JPS61194027A - 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 removing the non-mercaptoalbumin in the albumin by chromatography. CONSTITUTION:The objective albumin preparation can be produced by separating the albumin fraction from human plasma by fractional precipitation, separating the mercaptoalbumin from the non-mercaptoalbumin in albumin by chromatography, removing at least a part of the non-mercaptoalbumin, 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 albumin preparations.

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., and at the onset of edema, ascites, etc. 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).
Mercaptalbumin consists of mercaptalbumin, which consists of amino acid at the 34th position counting from the N-terminus of the albumin molecular chain -
Albumin is a cysteine residue having an SH group, and non-mercaptalbumin is a combination of this cysteine residue and cystine, oxidized glutathione, or other
A low molecular compound having a 5-S- bond has an intermolecular -3H2
Refers to albumin that is bonded by an -S-S- exchange reaction and does not have a -3H group.Hereinafter, the proportion of mercaptoalbumin in albumin will be expressed as P NMA and defined by formula (1).

P□1-(Q□a/(Q□, +Q□,))X100(%
) (1) Here, Q, IM, , QMNA are each HMA.

Represents the amount of HNA.

As a result of intensive research, the present inventors have found that the P NNA of albumin in albumin preparations manufactured based on conventional methods is
They found that the concentration was extremely low compared to that in the plasma of healthy individuals. Furthermore, PNMA shows changes over time after blood collection and decreases over time, and although it is generally said that conventional albumin production methods do not denature plasma proteins, in reality P□1 increases during the process. It was also found that the

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 PMMA content has already decreased at the raw material stage, and there is also a risk of fractional precipitation and By going through manufacturing processes such as filtration and heating, even more P□.

is getting lower.

The present inventors found that P□ of conventional albumin preparations.

Based on the fact that the binding ability of conventional albumin preparations to various drugs and metabolites is lower than that of healthy people, it was determined that the low binding ability of conventional albumin preparations to various drugs and metabolites is due to the low P-activity. However, conventional albumin preparations are manufactured with a focus solely on the purity of albumin in the final product, and there is no control over the raw plasma, the manufacturing process, or the finished product. 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 plasma as a raw material, which includes the steps of separating and obtaining an albumin fraction from human plasma by a fractional precipitation method, and separating mercaptalbumin and non-mercaptalbumin in albumin by chromatography. The mercaptalbumin content is much higher than that of conventional albumin preparations, which includes the steps of separating and removing at least a portion of non-mercaptalbumin, and sterilizing the albumin by filtration and heat treatment. The present invention provides a method for producing a high-albumin preparation.

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 "large 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.

In the present invention, the method of separating mercaptalbumin and non-mercaptalbumin by chromatography, removing at least a portion of the non-mercaptalbumin, and increasing the proportion of mercaptalbumin in albumin is a method for separating mercaptalbumin and non-mercaptalbumin. The method is not particularly limited as long as it is possible, but for example, an ion exchange chromatography method (R
Obert D, Hagervaier and J
oseph F.

Foster, (1971), Biochemis
Try, 10.637), Sepharose with a 2-pyridyldisulfide group fixed thereon was used as the stationary phase, and after adsorbing only mercaptoalbumin as shown in Figure 3, cobaleft was eluted with cysteine, reduced glutathione, etc. Chromatography methods (J, Carlsson a
nd A, 5renaon.

(1974) Ff! B.S. Lett, 42, 1
84), a high-performance liquid chromatography method using a hard fully porous granular crosslinked copolymer having an alcoholic water group bonded to the main chain as a stationary phase (Japanese Patent Application Laid-open No. 59-67456) can be considered. The last-mentioned high-performance liquid chromatography method is particularly preferred in terms of separation efficiency and rapidity.

The operation of separating mercaptalbumin and non-mercaptalbumin by chromatography and removing a portion of non-mercaptalbumin to increase the proportion of mercaptalbumin in albumin may be performed at any point during the production process of albumin preparations. However, since mercaptalbumin may convert to non-mercaptalbumin during the long manufacturing process, it is best to carry out this process as late as possible in the process, and immediately before sterile filtration and heat treatment after collecting the albumin fraction. preferable.

Sodium phosphate and sodium sulfate used in chromatography are removed by dialysis or the like. This dialysis operation
This may be carried out using a dialysis module using a normal dialysis membrane such as a Visking tube or a hollow fiber (with a pore size that does not allow complete permeation of compounds having a molecular weight of at least 10,000 or less).

By performing this dialysis operation, low molecular weight compounds that were mixed in the albumin solution, such as cystine with -5-S- bonds, oxidized glutathione, homocystine, cysteine-homocystine (mixed disulfide type), etc. are removed at the same time. Can be removed.

By removing compounds with a molecular weight of about 10.000 or less that have -5-S- bonds, mercaptoalbumin is prevented from being oxidized and converted to non-mercaptalbumin during the subsequent production process of albumin preparations, resulting in An albumin preparation with a high P□ can be obtained. Furthermore, the PMMA of the preparation obtained through this removal step is stable and does not decrease upon storage.

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□1 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 rv as a detection means, respectively. , ,A, and is obtained from equation (1). The packing material and mobile phase used in liquid chromatography can be selected arbitrarily as long as they can separate mercaptoalbumin and non-mercaptoalbumin.
High performance liquid chromatography using hard gel as a packing material is preferable because it is rapid. 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 -SH groups in the preparation has increased, and this -5) I group is directly involved in binding with certain drugs, metabolites, etc., or -SH groups are present on the albumin molecule. 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 individuals" PNMA was measured in plasma of 20 healthy individuals by high performance liquid chromatography under the conditions shown below.

