DE2416019A1 - MODIFICATION OF ORGANIC COMPOUNDS AND THEIR CLEANING - Google Patents

Description

Oipl -in i. P. '-'-' irin
'-r'. ·. ϊιϊ.ττ. ■■■; ■ "- - -ι · '. ;; . -..-.!. oiibsrg 0 '. ^ 1., ... ui. iV D. Gude!

ί "..- <v, '', i i.; cheiiiieimnr Str 39

Sandoz AG. 900-90 87

Basel - <

Switzerland

Modification of organic compounds and their purification

The invention relates to interferons, a method for their Purification and modification.

Interferons are known antiviral substances, those both in vivo of living organisms and in vitro of tissue cultures after exposure to more specific Inducers are formed, in particular by exposure to both viral and. also non-viral inducers. The interferons are well described in the literature. It has been shown that interferons are a broad spectrum of activity against different types of Have viruses, but do not have any toxicity themselves and also do not produce antibodies. You therefore pose

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an antiviral prophylactic and the rape utikurn with broad spectrum of activity, especially when compared with the limited number of synthetic Antiviral / effective drugs that are on the market compares «their therapeutic The possibility of use has not yet been used sufficiently, in particular because of the difficulties that arise opposed the production of interferon in sufficient quantities. These difficulties exist in particular in the fact that it has not previously been possible to obtain interferons in a sufficient degree of purity and In addition, the interferons have only a short half-life after parenteral administration *

The present invention relates to methods of modifying the interferon structure to increase the Half-life, with the antiviral activity is not substantially diminished. The present invention also relates to a method of cleaning of interferons as well as structurally modified interferons.

Very little has hitherto been known about the chemical nature of interferons, especially for this reason the interferons produced so far were obtained only in insufficient purity, and the chemical analysis was thereby falsified. Some structural characteristics

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could however with the help of various enzymatic and chemical investigations of the unpurified interferons as well as from the measurement of their antiviral Activity can be determined. It has been proven that interferons are, or at least, proteins Proteins received as main components, and that some of the molecules consist of carbohydrate residues. It it was also claimed that interferons are glycoproteins that contain a terminal sialylic acid residue (E. Schonne et al., Symp. Series Immunobiol. Standard JL4, 61 (1969)], but this hypothesis has not yet been conclusively confirmed. The following invention is based on or. supports the assumption that interferons are glycoproteins. The present Invention confirms that the penultimate saccharide residue is a galactose residue.

The present invention relates to methods of manufacture of interferon derivatives, which are characterized are that one

a) Introduces sialylic acids enzymatically into interferons or asialointerferons using specific Siylyl transferases,

b) the terminal galactose residue in asialo interferons enzymatically oxidized with the help of galactose oxidase,

c) the terminal galactose residue of the asialointerferons with the help of specific GalactosidasG enzymatically cleaved.

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The process described under a) can be carried out using known methods for the introduction of sialylic acid. in particular N-acetylneuraminic acid, carried out in glycoproteins or asialoglycoproteins. The specific sialy transferases used are also known for such processes and relate in particular to sialyl transferases from rat liver (RLST) [J. Hickman et al., J. Biol. Cheiru 245 , 759 '(197O)], the bovine colus (CCST) [BA Bartholomew and GW Jourden, Methods in Enzymology J>, 368 (1966)], the submaxillary gland of the sheep ( SSGST) [DM Carlson et al., Methods of Enzymology, JS, 361 (1956)], or the embryonic Kühnergehimes (ECBST) [B. Kaufman and S. Basu, Methods in Enzymology _8, 365 (1966)]. The conditions under which the process is carried out are known per se and vary according to the enzyme used. In general, the process is carried out at a pH between 5.5 and 8.0, but the general conditions depend on the enzyme used. Accordingly, if RLST, which is the preferred transferase used, is used, the pH is preferably from 6-8 using, for example, 2- (N-morpholino) ethanesulfonic acid or tri-KCl buffer. If CCST is used, the pH is preferably from 6.4-7.2 using, for example, phosphate buffer. When using SSGST transferase, the preferred pH is 5.8-6, using cacodylate buffer. There is no demonstrable metal requirement for these enzymes, although EDTA can enhance these effects.

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The Si aly acid donor is conveniently CMP-N-acetylneuraminic acid, but this can be done by CMP-N-glycolyl neuraminic acid replaced if SSGST is used as transferase. Temperatures and the remaining conditions are known per se and described in the literature.

