JP3778528B2 - Method for analyzing water-soluble polymer substances incorporated in micelles - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for analyzing a water-soluble polymer substance incorporated in a micelle.
[0002]
[Prior art]
Micelles such as polymer micelles, lipid micelles, and liposomes are attracting attention as effective means for drug delivery systems, and various devices have been devised to improve their safety, effectiveness, and stability. ing. For example, attempts have been made to introduce polyethylene glycol chains on the outer surface of the liposome for the purpose of maintaining the blood concentration of the liposome (for example, JP-A Nos. 1-249717, 2-149512, and JP-A No. 2-149512). 3-218309, JP-A-3-218309, JP-A-4-5242, FEBS letters 268, 235 (1990)).
[0003]
In addition, research on targeting vesicles by binding proteins such as antibodies to liposomes, and research and development of targeting therapeutic agents by adding functionality by complex modification of antibodies and polyethylene glycol (review on immunoliposomes, Immunomethods) 4, 259 (1994)).
Needless to say, when such micelles such as liposomes bound with polymer compounds are actually applied to pharmaceuticals, their binding amount (content) and purity are important factors for safety and effectiveness. There is a need for a system that can accurately and easily measure the content and purity of molecular substances.
[0004]
Taking polyethylene glycol as an example, many reports have been made on the correlation between polyethylene glycol content and blood maintenance effect. 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 the amount of polyethylene glycol actually introduced is not quantified at all. In addition, TM Allen et al. (BBA 1066, 29 (1991)) show that the added polyethylene glycol lipid derivative does not necessarily incorporate into the total amount of micelles (liposomes in this paper) and measures the amount actually incorporated. Method development is an indispensable technique for researching and developing targeting particles.
[0005]
TM Allen et al. (BBA 1066, 29 (1991)) calculated the amount of unbound polyethylene glycol indirectly by the dye binding method using Bio-Rad protein detection reagent for the determination of polyethylene glycol. The amount of bound polyethylene glycol is calculated. In the method of Allen et al., When proteins and phospholipids coexist, they become interfering substances, so that complicated pretreatment for separation is required, and accurate measurement is difficult. Moreover, it cannot be used when the drug encapsulated in the liposome has visible absorption used in the dye-binding method.
[0006]
Taking proteins such as antibodies as examples, methods using radiolabeled antibodies (BBA 1239, 133 (1995)), dye-binding methods, amino acid analysis methods, and electrophoresis methods can be considered. The dye binding method is still difficult to measure for the same reason as in the case of polyethylene glycol. The amino acid analysis method is complicated in operation and requires an expensive amino acid analyzer. Electrophoresis can be analyzed for purity by combination with a densitometer or the like, but the measured value varies greatly depending on the conditions of gel staining and washing, and lacks quantitativeness.
[0007]
[Problems to be solved by the invention]
The present invention finds a method for accurately and simply quantifying and purifying a water-soluble polymer substance incorporated in a micelle, for example, a lipid derivative such as polyethylene glycol and protein, which has solved such conventional problems. Is an issue.
[0008]
[Means for Solving the Problems]
As a result of intensive studies to develop an analysis method for water-soluble polymer substances incorporated in micelles such as liposomes, the present inventors have found that micelle dispersions are heated or ultrasonically coexisted with a solvent or a surfactant. It was found that the incorporated water-soluble polymer substance can be easily analyzed by solubilizing by the above-described treatment and separating the solubilized solution by gel filtration chromatography. The present invention has been completed based on these findings.
[0009]
That is, the present invention is characterized in that, in analyzing a water-soluble polymer substance incorporated in a micelle, the solubilized solution is separated by gel filtration chromatography after solubilization with a solvent capable of solubilizing the micelle itself. This is an analysis method.
