WO2005004902A1

WO2005004902A1 - Recombinant human serum albumin preparation accommodated in plastic container

Info

Publication number: WO2005004902A1
Application number: PCT/JP2004/010064
Authority: WO
Inventors: Yoshihisa Hama; Masakazu Mameda; Shinobu Mochizuki; Shouju Kameyama
Original assignee: Nipro Corporation; Mitsubishi Pharma Corporation
Priority date: 2003-07-10
Filing date: 2004-07-08
Publication date: 2005-01-20
Also published as: JPWO2005004902A1

Abstract

Recombinant human serum albumin preparations accommodated in a plastic container, comprising a plastic container in which liquid preparations of recombinant human serum albumin are accommodated and, packing the plastic container, a gas-barrier external packaging material having at least a layer containing an ethylene-vinyl alcohol copolymer. This product excels in long-term storability.

Description

Specification

Recombinant human serum albumin preparation in plastic container

Technical field

The present invention relates to a recombinant human serum albumin preparation in a plastic container.

Background art

Albumin, especially human serum albumin (HSA), is the most abundant protein in plasma and is made in the liver to maintain normal osmolarity in the bloodstream and to bind nutrients and metabolites. It has functions such as carrying these. Therefore, HSA is effective in treating trauma-related symptoms such as hemorrhagic shock patients and burn patients, hypoalbuminemia and fetal erythroblastosis. Such HSA is administered as a liquid formulation dissolved in purified water by injection or infusion into a human body at a clinical site.

Conventionally, a glass container has been used as a container for storing the HSA preparation. For example, it has been proposed to store the HSA preparation in a soft glass container which has been subjected to a dealkalization treatment (for example, see Japanese Patent Application Laid-Open No. 4-210106). No. 48). It has also been proposed that the HSA formulation be contained in a Type II glass pipe, for example, a silicate glass with standard surface treatment (ammonium salt or oxidized, Type II glass) (eg. (See Japanese Unexamined Patent Publication No. Hei 9-91 2 2 1 4 3 1). The purpose of each of these glass containers is to avoid the release of aluminum from the glass container. However, these glass containers have drawbacks such as being heavy and fragile. In addition, when injecting these HSA preparations into glass containers, there is a risk that bacteria and other contaminants in the air may be mixed into the HSA preparations in the containers, and these are not versatile. As a container that can be suitably substituted for a glass container in use, a lightweight and non-breakable plastic container is being studied. For example, Japanese Patent Application Laid-Open No. 2000-189492 discloses that a plastic container is used, and an HSA preparation is injected into the container in a system that does not come into contact with contaminants in the air. Open the container at extremely low temperature It is disclosed that by sealing the mouth end, it is possible to provide a plastic container containing an HSA preparation in which the generation of heat denatured albumin is completely suppressed. Japanese Unexamined Patent Publication No. 2003-107287 discloses that a plastic material is molded and processed so that the amount of water vapor transmission at a temperature of 25 ° C. and a relative humidity of 60% falls within a specific range. It is disclosed that the use of a permeable container allows the filled HSA formulation to maintain its properties with little or no long-term denaturation. Furthermore, Japanese Patent Application Laid-Open No. 2003-108287 discloses an outer packaging material having a low oxygen permeability by molding a plastic material so that the amount of oxygen permeation under the same conditions is within a specific range. It is also disclosed to use it together.

By the way, conventionally, HSA has been produced by fractionating blood collected from humans. However, such a production method is uneconomical and has a problem that it is difficult to supply blood. In addition, since blood contains an undesired substance such as hepatitis virus, development of a raw material that can be substituted for HSA has been desired.

Therefore, in recent years, mass production of albumin (recombinant human serum albumin; rHSA) by genetic manipulation and research and development of purification technology have been progressing, and therapeutic drugs by genetic recombination are about to be marketed.

In the case of HSA preparations, when formulated in the above plastic container (recombinant human serum albumin preparation in a plastic container), appearance changes and dimerization due to storage at 50 ° C for 60 days It has been newly found by the tests of the present inventors that the above-mentioned phenomenon of the formation of the rHSA polymer, the increase in the ammonia content, and the occurrence of such a phenomenon occur.

Although the details of why these phenomena occur are not known, the effects of oxygen exposure are of concern because the tests were performed in the presence of oxygen.

The above studies were performed to predict the stability of the drug product during long-term storage in a short period of time. This phenomenon of change in appearance, formation of polymer, and increase in ammonia content does not necessarily affect safety at the level of this test result. Hope to keep almost constant during the storage period Considered good.

As described above, the present inventors have developed a recombinant human serum albumin preparation in a plastic container that can minimize the change in appearance and the formation of a polymer, as well as minimize the increase in ammonia content, and have excellent long-term storage stability. The necessity was identified as a new issue to be solved.

Disclosure of the invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide a novel recombinant human serum albumin preparation in a plastic container excellent in long-term storage stability. is there.

The present inventors have conducted intensive studies to solve the above problems, and as a result, have completed the present invention. That is, the present invention is as follows.

(1) In a plastic container comprising a recombinant human 1, a plastic container containing a liquid preparation of serum albumin, and a gas-parallel outer packaging material having at least a layer containing an ethylene-butyl alcohol copolymer for packaging the plastic container Recombinant ぇ human serum albumin preparation.

(2) The preparation according to the above (1), wherein an oxygen scavenger is contained between the plastic container and the outer packaging material.

(3) The preparation according to the above (1) or (2), wherein the thickness of the layer containing the ethylene-vinyl alcohol copolymer of the outer packaging material is 5 to 50 m.

(4) The preparation according to any one of the above (1) to (3), wherein the thickness of the outer packaging material is 5 to 1000 m.

