DE1959603A1 - Crystalline combination of L-asparaginase and a metal or metalloid ion and process for their preparation - Google Patents

Description

PATENT LAWYERS

D R. I. M A A S

DR. W. PFEIFFER

DR. F. VOITHENLEITNER

3 MUNICH 23
UNG £ RERSTR. 25 - TEL. 39 02 36

81 492 '

Eli Lilly and Company, Indianapolis, Indiana, V.St.A.

Crystalline combination of L-asparaginase and a metal or metalloid ion and process for their preparation

The invention relates to a crystalline L-asparaginase preparation and relates in particular to a crystalline | Combination of L-asparaginase and a metal or metalloid, which is characterized by high enzymatic and oncolytic activity, as well as a process for their: production.

The crystalline combination according to the invention becomes from a crude L-asparaginase preparation, a soluble metal; or metalloid salt and a precipitant as follows manufactured: .

a) Dissolve a crude L-asparaginase preparation in water;

b) a suitable precipitant which is miscible with water (Antisolvena) clogs up to the onset of turbidity;

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c) gives an aqueous solution of a metal or metalloid salt which is soluble in the solvent-antisolvent mixture and when dissolved in water gives a pH of about 6.0 to about 9, in such an amount that the L-asparaginase crystallized and

d) separates the enzyme crystals obtained.

By repeating this. Stages, a higher purity of the crystals can be achieved.

It can be seen that steps (b) and (c) are not in the specified sequence must be carried out, but are exchanged without deviating from the essence = of the invention can. The resulting crystalline combination is characterized by superior purity and oncolytic activity as will be explained.

According to a particular embodiment of the method according to the invention, a crystalline combination of! -Asparaginase and magnesium ions can be produced by dissolving a crystalline combination of L-asparaginase and a metal or metalloid other than magnesium in water , the antiaolventing in any order until it begins to persist Turbidity and a magnesium salt, preferably magnesium acetate, are added and the crystalline product is then separated off.

In addition to the high enzymatic and oncolytic activity of the crystalline enzyme, the product is particularly suitable for pharmaceutical filling processes due to its size, smaller surface area and higher density of the crystals.

BATH ORIGINAL 009847/0889

partaking. Well-known L-asparaginase preparations are amorphous powders, which tend to be inconsistent in solution, and. are generally supplied as a dry powder together with a second bottle containing the necessary liquid for preparation. As a result of their high surface area, these amorphous powders tend to absorb impurities and static charges. A powder with such static charges causes great difficulties in the pharmaceutical industry. If one tries to prepare the amorphous material with inert fillers and the like, the material sticks on the walls of tung * Vorrich- and opposes the mixing. The material resists filling into ampoules, capsules, bottles and the like and tends to stick to the filling device or to fly off.

furthermore, powders of this type resist wetting by the liquid that is added during preparation before use. With the product according to the invention all of these known difficulties are effectively resolved.

The crystalline combination of L-asparaginase and a metal or metalloid salt according to the invention has an f specific activity of more than 350 international units (I.U.) L-asparagmase and is composed of a crude L-asparaginase preparation with a specific activity on the order of 60 I.U. produced according to the method described. j

The specific activity is a measure of the enzymatic ^ Activity, where an international unit is the amount of enzyme equivalent to the release of 1 micromole of asparaginate per minute in the following test.

1 to 100 Λ. the undiluted test solution is added to 1 ml v / aqueous 0.02 ml L-asparagiase solution and 1 ml was sr if _; Q _f 2 ,. Added in sodium acetate buffer (pH 5.0). That through

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T95ÄQ3

The asparaginate released in the following enzyme reaction is separated off on a gel carrier by high voltage electrophoresis in pyiidine acetate buffer at pH 6.3. For example, 20 μ of the sample is applied to Whatman No. 1 paper treated with buffer and the electrophoresis is carried out for 30 minutes at 2000 millivolts. The paper turns 10; Oven dried for minutes and then stained with ninhydrin reagent, forming dark blue spots corresponding to asparaginate. The density of the spots is determined with a densitometer. For each test, determinations with standard concentrations of asparaginate and with control samples, which only contain the reagents without the added [test solution, are carried out, in order to measure other than asparaginate released by enzyme activity. Alternatively, the enzyme activity can be determined by the method of Campbell et al. _t Bioehem., 6, 721 (1967); turn determined, in which: the enzyme is allowed to react with excess ii-asparagine and the ammonia released with Ifessler's reagent. The amount of ammonia released is measured by determining the change in the optical density of the Nessler reagent. '■

The production of crude L-asparaginase as described above is used for the purposes of the invention can include the following stages:

a) An L-asparaginase producing strain of E. coli, for that the A.T.C.G. 13706 is a typical example is in a nutrient medium containing assimilable carbohydrate, nitrogen and minerals, under normal conditions fermented aerobically for about 5 to 10 hours;

