IL33327A - A crystalline combination of l-asparaginase and a metal or ammonium or hydrazinium ion and method for preparing the same - Google Patents

A crystalline combination of l-asparaginase and a metal or ammonium or hydrazinium ion and method for preparing the same

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Publication number
IL33327A
IL33327A IL33327A IL3332769A IL33327A IL 33327 A IL33327 A IL 33327A IL 33327 A IL33327 A IL 33327A IL 3332769 A IL3332769 A IL 3332769A IL 33327 A IL33327 A IL 33327A
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Israel
Prior art keywords
asparaginase
ammonium
crystalline
metal
solution
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IL33327A
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IL33327A0 (en
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Lilly Co Eli
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Publication date
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Publication of IL33327A0 publication Critical patent/IL33327A0/en
Publication of IL33327A publication Critical patent/IL33327A/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y305/00Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001Asparaginase (3.5.1.1)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
    • A61K33/02Ammonia; Compounds thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
    • A61K33/06Aluminium, calcium or magnesium; Compounds thereof, e.g. clay
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00Medicinal preparations containing inorganic active ingredients
    • A61K33/24Heavy metals; Compounds thereof
    • A61K33/30Zinc; Compounds thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • A61K38/16Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46Hydrolases (3)
    • A61K38/50Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/78Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82Asparaginase (3.5.1.1)
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Inorganic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)

