JPH0348168B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a heat treatment method for inactivating hepatitis viruses that are likely to be contaminated in an aqueous solution containing α-fetoprotein. The ascites and plasma of primary liver cancer patients contain high concentrations of α-fetoprotein (AFP), which is used as a raw material (immunogen) for the production of anti-AFP antibodies used for the treatment and prevention of AFP-producing cancers. or affinity group for affinity chromatography)
It is used as a reagent material for liver cancer diagnosis. Anti-AFP antibodies bind to AFP-producing cells,
It has been shown that it exhibits cytotoxicity, and by applying this phenomenon, it becomes possible to treat or prevent AFP-producing cancers using anti-AFP antibodies or AFP antibodies conjugated with anticancer drugs. This anti-AFP
Antibodies are usually collected by administering purified AFP as an immunogen to a suitable animal (eg, sheep, horse), collecting the antiserum after inoculation, and collecting the antiserum. As a recovery method, it is produced by affinity chromatography using AFP, which is usually contained in ascites and plasma of primary liver cancer, as an affinity group. AFP can be purified from ascites or plasma of primary liver cancer patients by affinity chromatography, gel filtration, etc.
Cancer Res, 30 , 2507–2513
(1970), Biochemica et Biophysica Acta, 278 , 293
~298, 1972)] is known. However, in recent years, the onset of serum hepatitis associated with blood transfusions has become a major social problem, and it has been revealed that the cause is hepatitis viruses, and this problem is not included in individual human serum protein preparations obtained by fractionating plasma. ing. Although AFP is not directly applied clinically, it has been recognized that there is a high correlation between viral hepatitis and liver cancer (Hisaya Nishioka, “Australia antigen and ``Correlation of proteins'', History of Medicine, 80
(13), 816, 1972), there is concern about contamination with this hepatitis virus. In order to solve the problem of hepatitis virus development, human serum protein preparations in general, and albumin preparations in particular, are
It has been discovered that heat treatment at 60°C for 10 hours can inhibit the infectivity of hepatitis viruses without altering albumin, and albumin preparations subjected to this heat treatment are now safe for clinical use as pharmaceuticals. Because of this, the hepatitis virus inactivation method, which involves heat treatment at 60°C for 10 hours, is being applied to other human serum protein preparations. However, for materials to which this 60°C, 10 hour heat treatment method can be applied, the material itself must be stable against this treatment. However, when an aqueous solution containing AFP was heat-treated at 60°C for 10 hours, the AFP antigen titer significantly decreased. Therefore, the present inventors developed AFP
The positive aqueous solution contains a main stabilizer selected from aliphatic amino acids having 2 to 5 carbon atoms, monosaccharides, and sugar alcohols, and/or a stabilizer selected from specific amino acids and organic carboxylic acids. In particular, they discovered that the heating stabilization effect could be enhanced, and based on this new knowledge, they completed the present invention. The present invention heat-treats an AFP-containing aqueous solution at 50 to 80°C using at least one main stabilizer selected from aliphatic amino acids having 2 to 5 carbon atoms, monosaccharides, and sugar alcohols at 10 to 30% W/V. and/or heating for 3 to 15 hours in the presence or alone of one or more stabilizers selected from amino acids having 6 to 15 carbon atoms and organic carboxylic acids having 3 to 10 carbon atoms. Consists of processing methods. The AFP-containing aqueous solution to be subjected to heat treatment is not particularly limited, regardless of whether it is of human or animal origin. The concentration of AFP contained in the solution to be subjected to heat treatment is preferably within the range of 0.1 to 10 mg/dl. In addition, the pH of the solution to be heat treated is generally 5 to 10, preferably
It is preferably 6.5 to 8.5 and adjusted with an appropriate low salt concentration buffer. The aliphatic amino acids having 2 to 5 carbon atoms, monosaccharides, and sugar alcohols as main stabilizers will be explained. The aliphatic moiety in the above aliphatic amino acid may be either linear or branched, and may be saturated or unsaturated, but preferably linear or saturated, respectively. The aliphatic amino acids include glycine and alanine, the monosaccharides include glucose, xylose, and fructose, and the sugar alcohols include mannitol and
Examples include, but are not limited to, galactitol, glucosaminitol, sorbitol, and galactosaminitol. The amount of the main stabilizer added is 10 to 30% W/V. Further, the amino acid having 6 to 15 carbon atoms as a co-stabilizer used in the present invention may be any of neutral amino acids, basic amino acids, acidic amino acids, aromatic amino acids, and the like. The carboxyl group of the amino acid may be esterified, and examples of the ester include methyl ester, ethyl ester, n-propyl ester, inpropyl ester, n-butyl ester, t-butyl ester, etc. Examples include lower alkyl esters of No. 4. Specific examples of amino acids having 6 to 15 carbon atoms include tryptophan, lysine, and glutamine, which may be acetylated. The organic carboxylic acid having 3 to 10 carbon atoms refers to a hydrocarbon residue substituted with a carboxyl group, and the hydrocarbon residue may be saturated or unsaturated. Examples of the hydrocarbon residue include an alkyl group (preferably an alkyl group having 1 to 4 carbon atoms such as a methyl group, an ethyl group, and a t-butyl group);
