SU1364342A1 - Method of producing adenoviral antigens - Google Patents

Abstract

The invention relates to the field of medical and veterinary virology, in particular, to the technology for the production of adenovirus antigen preparations. The aim of the invention is to increase the yield of the target product and simplify the process for the production of adenoviral antigens. For this, adenoviruses are grown in human and animal cell culture, the infected cells are separated from the culture medium, the antigen is removed by distraction, first with sodium chloride buffer solution and then further extraction with 5M sodium chloride solution, after which the extract is treated with an organic solvent and further combined with the culture fluid , and purification and concentration are carried out by two-stage sorption chromatography, first on phenylsepharose, and then on DEAE-sepharose. As a result of these manipulations, a high frequency of adenoviral antigen () is achieved, and its output increases by 1.5-2 times. 1 tab. e (L W Odd Od 4) 00 4

Description

one

The invention relates to the field of medical and veterinary virology, in particular, to the technology for the production of adenovirus antigen preparations.

The purpose of the invention is to increase the yield of the target product and simplify the method.

Example 1: A monolayer wound with Q buffer (pH 7.2). Next, a column of washed culture of human cells of the line ner-2 is grown in rotating bottles of 2 liters in a device for roller culturing cells. Medium 199 with 10% bovine serum was used to purify cells. A human type 1 infectious adenovirus (Adhi) is applied to a formed monolayer of Hep-2 cells (20 bottles of 60 million cells) at a dose of 0.05 bln-forming units per cell in a full supporting medium (100 ml of medium 199 with 2% aminopeptide per bottle). After 72 hours, when the entire monolayer is damaged by cytopathic changes, the culture medium is drained and 10 ml of Hume sodium phosphate buffer, pH 7.2, are added to the bottles and the cells are removed from the glass with their simultaneous lysis by shaking.

A suspension of lysed cells (total volume 120 ml) is homogenized in a mechanical knife chopper for 2 minutes, after which

200 ml of 5 M NaCl solution and additionally homogenize for 2 minutes. 200 ml of carbon tetrachloride are added to the homogenate and shaken on a laboratory shaker for 5. minutes, after which the mixture is separated by centrifugation for 15 minutes at 1500 rpm. The upper aqueous phase (volume 395 ml) is collected and combined with the culture medium (volume 950 ml). The combined fraction is acidified to pH 6.4 by the addition of 1 M NaHjPOi, and applied to the column (diameter 5 cm, layer height of the sorbent 6 cm) containing CL-4B phenylsepharose (Pharmaceutical, Sweden) and equilibrated with 10 M sodium phosphate buffer (pH 6.4), at a rate of 1400 ml / h. The column is washed at the same rate with 1 l of UM sodium phosphate buffer (pH 7.2), after which the antigen is eluted with 30% ethanol in 10 M sodium phosphate buffer (pH 7.2) at a rate of 200 ml / h The presence of proteins is recorded in the eluate.

3643422

by absorption at 280 nm. The protein peak is combined into one fraction (volume 185 ml) and further purified.

For this, the eluate from phenyl sepharose is applied at a rate of 200 ml / h onto a DEAE-Sepharose column (2.6 x 6 cm), equilibrated with 10 M sodium phosphate

b

wash at the same rate 200 ml of 10 M sodium phosphate buffer (pH 7.2) containing 0.2 M NaCl; the elution of the antigen from DEAE-Sepharose is carried out

linear increase in concentration

NaCl from 0.2 to 0.4 M in 10 M sodium phosphate buffer (pH 7.2). The total volume of the gradient is 160 ml, the elution rate is 30 ml / h. Fractions with a volume of 3 ml are collected using an automatic collector of fractions. In the obtained fractions, the antigen content is determined by rocket immunoelectrophoresis in agarose with antiserum against

hexon adenovirus monkey n ad sim 16, the total protein content is determined by the Lauri method and the degree of purity of the antigen is obtained using a disacteliol in sodium dodecyl sulfate gel,

The results of the antigen preparation are illustrated in the table.

five

g.

The yield of purified antigen Ad hi corresponds to 16 mg per 10 cells (table, clause 5), while by a known method the maximum yield is 9 mg per 10 cells.

In addition to the main fraction of purified

0 antigen (table, point 5),; at the final stage, its additional fractions containing partially purified antigen (table, points 5 and 5), which can be obtained in

g of the purified state by repeated chromatography on an anion exchanger (Example 2). In a similar way, antigens of adenovirus monkeys and cattle can be purified,

PRI and MER 2, the monolayer of the primary culture of the kidney cells of a green monkey (PZM) is grown in 1.5 liter flat culture flasks in the medium indicated in example 1, on the formed layer of PZM cells (60 vessels of 25 million each, cells) infecting adenovirus lower monkeys SA7 (Ad sim 16 (clone MB-230) in

2 sponge in full supporting medium (150 ml of medium, according to example 1, per vial). After 96 h, the culture medium is drained, the cells are removed and the antigen is extracted from them, as indicated in Example 1. Chromatographic purification of the antigen from the combined fraction of the culture medium and cell extraction is carried out as indicated in Example 1, except that the combined fraction is acidified to pH 6.0, and the chromatographic dimensions

(volume 25 ml) and lyophilized. The output of the antigen is 80 mg, which corresponds to 53 mg per 10 cells.