Description

A CRYSTALLINE COMBINATION OF L- ASPAR AGINASE AND A METAL OR AMMONIUM OR HYDRAZINIUM ION AND METHOD FOR PREPARING THE SAME 11» I B nanDi Tflja 'AR-IS-OR- L U? *W*3A asm A This invention relates to a crystalline L-asparagin- ase preparation.
More particularly this invention pertains to a crystalline combination of L-as araglnase and a metal or a ..metal-r 5 loid possessing high enzymic and oncolytic activity and a method for its preparation.
The crystalline combination of this invention is prepared from a crude preparation of L-asparaginase, a soluble ammonium or hydrazinium metal or rhbtklLlUQbfal salt, and an antisolvent by the following 10 procedure: a) dissolving a crude preparation of L-asparaginase in water; b) adding an appropriate water-mlscible antisolvent to incipient cloudiness; c) adding a sufficient quantity of an aqueous solution of a metal or metalloid salt which is soluble in the solverit-antisolvent mixture, which salt when dissolved in water establishes a pH of about 6.0 to about 9, to cause crystallization of the L-asparaginase; 0 d) separating the resulting crystals of enzyme. Additional purity of the crystals can be achieved by a repeti- tlon .of the above steps.
It will be apparent that the steps (b) and (c) above need not be carried out in the order given but can be reversed 5 without; violating the spirit of the inventlpn. The resulting combination crystals possess a superior purity and oncolytic activity as hereafter described.
In a special embodiment of the process of this invention a crystalline combination of L-asparaginase and magnesium 0 ion can be prepared by dissolving a crystalline combination of slum in water and adding, in any order, the antisolvent until persistent incipient turbidity, and a magnesium salt, preferably magnesium acetate, followed by a separation of. the crysr talllne combination product.
In addition to the high enzymic and oncolytic activity of the crystalline enzyme,., the size, smaller surface area, and greater density make the crystals particularl use^ ful for pharmaceutical filling operations. L-Asparaginase preparations of the prior art are amorphous powders which tend to be unstable in solution, and are generally supplied as a dry powder accompanied by a second bottle containing the necessary fluid for reconstitution. Due to their high surface area, these amorphous powders tend to pick up impurities and static charges. A powder with such static charges causes many problems in the pharmaceutical Industry, When attempts are made to formulate the amorphous material with inert fillers and the like, the material clings to the walls of the apparatus and resists mixing. The material resists being filled into ampoules, capsules, bottles, and. the like; it tends to cling to the filling apparatus or become flyaway.
Additionally, powders of this type resist wetting by the liquid added during reconstitution prior to use. The product of this Invention effectively overcomes all of the above-mentioned prior-art difficulties.
The crystalline combination of L-as araginase and a metal or a metalloid salt of this invention possesses a spe-. cific activity of greater than 350 International Units (I.U.) of L-asparaginase and is prepared from a crude L-asparaginase preparation possessing a specific activity of the order of' 60 I.U. by the described method.
V.' tivity, one International Unit being the quantity of enzyme needed to release 1 micromole of aspartate per minute In the following test. , One to 100 Λ of undiluted sample of the test solu-tlon are added to 1 ml. of aqueous 0.02 M L-asparagine solution and 1 ml. of aqueous 0.2 M sodium acetate buffer (pH .0) . The aspartate liberated by the resulting enzyme reaction is separated by high voltage electrophoresis in pyridine -acetate buffer at pH 6.3 on a cellulose carrier. For example, twenty X of the sample are applied onto the buffer-treated Whatman No. 1 paper and the electrophoresis is carried out at 2, 000 mv. for 30 minutes. The paper is dried in the oven for 10 minutes, then stained with ninhydrin reagent to give dark blue spots corresponding to aspartate. The density of the spots is determined by a densitometer. Assays on standard concentrations of aspartate and controls containing only the reagents without the added test solution to measure aspartate other than that released by enzyme activity are carried out with each test. Alternatively, the enzyme activity can be as-sayed by the method of Campbell et al., Biochem. , 6, 724. (19β7) , wherein the enzyme is allowed to react with excess L asparaglne and the released ammonia is reacted with Nessler's reagent. The amount. of released ammonia is measured by determination of the change in optical density of the Nessler's re-agent.
Preparation of crude L-asparaginase useful in this invention, previously described, can comprise the following steps: a) aerobically fermenting an L-asparaginase-produc-ing strain of E. coll, of which A.T.C.C. 137.06 is a typical drate, nitrogen, and minerals under conventional conditions for about 5 to 10 hours; b) thereafter allowing the fermentation mixture to age under anaerobic conditions for about one hour; c) harvesting the cells; d) resuspending the cells in water; e) treating the resuspended cells with high frequency sound waves to rupture the cell walls; additionally or alternatively, treating the resuspended cells with lysozyme by the method of Cedar and Schwartz, J, Biol. Chem., 242, 3753 (1967 ) or additionally or alternatively further freezing and thawing the aqueous cell cake to disrupt the cell structure; f) adjusting the mixture to pH 5 and filtering to remove the cellular debris; g) adjusting the resulting solution to pH 8; h) adding to the filtrate, solid ammonium sulfate to a concentration of 45 percent (w./v.) and filtering the resulting mixture (the solid residue being discarded); 1 ) adding to the filtrate, solid ammonium sulfate to a concentration of 80 percent (w./v.) and filtering the resulting mixture; j) resuspending the solid residue In aqueous 10"^ M ammonium bicarbonate solution and dlalyzlng the resulting mlx-ture against water; k) adding one volume of ethanol to the aqueous solution of the dialyzed product and filtering the resulting mixture (the solid residue being discarded); 1 ) adding a second volume of ethanol to the aqueous filtrate; and filtering the resulting mixture; m) dissolving the residue in aqueous 10 J M ammonium n) suspending the resulting powder in water, adding solid ammonium sulfate to a final concentration of 5 percent (w./v.), and filtering (the solid being discarded); o) adding additional solid ammonium sulfate to a final concentration of 80 percent and filtering the mixture; p) dissolving the residue in 10 M ammonium bicarbonate, dialyzlng (water) and lyophilizing the solution to yield the final crude preparation of L-asparaginase .
In practicing the present invention, use of enzyme purified through step (p) is preferred although the enzyme preparation of step (m) above is also crystallizable by the process of this invention.
Thus, by the method of this invention, the crude L-asparaginase is first dissolved in water to any desired con-centration up to saturation (it being obvious that a relatively large quantity of water would not be advantageous owing primarily to mechanical losses alone).
An antisolvent is then added to the aqueous solution until incipient cloudiness has occurred. Appropriate water-misclble antlsolvents for the process of this invention include ethanol, acetone, isopropanol, and the like.
An aqueous solution of a metallic or a metalloid salt is then added dropwise to the diluted solution of L-asparaginase, causing immediate crystallization of the combination prod-uct of this invention. In general; ions of all metals of the periodic table as well as the metalloid ions ammonium and hy-drazinium produce a crystalline product with L-as araginase by the process of the invention, the only criteria for operability appearing to be that the salt must be soluble in water and in the water-antlsolvent mixture, and must impart thereto a hydro 6.0 and about 9.
