A method of obtaining a purified vaccine for tick-borne encephalitis

 

(57) Abstract:

The invention relates to the field of medical Virology. Active fractions antigen of the virus of tick-borne encephalitis elute from the material with the effective pH value of the starting and eluting buffer, predominantly at pH>7.5% as an eluting buffer can be used starting buffer with the addition of more than 0.1 M alkali metal halides such as KCl and/or NaCl, and/or KBr. Zone elution of the antigen is determined by any known method, mainly enzyme-linked immunosorbent assay (ELISA), and elution is advisable to choose the active fraction of the antigen of the virus of tick-borne encephalitis with isoelectric points with values of 7.8 and 10.3. If necessary erwerbende fraction of antigen are combined and subjected to further purification by gel filtration on a sorbent capable of absorbing low molecular weight impurities. As a result of receiving the vaccine, keeping efficiency at multiple dilutions of the current beginning. 4 C.p. f-crystals, 2 tab.

The invention relates to the field of medical industry and for the production of viral vaccines, including inactivated purified vaccine tick ultracentrifugation vaccinated product in the density gradient of sucrose [1. USSR author's certificate N 660389, With 12 N 7/04, With 12 To 5/00. Publ. 28.02.1980. B. I. No. 8; 2. Patent SU # 1318149. Publ. 15.06.1987, B. I. No. 22].

The disadvantages of these methods are the length of the process, the low yield of the target product (antigen of the virus of tick-borne encephalitis) and partial purification of the vaccine from the ballast impurities.

The closest analogue of the claimed invention is a method of obtaining a purified vaccine for tick-borne encephalitis, which provides for the cleaning of ballast substances by ultrafiltration, followed by gel-filtration on macroporous glass [3. Lyapustin Century. N. The dissertation on competition of a scientific degree Prof. Biol. Sciences. Moscow, 1993. The prototype]. Prototype method of receiving the vaccine has low efficiency and productivity, in this part of the antigen is lost as a result of irreversible sorption on porous glass. The known method does not clear the antigen of the virus of tick-borne encephalitis from harmful impurities to the required standards of parameters. As impurities derived vaccine contains formaldehyde, phenol red, kanamycin, foreign proteins of mouse brain tissue, chicken embryo, causing frequent allergic reactions when used, the loose connection of the immune system.

The objective of the invention is to increase the degree of purification of the vaccine from harmful impurities while simultaneously concentrating the current start - antigen of the virus of tick-borne encephalitis (AVCA).

The problem is solved by treating the semi-finished product by liquid chromatography using weak anion-exchanger in the concentration gradient of halides of alkali metals. Active fractions antigen of the virus of tick-borne encephalitis elute effective when the pH value of the starting and eluting buffer, predominantly at pH>7.5% as an eluting buffer can be used starting buffer with the addition of more than 0.1 M alkali metal halides such as KCl and/or NaCl, and/or CVG. The listed signs sufficient to obtain a higher degree of treatment than the degree of purification by the method prototype. Zone elution of the antigen is determined by any known method, however, the most effective method of enzyme-linked immunosorbent assay (ELISA), and for erwerbende it is advisable to choose the active fraction of the antigen of the virus of tick-borne encephalitis with isoelectric points with values of 7.8 and 10.3. If necessary erwerbende fraction of antigen are combined and subjected to additional get the vaccine, keeping efficiency at multiple dilutions of the current beginning.

The method can be carried out as follows. Prefabricated loaded into a chromatographic column Packed with a weak anionoobmennika, elute the starting buffer naservirujeme impurities and produce the active fraction of virus antigen with an isoelectric point of 10.3. Then include a linear gradient eluting buffer, which is used as the starting buffer with pH>7.5 by adding to it the halides of alkali metals with a concentration of more than 0.1 M, and elute the active fraction with an isoelectric point of 7.8. Active fractions of virus antigen determined by the method of enzyme immunoassay. Elyuirovaniya fraction with isoelectric point of 7.8 or the same faction, combined with elyuirovaniya faction with isoelectric point 10,3, is subjected, if necessary, further purification by gel filtration on a sorbent capable of absorbing low molecular weight impurities, such as Sephadex. The purified substance add stabilizer (human albumin) and adjuvant (aluminium hydroxide).

The method allows for the selection of immunogenic fragments of tick-borne encephalitis virus and conc is or sofjin". The method is also suitable for cleaning nishtanah (substandard) industrial parties vaccine that saves valuable biological material. An important advantage of the claimed method of obtaining a purified vaccine is that it allows you to select antigenic proteins of the virus in almost pure form. Minor impurities human serum albumin serve as both a stabilizer selected antigenic proteins.

