JP3802090B2 - Heat treatment method of apolipoprotein A-1 - Google Patents

Description

【００２５】
▲２▼カオトロピック剤
アポリポ蛋白Ａ-１の加熱処理に対して、チオシアン酸ナトリウム、塩酸グアニジン並びに尿素等のカオトロピック剤が顕著な安定化効果をもたらすことが判明した(表２参照)。
【００２６】
【表２】

【００２７】
▲３▼安定化効果に及ぼすカオトロピック物質濃度の検討
塩酸グアニジンについて０.４〜５.０Ｍの濃度域で、またチオシアン酸ナトリウムについては０.５〜３.０Ｍの濃度域で良好な安定化効果が観察された(表３参照)。
【００２８】
【表３】

【００２９】
▲４▼糖、糖アルコール類
アポリポ蛋白Ａ-１の加熱処理に対して、比較的高濃度の単糖類もしくはシュクロース等の二糖類を共存させた場合に顕著な安定化効果をもたらすことが判明した。とりわけ二糖類の効果は大きかった。一方、マンニトールで代表される糖アルコールでは大きな効果は認められなかった(表４、表５参照)。
【００３０】
【表４】

【００３１】
【表５】

【００３２】
▲５▼安定化効果に及ぼす糖濃度の検討
４０％(Ｗ／Ｗ)以上の高濃度域においてシュクロースはアポリポ蛋白Ａ-１に対する顕著な安定化効果をもたらした(表６参照)。
【００３３】
【表６】

【００３４】
▲６▼金属キレート剤
金属キレート剤について、１０ｍＭ程度の比較的低濃度の金属キレート剤にアポリポ蛋白Ａ-１に対する安定化効果を有することが判った。なお、ＥＤＴＡの単独の共存の場合、５０ｍＭ以上の高濃度域では期待される安定化効果を奏することはなかった(表７参照)。
【００３５】
【表７】

【００３６】
▲７▼ 安定化剤混合系
前出の安定化剤を組み合わせることによっても良好な安定化効果が観察された(表８参照)。
【００３７】
【表８】

【００３８】
実施例３
(ウイルス不活化効果)
手順: アポリポ蛋白Ａ-１を１０mg／ml含有する水溶液に、組成が５ミリモル／ｌのエチレンジアミン四酢酸二ナトリウム(ＥＤＴＡ)、５０ミリモル／ｌのトリス(ヒドロキシメチル)アミノメタン(Ｔｒｉｓ)、０.１モル／ｌの塩化ナトリウム、３モル／ｌの塩酸グアニジン、ｐＨ７.６となるように添加剤を加え、完全に溶解した。この溶液をガラス製バイアル瓶に分注し、ポリオ-Ｉ(強毒)、シンドビス、豚パルボウイルスを単独で添加し、６０℃で２０時間、経時的に水浴中にて加熱処理を施し、加熱後サンプルのウイルス感染価を測定した。培養細胞には、Ｖｅｒｏ細胞(ポリオ、シンドビス)、ＥＳＫ細胞(豚パルボ)を用い、さらに対照として２０％アルブミン製剤に豚パルボウイルスを添加し、６０℃、２０時間の加熱を行なった。
【００３９】
結果: ポリオ-Ｉ、シンドビスウイルスは、上記条件下で５時間の加熱により完全に不活化された。また熱抵抗性の豚パルボウイルスについては、臨床経験上、ウイルス伝播の危険性がないことが認められている２０％アルブミン製剤と比較して、同等の不活化効果が得られた(表９参照)。
【００４０】
【表９】

【００４１】
実施例４
(ウサギでの動物実験)
手順: ０.５％コレステロール食(日本農産)を１５週間、ニュージーランドホワイト種のウサギ(体重２〜２.５Ｋｇ:雄)に給餌し、その後正常食(日本農産)に切り替え、それと同時に非加熱及び本発明による方法で加熱されたアポリポ蛋白Ａ-１を静脈内に投与した。投与量は、週１回に４０ｍｇで４ヵ月間投与し、投与終了後、解剖し胸腹大動脈及び冠状動脈の動脈硬化病変部位の面積を非投与群と比較することで、効果の検討を行なった。
【００４２】
結果: 胸腹大動脈、冠状動脈共にアポリポ蛋白Ａ-１投与群では非投与群に比べて、有意に病変面積が小さく、さらに投与前と比べても病変面積は小さく、アポリポ蛋白Ａ-１の動脈硬化退縮・抑制作用が確認された(表１０参照)。また、非加熱及び加熱を施したアポリポ蛋白Ａ-１の間には、その効果に関しての差は認められず、加熱によるアポリポ蛋白Ａ-１の抗動脈硬化作用の低下は観られなかった。
【００４３】
【表１０】

