JPS61194026A - Production of albumin drug preparation - Google Patents

Abstract

PURPOSE:To obtain an albumin preparation having high mercaptoalbumin content, by separating albumin fraction from human plasma by fractional precipitation, and reducing the non-mercaptoalbumin in the albumin to mercaptoalbumin. CONSTITUTION:The objective albumin preparation can be produced by separating the albumin fraction from human plasma by fractional precipitation, reducing at least a part of the non-mercaptoalbumin in the albumin to mercaptoalbumin using a reducing agent having mercapto group (e.g. cysteine), removing the low-molecular compound (e.g. cystine) produced by the reduction process, and subjecting the albumin to aseptic filtration and heat-treatment. Since the amount of mercaptoalbumin in albumin can be increased, the capability of albumin to bond with various drugs and metabolic products and carrying the components which was insufficient in conventional albumin preparation can be increased to the level comparable to or higher than the albumin in the normal human plasma.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to a method for producing an albumin preparation.

More specifically, the present invention relates to a method for producing an albumin preparation, which includes a step of increasing the ratio of mercaptalbumin in albumin.

In recent years, when performing blood transfusions, the use of so-called component preparations, in which only necessary components of blood are used instead of using blood as it is, has become popular. Among them, plasma fraction preparations are effectively used for the treatment of various diseases.

Albumin preparations are one of the most commonly used plasma fraction preparations, and are used in the treatment of hypoproteinemia and plasma exchange therapy for liver diseases, etc. Recently, they have been used in the treatment of terminal cancer patients and during surgery. Large doses are administered before and after surgery.

Conventionally, albumin preparations have been mainly administered for the purpose of maintaining colloid osmotic pressure during shock due to rapid filtration of plasma proteins due to bleeding, burns, etc., or when edema, ascites, etc. develop. However, the fields in which albumin preparations will be used in the future will be
It is expected that albumin's physiological activity, that is, its ability to bind to various substances, will be used to advance and expand advanced treatments for various diseases.

(Prior art) The albumin preparation used in the present invention refers to "heated human plasma protein" defined in the "Biological Product Standards" of the Pharmaceutical Affairs Bureau of the Ministry of Health and Welfare.
and "human serum albumin", and the purity of albumin in these total proteins is over 80% and 97%, respectively.
% or more.

Conventional albumin preparations are usually produced by Cohn's cold ethanol fractionation method. The manufacturing process consists of the steps shown in FIG. 1 for heated human plasma protein and as shown in FIG. 2 for human serum albumin. That is,
In either case, human plasma separated from collected human whole blood is used as a raw material, and plasma proteins are analyzed according to ethanol concentration, temperature,
Taking advantage of differences in properties (solubility) with respect to hydrogen ion concentration, salt concentration, etc., each component is sequentially separated and precipitated to obtain an albumin fraction, and sterilization filtration and heating to remove bacteria and viruses such as hepatitis. It consists of a process of applying processing.

(Problems to be Solved by the Invention) Albumin has a very important role in binding and transporting various drugs and metabolites. It has been found that the albumin preparations used for this purpose have weak or almost no binding ability with drugs such as phenylbutacin, which albumin is known to bind and transport, and with biological components such as cystine and oxidized glutathione. Ta.

Normally, albumin in human plasma consists of mercaptoalbumin (abbreviated as HMA) and non-mercaptoalbumin (HNA).
(abbreviated as ). Mercaptalbumin is the 34th amino acid counting from the N-terminus of the albumin molecular chain.
3) Albumin is a cysteine residue having one group, and non-mercaptalbumin is an albumin in which this cysteine residue and cystine, oxidized glutathione, or other low-molecular compound having a -5-S- bond form a molecule. Between-5H
It is bonded by an exchange reaction of 1-S-S-, and -SH
Refers to albumin in which no groups are present. Hereinafter, the proportion of mercaptoalbumin in albumin will be expressed as P, and the formula (
Defined in 1).

