RU2230325C2 - Method for preparing purified preparation of botulinic toxin type a - Google Patents

Abstract

FIELD: microbiology, toxicology. SUBSTANCE: invention relates to methods for purification of preparations of botulinic toxin. The strain-producer Clostridium botulinum is cultured under anaerobic conditions. Toxic-containing culture is centrifuged and toxin is extracted from precipitate prepared. Isolation of neurotoxin is carried out under conditions optimal for dissociation of components of toxic complex at pH 7.9 and in the concentration of sodium phosphate salt 0.025 M. Method provides reducing time for preparing neurotoxin, amount of stages in isolation of the preparation and to elevate the yield of the end product. EFFECT: improved preparing method. 2 tbl, 4 ex

Description

The invention relates to methods for purifying botulinum toxin preparations by physicochemical methods and can be used in the production of antitoxic serum preparations, in particular for constructing an enzyme-linked immunosorbent assay system for the detection and identification of type A botulinum toxin.

It is known that botulinum toxins are a complex protein-nucleotide complex consisting of neurotoxin, hemagglutinin, non-toxic protein, and, in addition, preparations of botulinum toxin obtained by, for example, acid or salt deposition contain nutrient components and destroyed producer cells as ballast impurities . This makes it necessary to additionally purify botulinum toxin preparations for practical use, in particular, to obtain antitoxic sera that are specific for a particular type of botulinum toxin.

Known methods for producing a purified preparation of botulinum toxin in crystalline form, based on a multi-stage acid-salt and alcohol re-precipitation of the toxin from the culture fluid. For example, Lamanna S., McElroy O.E., Eklund H.W. The purification and crystallization of Clostridium botulinum type A toxin // Science. -1946. - Vol. 103. P. 613-621. According to this method, an acid-salt precipitate containing botulinum toxin is obtained, it is treated with a solution of acetic acid sodium, fractionated with various concentrations of methyl alcohol and ammonium sulfate of varying degrees of saturation.

In common with the claimed method is the stage of preparation of the strain of the producer of acid-salt sediment containing botulinum toxin from the culture fluid.

The disadvantages of this method include an insufficient degree of purification of the toxin. The preparation obtained by this method is inhomogeneous and is a complex of neurotoxin and hemagglutinin in a ratio of 1: 4 by weight (DasGupta BR, Boroff PA Separation of toxin and hemagglutinin from cristalline toxin of Clostridium botulinum type A by anion exchange chromatography and determination of their dimension by gelfiltration / / J. Biol. Chem. - 1968. - Vol. 243.- P.1065-1070).

Since the inclusions of hemagglutinin and nontoxic protein included in the toxic complex do not have type specificity, the antitoxic sera obtained on the basis of such a toxin preparation give cross-reactions with other types of toxins, i.e. the antibody preparation does not have absolute specificity with respect to type A toxin. the production of homogeneous botulinum neurotoxin type A, which has a clear type specificity, is a relatively high bond strength of individual protein co components of the botulinum toxin complex.

There is also a method for producing a homogeneous neurotoxin based on the use of multistage chromatographic purification of primary protein preparations (DasGupta BR, Sathyamoorthy V. Purification and amino acid composition of type A botulinum neurotoxin. - Toxicon, 1984, V.22, No. 3, P. 415 -424). This technique includes the preparation of an acid-salt precipitate of toxin using ammonium sulfate, treatment of the dissolved precipitate, sequential precipitation of ammonium sulfate, treatment of the precipitate with traziol and phenylmethylsulfonyl fluoride, multiple dialysis of the preparation, two chromatography steps on a column with DEAE-Sephadex A-50 chromatography, column with Sephadex G-100, chromatography on a column of SP-Sephadex C-50.

In common with the claimed method is the stage of preparation of the strain of the producer of acid-salt sediment containing botulinum toxin from the culture fluid, as well as the stages of fractionation of the drug by gel filtration and ion exchange chromatography.

