WO2004003184A1

WO2004003184A1 - A method for obtaining purified bacteriophage preparation s

Info

Publication number: WO2004003184A1
Application number: PCT/PL2003/000063
Authority: WO
Inventors: Janusz Boratynski; Andrzej Gorski; Danuta Syper; Beata Weber-Dabrowska
Original assignee: Instytut Immunologii I Terapii Doswiadczalnej Pan
Priority date: 2002-06-30
Filing date: 2003-06-30
Publication date: 2004-01-08
Also published as: AU2003256185A1; PL354822A1

Abstract

The invention concerns an use of polysaccharide or its partially esterified derivative in the purification of bacteriophage preparations as well as a method of obtaining purified bacteriophage preparations. The purified, endotoxin-free bacteriophage preparations are suitable for therapeutic application.

Description

A METHOD FOR OBTAINING PURIFIED BACTERIOPHAGE PREPARATIONS

The field of the invention is the biochemical methods of the purification of viral preparations. The invention involves the application of polysaccharide or its partially esterified derivative to purify bacteriophage preparations as well as a method for obtaining purified bacteriophage preparations. The purified, endotoxin-free preparations are suitable for, among other things, applications in therapeutics.

Bacteriophages (or phages) belong to a group of viruses which are widespread in nature, and their life cycle is connected with a bacteria cell. Four thousand phages active with regard to practically all species of bacteria have been described thus far. In a bacteriophage's lytic cycle, after it has been absorbed by and has injected its DNA into a bacterial cell susceptible to it, the bacteriophage multiplies, creating numerous copies of itself. After a period of maturation within the bacterial cell, the phage lyses the cell, setting free phage particles, together with fragments of the cell wall, into the surroundings.

Highly purified phage preparations are necessary both for research needs and for enabling the broad clinical application of bacteriophages. This is particularly desirable in the case of bacteriophage preparations intended for application in the treatment of humans and animals. Especially undesirable are impurities of bacterial endotoxin.

The purification of phage lysate can be performed by methods such as concentration- gradient separation using cesium chloride or sucrose (see Anderson T.F. et al. Virology, 1964, 23, 113-117; Eisenstark A.: Acad. Press, New York, 1967, 449; Yelton D. B. et al., J. Gen. Virol, 1979, 43, 29) or column chromatography using various solid-phase substrates (see Baylor M. B. et al., Virology , 1970, 40, 251-259, Roslansky P. F. et al., Virology, 1970, 40, 260-266, Sinsheimer R.L.: J. Mol. BioL, 1959, 1, 37-42). The methods of phage lysate purification hitherto employed, though, have not achieved a high degree of purification of these preparations, especially in regard to bacterial endotoxin.

The use of cellulose and its derivatives is well known, in particular the use of esterified derivatives of cellulose in the chromatographic purification of preparations of biological origin, including also those containing viruses. A known absorbent substrate is a sulfate derivative of cellulose which is characterized by its low level of esterification (8 micromole/ml of gel). Although the structure and properties of this material show many similarities with heparin, it does not contain any elements of animal origin. The hydrodynamic properties and chemical structure of it allow attaining equilibrium quickly during chromatography, at the same time assuring good flow conditions of the mobile phase. By fulfilling these conditions, the sulfate derivative of cellulose is suitable for industrial applications, including also work with biological material. A range of applications of such a substrate to remove endotoxin from biological material has been described. Some viruses or their components attach to a substrate containing the sulphate of cellulose (immune-deficient cat virus, BioTechnology 11 (1993), p. 173; surface antigen HBsAg, US patent No. 4,515,714; a subunit of the herpes virus gA, gB, US patent No. 4,725,547; flu virus, US patent No. 4,724, 201; Japanese brain inflammation or meningitis virus, US patent No. 4,725,546; rabies virus, US patent No. 4,725,547; papilloma virus, Virology 106 (1995), p. 126; and adenovirus-associated virus, Human Genome Therapy 7 (1996), p. 507).

There are, though, viruses which do not interact with a chromatographic substrate containing the sulphate of cellulose. This group includes the human adenoviruses, human Coxsackie virus, polio virus, and mouse leukemia virus. The level of affinity to the substrate is difficult to predict and does not seem to depend on the presence of DNA or RNA and the envelope, nor on the size of the viral particle. The goal of the invention is to obtain bacteriophage preparations of high purity which may by used for research and therapeutic purposes. Such preparations must in principle be free of bacterial endoxins.

Therefore, the goal of the invention is also to provide a method of purifying bacteriophage preparations as well as the agents employed to this end.

The object of the invention is a method for obtaining purified bacteriophage preparations characterized in that the mixture containing the bacteriophages is adsorbed onto a substrate containing cellulose or its partially esterified derivative, which is then washed with a solution to remove impurities, in particular endotoxin, after which the purified bacteriophages are rinsed out.

It was determined unexpectedly that the substrate does not react with the endotoxins contained in bacteriophage preparations such as raw or prefractionized phage lysates, which also shows its significant affinity to bacteriophages.