PHMA=(QIIM^/(QIIM^+QHNA)
)X100(%)(l) Here, Q■^, Ql (NA is HMA, respectively.

Represents the amount of HNA.

As a result of intensive research, the present inventors have found that the PHMA of albumin in albumin preparations manufactured based on conventional methods is
They found that it was extremely low compared to that of healthy human blood. Furthermore, the P content changes over time after blood collection and decreases over time, and although it is generally said that the conventional albumin production method does not denature plasma proteins, in reality, P production during the process It was also found that □ decreased.

In other words, albumin preparations manufactured based on conventional manufacturing methods are usually made from plasma that has been stored for a long time after blood collection, so the P□ is already low at the raw material stage, and it is difficult to separate it. By going through manufacturing processes such as precipitation, filtration, and heating, the PMMA becomes even lower.

The present inventors have developed a conventional albumin preparation.

Based on the fact that the PHMA is lower than that of healthy individuals, it was concluded that the low binding ability of conventional albumin preparations to various drugs and metabolites is due to the low PHMA. However, conventional albumin preparations are manufactured with focus only on the purity of albumin in the final product, and the purity of albumin in the raw plasma, during the manufacturing process, and the finished product is
There was no control of P IINA, and therefore, there was no idea of increasing the proportion of mercaptalbumin during the manufacturing process of conventional albumin preparations.

(Means for Solving the Problems) In order to solve the above-mentioned problems of conventional albumin preparations, the present inventors have actively adjusted the ratio of mercaptoalbumin in albumin during the manufacturing process of conventional albumin preparations. Added a process to raise the temperature. That is, the present invention is a method for producing an albumin preparation using human serum as a raw material, which includes a step of separating and obtaining an albumin fraction from human plasma by a fractional precipitation method, and a step of separating and obtaining an albumin fraction from human plasma using a reducing agent having a mercapto group. It is characterized by comprising a step of reducing at least a portion of non-mercaptalbumin to mercaptalbumin, a step of removing a low molecular weight compound generated by the reduction reaction of non-mercaptalbumin, and a step of sterilizing filtration and heat treatment of albumin. The present invention provides a method for producing an albumin preparation that has a much higher mercaptoalbumin content than conventional albumin preparations.

The present invention will be explained in detail below.

In the step of separating and obtaining the albumin fraction from human plasma by the fractional precipitation method, the conventionally used Cohn's low-temperature ethanol fractionation method is used, that is, the first step of the "heated human plasma protein production process" shown in Figure 1. A multi-stage method such as a fractional precipitation method from stage to stage 4 or a fractional precipitation method from stage 1 to stage 4 of the "human serum albumin production process" shown in Figure 2 may be adopted, The ionic strength, hydrogen ion concentration, temperature, ethanol concentration, and protein concentration may be adjusted in order to precipitate fibrinogen and immunoglobulin all together without separating them individually to obtain an albumin fraction.

The reducing agent used in the step of reducing at least a portion of non-mercaptoalbumin in albumin to mercaptoalbumin includes reducing amino acids such as reduced glutathione, cysteine, dithiothreitol having a mercapto group, mercaptoacetic acid, etc. Reduced amino acids, which are biological components, are preferred from the viewpoint of safety, but only the -5-S- bond formed between the cysteine residue, which is the 34th amino acid from the N-terminus, and the sulfur-containing compound. It is not particularly limited as long as it selectively reduces and does not reduce the 17 -5-8- bonds that other albumins originally have. Considering the shortening of reaction time, the amount of the reducing agent added is preferably from equimolar to 5 times the mole of albumin, preferably 2 to 3 times the molar amount. Further, the reaction time may vary somewhat depending on the reducing agent used, but in general, it may be several tens of minutes. In addition, the reaction temperature is 25-3
The temperature is preferably 7°C, and more preferably 35 to 37°C because the reaction time can be shortened.

