DE2009492C3 - Process for the separation of pyrogens from preparations of L-asparaginase - Google Patents

Description

10

L-asparaginase is an enzyme that has the amide group of L-asparagine is hydrolyzed, producing L-aspartic acid and ammonia. The enzyme has to fight of malignant growth are of great importance in medicine.

L-asparaginase suitable for chemotherapeutic purposes is obtained from Escherichia coli by methods that z. B. von Campbell et al. (Biochemistry 6, 721 j 1967J) have been described. The enzyme produced in this way with high purity but still contains pyrogenic substances, which are likely to come from the bacterial cells themselves, and is therefore unsuitable for therapeutic use.

The removal of the pyrogens has hitherto been carried out by the heating process according to the German Offenlegungsschriften 1792043 and 18 16901 or by column chromatography on cross-linked dextranjo gel with diethylaminoethy! groups, e.g. B. according to DE-OS Π 67 389 with na "! Following precipitation with polyethylene glycol performed. The pyrogen content of individual batches is very different. Games with low pyrogen content can be achieved by ii the heating process with good yields of free the pyrogen. If the pyrogen content is high, the process must be repeated several times large losses of L-asparaginase then occur.

The säulenchromalographische process according to DE-OS 17 67 389 with the anionic cross-linked dextran gel with Diäthylaminoäthylgruppen has to be to the detriment of the greater technical complexity, especially as the column in-4> follow the strong volume contraction during elution with salts refilled for each approach.

According to BE-PS 7 37 859, the contact of L-asparaginase- _{containing solutions in AI (OH) 1} -CJeI is proposed to remove pyrogens from L-asparaginase preparations.

Due to the physical properties of Al (OH) ₁ - (JeIs (rapid aging, poorly reproducible properties), the method of operation has some disadvantages

You cannot work with a chromatographic column that is technically easy to handle. AI (OH) ₁ can only be used in the status nascendi because of its rapid aging.

The Al (() H) i -Callcrte adsorbs a considerable amount of wi borrowed for the / u adsorbing substance (here! .- asparaginase) irreversible, and excessively high total * losses of L-asparagiriase occur,

The method according to the invention characterized in the claim provides the above-described Avoid disadvantages of the known method.

Surprisingly, it has been shown that one can use Asparagiriaseparlien with a high pyrogen content, the after heating once according to DE-OS 18 16901 are not yet usable by chromatography on uncharged acrylamide or dextr gels, in particular on cross-linked dextr gels with exclusion limits for molecular weights> 150,000 Dalton or> 200Q0Q Dalton can remove the remaining pyrogens. The pyrogens leave the Column together with higher molecular weight accompanying substances in front of the asparaginase fraction. Be at the same time separated by the process of higher molecular associates of asparaginase and some dyes, whereby the specific activity is also further improved. The whole process depends on the type and composition of the eluting buffer not changed. So that there is no shrinkage of the gel bed, so that σ-.an can pass one batch after the other through the same column without further regeneration.

The separation of the pyrogens succeeds equally well with both types of dextran gel, although three separation the higher molecular weight associates of asparaginase on cross-linked dextran gel with a reject limit for molecular weights> 200,000 Dalton is even better than on cross-linked dextran gel an exclusion limit for molecular weights> 150,000 Daltons. Because of the cheaper physical Properties is cross-linked dextran gel with an exclusion limit for technical use used for molecular weights> 150,000 Dalton.

For chromatographic purification, the enzyme is used in a concentration of 10 to 20 percent in one suitable buffer, e.g. B. Phosphate buffer, Tris buffer or glycine, dissolved at pH 6 to 8.5, with a little polyethylene glycol for stabilization and prevention Thimerosal has been added to bacterial growth.

The dextran gel was previously swollen in the same buffer and filled into columns, with the ratio from column diameter to column height is 1: 3 to I: IO. The enzyme solution is applied to the column and eluted at a rate of 1 to 3 ml per cnv column cross-section and collected in a fractionated manner. At the outlet of the column, the absorption at 254 or 280 nm and the L-asparaginase reactivity measured (figure).

Depending on the purity and the type of pretreatment, the L-asparaginase preparation contains a greater or lesser proportion of asocials living in a pre-fraction together with impurities be united and separated. The proportion of L-asparaginase activity in the preliminary fraction can 10 to 40 percent of the total activity. This Ar'eil can be a great one through Rechromatographic / u Part to be recovered in the main section. The main fraction consists of L-asparaginase with a molecular weight of about 120,000. The fractions making up the main part of enzyme activity are also combined and the enzyme by known methods, such as /. B. Precipitation with acetone, alcohol or polyethylene glycol, isolated in solid, mostly crystalline form. Based on an enzyme preparation from 150 to 200 U / mg initial weight, the enzyme is obtained with a molecular weight of about 120,000 in Bulging from 50 to 75 percent and with an activity of 200 to 240 U / mg sample weight or 220 to 270 U / mg protein, the fever-provoking effect measured on rabbits with 500 and 1000 U / kg animal a temperature can be added in the initial preparation

increase of 1 to 2 C. After the column run the initial fraction of the enzyme in the same dosage corresponds to the requirements of German Pharmacopoeia VII Pur pyrogen-free, that is, the sum of the squares of the temperature increase of three animals is less than 0.6.

The figure shows the chromatography of L-asparaginases on cross-linked dexl rod with a Exclusion limit for molecular weights> 200,000 Dalton, according to Example 1.

On the left ordinate is the L-asparaginase activity in U / ml, on the right ordinate the UV absorption at 280 or 254 nm is given in%.