[0001]
[Industrial application fields]
The present invention relates to a heat treatment method for a plasma fraction preparation. More specifically, the present invention relates to a heat treatment method for inactivating contaminated viruses of apolipoprotein A-1 containing products.
[0002]
[Background Art and Problems to be Solved by the Invention]
In recent years, with the westernization of lifestyle habits such as meals, an increase in the onset of ischemic heart disease due to arteriosclerosis in the 30s to 40s has become a problem. Atherosclerosis is pathologically classified into three types of atherosclerosis, Monskeberg-type sclerosis, and arteriole sclerosis. Above all, atherosclerosis is a basic lesion of ischemic heart disease. .
[0003]
Various proteins are contained in blood, and it has various functions necessary for maintaining vital activities. Among these, lipoprotein is a protein deeply involved in lipid metabolism. Lipoproteins exist as lipid-protein complex particles by combining lipids insoluble in plasma such as phospholipids, cholesterol, and triglycerides with a protein called apolipoprotein. Usually, due to its specific gravity, very low density lipoprotein (VLDL: d <1.006), intermediate density lipoprotein (IDL: 1.006 <d <1.019), low density lipoprotein (LDL: 1.019 <d <1.063), high density lipoprotein It is classified into (HDL: 1.063 <d <1.21) and fractionated by ultracentrifugation.
VLDL synthesized and secreted in the liver is degraded in the blood by the action of the enzyme lipoprotein lipase (LPL), and cholesterol is supplied to peripheral tissues in a form catalyzed to IDL and LDL. One possible cause of the establishment of the above-mentioned atherosclerotic lesion is the accumulation of LDL denatured due to abnormal lipid metabolism in peripheral tissues. That is, atherosclerotic lesions are characterized by foam cells that have accumulated esterified cholesterol. These foam cells are mainly monocyte-derived macrophages that have infiltrated under vascular endothelial cells. Macrophages take in LDL that has been denatured through receptors (scavenger receptors) and decompose them with lysosomes. As a result, cholesterol is released into the macrophages, and cholesterol is accumulated (foamed) in the esterified form under the action of the enzyme acyl CoA: cholesterol acyltransferase (ACAT) to form foam cells.
[0004]
On the other hand, there is a pathway for transporting cholesterol from peripheral tissues to the liver (reverse cholesterol transfer pathway), and lipoprotein that plays a major role in this pathway is HDL, which suppresses the progression of arteriosclerosis. It is thought to work. Furthermore, from an epidemiological survey, there is an inverse correlation between the incidence of ischemic heart disease and HDL cholesterol, and it is considered that HDL has an anti-arteriosclerotic effect.
[0005]
The anti-atherosclerotic action of HDL activates the enzyme lecithin / cholesterol acyltransferase (LCAT) and incorporates cholesterol accumulated in peripheral tissues into the particles in an esterified form (cholesteryl ester), which acts as a reverse transfer pathway. It depends on transport to the liver. It is apolipoprotein A-1, which is a main constituent protein of HDL, that contributes to the activation of the enzyme LCAT. Recently, EMRubin et al. Have shown that HDL actually reduces the risk of atherosclerosis (Nature: Nature 353 , p. 265-267 (1991)). They introduced the gene for apolipoprotein A-1, which is a major component of human HDL, into mice that are genetically susceptible to atherosclerosis. It has been reported that the regression of fatty plaque tissue that appeared in This suggests that apolipoprotein A-1, which is a main component protein of HDL, can be effective as a therapeutic agent for arteriosclerosis, that is, a therapeutic agent for ischemic heart disease.
[0006]
Conventionally, various drugs (hyperlipidemic drugs) that lower blood total cholesterol have been widely used as preventive drugs for the development of ischemic heart disease. However, these drugs have to be taken over a long period of time, and their side effects are unavoidable. In addition, there has been no fundamental treatment for ischemic heart disease, that is, an action to regress arteriosclerosis occurring in the coronary artery. Under such circumstances, apolipoprotein A-1 has an action to regress arteriosclerosis as described above, and is highly expected in the field of ischemic heart disease treatment in the future.
[0007]