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; And the above method for purity analysis; the water-soluble polymer substance is a protein lipid derivative and / or a polyethylene glycol lipid derivative; and the water-soluble polymer substance is a protein lipid derivative and a polyethylene glycol lipid derivative. Methods or methods are provided wherein the lipid derivative of the method or protein is a lipid derivative of an antibody.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
In this specification, the micelle means water-soluble particles obtained by aggregation of amphiphilic molecules containing a hydrophilic part and a hydrophobic part in the molecule. Such micelles can take the form of globules, ellipsoids or long cylinders and can also be bilayers consisting of two parallel layers of amphiphilic molecules described below, ie amphiphilic micelle bilayers. . A micelle having a closed vesicle structure is referred to as a liposome.
[0011]
The amphiphilic molecule constituting the micelle may be any one as long as it contains a hydrophilic portion and a hydrophobic portion and can form a micelle by a commonly used method known per se. As a suitable amphiphilic molecule, lipid can be exemplified.
Examples of lipids capable of forming micelles in the present invention include natural phosphatidylcholine such as egg yolk phosphatidylcholine (EYPC), soybean phosphatidylcholine and the like; phosphatidylcholine (PC) such as dipalmitoylphosphatidylcholine (DPPC), dimyristoylphosphatidylcholine (DMPC), distearoyl Phosphatidylcholine (DSPC), dioleyl phosphatidylcholine (DOPC), etc .; natural phosphatidylethanolamine, such as egg yolk phosphatidylethanolamine (EYPE); phosphatidylethanolamine (PE), such as dimyristoyl phosphatidylethanolamine (DMPE), etc .; phosphatidylglycerol (PG) E.g. dipalmitoylphosphatidylglycerol (DPPG) Etc .; it can be cited phospholipids, such as dimyristoyl lysophosphatidic acid (DMPA), glycosphingolipid, glycolipids such as glyceroglycolipids; phosphatidylserine (PS); phosphatidylinositol (PI). These lipids may be used alone or in combination of two or more, or in combination with a nonpolar substance such as cholesterol.
[0012]
The micelle in the present invention is formed by incorporating a water-soluble polymer substance such as a lipid derivative in which a protein such as an antibody or a water-soluble polymer such as polyethylene glycol is bound to the lipid, in addition to the lipid or nonpolar substance. is there.
The micelle in the present invention may be manufactured by any method, but can be manufactured by using a commonly used manufacturing technique known per se, for example, using the above-mentioned materials. For example, multilamellar liposomes (MLV) formed by adding an aqueous solution to a lipid thin film attached to a glass wall and mechanically shaking, or small unilamellar liposomes (SUVs) obtained by ultrasonic treatment, ethanol injection, or French press. ), Surfactant removal method, reverse phase evaporation method (liposome Junzo Sanamoto et al., Minamiedo 1988), large unilamellar liposome (LUV) obtained by extruding MLV by pressing a membrane having a uniform pore size ) (Liposome Technology, Vol.1 2nd Edition).
[0013]
Incorporation of the water-soluble polymer substance into the micelle may be carried out by preparing a lipid derivative of the water-soluble polymer in advance and incorporating it in the production of the micelle according to the above method. A water-soluble polymer may be bound (see JP-A-4-346918).
Furthermore, a drug or a diagnostic agent may be introduced into the micelle.
[0014]
Drugs that can be introduced into micelles include: anti-tumor agents such as adriamycin, daunorubicin, vinblastine, cisplatin, mitomycin, bleomycin, actinomycin, 5-FU (fluorouracil), adrenergic blockers such as timolol, hypertensive agents such as clonidine, prokaiamide Anti-malarial agents such as chloroquinine, and pharmaceutically acceptable salts and derivatives thereof; toxin proteins such as ricin A and diphleriatoxin and DNA encoding the same, and cytokine genes such as TNF Nucleotides such as DNA, antisense DNA and the like. Examples of the pharmaceutically acceptable salt of the antitumor agent and the like include salts with pharmaceutically acceptable polyanionic substances such as citrate, tartrate, glutamate, and derivatives thereof. Is preferred.
[0015]
Examples of diagnostic agents that can be introduced into micelles include radiopharmaceuticals, for example, imaging agents such as indium and technesum; enzymes such as horseradish peroxidase, alkaline phosphatase and β-galaclosidase; phosphors such as europium derivatives; N-methyl Examples thereof include light emitters such as an acridium derivative.
