JPH087219B2 - Method for quantifying liposome-encapsulated bioactive protein - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for quantifying a physiologically active protein encapsulated in a liposome with high accuracy and efficiency. This method can be usefully applied in the production of pharmaceuticals or therapeutically.

[0002]

2. Description of the Related Art Liposomes are a type of lipid artificial membrane. In order to produce liposomes, many phospholipids and glyceroglycolipids are suspended in at least 50% or more of water above the gel-liquid crystal phase transition temperature of the lipid, and automatically closed vesicles composed of a lipid bilayer. Is formed. This vesicle is called a liposome. In addition, vesicles with multiple concentric lamellae (bilayers) with a diameter of 0.1 to 10 μm are created by applying mechanical vibration. In a narrow sense, this vesicle may be called a liposome. Often referred to as multilamellar vesicles (MLV). Vesicles generated when MLV is sonicated, lipids dissolved in ethanol are rapidly mixed with water, or only cholic acid is removed from a mixed solution of cholic acid and lipid by dialysis, etc. It consists of a membrane. Called small unilamellar vesicles (SUVs). Large lamellas (LUV) with a diameter of about 1 μm can also be produced. An aqueous solution or aqueous dispersion can be enclosed in such a lipid membrane. In addition, liposomes are also called by the name based on their manufacturing method, especially when prepared by mixing an organic solvent solution of lipid with water, removing the organic solvent, and performing phase inversion REV (reverse-phase). evaporation vesi
cle).

Various substances can be retained in the aqueous layer or the lipid bilayer inside the liposome, and they are often used as microcapsules. It is also protected by lipid membranes and is used for its effect on the stability of encapsulated substances and for improving the accessibility of target tissues such as hormones. In the formulation of liposomes such as pharmaceuticals, the above RE
V is often used, and it is prepared as a liposome preparation by dissolving or dispersing a physiologically active substance such as protein or hormone in an aqueous solution. In order to quantify the substance encapsulated in the liposome, it is necessary to physically break the liposome for measurement.

[0004]

[Problems to be Solved by the Invention] When quantifying a physiologically active protein encapsulated in a liposome, an immunoassay is often used, for example, a method using an antibody such as enzyme immunoassay (EIA) or radioimmunoassay (RIA). Is adopted. The liposome has a diameter of 0.1 to
Although they are small vesicles of 10 μm, a considerable amount of bioactive protein is adsorbed on the liposome surface due to the step of encapsulating bioactive protein. The physiologically active protein adsorbed on the liposome surface and the protein encapsulated in the liposome behave differently when used in the human body as a drug.Therefore, when a liposome preparation is used as a drug, it is encapsulated in the liposome. It is necessary to accurately quantify bioactive proteins. For this reason, first, the physiologically active protein adsorbed on the liposome surface and present in the external phase liquid was first measured, and then the liposome was destroyed, and then measured again, and the difference between the value after destruction and the value before destruction was encapsulated. The amount of protein is used. However, in the measurement method using an antibody such as the EIA method and the RIA method, which are considered to have the highest accuracy as a method for quantifying a bioactive protein, the liposome is much larger than the antigen-binding site of the antibody. Therefore, steric hindrance appears in the measurement. Therefore, it has been difficult to accurately measure the amount of bioactive protein encapsulated in the liposome.

As a result of further studies on a method for accurately quantifying the amount of physiologically active protein encapsulated in such liposomes, the present invention has found a method for quantifying the amount of liposome-encapsulated protein more accurately than ever before. . An object of the present invention is to provide a method for quantifying a physiologically active protein encapsulated in liposomes.