Examples include aryl groups (for example, Feniel group), aralkyl groups (such as benzyl group), and the like. The organic carboxylic acid may have a plurality of carboxyl groups, but preferably one or two carboxyl groups. Further, the organic carboxylic acid may be substituted with a hydroxyl group. There are no particular limitations on the salt in the organic carboxylate as long as it is physiologically acceptable, and preferred ones include:
Alkali metal salts (sodium salts, potassium salts, etc.), alkaline earth metal salts (calcium salts, etc.),
Particularly preferred are sodium salts and potassium salts. Specific examples of the organic carboxylates include physiologically acceptable acids such as propanoic acid, butanoic acid, pentanoic acid, caprylic acid, caproic acid, malonic acid, succinic acid, glutaric acid, adipic acid, citric acid, and mandelic acid. salts, especially alkali metal salts (sodium salts, potassium salts). The total amount of the auxiliary stabilizer added alone or in combination is 0.06 to 3% W/V. The heating temperature is 50-80℃, preferably 60℃,
The heating time is 3 to 15 hours, preferably 10 hours. The electrical conductivity of the AFP-containing aqueous solution heat-treated in this manner is two to three times that of a normal solution. Therefore, in order to recover AFP, dialysis treatment,
The aqueous solution is either diluted or centrifuged to recover AFP as a precipitate, which is then combined with known treatment steps. In order to examine the heating effect of the present invention, HBsAg
Human-derived AFP-containing aqueous solutions recovered from ascites and plasma of primary liver cancer patients (hepatitis virus antigen) positive were respectively heat-treated at 60°C for 10 hours under the conditions of adding a stabilizer according to the method of the present invention. of
5 ml of AFP antigen with a concentration of 10 mg/dl,
As a result of inoculating chimpanzees and investigating the onset of hepatitis, no onset of hepatitis was observed even after one month. The method of the present invention thus provided eliminates the infectivity of hepatitis viruses that may be contaminated in preparations without causing loss of antigenic titer of AFP, which can be an immunogen for anti-AFP antibodies, which are valuable biological preparations. Because it is activated, it has great effects in industrial production of anti-AFP antibody preparations that include a virus inactivation step. The present invention will be explained below using examples, but the present invention is not limited thereto. Example Ascites of a primary liver cancer patient with pH 7.0 (AFP concentration: 7
mg/dl) 1 contains 200 g of glycine, 984 mg of acetyltryptophan, and 664 mg of sodium caprylate.
mg was added. After stirring and dissolving this well,
Heat at 60°C for 10 hours. After cooling, centrifuge and collect the supernatant, which contains 0.85% sodium chloride.
Dialyze against 0.02M phosphate buffer (PH7.2) to obtain a clear solution. After this, separately prepared anti-
Affinity chromatography was performed by passing the above solution through an AFP antiserum/Sepharose column, followed by Sephadex G-200 gel filtration to obtain 50 mg of purified AFP. The recovery rate was 71.4%. Experimental Example An experiment was conducted to confirm the stabilizing effect of various additives during heat treatment. For the experiment, AFP
Using 1 piece of ascites from a primary liver cancer patient with a concentration of 7 mg/dl, after adding various additives (the amount added is specified in the table), heat treatment was performed at 60°C for 10 hours, and the total AFP was compared with that before heat treatment. The residual rate of antigen concentration is shown in Table 1. As a result, the heat stability of AFP is increased by adding a co-stabilizer to each main stabilizer.

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Claims (1)

[Scope of Claims] 1. 50% of an aqueous solution containing α-fetoprotein
Heat treatment at ~80°C with 10-30 w/v% of one or more stabilizers selected from aliphatic amino acids having 2 to 5 carbon atoms, monosaccharides, and sugar alcohols and/or carbon atoms that may be esterified. Number 6 to 15 amino acids,
In the presence of one or more stabilizers selected from organic carboxylic acids having 3 to 10 carbon atoms or salts thereof, 3 to
A heat treatment method characterized by being carried out for 15 hours. 2 50% of aqueous solution containing α-fetoprotein
Heat treatment at ~80°C is performed with 10 to 30 w/v% of one or more main stabilizers selected from aliphatic amino acids having 2 to 5 carbon atoms, monosaccharides, and sugar alcohols, and carbon atoms that may be esterified. Claim 1, characterized in that the process is carried out for 3 to 15 hours in the presence of one or more stabilizers selected from amino acids having 6 to 15 carbon atoms, organic carboxylic acids having 3 to 10 carbon atoms, or salts thereof. The heat treatment method described in section. 3 The organic carboxylic acid or its salt is propanoic acid,
butanoic acid, pentanoic acid, caprylic acid, caproic acid, malonic acid, succinic acid, glutaric acid, adipic acid, citric acid, mandelic acid or a physiologically acceptable salt thereof; The heat treatment method according to item 1 or 2.