The disadvantage of this method is its complexity, the multi-stage preparation of the drug (the method includes nine stages), the low yield of the final product (about 60 mg of neurotoxin was obtained from 64 liters of culture fluid), due to both physical losses at individual stages and partial inactivation of neurotoxin associated with its denaturation during isolation and purification.

There is also known a method for producing a purified preparation of botulinum neurotoxin based on the sequential use of stages of ion exchange chromatography on DEAE-sephacel and high performance liquid chromatography (HPLC) (Matsuda M., Orutsumi S. Rapid method for purification of Clostridium botulinum type C neurotoxin by high performance liquid chromatography // Eur. J. Epidemiol. - 1986. - Vol. 2. - P. 265-271).

In common with the claimed method is the stage of preparation of a strain of a producer of acid-salt sediment containing botulinum toxin from the culture fluid, as well as the implementation of ion exchange chromatography as one of the steps.

The disadvantage of this method is the high cost of materials and equipment used for HPLC, in addition, this method of isolation of neurotoxin is suitable for the preparation of the drug only for analytical purposes.

Closest to the claimed solution is a method for the isolation of type A neurotoxin from the culture fluid, including a series of successive gel filtration steps and ion exchange chromatography of the toxin preparation (Preparation of Cl. Botulinum type A neurotoxin and hemagglutinin and neurotoxin characterization / I.D. Vinogradova, R.N. Uvarova, N.N. Ivanova and others. // Biochemistry. - 1982. - T. 48. - Issue 5. - S. 788-795).

The method includes the following steps:

- cultivation of a producer strain of botulinum toxin;

- obtaining from the culture fluid acid-salt sediment containing botulinum toxin;

- gel filtration of the dissolved precipitate on a column with Sephadex G-l00;

- chromatography of the fraction of the first peak obtained after gel filtration on DEAE-32 cellulose;

- precipitation of the toxin from the eluate by adding a saturated solution of ammonium sulfate to a final concentration of 60%;

- ascending gel filtration on a column with Sephadex G-200;

- transfer of the obtained fractions to another buffer system by gel filtration through Sephadex G-25;

- chromatography of the toxin on DEAE-Sephadex A-50.

Moreover, Cl. botulinum type A strain 501 is grown under anaerobic conditions in an environment containing an acidic hydrolyzate of whale flour with corn extract. After cultivation, the microbial cells are separated by sterile filtration. The microbial-free culture fluid is cooled to 7-10 ° C, acidified with hydrochloric acid to pH 5.6, dry ammonium sulfate is added to a final degree of saturation of 40% and incubated for 36 hours at 7 ° C to form a protein precipitate, which is then collected by centrifugation.

To extract the toxin, the acid-salt precipitate is dissolved in a pH of 6.8 in sodium phosphate buffer. Insoluble particles are removed by centrifugation. The extract is applied to a Sephadex G-100 column, equilibrated with citrate phosphate buffer, pH 5.6, and the protein is eluted with the same buffer. Chromatography of the fractions of the first peak obtained after gel filtration is carried out on DEAE-32 cellulose in citrate buffer buffer, pH 5.6. The toxin eluting from the column with one protein peak is precipitated by the addition of a saturated solution of ammonium sulfate to a final degree of saturation of 60%. The mixture is centrifuged, the precipitate is dissolved in sodium phosphate buffer pH 5.6. The toxin solution is added to a column with Sephadex G-200, equilibrated with Tris-HCl buffer, pH 7.9, and elution is carried out with the same buffer using an ascending gel filtration method. The obtained fractions of the second peak, including the toxic protein, are combined and gel filtered through Sephadex G-25 in sodium phosphate buffer pH 7.9. At the last stage of purification, the toxin preparation is chromatographed on DEAE-Sephadex A-50, equilibrated in sodium phosphate buffer pH 7.9 by feeding a NaCl solution with a linear gradient at a concentration of 0-0.5 M.

