SU1071306A1 - Method of preparing sorbent for cleaning viral suspensions - Google Patents

Abstract

1. METHOD FOR OBTAINING SORBENT FOR CLEANING VIRAL SUSPENSIONS, including treatment of silica-containing material - immiscible, and sodium nitrite in the presence of acid, in order to reduce the sorption capacity of the virus, in order to reduce the sorption capacity of the virus, in order to reduce sorption capacity for the virus, in order to reduce sorption capacity for the virus, after being treated with a mixture of sodium nitrite and acid, the silica-containing A / material is reacted with N-vinyl pyrrolidone or its aqueous solution in the presence of hydrochloric acid. ; 2. The method according to p. 1, about tl and h a y i and with the fact that you use an aqueous solution of N-vinylairrolidone concentration e below 30%.

Description

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The invention relates to a method for the preparation of a sorbent for the purification of viral suspensions and can be used in the production of viral antigens and vaccines.

A known method for the preparation of a sorbent for cleansing viral cpd: penzky includes multiple processing of macroporous silica with a solution of polyvinylpyrrolidone followed by heat treatment for 24 hours 1.

However, the known method is long lasting. Insufficiency of the resulting sorbent, due to the indirect linkage of silica, leads to the aggravation of the virus suspension with the odifferent agent.

The closest to the technical essence and the achieved result is the method of obtaining the njrreM sorbent for the superficial treatment of glass Jf-aminotryththoxysilane and a mixture of sodium nitrite and hydrochloric acid {2J.20

The resulting sorbent with hydroxypropyl. 1 rups does not only absorb impurity proteins from the viral suspension being purified, but also virus-: ttbie particles, which leads to a decrease in virus yields.25

The purpose of the invention is to reduce the sorption capacity for viruses.

This goal is achieved by the fact that according to the method of obtaining a sorbent for the purification of BHpyctox suspensions, including the processing of a cremiezem-containing material, α-aminopro; shstriethoxysilane and sodium in the presence of acid, after treatment with a mixture of BMTpHta sodium and acid, the silica-containing material is reacted with N-viylpyrrolidone or its aqueous solution in the presence of hydrochloric acid.

A water solution of N-vinylprolidone with a concentration of at least 30% is used;

Silica-containing material with hydroxyprocyl groups can also be obtained by treating with hydroxypropylsilane in the form of trimethylsilyl sephira or in the form of efuf3 of sulfuric acid, followed by the removal of the protective group.

Example 1. To 50 ml of glass MPS-. 2000 To add 50 ml of a 5% solution, j-aminoproshtriethoxysilane in acetone, mix and keep at 100 for 6 hours. To a modified glass, 100 ml of 0.1N are heated at -50. A solution of 2 g of a sodium syringe in 10 ml of water is added in portions over a period of 0.5 h. The solution is stirred for another 2 h and washed with water. The amount of hydroxypropyl groups, determined from the difference between the 55 starting and unreacted aminoTpymt reactions, is 0.4 mg-ssv / g. Unreacted amino groups are absent.

To, 30 ml of the modified MPS-2000 st row, 30 ml of N-vinyl pyrrolidone and 0.1 ml of concentrated hydrochloric acid are added, stirred for 2 hours, washed with water, sodium bicarbonate solution and again with water. Monitoring the washing of the modified sorbent from unreacted N-vinshirrolidone ved} yy by the absence of absorption at 230 nm. Modified glass is discharged in air at bSRC. Nitrogen content is 0.59%. The IR spectrum has an intense absorption band at 1650 cm (CO-MR).

The resulting sorbent is introduced into a 1 × 10 cm chromatographic column, which is equilibrated with M g / cc-HCl buffer with a pH of 7.8. 1 ml of allantoic fluid containing the A j Texas influenza virus is injected into the column, and elution is carried out with buffer, feeding it at a rate of 2 ML / MINsg. At the outlet of the column, 1 ml frakddy is collected.

In the 4th and 5th fractions of the zljuat, 90% of the polluted influenza virus is collected, impurity proteins and low molecular weight substances are zoned in 6-8 fractions of the eluate. The content of the impurity protein in the purified suspension of influenza B1fus is 0.01 ml / ml (in the initial alantoic liquid, 2.5 mg / ml), the purification rate is 99.6%. There is no loss of viral material,

Similarly, virus A group / Victory / 72 is carried out. The virus yield is 90%, the degree of purification from impurity proteins is 99.2%.