Introduction of these drugs and diagnostic drugs into micelles can be carried out by a commonly used method known per se. For example, it may be added and enclosed as an aqueous solution when forming micelles such as liposomes, and a concentration gradient such as a pH gradient is formed inside and outside the vesicle after micelle formation, and a drug capable of being ionized is taken in by using this potential as a driving force ( Cancer Res. 49 , 5922 (1989)) may be used.
[0016]
In the analysis method of the present invention, a micelle incorporating a water-soluble polymer substance is solubilized with a solvent that can be solubilized by the micelle itself, and then the solubilized solution is separated by gel filtration chromatography. Functional polymer substance 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. As the surfactant, any of nonionic, anionic, cationic and zwitterionic can be used. Suitable surfactants include, for example, sodium dodecyl sulfate, sodium deoxycholate, polyoxyethylene sorbitan monolaurate (Tween 20), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene octyl phenyl ether (Triton) X-100), N-cyclohexyl-3-aminopropanesulfonic acid (CAPS), 3-[(3-colamidopropyl) dimethylammonio] -1-propanesulfonate (CHAPS), cetylpyridium chloride (CPCl) And tetradecyltrimethylammonium bromide (TDMABr). Of these, sodium dodecyl sulfate is most preferred. The concentration of the surfactant is not particularly limited, but a final concentration of 0.5 to 10% by weight is appropriate.
[0017]
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, it is usually 30 to 100% by volume, preferably 50 to 80% by volume. Further, when the water-soluble polymer substance is a lipid derivative of a protein, it is preferable to use water that does not contain an organic solvent.
[0018]
The pH of the solvent is not particularly limited, but is usually 4.0 to 9.0, preferably 6.0 to 8.0. The aqueous solvent may be buffered with a suitable buffer such as a phosphate buffer, a Tris-HCl buffer, an acetate buffer, or the like. The concentration of the buffer is usually 20 to 50 mM.
[0019]
The micelle is easily solubilized by mixing the micelle with the solvent and heating as necessary. The heating conditions are not particularly limited, but it is usually appropriate to carry out at a temperature of 50 to 80 ° C. for 15 to 45 minutes.
The solubilized solution of micelles thus obtained is separated by gel filtration chromatography to detect a water-soluble polymer substance.
[0020]
Examples of the column for gel filtration chromatography include a column filled with a hydrophilic polymer or silica gel. For example, TSK gels G2000PW _XL , G3000PW _XL, and G4000PW _XL (commercially available from Tosoh Corporation) are listed as polymer columns, and MCI gels CQS10 and CQS30 (commercially available from Mitsubishi Chemical Corporation) as silica gel columns, TSK gel G2000SW _XL, (commercially available from Tosoh Corporation) G3000SW _XL and G4000SW _XL, and the like. In particular, when analyzing a lipid derivative of a protein, a silica gel column is preferable.
[0021]
The mobile phase of gel filtration chromatography is not particularly limited, but an aqueous solution containing a surfactant is suitable. Examples of the surfactant include those described above. 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, it is usually 30 to 100% by volume, preferably 50 to 80% by volume, and the analysis target is a protein lipid derivative. In some cases, it is preferable to use an aqueous solution that does not contain an organic solvent.
[0022]
The solution used for the mobile phase may further contain an appropriate salt such as 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 about 100 to 300 mM. 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.
[0023]
The pH of the solution used for the mobile phase is not particularly limited, but usually pH 6.0 to 8.0 is appropriate. If the analyte is a lipid derivative of a protein, the pH is 6.5 to 7.5, and the analyte is a lipid of polyethylene glycol. In the case of a derivative, pH 7.0 to 8.0 is most preferable. The solution may be buffered with the appropriate buffer described above, and the concentration of the buffer is usually 20 to 50 mM.