The process described under b) is carried out using known methods for the oxidation of terminal Galactose residues in glycoproteins usual oxidation methods, using an enzymatic galactose oxidase (which is commercially available). The oxidation conditions are known. For example, the Oxydaiton can be carried out at pH values of 7-8, where for example phosphate buffers can be used. The extent the oxidation can be carried out by subsequent reduction of the C6 aldehyde group formed with labeled, for example checked with tritiated sodium borohydride. After hydrolysis of the reduced material, for example with hydrochloric acid, the obtained becomes labeled Galactose in the hydrolyzate determined using paper chromatography. ^

Process c) is based on a customary method for the cleavage of terminal galactose residues in glycoproteins Manner carried out, the known for this

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2416013

β-galactosidase is used. This β-galactosidase can either be of bacterial or animal origin and, for example, from Dipplococcus pneumoniae (DPG) [RC Hughes and RW Jeanloz, Biochem. ^ 3, 1535 (1964)], from the Concanavalia ensiformis (CEG) [Y.ΐ. Li and S ₁ C. Li _f Methods in Enzymology 2_8 / 702 (1972)], voia Phaseus vulgaris (PVG) [KML Agranal and OP Bahl, Methods in Enzymology 2j3, 720 (19 72)], from Aspergillus niger (ANG) [OP Bahl and KML Agranal, Methods in Enzymology 28, 728 (1972)] or from Clostridium perfringens (CPG) [EG McGuire et al., Methods in Enzymology 2δΐ, 755 (19 72)]. The conditions of the process are known per se and depend in particular on the enzyme used. For example, the process can generally be carried out at a pH of 3.5-6.5, the preferred pH values depending on the specific enzymes used. For example, when using DPG, the preferred enzyme, the pH should be 6.3-6.5, with phosphate buffers being used, with CEG the pH should be 3.5-4.5, with PVG the pH value of 3.5-4.8 and for ANG the pH value of 3.8-4.6. The cleavage of the galactose residue can be determined with the aid of a tritium-containing asialointerfex-on. This is treated with ß-galactosidase, dialyzed and hydrolyzed, and the tritium-containing galactose is found in the hydrolyzate.

The interferon derivatives obtained can be isolated and purified in a manner known per se, for example by dialysis and centrifugation.

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The asialointerferons are interferons in which the terminal sialylic acid residues have been wholly or partly split off. These compounds used as starting compounds in processes a), b) and c) are either known [E. Schonne et al. Syiap. Series Imminobiol. Standard JL4, 61 {1969}] or can be prepared in a manner known per se by removing terminal sialylic acid residues from glycoproteins. They can be obtained by mild acid hydrolysis of the interferons, by prolonged incubation at pH = 2 in the cold, e.g. at 4 ° C for 1 week. obtained for example from Vibrio cholerae, Clostridium perfringens or Diplococcus pneumoniae [Brzenieck R., Current Topicx in Microbiology and Immunology 59_j 35 (1972)], or treated from the rat heart. The incubation conditions are known and depend on the nauraminidase used for iieuraminidase from Vibrio cholerae, a pH of 5.5 is preferred, using, for example, an acetate buffer, and for IteuraiBinidase from Diplococcus pneumoniae, a pH of 6.5 is preferred.

If desired, the two methods of cleavage can be used of the sialyl radicals are used, whereby a higher degree of cleavage of the groups is obtained.

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The asialo interferons obtained can be isolated and purified in a manner known per se.

The interferons which are used as starting compounds are well described in the literature and can produced by the interaction of inducers such as myxoviruses, arboviruses, enteroviruses, DNA viruses, and various others other viruses, for example foot and mouth disease viruses, which do not belong to any group than also of non-viral inducers such as microorganisms and bacterial endotoxins with cells in vivo and in vitro. Specific interferons that may be mentioned include virus-induced animal interferon, for example Rabbit interferon, duck interferon, mouse interferon, monkey interferon, calf interferon and human Interferon.

The present invention also relates to a biosynthetic method for producing structurally modified interferons due to incomplete synthesis the carbohydrate content of the interferons. In particular concerns: the invention a process for the production of structurally modified interferon, which inhibits the Carbohydrate synthesis during interferon biosynthesis by incorporating a glycosyl transferase, preferably of 2-deoxyglucose or 2-deoxy-2-aminoglucose in the Contains reaction medium. The method is known per se, for the inhibition of the carbohydrate synthesis

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customary manner using, for example, 2-deoxyglucose or 2-deoxy-2-amino ~ glucose carried out. The process is expediently carried out at temperatures of 30-40, preferably at 37.degree. The product of the process is changed in such a way that there are no longer any terminal galactose residues seems to contain.