By the way, in formulating apolipoprotein A-1 as a therapeutic agent for ischemic heart disease, since it is a plasma-derived protein and positioned as a plasma fractionation product, blood-derived viruses (hepatitis, HIV, etc.) In order to prevent infection, the virus must be inactivated by heating. It has been found that plasma protein preparations, particularly albumin, can be treated by heating at 60 ° C. for 10 hours to prevent infectivity of contaminating viruses without denaturing plasma proteins in the preparations. Virus inactivation methods are common. However, a substance to which the method of heat treatment at 60 ° C. for 10 hours can be applied must be stable to the treatment.
[0008]
However, the apolipoprotein A-1 which is the object of the present invention has an amphiphile in which polar amino acids (hydrophilic) are distributed on the front side and nonpolar amino acids (hydrophobic) are distributed on the back side with respect to the molecular axis. It is known that it has a sexual helix structure and self-associates in an aqueous solution concentration-dependently to form an aggregate. In addition, self-association is caused by heating and is an irreversible association that does not return to its original state once an aggregate is formed. Therefore, heat treatment of apolipoprotein A-1 in a solution state for the purpose of virus inactivation must be carried out under conditions that suppress the formation of aggregates by heating and reduce the decrease in activity.
[0009]
[Means for Solving the Problems]
The present invention stabilizes apolipoprotein A-1, which is a main constituent protein of HDL involved in the suppression of the progression of arteriosclerosis and regression, and heat treatment, so that hepatitis, HIV, etc. The purpose is to eliminate the possibility of virus transmission. Heretofore, no report has been found on the method of heat inactivation of contaminating virus of apolipoprotein A-1 with the above-mentioned purpose.
The present inventors have stabilized the heat of apolipoprotein A-1 when a chaotropic substance, a saccharide or a metal chelating agent is used alone or in combination as a stabilizer during the heat inactivation treatment of a contaminating virus. Was found to be remarkably enhanced, and the present invention was completed based on this new finding.
[0010]
The present invention provides a solution containing apolipoprotein A-1 or a chaotropic substance, a saccharide or a metal chelate as a stabilizer when infecting a transmissible virus that is likely to be mixed in the fraction. It is characterized in that the heat treatment is carried out in the presence of agents alone or in combination. Specifically, when heating at 30 to 100 ° C. for 1 minute to 20 hours, the chaotropic substance at a concentration of 0.1 to 8.0 M, the saccharide at a concentration of 40% (W / W) or more, or a metal chelate The agents are used alone or in combination. As the chaotropic substance used in the present invention, guanidine hydrochloride, sodium thiocyanate and urea are used, saccharides are monosaccharides such as glucose, xylose and fructose, disaccharides such as sucrose, trehalose and lactose, and metaldiamine is ethylenediamine. Examples of chelating agents that are easily soluble in water, such as tetraacetic acid (EDTA), ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (EGTA), nitrilotriacetic acid, ethylenediamine di (o-hydroxyphenylacetic acid), Of these, guanidine hydrochloride, sucrose, and EDTA are preferred embodiments, but are not limited thereto. Furthermore, the aforementioned stabilizing effect is expected to be further enhanced by the combined use of amino acids such as arginine, histidine, lysine, leucine, or salts thereof, and sodium chloride.
Apolipoprotein A-1, which is the subject of the present invention, is mainly contained in human plasma, and a method for separating and purifying human plasma as a starting material is already known (method using ultracentrifugation: Havel, RJ et al. al., Journal of Clinical Investment: J. Clin. Invest., 34 , p. 1345-1353 (1955), Scanu, A., Journal of Lipid Research: J. Lipid Res., 7 , p. 295 306 (1966), ion exchange chromatography: Edelstein, C. et al., Journal of Biological Chemistry: J. Biol. Chem., 247 , p. 5842-5849 (1972), hydrophobic chromatography: Carson, SD Biokimika Eto Biophysica Actor: Biochim. Biophys. Acta, 750 , p. 317-321 (1982), Poss, SE et al., Analytical Biochemistry: Anal. Biochem., 149 , p. 166-168 (1985) )) However, in the present invention, no matter what purification method is used, there are no special restrictions on the preparation. The pH of the solution to be heat inactivated is generally 5.0 to 10.0, preferably 6.0 to 8.0, and the content of the apolipoprotein A-1 in the solution or fraction is: About 0.1-30 mg / ml is preferable.
