JPS5840930B2 - Manufacturing method for hepatitis B vaccine - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to an inactivated hepatitis B virus (HBV) vaccine, and more specifically, it has high antigenicity and has been subjected to heat inactivation treatment sufficient to eliminate infectivity. High purity B
This article relates to a hepatitis vaccine and its manufacturing method.

The so-called HBV-related substances were discovered as Australian antigens by Blumberg in 1964, and since then they have been the causative agent of hepatitis that often occurs when human plasma or human plasma fractions obtained from it are injected into humans. As a result, it has developed into a major social problem.

Furthermore, it has recently become known that HBV is present not only in plasma but also in urine, feces, saliva, and semen, and that it can be transmitted from mother to fetus through the placenta.
The problem is getting bigger.

Now, HBV has a core antigen (HBcAg) and a surface antigen (HBcAg).
It is known that there are large particles with a diameter of about 49Hm consisting of HBsAg) and small particles with a diameter of about 20nm consisting only of HBsAg (WHO Technical Report Series 570, Spiral Hebatiteis (WHO Technical Report 5).
eries /16570, Viralhepat
itis) 1975), confirmation of the existence of Rano,
It is mainly performed using an immunoassay method based on the reaction between HBsAg and HBs antibodies against it.

Furthermore, it has already been revealed that a solution containing HBsAg subjected to HBV inactivation treatment is effective as a vaccine against hepatitis B.

Inactivation by heat is performed to eliminate HBV infectivity, and plasma albumin fraction preparations are generally heat-treated at 60° C. for 10 hours to suppress the occurrence of hepatitis during administration.

However, when administered, large amounts of antibodies are not produced in the human body.

Cultivation of HBV has not yet been successful. On the other hand, diluted serum from a patient with serum hepatitis was shaken for 1 to 3 minutes (1 to 3 minutes at 98°C).
It has been proposed to produce an inactivated vaccine (U.S. Pat. No. 3,735,004).

Although intramuscular administration of this vaccine does reduce the incidence of hepatitis, and even if it does occur, the disease is mild.
In general, heating also reduces antigenicity, so for more complete prevention and treatment, it is desirable to develop inactivated vaccines that have higher antigenicity and are highly purified and concentrated in terms of infectivity, efficacy, and side effects. It was because of this.

In order to obtain a heat-treated vaccine that does not reduce antigenicity, we first prepared a solution of human plasma proteins containing HBV in an aqueous medium with monosaccharides, trisaccharides, sugars as stabilizers. We have invented a method for producing an HBV inactivated vaccine that is sufficient to destroy infectivity in the presence of alcohols or mixtures thereof, but which requires heating at 60 to 120°C for a time that does not cause loss of antigenicity. Japanese Patent Application Publication No. 5
No. 0-95420, hereinafter referred to as (A)].

Furthermore, we have determined that solutions of HBV-containing human plasma proteins in aqueous media, in the presence of protein denaturing agents such as urea or guanidine, are sufficient to destroy infectivity, but not antigenic. Invented a method for producing a hepatitis virus vaccine with high antibody production ability that requires heating at 60 to 120°C for an increasing period of time.
No. 5817, hereinafter referred to as (B)].

These methods are compared to heat treatment at 60°C for 10 hours, which has been confirmed to be safe, or heat treatment at 98°C, for 1 to 3 minutes, which has been proven to be a safe vaccine treatment. Although the treatment is performed at a higher temperature for a longer period of time, it is safer and therefore can be administered in larger quantities.

In the method (B), for example, in an aqueous solution containing HBV, ? Add urea to a molar concentration and heat at 105℃ for 1
The doubling of the HBs antigen titer by heating for a period of time indicates that new antigenicity has been exposed.

Another feature of heat denaturation of HBV is enhancement of antibody production ability.

If we consider the antigenic part of HBV to be a butene and the denatured part to be a carrier, the denatured HBV itself is considered to be a combination of butene and a carrier.