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b) then the fermentation mixture is under anaerobic conditions aged for about 1 hour;

o) the cells are harvested;

d) the cells are resuspended in water;

e) the resuspended cells are high frequency. Treats sound waves to break down cell walls additionally or alternatively, the resuspended cells are coated with lysocyme according to the method of Cedar and Schwartz, J. Biöl. Chem. 242, 3753 (196?) or - additionally or alternatively, the aqueous cell cake is frozen and thawed in order to maintain the cell structure break up;

f) the mixture is adjusted to pH 5 and filtered to remove the cell debris; ·

g) the solution obtained is adjusted to pH 8;

h) the filtrate is mixed with solid ammonium sulfate up to a concentration of 45 $ (weight / volume), and the mixture obtained is filtered (the solid residue is discarded); ^x

I) solid ammonium sulphate is added to the filtrate to a concentration of 80 fo (weight / volume), and the mixture obtained is filtered;

j) the solid residue is dissolved in 10 M aqueous ammonium bicarbonate solution r «suspended and the mixture obtained is dialyzed against Waaser;

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k) the aqueous solution of the dialyzed “product is mixed with the same volume of ethanol, and the mixture obtained is filtered (the solid residue is discarded); ΐ- .. '■' ■■■■

1) the aqueous filtrate is washed a second time with the same Volume of ethanol is added and the mixture obtained is filtered; · .. ··. ·. ...

m) the residue is' in aqueous 10 M ammonium bicarbonate solution dissolved and the resulting solution is freeze-dried; '- · ·' ■■■■ '"«'

n) the powder obtained is suspended in water, and the Suspension is made up to one with solid ammonium sulfate Final concentration of $ 45 (weight / volume) added and filtered (the solid is discarded);

o) the filtrate is mixed with further solid ammonium sulfate up to a final concentration of 80 % and the mixture is filtered;

p) the residue is dissolved in 10 M ammonium bicarbonate solution, the solution is dialyzed. (Water) and the solution is lyophilized, making the finished crude L-asparaginase preparation is obtained.

For the purposes of the invention, the use of Preferred enzyme that is purified up to step (p), whether-. probably also the enzyme preparation from stage (m) can be crystallized by the process according to the invention.

'Ifach the method according to the invention is first the " raw L-asparaginase in water in any desired concentration

tration up to the total saturation value (it makes sense that. that a relatively large amount of water would not be advantageous, if only because of the mechanical losses).

Then the aqueous solution is mixed with an an'tisolvens Ms for the onset of turbidity. Suitable water-miscible antisolvents oüer]? al treatment means for the invention _; Processes are, for example, ethanol, acetone and Isopro- ^. panol.

This becomes the diluted solution of L-asparaginase. ^ An aqueous solution of a metal or metalloid salt is added dropwise, which leads to immediate crystallization of the inventive Combination product leads. In general, ions of all metals of the periodic system as well Ketalloid ions such as ammonium and hydrazinium ions crystalline P: oduct with L-asparaginase according to the invention Procedure. Crucial for the implementation of the invention Procedure is apparently just that the salt be soluble in water and in the water-antisolvent coating ' must and generate a hydrogen ion concentration therein which corresponds to a pK value between about 6.0 and about 9. '

The preferred embodiment of the invention are crystals which ^{contain the metalloid ions KEL +} and ^ 2 ^ 5 ^{+ unö di} v ^e metal ions

25 Na ⁺ , K ⁺ , Id ⁺ , Mg ⁺⁺ , Ba ⁺⁺ , Sr ⁺⁺ _{> Mn} ++, Ca ⁺⁺ , Zn ⁺⁺ , Gd ++, jj

Contains Co ⁺⁺ , Ni ⁺⁺ _f Cr ++, 3? E + 3, Pb ++, Al + 5, or Cu ⁺⁺ . Is the depot form of the enzyme obtained in crystallization with Zn ++ a particularly valuable pharmaceutical Eorm .. W _e nn one l'ÜR the enzyme has a molecular weight of 180 000, accepts, s ^ nd generally about 150 alkali metal or metalloid ions per enzyme molecule, about 30 alkaline earth metal ions per enzyme molecule and about 4 to 6 transition metal ions per enzyme molecule are required for crystallization of the crystalline combination product.

It is true that, as stated above, crystallization begins immediately 'after treatment of the enzyme-' containing solution with the metal or metalloid salt, in order to achieve the highest possible yield, however, it is preferred let the crystallizing solution stand at room temperature or below until crystallization is practically complete. The crystalline product can be obtained by decanting, filtering, centrifuging or ahn-_ borrowed separation methods are separated.