[0011]
As an index for judging the stability of apolipoprotein A-1 by heating, the following phospholipid binding ability and antifoaming ability using macrophages, which are considered to be one of the biological activities of apolipoprotein, were used. In addition, the sample used for confirmation of stability used what removed the above-mentioned stabilizer by dialysis after heat inactivation processing.
Phospholipid binding ability is the ability of apolipoprotein A-1 to bind to phospholipids resulting from the amphiphilic helix structure that is characteristic of its structure. For details, see the paper by HJPownall et al. (Biochemistry: Biochemistry 18 , pp. 574-579, (1979)). The measurement principle is to measure the decrease in turbidity due to the addition of apolipoprotein A-1 to dimyristoylphosphatidylcholine (DMPC) / cholesterol liposomes.
[0012]
The decrease in turbidity is observed because the addition of apolipoprotein A-1 to DMPC / cholesterol liposomes causes binding between apolipoprotein A-1 and DMPC, resulting in the formation of smaller particle liposomes. Is.
In the heat inactivation treatment of apolipoprotein A-1 by the stabilization method described in the present invention, as a result of measuring the phospholipid binding ability of apolipoprotein A-1 before and after heating, the decrease in phospholipid binding ability by heating Was effectively suppressed.
[0013]
The anti-foaming ability of apolipoprotein A-1 using macrophages is an indicator of how much apolipoprotein A-1 suppresses uptake of denatured LDL (acetyl LDL) by macrophages. For details on the measurement method, see Miyazaki et al. (Miyazaki, A. et al., Biochim. Biophys. Acta, 1082 , p.143-151 (1991); Miyazaki, A. et al. , Journal of Biological Chemistry: J. Biol. Chem., 269 , p. 5264-5269 (1994)). In the heat inactivation treatment method according to the stabilization method described in the present invention, apolipoprotein A-1 suppressed the decrease in antifoaming ability before and after heating, and some obtained the same results as before heating.
[0014]
In the present invention, several viruses having various forms were selected as models for inactivation of contaminating viruses, and the effects were confirmed in vitro. Three types of viruses were used: a porcine parvovirus with a single-stranded DNA virus and no envelope, a polio-I virus with a single-stranded RNA virus and no envelope, and a Sindbis virus with a single-stranded RNA virus and an envelope. Each virus suspension having a predetermined virus content was added to an apolipoprotein A-1 solution containing a stabilizer, and after heat treatment, the virus infectivity in the preparation was measured. Viral inactivation was measured using Vero cells for polio and Sindbis viruses, and ESK cells for porcine parvovirus, using 50% infectivity (TCID ₅₀ ) as an index. As a result, it was confirmed that the heat inactivation treatment of the present invention brings about a sufficient inactivation effect on the model virus under the condition that the activity of apolipoprotein A-1 is maintained.
[0015]
Furthermore, it was confirmed that apolipoprotein A-1 heated according to the present invention has an inhibitory effect on the progression of coronary arteriosclerosis when injected intravenously into a rabbit loaded with a high fat diet (high cholesterol diet). It was done. Therefore, it is expected that this apolipoprotein A-1 reduces the risk of blood-borne virus infection and can be used clinically very safely as a therapeutic agent for arteriosclerosis or an arteriosclerosis inhibitor.
[0016]
[Operation and effect of the invention]
The method of inactivating virus by heating apolipoprotein A-1 provided by the present invention stabilizes apolipoprotein A-1 against heat, and in the use of the preparation after heat inactivation treatment, hepatitis, AIDS It is extremely useful to eliminate the possibility of infection.
The heating conditions were as follows. A solution containing apolipoprotein A-1 was mixed with a chaotropic agent such as guanidine hydrochloride and sodium thiocyanate at a concentration of 0.1 to 8.0 M and 40% (W / W) or higher, or a metal chelating agent such as EDTA, alone or in combination, and heated at 30 to 100 ° C. for 1 minute to 20 hours, and biological activity of apolipoprotein A-1 by heating The decrease in phospholipid-binding ability and anti-foaming ability was significantly suppressed. It was also confirmed that inactivation of contaminating viruses was achieved to such an extent that practically no problem was observed under the above heating conditions.