[0024]
In the solution used for the above mobile phase, when the analyte is a lipid derivative of polyethylene glycol, it contains 50 to 80% by volume of an organic solvent such as methanol or acetonitrile and 0.05 to 0.5% by weight of sodium dodecyl sulfate. 20-50 mM sodium phosphate buffer (pH 7.0-8.0) is preferred. When the analyte is a lipid derivative of a protein, 20 to 50 mM sodium phosphate buffer (pH 6.5 to 7.5) containing 100 to 300 mM salt and 0.05 to 0.5 wt% sodium dodecyl sulfate is used. preferable.
[0025]
For detection of water-soluble polymer substances separated by gel filtration chromatography, for example, when the analyte is a lipid derivative of polyethylene glycol, ultraviolet absorption method, suggested refractive index method, etc .; When it is a derivative, it can be easily performed by an ultraviolet absorption method or the like.
[0026]
【The invention's effect】
According to the analysis method of the present invention, the water-soluble polymer substance incorporated in the micelle can be analyzed accurately and simply. Further, the analysis method of the present invention can easily and directly analyze not only the content of water-soluble polymer substances but also the molecular weight distribution even in the presence of excess micelle components such as lipids or in the presence of a large amount of encapsulated drugs. Is possible. Furthermore, fractional quantification is possible even in micelles incorporating a plurality of water-soluble polymer substances by slightly changing solubilization conditions and column separation conditions.
[0027]
【Example】
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 liposomes 1 ml of 0.3 M per 100 mg of solid lipid mixture consisting of dipalmitoylphosphatidylcholine (DPPC) / cholesterol (Chol) / maleimidated dipalmitoylphosphatidylethanolamine (DPPE) (18/10 / 0.5) Citrate buffer pH 4.0 was added and hydrated with a vortex mixer. Furthermore, multilamellar liposomes were prepared by repeating freeze-thaw 5 times. Next, the liposomes were sequentially filtered under pressure while heating at 60 ° C. with an extruder (manufactured by NOF Liposome Co., Ltd.) equipped with 200 nm and 100 nm pore size polycarbonate membranes to prepare sized liposomes.
[0028]
The obtained liposome solution was neutralized with 1M NaOH solution, and then 1/10 weight of adriamycin (Kyowa Hakko) was added. As a result, adriamycin-encapsulated liposomes encapsulating 97% or more of adriamycin were obtained.
Furthermore, a F (ab ′) type 2 human monoclonal antibody in which a thiol group was introduced into this liposome by iminothiolane (Traut, RR et al., Biochemistry 12, 3266 (1973)) by the method of Traut et al. Were added to react to obtain antibody-bound liposomes. Next, a thiolated polyethylene glycol prepared from 2,4-bis (polyethylene glycol) -6-chloro-S-triazine (Seikagaku Corporation) is added to the above antibody-bound liposome solution and reacted with maleimide to produce an antibody. And liposomes with polyethylene glycol were prepared. Unreacted antibody and polyethylene glycol were purified and removed by gel filtration with Sepharose CL6B.
[0029]
Example 1 Quantitative determination and purity analysis of polyethylene glycol chain An equal volume of the liposome dispersion prepared in Reference Example 1 (lipid concentration of about 10 mg / mL) and 4 wt% sodium dodecyl sulfate aqueous solution were mixed, and 50 Heat at 30 ° C. for 30 minutes. The resulting 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.
[0030]
Operating conditions and detector: UV absorption photometer (measurement wavelength: 215 nm)
Column: A stainless steel tube having an inner diameter of about 8 mm and a length of about 30 cm is packed with silica gel for aqueous solvent gel filtration chromatography in which a hydrophilic group of about 5 μm is chemically bonded (using TSK gel G2000SW _XL ).
Precolumn: Fill a stainless steel tube with an inner diameter of about 6 mm and a length of about 4 cm with the same packing material as the column (use TSK gel guardcolumn SW _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 10 wt% sodium dodecyl sulfate solution, then 20 wt% Add sodium hydroxide solution to adjust pH to 7.5, and add water to make 1,000 mL.
・ Flow rate: 0.5mL / min.
・ Area measurement range: 55 minutes from the start of measurement.