The structurally modified interferons obtained in this way can be isolated and purified in a manner known per se.

The invention also relates to a cleaning method for interferons, asialo interferons or interferons, which are modified according to the above invention. The purification process includes chromatography over an immobilized agglutin and a subsequent desorption of the agglutin-bound interferon, asialointerferon or modified interferons. The affinity chromatographic Cleaning can be carried out in a manner known per se. This is how the agglutin fraction is expedient on a mainly inert solid support

immobilized. The carriers suitable for the process depend on the agglutin used. A "preferred carrier is agarose, which is activated by known methods and to which the agglutin is covalently bound (P. Cuatrecasas et al., Biochemistry IJ _- , 2291-2299).

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The interferons are "specifically adsorbed on the agglutin via their cobJ hydrate content, while most of the impurities pass through. The adsorbed interferon is eluted with the aid of a suitable eluent . Suitable agglutins are those that are necessary for glycoprotein purification, in particular phytonera agglutins, such as Concanavalin A phytohemagglutinin, the usual Lentil phytohemagglutinin, castor bean phytohemagglutinin and, preferably the first phase, Vulgaris phytohein agglutinin Mono-, oligo- or polysaccharides or other glycoproteins , preferably a glycoprotein or its fragments, obtained from human erythrocytes [S. Kornfeldt et al., Proc. Nat. Acad. Sci. (USA) 6J3, 1439-1446 3. Likewise, a desorption of the interferon can be effected by changing the pH to a value of 2,

The modified interferons of the present invention have similar antiviral activities as the unmodified interferons, but have their half-life is longer. In particular, they are active against herpes Simplex virus as seen in New Zealand rabbits (1-2 kg) after injection with 10 "pfu herpes simplex virus has shown. 3 to 6 days after administration

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the rabbits develop ascending paralysis and about 2/3 of the animals develop encephalytis with a usually fatal outcome. 10 units The interferon preparation should be given in a single dose at the beginning of the infection or divided into 4 doses 6-hour interval, and the results observed in the animals.

The modified interferons of the present invention are indicated as having antivirals in particular prophylactic use. The amount to be used daily should be from 5 χ 10 to -200 χ 10 units, which are conveniently in smaller doses 2-4 times a day ve r ab r e i t we r de η.

The modified interferons can zv / eekmäss :! wise can be mixed together with inert liquid diluents and parenterally in the form of sterile injection solutions or suspensions are administered.

The following examples describe the invention. The examples are based on isoelectric focusing hingev / iesen. This is in a LKB 7900 Uniphor column electrophoresis system with a volume of 220 ml with a pll-fourth of 3-10 carried out, the isoelectric Focusing is carried out according to the LKB instruction manual. All operations are done at 2 ° C. After 36 hours, 5 ml of each fraction are collected and the pH is determined immediately.

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Example 1: Manufacture of interferon

Interferon is obtained from primary rabbit kidney cell cultures using the method of Tan et al (from Proc. Nat. Acad. Sci. 6-7 464-471) with the addition of the following Modifications made: individual layers are mixed with 200 // g / ml of poly (1) poly (C) for 1 hour incubated at 37 ° C. After removing the inductor, the cells are washed twice with a buffered saline solution (Hanks) and 10 1/2 g / ml cycloheximide in Eagle's solution containing 2% fetal calf serum was added. The cultures ground incubated for 3 1/2 hours at 37 ° C and then treated with 3 Wg / ml with actinomycin D and the incubation continued for another half hour. The antimetabolites are removed from the cells 5 times with a buffered saline solution (Hanks) and covered with the fresh medium without serum. After 8-10 hours the supernatant is lifted off, centrifuged and stored at -70 ° C.

20 liters of the excess ground 200 times concentrated by ultrafiltration through Diaflo PM-10 membranes, against dialyzed dilute acetic acid (pH 3) and freed from precipitated proteins by centrifugation.

The plaque reduction test on primary rabbit kidney cells is used to detect interferon. Single layers are treated in 6 cm Petri dishes for 18 hours with 2 ml of interferon solutions and then with

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50-80 plaque-forming units of vesicular stomatitis Virus brought into contact. The titers are defined as the interferon dilution that results in a 50% plaque reduction causes. Each series of experiments is based on a laboratory standard and all results are in international units corrected and expressed as laboratory units per 2 ml.