Those obtainable by the process according to the invention Magnesium-L-asparaginase crystals belong to the orthorhombic System and have the following unit cell parameters:

a = 153 ° A

b = 64 A

c = 127 A

ν = 1 224 153 A ⁵ = 122 415 x'10 " ²¹ 'cm ³

To prove that the enzyme preparation purified by the process according to the invention contains oncolytically effective asparaginase, the antitumor activity of the isolated product is tested by the following method.

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AD OFHOiNAL.

Lymphosarcoin G-aranoi'j a solid, non-metastatic tumor, is implanted subcutaneously in mice through a trocar in the axillary region. Treatment is started 24 hours after implantation. The animals are treated by intramuscular administration of the enzyme daily for a total of 10 days. Activity is determined by comparing the tumor size in test animals to the size in control animals that receive no treatment after implantation of the tumor. Each test group consists of 10 animals. Which he- . % results are reported as the mean value of the tumor size observed in each animal. Table I shows the dose-effect relationship for L-asparaginase dieoiirii test system. In column 1 of the table is the dose in I. Ii. named per mouse, in which the enzyme is administered daily. For this purpose, the appropriate amount of organometallic crystalline enzyme is dissolved in sterile saline solution before each daily injection. Column 2 gives the activity of the active ingredient in the test system. where the first number corresponds to the reduction in tumor size compared to the control animals in percent and the number in brackets indicates the number of surviving animals in each test group at the end of the period. None of the 10 control animals survived the test period.

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- yt - I. activity (8th) 100 (8th) Tabel 100 (8th) '· - 100 (8th) 98 (9) ■ 68

I.U./Mouse

Similar results are obtained with subcutaneous administration of Snzyin.

Ex iel .1

5 gL asparaginase in the form of a lyophilized powder with an activity of 81 I.II. per milligram of protein are dissolved in 60 ml of distilled water. The solution is used with 0.1 η sodium hydroxide solution adjusted to pH 7.0 and about -

-3

Dialyzed for 18 hours against 10m aqueous ammonium bicarbonate solution. The solution obtained is clarified by centrifugation, and the clear yellow supernatant liquid is brought to a total volume of 80 ml and a protein concentration of 38 mg per ml by adding distilled water. The mixture is stirred and 80 ml of absolute ethanol are added. The solution is cooled to ⁰ G 4, held for 1 hour at 4 ° 0 and then treated with 1 ml of 1M potassium phosphate solution. The mixture is cooled to -20 ⁰ G and held for 2 hours at this temperature. A white precipitate of potassium L-asparaginase is deposited,

8ADORtGIKlAL 009847/0889

which is isolated by centrifugation at −10 ° C. The precipitate is stirred with 20 ml of distilled water and the suspension is clarified by centrifugation. The protruding ! Ethanol is added drop by drop to the liquid, "until the cloudiness" remains ".-- The solution will be 18 hours stored at 4 ° C. During this time a white crystalline enzyme preparation separates out. The crystals will be centrifuged off. Yield 51.5%, specific activity 380 I.TJ. per mg of protein. Analytical ultracentrifugation results in a product homogeneity of 99 »0 ^.

Example 2

L-asparaginase is purified and crystallized according to the method of Example 1, with the exception that the aqueous Ethanol solution of the crude L-asparaginase 1 ml of aqueous 1 M ammonium sulfate is added instead of the potassium phosphate. Yield 61 #, specific activity 382 I.U./mg protein.

Example 3

The product from Example 2 is dissolved in 10 ml of water, 25 λ is added. 1 M aqueous magnesium acetate solution is added and alcohol is added dropwise until the cloudiness persists. The mixture obtained is left to stand at room temperature for 2 hours and at 4 ° C. overnight. The resulting rectangular crystals are isolated by centrifugation and decanting and then washed twice with 50% aqueous alcohol. The yield is 55 i ° of theory, based on the original crude L-asparaginase of Example 2. Specific activity: 425 I.IT./mg protein.

Example 4

5 g of L-asparaginase in the form of a lyophilized powder with an activity of 81 I.TJ. per mg protein are in 60 ml

009847/0883

dissolved in distilled water. The solution is adjusted to pH 7.0 with aqueous 0.1 η sodium hydroxide solution and dialyzed for about 18 hours against aqueous -10 M ammonium bicarbonate solution. The resulting solution is clarified by centrifugation, and the clear, yellow supernatant liquid is brought to a total volume of 80 ml and a protein concentration of 38 mg per ml by adding distilled water. Then 80 ml of absolute ethanol are added with stirring. The solution is cooled to 4 ° C, held for 1 hours at 4 ⁰ C and then added with 1 ml of 1 m Hatriumehloridlösung. The mixture is cooled to -20 ⁰ C and held for 2 hours at this temperature. It separates out a ■ white precipitate of sodium L-Äsparaginase, which is isolated by centrifugation at -10 ^0C. The precipitate is stirred with 20 ml of distilled water. The solution obtained is clarified by centrifugation. 1 ml of aqueous 1 M magnesium acetate solution is added to the supernatant liquid. By adding ethanol drop by drop until the onset of turbidity and then storing for 18 hours at 4 ° C., white crystals of magnesium L-asparaginase are formed which ^{contain 5 moles of Mg +} per mole of enzyme. The crystals are collected by centrifugation. Yield $ 80, specific activity 382 IU per mg protein.