[0031]
By heating the liposome in the sodium dodecyl sulfate solution (solubilizing solvent), the liposome was disintegrated and each component was in a solubilized state. The 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 (polyethylene glycol lipid derivatives) were detected as peaks separated from lipids and drugs. FIG. 1 shows the elution pattern. The polyethylene glycol chain peak was good in both linearity and reproducibility, 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 weight is about 20,000, about 10,000, and about 50,00 in order of elution. From this, it was found that the purity analysis of the polyethylene glycol chain was possible. In this condition, since the protein bound to the liposomes is not eluted, the polyethylene glycol chain can be quantified and analyzed for purity even when the molecular weight of the polyethylene glycol chain is equal to that of the protein.
[0032]
Example 2 Antibody quantification and purity analysis An equal volume of a liposome dispersion (lipid concentration of about 10 mg / mL) prepared in the same manner as in Reference Example 1 and a 4% by weight aqueous sodium dodecyl sulfate solution were mixed together. Heat at 30 ° C. for 30 minutes. The resulting liposome solubilized solution was analyzed by high performance liquid chromatography under the following operating conditions. For quantification, an antibody solution having a clear concentration was used as a standard solution.
[0033]
Operating conditions and detector: UV absorption photometer (measurement wavelength: 280 nm)
Column: A stainless steel tube having an inner diameter of about 8 mm and a length of about 30 cm is packed with silica gel for aqueous solvent gel filtration chromatography in which a hydrophilic group of about 5 μm is chemically bonded (using TSK gel G3000SW _XL ).
Precolumn: Fill a stainless steel tube with an inner diameter of about 6 mm and a length of about 4 cm with the same packing material as the column (use TSK gel guardcolumn SW _XL ).
-Column temperature: A 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 10 wt% sodium dodecyl sulfate solution, and then add 20 wt% sodium hydroxide solution to adjust the pH to 7.0 And add water to make 1,000 mL.
・ Flow rate: 0.5mL / min.
・ Area measurement range: 50 minutes from the start of measurement.
[0034]
By heating the liposome in the 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 is negatively charged and denatured by sodium dodecyl sulfate. The 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.
[0035]
As a result, the antibody (lipid derivative of the antibody) was detected as a peak separated from the lipid or drug. FIG. 3 shows the elution pattern. The three peaks in FIG. 3 indicate the peaks of polymerized antibody, antibody, and decomposed antibody, respectively. These antibody peaks had good linearity and reproducibility, and quantification using a standard antibody solution was possible.
[Brief description of the drawings]
BRIEF DESCRIPTION OF DRAWINGS FIG. 1 is a diagram showing an elution pattern of solubilized liposomes by gel filtration chromatography. In the figure, the horizontal axis indicates time (minutes) and the vertical axis indicates absorbance.
FIG. 2 is a diagram showing an elution pattern of solubilized liposomes by gel filtration chromatography. In the figure, the horizontal axis indicates time (minutes) and the vertical axis indicates absorbance.
FIG. 3 is a diagram showing an elution pattern of solubilized liposomes by gel filtration chromatography. In the figure, the horizontal axis indicates time (minutes) and the vertical axis indicates absorbance.

Claims (7)

When analyzing a water-soluble polymer substance incorporated in a micelle, the solubilized solution is separated by gel filtration chromatography after being solubilized with an aqueous solvent containing a surfactant that can be solubilized by the micelle itself. Analysis method to do. The analysis method according to claim 1, wherein the surfactant is sodium dodecyl sulfate. The analysis method according to claim 1, wherein the micelle is a liposome. The analysis method according to any one of claims 1 to 3, wherein the analysis is a quantitative and purity analysis of a water-soluble polymer substance. The analysis method according to any one of claims 1 to 4, wherein the water-soluble polymer substance is a lipid derivative of protein and / or a lipid derivative of polyethylene glycol. 6. The analysis method according to claim 5, wherein the water-soluble polymer substance is a lipid derivative of protein and a lipid derivative of polyethylene glycol. The analysis method according to any one of claims 5 to 6, wherein the lipid derivative of the protein is a lipid derivative of an antibody.

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