equilibrated with aqueous buffer solution having a pH of between about 6.5 and 7.5;
(b) eluting the chymopapain retained on the exchanger with a similar aqueous buffer solution having a pH in the same pH as the buffer used for equilibrating the carboxymethyl substituted agarose gel, but having a linearly increasing ionic concentration of a compatible, non-reactive, water-soluble, pharmaceutically acceptable, neutral inorganic salt with respect to eluent volume;
(c) collecting and discarding a first series of fractions of eluent from said exchanger containing an initial protein component from crude chymopapain until the molarity of the eluent with respect to said soluble salt increases to and reaches the range of 0.25 to 0.4;
(d) continuously collecting and retaining a further series of fractions of chymopapain eluted from the exchanger comprising two proteolytically active chymopapain components at increasing soluble salt concentrations greater than about 0.25 to 0.4 molar, until substantially all of the said absorbed cymopapain is recovered;
(e) treating said retained fractions containing proteolytically active components of the chymopapain to remove the dissolved ionic inorganic salts, and buffer components; and
(f) lyophilizing the essentially salt-free chymopapain solution to produce a dry, purified proteolytically active chymopapain essentially free of proteolytically inactive or toxic components.
2. The process according to claim 1 wherein the eluent buffer solution employed contains a water-soluble inorganic salt concentration gradient of from zero to 1 molar.
3. The process according to claim 1 wherein the pH of the exchanger is equilibrated to between 7.3 and about 7.5.
4. The process according to claim 1 wherein the aqueous solution of crude chymopapain, the buffer used to equilibrate the exchanger and the eluent are adjusted to a pH of about 7.4.
5. The process according to claim 1 wherein the water-soluble salt employed in the eluting solution is sodium chloride.
6. A process according to claim 1 wherein the soluble salt concentration in step (c) reaches a molarity of about 0.3.
7. A process according to claim 1 wherein the steps (a) through (d) are carried out at a temperature of from about 2.degree. to 8.degree. C.
8. A process according to claim 1 wherein the carboxymethyl substituted cross-linked agarose gel cationic exchanger have a neutralizing capacity of from about 0.02 to about 0.10 milliequivalents of sodium hydroxide/cc.
9. A process according to claim 1 wherein the purified chymopapain recovered is characterized by a failure to form a precipitate with acidified barium chloride solution.
10. A process according to claim 1 wherein the purified chymopapain is essentially free of colored materials.
11. A process according to claim 1 wherein the buffer solution used is a phosphate buffer.
12. A process according to claim 1 wherein a crude colored chymopapain is dissolved in a phosphate buffer at a concentration (weight to volume) of 12 to 18% and the buffer concentration with respect to phosphate is about 0.05 molar.
13. A process according to claim 1 wherein the soluble ionic inorganic salts are removed from the chymopapain solution in step (e) by dialysis.
14. A process according to claim 1 wherein step (e) also includes the steps of filtration and sterilization.
15. A process according to claim 1 wherein a sufficient amount of the colored protein component of the crude chymopapain initially eluted from the exchange is removed so that an acidified aqueous solution of the remainder of the proteolytically active chymopapain components eluted and recovered from said exchanger do not cause a precipitate when treated with barium chloride solution (USP).
16. A process according to claim 1 wherein the concentrations of salt in step (d) are gradually increased up to about 1 molar.
17. A process according to claim 16 wherein the purified chymopapain is recovered in the eluent fractions from the column at increasing salt concentrations of from about 0.3 to about 0.8 molar.
18. A process according to claim 17 wherein the purified chymopapain is essentially fully proteolytically active and is essentially free of colored, odoriferous and toxic antigens and is further characterized by its failure to form as precipitate with acidified barium chloride.
19. An essentially continuous method according to claim 1 wherein after the recovery of purified chymopapain from said eluent, residual salt is removed from the column by application thereto of salt-free buffer solution sufficient in volume to remove salt residual salt and equilibrate salt column and repeating the said process of claim 1.
20. A method of identifying a purified chymopapain essentially free of proteolytically inactive, antigenic and colored material, which comprises the essential absence of a precipitate formation of an acidified solution of said purified chymopapain when contacted with a U.S.P. test solution barium chloride.