JPH10142214A - Method for analysing water-soluble polymeric substance incorporated in micelle - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an accurate and simple method for analysis in quantity and purity of water-soluble polymeric substance incorporated in a micelle, such as lipid inducer included in polyethylene glycol or protein. SOLUTION: The present method is for analyzing a water-soluble polymeric substance incorporated in a micelle, such as a liposome. In the method, after solubilizing a micelle with a solvent by which the micelle is soluble, such as a water-soluble solvent including a surfactant, the solubilized solution is separated by gel-filtration chromatography. The method realizes an accurate and simple analysis for water-soluble polymeric substances. In addition, under the condition in which the micelle component such as lipid is over-mixed or a great amount of a sealed-in drug is co-existed, said method enables to analyze not only the amount of the water-soluble polymeric substance but also distribution of molecular weight all simplicity. Furthermore, an individual quantitative analysis of the micelle which combines a plurality of water-soluble polymeric substances can be performed with minor change of solubilization and column separation conditions.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for analyzing a water-soluble polymer substance incorporated in a micelle.

[0002]

2. Description of the Related Art Micelles such as polymer micelles, lipid micelles, and liposomes have been attracting attention as an effective means of drug delivery systems, and various methods have been aimed at improving their safety, efficacy, and stability. Have been devised. For example, attempts have been made to introduce a polyethylene glycol chain into the outer surface of the liposome in order to maintain the blood concentration of the liposome (for example, JP-A-1-249717, JP-A-2-149512, JP-A-2-149512). 3-218309, JP-A-3-218309, JP-A-4-5242, FEBS lette
rs 268, 235 (1990)).

[0003] Also, research on targeting vesicles by binding proteins such as antibodies to liposomes,
Furthermore, targeting therapeutic agents by imparting functionality by complex modification of antibodies and polyethylene glycol have been researched and developed (reviews on immunoliposomes, Im
munomethods 4, 259 (1994)). When micelles such as liposomes to which such a high molecular compound is bound are actually applied to pharmaceuticals, the binding amount (content) and purity are safe,
Needless to say, it is an important factor of effectiveness, and a system is required that can accurately and easily measure the content and purity of these high-molecular substances.

[0004] Taking polyethylene glycol as an example, many reports have been made on the correlation between the content of polyethylene glycol and the maintenance effect in blood. However, most of them are only quantitatively defined by the addition ratio of the polyethylene glycol lipid derivative added to the constituent lipid components, and are not quantified at all with respect to the amount of polyethylene glycol actually introduced. Furthermore, TM Allen et al. (BBA 1066, 29 (1991)) show that the added polyethylene glycol lipid derivative is not always incorporated into micelles (liposomes in this paper), and the amount actually incorporated is measured. Method development is an indispensable technology for research and development of targeting particles.

[0005] TM Allen et al. (BBA 1066, 29 (1991)).
Is based on the dye-binding method using Bio-Rad protein detection reagent for the determination of polyethylene glycol.
o et al. calculate unbound polyethylene glycol and indirectly calculate the amount of bound polyethylene glycol.
In the method of Allen et al., When proteins and phospholipids coexist, these interfere with each other and require complicated pretreatment for separation, making accurate measurement difficult. Further, when the drug encapsulated in the liposome has visible absorption used in the dye binding method, it cannot be used.

Taking a protein such as an antibody as an example, a method using a radiolabeled antibody (BBA1239, 133 (1995))
And a dye binding method, an amino acid analysis method, and an electrophoresis method. The dye binding method is also difficult to measure for the same reason as for polyethylene glycol. The amino acid analysis method is complicated and requires an expensive amino acid analyzer. Although the electrophoresis method enables purity analysis by combination with a densitometer or the like, the measured values vary greatly depending on the conditions of gel staining and washing, and the quantitativeness is lacking.

[0007]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned conventional problems and provides a method for quantification and purity analysis of water-soluble polymer substances incorporated in micelles, for example, lipid derivatives such as polyethylene glycol and protein. It is an object to find an accurate and simple method.

[0008]

Means for Solving the Problems The present inventors have intensively studied to develop a method for analyzing a water-soluble polymer substance incorporated in a micelle such as a liposome. As a result, the micelle dispersion was found to be a solvent or a surfactant. In the coexistence with, it was found that it was possible to analyze the incorporated water-soluble polymer substance easily by solubilization by treatment such as heating or ultrasonic waves, and separating the solubilized solution by gel filtration chromatography. . The present invention has been completed based on these findings.