The crystalline magnesium L-asparaginase is

sen recrystallized in water, adding alcohol until the onset of turbidity and leaving to stand at room temperature for 2 hours and at -4 ° C overnight in 69 i ° yield to magnesium L-asparaginase with a specific activity of 400 I.TJ./mg . The purified product also contains 5 moles of Mg ⁺⁺ per mole of enzyme. .

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Example 5

Sin rones L-Asparaginase product as in Example 4- is over Kacht against aqueous 10 m ammonium bicarbonate solution dialyzed. The dialyzed enzyme solution contains about 10 ppm Mg. An attempt to induce crystallization by adding ethanol only gives a non-crystalline one Precipitation.

Aliquots of this solution of 1 ml each are successively with 20 Λ. an aqueous 0.1 M solution of metal or Metalloid salt and alcohol are added until crystallization takes place after standing over fold at 4 ° C. The amount of Metal or metalloid ions required to induce crystallization are estimated based on a Molecular weight of 180,000 for L-asparaginase was determined.

The results of the various tests are summarized in the following table ".

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Metal or metalloid cation. Cation: protein molar ratio

₄ ⁺ '150

N ₂ H ₅ ⁺ 150

Na ⁺ 150

K ⁺ 150

Li ⁺ , 30

Mg ⁺⁺ . 6th

Ba ⁺⁺ '6

Sr ⁺⁺ ■. ■ 'δ

Ca ⁺⁺ 6

Mn ⁺⁺ 6

Zn ⁺⁺ 6

0d ⁺⁺ 6

0o ⁺⁺ 6

Cr ⁺⁺ δ

Pb ⁺⁺ - β

Cu ⁺⁺ 6

Al ⁺³ "" ■■ '"' 6

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The metals and metalloids are used in the form of their acetates, h citrates, phosphates, bicarbonates or sulfates. ''

It can be seen from this that the method according to the invention for the purification of crude preparations from L-asparaginase by crystallization a combination product and then dialyzing the crystalline communication product is suitable for removing the metal ions. In this way, a metal-free product of high purity can be obtained will.

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Claims (1)

Patent to τ> rü che 1, Crystalline combinations of! ^ Asparaginase and metal * - or metalloid ions. 2. Crystalline combination according to claim 1, characterized in that the metallianes from \ Alka.limetallionen stand, ■ 5, - ^ Crystalline combination according to claim 1 \, characterized in that the metal ions
confess '.' 4, Crystalline combination according to. Claim 3 _:. » characterized in that the metal * - or metalol ions ..from W ⁺ ; K ⁺ , ' M * ,, , Zn ^++,, i »b ⁺⁺ , ^ H ₄ ⁺ or H 3 * crystalline combination -according to claim ¹ I or 4, characterized. Marks that the metal ions consist of zinc ions. 6. Crystalline combination make claim 1, 3 or 4, characterized that the MetaO-lionen from magnesium ions "exist. ? .. Crystalline combination according to claim 1 pÄ @ r 4, characterized ,, that the Metalloiäionen from ammonium ions exist. 0098A7 / 0889 If 8 "Crystalline combination according to claim 1, 2 or 4, characterized in that the metal ions consist of potassium ions ". 9. Process for the preparation of crystalline combinations of L-asparaginase and metal or metalloid ions, characterized in that L-asparaginase is in water solves and in any order a) an antisolvent or sedative up to persistent ^ incipient turbidity and b) a metal or metalloid salt that is in the water-antisοIvens mixture is soluble and when dissolved in water gives a pH of about 6.0 to about 9.0, adds and separates the crystalline combination product. · ' 10. The method according to claim 9 for producing a crystalline combination of L-asparaginase and magnesium ions, thereby characterized in that one has a crystalline combination of L-asparaginase and other metal or Metalloiö ions dissolves as magnesium ions in water and in any f ger order ä) the antisolvent up to persistent onset of opacity and b) a magnesium salt adds and the crystalline combination of L-asparaginase and separates magnetic ions. 0 98 4 77 088g 11. The method according to claim 10, characterized in that one uses as magnesium sala magnesxum acetate. 009847/0889