(5) oxygen permeability of the outer wrapping material, the temperature 25 ° C, at a relative humidity of ^{60%, 50 cm 3, !!!} 2, a day. 1 0 1 3. less than 25 h P a, the (1 The preparation according to any one of (1) to (4).

(6) The outer packaging material has a multilayer structure having an innermost layer formed of polyethylene, an intermediate layer formed of an ethylene-vinyl alcohol copolymer, and an outermost layer formed of nylon. The preparation according to any one of (1) to (5). (7) The preparation according to any one of (1) to (6) above, wherein the recombinant human serum albumin concentration in the liquid preparation of recombinant human serum albumin is 1 to 50 OmgZmL.

(8) The preparation according to the above (1), wherein the material of the plastic container is polyolefin, polychlorinated vinyl, or ethylene monoacetate vinyl copolymer.

(9) The preparation according to (1) above, wherein the liquid preparation of recombinant human serum albumin is sealed in a plastic container under aseptic conditions.

(10) After the plastic container is molded in the mold, while the plastic container is still in the mold, a liquid preparation of recombinant human serum albumin is injected into the plastic container, The preparation according to the above (9), wherein the opening is pressed and sealed by a mold.

(1 1) Any of the above (1) to (10), wherein the content of the recombinant human serum albumin polymer of at least dimer in the liquid preparation of recombinant human serum albumin is 3.8% or less. The preparation according to any one of the above.

(12) The ammonia content in the liquid preparation of recombinant human serum albumin is 4.6

The preparation according to any one of the above (1) to (11), wherein the preparation is 0 μgZmL or less.

Brief Description of Drawings

FIG. 1 is a simplified diagram showing a preferred example of a recombinant human serum albumin preparation 1 in a plastic container according to the present invention. The symbols in the figure indicate 1: recombinant human serum albumin in a plastic container, 2: plastic container, 3: r HSA preparation, 15: outer packaging material, respectively.

FIG. 2 is a diagram showing stepwise a preferred example of a method for producing a plastic container containing an rHSA preparation according to the present invention.

BEST MODE FOR CARRYING OUT THE INVENTION

The recombinant human serum albumin preparation in a plastic container of the present invention comprises a plastic container containing a liquid preparation of recombinant human serum albumin (rHSA preparation), and a layer containing the ethylene-butyl alcohol copolymer for packaging the plastic container. To At least a gas-paria outer packaging material.

According to the present invention having such a constitution, a recombinant human serum albumin preparation in a plastic container which is excellent in long-term storage stability is provided, while having a constitution in which a liquid preparation of recombinant human serum albumin is contained in a plastic container and formulated. can do. -Here, "excellent in long-term storage stability" means that the above-mentioned rHSA preparation in a plastic container is stored for 2 years at room temperature (30 ° C or less, specifically 10-30 ° C). This means that there is no significant change in the quality and properties including appearance, polymer formation, and ammonia content compared to the time of storage (meaning that HSA products (derived from plasma) Validity period).

As a means for predicting the long-term storage stability, the recombinant human serum albumin preparation (5 (w / v)%) in the plastic container was used. After standing for 60 days under the conditions of C and 75% RH, the appearance of the rHSA preparation is pale yellow and clear, and the rHSA polymer contains at least 3.8% or less of the dimer or more rHSA polymer contained in the rHSA preparation. If the ammonia content in the HS A preparation is 4.60 μg / mL or less, preferably 3.35 μg ZmL or less, the effect is equivalent to the above long-term storage stability. Can be presumed to be obtained.

The polymer formation in the rHSA preparation after the standing can be measured by calculating a high molecular weight fraction of dimer or more as an rHSA polymer by HP LC gel filtration. The ammonia content of the rHSA preparation after the standing was determined by ultrafiltration of a 5-fold dilution of the rHSA preparation through an ultrafiltration membrane (30,000 molecular weight cut). The solution can be calculated by measuring the absorbance of indophenol (; imax, 640 nm) and quantifying it using the Ammourea test method of the JP / General Test Method.

Hereinafter, the [1] recombinant human serum albumin preparation, [2] plastic container, and [3] outer packaging material, which constitute the present invention, will be described in detail.

[1] Recombinant human serum albumin preparation (rHS A preparation) The recombinant human serum albumin (rHSA) used in the present invention is human serum albumin having a molecular weight of about 67,000 produced by a genetic engineering technique. As rHSA, a gene encoding human serum albumin is isolated, inserted into an appropriate vector, and the obtained recombinant vector is introduced into an appropriate host to obtain a transformant, and the transformant is cultured. However, examples include rHSA, which is a culture extract purified by various purification techniques after culturing.

In the present invention, a liquid preparation (rHSA preparation) of recombinant human serum albumin prepared by formulating the above rHSA by a conventionally known method is contained in a plastic container. Such an rHSA preparation is, for example, a solution having an rHSA concentration of 1 to 50 Omg mL (preferably 10 to 100 mg L) dissolved in water for injection. The rHSA preparation of the present invention preferably contains a stabilizer such as acetyl tryptophan salt, an organic carboxylic acid having 6 to 18 carbon atoms or a salt thereof. Preferably, such a stabilizer, for example, acetyltryptophan, is present in an amount of about 20-6 mg per gram of rHSA contained in the rHSA formulation. Examples of the organic carboxylic acids having 6 to 18 carbon atoms include cabronic acid, caprylic acid, capric acid, lauric acid, palmitic acid, and oleic acid, and the salts thereof include alkali metal salts such as sodium and potassium. And alkaline earth metal salts such as calcium.