the content of hexane Ad sim 16 - 97%, Froze. The MDVC cell culture monolayer is grown in rotating bottles of 500 ml in the device 10 for roller culturing cells in the medium indicated in Example 1. The cells (20 bottles of 20 million cells each) are infected with an infectious bovine adenovirus column. - is Ad) (0.01 PFU / cell) in support, respectively, 10 and 15 cm for living medium (50 ml per vial). Chephenyls and DEAE-sepharoses). For 120 hours, the medium is drained, and the antigen from the DEAE-Sepharose flask is washed with 500 ml of cells extracted as indicated by 10 M sodium phosphate buffer (pH in Example 1, Chromatographic Purification 7.2), containing 0.07 M NaCl , and the elution of antigen is carried out as described, with a gradient of NaCl concentration from 0.07 to 0.2 M per 10 M sodium phosphate buffer (total volume of 350 ml gradient). Collect 7 ml fractions. The peak of the purified antigen (fractions 25-36) is pooled. Fractions of partially purified antigen (21-24 and 37-42) are combined (total volume 68 ml), diluted to 200 ml with 10 M sodium phosphate buffer and applied to a column of 2.6 x 8 cm DEAE-Sepharose, equilibrated YuM sodium phosphate buffer containing 0.07 M NaCl. The column was washed with 150 ml of the same buffer, after which the antigen was eluted with a linear gradient of NaCl concentration of 0.07–0.2 M on YuM sodium phosphate, 20 mg / 10 cells, purity 96%.

The proposed method for producing an antigen provides an increase in the yield of the drug by 2-5 times as compared with the known method. . Obtained by the proposed method

Adenovirus antigens retain their full antigenicity and immunogenicity, and 45 can be used as diagnostic and vaccine preparations.

in Example 1, with the exception of the fact that the elution of antigen from phenyl sepharose is carried out with 20% isopropanol on 10 M sodium phosphate buffer (pH 7.2). On

In stage 25 of DEAE-Sepharose, the NaCl concentrations indicated in Example 2 are used. The purified Ad bos 3 antigen (volume 38 ml) is concentrated. For this, 100 ml of cooled to acetone is added to the preparation, mixed, and the mixture is left for 1 hour at -20 ° C. After that, the precipitate of antigen is separated by centrifugation (20 minutes at 3000 rpm at 0 s) and dissolved in

.- 3 ml of 0.15 M NaCl. Exit antigen o.

 Ad bos 3 - 81 mg (which corresponds to

buffer (pH 7.2). The total volume of the gradient is 200 ml; fractions of 5 ml are collected. Purified antigen is released in fractions 21-26. These fractions are combined with the purified antigen from the 1st column, DEAE-Sepharose, Total volume of the preparation, purified hexon antigen 110 ml, the content of hexone Ad sim 16 tons 84 mg.

Fenipsepharose chromatography is carried out to remove salts and concentrate the purified antigen. For this, the combined fraction is applied. on a column of phenylsepharose (1.6 x X 12 cm), equilibrated with 10 M sodium phosphate buffer (pH 6.4) at a rate of 10 ml / h. The column is washed with

Claims (2)

Invention Formula gQ A method of producing adenoviral antigens, which includes growing adenoviruses in cell culture, separating infected cells from the culture fluid, destroying cells at the same rate of 50 ml of 5 M NH COOCH, gg by lysis and removing adenovirus, after which the concentrated antigen is eluted by extraction a buffer antigen with 30% ethanol on a disodium chloride solution; treat tilated water at an extract rate of 20 ml / h with an organic solvent. Protein peak (by absorption followed by purification and concentrated (volume 25 ml) and dried by lyophilization. Antigen yield 80 mg, which corresponds to 53 mg per 10 cells. the content of hexane Ad sim 16 - 97%, Froze. The MDVC cell culture monolayer is grown in rotating bottles of 500 ml in the device 10 for roller culturing cells in the medium indicated in Example 1. The cells (20 bottles of 20 million cells each) are infected with an infectious bovine adenovirus is Ad) (0, 01 PFU / cell) in support medium (50 ml per vial). After 120 hours, the medium is drained, and the antigen is extracted from the cells, as indicated in Example 1, Chromatographic purification of the antigen is carried out, as described, 20 mg / 10 cells), purity 96%. in Example 1, with the exception of the fact that the elution of antigen from phenyl sepharose is carried out with 20% isopropanol on 10 M sodium phosphate buffer (pH 7.2). On Stage 5 DEAE-Sepharose use NaCl concentrations indicated in the example. 2. Purified Ad bos 3 antigen (volume 38 ml) is concentrated. For this, 100 ml of cooled to acetone is added to the preparation, mixed, and the mixture is left for 1 hour at -20 ° C. After that, the precipitate of antigen is separated by centrifugation (20 minutes at 3000 rpm at 0 C) and dissolved in .- 3 ml of 0.15 M NaCl. The output of antigen b.  Ad bos 3 - 81 mg (which corresponds to Invention Formula five . 136L3426 sorption chromatography, ot chromatography is carried out with the use of the fact that, with the use of phenylsepharose with a pH of 6.0, the goal of increasing the yield of the target pro- gram is 6, while elution is carried out and the process is simplified; ethanol or 20% treatment of adenoviral antigen with an isopropanol solution followed by additional extraction by sorption of eluent on DEAE-sepharose and 5 M sodium chloride solution, after elution of the antigen 10 M sodium phosphate treatment of the extract with organic pH buffer 7.2 with a linear enhancer; additionally combining it with the concentration of chloride in it with the culture fluid, sodium sorbate from 0.2 to 0.4 M. Cultivation medium 950 3230 23 Cell Extract395 350 25 Combined parent drug. 1345 3580 48 4 Eluate from phenylsepharose 185 292 5 Eluate with DEL-; sepharose a) fraction 25-27 8.5 14.0 7.6 51 15 20.5 20.0 19.2 95 40 17.0 27.8 10.1 36 21 d) amount (a + b + c) (46) (61.8) (36.9) (74) (76) 0.7148 6,952 1,3100 14,388