That is, in the present invention, in analyzing a water-soluble polymer substance incorporated in a micelle, the solubilized liquid is separated by gel filtration chromatography after solubilization with a solvent capable of solubilizing the micelle itself. An analysis method characterized by the following. According to a preferred embodiment of the above invention, the above method wherein the solvent is an aqueous solvent containing a surfactant; the above method wherein the surfactant is sodium dodecyl sulfate; the above method wherein the micelle is a liposome; Or a lipid derivative of polyethylene glycol.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION In the present specification, a micelle is
It means a water-soluble particle obtained by aggregation of an amphipathic molecule containing a hydrophilic part and a hydrophobic part in the molecule. Such micelles may take the form of small spheres, ellipsoids or long cylinders, and may also be a bilayer consisting of two parallel layers of the amphipathic molecules described below, i.e. the amphiphilic micelle bilayer. . Further, the micelle having a closed vesicle structure is called a liposome.

The amphipathic molecules constituting micelles include:
Any substance containing a hydrophilic portion and a hydrophobic portion and capable of forming micelles by a commonly used method known per se may be used, and among them, a preferable amphipathic molecule is a lipid. Can be mentioned. Examples of the lipid capable of forming micelles in the present invention include natural phosphatidylcholines, such as egg yolk phosphatidylcholine (EYPC), soybean phosphatidylcholine, etc .; Phosphatidylcholine (DSPC), dioleylphosphatidylcholine (DOPC), etc .; natural phosphatidylethanolamines, such as egg yolk phosphatidylethanolamine (EYPE); phosphatidylethanolamine (P
E), such as dimyristoyl phosphatidylethanolamine (DMPE); phosphatidylglycerol (PG), such as dipalmitoyl phosphatidylglycerol (DPPG); phosphatidylserine (P)
S); phosphatidylinositol (PI); phospholipids such as dimyristoyl phosphatidic acid (DMPA), and glycolipids such as glycosphingolipid and glyceroglycolipid. These lipids may be used alone or in combination of two or more, or in combination with a nonpolar substance such as cholesterol.

The micelles of the present invention incorporate, in addition to the lipids and nonpolar substances described above, water-soluble polymer substances such as lipid derivatives and the like in which proteins such as antibodies and water-soluble polymers such as polyethylene glycol are bonded to lipids. It consists of The micelle in the present invention may be a product produced by any method, and for example, can be produced using the above-mentioned materials and using a commonly used production technique known per se. For example,
Multilamellar liposome (MLV) formed by adding an aqueous solution to a lipid thin film adhered to a glass wall and subjecting it to mechanical shaking
And small unilamellar liposomes (SU) obtained by sonication, ethanol injection, and French press
V), a surfactant removal method, a reverse-phase evaporation method (liposomes, Junzo Sunamoto et al., Nanedo 1988), a large unilamellar liposome obtained by an extrusion method in which MLV is extruded by pressing a membrane having a uniform pore size ( L
UV) can be used (Liposome Technology, V
ol.1 2nd Edition).

The incorporation of the water-soluble polymer substance into the micelle may be carried out by preparing a lipid derivative of the above-mentioned water-soluble polymer in advance and incorporating it into the micelle according to the above-mentioned method. A water-soluble polymer may be bound via the compound (see JP-A-4-346918). Furthermore, pharmaceuticals and diagnostic agents may be introduced into the micelles.

Pharmaceuticals which can be introduced into micelles include anti-tumor agents such as adriamycin, daunorubicin, vinblastine, cisplatin, mitomycin, bleomycin, actinomycin, 5-FU (fluorouracil), adrenaline blockers such as timolol, and hypertension such as clonidine. Agents, antiemetic agents such as prokaiamide, antimalarial agents such as chloroquinine, and pharmaceutically acceptable salts and derivatives thereof; toxin proteins such as ricin A and dyfreria toxin, and DNAs encoding the same and cytokines such as TNF DNA encoding a gene, antisense DN
A and the like. As the pharmaceutically acceptable salts of the above antitumor agents and the like, salts with pharmaceutically acceptable polyvalent anionic substances, such as salts with citrate, tartrate, glutamate, and derivatives thereof Is preferred.

Diagnostic agents that can be introduced into micelles include:
Radioactive elements, for example, imaging agents such as indium and technesium; enzymes such as horseradish peroxidase, alkaline phosphatase and β-galaclosidase; phosphors such as europium derivatives; luminous bodies such as N-methylacridium derivatives and the like. . The introduction of these drugs and diagnostic agents into micelles can be carried out by a commonly used method known per se. For example, an aqueous solution may be added and encapsulated when forming micelles such as liposomes, or a concentration gradient such as a pH gradient may be formed inside and outside the vesicle after micelle formation, and the potential may be used as a driving force to incorporate an ionizable drug ( Cancer Re
s. 49 , 5922 (1989)).

