NO300421B1 - Complex suitable for purification of pre-S hepatitis B virus surface antigen - Google Patents

Abstract

The invention relates to a complex consisting of an insoluble polymeric support to which monomeric human albumin is covalently bonded, and of a pre-S hepatitis B virus surface antigen which is bound via its pre-S(2) and/or pre-S(1) part to the monomeric human albumin in a form which can be eluted. This complex can be used for therapeutic and diagnostic purposes and allows rapid and efficient purification of pre-S HBsAg by affinity chromatography.

Description

The present invention relates to a complex which is characterized by the fact that it consists of an insoluble polymer carrier, especially on an agarose or dextrose basis to which human albumin is covalently bound, and a pre-S hepatitis B virus surface antigen (pre-S -HBsAg), which over its pre-S(2) part and/or pre-S(1) part is bound to the monomeric human albumin in eluable form, as well as its use in the purification of pre-S-hepatitis-/3 - virus surface antigen and production of diagnostics and vaccines.

From EP-A2-0 243 103, a method for purifying pre-S-HBsAg is known, which method consists in yeast cells expressing recombinant pre-S-HBsAg being burst, and the pre-S-HBsAg is separated from the cell contents by affinity chromatography known as . Polymerized human serum albumin (polyalbumin), which is bound covalently to a matrix, acts as an adsorbent for pre-S-HBsAg. This polyalbumin is one with cross-links, e.g. glutaraldehyde, in high molecular form artificially produced product. Pre-S-HBsAg is adsorbed over its pre-S-(2) part on polyalbumin and is eluted from the matrix after interfering substances have been washed out, and is subjected to a further purification step.

It was demonstrated that in the pre-S part of HBsAg there is an active receptor for polyalbumin which consists of a polypeptide of 55 amino acids, and which is encoded by a section of hepatitis virus DNA that is immediately stored in the S region ( "pre-S2") (Valenzuela et al., Bio/Technology 3: 317-320, 1985). It was further demonstrated that both this immediately stored region (pre-S2) and also the total pre-S region in connection with the S region (pre-S1) code for surface proteins which constitute receptors for hepatitis B virus (HBV ) for binding to the cell membrane of liver cells.

HBsAg containing a pre-S part was first recovered in 1979 from infected human plasma (Neurath and Strick, Arch. Virol. 60: 79-81, 1979) and was later also produced by genetic engineering by Valenzuela et al. (Bio/Technology 3: 317, 1985, and Nature 311: 67, 1984) and Paoletti et al.

(PNAS 81: 193, 1984).

Later, various recombinantly expressed pre-S-containing surface antigens were proposed as a vaccine to induce antibody formation against HBV. These antibodies target the virus's receptor and thus prevent it from binding to the liver cell, thus preventing infection.

The affinity chromatographic purification of the antigens over polyalbumin has the disadvantage that when cross-linkers are used in the production of polymerization products, toxic substances, such as glutaraldehyde, can be introduced into the eluate.

The present invention aims to eliminate this difficulty and provide a complex which, in addition to the possibility of a fast and effective affinity chromatographic purification of pre-S-HBsAg, opens up possibilities of use for therapeutic and diagnostic purposes.

The complex according to the invention consists of an insoluble, polymeric carrier, especially on an agarose or dextran basis, to which carrier monomeric human albumin is covalently bound, and a pre-S-hepatitis-B virus surface antigen which is bound in eluable form above its pre-S (2)- and/or pre-S(l) part of the monomeric human albumin.

According to the invention, it was determined that pre-S-HBsAg forms a complex with the monomeric human albumin in sufficient strength for affinity chromatographic purification only when this is bound covalently to the carrier. If the albumin is bound only adsorptively, pre-S-HBsAg cannot - or only to a small extent - form a complex with the albumin, so that an affinity chromatographic purification is not possible. A further advantage of the complex according to the invention is that pre-S-HBsAg is easily eluted, whereby the protein can be extracted more gently and in higher yield.

For the formation of the complex according to the invention, an insoluble carrier is required to which the monomeric human albumin is covalently bound. For the production of the insoluble, polymeric carrier, any polymer can be used which, after corresponding activation, is capable of covalently binding proteins. Current carrier materials are

organic polymers, such as polyamides and vinyl polymers (polyacrylamide, polystyrene and polyvinyl alcohols

and their derivatives), further

natural polymers, such as cellulose, dextrans,

agarose, chitin and polyamino acids, and

inorganic polymers, such as silica gel, glass and metal hydroxides.

These carrier materials are used in the form of particles, e.g. molecular sieve, in the form of membranes or plates, e.g. microtiter plates.