After the reduction reaction is completed, excess reducing agent and low molecular weight compounds produced by the reaction are removed. The low molecular weight compounds produced by the reduction reaction include, for example, cystine and cysteine-glucthione (mixed disulfide type) when cysteine is the reducing agent, and cysteine-glutathione (mixed disulfide type) when reduced glutathione is the reducing agent. disulfide type) and oxidized glutathione. The method for removing these low molecular weight compounds having -5-S- bonds is not particularly limited, but for example, Sephadec G
A column was filled with gel such as -5
Chromatography in which separation is performed by developing with a developing solution that does not contain a low molecular weight compound having an -S- bond can be used.

Low molecular weight compounds with -5-S-bonds with albumin, i.e. cystine, oxidized glutathione, homocystine, cysteine-homocystine (mixed disulfide type)
Since there is a large difference in the elution volume of the two, it is possible to separate them almost completely.

In addition, ultrafiltration membranes having a large number of pores with a pore size that allows substances with a molecular weight of about io, ooo or less to pass through, but does not allow albumin to pass through, such as collodion membranes, gelatin membranes,
Using a cellophane membrane etc., albumin and -8-
Ultrafiltration methods can also be used to separate low molecular weight compounds with 3-bonds.

In addition, dialysis modules using regular dialysis membranes such as Visking tubes or hollow fibers (which have pores that allow substances with a molecular weight of about 10,000 or less to pass through, but not albumin) and -5- A dialysis method using a dialysate that does not contain compounds with S-bonds is also effective.

Of the three methods mentioned above, namely chromatography, ultrafiltration, and dialysis, hollow is the most preferred method because of its high albumin recovery rate, economic efficiency, operability, and the possibility of continuous processing, especially in the case of large-scale processing. Particularly preferred is a dialysis method using fibers.

By performing an operation aimed at removing low molecular weight compounds produced by the reduction reaction, compounds with a molecular weight of about io, ooo or less having -5-S- bonds that were mixed in the albumin solution before the reduction reaction, i.e. , cystine, oxidized glutathione, homocystine, cystine-homocystine (mixed disulfite type), etc. can also be removed at the same time. Molecular weight approximately 10,00 with -5-S- bond
By removing 0 or less compounds, mercaptoalbumin will not be oxidized and converted to non-mercaptalbumin during the subsequent production process of the albumin preparation, resulting in an albumin preparation with high PHMA. In addition, the P of the preparation obtained through this removal step
IA is stable and does not deteriorate upon storage.

The step of reducing non-mercaptalbumin to mercaptalbumin and the subsequent step of removing low molecular weight compounds produced by the reduction reaction are preferably carried out after isolating only the albumin fraction from human plasma. Alternatively, it is also possible to add a reducing agent to serum for reduction.

However, as mercaptoalbumin may convert to non-mercaptoalbumin during the long manufacturing process,
The reduction operation is preferably carried out as late as possible in the entire manufacturing process. However, it is preferable that the reduction reaction and subsequent removal of low molecular weight compounds be completed before the sterilization filtration and heat treatment steps.

It goes without saying that when performing these operations, sufficient care must be taken to ensure that each step satisfies the test items of the "Biological Product Standards."

P□ of plasma, albumin preparations, etc. is the peak area or peak height of mercaptoalbumin and non-mercaptoalbumin in a chromatogram obtained by liquid chromatography using ultraviolet absorption at a wavelength of 280 nm as a detection means.Q) IMA - , Q) INA, (1)
It can be found from Eq. The packing material and mobile phase used in liquid chromatography can be selected as long as they can separate mercaptoalbumin from non-mercaptoalbumin, but high performance liquid chromatography using hard gel as a packing material is superior in speed. This is preferable.

Such conditions are shown in, for example, Japanese Unexamined Patent Publication No. 59-67456.