The proposed method for obtaining purified vaccine for tick-borne encephalitis is illustrated by the following examples.

Example 1. Chromatographic column XK 26/20 fill weak anionoobmennika type "DAE Sepharose Fast Flow" or similar to the height of the layer 16 cm and balance the starting buffer with a pH value of 8.4% with a linear flow rate of 70 cm/h In a column through the "superpacs" load 180 CC semi vaccines and elute naservirujeme impurities within 25 minutes. Next elute sorbed impurities eluting buffer by creating a linear gradient of halides of alkali metals with a change in concentration from 0 to 100%. As an eluting buffer is usually used starting buffer + 1 M Nad. By ELISA to determine the zone of cutting the military faction can optionally be subjected to a second stage purification by gel-filtration using a chromatographic column. This stage is carried out on the sorbent capable of absorbing low molecular weight impurities, such as Sephadex G-25 F. Zone cut and control the activity of the fractions is also determined by ELISA. The purified substance by known methods add stabilizer (human albumin) and adjuvant (aluminium hydroxide). The obtained purified vaccine encephalitis volume of 40 ml has increased 4 times activity (using ELISA) in comparison with the vaccine, obtained by the method prototype.

Example 2. The chromatographic column is filled with a weak anionoobmennika type "DEAE Sepharose Fast Flow" at the height of the layer 18 see Column balance starting buffer with a pH of 9.4% with a linear flow rate of 70 cm/H. Then into the column through the "superpacs" load 180 CC vaccinated material followed by washing nenormiruemym impurities within 20 minutes. Next, create a linear gradient eluting buffer. As an eluting buffer use the starting buffer of + 1.2 M KCl with a change in concentration from 0 to 100%. Zone cut and control activity determined by ELISA. Elute the active fraction of the antigen of the virus of tick-borne encephalitis with an isoelectric point of 7.8 and 10.3 and unite them by the stir (aluminium hydroxide). The obtained purified vaccine encephalitis in 1,3-1,5 times more vaccine produced by known methods. Adverse effects of its application is not detected. If necessary combined fractions are subjected to additional purification by gel-filtration using a chromatographic column XK 26/40 filled with sorbent type Sephadex G-25 F. This concentrated vaccine volume of 60 ml has increased in 3 times activity (using ELISA) in comparison with the vaccine, obtained by the method prototype. Get the drug, after the introduction of the necessary additives, is prepared inactivated tick-borne encephalitis vaccine.

The quantitative results obtained when testing the effectiveness of the inventive method, are illustrated by the tables.

Table 1 shows the comparative characteristics of the content of the ballast and contaminants in vaccines produced according to the method of the prior art and the claimed method. Table 2 shows the comparative characteristics of these vaccines for antigenic activity and immunogenicity. The data tables show that the inventive method provides a high degree of purification protein and other nizkomolekulyarnykh impurities, as kanamycin, formaldehyde, phenol red, three times to reduce the amount of chicken protein and freed from the proteins of the mouse brain, without compromising immunogenic activity. The vaccine is stable during storage and transport.

LITERATURE

1. Auth. mon. USSR N 660389, WITH 12 N 7/04, WITH 12 TO 5/00. Publ. 28.02.1980, B. I. No. 8.

2. Patent SU # 1318149, C 12 N 7/04. Publ. 15.06.1987, B. I. No. 22.

3. Lyapustin Century. N. The dissertation on competition of a scientific degree Prof. Biol. Sciences. Moscow, 1993.

1. A method of obtaining a purified vaccine encephalitis, including cleaning of prefabricated vaccine encephalitis from the ballast and harmful impurities, adding a stabilizer and adjuvant, characterized in that the cleaning of prefabricated carried out by liquid chromatography using weak anion-exchanger, creating a concentration gradient of halides of alkali metals and elwira active fraction of virus antigen in an effective pH value of the starting and eluting buffers.

2. The method according to p. 1, characterized in that the area of the elution determine enzyme-linked immunosorbent assay.

3. The method according to PP.1 and 2, characterized in that as an eluting buffer Hispalis.1-3, characterized in that elyuirovaniya fraction of the antigen of the virus of tick-borne encephalitis with isoelectric point with a value of 7, 8 or the same faction, combined with elyuirovaniya active fraction of virus antigen with an isoelectric point value of 10, 3, is subjected to additional purification by gel-filtration on a sorbent capable of absorbing low molecular weight impurities.

5. The method according to PP.1-4, characterized in that as halides of alkali metals using NaCl and/or KCl, and/or CVG.

 

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