M £ i £ EiiüLJL ^: - Purification procedure for interferon

The phytoagglutinin required for the procedure from Pha ~ seolus vulgaris is available as Bacto-Phyterciaagglutinin and. v / urde, v / ie described by T. Weber et al., Scand. J, Hemat. 4 / 77-80 / cleaned. Of the. erythroagglutination portion vmrde gel-filtered on Sephadex G-1.50 and then on the N-hydroxysuccinimide ester of succinylated aminoalkyl cigar rose coupled- (P. Cuatrecasas et al., Biochemistry; 11 / 2291-2299). . '- -,. '-' ·. '"·' -

This agglutinin shows a specific reaction with the ■ Oligosaccharide sequence galactose -j N-acetylglucosamine -r-5-. ■ Mannose, found in many glycoproteins as a structural feature is to be found. As can be seen from Figure 1, adsor- ".

3.

[H] -labeled asialointerferon is very strong on this agarose coupled lectin. -. '. ·.

It vmrde a total of lo, ooo units of interferon '

with 12o.oqo dpm [. H] activity is used. About 50% of the radioactivity does not adsorb, a further 20% could eluted with 0.1 M galactose. However, this material showed no biological activity. Complete desorption could with a Glykopropteinfragiuent from human

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- Erythrocytes can be reached. This Glykoproteinfragiaeni: is from the membrane of human erythrocytes after treatment with trypsin according to the method described by S. Kornfeld et al. _f Proc.Nat.Acad.Sci. (USA) J53 / 1439-1446.

After adding this glycoprotein fragment to the elution medium is generally given a sharp maximum (see FIG. 1). All remaining radioactivity is eluted, i.e. that the interferon is desorbed in a sharply delimited area.

BATH

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Example 3: Incorporation of N-acetylneuraminic acid _; iη interferon

Reagents: CMP-N-acetylneuraminic acid was converted from CTP-N-acetylneui-ariiic acid with the help of the enzyme CMP-sialylic acid synthctase from salivary glands according to the instructions of ELKean and S.Rosenuui, Methods in Enzymology j> 208 ( 1966) synthesized. The enzyme Sia IyI transferase was obtained from rat liver according to J, Hickman et al., J. B i o) "Chern . .245 759 (1970) or bovine colostrum according to BABartho! Omew and GW Jourdian, Methods in Enzymology 8 368 (1966) obtained,

4 ra! a solution of rabbit interferon which contains 2 la units of interferon in 0.05 M Tris HCl buffer pH 7.5, -IO ηΰ · ϊ EDTA, 5 mM magnesium acetate, are mixed with 2 roM CMP-ϊ-ϊ-acetyl Neuraminic acid and 500 units of sialyl transferase were incubated. Wax 2 hours the solution to O _r l N acetic acid and then against water is destil3.iertes alysiert CII and the precipitate removed by -2entrifugation. The supernatant contains interferon, in which! Additional N-acetyl-nc-uraminic acid is incorporated »

The enzymatic transfer is through an isoelectric Focus verified. While before the appointment "Interferon in several molecular species from pi 6.3; 5.8? 5.3 is present, the newly created product has the largest Part of a pi of 4.8- Furthermore, the installation is due to the

Transmission of radioactive N-acetyl-neuraraic acid is bestall
does. If CMP-L '"Cj-N-acetyl-neuraic acid is used in the enzymatic reaction, the pI-4,8 fraction shows a high incorporation of [" C] activity, that of labeled

hey-stirs »

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A solution of 40 nil rabbit interferon with 5.10 units of interferon is _{treated in 0.05 M Na acetate buffer pH 5.5 r} 0.15 M NaCl, 20 κΜ CaCl ^ with one international unit of Kourajp-inidase from Vibrio cholerae « After 4 hours, the material is dialyzed against 0.1 N acetic acid and then against Viasser. Asialointerferoia is in the supernatant. If such a preparation is subjected to isoelectric focusing, it becomes apparent, as already described earlier (E. Schonne et al., Synip. Series immunobiol. Standard. 14_ / 61-6S), - that the charge heterogeneity has disappeared and a Uniform product was formed with a pi of 6.3. This asialointerferon is oxidized with the enzyme galactose oxidase. are incubated with 500 units of galactose acid for 20 hours After dialysis against 0.1 U acetic acid, centrifugation is carried out.