On the other hand, cystine, oxidized glutathione, homocystine,
Analysis of low molecular weight compounds with -5-S-bonds, such as cysteine-homocysteine (mixed disulfide type), is carried out using conventional amino acid analysis, which involves separating them by ion exchange chromatography and detecting them by reacting them with ninhydrin and various fluorescent substances. This can be done by meter.

The reason why albumin preparations with excellent binding ability for drugs, metabolites, etc. can be produced by the method for producing albumin preparations performed in the present invention is not necessarily clear, but by increasing the ratio of mercaptoalbumin in albumin, albumin preparations with excellent binding ability for drugs, metabolites, etc. The number of highly reactive -3H groups in pharmaceutical preparations has increased, and these -3H groups are directly involved in binding with certain drugs, metabolites, etc., or -5)I groups on albumin molecules have increased. As a result of the formation, the conformation of albumin molecules in the vicinity changes,
This is presumed to be due to the formation of binding sites for certain drugs or metabolites, which non-mercaptalbumin does not have.

(Example) Below, experiments and examples conducted in connection with the present invention will be shown.
It goes without saying that the scope of the present invention is not limited to these examples.

Reference Example 1 "Analysis of Albumin in Plasma of Healthy People" P and □ of 20 healthy people's plasma were measured by high performance liquid chromatography under the conditions shown below.

<Conditions) Column Asahipak GS 520+ Inner diameter 7.5 mm.

Length 50cm, 4 [Asahi Kasei Kogyo ■] Pump JASCO Corporation (II Twincle detector UV detector (280 nm) JASCO Corporation ■ UVIDEC Sample Healthy human plasma (20μi) within 1 day after blood collection Mobile phase 0.1M phosphorus Sodium chloride + 0.3M sodium chloride (pH 6,9) Flow rate 0.5 mj2/min Temperature 28℃ <Results> Calculate the areas of peak A (mercaptalbumin) and peak B (non-mercaptalbumin) in the obtained chromatogram. They were determined as QHMA and QH□, respectively, and PIIMA was obtained by calculating according to (1) 2.

Table 1 As shown in Table 1, the average PHMA of 20 healthy subjects' plasma was 72±4%.

Reference Example 2 "Analysis of albumin in plasma of dialysis patients" Using high performance liquid chromatography under the same conditions as Reference Example 1,
When PHMA was measured in the plasma of 112 dialysis patients before dialysis, the average was 45±8%.

Reference Example 3 "Analysis of Albumin in Plasma of Liver Cirrhosis Patients" Using high performance liquid chromatography under the same conditions as in Reference Example 1, PHMA in the plasma of 8 patients with liver cirrhosis was measured.

The results are shown in Table 2.

Table 2 As shown in Table 2, the average P of the plasma of 8 patients with liver cirrhosis was 48±6%.

Reference Example 4 "Decrease in PHMA due to Plasma Storage" The PHMA of healthy human plasma collected within 1 day after blood collection and the same plasma stored at 3°C for 30 days was subjected to high performance liquid chromatography under the same conditions as Reference Example 1. It was measured using P□, which was 74% before storage, became 58%.

Reference Example 5 "Analysis of Conventionally Commercially Available Albumin Preparations" Using high performance liquid chromatography under the same conditions as Reference Example 1, the PHMA of 17 conventional albumin preparations was measured. As shown in Table 3, their average value is 42±1
It was 4%.

Example 1 Blood collected from a healthy person was immediately centrifuged at 5°C to obtain plasma. When the PHMA in this blood was measured under the same conditions as in Reference Example 1, it was 70%.

Using this plasma, fractionation was performed by Cohn's cold ethanol method as shown below, and the albumin fraction (fraction
) was obtained. That is, the raw plasma has an ionic strength of 0.14,
The pH was adjusted to 7.2, the temperature was -2°C, the ethanol concentration was 8%, and the protein concentration was 5.1%, and the precipitate (fibrinogen fraction) was removed by centrifugation.