[0006]

MEANS FOR SOLVING THE PROBLEMS The present invention provides a method for quantifying a physiologically active protein encapsulated in a liposome (1)
After treating the physiologically active protein adsorbed on the liposome surface with a protease until the antigenic properties of the protein are lost,
(2) A protease inhibitor is added to inactivate the protease, (3) the liposome is then destroyed, and the physiologically active protein encapsulated in the liposome is eluted outside the liposome, and (4) the physiology eluted outside the liposome. The present invention relates to a method for quantifying liposome-encapsulated physiologically active protein, which comprises quantifying active protein by an immunoassay. In the present invention, the liposome refers to a vesicle composed of the above-mentioned lipid artificial membrane. When such a liposome-enclosed physiologically active protein is measured, it can be easily and accurately measured by using the method of the present invention. Examples of the physiologically active protein include erythropoietin, TNF, t-PA, CSF and the like.
The liposome encapsulating the physiologically active protein may be prepared by any method and any lipid. The liposome is suspended in a buffer solution such as PBS and then a protease is added to inactivate the physiologically active protein adsorbed on the liposome surface and the physiologically active protein present in the external phase liquid by the protease. In this case, the conditions for enzyme treatment are optimal conditions for the protease used. A protease inhibitor is then added to the liposome suspension to inhibit protease activity. Examples of proteases and protease inhibitors used are as follows.
Table 1 shows examples of combinations of protease and protease inhibitor. However, the protease, the protease inhibitor, and the combination thereof are not limited to the present examples, and any protease and protease inhibitor may be used and any combination may be used as long as the object of the present invention is achieved.

Proteases; pepsin, trypsin, papain, ficin, promeline, plasmin, thrombin, thermolysin, subtilisin, elastase, collagenase, factor Xa, aminopeptidase, chymotrypsin A, endoproteinase, carboxypeptidase, leucine aminopeptidase protease inhibitor; α ₂ -macroglobulin,
Aprotinin, α ₁ -antitrypsin, ethylenediaminetetraacetic acid (EDTA), trypsin inhibitor, TPK
, TLCK, leupeptin, pepstantin

[0008]

[Table 1]

After inhibiting the protease with a protease inhibitor, the liposome is destroyed. To destroy the liposome, methods such as osmotic pressure change, protein toxin treatment, and surfactant treatment are used. However, a method in which a surfactant is added to a solution in which liposomes are suspended to destroy the liposomes is desirable because it is possible to elute the physiologically active protein outside the liposomes by reducing impurities. As the surfactant, Tween series, Span series, polyoxyethylene polyoxypropylene glycol (Pluronic), etc. are used, but those that disrupt the liposome membrane and do not affect the quantification of bioactive proteins Any surfactant can be used as long as it is. As such a surfactant, octoxynol (trade name: Triton X
-100) etc. can be illustrated. A surfactant is added and gently mixed with stirring to break the liposomes. Then, the EIA method using an antibody that binds to a bioactive protein or
Quantify by RIA method.

An example of the EIA method for measuring erythropoietin is shown below. (1) Coating of primary antibody (anti-EPO antibody) Anti-EPO antibody was diluted with the following buffer solution to an antibody concentration of 10 μg / ml, and 100 μl of this was added to each well of the microplate for 2 hours at room temperature or Let stand for half a day at 4 ° C. Coating buffer composition: NaHCO ₃ 2.1g NaN ₃ 0.1g 1N NaOH (pH 9.6) About 5ml / 500ml

(2) Washing The plate prepared in (1) is washed three times with the above buffer solution. (3) Blocking Add 200 μl of blocking agent of the following composition to each well and incubate at 4 ° C for 16 hours or more. Blocking solution _{composition: NaH 2 PO 4 · 2H 2} O 0.436g Na 2 HPO 4 · 12H 2 O 2.575g NaCl 8.5g NaN 3 0.2g BSA 10g / l

(4) Dilution of sample Dilute so that the EPO concentration is about 0.1 U / ml. The following buffer is used as the diluent. Diluting buffer composition: NaH ₂ PO ₄・ 2H ₂ O 0.436g Na ₂ HPO ₄・ 12H ₂ O 2.575g NaCl 8.5g NaN ₃ 0.2g BSA 2g 0.25M EDTA (pH8) 8ml Tween20 1ml / l

(5) Washing of plate The plate is washed again three times with the following washing solution. The cleaning _{solution: pH7.4 NaH 2 PO 4 · 2H} 2 O 0.436g Na 2 HPO 4 · 12H 2 O 2.575g NaCl 8.5g NaN 3 0.2g Tween20 1ml / l

(6) Preparation and addition of standard solution EPO standard product was prepared using the buffer solution described in (4),
Create a concentration series of 0,160,200,240,300 mU / ml, 50 μl each
Put in the well.