Specifically, rHSA in the present invention is prepared and formulated into the following method.

(i) Preparation of rHSA by genetic manipulation

The rHSA-producing host prepared by the genetic manipulation used in the present invention is not particularly limited as long as it is prepared through the genetic manipulation. Can be used. Specific examples include bacteria (eg, Escherichia coli, yeast, Bacillus subtilis, etc.) that have been made rHSA-producing through genetic manipulation, animal cells, and the like. In particular, yeast, especially Saccharomyces cerevisiae (for example, Saccharomyces cerevisiae) or Pichia (for example, Pichia pastoris) is used as a host. Is preferred. An auxotrophic strain or an antibiotic-sensitive strain may also be used. More preferably, Saccharomyces cerevisiae AH 22 strain (a, his 4, leu 2, can 1) and Pichia pastoris GTS 115 strain (his 4) are used.

The method for preparing these rHSA-producing hosts, the method for producing rHSA by culturing the host, and the method for separating and collecting rHSA from the culture, if known, should employ the same methods as those described above. Can be implemented. As a method for preparing an rHSA-producing host, for example, a method using an ordinary HSA gene (Japanese Patent Application Laid-Open Nos. 58-56684, 58-90515, and 58-150517) is described. ), A method using a novel HSA gene (see JP-A-62-29985 and JP-A-1-98486), and a method using a synthetic signal sequence (see JP-A-11-240191). A method using a serum albumin signal sequence (Japanese Unexamined Patent Application Publication No.

-1), a method of integrating a recombinant plasmid into a chromosome (see Japanese Patent Application Laid-Open No. 3-72889), a method of fusing hosts with each other (see Japanese Patent Application Laid-Open No. 3-53877), a medium containing methanol. And a method using a mutated AOX2 promoter (JP-A-6-90768, JP-A-4-90768).

), Expression of HS A by Bacillus subtilis (see JP-A-62-25133), Expression of HS A by yeast (JP-A-60-41487, JP-A-63 -39576, JP-A-63-74493), and expression of HSA by Pichia yeast (see JP-A-2-104290).

Among them, the method of causing mutation in a medium containing methanol is specifically performed as follows. That is, first, transcription in which HS A is expressed under the control of AOX1 promoter in the AOX1 gene region of an appropriate host, preferably Pichia yeast, specifically, GTS115 strain (NRRL Accession No. Y-15851) by a conventional method. A transformant is obtained by introducing a plasmid having a cut (see JP-A-2-104290). This transformant has a weak growth ability in a methanol medium. Therefore, this transformant is cultured in a medium containing methanol to cause mutation, and only viable strains are recovered. Get it. At this time, the methanol concentration is, for example, about 0.001 to 5%. The medium may be an artificial medium or a natural medium. The culture conditions are 15 to 40 ° C; For example, about 1000 hours.

In addition to the methods described in the above publications, rHSA-producing hosts can be produced by feeding a high concentration of glucose, methanol, or the like in an appropriate small amount by fed patch culture (half-batch culture). A method for obtaining a high concentration of cells and a product by avoiding high concentration substrate inhibition of the cells (see Japanese Patent Application Laid-Open No. 3-83595) is a method of adding rFSA by adding fatty acids to a medium. For example, a method for enhancing production (see Japanese Patent Application Laid-Open No. H4-293495) is exemplified.

(ii) Purification of rHSA

Various researches have been conducted on methods for isolating and purifying rHSA produced by the culture treatment from components derived from host cells and culture components with sufficient accuracy. For example, as a conventional method, a yeast culture solution containing rHSA is subjected to squeezing → ultrafiltration membrane treatment → heat treatment-> ultrafiltration membrane treatment, and then a cation exchanger and a hydrophobic And a method of subjecting it to a step such as column chromatography of a mat or anion exchanger (Japanese Unexamined Patent Publication (Kokai) No. 5-317979, Biotechnology of Blood Proteins. 1993, Vol. 227, 293-298). See). In addition, a method has also been reported in which the above-mentioned conventional method is further subjected to a step of chelating resin treatment or boric acid << salt treatment (JP-A-6-56883, JP-A-6-24585). See 789 publication). Further, a stream line method or the like using an adsorption fluidized bed technique after heat treatment of the yeast culture solution can be used (see Japanese Patent Application Laid-Open No. 8-116895). That is, a heat treatment is performed on a culture solution containing a host producing rHSA, the heat treatment solution is brought into contact with adsorbent particles floating in a fluidized bed, and the adsorbed fraction is collected. is there.

(iii) Properties of highly purified rHSA

RHSA in the present invention is a single substance having a molecular weight of about 67,000 and an isoelectric point of about 4.9. The rHSA is composed of a monomer and a trace amount of a dimer, and does not substantially contain a polymer or a decomposition product of a trimer or more. Here, the above “substantially does not include” At least immediately after production means that polymers or degraded products of the above trimer or more are not detected by techniques commonly used in the relevant field (SD S-PAGE, gel filtration, reverse phase chromatography, etc.). . In addition, rHSA in the present invention is substantially free of contaminants derived from the production host. Specifically, in the case of a r HSA25 (w / v)% solution, the host-derived contaminant component having an antigenicity of 1 ngZmL or less is exemplified. In the same case, Pai Gen is 1 EU_ / mL or less.

Coloring degree in the case of 2 5 (w / v)% of r HS A formulation, eight _35. No ₂₈ . But usually 0.5 0 1 5 or less, preferably 0.5 at 0 1 3 or less (specifically, A ₃₅₀ Bruno A ₂₈₀ is 0. 0 1 to 0.0 1 of about 5 is illustrated.) . The coloring degree was further suppressed by using known purification methods in further appropriate combination, that is, A ₃₅ . The value of Roh A ₂₈ 0 it is possible to obtain a low r HSA.