In common with the claimed method is the stage of preparation from the culture fluid of the producer strain of acid-salt sediment containing botulinum toxin, as well as the use of combinations and methods of gel filtration and ion exchange chromatography.

The disadvantages of this method include the duration of the process of obtaining the drug, due to the multi-stage process and the imperfection of the matrices used for gel filtration and ion exchange chromatography. So, in the method, G-100 and G-200 Sephadexes are used at the gel-filtration stages, and DEAE-32 and DEAE Sephadex A-50 cellulose are used at the stages of ion exchange chromatography. However, it is known that Sephadex granules are relatively elastic and easily compressible under the influence of hydrodynamic influences, which imposes significant restrictions on the permissible values of the chromatographic elution rates of fractions. DEAE-32 cellulose powder is also quite easily compressed at high flow rates of the eluting solutions and, in addition, with slight random deviations of the pH and ionic strength of the eluent, irreversible granule deformations can occur, which leads to a sharp deterioration in the flow conditions of the eluent (Osterman A.A. Chromatography proteins and nucleic acids. - M .: Nauka, 1985). It is also known that DEAE-32 cellulose and Sephadex are produced in the form of dry powders, and therefore they must be subjected to lengthy preparation for work: Sephadex is soaked for swelling for two days, followed by deaeration, the gel layer is converted in the column and washed. Cellulose powder is also soaked for one hour, followed by elutriation of fine particles and the transfer of the ion exchanger into the desired ion exchange form.

The imperfection of the carriers and the multi-stage technique significantly complicate the process of chromatographic purification of botulinum toxin and lengthen its time; this is the reason for the loss of serological activity of the drug, both due to the physical loss of part of a specific protein, and due to its partial denaturation during the multi-stage chromatographic process.

The objective of the invention is the preparation of a purified preparation of botulinum neurotoxin type A, suitable for producing highly specific antitoxic sera by destroying the toxin complex and completely removing hemagglutinin and other cross-reacting compounds with antibodies of antitoxic serum from the botulinum toxin preparation in a short time, with fewer purification steps and in conditions minimizing the effects adversely affecting the antigenic properties of the mole cool purified neurotoxin.

This task is a prerequisite for the preparation of serologically pure toxoids intended for the immunization of animals and the production of strictly type-specific antibodies to botulinum toxin type A.

The problem is solved due to the fact that the preliminary purification of the toxin concentrate is carried out by reprecipitation with ammonium sulfate at a saturation degree of 20% (in order to remove large molecular weight ballast with sediment), followed by reprecipitation of proteins at 40% saturation with the same salt to remove low molecular weight compounds remaining in the supernatant from cultural environment. As a result, the resulting preparation in a smaller volume contains a larger amount of a specific component, which provides a more effective further chromatographic purification from ballast impurities.

The chromatographic process at pH 7.9 ensures the destruction of the toxin complex already at the first stage, which allows you to immediately get rid of the main amount of hemagglutinin and nontoxic protein impurities during gel filtration on Sephacryl S-300, the fractionation region of which lies in the range from 10 to 1500 thousand Dalton.

The use of adsorption chromatography on hydroxyapatite eliminates the additional stage of ion exchange chromatography and upward gel filtration. At this stage, the neurotoxin is completely purified from residual hemagglutinin inclusions due to the difference in their sorption ability.

In addition, the procedure for performing the steps of isolating a homogeneous neurotoxin is selected in such a way that there is no need for intermediate operations to concentrate the toxin and transfer the collected protein to another buffer system using dialysis or gel filtration methods.