Preferably, the insoluble, polymeric carrier is constructed on an agarose or dextrose basis.

The complex according to the invention can be stored for a longer period of time, thus both in an aqueous phase where the carrier is in a swollen state (e.g. as an affinity resin) and also in a lyophilized state (e.g. as a membrane or microtiter plate). A lyophilization is preferably carried out from a volatile buffer containing glycine or glucose.

From the complex according to the invention, pre-S-HBsAg can be isolated with high purity. The invention also relates to the use of the complex for the production of diagnostics or vaccines. The vaccines can be used both for active immunization against hepatitis B and for the extraction of specific immunoglobulin from donors who have been immunized with such a vaccine, but such a vaccine does not in itself form part of the present invention.

The carrier according to the invention can be loaded in a simple way with S-hepatitis B virus surface antigen by bringing an aqueous solution of hepatitis B virus surface antigen into contact with the carrier, whereby the pre-S containing parts of the hepatitis B surface antigen are adsorbed on the albumin molecules.

The use of the complex according to the invention in the purification of pre-S-hepatitis-B virus surface antigen comprises that the complex is washed with buffer solution to remove

any contaminants present, and the pre-S-hepatitis-B virus surface antigen that is selectively adsorbed on the monomeric human albumin, are separated and recovered by either treating the complex with an eluent containing chaotropic substances, such as urea, guanidine hydrochloride, thiocyanate, potassium chloride, magnesium chloride or potassium iodide; or

the complex is treated with detergents; or the complex is treated with a pH-changing agent, after which a further purification is possibly carried out.

A preferred application of the complex according to the invention is where ionic detergents are used as detergents, especially sodium deoxycholate and taurodeoxycholic acid, or zwitterionic detergents, especially 3[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 3[(3-cholamidopropyl)-dimethylammonio ]-2-hydroxy-1-propanesulfonate, or non-ionic detergents, especially octyl-glucopyranosides.

It has been shown that using these detergents does not impair the quaternary structure of eluted pre-S-HBsAg.

Preferably, the further purification after the elution consists of gel filtration, ultrasound centrifugation, hydrophobic chromatography, lectin affinity chromatography or ion exchange chromatography. Each of these cleaning steps leads to a purity of pre-S-HBsAg of more than 90%.

In the following, the invention is described in more detail: Pre-S-HBsAg can be expressed from recombinant Vaccinia virus which contains the genetic information for pre-S-HBsAg. This technique is described in the literature (Moss et al., Nature 311: 67 (1984). First, a high-titer virus standard solution is prepared, in which Vero cells are infected with 1 pfu/cell. After 2 to 3 days of incubation, the infected cells are transferred to the medium and pelleted by centrifugation for 20 minutes at 5,000 g. The liquid is poured off and stored at 4°C. The cells are washed three times with PBS, after which a trypsin solution is added to the cell suspension until a final trypsin concentration of 0.025% by weight is achieved. The suspension is then stirred. easily for 30 min at 37° C, combined with the supernatant, distributed in aliquoted ampoules and deep-frozen at -80° C. With this method, the virus titer is increased to approximately tenfold what is otherwise achieved in the cell medium.

The Vero cell starter culture is prepared by growing the cells in Roux bottles and in plastic Roller bottles (Nunc) to produce a sufficient number of cells to be inoculated into a 6 liter fermentation vessel which then in turn serves as a starter culture for a 40 liter rate. For this purpose, a single vial of Vero cells of a specific generation number is first harvested and further cultured to complete twelve densely packed Roller bottles.

The cells are trypsinized, resuspended in medium 199 with 5% by weight fetal calf serum, mixed with a suspension of microcarriers (Cytodex 3, Pharmacia) and pumped into the fermentation vessel, whereby a final concentration of 2x10<8> cells and 5 g of microcarrier per litres. At this stage additional medium is added in an amount of one third of the final volume.

During three hours, the cells are given the opportunity to bind to the microcarrier, whereby the suspension is simultaneously slowly stirred. Additional medium (DMEM containing 5 wt% fetal calf serum) is then added to achieve the final volume. As soon as a cell density of 6 to 8x10<8>/liter is established, a continuous perfusion with DMEM (containing 5 wt% fetal calf serum) is started. After a cell density of approx. 5x10<9>/litre has been achieved, the microcarriers are trypsinised, and the cells and the microcarriers are pumped into the 50 liter fermentation container which contains an additional 5g of microcarrier per litres. After the adsorption, the container is filled up to 40 litres, and the cultivation is continued as described above.