(7) Addition of sample Add 50 μl of the sample prepared in (4) to the well. (8) Preparation and addition of secondary antibody Anti-EPO antibody was added to alkaline phosphatase (ALP) by a conventional method.
The secondary antibody modified with is diluted with an antibody dilution buffer. Antibody dilution buffer composition: NaH ₂ PO ₄・ 2H ₂ O 0.436g Na ₂ HPO ₄・ 12H ₂ O 2.575g NaCl 8.5g NaN ₃ 0.2g / l Add 50 μl of diluted antibody to each well and leave at room temperature for 2 hours To do.

(9) Washing of plate After 2 hours, wash the plate 8 times with the washing solution described in (5). (10) Preparation and Addition of Substrate Solution The substrate p-nitrophenyl phosphate disodium (Sigma) is diluted with the following substrate buffer solution to prepare a 2 mg / ml solution. Substrate buffer composition: Diethanolamine 52.57g (pH 9.8) NaN ₃ 0.1g 1M MgCl ₂ 250 μl / 500 ml Adjust the pH to 9.8 with hydrochloric acid. Add 100 μl of the prepared substrate solution to each well and let stand for 15 minutes to allow reaction. (11) Stop the reaction Add 50 μl of 3N NaOH aqueous solution to the wells to stop the reaction. (12) Quantification Quantification is performed by measuring the absorbance at 405 nm using an immunoreader.

The present invention will be described in more detail below by showing Examples and Comparative Examples.

Example 1 Preparation of liposomes encapsulating erythropoietin Egg yolk lecithin (179 mg), cholesterol (90 mg) and dicetyl phosphate (12.7 mg) were dissolved in chloroform (15 ml), and then the solvent was distilled off by concentration under reduced pressure to form a lipid thin film. This thin film was dissolved in 15 ml of diethyl ether, this solution was transferred to a test tube, and 5 ml of an erythropoietin (EPO) solution dissolved in PBS at a concentration of 6000 U / ml was added and mixed well. After that, sonicate with a tip-type sonicator and emulsion (W / O
Type) This solution was placed in an eggplant-shaped flask, the solvent was again distilled off under reduced pressure, and the obtained gel-like substance was shaken using a vortexter (manufacturer name) to carry out phase inversion. This was again subjected to reduced pressure treatment with an evaporator. By the above operation, 2 ml of EPO-encapsulated REV type liposomes were obtained. Dilute this liposome about 50 times with PBS,
Centrifugation was performed at 11000 rpm and 15000 g for 60 minutes to collect the precipitate. This washing and centrifugation operation was repeated 4 times to remove the EPO not encapsulated in the liposome. The EPO-encapsulated liposome was diluted with 10 ml of PBS to obtain 10 ml of a purified liposome solution.

[0018]

Example 2 Measurement of EPO Activity Encapsulated in Liposomes As described above, optimum protease conditions for deactivating EPO adsorbed on the liposome surface by protease treatment of liposomes and protease inhibitor treatment conditions were examined. (1) Inactivation condition of EPO by trypsin treatment 0.1 mg / ml trypsin PB in 2 ml of 298 U / ml EPO PBS solution
Mix 2 ml of S solution and incubate at 30 ° C for approx. 1
EPO was measured by EIA method by sampling at minute intervals. The measurement results are shown in FIG. It was confirmed that EPO completely disappeared after 10 minutes of enzyme treatment. (2) Inhibition of trypsin activity by protease inhibitor Aprotinin was selected as an inhibitor for trypsin used in (1) above, and the trypsin activity inhibitory effect of aprotinin was measured. 0.1 mg / ml trypsin in PBS
To 1 ml, 1 ml of PBS solution in which aprotinin was adjusted to concentrations of 1 nM / ml, 10 nM / ml and 100 nM / ml was added and mixed, and the mixture was reacted at 30 ° C. for 10 minutes. Next, add EPO solution adjusted so that the final concentration of EPO will be about 50 U / ml, mix, and then mix with EP by EIA method.
O 2 activity was measured. The results are shown in Table 2.