Furthermore, it was also confirmed by animal experiments using rabbits that apolipoprotein A-1 heated under the above conditions has an anti-arteriosclerotic action. Therefore, apolipoprotein A-1 heated by the method according to the present invention can be used for the purpose of prevention or treatment of cardiovascular disease and post-cardiac surgery disease.
[0017]
Hereinafter, the present invention will be specifically described with reference to test examples and examples, but the present invention is not limited to these examples.
[0018]
【Example】
Example 1
(Preparation of apolipoprotein A-1)
The HDL fraction was collected from plasma by the ultracentrifugation method of RJHavel et al. First, the plasma was centrifuged at 30,000 rpm for 1 hour, and then the surface cream layer (chylomicron) was removed. Sodium chloride was added to the remaining plasma to adjust the specific gravity to 1.063, and this was centrifuged overnight at 30,000 rpm to remove the upper layer (VLDL, LDL), and sodium bromide was added to the remaining plasma to adjust the specific gravity to 1.21. After that, it was centrifuged again at 30,000 rpm for a whole day and night. After completion of the centrifugation, the upper layer (HDL) is collected, and this fraction is degreased with only ethanol / ethyl ether (EtOH / Ether = 3/2) mixed solution and ethyl ether, and then dried in a nitrogen stream. Apolipoprotein A-1 powder was prepared. This powder was dissolved in 3M guanidine hydrochloride solution and dialyzed with distilled water to prepare an apolipoprotein A-1 solution.
[0019]
Example 2
(Liquid heating)
Various stabilizers were allowed to coexist in the apolipoprotein A-1 solution prepared according to Example 1 alone or in combination, and liquid heating was performed. In the experiment, 10 ml of an aqueous solution containing 10 mg / ml apolipoprotein A-1 was used, and various stabilizers were added at predetermined concentrations, followed by heat treatment under predetermined conditions. By removing the stabilizer and using the phospholipid binding ability and antifoaming ability shown below as an index, the residual ratio of the activity before the heat treatment (value after heating at a ratio where the value before heating is 100) is Asked. The results are shown in the table below.
[0020]
(Measurement of phospholipid binding ability)
For the preparation of DMPC / cholesterol liposomes, first, 20 ml / ml DMPC in chloroform was mixed with 1 ml and 10 mg / ml cholesterol in chloroform at a ratio of 79.4 μl, and finally adjusted to a cholesterol concentration of 7 mol%. From this solution, a chloroform flask was used to remove the solvent chloroform in a nitrogen stream, and the remaining solvent was completely removed under reduced pressure to form a DMPC / cholesterol thin film in the flask. 1 ml of a buffer solution (8.5% potassium bromide / 0.01% sodium azide / 0.01% EDTA / 0.01M tris (hydroxymethyl) aminomethane, pH 7.4) was added to the thin film, A liposome solution of 20 mg DMPC / 7 mol% cholesterol was prepared. This liposome solution was diluted 40-fold with the above buffer solution to obtain 0.5 mg DMPC / cholesterol liposome, which was used as a substrate solution for measurement of phospholipid binding ability.
[0021]
The measurement was performed by adding 150 μl of a sample adjusted to apolipoprotein A-1 concentration of 2 mg / ml to 3 ml of the substrate solution at 23 ° C., and monitoring the decrease in turbidity of the solution at an absorbance of 325 nm for 30 minutes. . The phospholipid binding ability was determined from the turbidity value after 30 minutes.
[0022]
(Measurement of antifoaming ability)
Rat peritoneal macrophages (3 × 10 ⁶ ) were added with 25 μg / ml acetyl LDL and 250 μg / ml apolipoprotein A-1 and incubated for 18 hours, and then the amount of cholesteryl ester in the macrophages was determined by the method of Miyazaki et al. Quantification was carried out to determine the effect of apolipoprotein A-1 on the suppression of foaming of macrophages (antifoaming ability).
[0023]
(1) In the absence of stabilizer In the absence of stabilizer, it was found that 70% or more of the activity of apolipoprotein A-1 was already lost by heating at 60 ° C. for 2 hours. It was also found that a substantially correlated result was obtained between the phospholipid binding ability and the antifoaming ability as an index of the activity of apolipoprotein A-1 (see Table 1).
[0024]
[Table 1]