When the cell density is 5xl0<9>/litre, the medium is pumped away after the microcarriers have settled. Five liters of the medium containing the recombinant microorganism is pumped into the fermentation vessel to give an m.o.i. value of 2 pfu per recombinant cell. After adsorption of the virus, the fermentation container is filled up to 40 liters with medium 199 (containing 5% by weight fetal calf serum) and perfused with 40 liters of the same medium for 40 hours. After this period of time, approximately 80% of the cells have detached from the microcarriers and are pumped away with the medium.

The remaining cells that are still attached to the microcarriers are dissolved by washing with medium under rapid stirring and combined with the first part. The microcarriers are removed using a 70 µm sieve. By centrifugation in a Beckmann JFC-Z flow-through rotor, the cells are pelleted at 16,000 g. The medium is concentrated by ultrafiltration in a Pellikon system (Millipore-Waters). Urea is added to the concentrated medium until an 8M solution is obtained. This solution is dialysed.

The concentrated Vero cell supernatant is transferred to a column which is filled with an insoluble polymeric carrier material to which human serum albumin is covalently bound. As is known, the coupling method depends on the chemical nature of the carrier. When using "Sepharose" activated with e.g. CNBr, the monomeric human serum albumin is coupled to "Sepharose".

After the column was washed with buffer 1 consisting of 0.2 M sodium acetate (pH 4.0) and 0.5 M NaCl, with buffer 2 consisting of 0.1 M Tris (pH 8.0) and 0.5 M NaCl, with solution 3 consisting of 8 M urea, and finally with buffer 3 consisting of 0.02 M Tris (pH 7.4), the Vero cell supernatant is transferred to the column. As soon as the supernatant is taken up by the column, the non-adsorbed proteins are washed from the column with buffer 4, which consists of 0.02 M Tris (pH 7.4) and 0.5 M NaCl. The adsorbed pre-S-(2)-HBsAg is eluted with sodium thiocyanate in buffer 4 (1 to 4 M, preferably 3 M) at a pH value of 6 to about 8, preferably at pH 7 (buffer 5) or with urea in buffer 4, preferably with 4 M urea (buffer 6). This separation according to the invention with monomeric human albumin leads to a 145-fold enrichment of pre-S-HBsAg. Its identity can be demonstrated by the immunoblot technique (Burnette, Anal. Biochem., 112, 195, 1981), whereby the purity amounts to 80%.

Pre-S(2)-HBsAg can be further purified by gel filtration on "Sepharose" (Pharmacia). For this purpose, the fraction obtained above is dialyzed in a buffer consisting of 0.02 M Tris (pH 7) and applied to the "Sepharose" column activated with 0.02 M Tris (pH 7). With the "Silverstaining" and "Immunoblotting" technique, a purity of more than 90% can be demonstrated for pre-S-HBsAg.

In the following execution examples, the execution of is described

the procedure in more detail:

Example 1

Affinity chromatographic purification of pre-S(2)-HBsAg using monomeric human albumin

The recombinant Vaccinia virus with the genetic information for pre-S(2)-HBsAg was obtained using the techniques described by Moss et al. (Nature 311: 67, 1984). The high titer standard solution of the recombinant Vaccinia virus was prepared as described above. The Vero cells for infection with the recombinant virus were cultured under the same conditions as described above. The concentrated, dialyzed medium with pre-S(2)-HBsAg was also prepared as previously described.

Monomeric human albumin was covalently bound to CNBr-activated "Sepharos 4B" (Pharmacia) according to the manufacturer's instructions. Then a column with a volume of 50 ml was filled and washed with 500 ml of buffer solution 1, 500 ml of solution 3 and finally with 500 ml of buffer solution 4.

The clear vero cell supernatant contained 8500 mg of protein and 95 mg of pre-S(2)-HBsAg and was pumped into the prepared column at a flow rate of 100 ml/hour. After the entire amount had been transferred to the column, the non-adsorbed proteins were washed out of the column with buffer solution 4. Pre-S(2)-HBsAg was eluted with 3 M sodium thiocyanate, pH 7 (buffer solution 5). The identification and purity investigation1-sen took place with the "silver-staining" method and immunoblotting after a polyacrylamide gel electrophoresis.

The fractions eluted from the column were split in half and incubated for 15 minutes at 100°C in a buffer consisting of 2 wt% sodium dodecyl sulfate (SDS), 0.125 M Tris-HCl (pH 6.8) and 100 mM dithiothreitol. The samples were separated electrophoretically over 12.5% by weight polyacrylamide separation gel for 2.5 hours at 65 mA/gel (Laemmli, Nature 227: 680, 1971). One part of the gel samples was stained with silver nitrate to make the polypeptides visible. The second part of the samples was examined by means of the immunoblotting technique, whereby rabbit antiserum was used.