Generally speaking, if a small molecule peptide that cannot exhibit antigenicity is used as a hapten and is bound to a carrier protein such as γ-globulin to form a habten-carrier conjugate, when this is administered to an animal, the hapten Starts producing antibodies.

Therefore, when denatured HBV is administered to humans as a vaccine based on the same principle, it is thought that the ability to produce antibodies against HBs antigens is increased.

This has been demonstrated in domestic rabbits, as shown in (B), where the antibody production ability was enhanced several times.

Although it is already clear that such an HBV inactivated vaccine is useful, if it is used as is, it may be accompanied by side effects that are not directly related to HBV.

In other words, when an aqueous solution containing HBV obtained from HBs antigen-positive plasma is heat-treated, not only HBV but also plasma protein components coexisting in the solution undergo denaturation, resulting in new antigenicity. It became like this,
If this substance is administered to humans together with a vaccine, antibodies against new antigenic parts will be produced, which will cause skin reactions and other side effects based on antigen-antibody reactions.

Therefore, the fact that most of the plasma components are removed from the hepatitis B vaccine, which contains such denatured proteins, suggests that H.
This is extremely important not only from the safety point of view that BV is inactivated, but also from the viewpoint of safety as a drug.

Another feature of the removal of plasma components from HBV vaccines is to reduce competition for antibody production and encourage the production of more antibodies against HBV.

In the case of a vaccine containing denatured plasma components, when administered to humans, not only will HBs resistance be produced, but at the same time antibodies against the new antigenic properties of the denatured protein will be produced, and antibodies against HBV will be produced due to competition. Production is attenuated.

In this sense, it is important to remove plasma components that cause side effects from the prior art inactivated vaccines, especially the vaccines (A) and (B).

Until now, HBs antigen purification has mostly been carried out by density gradient ultracentrifugation or affinity chromatography, but these methods require equipment and advanced technology, and are large-scale. It is difficult to perform this process, is extremely expensive, and is difficult to manufacture in large quantities at low cost.

The present inventors took advantage of the fact that the aqueous solution containing HBV is subjected to heat treatment, and used simple methods that have been used on an industrial scale to fractionate plasma proteins, such as ammonium sulfate fractionation. It is a simple method that combines a gel filtration method using a gel suitable for fractionating high-molecular proteins such as Sepharose, and a method that can be obtained using conventional density gradient supercentrifugation or affinity chromatography. High purity HB
We succeeded in obtaining a highly pure hepatitis B vaccine that was applied to the s antigen solution.

According to the present invention, an aqueous solution of human plasma proteins containing HBV is subjected to a heat treatment that causes loss of HBV infectivity but not antigenicity, and ammonium sulfate is removed from the treated aqueous solution.
Collect the fractions that precipitate at 7 to 50% saturation, filter the aqueous solution using a gel filtration material that can be used to separate substances with molecular weights ranging from several hundred thousand to several million, and collect the HBs antigen protein fraction. A method for producing a hepatitis B vaccine is provided.

The human plasma protein used in the present invention is HBV
In other words, it can be anything that can actually cause hepatitis in humans, as long as HBs antigen can be detected.

That is, plasma, serum, and various protein fractions obtained by known plasma protein fractionation methods are used, but among the raw materials used in the present invention, the most preferred raw material is α, which has a relatively large distribution of HBs antigen. - and β-globulin fractions.

It is also convenient for these two fractions to be used as raw materials because they are by-products when separating other useful plasma protein fractions.

The plasma protein fraction used was determined by ammonium sulfate precipitation method, Cohn's cold ethanol fractionation method [Cohn's E, J0, Strong's L,
E., Hughes, W.L., Malfoord, D.J1, Ashworth, J., N.O., Melin, M., and Tiller, H.L.
(Cohn E, J, Strong L, E
,, Hughes W., L., Mulford D.
, J., , Ashworth J., N6, Melin M.
and Taylor H,L, ): Journal
Of the American Chemical Society (Jou
rnal of the American Chem
icalSociety) Volume 68, Page 459,
[1946] and other well-known plasma protein fractionation methods.