Crystals containing metalloid Ions of the group consisting of H^+ and NgH^ and the metal ions of the group consisting of Na+, K+, Li+, Mg" ", Ba++, Sr++, Mn++, Ca++, Zn++, Cd++, Co++, Ni++, Cr"1" , Fe+3, Pb"1*, Al+3, or Cu++ are the preferred embodiments of the invention. The depot form of the enzyme when crystallized to include ¾n++ is an especially valuable pharmaceutical form. In general, assuming a molecular weight of 180,000 for the enzyme, about 150 ions per enzyme molecule of alkali metal or metalloid ions, about 30 "ions per enzyme molecule of alkaline eart ions, and about 4-6 ions per enzyme molecule of the transition metal ions are needed to effect crystallization of the crystalline combination product.
Although, as has been previously disclosed herein, crystallization begins Immediately upon treating the antisol-vent -diluted enzyme -containing solution with the metallic or metalloid salt, it is preferable to maximize the yield by allowing the crystallizing solution to stand at room temperature or below until crystallization is substantially complete. The crystalline product can be separated by decantation, filtration, centrifugation, or like separative techniques.
Magnesium L-asparaginase crystals produced by the process of this invention belong to the orthorhombic system and have the following cell paramaters. a = 153°A b = 63 A c « 127 A v = 1,224,153 A = 122,415 x 10"21 cm.3 In order to determine that the enzyme preparation purified by the process of this invention contains an oncolytic was tested by the following experiment.
Gardner lymphosarcoma, a solid non-metastasizing tumor was implanted subcutaneously b trocar into the axillary region of mice. Treatment was begun 24 hours after im-plantation and the animals were treated by daily intramuscular administration of the enzyme for a total of 10 days. Activity was determined by comparison of the tumor size in test animals to the size in control animals which received no treatment after implantation of the tumor. Each test group consisted of ten animals and the results were expressed as the average of the tumor size observed in each animal. Table I shows the dose-response relationship of L-asparaginase in this test system. In the Table, column 1 lists the dose in I.U. per mouse at which the enzyme was given daily, the, appropriate quantity of metallo-organic crystalline enzyme being dissolved in sterile saline prior to each daily Injection. Column 2 indicates the activity of the drug in the test system, the first number representing the percent reduction in tumor size over the control animals and the number in parentheses showing the number of survivors in each test group at the end of the test period. None of the ten control animals survived the test period .
TABLE I • Dose I.y./mouse Activity 2 , 0 100 (8) 1.0 100 (8) 0.5 100 (8) 0.2 98 (8) 0.1 68 (9) of the enzyme.
EXAMPLE I Five grams of L-asparaginase as a lyophilized powder . o with an activity of 81 I. U. per milligram of protein were dls-solved In 60 ml. of distilled water. The solution was adjusted to pH 7.0 with 0.1 N sodium hydroxide and dlalyzed against aqueous 10-3 M ammonium bicarbonate solution for about 18 hours. The, resulting solution was clarified by centrifugation and the clear, yellow supernatant was brought to a total vol-ume of 80 ml. and a protein concentration of 38 mg. per ml. by the addition of distilled water. The mixture was stirred and 80 ml. of absolute ethanol were added. The solution was cooled to °C, held at °C. for one hour, then 1 ml. of 1 M potassium phosphate was added. The mixture was cooled to -20°C. and held at that temperature for two hours, A white precipitate of potassium L-as araginase separated and was isolated by centrifugation at -10?C. The precipitate was agitated with 20 ml. of distilled water and the suspension was clarified by centrifugation. Ethanol was added dropwise to the supernatant until turbidity persisted, and the solutipn was stored for 18 hours at 4°C, during which time a white crystalline enzyme preparation separated. The crystals were removed by centrifu-gation. Yield, 5I.5 percent; specific activity, 38Ο I.U. per mg. of protein. Analytical ultracentrifugation showed 99.0 percent homogeneity of product..
EXAMPLE II L-Asparaglnase was purified and crystallized by the method of Example I except that 1 ml. of aqueous 1 M ammonium sulfate was added to the aqueous ethanol solution of the crude crystalline ammonium L-asparaginase L-asparaginase in place of the potassium phosphate. Yield 61$/ EXAMPLE III The product of Example II was dissolved In 10 ml. of water, 25 of aqueous 1 M magnesium acetate solution were added, and alcohol was added dropwlse until cloudiness persist - ed. The resulting mixture was allowed to s,tand for 2 hours at room temperature and overnight at 4°C. The rectangular crystals which formed were collected by centrlfugatlon and decanta- crystalline magnesium L-asparaginase tlon, then washed twice with 50$ aqueous alcohol. Yield, $ / of theory based upon the original crude L-asparaginase of Ex-ample II, Specific activity, 25 I.U./mg. of protein.
EXAMPLE IV Five grams of L-asparaginase as a lyophilized powder with an activity of 81 I.U. per milligram of protein were dissolved in 60 ml. of distilled water. The solution was adjust-ed to pH 7.0 with aqueous 0.1 N sodium hydroxide solution and dialyzed against aqueous 10 -3 M ammonium bicarbonate solution for about 18 hours. The resulting solution was clarified by centrlfugatlon and the clear, yellow supernatant was brought to a total volume of 80 ml. and a protein concentration of 38 mg. per ml. by the addition of distilled . water , With stirring, 80 ml. of absolute ethanol were added. The solution was coolr ed to 4°C. and held at 4°C. for one hour, then 1 ml. of 1 M sodium chloride was added. The mixture was cooled to -20°C. and held at that temperature for two hours. A white precipir-tate comprising sodium L-asparaglnase separated and- was Isolated by centrlfugatlon at -10°C. The precipitate was agitated with 20 ml. of distilled water; the resulting solution was clarified by centrlfugatlon; and 1 ml., of aqueous 1 M magnesium acetate solution was added to the supernatant. Dropwlse addi-r tlon of ethanol to incipient turbidity followed by stora e for asparaginase containing 5 moles of Mg per mole of enzyme.
The crystals were recovered by centrif ugation, Yield, 80 percent; specific activity, 382 I.U. per mg. of protein.
The crystalline magnesium L-as araglnase was re- crystallized by dissolving in water, adding alcohol to in^ cipient cloudiness, and allowing to stand at room .temperature for 2 hours and at - °C. overnight to yield 69# magnesium L- asparaginase with a specific activity of 400 J.U./mg. The purified product also contained 5. moles of Mg ++ per mole of 0 enzyme.
EXAMPLE V A crude L-asparaginase product as in Example Iv was -4 dialyzed overnight against aqueous 10 M ammonium biqarbonate solution. The dialyzed enzyme solution contained approximately 5 10 ppm. of Mg"*"4". Attempted crystallization by the addition of ethanol produced only a non-crystalline precipitate.
One milliliter aliquots of this solution were treaty ed with 20 ^ of metal or metalloid salt in aqueous 0.1 M solution and alcohol sequentjlally until crystallization occurred 0 after standing at 4°C. overnight. The amount of metal or ammonium or hydrazinium ifiQ ffl/Lpfiji ion necessary to induce crystallization was determined based upon an estimated molecular weight of 180,000 for the L-asparaginase.
The following table lists the results of the several 5 experiments.
Metal or Cation: protein rat Metalloid cation mole/mole N¾+ 150 ¾H5+ 150 Na+ 150 K+ 150 Li+ 30 MS 6 Ba" " 6 Sr++ 6 Ca"^ .6 Mn++ 6 Zn^ ' 6 .Cd"^ 6 Co++ 6 N1++ 6 Cr"1-*" 6 P ^ 6 Cu"^ 6 Fe+3 6 Al+3 6 ammonium or hydrazinium compounds Metals and tnk/tla/llll(j$ It can be seen from the above description an specl-fic examples that the process of this invention can be used to purify L-asparaginase frpm a crude preparation thereof by crys tallizing a combination product and thereafter dialy zing the crystalline combination product to remove the metal ions . In this manner a non-metallic product of high purity can be a-chieved .