The eluted fractions contained 42 mg of protein and 68 mg of pre-S(2)-HBsAg. With this, a 145-fold enrichment of pre-S(2)-HBsAg was achieved.

The following examples 2 and 3 describe the further purification of the fraction obtained in example 1.

Example 2

A pre-S(2)-HBsAg-containing solution obtained according to example 1 was further purified by means of column chromatography on "Sepharose 4B" (Pharmacia). For this, the column was first prepared and washed with 500 ml of 0.02 M Tris (pH 7.0) at a flow rate of 30 ml/h. The pre-S(2)-HBsAg-containing solution was dialyzed against this buffer, and the dialyzed antigen present at a concentration of 10 mg/12 ml next to 6.2 mg protein/12 ml was transferred to the column. The column was activated with 0.02 M Tris (pH 7.0), and the eluted pre-S(2)-HBsAg-containing fraction contained 4.8 mg of protein and 8.2 mg of pre-S(2)-HBsAg. Silver staining and immunoblotting of the polyacrylamide gels performed as described in Example 1 gave a purity of pre-S(2)-HBsAg of more than 90%.

Example 3

A pre-S(2)-HBsAg-containing solution obtained according to example 1 was further purified by means of column chromatography on "Lentil-Lectin-Sepharose 4B" (Pharmacia). For this, the column was first prepared and washed with 100 ml of 0.02 M Tris (pH 7.0) at a flow rate of 50 ml/h. It

pre-S(2)-HBsAg-containing solution was dialyzed against this

buffer, and the dialyzed antigen present at a concentration of 21 mg/25 ml next to 13 mg protein/25 ml was transferred to the column at a flow rate of 25 ml/h. Then, the non-adsorbed material was washed with Tris buffer solution, and pre-S(2)-HBsAg was eluted with Tris buffer solution, which contained 5 wt% alpha-methyl-mannoside. The eluate contained 9.1 mg of protein and 15.6 mg of pre-S(2)-HBsAg. "Silver-staining" and immunoblotting of the polyacrylamide gels was performed as described in Example 1, and gave a purity for pre-S(2)-HBsAg of more than 90%.

The purification of hepatitis B surface antigens which instead of the pre-S(2) part carry the pre-S(1) part, respectively the pre-S(2) and pre-S(1) parts, takes place analogously to the described embodiment examples. The method according to the invention can also be used for the purification of any protein that carries a pre-S(2) part of HBsAg.

The following examples 4 and 6 describe the use of different carrier materials and forms as well as connection methods that correspond to the desired application purpose.

Example 4

Coupling of monomeric human albumin to silicate carriers

As carriers, either silica gels or microglass spheres (Controlled Pre Glass = CPG) come into question. 10 g of an aminated glass support (aminopropyl-CPG, Pierce) is shaken with 100 ml of a 2.5% aqueous glutaraldehyde solution for 4 hours at room temperature. After activation, the carrier is thoroughly washed with deionized water until it is aldehyde-free. The thus activated carrier is incubated with 200 mg of human serum albumin in 0.1 M phosphate buffer (pH 8.0) for one hour. After blocking with 1 M ethanolamine and thorough washing with phosphate buffer, the carrier is ready for use.

200 ml of a pre-S(2)-HBsAg-containing cell culture supernatant (11 mg pre-S(2)-HBsAg, 950 mg protein) is incubated with 10 ml of the affinity carrier for 2 hours at 14°C. Unbound protein is removed by washing with buffer on a sinter filter, and the specifically bound pre-S(2)-HBsAg is eluted by incubating the charged carrier in 10 ml of 8 M urea solution. This experiment yielded 6.8 mg of highly purified pre-S(2)-HBsAg with a total protein amount of 5.4 mg (determination according to Bradford).

Example 5

Binding of monomeric human albumin to membranes

Current as carriers are membranes of e.g. nylon, polyvinylidene difluoride or cellulose-polyacrylamide blend polymers. Nylon membrane (e.g. Zetabind, CUNO) is partially hydrolysed by incubation in HC1/H20. The thereby released amino groups are reacted with 1 M oxaldialdehyde in the presence of 0.1 M sodium cyanoborohydride at pH = 7 for two hours. After washing with deionized water, human serum albumin (10 mg/ml in 0.1 M phosphate buffer pH = • 7) is connected to this membrane activated with reactive aldehyde groups in the presence of 0.1 M sodium cyanoborohydride. Excess reactive groups are blocked by the addition of 1 M ethanolamine. After washing with phosphate buffer (0.1 M; pH = 8), the membrane can be used for selective binding of pre-S(2)-containing proteins.