(iv) Formulation

The obtained rHSA can be formulated by known methods (for example, heat sterilization at 50 to 80 ° C for 30 minutes or more, ultrafiltration, sterilization filtration, dispensing, lyophilization, etc.). it can. Specifically, the formulation contains rHSA in a 5 to 25 (w / v)% solution (5 to 12.5 g as the amount of rH3) and has a pH of 6.4 to A liquid preparation having a ratio of about 7.4 and an osmotic pressure ratio of about 1 (as a ratio to physiological saline) is exemplified. The rHSA preparation of the present invention preferably contains sodium caprylate and / or acetyltryptophan as a stabilizer. The amount of the stabilizer to be added is, for example, about 20 to 6 Omg per 1 g of rHSA. The sodium content is, for example, 3.7 mgZmL or less. Usually, the stabilizer is added before heat sterilization, ultrafiltration, sterilization filtration, dispensing, freeze-drying, or the like. Thus, rHSA is not only stable in storage, but also stabilized in the formulation process.

[2] r Plastic container containing HS A preparation

FIG. 1 is a simplified cross-sectional view showing a preferred example of a recombinant human serum albumin preparation 1 in a plastic container according to the present invention. In the present invention, r HS A The shape of the plastic container 2 for accommodating the agent 3 is not particularly limited as long as it has an internal space capable of accommodating a liquid material, but a cylindrical shape having an open end 4 only on one side. (The part of the plastic container except for the open end 4 is sometimes called the “container body 5”.) Here, the open end 4 of the plastic container 2 in the present invention is an open end for accommodating the rHSA preparation in the container, and may be sealed to form the container head 6 as described later. Good (when used, open end 4 is opened, from which rHSA formulation is provided). In the plastic container 2 according to the present invention, the container body 5 is formed such that the cross-sectional area of the container body 5 is gradually reduced in the vicinity of the opening end 4 (hereinafter referred to as “shoulder 7”) and the above. It is preferable that the shoulder portion 7 and the neck portion 8 are formed so as to have a portion (hereinafter, referred to as a “neck portion 8 J”) extending to the opening end 4 with the same cross-sectional area as the shoulder portion 7. In the plastic container 2 of the present invention, the neck 8 and the open end 4 may be formed in a container. It may be formed integrally with the main body, or may be formed separately (for example, using a resin material different from that of the container main body).

The plastic container 2 in the present invention has a length D (a distance from the shoulder side end of the neck 7 to the open end) of the neck 8 and a length L of the container body (from the bottom of the container to the end of the shoulder 7 on the neck 8 side). (Preferably the distance from the bottom of the container to the flange 9)), and the ratio D L is preferably 0.1 to 0.5, more preferably 0.1 to 0.3, and particularly preferably 0 to 0.3. 1 to 0.15.

Further, the ratio of the container body 5 to the horizontal cut surface of the opening end 4 varies depending on the capacity of the target rHSA preparation 3, but is preferably 0.01 to 0.5, more preferably 0.5 to 0.5. It is 1 to 0.2, most preferably 0.1 to 0.15.

There is no particular limitation on the horizontal cut surface of the container body 5 and the opening end 4 of the plastic container 2 in the present invention, and examples thereof include a circular shape (true circular shape, elliptical shape) and a square shape (square shape, rectangular shape). Among them, a circular shape or a square shape is preferable. There is no particular limitation on the size of the plastic container 2 according to the present invention. Depending on the amount, various selections can be made.For example, when the horizontal cross-sectional shape of the container body 5 is a perfect circle, the diameter is preferably 10 to 15 Omm, and preferably 50 to 100 mm. More preferably, it is particularly preferably 60 to 8 Omm. When the horizontal cross-sectional shape of the container body is elliptical, the ratio of the major axis to the minor axis is preferably 1.1 or more, more preferably 1.5 to 5.0, and 2.0. It is particularly preferable that the value is from 3.0 to 3.0. When the horizontal cross-sectional shape of the container body 5 is rectangular, the ratio of the long side / short side is preferably 1.1 or more, more preferably 1.5 to 5.0, and 2 It is particularly preferred that it is from 0 to 3.0.

Further, the thickness of the container body 5 is usually 0.2 to 2.5πιπι, preferably 0.3 to 1.5 mm, most preferably 0.4 to 0.7 mm, and the thickness of the container neck 8 is Usually, 0.3 to 3. Omm, preferably 0.8 to 2. Omm, most preferably 1.0 to 1.5 mm.

The plastic container 2 may have a single-layer structure or a multi-layer structure. The material for forming the plastic container 2 is not particularly limited, and a known resin material can be appropriately used. R The temperature of the heat sterilization of the HSA preparation (40 to 60 ° C) A resin material that can withstand C) and that can be sealed by pressing with a mold at the head at a temperature of 40 to 70 ° C at the open end of the plastic container 2 containing the HS A preparation 3 as described below is preferable. . Examples of such a resin material include polyolefin such as polyethylene and polypropylene, polychlorinated vinyl, and ethylene monoacetate biel copolymer. Specifically, the melting point is 90-140. And polyolefins such as low-density polyethylene having a density of 0.890 to 0.940.