In the analysis method of the present invention, micelles incorporating a water-soluble polymer substance are solubilized with a solvent capable of solubilizing the micelles themselves, and the solubilized solution is separated by gel filtration chromatography. The water-soluble polymer substance detected is detected. The solvent that can be used is not particularly limited as long as it can solubilize micelles, and examples thereof include an aqueous solvent containing a surfactant. Surfactants can be any of non-ionic, anionic, cationic and zwitterionic. Suitable surfactants include, for example, sodium dodecyl sulfate, sodium deoxycholate, polyoxyethylene sorbitan monolaurate (Tween 20),
Polyoxyethylene sorbitan monooleate (Tween 8
0), polyoxyethylene octyl phenyl ether (T
riton X-100), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), cetylpyridium chloride (C
PCl), tetradecyltrimethylammonium bromide (TDMABr) and the like. Of these, sodium dodecyl sulfate is most preferred. The concentration of the surfactant is not particularly limited, but the final concentration is 0.5 to 10
% By weight is appropriate.

The aqueous solvent may further contain a suitable organic solvent such as acetonitrile, methanol, isopropanol, acetone, tetrahydrofuran and the like.
The concentration of the organic solvent is not particularly limited. For example, when the water-soluble polymer substance is a lipid derivative of polyethylene glycol, the concentration is usually 30 to 100% by volume, preferably 50 to 80% by volume. When the water-soluble polymer substance is a lipid derivative of a protein, it is preferable to use water containing no organic solvent.

The pH of the solvent is not particularly limited, but is usually 4.
0 to 9.0, preferably 6.0 to 8.0 is suitable. The aqueous solvent may be buffered with a suitable buffer such as a phosphate buffer, a Tris-HCl buffer, an acetate buffer and the like. The appropriate concentration of the buffer is usually 20 to 50 mM.

The micelles are easily solubilized by mixing the micelles with the above-mentioned solvent and heating if necessary. The heating conditions are not particularly limited, but are usually at a temperature of 50 to 80 ° C. and 15
Appropriately for ~ 45 minutes. The micelle solubilized solution thus obtained is separated by gel filtration chromatography, and the water-soluble polymer substance is detected.

Examples of the column for gel filtration chromatography include a column filled with a hydrophilic polymer or silica gel. For example, TSK gel G2000PW _XL , G3000PW _XL and G4000PW _XL (commercially available from Tosoh Corporation) and the like are listed as polymer columns, and MCI gel CQS is used as a silica gel column.
10 and CQS30 (commercially available from Mitsubishi Chemical Corporation), T
SK gel G2000SW _XL, G3000SW _XL and G4
000SW _XL (commercially available from Tosoh Corporation). In particular, when analyzing lipid derivatives of proteins, silica gel columns are preferred.

The mobile phase for gel filtration chromatography is not particularly limited, but an aqueous solution containing a surfactant is suitable. Examples of the surfactant include those described above, and among them, sodium dodecyl sulfate is preferably used. The concentration of the surfactant is usually 0.05 to 0.5% by weight, preferably 0.1 to 0.2% by weight. The aqueous solution used for the mobile phase may further contain a suitable organic solvent such as acetonitrile, methanol, isopropanol, acetone, tetrahydrofuran and the like. The concentration of the organic solvent is not particularly limited, but when the analysis target is a lipid derivative of polyethylene glycol, usually 30 to 10
0% by volume, preferably 50 to 80% by volume is appropriate. When the analysis target is a lipid derivative of a protein, it is preferable to use an aqueous solution containing no organic solvent.

The solution used for the mobile phase may further comprise a suitable salt,
For example, it may contain sodium sulfate, sodium chloride, potassium chloride, ammonium sulfate and the like. The concentration of the salt is not particularly limited, but is usually 50 to 500 mM, preferably 1 to 500 mM.
About 00 to 300 mM is appropriate. In particular, when the analyte is a lipid derivative of a protein, it is preferable to use a solution containing the salt at the above concentration.

The pH of the solution used for the mobile phase is not particularly limited, but usually pH 6.0 to 8.0 is appropriate. When the analyte is a lipid derivative of a protein, the pH is 6.5 to 7.5, and the analyte is polyethylene. When the lipid derivative is a glycol derivative, the pH is most preferably 7.0 to 8.0. The solution may be buffered with a suitable buffer as described above, the concentration of the buffer being:
Usually, 20 to 50 mM is appropriate.