When the trypsin concentration was 0.1 mg / ml, the enzyme activity could be inhibited by mixing equal amounts of aprotinin 10 nM / ml solution. When the trypsin concentration was 1.0 mg / ml, the enzyme activity could be inhibited by mixing equal amounts of aprotinin 100 nM / ml solution.

[0020]

[Table 2]

(3) Measurement of Encapsulation Amount of EPO Encapsulated in Liposomes 1 ml of liposomes diluted 10 times with PBS was mixed with 1 ml of trypsin-PBS solution having a concentration of 0.1 mg / ml, incubated at 30 ° C. for 10 minutes, and then 10 nM aprotinin was added. PBS solution 1 ml
Was added, mixed, and further incubated at 30 ° C. for 10 minutes. To 1 ml of the enzyme-treated sample solution, add 1 ml of 2% Triton X-100 prepared by diluting PIERCE's 10% Triton X-100 solution 5 times with PBS, and add the liposomes. I made it collapse. It was further diluted 50 times with PBS and EIA measurement was performed. 0.1 mg / ml for 1 ml of liposome
After mixing with 1 ml of trypsin-PBS solution at the following concentration and incubating at 30 ℃ for 10 minutes, a further 10 nM aprotinin PBS was added.
1 ml of the solution was added, mixed and further incubated at 30 ° C for 10 minutes. 2% Triton X- was added to the treatment solution thus obtained.
100 1 ml was added to disrupt the liposomes. This solution was diluted 50 times with PBS and used as a sample to measure the EPO activity by the EIA method. This measured value was set as ENZ.
The amount of EPO existing on the liposome surface and in the external phase was also measured by the direct EIA method using 1 ml of the liposome as a sample. The liposome solution diluted 10 times with PBS was further added to PB.
It was diluted 100 times with S and measured by the EIA method. FR this value
EE. Furthermore, operate EPO in the same way as the ENZ measurement sample,
The liposomes were disrupted and EPO was measured. This measurement is To
It was tal. In conventional measurement, the difference between Total and FREE is included
It was the EPO. The measurement results are shown in Table 3. Measurement is A, B
The same sample was measured twice.

[0022]

[Table 3]

(4) Calculation of encapsulation rate The encapsulation rate was calculated from the measured values for the total EPO. From the measured values shown in Table 3, as a conventional method, the difference between Total and Free and the ENZ method of the present invention as the encapsulation rate are shown in Table 4 below. This measurement method showed a lower value than the conventional method. In this measurement method, only EPO in the liposome is selectively measured, whereas in the conventional method, the external phase liquid and the liposome-adsorbed EPO are also measured. However, when measured as Free, it is lower than the actual value due to steric hindrance of the liposome. The conventional method does not reflect the true value.

[0024]

[Table 4]

[0025]

[Example 3] Measurement of encapsulation rate Comparative encapsulation rates of EPO liposomes with various encapsulation amounts prepared in the same manner as in Example 1 were measured for a large number of samples. The conventional method and the ENZ method of the present invention were compared, and Table 5 It was shown to. It was confirmed that the method of the present invention always calculates the encapsulation rate lower than that of the conventional method. When these encapsulated EPOs were administered to animals, biological activity was obtained that was consistent with the EPO encapsulation rate measured by the method of the present invention. From this, it was confirmed that the present measurement method represents the true encapsulation rate.