For example, the α and β-globulin fractions can be obtained as ■ and ■-1 fractions by Cohn's fractionation method, and as 35% by ammonium sulfate precipitation method.
It is obtained as a fraction that precipitates from saturation to 50% saturation.

If the aqueous solution of the protein fraction is cloudy, it can be clarified to facilitate subsequent operations.

Clarification can be accomplished, for example, by centrifugation, or at about 60°C in neutral conditions.
This is carried out by heating and removing the formed precipitate.

The solution of hepatitis B vaccine material thus obtained may be subjected to heat treatment by any method for achieving the above-mentioned inactivation.

A preferable heat treatment is performed by either method (A) or (B) described above.

The heat-treated solution is clarified by centrifugation, if necessary, and then the precipitate produced by saturation with ammonium sulfate from 27 to 50% is collected.

If the amount of precipitant added exceeds the upper limit, it will contain so much contaminant protein that the subsequent purification will not be sufficiently effective, and if it is less than the lower limit, it will cause a decrease in yield. Not industrial.

The precipitate is collected by a suitable method such as centrifugation and dissolved in a small amount of water.

The resulting aqueous solution is dialyzed to remove the stabilizer, ammonium sulfate, etc. added during the heating.

If precipitates occur during dialysis, they are removed and finally,
A clear aqueous solution of phosphate buffered saline (PBS), pH 7.2, is obtained.

Regarding this, gels that can be applied to substances with molecular weights between hundreds of thousands and millions of millions, such as Sepharose 6 B (5e
pharose 6B), sepharose 4B (S e
Gel 1 hyperfractionation is performed by column using a commercially available suitable gel such as Iilarose 4 B).

Minute images are absorbance at 280 nm (E280 nm)
When determined by the equation, it is shown that a small amount of protein first flows out to almost the void volume, followed by some small fractions, and then most of the protein flows out.

The first fraction is collected and anti-human plasma rabbit serum is assayed for the presence of human plasma components by an immunological method such as two-way immunodiffusion or immunoelectrophoresis.

If the presence of human plasma components is detected, the gel filtration is repeated in the same manner.

Normally, human plasma components are no longer recognized after gel filtration occurs three times or less.

If human plasma components are found even after repeating gel filtration three times, or if a relatively large amount of human plasma components are found as a result of the first gel filtration, heat treatment is performed again and then the same method is performed again. By gel filtration, a vaccine aqueous solution free of human plasma components is finally obtained, and a sterile physiological isotonic solution is obtained to obtain a hepatitis B vaccine.

Examples of the present invention are shown below, but the present invention is not limited to these Examples.

In the examples, the RPHA titer is the value determined by measuring the HBs antigen titer of the sample using the Au5cell Kit (Guinapot Radioisotope Research Institute) by the reverse passive hemagglutination method (Japanese Patent Application Laid-open No. 12227/1983). be.

Example I A fraction consisting mainly of α- and β-globulins obtained by fractionating pooled plasma (200 pools) showing an HBs antigen titer of 1:512 by the RPHA method according to Cohn's low-temperature ethanol method. 300TL
After dialysis, most of the insoluble matter is removed by centrifugation.

After heating this solution at 60°C for 10 hours, the resulting precipitate is removed by centrifugation.

Ammonium sulfate was added to the resulting clear solution to make it 50% saturated, and the resulting precipitate was collected by centrifugation and suspended in a small amount of water.
Dialyze to remove ammonium sulfate.

At this time, a part of the denatured protein precipitates, so the precipitate is dialyzed until all the heat is removed, and the precipitate is removed by centrifugation to obtain a clear PBS solution with a pH of 7.2.

The clear solution thus obtained is fractionated by gel filtration using a 10×90 cIrL Sepharose 4B column.

The minute images are as shown in the attached drawings.