Trials of the Modified Enzymatic Carrier show that the release of the hrshrsh virus during cleaning does not depend on the market.

When using oxypropyl glass MPS-2000B in similar conditions of the flu virus's sorption capacity reaches 95%, respectively, the virus yield is 5%. Sequentially, washing the column with 0.1 M Tpuc-UCl buffer with a pH of 8.6 and containing 0.5 M MaCJj leads to desorption of 40 M % of virus added to the column. Loss of viral material 55%,

Example 2. When modifying the glass of MPS-1000 V under the conditions of example 1, a glass is obtained with a content of hydroxypropyl groups of 0.26 meq / g.

20 ml of a 50% aqueous solution of M-vinyl pyrrolidone and 0.2 ml of concentrated hydrochloric acid are added to 10 ml of glass MPS-1000 V with the content of hydroxypropyl groups of 0.26 mg eq / g and concentrated treatment is carried out in analogy to Example 1. a sorbent with a nitrogen content of 0.4%, which is used to isolate tick-borne virus infection from a culture suspension. For this, a 0.8 x 12 cm column filled with the resulting sorbent is equilibrated with 0.1 M to ipuc-HCl buffer with a pH of 8.0. A 1 ml bottle & suspensions and zlyuyut specified, the speed of zlyuira-3107 2 ml / min. In the 3 and 4 fractions of the eluate the virus is collected, in the 6-8th fractions of the viral proteins. The output of the virus from the column is 95%. The content of impurity protein in the purified preparation of tick-borne encephalitis virus is 0.007 mg / ml (in the initial suspension of 1.1 mg / ml) the purification rate is 99.6%. When using oxen ropilstekla in anala; Under favorable conditions, the virus is tick-borne encephalitis virus 50%. Accordingly, the yield of the purified virus causes half of the ot. It is added to the column with more stringent conditions (when using 0.1 M Tris-IIS buffer with pH 8.5, containing Oi5 M NaCl for eyuirovania virus) desorption of the virus is not observed . 1 Example 3. To 50 ml of silochrome C-80, add 50 ml of a 7% solution of gc-amino-protona and triethoc (Lansilan in acetone and then proceed as in Example 1, the amount of oxyllope1X groups is 0.49 mg-eq / g.2 K , 30 ml of modified silochrome C-80 are added 60 ml of a 30% aqueous solution of N-jinshI6 pyrrolidone and 0.2 ml of concentrated hydrochloric acid, then the treatment is carried out similarly to Example 1. A sorbent with a nitrogen content of 0.7% is obtained, by which The column is loaded with a size of 0.8x12 cm. The column is equilibrated with 0.1 M phosphate buffer with a pH of 8.0 and injected with 1 ml of inactivated suspension. Tick-borne cephalitis virus (immunogenicity is 27 units of PR. protein concentration 870 µg / ml). The virus is eluted with phosphate buffer, the KOTOpbnti column is balanced, the eluate is collected in a 1 ml fraction. Purified virus is collected in the 3rd and 4th fraction of the eluate (23 units And, the protein concentration is 6 µg / ml. In the 6-8th fraction, impurity proteins are eluted. The virus yield from the column is 85%, the degree of purification from proteins is 99.2%. Thus, the sorbent is prepared by the proposed method , allows for efficient purification of viral suspensions without loss of viral material with a high degree cleaning of impurity proteins.

Claims (2)

1. METHOD OF PRODUCING SORBENT FOR CLEANING VIRAL SUSPENSIONS, including the treatment of silica-containing material with aminopropyltriethoxysilane and sodium nitrite in the presence of acid, they differ in that, in order to reduce the sorption capacity for viruses, after treatment with a mixture of sodium nitrite , silica-containing material is reacted with N-vinylpyrrolidone or its aqueous solution in the presence of hydrochloric acid. 2. Ogosb by π. 1, characterized in that the use of an aqueous solution of N-vinylpyrrolidone with a concentration of not less than 30%. Ω _m SU ,,., 1071306