The essence of the proposed method lies in the fact that to increase the degree of antigenic purity of the botulinum neurotoxin preparation and reduce the time and cost of isolation, acid-salt precipitation stages of proteins precipitated from the culture fluid, one-step gel filtration on Sephacryl S-300 under conditions ensuring the destruction of the toxin are used complex, what is achieved by removing the main amount of impurities of cross-reacting antigens. Removal of hemagglutinin residues from the precipitate is achieved at the stage of adsorption chromatography on hydroxyapatite, and nucleic acid impurities by ion-exchange chromatography on DEAE-Sephacel.

The method is as follows.

The producer strain Cl. botulinum type A is grown under anaerobic conditions on a medium containing an acid hydrolyzate of fish meal with the addition of corn extract. After cultivation, microbial cells are separated by centrifugation. The centrate of the culture fluid is cooled, acidified with hydrochloric acid, dry ammonium sulfate is added to a final degree of saturation of 60% and incubated to form a protein precipitate, which is collected by centrifugation. To extract the toxin, one part of the obtained acid-salt precipitate is dissolved in ten parts by weight of 0.05 M phosphate buffer solution (FBI) pH 7.9. A saturated solution of ammonium sulfate is poured to the obtained extract to a final degree of saturation of 20% and clarified by centrifugation. A saturated solution of ammonium sulfate is poured to the supernatant to a final degree of saturation of 40%. The resulting mixture was centrifuged, the supernatant was decanted, the precipitate was resuspended in PBS, pH 7.9. Proteins are dissolved with constant stirring on a magnetic stirrer and clarified by centrifugation. The clarified toxin preparation is applied to Sefacryl S-300 gel balanced with 0.025 M PBS with 0.3 M KCl pH 7.9 filling the chromatographic column. Gel filtration is carried out under feed conditions of 0.025 M PBS with 0.3 M KC1 as an eluate by means of a peristaltic pump. The eluate is collected in fractions. The obtained fractions of the second and third peaks are analyzed in the Ouchterlooney diffuse precipitation (RDP) reaction with mono- and polyspecific hyperimmune sera to type A toxin. Fractions with activity of at least 4 EA / ml are combined. The combined fractions are applied to a balanced 0.025 M PBS, pH 7.9 hydroxyapatite (HAP), fill the chromatographic column. The gel is washed with 0.05 M PBS until extinction ≤0.01 units. The toxin is eluted with a HAP of 0.2 M PBS, pH 7.9. The resulting toxin solution was diluted four times with distilled water, applied to a balanced 0.025 M PBS, pH 7.9, DEAE-Sephacel cellulose filling the chromatographic column. The protein is eluted from an ion exchanger 0.11 M PBS, pH 7.9. From the start of elution, the liquid exiting the column is collected in fractions. The obtained fractions are analyzed in RDP with mono-and multispecific hyperimmune sera to type A toxin. Fractions with activity not lower than 4 EA / ml are combined.

To compare the effectiveness of the proposed method and the prototype, each of the methods obtained preparations of purified neurotoxin and the use of the same initial concentrate of botulinum toxin type A.

During the process, the time of purification of the drug was taken into account, the yield of the finished product was estimated by the total protein content, its serological activity, electrophoretic and serological homogeneity, and toxicity. The total protein content in the finished preparations was determined by the Lowry method, their serological activity and homogeneity were studied in the Ouchterloni diffuse precipitation reaction by titration of preparations in steps of two using mono- and polyspecific serums to botulinum toxins of types A, B, E. Electrophoretic homogeneity was determined by staging electrophoresis of the studied drugs in a gradient polyacrylamide gel (PAAG) (concentration gradient 10-15%) under denaturing conditions in the presence of sodium salt of dodecylsul Veil (DS-Na). The toxicity of the obtained preparations was determined by titration on white mice by intraperitoneal injection of an toxin solution into the animal and expressed in LD ₅₀ in 1 ml Comparative studies show that the claimed method provides, in comparison with the prototype, the process of preparing a purified preparation of botulinum toxin in a significantly shorter time and allows you to get the finished drug with greater toxicity and serological activity (Table 1).

