CS236992B1 - Purificattion method of surface antigen of hepatitide b - Google Patents

Abstract

The invention relates to the production of standard hepatitis B immunization antigen (HBaAg) for the preparation of an immunoreaction for detection of HBs-antigen and anti-HBs antibodies by sensitive 3rd generation methods for other purposes, such as preparation different types of vaccines for hepatitis, monoclonal antibodies to determinants HBaAg et al. HRsAg isolation and purification technology it is conceived from the processing of the starting material / plasma, sera) through preparation roughly petrified concentrate until ready and performing final purification. The process provides high yield and preserves HBsAg antigenicity. Velká joéče is dedicated to ensuring work safety with virulent material. They are in subsequent purification steps. selectively removes infectious virions in. and the purification process can be performed in closed systems.

Description

The present invention relates to the preparation of a standard hepatitis B surface antigen (HBsAg) for immunization and the preparation of immunoreactivities for the detection of HBs-antigen and α-β-HBs by sensitive third generation methods, or for other purposes such as preparation of various types of hepatitis B vaccines. , monoclonal antibodies and biochemical and immunochemical studies.

Human serum or converted plasma of HBsAg carriers selected predominantly from the transfusion service capture, or the culture medium of tissue culture production lines, is used as a starting material for the production of standard HBsAg immunization materials.

The biochemical value of the starting materials for HBsAg purification is determined by the HBsAg titer in upstream immunoelectrophoresis, which is at least 1: 4 and above, or by determining the HBsAg concentration in the radial immunodiffusion, which must be at least 0.05 mg HBsAg in 1 ml.

HBsAg purities of the highest partial purity and at the same time the least denatured for antigenic properties are obtained by various precipitation, centrifugation and / or centrifugation processes. by chromatographic methods. Due to the complex composition of the starting material, the desired quality of HBsAg can be achieved only by the combined technology of these methods, although we are offered the use of a simple principle of affinity chromatography. Using affinity chromatography under operating conditions would be too demanding and uneconomical.

The purification procedure proposed by us is variable

238,882 compositions of the starting material, in particular plasma or serum derived from HBsAg carriers. Variable abundance of immunocomplexes and heterogeneous profile of high molecular weight and lipid components of plasma or serum are anticipated · These components may, due to their physicochemical properties, significantly interfere with HBsAg behavior and thus affect the efficiency of the purification process ·

The technology, isolation and purification of HBsAg is designed from the processing of the starting material through the preparation of a coarse-purified concentrate obtained with polyethylene glycols to the final purification using density and rate fractionation. The proposed process can achieve a morphologically homogeneous,. immunochemically pure, immunogenic and highly concentrated HBsAg purified ·

The procedure ensures 15 and 25% recovery, fully preserving the antigenicity of HBsAg. since the denaturing disintegration of the particles is minimized. We have also taken great care to ensure the safety of this virulent material where potentially infectious virions are selectively removed during purification as a component of balsa fractions. Gradually, they are completely removed, with an unchanged recovery of the desired product, which is non-infectious HBsAg.

The workflow consists of the following stages:

-defibrinogenization of HBsAg positive plasma by recalcification in the presence of thrombin followed by separation of the formed fibrin by centrifugation.

Of course, the use of serum defibrinogenization is not necessary.

- treating the supernatant with polyethylene glycol 6000, which is added

- 3 23β 982 to a final concentration of 4 to 7 volume percent, preferably 5.5 volume percent, at pH 6.8 to 7.4, preferably pH = 7.0. In this step, most of the high molecular weight components, including infectious virions and immunocomplexes, are removed from the feedstock. Said undesirable components are part of the precipitate which is removed by centrifugation # We further process the supernatant, which is saturated by adding polyethylene glycol to a final concentration of 6 to 15% by volume, preferably 7.9% by volume, and adjusting the pH to 3.5 to 6.5, preferably pH = 4.5. After centrifugation of this mixture, HBsAg is present in the sediment and is thereby concentrated. The main components of the supernatant are low molecular weight proteins, especially albumin.

delipidation and density fractionation in a cesium chloride medium having a density of 1.2 to 1.35 g / cm @ 2 is the most efficient step in the purification process and achieves the highest purification efficiency. High efficiency of density fractionation is achieved by application of H3sAg solution, by which we layer cesium chloride gradient. Thus, the gravitational field of the centrifuge separates HBsAg from the ballast protein residue by flotation. HBsAg travels in the high density region upstream of the centrifugal force, and ballast proteins having a substantially higher density travel in the opposite direction. A cryogenic process, which is also part of the present invention, can be used to prepare the cesium chloride gradient.

the rate fractionation in sucrose medium is the last purification step in which the final removal of ballast components is achieved based on different sedimentation constants.