[0026]

[Table 5]

[0027]

[Experimental Example 1] Calculation of true encapsulation rate using aqueous phase marker In order to confirm the difference between the encapsulation rate measured by the present invention and the conventional method, the 5 (6) -carboxyl fluoro was used. The liposome encapsulation rate was measured using cein. (1) Preparation of Aqueous Phase Marker-Encapsulated Liposomes 179 mg of egg yolk lecithin, 90 mg of cholesterol, and 12.7 mg of dicetyl phosphate were dissolved in 15 ml of chloroform, and then the solvent was removed by concentration under reduced pressure to form a lipid thin film. This thin film was dissolved with 15 ml of diethyl ether, transferred to a test tube, and then 5 ml of a solution of 5- (6) -carboxyfluoroscein (CF) dissolved in PBS at a concentration of 2 mM was added and mixed well.
After that, ultrasonic treatment was performed with a chip type sonicator to obtain a W / O emulsion. Put this solution in an eggplant-shaped flask,
The solvent was distilled off again under reduced pressure, and the obtained gel-like substance was shaken using a vortexer to carry out phase inversion. This was again subjected to reduced pressure treatment with an evaporator. With the above operation, CF
2 ml of encapsulated REV type liposomes were obtained. This liposome was diluted about 50 times with PBS and centrifuged at 11000 rpm and 1500 G for 60 minutes to recover the precipitate. This operation was repeated 4 times, and the liposomes were thoroughly washed to remove CF not encapsulated in the liposomes. Precipitated CF-encapsulated liposomes in PBS
It was diluted with 10 ml to obtain 10 ml of purified liposomes.

(2) Measurement of Fluorescence Intensity of Aqueous Phase Marker-Encapsulated Liposomes Measurements were performed using a fluorescence spectrophotometer. Excitation was carried out at a wavelength of 490 nm, and the fluorescence intensity at 520 nm was measured. Before the measurement, a calibration curve (CF concentration 0-5 μM / ml) was prepared and measured. The measurement measures CF existing on the liposome surface and the external phase,
Then, the liposome was destroyed with a surfactant and the CF concentration was measured.

(3) Measurement Results The measurement results are shown in Table 6 below. Quantitation by fluorescence method is EI
Unlike the method using an antibody such as A, there is no steric hindrance, and therefore substances present on the surface of the liposome or on the outer phase can be accurately measured. Therefore, it can be said that the amount of the substance encapsulated in the liposome is equal to the value obtained by measuring the liposome by breaking it and the value obtained by measuring the outer phase and the liposome surface. In each of the measurement examples of EPO shown in the examples, the free phase corresponding to the amount adsorbed on the outer phase and the liposome surface was used.
It was estimated that the value of was measured low. When considering steric hindrance like the EIA method, it is expected that the amount corresponding to the Free value is measured low. Therefore, EI
It was predicted that the method of accurately measuring the amount of protein encapsulating liposome using an antibody such as method A would have a problem in measurement accuracy.

[0030]

[Table 6]

[0031]

By carrying out the present invention, it becomes possible to accurately measure the amount of the physiologically active protein encapsulated in the liposome. This measurement method can be used for product management of pharmaceutical products and contributes to stable product quality.

[Brief description of drawings]

FIG. 1 shows an example of measurement of inactivation of EPO activity by trypsin treatment.

Continuation of the front page (56) References JP-A 61-55561 (JP, A) JP-A 62-242856 (JP, A) JP-A 61-501921 (JP, A)

Claims (3)

[Claims] 1. When quantifying a physiologically active protein encapsulated in a liposome, the physiologically active protein adsorbed on the liposome surface is treated with a protease until the antigen specificity of the protein is lost, and then a protease inhibitor is added. The protease is inactivated, the liposome is then destroyed, the physiologically active protein encapsulated in the liposome is eluted outside the liposome, and the physiologically active protein eluted outside the liposome is quantified by an immunoassay method. Method for quantifying liposome-encapsulated bioactive protein. 2. The method according to claim 1, wherein the destruction of the liposome is performed by adding a surfactant to the liposome-containing solution. 3. The method according to claim 1, wherein the physiologically active protein is erythropoietin.