Plastic containers 2 in the present invention is the 10 1 3. 25 hP a pressure, the water vapor permeation amount of surface area lm ^2, per 24 hours, at a temperature 25 ° C and a relative humidity of ^{6 0%, 1. 5 gZm 2} / It is preferred that it be less than 10 .1 3.25 hPa per day (that is, 6 · 25 X 10—SmgZcm ² · 1013.25 hPa or less). For containers with a water vapor transmission rate exceeding this value, the rHSA concentration in the rHSA preparation may be 1 1 An excessive concentration of 0% or more is not preferable. The measurement of the water vapor transmission rate can be performed by a gravimetric method in accordance with the provisions of AST ME 96. Examples of the material that can achieve such a water vapor permeation amount include at least one resin material selected from polyolefin (polyethylene, polypropylene, etc.), polychlorinated vinyl, ethylene monoacetate vinyl copolymer, and the like. Among them, polyethylene is preferable, and a resin mixture of linear low-density polyethylene and low-density polyethylene is more preferable.

Further, when the plastic container is hermetically formed by heat sealing, the innermost layer is made of a material that can be heat-sealed at a relatively low temperature (about 40 to 70 ° C), for example, polyethylene or polypropylene. It is desirable to be formed from polyolefin, polyvinyl chloride, or ethylene / vinyl acetate copolymer. After the rHSA preparation is placed in a plastic container, it can be sterilized with steam or hot water.In this case, the temperature must be a temperature that does not denature rHSA (70 ° C or less). .

As a method for producing a plastic container, there are a professional molding method and an inflation method (extrusion molding method). To produce the plastic container of the present invention, any production method may be employed. The injection of the plastic container of the rHSA preparation can be performed at any stage during or after the production of the plastic container, provided that the plastic container is capable of accommodating the rHSA preparation at a temperature that does not denature the preparation. However, it is preferable to form the plastic container and inject the rHSA preparation together by the following method described in Japanese Patent Publication No. 2000-189492.

FIG. 2 is a diagram showing step by step the method described in Japanese Patent Application Laid-Open No. 2000-189492.

First, (1) a cylindrical plastic molded body (parison) 21 having one end closed is melt-molded, and (2) the molded body 21 is set inside the main body mold 22 (FIGS. 2 (a) and 2 (b)). (3) The mandrel 23 is pushed in from the upper opening end of the molded body 21, and a compressed gas is blown into the molded body 21 to form a container 24 (FIG. 2 (c)), (4) Insert the chemical liquid injection nozzle 25 into the mandrel 23 from the open end of the molded container 24, lower it to the bottom of the container 24, and inject the HSA preparation 3 (Fig. 2 (d)). Next, (5) the opening end of the obtained container 24 is pressed and sealed with a head mold 26. Instead of the step of blowing the compressed gas to form the container, it is also possible to form the container by evacuating the inside of the main body mold 22 from the suction hole 27 of the head mold 26.

The cylindrical plastic molded body 21 is melt-extruded into the main body mold 22 opened as described above, and a cylindrical plastic molded body (parison) having one end closed in the mold is produced. Compressed gas is blown into the molded body 21 in the mold 22 or the inside of the main body mold 22 is evacuated to form a container 24, and the container 24 is still in the main body mold 22. Injection of rHSA Formulation 3 into the inside of the container from the open end of the container allows the rHSA Formulation 3 to be contained in a plastic container in an aseptic condition with almost no exposure to air. It becomes. Further, after the container body 24 is rapidly cooled by cooling the main body mold 22, the opening end of the plastic container is pressed with the head mold 26 to seal it, and the container head is cooled by cooling the head mold 26. By cooling, it is possible to completely suppress the formation of denatured aluminin denatured product and form a plastic container containing the rHSA preparation.

Examples of an apparatus for performing the method for producing a plastic container containing the above-mentioned rHSA preparation include, but are not limited to, a blow-fill seal system such as a potter pack machine of Lomerag Inc. .

The conditions for producing the container are, for example, as follows.

(1) In order to melt-mold a cylindrical plastic molded body 21 having one end closed, first, a plastic material is heated to a melting temperature or higher and extruded from an extruder having a cylindrical orifice. Extrude so that it hangs between the opened molds.

(2) In order to install the molded body 21 inside the main body mold 22, the molten cylinder is placed in the main body mold 22 having a main body mold 22 and a head mold 26. The plastic molded body 21 is suspended by gravity and installed. At this time, the main body mold 22 and The temperature of the head mold 26 is normally controlled at 5 to 30 ° C. Therefore, the temperature of the cylindrical plastic molded body 21 that has hung in the molten state has dropped to about 100 to 150 ° C.

(3) In order to blow the compressed gas into the molded body 21 to form the container 24, a compressed gas, for example, air is blown from the open end of the cylindrical plastic molded body 21.

Then, the molded body 21 is expanded and brought into contact with the inner surface of the main body mold 22 to form the container 24. At this time, since the temperature of the main body mold 22 is normally controlled at 5 to 30 ° C., the temperature of the cylindrical plastic molded body 21 drops to, for example, 10 to 70 ° C. However, the portion (open end) of the upper part of the cylindrical plastic molded body 21 which does not contact the main body mold 22 is usually in a state of 100 to 150 ° C. Or, to mold the inside of the mold with a vacuum, follow an ordinary method. Also in this case, the portion (open end) of the upper part of the cylindrical plastic molded body 21 that does not contact the main body mold 22 is usually in a state of 100 to 150 ° C.

(4) Injecting the rHSA preparation 3 from the open end of the molded container 24 is carried out as quickly as possible after molding the container from the cylindrical plastic molded article. Since the temperature of the rHSA preparation 3 to be injected is usually close to 5 to 25 ° C., the temperature of the molded container body by injection of the rHSA preparation is 10 to 60. C. However, the inner surface temperature of the container opening remains at 50 to 90 ° C.

(5) The open end of the obtained plastic container is pressed and sealed with a head mold 26. This is preferably performed when the inner surface temperature of the container opening end is 40 to 70 ° C.