This supernatant was collected at an ionic strength of 0.12, a pH of 6.8, and a temperature of -6.
℃, ethanol concentration 2%, protein concentration 3.0%,
After removing the precipitate (immunoglobulin fraction) 2 by centrifugation,
The supernatant was heated to an ionic strength of 0.11, a pH of 5.2, and a temperature of -6°C.
The ethanol concentration was adjusted to 19% and the protein concentration was adjusted to 1.6%, and the precipitate was removed by centrifugation.

Next, this supernatant was mixed with an ionic strength of 0.094 and a pH of 5.8.
Temperature -5℃, ethanol concentration 40%, protein concentration 1.0%
After removing the precipitate by centrifugation, the supernatant was adjusted to ionic strength 0.11, pH 4.8, temperature -5°C, ethanol concentration 40%, and protein concentration 0.8%, and centrifuged. .

This precipitate was dissolved at pH 4.5, temperature -3°C, ethanol concentration 10%, and protein concentration 3%. After removing the precipitate by centrifugation, the supernatant was collected at an ionic strength of 0.01 and a pH of 5.2.
The mixture was adjusted to a temperature of -5°C and an ethanol concentration of 40%, and centrifuged to collect a precipitate [albumin fraction (fraction V)].

To this fraction V (5% solution), 3 times the molar amount of reduced glutathione relative to albumin is added, and after standing at 37°C for 1 hour, excess reduced glutathione and low molecular weight compounds generated by the reaction are removed. Therefore, dialysis was performed using a hemodialysis module. The dialysis module used here is an AM-Neo 150 (with a membrane area of 1.5 d) using cupro ammonium rayon hollow fibers (inner diameter 230 μm, membrane thickness 7 μm).
Manufactured by Asahi Medical.

Before starting dialysis, apply 100ml/l1lin of physiological saline inside the hollow fiber as a priming.
It ran for 1 minute. Subsequently, 250n+42 plasma was applied to the inside of the hollow fiber using a peristaltic pump.
The dialysate (0.08 M
NaCl + ionic strength 0.02 (7) sodium I77 acid buffer (pH 7,54)) at 500 m
! ! /min. This dialysis was performed in a low temperature room at 4° C. for 1 hour. As a result of analysis using an amino acid analyzer, cystine and oxidized glutathione were not detected in the plasma after dialysis.

Sodium acetyltryptophan and sodium caprylate were added to the thus obtained dialysis fraction (1) solution at a concentration of 0.08mM,
After making a solution with an albumin concentration of 5%, the pore size is 0.
．． The mixture was sterilized by filtration using a 22 μm filter and heated at 60° C. for 10 hours to obtain a preparation.

The P□ of this preparation was 84%.

Example 2 Using the plasma (PIIMA = 70%) used in Example 1 as a raw material, Fraction V (5% solution) obtained by Cohn's cold ethanol fractionation method similar to Example 1 was added to Add 3 times the molar amount of cysteine and heat to 37°C.
After standing for 1 hour, excess cysteine and low molecular weight compounds produced by the reaction were removed by dialysis in the same manner as in Example 1. The thus obtained post-dialysis fraction V
The solution was filtered and sterilized and heated in the same manner as in Example 1 to obtain a preparation.

The PM□ of this formulation was 75%.

Comparative Example Using the plasma (PI4MA = 70%) used in Example 1 as a raw material, fraction V obtained by Cohn's cold ethanol fractionation method, which is subjected to special treatment, was added to a sodium acetyltryptophan/sodium caprylate solution (each with 0% .08mM), the albumin concentration was set at 5%, and the same procedure as in Example 1 was performed to sterilize by filtration and heat treatment to prepare a preparation. The PHMA of this formulation was 58%.

Reference Example 6 Among the 17 commercially available albumin preparations whose P□ was measured in Reference Example 5, sample No. 10.14. and 17
The binding ability to phenylbutacin (an anti-rheumatic drug, abbreviated as PB) was compared for each of the albumin preparations obtained in Example 1 and Comparative Example. The method is shown below.