An effective elution of endotoxin is obtained with water, a solution of a non-dissociating substance, or a saline solution of a concentration not greater that 0.1 M, preferably buffered, while the elution of the bacteriophage fraction is obtained with a solution of a non-dissociating substance, or any buffer, or a saline solution of a concentration not greater that 0.05 M, preferably buffered. Both the elution of endotoxin and that of the bacteriophages are best carried out at a temperature of from -25°C to 100°C.

In accordance to the invention, advantageous is when the elution of endotoxin and bacteriophages is accomplished using an aqueous solution of salt containing an organic solvent which, to realize the invention, is best selected from the group containing dimethylsulphoxide, dimethylformamide, isopropanol, and acetone. As the substrate, preferred is cellulose partially esterified by an organic or inorganic acid, best by acetic acid, nitric acid, sulphuric acid, or phosphoric acid. Particularly the use of cellulose as the substrate in which from 0.01% to 5% of the glucose molecules, advantageously from 0.25 to 1% of the glucose molecules, optimally from 0.5% to 1% of the glucose molecules, are esterified.

Another object according to the invention is the application of polysaccharide or its partially esterified derivative to obtain the substrate to remove the endotoxin from the bateriophage preparation. Preferred is the use of polysaccharide of non-spherical and irregular structure of the dimensions or 30 to 200 microns, characterized by sufficient mechanical durability to allow it to be used in a chromatographic apparatus without pressure or at pressures exceeding 50 psi. The sorptive capacity of the substrate for the avidin must be from 2 to 5 mg/ml. In this application the preferred polysaccharide is cellulose, preferably partially esterified by an organic or inorganic acid, particularly by acetic acid, nitric acid, sulphuric acid, or phosphoric acid. Preferred is the use of cellulose in which from 0.01% to 5% of the glucose molecules, advantageously from 0.25 to 1% of the glucose molecules, optimally from 0.5% to 1% of the glucose molecules, are esterified.

Another object according to the invention is the application of the same polysaccharide or its partially esterified derivative to remove the endotoxin from the bateriophage preparation. In this application the preferred polysaccharide is cellulose, preferably partially esterified by an organic or inorganic acid, particularly by acetic acid, nitric acid, sulphuric acid, or phosphoric acid. Preferred is the use of cellulose in which from 0.01% to 5% of the glucose molecules, advantageously from 0.25 to 1% of the glucose molecules, optimally from 0.5% to 1% of the glucose molecules, are esterified. In particular, the preferred realization of this invention are bacteriophage preparations intended for bacterial infections. In this case, raw or prefractionized phage lysates containing strains of determined therapeutic characteristics should be used. Examples of such strains appear in the Polish patent application P 348740.

This invention permits the user to obtain highly purified bacteriophage preparations which may be for research as well as therapeutic purposes. The main advantage of the bacteriophage preparations obtained in accordance with this invention is the lack of natural, difficult to remove impurities, in particular bacterial endotoxins.

Example 1. Obtaining the substrate to purify bacteriophage preparations.

Five grams of sawdust, for example beech wood, was suspended in 200 ml water and boiled for 1 hour. Then the particles were strained and suspended in 200 ml of 0.5 M NaOH. After 5 hours the suspension was strained, washed with 200 ml of 1% citric acid, 200 ml ethyl alcohol, and water until a neutral pH value of the rinse was obtained. The leached sawdust was then freeze-dried and suspended in dichlormethane and chlorosulphuric acid was added.

The reaction achieved a medium level of esterification, corresponding to the esterification of from 0.01% to 5% of the glucose molecules included in the matrix, more advantageously from 0.25% to 1%) of the glucose molecules in the matrix, optimally from 0.5% to 1% of the glucose molecules in the matrix. This means in particular that a substrate is obtained in which on average every 100th to 200th or every 400th glucose molecule in the matrix becomes esterified. Using commonly known methods of the chemical esterification of cellulose, one can also obtain other inorganic or organic esters of cellulose, such as the phosphate, nitrate and acetate of cellulose.

The modified sawdust was rinsed with dichlormethane and dried in the air.

This procedure resulted in a substance of non-spherical and irregular structure of the dimensions or 30 to 200 microns and of sufficient mechanical durability to allowed it to be used in a chromatographic apparatus without pressure or at pressures exceeding 50 psi. The absorbent power of the substrate for the avidin was from 2 to 5 mg/ml. Other known polysaccharides can also be used to obtain a substrate of such physical and chemical properties.

It has been stated that one can also use some commercially available chromatographic substrates containing esterified cellulose possessing the required physical and chemical properties described above for the purification of bacteriophage preparations, in particular of endotoxin impurities. An example of such is a sulfate derivative of cellulose which is characterized by a low level of esterification (8 micromole/ml of gel), available under the trade name Matrex® CellufineTM Sulfate Millipore.

Example 2. The purification of bacteriophages; removing endotoxins from a mixture contaimng bacteriophages.

In the method of the invention, the affinity of bacteriophages to a substrate containing cellulose, or, preferably, its esterified derivative, was employed. In this example of the realization, a commercially available sulphate derivative of cellulose characterized by a low level of esterification (8 micromole/ml of gel) was used. The substrate was used to remove endotoxins from a mixture containing bacteriophages. After rinsing the substrate and removing the endotoxins, the next step of purification was the elution of the adsorbed bacteriophages.