In the present invention, the plastic container containing the rHSA preparation needs to be subjected to heat sterilization as necessary to inactivate microorganisms and the like that may be mixed into the rHSA preparation. As for sterilization conditions, it is desirable to perform heating at 50 to 80 for 30 minutes or more.

r The storage conditions for the plastic container containing the HSA preparation should be a temperature of 5 ° C to 50 ° C and a relative humidity of 10% or more to avoid freezing and denaturation. In the plastic container according to the present invention, a rubber-like elastic body 10 may be fixed to the upper surface of the container head 6 as in the example simplified in FIG. When the flange 9 protrudes outwardly in the circumferential direction between the shoulder portion 7 and the neck portion 8 or at the neck portion 8 as described above, the caulking member 12 is used. The rubber-like elastic body 10 may be fixed to the container head 6 by welding to the bottom end of the container. r When the rubber-like elastic body 10 is fixed on the upper surface of the container head of the plastic container 2 containing the HSA product 3, even if the puncture needle punctures the rubber-like elastic body 10 and the container head, Since a restoring force acts in the closing direction of the needle hole punctured in the elastic body 10, there is an advantage that the puncture needle abuts the needle hole and the HSA preparation 3 does not leak to the outside. When the rubber-like elastic body 10 is provided, it is preferable that the outer surface of the rubber-like elastic body 10 is covered with the heat-shrinkable protective film 11. As a result, adhesion of air pollutants to the rubber-like elastic body 10 can be prevented. Examples of the material for forming the rubber-like elastic body 10 include synthetic rubber such as butyl rubber, polyisobutylene rubber, silicone rubber, and ethylene-propylene rubber, and natural rubber. In addition, as a material for forming the heat shrink protection film 11, for example, polyethylene terephthalate, styrene, butyl chloride, biaxially oriented polypropylene, or the like can be used, and among them, polyethylene terephthalate is preferable. The material of the caulking member 12 is preferably a plastic such as polyolefin, polyester, or polyamide. Metal such as aluminum or stainless steel, ceramic, or the like may be used.

[3] Outer packaging material

In the present invention, the outer wrapping material 15 for wrapping the plastic container 2 is made of plastic having at least a layer containing an ethylene-butyl alcohol copolymer, and is characterized by having gas barrier properties. By using the outer packaging material 15 having at least the layer containing the ethylene-vinyl alcohol copolymer, the above-mentioned recombinant human serum albumin preparation in a plastic container excellent in long-term storage stability can be obtained. be able to. Here, the outer wrapping material 15 of the present invention has an oxygen permeability at a pressure of 10 13 .25 hPa, a temperature of 25 ° C, and a relative humidity of 60%. The overdose is usually less than 50 cm ³ / ni ² / day and less than 10 13 .25 hPa, preferably less than 5 cmm B-10 13 .25 hPa, more preferably Is less than 1 cm ³ m ² Z days · 10 13 .25 hPa. Such an oxygen permeation amount can be measured by a differential pressure method in accordance with the provisions of JISK712.

In addition, by using the outer packaging material 15 having at least a layer containing an ethylene-vinyl alcohol copolymer, it exhibits gas barrier properties even with a relatively small thickness, so that it is easier to handle than other gas barrier materials. This method has the advantage that it is possible to obtain a recombinant human serum albumin preparation in a plastic container which is excellent in operability and inexpensive and has excellent long-term storage stability as described above.

The outer packaging material 15 may be a single layer formed of the ethylene-vinyl alcohol copolymer as long as the outer packaging material 15 has at least a layer containing the ethylene-butyl alcohol copolymer. However, a plurality of layers further including a layer formed of another resin material may be used.

When the outer packaging material 15 is composed of a single layer, it may be prepared only with an ethylene-vinyl alcohol copolymer, or may be made of polyethylene, polypropylene, polyvinyl chloride, cross-linked ethylene-vinyl acetate copolymer, or poly (ethylene-vinyl alcohol). It may be prepared from a mixture of at least one (preferably two or more) selected from vinylidene chloride, polybutene, polyester, and ethylene copolymer, and an ethylene-butyl alcohol copolymer. The single-layer outer packaging material made of only ethylene-vinyl alcohol copolymer also has the advantage of being superior in transparency as compared to the case where it is made of other gas barrier materials, depending on its thickness. .

When the outer packaging material 15 has a plurality of layers, the (resin) layer containing the ethylene-vinyl alcohol copolymer may be located in any of the outermost layer, the innermost layer, and the intermediate layer (intermediate layer). If there are multiple layers, it may be at any position.) In such a case, as a material for forming a layer other than the layer containing the ethylene-vinyl alcohol copolymer, those exemplified above as the resin which may be mixed with the above-mentioned ethylene-vinyl alcohol copolymer are preferably used. be able to. If the outer packaging material has multiple layers, Specifically, a multi-layer outer packaging material having an innermost layer formed of polyethylene, an intermediate layer formed of an ethylene-butyl alcohol copolymer, and an outermost layer formed of nylon is preferable. As an example.

When the outer packaging material 15 has a plurality of layers (the innermost layer, the outermost layer, and one or more intermediate layers as necessary) as described above, the thickness of each layer can be variously selected. The thickness of at least the layer formed of the ethylene-biel alcohol copolymer is selected so as to satisfy the above-mentioned oxygen permeation amount (preferably 5 to 5 μμηη, more preferably 10 to 30 / zm).