The conversion is demonstrated by reducing the newly formed C-6 aldehyc group of the terminal galactose by tritiated NaBK ₄ . If the reduced material is electrofocused, the pI-6,3-1'iiterferon fraction shows an upper left incorporation of Tritiuia. This material is hydrolyzed in 2N HCl for 2 hours at 100 ° C. _{, neutralized with Ag 9} CO ,, and freed from ions by ion exchangers. In the hydrolyzate can by Fa-pierchroiaatographie. tritiurainelabeled galactose can be detected free of charge (see table).

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BATH ORIGINAL

J * -

/ η.

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T-9

Reduction rait NaB [ ³ H ₄ ] -

Tabel

3 3

Incubation with total [H] - [H] incorporation

Galactose oxidase

Installation of clpm // ig in galactose Protein. dpm / ng protein

Interferon Interferon Asialointerfex'on Asialointerferon

39.115 24; 765 26,593

113.135

.2,182 6,224 4,080

77-724

iper galactose

10 units of rabbit asialointerferon in 0.01 M Na phosphate buffer, pH G, 5 are 1000 units β-galactosidase from Diplococcus pneumonias after R.C, Kughes and R.W. Jeanloz, Biochem. _3 1535 (19Gi-). Incubated for 3 hours. Then against 0.1 N acetic acid dialysed and the precipitate by centrifugation severed. The supernatant contains galactointarreron. The evidence of the splitting off of galactose can be traced back to Bring tritiated asialointerferon. This material is treated with β-galactosidase and dialyzed extensively and. then subjected to acid hydrolysis, no tritiated galactose can be detected in the hydrolyzate.

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Example 6: Representation of biosyn thetical mod if ί ζ i er tem i nterfero n

Interferon was induced in primary rabbit kidney cultures with 200 μg / ml poly I: C. For a further 60 min incubation, the inductor was aspirated, the cells were washed 3 MSI and rn it fresh medium added containing 40 niM 2 - deoxyglucose or 40 mM 2 ~ deoxy-2-aminog Iucose contained "After 10 hours incubation at 37 ⁰ C the interferon in the supernatant was harvested.

If this interferon is subjected to isoelectric focusing, material with biological activity can only be found in the pH range 6.3 - 6.8, while native interferon binds in several pH ranges. If this material with a uniform charge does not contain any terminal galactose, it was demonstrated as follows: 10 units of biosynthetically modified interferon were as in Example 1 in 0.05 M Tris HCl buffer pH 7.5, 10 mM EDTA, 5 mM magnesium acetate and 2 mM CMP-N-Acety 1 neuramic acid and 500 units Si a Iy [transferase incubated. In contrast to Asia! O ~ interferon, the bi-synthetically modified interferon cannot absorb N-acetylneuraminic acid. The molecular species with pi 6.3 6.8 is therefore retained even after treatment with Si alyI transferase and no additional fractions with a lower pi occur. Since sialyic acid is transferred to terminal galactose residues according to Example 1, this experiment proves that biosynthetically modified interferon does not contain any terminal galactose as an acceptor for Si a IyI acid.

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ORIGlNAL BATHROOM

Claims (8)

900-9087 patent pending 1. Process for the production of structurally ir.oüified Interferons characterized in that one a) Sialylic acid residues in interferons or asialointer- 'ferone with the help of specific Sialy! transferases introduces enzymatically, b) terminal galactose residues in asialointerferon with Galactose oxidase is enzymatically oxidized, c) terrain galactose residues of the asialointerferons with With the help of specific galactosidase it is enzymatically split off ., 2. Process for the production of structurally codified persons Interferons thereby cffckennzcd.chnet that ΐΛ-an in the biosynthesis of; Interferons the carbohydrate synthesis by binding a Glyko ^ y! Transferase in Inhibits reaction direction. 3. Process for the purification of interferons _r Asian interferons and structurally modified interferons !! characterized in that the Ii '· tor is fei. one is adsorbed on the immobilized agglutin and then the agglutin-bound interferons are desorbiorted. 4 0 9 8 4 3/1 1 1 p BATH ORIGINAL 900-9087 Λ * Germany. 4. A structurally modified interferon _r obtained by the method of claims 1 or 2. 5. An interferon containing sialylic acid residues or Asialointerferon. 6. Asialointerferons / their terminal galactose residues are away. 7. An interferon / asialointerferon or structurally modified interferon, purified according to the procedure of claim 3. 8. Therapeutic mixtures containing interferon, asialointerferon or modified interferon obtained or cleaned according to one of claims 1, 2 or 3, 3700 / ST / SE SANDOZ AG. 409843/1113