In the above-mentioned solution used for the mobile phase, when the analyte is a lipid derivative of polyethylene glycol, 50 to 80% by volume of an organic solvent such as methanol or acetonitrile and 0.05 to 0.5% by weight of dodecyl sulfate are used. A sodium-containing 20 to 50 mM sodium phosphate buffer (pH 7.0 to 8.0) is preferred. When the analyte is a lipid derivative of a protein, 100 to 300 mM salt and 0.1% are used.
20 containing from 0.5 to 0.5% by weight of sodium dodecyl sulfate
〜50 mM sodium phosphate buffer (pH 6.5-7.5) is preferred.

The detection of a water-soluble polymer substance separated by gel filtration chromatography can be performed, for example, when the analyte is a lipid derivative of polyethylene glycol, such as an ultraviolet absorption method or a suggested refractive index method; When it is a lipid derivative of a protein, it can be easily carried out by an ultraviolet absorption method or the like.

[0026]

According to the analysis method of the present invention, a water-soluble polymer substance incorporated in a micelle can be analyzed accurately and easily. In addition, the analysis method of the present invention can easily directly analyze not only the content of the water-soluble polymer substance but also the molecular weight distribution even in the presence of excess micelle components such as lipids or a large amount of encapsulated drug. It is possible. Furthermore, fractional quantification is possible even in micelles incorporating a plurality of water-soluble polymer substances by slightly changing the solubilization conditions and column separation conditions.

[0027]

The present invention will be described in more detail with reference to examples, but these examples do not limit the scope of the present invention. Reference Example 1 Preparation of liposome 1 ml of 0.3 M citrate buffer per 100 mg of a solid lipid mixture composed of dipalmitoylphosphatidylcholine (DPPC) / cholesterol (Chol) / maleimidated dipalmitoylphosphatidylethanolamine (DPPE) (18/10 / 0.5) pH 4.
0 was added and hydrated with a vortex mixer. Furthermore, multilamellar liposomes were prepared by repeating freeze-thaw five times. Then, the liposomes were sequentially added at 200 nm and 1 nm.
Extr equipped with 00nm pore size polycarbonate membrane
Pressure filtration was performed while heating at 60 ° C. with uder (manufactured by NOF Liposomes) to produce sized liposomes.

After the obtained liposome solution was neutralized with a 1 M NaOH solution, adriamycin (Kyowa Hakko) of 1/10 weight of the lipid weight was added. As a result, adriamycin-encapsulated liposomes encapsulating 97% or more of adriamycin were obtained. Furthermore, a thiol group was introduced into this liposome with iminothiolane by the method of Traut et al. (Traut, RR et
al., Biochemistry 12, 3266 (1973)), an F (ab ') 2 type human monoclonal antibody (JP-A-5-304987) was added and reacted to obtain antibody-bound liposomes. Next, 2,4-
A thiolated polyethylene glycol prepared from bis (polyethylene glycol) -6-chloro-S-triazine (Seikagaku Corporation) is added to the antibody-bound liposome solution described above, and reacted with maleimide to form a liposome having an antibody and polyethylene glycol. Produced. Unreacted antibody, polyethylene glycol is Sepharose CL6B
The product was purified and removed by gel filtration.

Example 1 Determination of polyethylene glycol chain
Analysis of Amount and Purity An equal volume of a liposome dispersion (lipid concentration: about 10 mg / mL) prepared in Reference Example 1 and a 4% by weight aqueous solution of sodium dodecyl sulfate were mixed and heated at 50 ° C. for 30 minutes. The obtained liposome solubilized solution was analyzed by high performance liquid chromatography under the following operating conditions. In the determination, a lipid derivative of polyethylene glycol was used as a standard substance.

Operating conditions Detector: UV absorption spectrophotometer (measurement wavelength: 215 nm) Column: about 5 mm in a stainless steel tube of about 8 mm in inner diameter and about 30 cm in length
Packing with silica gel for aqueous solvent gel filtration chromatography in which a hydrophilic group of μm is chemically bonded (TSK gel G2000)
Use SW _XL ).・ Pre-column: A stainless steel tube with an inner diameter of about 6 mm and a length of about 4 cm is filled with the same packing material as the column (TSK gel guardcol)
Use umnSW _XL ).・ Column temperature: constant temperature around 30 ° C. ・ Mobile phase: After dissolving 7.8 g of sodium dihydrogen phosphate in 300 mL of water, add 600 mL of methanol and 10 mL of a 10% by weight sodium dodecyl sulfate solution, and then add 20% by weight. Sodium hydroxide solution to adjust the pH to 7.5, and add water
Make up to 1,000 mL.・ Flow rate: 0.5 mL / min. -Area measurement range: 55 minutes from the start of measurement.