High yield of the proposed purification process and achieved

The purity of the product, i.e. the hepatitis B surface antigen (HBsAg), during purification is ensured by continuous analysis of the fractions for both HBsAg and serum protein by a single radial immunodiffusion method. Fractions that do not meet purity and contain HBsAg are used for refractionation.

The use of the manufacturing process according to the invention solves the basic problem of the production of purified, immunogenic HBsAg antigen necessary for the production of avid and specific antibodies.

The obtained antisera are by their avidity suitable for use in enzyme immunoassay or radioimmunoassay. They are suitable for conjugation with horseradish peroxidase as well as for radioactive iodine labeling for the detection of HBsAg, resp. of anti-HBsAg antibodies by third generation methods.

Purified HBsAg may also be a starting point in the eventual development of various types of hepatitis B vaccines or other purposes, for example for the preparation of mondclonal antibodies or immunochemical and biochemical assays.

Exemplary embodiment

The purification procedure is divided into five stages of ten working years. Materials from individual donors - preferably up to step

3.2 inclusive - are processed separately to avoid disturbing interactions of protein material obtained from different donors. From step 3.3. it is preferable to process the semi-purified from the individual starting plasma or serum together.

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Starting material

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To purify HBsAg from human plasma or serum from HBsAg carriers, the HBsAg titer in the antisense immunoelectrophoresis must be at least 1: 4 or radioimmunoassay of at least 0.05 mg / ml.

Defibrinogenation of HBsAg -positive plasma

1.1. To 100 ml of plasma are added 4 ml of 1 molar calcium chloride and 1 ml of Topostazine / 70 N.I.H./ml/ -thrombin solution. After 30 minutes incubation at 37 ° C, the mixture is stored at + 4 ° C for 16 hours. After the following centrifugation (Janetzki K 60, 5,000 rpm, 60 minutes, + 4 ° C), only the supernatant is continuously processed.

Differential precipitation with polyethylene glycol 6000 / PEG 6000 /

2.1. To 100 ml of supernatant 1.1. with stirring, 37.93 ml of 20% by volume of a solution of poly (ethylene glycol 6000) in distilled water is added to give a serum concentration of 5.5% by volume of ethylene glycol 6000, the pH of the mixture being neutral.

The mixture is stirred for 60 minutes at room temperature and then left overnight at + 4 ° C.

Following centrifugation under the same conditions as in step 1.1. the supernatant labeled * PEG A 'is further processed.

.2. To 100 ml of the supernatant 2.1. PEG A 'is slowly added with stirring, 20 ml of a solution of ethylene glycol 6000 poles in distilled water at a concentration of 20% by volume. This will saturate the concentration

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7.9% by volume of polyethylene glycol 6000. After complete mixing of both components, the pH is lowered to 4.5 by adding 4 N acetic acid (average acetic acid consumption is 1.4 ml per 100 ml volume of starting plasma). The mixture is further stirred for 60 minutes at room temperature and then left to stand at + 4 ° C for 16 hours.

After subsequent centrifugation under the same conditions as in step

1.1. only the sediment which is resuspended in 0.01 M trihydroxymethylaminomethane (TRIS) buffer, 0.15 M sodium chloride, 0.001 M ethylenediaminotetacetic acid, pH 7.2 (TENa buffer) to 1/50 of the starting volume of the raw material is further processed and the dialyzone at room temperature against distilled water (2 times the volume of distilled water exchanged is equal to the volume of the starting plasma or serum).

After the following centrifugation (Janetzki K 27.7000 rpm, 90 minutes, + 4 ° C), a supernatant that can be combined with an aqueous sediment flush (to achieve a higher yield) is processed.

The supernatant obtained is stored at -20 ° C and labeled with PEG B until further processing.

Delipidation and fractionation in cesium chloride density gradient (CsCl) .1. Κ 260 ml of supernatant 2.2. PEG B is added 110 g solid cesium chloride (288 ml solution is formed). After reconstitution, the material is filled 8 times by 36 ml into Befcman angle rotor centrifuge tubes, type 42.1. and topped with water (3ml) to replenish the centrifuge volume.

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After subsequent centrifugation (25,000 rpm, + 10 ° C, 60 minutes), only clear, delipidated supernatant is removed from each cuvette, which is used for the subsequent preparation of the ces chloride gradient. us.

3.2. Delipidated supernatant from 3.1. is filled by 28 ml into eight Beckman vertical rotor type cuvettes: VTi 50 and the opposite pairs are carefully balanced.

The following is a cryogenic preparation of a cesium chloride gradient in situ ”: centrifuge tubes with filled solution are stored in a vertical position for 24 hours at -20 ° C. Before centrifugation, the cuvettes are transferred for 16 hours to + 10 ° C, where a freeze-thawing produces a cesium chloride gradient.