Claims (1)

1. 33327/2 'Μ which comprises dissolving a crystalline combination of. L- asparaginase and a metal or ammonium or hydrazinium ion other than magnesium ion in water and adding, in any order; 33327/2 a) the antisolvent until persistent Incipient turbidity, and b) a magnesium salt, and '" ' ' separating the crystalline combination of L- asparaginase and magnesium ion. ' < ' 11. The process of claim 10 wherein the magnesium salt is magnesium acetate. ■ 12. A crystalline combination of L-asparaginase and a metail or ammonium or hydrazinium ion substantially as hereinbefore described with particular reference to the examples. ^ 13, A process of preparing a crystalline combination of L- asparaginase and a metal ammonium or hydrazinium ion substantially as hereinbefore described with particular reference to the examples. S. HOROWITZ & CO. AGENTS FOR APPLICANTS
IL33327A 1968-11-29 1969-11-07 A crystalline combination of l-asparaginase and a metal or ammonium or hydrazinium ion and method for preparing the same IL33327A (en)

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US78025168A 1968-11-29 1968-11-29

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AT (1) AT295452B (en)
BE (1) BE742215A (en)
DE (1) DE1959603A1 (en)
FR (1) FR2024571B1 (en)
GB (1) GB1242976A (en)
IL (1) IL33327A (en)
NL (1) NL6917822A (en)

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Publication number Priority date Publication date Assignee Title
DE3542086A1 (en) * 1985-11-28 1987-06-04 Berker Geb Switch construction for a switch, especially a built-in apparatus switch
JP2975109B2 (en) * 1989-12-21 1999-11-10 ノボ ノルディスク アクティーゼルスカブ Enzyme crystallization method
WO1991009941A1 (en) * 1989-12-21 1991-07-11 Novo Nordisk A/S Enzyme containing preparation and detergent containing such preparation

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DE1959603A1 (en) 1970-11-19
BE742215A (en) 1970-05-26
IL33327A0 (en) 1970-01-29
NL6917822A (en) 1970-06-02
GB1242976A (en) 1971-08-18
FR2024571A1 (en) 1970-08-28
AT295452B (en) 1972-01-10
FR2024571B1 (en) 1974-02-01

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