The upper row of numbers on the abscissa gives the amount of eluate in liters, the lower row of numbers on the abscissa the fraction number. Fraction 6 to 22 is the preliminary fraction, fraction 23 to 36 is the main fraction and Fnaktion 37 to 40 the post-fraction. The solid one The curve shows the L-asparaginase activity, the dashed curve shows the UV absorption.

example 1

2800 g dextran gel (cross-linked dextran gel with an exclusion limit for molecular weights > 200,000 Dalton) vmrden in 150 liters of buffer with the following composition pre-swollen:

Glycine 30 g / liter (0.4 M),
Polyethylene glycol (with a molecular weight of approx. 1550 Dalton) 10 g / liter,
Na ethyl mercurithio salicylate 12.5 mg / liter.

This solution was adjusted to pH 8.0 with 4N sodium hydroxide solution. The gel was then placed in a column with 28 cm in diameter and 120 cm in height, taking care to keep the flow rate Does not exceed 0.8 liters per hour. The height of the gel bed was 112 cm.

190 g of an L-asparagenase preparation with 201 U / mg, that is 38.2 MU (MU = mega-units, pyrogenicity of the substance see Table 1) was in 1.3 liters of the dissolved above buffer, applied to the column and chromatographed with the same buffer (figure). The throughput speed was initially 0.50 liters / hour, later 0.45 liters / hour. The templates were automatically changed every 2 hours. The Επί "enzyme fractions were determined for their L-asnaraginase activity examined and then united, jo that the associates the L-asparaginase were separated off as a preliminary fraction. For the extraction of dry substances were the solutions at pH 5.1 by adding >> polyethylene glycol with a molecular weight of approx. 155CDalton precipitated or crystallized. The precipitates were washed with acetone and with acetone dried. Table 2 shows the yields.

Table 1

Pyrogenicity of the preparations from Example 1, tested on the rabbit

preparations dose Temperature increase 2nd animal ; (Π 3rd animal DAB Werl *) U / kg animal 1st animal 0.3 0.6 Source material 500 0.6 1.5 1.1 0.81 1st day 1000 1.0 0 3 0.7 4.46 2 day 500 0.5 1.2 0.9 0.83 1000 U 0.7 0.4 3.46 Pre-fraction 500 0.4 0.8 0.5 0.81 1st day 1000 1.0 0.3 0.4 1.89 main fraction 500 0.5 0.5 0.1 0.50 1st day 1000 0.5 0.3 0.4 0.51 2 day 500 0.5 0.1 0.0 0.50 1000 O ₁ O 0.01

*) The DAB-Wcri is the sum of the squares of the temperature increase of three animals.

Table 2

Bulging and L-asparaginase activity of the preparations of Example I.

preparation Faction volume In / ymaklivil.it *) (MIl) Bulge number 38.2 (L) lU / mgr 8.86 (X) Source material - - 201 26.18 = 100 Pre-Fraklion 6 to 22 15.7 76 0.3 "! 23.2 Main Fraklion 23 to 36 12.9 222 68.5 Post-fraction 37 to 40 3.6 115 0.9

*) The bnits were weighed in on mg of the precipitated or crystallized and dried preparations based. The protein content (determination) is 87.1% in the main fraction and thus the specific activity is 255 U / mg protein.

Example 2

3 kg dextran gel (cross-linked dextran gel with a Exclusion limit for molecular weights> 150,000 Dalton) were pretreated as described in Example I. and filled into a column with a diameter of 28 cm and a height of 120 cm. The flow rate can be up to 2 liters / hour here. The height of the gel bed would be 110 cm.

275 g of an L-asparaginase preparation with 216 U / mg were dissolved in 1800 ml of the buffer from Example 1 • and on the above column with one pass speed of 850 ml / hour chromatographies. The template was automatically changed every two hours. The enzyme-containing fractions became a preliminary fraction with the associations of L-asparaginasc and impurities, a major fraction with the enzyme having the molecular weight of about 120,000 and a small fraction grouped together. The solutions were worked up to dry preparations as indicated in Example 1. the Testing of the preparations for pyrogens is shown in Table 3, the yields and enzyme activities in Table 4; stirred up.

Table 3

Pyrogenicity of the preparations from Example 2, tested on rabbits

['räparal

Dose temperature increase ("C)

U / kg animal!. Animal 2nd animal 3rd animal

DAB value *)

Source material 500
1000 Pre-fraction 1000 Main fraction solution 1000 ! Crist. preparation
1st day 500
1000 2 day 500
1000 Post-fraction 1000 *) The DAB value is the Sum ύ Table 4

ι, ο
1.5 0.9
0.9 1.2
i, 4 3.25
5.02 1.2 1.1 0.8 3.29 0.3 0.3 0.2 0.22 0.3
0.2 0.4
05 0.4
0.3 0.41
0.38 0.3
0.0 0.1
0.4 0.2
0.2 0.14
0.20 0.6 0.7 o:> 1.10

Yields and L-asparaginase activity of the preparations from Example 2

preparation

Enzyme activity (U / mg) *) MU yield
in percent

Source material 216 59.4 100 Pre-fraction 150 17.8 30th Main faction 250 39.8 67 Post-fraction 188 1.2 2

*) The units are based on the mg weight of the dried substance. The protein content of the main fraction was determined to be 92% (Biuret test), the specific activity of this fraction is therefore 272 U / mg protein.

1 sheet of drawings

Claims (1)

Claim: Process for the separation of pyrogens from preparations of L-asparaginase, characterized in that that one can use these preparations on uncharged or uncharged dextran gels Acrylamide gel chromatographies.