The first fraction containing HBs antigen (4-4 in the figure)
．． 41) was collected, concentrated by vacuum dialysis, and then 1.5×9
Gel filtration is performed using a 0 cm Sepharose 4B column.

At this time, the first fraction represents the main fraction, which was collected and subjected to vacuum dialysis against physiological saline, resulting in an RPHA value of 1:3.
A solution showing a concentration of 2000 was obtained and filtered for sterilization to obtain 54 ml of hepatitis B vaccine solution used for injection.

The obtained vaccine solution showed a negative precipitation reaction with anti-normal human plasma rabbit serum.

Further, no large particles of 40 nm were observed by electron microscopy.

Example 2 Fraction IV150P obtained in Example 1 was suspended in distilled water at 40°C and dialyzed, then insoluble materials were removed and the suspension was heated at 300 r.
An aqueous solution of rtl was obtained.

This aqueous solution was heated at 60°C for 1 hour to remove the formed precipitate, and then dialyzed against a phosphate buffer solution of pH 7.6 to
280 TLl of a buffer solution of 7,6 was obtained.

Mannitol was dissolved in this to a concentration of 20% and heated in a sealed tube in an oil bath at 105° C. for 1 hour.

The resulting precipitate was removed by centrifugation, and ammonium sulfate was added to the resulting clear solution to make it 50% saturated.
In the same manner as above, a solution having an RPHA value of 1:4000 was obtained, and sterilized and filtered to obtain 480 TIll of hepatitis B vaccine solution for use in injection.

No precipitation reaction was observed in the obtained concentrated vaccine solution with anti-normal human plasma rabbit serum in a two-way immunodiffusion method.

Furthermore, no large particles of about 40 Hm were observed by electron microscopy.

Example 3 After adding 1000 TI'Ll of distilled water vertically to 1000 pooled plasma consisting of 12 samples showing RPHA titer 1: 8000, the mixture was adjusted to pH 7.0 using 1N hydrochloric acid or hydroxide, and then diluted with ammonium sulfate. was added and dissolved until the degree of saturation reached 35%.

After the resulting fibrinogen and γ-globulin precipitates were removed by centrifugation, the ammonium sulfate saturation was increased to 50%, and the resulting precipitates were collected by centrifugation.

This was mixed with a phosphate buffer solution with a pH of 7.2, 0.06 molar and
After time dialysis, a solution of 200 rI'Ll was obtained.

Urea was added to this solution to give a 2 molar concentration, and 105
Heated at ℃ for 1 hour.

Urea is removed by dialysis, and the precipitated denatured protein is precipitated and removed by centrifugation.Ammonium sulfate is added to the clear solution to make it 50% saturated, the precipitate is collected by centrifugation, and a small amount of After suspending in water, the ammonium sulfate was removed by dialysis, and the precipitated denatured protein was removed by centrifugation to obtain a clear physiological saline solution.

3.5 x 90 crrL of the clear solution thus obtained
Gel filtration was performed using a column of Sepharose 6B, and the first two fractions were collected, concentrated, and sterilized by filtration to obtain 50 TL of a hepatitis B vaccine solution used for injection and having an RPHA titer of 1:320,000.

The obtained vaccine solution was tested against anti-normal human plasma rabbit serum.
The sedimentation reaction was negative.

Furthermore, no large particles of 4QHm were observed by electron microscopy.

[Brief explanation of the drawing]

The figure is a graph showing a filtration image of gel filtration using Sepharose 4B in the embodiment of the present invention described in Example 1.

Claims (1)

[Claims] 1. Heat treatment is applied to an aqueous solution of human plasma proteins containing hepatitis B virus (HBV), which causes the infectivity of HBV to be lost but not antigenicity, and the treated aqueous solution is precipitated by ammonium sulfate at 27 to 50% saturation. Proteins are collected, and the aqueous solution is gel-filtered using a gel filtration material that can be used to separate substances with molecular weights ranging from several hundred thousand to several million.
A method for producing a hepatitis B vaccine, which comprises collecting both components containing the s antigen.