A positive effect was obtained due to the fact that when using the inventive method, the isolation of a homogeneous neurotoxin occurs using only three stages of chromatographic purification. The proposed procedure allowed operations to completely abandon the additional step of transferring partially purified botulinum toxin to another buffer system. The conditions of the cleaning operations and the reduction in the duration of the procedure allowed to significantly reduce protein loss at the stage of the chromatographic process, as well as the toxic and serological activity of the final product (Table 1).

The possibility of implementing the proposed method is confirmed by the following examples.

Example 1. Isolation of a purified preparation of botulinum toxin type A from Cl. botulinum strain 98A. The drug was isolated from 14 liters of a 6-day-old Cl. botulinum production strain 98A, grown under anaerobic conditions in a nutrient medium based on 1% hydrochloric acid hydrolyzate of casein with the addition of 2% corn extract at a temperature of 34 ° C. The culture was clarified by centrifugation (8000 g, 20 min, 4 ° C). The toxin was precipitated with ammonium sulfate at a degree of saturation of 60%, separated from the supernatant by centrifugation under the same conditions and homogenized. The resulting paste was stored at a temperature of 20 ° C, taking samples as necessary to clean the toxin.

A portion of a concentrated paste weighing 5 g was extracted in 0.05 M PBS, pH 7.9.

At each stage of toxin purification, the toxicity of the preparation was determined by titration on white mice, and the preparations obtained were studied by the method of disk electrophoresis in gradient polyacrylamide gel (PLAT) (concentration gradient 10-15%) in the presence of sodium dodecyl sulfate (DS-Na) under deionizing conditions. Fractional protein deposition of toxin was carried out with ammonium sulfate at a final degree of saturation of 20 and 40%. When examining the preparation obtained by the method of disk electrophoresis, it was revealed that at this stage there was a removal of large molecular weight ballast and low molecular weight compounds of the culture medium from the extract. During subsequent gel filtration of the clarified preparation through Sephacryl S-300, the toxin was completely released from the low molecular weight components of the culture medium. Partially purified toxin was eluted in fractions of the second and third peaks. When adsorption chromatography on hydroxyapatite was achieved almost complete removal of hemagglutinin. The toxin was eluted with a HAP 0.2 M FBI, pH 7.9, with one protein peak.

At the last stage of purification as a result of ion-exchange chromatography on DEAE-Sephacel cellulose, a homogeneous neurotoxin preparation was eluted with 0.11 M PBS, pH 7.9. The specific toxicity of the drug at this stage was l, 8 · 10 ⁸ LD ₅₀ / mg, serological activity - 16 EA / ml.

In addition to proteins, the presence of nucleic acids was not registered in the preparation (the ratio of extinctions of the ultraviolet spectrum A ₂₆₀ / A ₂₈₀ = 0.1). The purified toxin in the RDP did not produce precipitation lines with hyperimmune sera against other types of botulinum toxins (B, C, E). From 14 liters of culture fluid, 34 mg of a homogeneous preparation of botulinum neurotoxin type A was obtained. When setting the Ouchterloni RDP, it was shown that the preparation is serologically homogeneous.

Example 2. The selection of the purified preparation of botulinum neurotoxin from a culture of Cl. botulinum strain 316. (The toxicity of the culture fluid obtained on Kitta-Tarozzi medium is 0.5 · 10 ⁵ LD ₅₀ / ml). The stages of the method for purification of botulinum toxin are similar to those described in example 1. During gel filtration of the toxin on Sephacryl S-300, the toxin was contained in fractions of only the second peak. During adsorption chromatography on HAP, the toxin was eluted with a flatter protein peak. At the last stage of purification, the drug had a specific toxicity of 1.1 · 10 ⁷ LD ₅₀ / mg, serological activity of 8 EA / ml. From 14 liters of culture fluid, 19 mg of homogeneous botulinum neurotoxin was obtained. When setting up the RDP, it was shown that the resulting preparation gives one clear precipitation line with homologous hyperimmune serums and does not give precipitation lines with hyperimmune serums against botulinum toxins of other types.