[0025]
{Circle around (2)} It was found that chaotropic agents such as sodium thiocyanate, guanidine hydrochloride and urea have a remarkable stabilizing effect on the heat treatment of the chaotropic agent apolipoprotein A-1 (see Table 2).
[0026]
[Table 2]

[0027]
(3) Examination of chaotropic substance concentration on stabilization effect Good stabilization effect in the concentration range of 0.4 to 5.0 M for guanidine hydrochloride and 0.5 to 3.0 M for sodium thiocyanate. Was observed (see Table 3).
[0028]
[Table 3]

[0029]
(4) Sugar and sugar alcohols Apolipoprotein A-1 was found to have a remarkable stabilizing effect when heat-treated with a relatively high concentration of monosaccharides or disaccharides such as sucrose. did. In particular, the effect of disaccharides was great. On the other hand, no significant effect was observed with sugar alcohols typified by mannitol (see Tables 4 and 5).
[0030]
[Table 4]

[0031]
[Table 5]

[0032]
(5) Examination of sugar concentration on stabilizing effect In the high concentration range of 40% (W / W) or more, sucrose brought a remarkable stabilizing effect on apolipoprotein A-1 (see Table 6).
[0033]
[Table 6]

[0034]
(6) Metal chelating agent It was found that a metal chelating agent having a relatively low concentration of about 10 mM has a stabilizing effect on apolipoprotein A-1. In the case of coexistence of EDTA alone, the stabilization effect expected in a high concentration range of 50 mM or higher was not achieved (see Table 7).
[0035]
[Table 7]

[0036]
{Circle around (7)} Stabilizer mixing system A good stabilizing effect was also observed by combining the above-mentioned stabilizers (see Table 8).
[0037]
[Table 8]

[0038]
Example 3
(Virus inactivation effect)
Procedure: An aqueous solution containing 10 mg / ml of apolipoprotein A-1 was added to disodium ethylenediaminetetraacetate (EDTA) having a composition of 5 mmol / l, tris (hydroxymethyl) aminomethane (Tris), 0.5 mmol / l. Additives were added to achieve 1 mol / l sodium chloride, 3 mol / l guanidine hydrochloride, pH 7.6 and dissolved completely. Dispense this solution into a glass vial, add polio-I (strongly toxic), Sindbis, and porcine parvovirus alone, heat at 60 ° C for 20 hours, and heat in a water bath over time. The virus infectivity value of the post sample was measured. As cultured cells, Vero cells (Polio, Sindbis) and ESK cells (pig parvo) were used, and as a control, porcine parvovirus was added to a 20% albumin preparation and heated at 60 ° C. for 20 hours.
[0039]
Results: Polio-I, Sindbis virus was completely inactivated by heating for 5 hours under the above conditions. In addition, the heat-resistant porcine parvovirus showed the same inactivation effect as compared to the 20% albumin preparation, which has been found in clinical experience to have no risk of virus transmission (see Table 9). ).
[0040]
[Table 9]

[0041]
Example 4
(Animal experiments with rabbits)
Procedure: Feed a 0.5% cholesterol diet (Japanese agricultural product) to New Zealand white rabbits (weight 2 to 2.5 Kg: male) for 15 weeks, then switch to normal diet (Japanese agricultural product), at the same time without heating and Apolipoprotein A-1 heated by the method according to the present invention was administered intravenously. The dose is 40 mg once a week for 4 months. After completion of the administration, the effect is examined by dissecting and comparing the area of the arteriosclerotic lesion of the thoracoabdominal aorta and coronary artery with the non-administration group. It was.
[0042]
Results: Both the thoracoabdominal aorta and coronary artery were significantly smaller in the apolipoprotein A-1 administration group than in the non-administration group, and the lesion area was smaller than before administration, and the apolipoprotein A-1 artery Curing regression / inhibition action was confirmed (see Table 10). Further, there was no difference in the effect between non-heated and heated apolipoprotein A-1, and no decrease in the anti-atherosclerotic action of apolipoprotein A-1 due to heating was observed.
[0043]
[Table 10]

Claims (5)

  When performing heat treatment for virus inactivation which contaminates a solution or fraction containing apolipoprotein A-1, a stabilizer selected from a chaotropic substance and a metal chelating agent is used alone or in combination. A method for heat-treating apolipoprotein A-1.   The method for heat-treating apolipoprotein A-1 according to claim 1, wherein the chaotropic substance is selected from guanidine hydrochloride, sodium thiocyanate, and urea.   The method for heat-treating apolipoprotein A-1 according to claim 1 or 2, wherein a chaotropic substance is present as a stabilizer at a concentration of 0.1 to 8.0 M.   The heat treatment of apolipoprotein A-1 according to claim 1, wherein the metal chelating agent is selected from ethylenediaminetetraacetic acid (hereinafter referred to as EDTA), ethylene glycol bis (2-aminoethyl ether) tetraacetic acid (hereinafter referred to as EGTA), and nitrilotriacetic acid. Law. The method for heat-treating apolipoprotein A-1 according to claim 4, wherein the metal chelating agent is EDTA.
more than