By heating the liposome in the above-mentioned sodium dodecyl sulfate solution (solubilizing solvent), the liposome was disintegrated and each component was in a solubilized state. This solubilized solution was separated by high performance liquid chromatography using a column for gel filtration chromatography, and the separated product was detected by an ultraviolet absorption method. As a result, polyethylene glycol chains (lipid derivatives of polyethylene glycol) were detected as peaks separated from lipids and drugs. FIG. 1 shows the elution pattern. The peak of the polyethylene glycol chain is linear,
The reproducibility was good, and quantification using a standard substance was possible. FIG. 2 shows the analysis results (elution pattern) of liposomes prepared using polyethylene glycol chains having different molecular weights in the same manner as in Reference Example 1. The molecular weights are about 20,000, about 10,000, and about 50,000 in the order of elution.
From this, it was found that the purity analysis of the polyethylene glycol chain was possible. Under these conditions, the protein bound to the liposome was not eluted, so that the quantification and purity analysis of the polyethylene glycol chain was possible even when the molecular weight of the polyethylene glycol chain was the same as that of the protein.

Example 2 Antibody Quantification and Purity Analysis An equal volume of a liposome dispersion (lipid concentration: about 10 mg / mL) and a 4% by weight aqueous solution of sodium dodecyl sulfate were mixed at 50 ° C. For 30 minutes. The obtained liposome solubilized solution was analyzed by high performance liquid chromatography under the following operating conditions. In addition, at the time of quantification, an antibody solution having a clear concentration was used as a standard solution.

Operating conditions Detector: UV absorption spectrophotometer (measuring wavelength: 280 nm) Column: about 5 mm in a stainless steel tube of about 8 mm in inner diameter and about 30 cm in length
Packing with silica gel for aqueous solvent gel filtration chromatography, in which a hydrophilic group of μm is chemically bonded (TSK gel G3000)
Use SW _XL ).・ Pre-column: A stainless steel tube with an inner diameter of about 6 mm and a length of about 4 cm is filled with the same packing material as the column (TSK gel guardcol)
Use umnSW _XL ). Column temperature: constant temperature around 30 ° C. -Mobile phase: Dissolve 7.8 g of sodium dihydrogen phosphate and 28.4 g of sodium sulfate in 900 mL of water, add 10 mL of a 10% by weight sodium dodecyl sulfate solution, and add a 20% by weight sodium hydroxide solution to adjust the pH to 7.0. And add water to 1,000
mL.・ Flow rate: 0.5 mL / min. -Area measurement range: 50 minutes from the start of measurement.

By heating the liposome in the above-mentioned sodium dodecyl sulfate solution (solubilizing solvent), the liposome was disintegrated and each component was in a solubilized state. At the same time, the antibody bound to the liposome becomes negatively charged and denatured by sodium dodecyl sulfate. This solubilized solution was separated by high performance liquid chromatography using a column for gel filtration chromatography, and the separated product was detected by an ultraviolet absorption method.

As a result, the antibody (lipid derivative of the antibody) was detected as a peak separated from the lipid and the drug. FIG. 3 shows the elution pattern. The three peaks in FIG. 3 indicate the peaks of the polymerized antibody, the antibody, and the decomposed antibody, respectively. The peaks of these antibodies were good in both linearity and reproducibility, and quantification using a standard solution of the antibody was possible.

[Brief description of the drawings]

FIG. 1 is a diagram showing an elution pattern of solubilized liposomes by gel filtration chromatography. In the figure, the horizontal axis represents time (minutes), and the vertical axis represents absorbance.

FIG. 2 is a diagram showing an elution pattern of solubilized liposomes by gel filtration chromatography. In the figure, the horizontal axis represents time (minutes), and the vertical axis represents absorbance.

FIG. 3 is a diagram showing an elution pattern of solubilized liposomes by gel filtration chromatography. In the figure, the horizontal axis represents time (minutes), and the vertical axis represents absorbance.

Claims (6)

[Claims] 1. A method for analyzing a water-soluble polymer substance incorporated in a micelle, comprising the steps of: solubilizing the micelle itself with a solvent capable of solubilizing the micelle; and separating the solubilized solution by gel filtration chromatography. Analysis method. 2. The method according to claim 1, wherein the solvent is an aqueous solvent containing a surfactant. 3. The method according to claim 2, wherein the surfactant is sodium dodecyl sulfate. 4. The analysis method according to claim 1, wherein the micelle is a liposome. 5. The analysis method according to claim 1, wherein the water-soluble polymer substance is a lipid derivative of a protein. 6. The analysis method according to claim 1, wherein the water-soluble polymer substance is a lipid derivative of polyethylene glycol.