The binding capacity of a membrane prepared in this way is 5 to 10 µg pre-S(2)-HBsAg/cm 2 .

Example 6

Binding of monomeric human albumin to microtiter plates

Functionalized polystyrene sheets can be used for this, such as e.g. an amino plate (with primary amino groups) or a carbo plate (with carboxyl groups) (Nissho Iwai, Tokyo) or similar materials.

100 µl of a solution of N-ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline (40 mM in 50% aqueous ethanol) is pipetted into each well of a 96-well microtiter plate and incubated for 2 hours at 40°C. . After thorough washing with ethanol and deionized water, 200 µl of a 1% aqueous solution of human serum albumin is pipetted into each well and incubated overnight at +4°C. After blocking with respectively 1 M ethanolamine and 1 M acetate buffer pH = 4 and subsequent washing, the plate is ready for use.

100 µl of a pre-S (2)-HBsAg-containing cell culture supernatant is pipetted with increasing dilutions into the prepared microtiter plate and incubated for 2 hours at +4°C. After washing with buffer and incubation with enzyme-labelled anti-pre-S(2)-HBsAg (monoclonal antibodies) according to the known ELISA technique, the bound amount of pre-S (2) protein is determined by substrate addition by absorption spectrometry according to known methods.

If the microtiter plate is loaded with SH-denatured pre-S(2)-HBsAG or synthetic pre-S(2)-peptide, a plate thus treated can be used for the determination of anti-pre-S(2)-antibodies (e.g. of patient serum). 10 mg pre-S (2)-HBsAg in 10 ml phosphate buffer (0.1 M; pH = 7.0) is incubated with 100 µl mercaptoethanol 9 10% sodium dodecyl sulfate (SDS) for 5 min at 100°C. Thereby, the immunologically active S-antigen is destroyed by HBsAg (Milich D.R. et al.,

"Enhanced immunogenicity of the Pre-S region of hepatitis B surface antigen", Science 227: 1195-1199 (1985)). After blocking the SH groups with 500 iodoacetamide for one hour, excess reaction products are removed by dialysis, and

the pre-S(2) antigen is diluted with phosphate buffer pH = 7.4 to 100 µg protein/ml.

A prepared HSA microtiter plate is coated with this pre-S(2) solution and then washed. Thus prepared

plates can also be stored in dry form.

100 µl of an anti-pre-S (2)-containing rabbit serum is pipetted at increasing dilutions into the wells. After incubation at +4°C overnight, bound pre-S(2)-antibody is determined absorption spectrometrically by incubation with enzyme-labeled anti-rabbit serum and subsequent substrate addition.

Example 7

Binding capacity and yield of pre-S(2)-HBsAg using different albumin-carrier complexes.

Respectively, human serum albumin (HSA) and polymerized human serum albumin (poly-HSA) were bound, respectively covalently and adsorptively in comparable amounts, to different carrier materials. The binding capacity and yield of pre-S(2)-HBsAg after elution was determined using an HBsAg-specific ELISA method and is summarized in the following table.

From the table, it can be seen that in the present embodiment a higher yield of pre-S(2)-HBsAg is achieved with the insoluble polymeric carrier of the invention, where the monomeric human albumin is covalently bound to Sepharose, than with Sepharose to which polyalbumin is covalently bound .

These clearly higher yields can be traced back to the gentler elution conditions that are possible when using the carrier in the invention. Furthermore, it can be seen that precisely the covalent binding of HSA to the carrier gives the invention the property of pre-S(2) affinity, and a molecular sieve to which the albumin is only adsorptively bound is not suitable for

Claims (5)

1. Complex, characterized in that it consists of an insoluble, polymeric carrier, especially on an agarose or dextrose basis, to which monomeric human albumin is covalently bound, and a pre-S-hepatitis-B virus surface antigen, which over its pre- The S(2) part and/or pre-S(1) part is bound to the monomeric human albumin in eluable form. 2. Use of complex according to claim 1 in the purification of pre-S-hepatitis I/3 virus surface antigen. 3. Use according to claim 2 while simultaneously using a detergent. 4. Use according to claim 2 or 3, where the purification comprises gel filtration, ultracentrifugation, hydrophobic chromatography, lectin affinity chromatography or ion exchange chromatography. 5. Use of a complex according to claim 1 in the production of diagnostics and vaccines.