The gradient formed is overcoated to the final volume of the cuvette (39ml) with a solution of esium chloride in TENa buffer (composition as in step 2.2) with 24% eq of cesium chloride. After cuvettes are sealed, the material is ready for density fractionation at 35,000 rpm, + 10 ° C, 8 hours in a Beckman type: VTi 50 vertical rotor.

Upon completion of the centrifugation, a solution of up to 1.25 g / cnA containing HBsAg, labeled " CsCl A, is withdrawn from the top of the centrifuge tube.

The higher density material contains most undesirable serum proteins and is not further processed.

3.3. A Beckman type: SW 40 rotor centrifuge cuvette is prepared after 1 ml of a linear gradient of cesium chloride in TENa buffer (composition identical to step 2.2 *), 8 to 32 percent cesium probe.

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The gradient thus prepared is coated with 3 ml of the active fraction

CsCl A ”from step 3.2., Concentrated to one fifth of the volume obtained and pretreated to a density of 32 g / cm3 by addition of solid cesium chloride

The samples are centrifuged in a SW 40 rotor at 35,000 rpm, + 5 ° C, for 20 hours.

Alternatively, a larger VTi 50 rotor can be used at larger volumes. A 30 ml linear cesium chloride gradient (3 to 32 weight percent) is prepared into centrifuge tubes of a declared volume of 39 ml. The gradient is layered with 9 ml of the active fraction CsCl A, pre-treated as before, to a density of 1.32 g / cm. Thus, it is possible to fractionate at 35,000 rpm for 8 hours, + 10 ° C, 72 ml of active CsCl A fraction at a time.

After centrifugation, the material is fractionated from above to 50 fractions. Individual fractions are analyzed for the presence of HBsAg and serum proteins by radial immunodiffusion.

Active fractions with a minimum serum protein content are pooled and dialyzed against TENa buffer (composition as in step 2.2./) and concentrated to 1/20 volume of pooled fractions. The concentrate obtained is labeled CsCl B and is stored at -20 ° C.

Speed fractionation in sucrose gradient

4.1. The active CsCl B concentrate is applied in 0.2 ml volumes to the top of the stabilized (18-24 hours at + 4 ° C) linear sucrose gr ₉ dient (9 to 21 volume percent sucrose in TENa buffer (same composition as in step 2.2.) , prepared in polyallomeric

- 9 236 992 or Beckman rotor nitrocellulose tubes. Type: SW 40

After subsequent centrifugation in a SW 40 rotor. At 40,000 rpm, θ 29 * +50, wt 300 x 10, the material is fractionated either from above as from the previous case or from the bottom and analyzed in the same manner as in step 3.3.

The active fractions not containing serum proteins are pooled and dialyzed against buffer: 0.01 M phosphate, 0.15 M sodium chloride, pH 7.2 (PBS).

Adjustment and analysis of HBsAg r

5.1. The final purified after dialysis against PBS buffer (composition as in step 4.1./) is analyzed by spectrophotometry and adjusted for protein content: 1mg (1ml / 37.3), filled in aliquots

1mg and stored at -20 ° C.

5.2. The quality of the purified product is verified spectrophotometrically by the δ assay

tryptophan / tyrosine amino acid ratio, determination of protein concentration by Lowry ·, concentration of HBsAg by radioimmunodiffusion and specific concentration of HBsAg in the product.

♦

The high content of tryptophan detected, the high extinction coefficient and the high specific concentration are indicative of high purity quality.

Claims (5)

BEFORE THE INVENTION 238 392 I. A method for purifying hepatitis 3 / HBsAg / j surface antigen comprising treating the hepatitis B surface antigen from human serum, converted plasma or culture medium with tissue culture lines with a final concentration of 4-7; % by volume, at pH 6.8 to 7.4 and after centrifugation, treated with polyethylene glycol at a final concentration of 6 to 15% by volume, at pH 3.5 to 7.0 and after centrifugation, the sediment is treated twice by density fractionation in cesium chloride medium having a density of 1.2 to 1.35 g / cm < 3 > and after centrifugation, the sediment is subjected to speed fractionation in sucrose medium. 2. The method of claim 1, wherein the first concentration of polyethylene glycol is 5.5 volume percent at pH 7.2. 3. The method of claim 1, wherein the second concentration of polyethylene glycol has a final concentration in the polyethylene glycol. 7.9% by volume at pH 4.5. % 4. The method of claim 1, wherein the fractionated material is layered in such a way that fractionation is achieved by flotation. 5. The purification method of claim 1, wherein the cesium chloride density medium gradient is prepared by freezing the medium to below -10 [deg.] C and then thawing at a temperature of *. -5 to + 50 ° C, preferably at + 10 ° C.