In the recombinant human serum albumin preparation 1 in a plastic container of the present invention, the plastic container 2 containing the rHSA preparation 3 is packaged in the outer packaging material 15. Here, `` packing '' means that the outer packaging material covers and wraps at least the body of the plastic container containing the rSA preparation, preferably the entire plastic container, so as to be completely contained therein. Refers to The shape of the outer packaging material 15 is not particularly limited, but is preferably a thin film from the viewpoint of handleability. The thickness (in the case of a multilayer), the total thickness of all the layers is preferably from 5 to 100; ζιη, more preferably from 10 to 500 m.

The outer wrapping material 15 can be manufactured using a conventionally known appropriate manufacturing method, for example, a professional molding method, an inflation method (extrusion molding method), or the like. In addition, as a method of packaging the plastic container 2 containing the HSA preparation 3 in the outer packaging material 15, a conventionally known appropriate method, for example, sealing the opening of the outer packaging material after storing the plastic container And the like.

In the present invention, when the plastic container 2 containing the rHSA preparation 3 is packed in the outer packaging material 15 as described above, a plastic container is required to further suppress the effect of oxygen on the rHSA preparation 3. It is preferable that an oxygen absorber is contained in a space 16 between the outer packaging material 15 and the outer packaging material 15. As the oxygen scavenger, conventionally known suitable commercial products, for example, Ageless (manufactured by Mitsubishi Gas Chemical Co., Ltd.), Tamotsu (manufactured by Oji Tack Co., Ltd.) and the like can be used without particular limitation. Further exposure to oxygen An oxygen detector (trade name: Ageless Eye (manufactured by Mitsubishi Gas Chemical Co., Ltd.)) or the like may be stored to confirm whether or not the gas has been emitted.

Example

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited to these examples.

Example 1

(1) Preparation of rHSA

r Acquisition of HS A-producing yeast Pichia pastoris and its cultivation were carried out according to the method described in Japanese Patent Application Laid-Open No. 5-317079. RHSA is recovered from the obtained culture solution. Purification is performed by heating the culture solution → adsorbent particle treatment (streamline SP treatment) according to the method described in JP-A-8-16985.

) → heat treatment → hydrophobic chromatography treatment → anion exchanger treatment → chelate resin treatment → boric acid salt treatment.

The prepared rHSA was adjusted to a 5 (w / v)% solution, and sodium caprylate and acetyl tryptophan sodium were added in amounts of 66.5 mg and 107.3 mg, respectively, per 10 OmL of the solution. After that, rHSA can be used as an injection by filtering the bacteria with a 0.22 μm filter (manufactured by Millipore). Its composition was sodium chloride content of 3.7 _mg mL or less, pH 6.4 to 7.4, and osmotic pressure ratio of about 1 (ratio to physiological saline).

(2) Properties of purified rHSA (containing composition)

(HPLC analysis)

rHSA was analyzed by HPLC gel filtration. Gel filtration analysis line Tsu under the following conditions: 7 this ₀

(a) Column: TSK gel G 3000 SW (manufactured by Tosoh Corporation)

(b) Developing solution: 0.1 M KH ₂ PO ₄ /0.3 M Na C 1 buffer

(c) Detection: absorbance at wavelength 280 nm

The main peak in the purified rHSA-containing composition corresponds to the monomer of rHSA. It was.

(Yeast-derived component analysis)

A culture supernatant of a non-HSA-producing yeast is roughly purified in the same manner as this method, and immunized to rabbits. Purified using the obtained antiserum r Detection of yeast-derived components present in HSA-containing composition Was done. The measurement was performed by enzyme immunoassay (EIA method). The amount of yeast-derived components was less than 1 ng per 250 mg of rHSA.

(Molecular weight)

The molecular weight was measured by the HPLC gel filtration method described above. The molecular weight of rHSA in the purified rHSA-containing composition of the present invention was about 67,000.

(Isoelectric point)

The isoelectric point was measured using a thin-layer polyacrylamide gel according to the method of Allen et al. [J. Chromatog., 146, pi (1978)]. The isoelectric point of rHSA in the purified rHSA-containing composition of the present invention was about 4.9.

(Coloring degree)

Degree of coloration was measured absorbance at 280nm, 350 nm, 450 nm, 500 η m, A 3S0 ZA 280, A 450 ZA 280, A 500 / A 28. Was calculated. The coloring degree of the purified rHSA-containing composition of the present invention is _A35 . / A ₂₈ . About 0.015, A ₄₅₀ / A ₂₈₀ is about 0.01,

. Was about 0.002.

[Measurement of pyrogen]

The amount of pyrogen was less than 0.5 EU per 25 mg of rHSA25O, as measured using Seikagaku's Endoscopy.

(3) Molding of plastic container r Injection of HS A preparation

A low-density polyethylene having a density of 0.906 g / cm ³ (DEFD-1137, manufactured by Nippon Tunicer Co., Ltd.) is melted and extruded into a cylindrical shape at an extrusion temperature of 190 ° C. from an extrusion nozzle. (Parison) was manufactured. The parison 21 was lowered between the split molds (FIG. 2 (a)), and the main body mold 22 was clamped (FIG. 2 (b)). Next, push the mandrel 23 from the upper open end of the parison 21 and pressurize. Pressurized air was blown at 3 kg / cm ² to form a container body 24. At this time, the temperature of the main body mold 22 was 50 ° C (Fig. 2 (c)). Thereafter, the tip of the chemical solution injection nozzle 25 was lowered to the bottom of the container body 24, and 25 OmL of a 5 (w / v)% r HSA aqueous solution was injected into the container body 24 (FIG. 2 (d)). Next, the chemical injection nozzle 25 is raised, and at the opening end inner surface temperature of 40 ° C., the head mold 26 is closed, and the container head 6 is formed by vacuum forming from the suction hole 27 to form the container head 6. Open end 4 was sealed (Fig. 2 (e), Fig. 2 (f)).