Example 3. The selection of the purified preparation of botulinum neurotoxin from cultures of Cl. botulinum strain 333 (the strain does not have hemagglutinating activity, the toxicity of the culture fluid obtained in Kitta-Tarozzi medium is 1.0 · 10 ⁶ LD ₅₀ / ml).

The stages of the method for purifying botulinum toxin are similar to those described in Example 1. During gel filtration of the toxin concentrate on Sephadex S-300, the toxin was determined in fractions of only the second peak, and the average serological activity of the fractions was 4 EA / ml. Since the electrophoregram in gradient PAGE (concentration gradient 10-15%) under denaturing conditions in the presence of DS-Na after the gel filtration step on Sephacryl S-300 did not reveal any traces of hemagglutinin, the chromatography step on HAP was not performed. The toxin solution obtained after gel filtration was applied onto a balanced 0.05 M PBS, pH 7.9 cellulose DEAE-Sephacel. Protein was suirable from the ion exchanger 0.11 M FBI pH 7.9. The toxin exited the column with one protein peak. Fractions with activity not lower than 8 EA / ml were combined. The resulting toxin preparation had a specific toxicity of l, 3 · 10 ⁸ LD ₅₀ / ml, serological activity of 16 EA / ml. When examining the resulting preparation in gradient PAGE under the same conditions, it was shown that the resulting preparation is homogeneous. Two protein bands of a molecular weight of approximately 50 and 100 kDt were detected on the electrophoregram, which corresponds to the molecular weight of the light and heavy chains of botulinum toxin.

When setting the RDP, the resulting preparation did not produce precipitation lines with hyperimmune sera against botulinum toxins of other types. From 14 liters of culture fluid, 27 mg of homogeneous botulinum neurotoxin was obtained.

Example 4. Obtaining on the basis of the preparation of a homogeneous neurotoxin isolated by the present method, hyperimmune sera.

Preparation of botulinum toxoid. Immunization of rabbits was carried out according to previously developed methods. 7-8 days after the end of the immunization period, a test blood sample was taken from the marginal ear vein. The obtained blood serum was analyzed in the Ouchterloni RDP. In the presence of specific antibodies in the serum in a titer of 1:32 and higher, blood was drawn from the heart to isolate immunoglobulins. The cylinder with blood was placed in a thermostat and kept at a temperature of 36-38 ° C for one hour. The resulting serum was poured into centrifuge glasses and centrifuged for 30 min at 3000 r / s and t 4 ° C. To the resulting serum was added a 2% sodium sulfate solution to a final concentration of 0.05%. In the RDP it is shown that the obtained serum does not produce precipitation lines with other types of botulinum toxins.

The preparations of immunoglobulins obtained from the prepared monoserum for type A toxin do not cross-react with heterologous toxins when used in enzyme-linked immunosorbent assay (TIFA) (Table 2).

Claims (1)

A method for preparing a purified preparation of botulinum toxin type A, including culturing the producer strain Clostridium botulinum in a liquid nutrient medium, separating the toxin-containing components from the culture fluid, acid-salt precipitation of proteins from the supernatant, extracting the toxin from the precipitate, fractionating the components of the preparation by gel filtration and anion exchange chromatography, characterized in that before the chromatography step, proteins from the toxin extract are precipitated twice in the presence of ammonium sulfate up to a saturation degree of 20% and 40%, respectively, gel filtration and anion exchange chromatography are performed under conditions optimal for the dissociation of the components of the toxic complex — at pH 7.9 and a concentration of sodium phosphoric acid salt of 0.025 M, adsorption chromatography is performed before the anion exchange stage chromatography, and hydroxyapatite is used as an adsorbent at the stage of adsorption chromatography.

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