One milliliter of substrate containing the sulphate derivative of cellulose was filled into a sterile chromatograph column. Glass wool soaked in 70% ethanol sealing the bottom of the column prevented the efflux of the substrate from the column.

The following elution buffers were prepared:

Buffer 1 : 0.01 M phosphate buffer, pH 7.6;

Buffer 2: 0.01 M phosphate buffer, pH 7.6, containing 1 M sodium chloride.

The salts which were parts of the eluent are baked for 1 hour at 145°C.

The solutions were prepared using apyrogenic distilled water

Before chromatography, 1 ml of the substrate was filled into the chromatographic column and rinsed with 5 ml of the 0.01 M phosphate buffer of pH 7.6 followed by rinsing with 5 ml of the 0.01 M phosphate buffer of pH 7.6 containing 1 M sodium chloride.

The column was prepared for the actual chromatography by washing it with 10 ml of the 0.01 M phosphate buffer of pH 7.6. The removal of endotoxins was carried out using the high-molecular fraction obtained in the procedure of molecular sieving onto Sepharoze 4B of condensed lysate of the bacteriophage

Ps F-8 obtained by the method described in the application to the Polish patent P 348740.

0.2 ml of the mixture of bacteriophages and endotoxin were put into the column containing the 1 ml substrate contaimng the sulphate derivative of cellulose. A fraction of a volume of 0.2 ml was collected. Fraction 1 was eluted with 3 ml of Buffer 1. Under these conditions, non-associated endotoxins flowed from the column. Fraction 2 contained purified bacteriophages.

The balance of endotoxic units in the fraction:

The material submitted to chromatography on the substrate containing the sulfate derivative of cellulose contained 2500 units of endotoxin/ml. The fraction eluted by buffer 1 contained 600 - 1000 units of endotoxin/ml, and that eluted by buffer 2 contained bacteriophages devoid of endotoxin (less than 10 unit/ml).

The content of endotoxin in the bacteriophage preparations were determined using the gel method of the company Charles River Endosafe, Charleston, U.S.A.

Claims

1. The method of obtaining purified bacteriophage preparations characterized in that the mixture containing bacteriophages is contacted with a substrate containing esterified cellulose or its partially esterified derivative, is then washed with a solution to remove impurities, in particular endotoxin, after which the purified bacteriophages are rinsed out.

2. The method according to claim 1, wherein the elution of endotoxin is accomplished using water, a solution of a non-dissociating substance, or a saline solution of a concentration not greater than 0.1 M, preferably buffered.

3. The method according to claim 1, wherein the elution of the bacteriophage fraction is accomplished using a solution of a non-dissociating substance or any buffer or a saline solution of a concentration greater than 0.05 M, preferably buffered.

4. The method according to claim 1, wherein the elution of endotoxin and bacteriophages is accomplished at a temperature from -25°C to 100°C.

5. The method according to claim 1, wherein the elution of endotoxin and bacteriophages is accomplished using an aqueous solution of salt containing an organic solvent.

6. The method according to claim 5, characterized in that the organic solvent is selected from the group containing dimethylsulphoxide, dimethylformamide, isopropanol, and acetone.

7. The method according to claim 1, wherein the cellulose partially esterified by an organic or inorganic acid is used as the substrate.

8. The method according to claim 1 wherein the cellulose used as the substrate is partially esterified by acetic acid, nitric acid, sulphuric acid, or phosphoric acid.

9. The method according to claim 1 wherein the cellulose used as the substrate in which from 0.01% to 5% of the glucose molecules, advantageously from 0.25 to 1% of the glucose molecules, optimally from 0.5% to 1% of the glucose molecules, are esterified.

10. Use of polysaccharide or its partially esterified derivative to obtain the substrate to remove the endotoxin from the bateriophage preparation.

11. The use according to claim 10, wherein the polysaccharide is cellulose, preferably partially esterified by an organic or inorganic acid.

12. The use according to claim 10, wherein the polysaccharide is cellulose partially esterified by acetic acid, nitric acid, sulfuric acid, or phosphoric acid.

13. The use according to claim 10, wherein the polysaccharide is cellulose in which from 0.01% to 5% of the glucose molecules, advantageously from 0.25 to 1% of the glucose molecules, optimally from 0.5% to 1% of the glucose molecules, are esterified.

14. Use of polysaccharide or its esterified derivative for removing endotoxin from the bacteriophage preparation.

15. The use according to claim 14, wherein the polysaccharide is cellulose, preferably partially esterified by an organic or inorganic acid.

16. The use according to claim 14, wherein the polysaccharide is cellulose partially esterified by acetic acid, nitric acid, sulphuric acid, or phosphoric acid.

17. The use according to claim 14, wherein the polysaccharide is cellulose in which from 0.01% to 5% of the glucose molecules, advantageously from 0.25 to 1% of the glucose molecules, optimally from 0.5% to 1% of the glucose molecules, are esterified.

18. The use according to claim 14, wherein the bacteriophage preparation is intended for the treatment of bacterial infections.