(4) Packaging with outer packaging material

1st layer (innermost layer) Linear low-density polyethylene 50 μπι, 2nd layer ethylene-vinyl alcohol copolymer 15 m, 3rd layer (outermost layer) Nylon 15 / m Packaging materials (Fujimori Kogyo, outer dimensions: about 16 OmmX 26 Omm (seal width: 1 Omm), inner dimensions: about 14 OmmX 24 Omm) were prepared. The outer packaging material has a gas permeability (oxygen permeation at 25 ° C and 60% relative humidity (RH) of about 0.5 cm ³ / m ² · 24 hr · 10 1 3.25 hP a or less (measured by the differential pressure method in accordance with the provisions of JISK 7126)). The plastic container containing 5 OmL of 5% (w / v) r HSA preparation prepared in (3) above is packed in the outer packaging material, and at that time, the plastic container and the outer packaging material are An oxygen absorber (Ageless ZH100, manufactured by Mitsubishi Gas Chemical Co., Ltd.) was housed in the tank.

Thereafter, ripening and sterilization were performed at 60 ° C for 30 minutes to obtain a recombinant human serum albumin preparation in a plastic container of the present invention.

Comparative Example 1

Example 1 was repeated except that the plastic container was not wrapped with the outer wrapping material.

〔Evaluation test〕

After the recombinant human serum albumin preparations in plastic containers prepared in Example 1 and Comparative Example 1 were left at 50 ° C and 75% relative humidity for 60 days, the following three items were evaluated. . 'appearance

The appearance was visually evaluated.

• rHSA polymer content

Each rHSA formulation was measured by the HP LC gel filtration method described above, and the high molecular weight fraction of the dimer or higher was calculated as the polymer.

• Ammonia content

A 5-fold dilution of each rHSA preparation was ultrafiltered with an ultrafiltration membrane (molecular weight 30,000 cut, Centricon-1 30 manufactured by Amicon), and the obtained filtrate was used as a sample solution. The test was performed by the ammonia test method of the Japanese Pharmacopoeia's general test method, and quantified by measuring the absorbance of indophenol (! Π3Χ, 640 nm).

The results are shown in Table 1. The ND in the table is below the quantification limit (1. eS / z gZmL), meaning that quantification was not possible.

table 1

As a result, it was possible to suppress the change in appearance and the increase in ammonia content and polymer content by wrapping it in a gas-paria outer wrapping material together with the oxygen scavenger. That is, this effect was presumed to be due to the suppression of the effects of oxygen exposure.In addition, except that plasma-derived HSA was used instead of the rHSA preparation, the results were the same as in Example 1 and Comparative Example 1, respectively. When similar samples were prepared and subjected to the same evaluation test, the presence or absence of the exterior material did not affect the polymer content. Industrial applications

According to the present invention, as the outer wrapping material, a plastic material having a gas barrier property, in particular, a low oxygen permeability, having a layer containing an ethylene-butyl alcohol copolymer is used, and is further combined with a deoxidizer. However, even if recombinant human serum albumin preparations prepared using genetic recombination technology are stored in plastic containers and formulated, the formation of polymers in recombinant human serum albumin preparations and an increase in ammonia content are higher than before. It was able to reduce the amount significantly, and it became possible to store it stably for a long period of time. Therefore, it is possible to provide a more convenient recombinant human serum albumin preparation to medical practice.

This application is based on a patent application No. 2003-19591 filed in Japan, the contents of which are incorporated in full herein.

Claims

The scope of the claims

1. A package containing a plastic container containing a liquid preparation of a recombinant human serum albumin, and a gas-parisable outer packaging material having at least a layer containing an ethylene-butyl alcohol copolymer for packaging the plastic container. Recombinant human serum albumin preparation.

2. The preparation according to claim 1, wherein an oxygen scavenger is contained between the plastic container and the outer packaging material.

3. The preparation according to claim 1, wherein the thickness of the layer containing the ethylene-butyl alcohol copolymer of the outer packaging material is 5 to 50 μm.

4. The preparation according to any one of claims 1 to 3, wherein the thickness of the outer packaging material is 5 to 1000 µπι.

5. The oxygen permeation amount of the outer packaging material is less than 50 cm sZni ² days.10 1 3.25 hPa at a temperature of 25 ° C and a relative humidity of 60%. Preparations described in

6. The outer packaging material has a multilayer structure having an innermost layer formed of polyethylene, an intermediate layer formed of an ethylene-vinyl alcohol copolymer, and an outermost layer formed of nylon. The preparation according to any one of 1 to 5.

7. The preparation according to any one of claims 1 to 6, wherein the concentration of the recombinant human serum albumin in the liquid preparation of recombinant human serum albumin is 1 to 50 Omg / mL.

8. The formulation according to claim 1, wherein the material of the plastic container is polyolefin, polyvinyl chloride, or an ethylene-vinyl acetate copolymer.

9. The formulation of claim 1, wherein the liquid formulation of recombinant human serum albumin is aseptically sealed in a plastic container.

10. After the plastic container is molded in the mold, while the plastic container is still in the mold, a liquid preparation of recombinant human serum albumin is injected into the plastic container, and 10. The preparation according to claim 9, wherein the opening is pressed and sealed by a mold.

11. The method according to any one of claims 1 to 10, wherein a content of the recombinant human serum albumin polymer of at least dimer in the liquid preparation of the recombinant human serum albumin is 3.8% or less. Preparations.

12. The formulation according to any one of claims 1 to 11, wherein the recombinant human serum albumin liquid formulation has an ammonia content of 4.60 µgZmL or less.