Conditions> Column Asahipak GS 520+ inner diameter 7.5 mm.

Length 50cta + 4 pumps [Asahi Kasei ■]
JASCO Corporation ■ Twincle detector UV detector (280nm) JASCO Corporation @ UVIDEG Sample Healthy human blood fi (20μm) within 1 day after blood collection
) Mobile phase 0.1M sodium phosphate + 0.3M sodium chloride (pH 6,9) Flow rate 0.5 mA! /akin Temperature: 28℃ Results> The areas of peak A (mercaptoalbumin) and peak B (non-mercaptoalbumin) in the obtained chromatogram, respectively. ,stomach. A □ was obtained, and PMMA was obtained by calculation using equation (1).

Table 1 As shown in Table 1, the average of P and □ of 20 cases of healthy human 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, P and □ of plasma of 8 patients with liver cirrhosis were measured. The results are shown in Table 2.

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

Reference example 4 “Decrease in PHMA due to plasma storage” After blood collection 1
The P concentration of healthy human plasma and the blood sputum of the same plasma stored at 3° C. for 30 days were measured using high performance liquid chromatography under the same conditions as in Reference Example 1. 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 ptrs^ of 17 conventional albumin preparations was measured. As shown in Table 3, their average value is 42±1
It was 4%.

Table 3 Example Blood collected from a healthy person was immediately centrifuged at 5°C to obtain plasma. When P□ 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 was fraction V.
) 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) 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)].

This fraction V (5% solution) was separated into mercaptoalbumin and non-mercaptoalbumin by high performance liquid chromatography under the conditions shown below, and only mercaptoalbumin was collected.

(Conditions) Column Asahi Kasei @ Asahipak GS
520P inner diameter 7.5ms+, length 50cII+ (
2) Pump JASCO Corporation @ Twincle detector JASCO Corporation @ UVIDEC (wavelength 2B0
na+) Mobile phase 30mM sodium phosphate + 150mM sodium sulfate (pH 6,9) Flow rate 3.0mβ/min Temperature 25°C The albumin thus separated and collected is removed to remove excess low molecular weight compounds such as sodium phosphate and sodium sulfate. I underwent dialysis for this purpose. The dialysis module used here is AM-Neo-150 (manufactured by Asahi Medical ■), which uses cuproammonium rayon hollow fibers (inner diameter: 230 μm, membrane thickness: 7 μm) and has a membrane area of 1.5 μm.

Before starting dialysis, apply 100 tall/n+in of physiological saline inside the hollow fiber as a priming.
It ran for 1 minute. Subsequently, plasma 250+AI was applied to the inside of the hollow fiber at 100 v using a peristaltic pump.
It was circulated at a flow rate of a l /+sin, and dialysate (0.08M
NaCJ! + Sodium phosphate buffer (pH 7,54) with ionic strength 0.02) at 500 all/s
I flushed it with +in. This dialysis was performed in a low temperature room at 4° C. for 1 hour.

After freeze-drying the obtained albumin, it was dissolved in sodium acetyltryptophan/sodium caprylate solution (0.08mM each) to create a solution with an albumin concentration of 5%, which was then sterilized by filtration and heat treated using the method described above. A formulation was obtained. The P□1 of this preparation was 91%.

Comparative Example Using the plasma (PMM & -70%) used in the example as a raw material, Fraxiquatin V obtained by Cobn's cold ethanol fractionation method without any special treatment was added to a sodium acetyltryptophan/sodium caprylate solution (respectively). 0.0
8mM), the albumin concentration was set at 5%, and a preparation was prepared by filtering and sterilizing and heating in the same manner as in the example. The P□1 of this formulation was 58%.

Reference Example 6 Sample No. 10+ among the 17 commercially available albumin preparations for which P was measured in Reference Example 5. and 1
The binding ability to phenylbutacin (an anti-rheumatic drug, abbreviated as PB) was compared for each of the albumin preparations obtained from Example 7 and Examples and Comparative Examples. 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 15,000.

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 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 left 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, it is known that cis-DDP (an anticancer drug) binds to albumin and is transported, but non-mercaptalbumin has no binding ability to this drug (50%).
983) J.

Biological Chemistry+ 2
Dandan-, No, 9. 5764) -pi.

Therefore, for example, when using anticancer drugs that have very large side effects, the albumin preparation produced by the production method of the present invention is effectively used in combination.

Enzymes such as Mata, pyruvate kinase, hexokinase, glycogen synthetase D1 phosphorylase phosphatase, adenylate cyclase, ribonucleotide reductase, γ-glutamylcysteine synthetase, etc.
It is known that low molecular weight compounds with 3-bonds inhibit their enzyme activity* (Ben
gt Mannervikand Kent Ax
elsson (1980) Biocheag,
J-+ 190 +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]

Figure 1 is a flow sheet showing the conventional manufacturing process of "heated human plasma protein", Figure 2 is a flow sheet showing the conventional manufacturing process of "human serum albumin", and Figure 3 is a flow sheet showing the conventional manufacturing process of "human serum albumin". 31 SΩ 10x-ss-smi Blue

Claims (1)

[Claims] A method for producing an albumin preparation using human plasma as a raw material, which includes the steps of separating and obtaining an albumin fraction from human plasma by a fractional precipitation method, and separating mercaptoalbumin and non-mercaptoalbumin in albumin by chromatography. 1. A method for producing an albumin preparation, comprising the steps of removing at least a portion of albumin, and sterilizing and filtrating albumin and heat-treating it.