The albumin concentration of the above five albumin preparations is approximately 4%.
4 times the molar amount of phenylbutacin relative to albumin was added. After standing at 37°C for 30 minutes, centrifugally filtered,
The amount of unbound PB was determined by measuring the absorbance of the filtrate at 265 nm (the maximum wavelength of PB).

For centrifugal filtration, a centrifugal protein binding tester (cent-free) manufactured by Amicon was used. Note that the molecular weight cutoff of the membrane of this tester was 1,50,000.

Table 4 lists the number of moles of PB bound to 1 mole of each albumin (abbreviated as Alb).

Reference Example 7 The same five types of albumin preparations as in Reference Example 6 were compared in their binding ability to cystine. The method is shown below.

The albumin concentrations of the five albumin preparations were made the same at about 4%, and cystine was added in a molar amount 3 times that of albumin. This was sterilized by filtration using a filter with a pore size of 0.22 μm (manufactured by Millipore), and allowed to stand at 37° C. for 2 days. Centrifugal filtration was performed in the same manner as in Reference Example 6, and the amount of cystine in the filtrate was measured using an amino acid analyzer.

Table 5 lists the number of moles of cystine bound to each mole of albumin.

(Effect of the invention) By using the method for producing an albumin preparation of the present invention, it is possible to increase the ratio of mercaptoalbumin in albumin, so that albumin binds with various drugs, metabolites, etc., which were lacking in conventional albumin preparations. , it is possible to obtain an albumin preparation whose transport ability is as high as or higher than that of albumin in plasma of a healthy person.

In addition, it has been found that the binding ability of albumin with drugs, etc. is approximately proportional to the ratio of mercaptalbumin in albumin. The ratio of mercaptalbumin therein can be adjusted as desired, and as a result, albumin preparations with various levels of drug-binding ability can be produced.

Since the albumin preparation obtained according to the production method of the present invention is rich in mercaptoalbumin, it has excellent binding ability with certain drugs.

For example, cis-DDP (an anticancer drug) is known to bind to albumin and be transported, but non-mercaptalbumin has no binding ability for this drug (5
Teven L. Gonias et al (198
3) J.

Biological Chemistry, 258
+ No, 9+ 57643 m Therefore, for example, when using anticancer drugs etc. that have very large side effects, the albumin preparation produced by the production method of the present invention is effectively used in combination.

In addition, enzymes such as pyruvate kinase, hexokinase, glycogen synthetase D, phosphorylase phosphatase, adenylate cyclase, ribonucleotide reductase, and γ-glutamylcysteine synthetase are capable of producing -5- oxidized glutathione, cystamine, etc.
It is known that low molecular weight compounds having 3-bonds inhibit their enzymatic activity. (Bengt
Mannervikand Kent Axelss
on (1980) Biochem, J., 19
0 +125~130].

Therefore, since the albumin preparation obtained by the production method of the present invention has excellent reducing ability, it can be administered to patients whose body fluid redox balance is tilted toward oxidation, such as hemodialysis patients and other renal disease patients. It is also extremely effective to do so.

[Brief explanation of drawings]

FIG. 1 is a flow sheet showing the conventional manufacturing process of "heated human plasma protein", and FIG. 2 is a flow sheet showing the conventional manufacturing process of "macro serum albumin". Built/1a Kasha 5 Shino 2t

Claims (1)

[Claims] A method for producing an albumin preparation using human plasma as a raw material, comprising the steps of separating and obtaining an albumin fraction from human plasma by a fractional precipitation method, and using a reducing agent having a mercapto group to reduce at least one non-mercapto albumin in the albumin. A method for producing an albumin preparation, comprising the steps of: reducing non-mercaptalbumin to mercaptalbumin; removing low molecular weight compounds produced by the reduction reaction of non-mercaptalbumin; and sterilizing and heat-treating albumin. .