阅读说明：本技术 一种在微球制备过程中准确监控plg分子量变化的方法 (Method for accurately monitoring molecular weight change of PLG in microsphere preparation process ) 是由 朱树兰 李深正 方晓云 于 2019-11-13 设计创作，主要内容包括：本发明涉及药物分析领域,公开了一种在微球制备过程中准确监控PLG分子量变化的方法,包括：1)第一油相溶液的制备；2)第二油相溶液的制备；3)在搅拌及水浴加热条件下,将第二油相溶液快速加入到第一油相溶液中,得到混合溶液；4)设置取样时间点,从溶液混合之后开始计时,在各个设置的时间点取混合溶液作为样品,直接进行凝胶渗透色谱法分析,检测PLG重均分子量。本发明操作简便,样品随取随测,无需甲醇水体系进行沉淀,也无需经过干燥步骤,不仅效率高,而且将PLG再次降解的风险彻底化解。采用本发明方法可以快速高效地得出结论,指导下一步工艺；同时由于降低了甲醇这一毒性物质的使用量,对环境及操作人员也更友好。(The invention relates to the field of pharmaceutical analysis, and discloses a method for accurately monitoring the change of PLG molecular weight in the preparation process of microspheres, which comprises the following steps: 1) preparing a first oil phase solution; 2) preparing a second oil phase solution; 3) under the conditions of stirring and water bath heating, quickly adding the second oil phase solution into the first oil phase solution to obtain a mixed solution; 4) sampling time points were set, timing was started after the solutions were mixed, and the mixed solution was sampled at each set time point and directly subjected to gel permeation chromatography analysis to detect the weight average molecular weight of PLG. The invention has simple and convenient operation, samples are taken and measured at any time, a methanol-water system is not needed for precipitation, a drying step is not needed, the efficiency is high, and the risk of degrading the PLG again is thoroughly solved. By adopting the method, the conclusion can be rapidly and efficiently obtained, and the next process is guided; meanwhile, the use amount of methanol which is a toxic substance is reduced, so that the method is more friendly to the environment and operators.)

1. A method for accurately monitoring the change of the molecular weight of PLG in the preparation process of microspheres is characterized by comprising the following steps:

1) preparing a first oil phase solution, namely a PLG solution;

2) preparing a second oil phase solution, namely a raw material medicine solution;

3) under the conditions of stirring and water bath heating, quickly adding the second oil phase solution into the first oil phase solution to obtain a mixed solution of the two;

4) sampling time points were set, timing was started after the solutions were mixed, and the mixed solution was sampled at each set time point and directly subjected to gel permeation chromatography analysis to detect the weight average molecular weight of PLG.

2. The method of claim 1, wherein the microspheres are risperidone microspheres.

3. The method as claimed in claim 2, wherein the step 1) comprises the following specific processes: weighing the PLG and the ethyl acetate, mixing, heating in a water bath at 20-30 ℃ in a dark condition, stirring for 2-4h until dissolving to obtain a first oil phase solution with the PLG concentration of 15-18.5%.

4. The method of claim 3, wherein in step 1), the PLG has a type of 7525 and a weight average molecular weight of 7000 to 170000 Da.

5. The method as claimed in claim 2, 3 or 4, wherein the step 2) comprises the following specific processes: weighing risperidone and benzyl alcohol, mixing, heating in water bath at 20-30 ℃, stirring and dissolving to obtain a second oil phase solution with the concentration of 21.5-26.5%.

6. The method of claim 1, wherein in step 3), the water bath heating temperature is 20-30 ℃.

7. The method as set forth in claim 1, wherein the sampling is once set at 0min in the step 4), and the sampling time point is set again within a time not longer than 30 min.

8. The method of claim 1 or 7, wherein in step 4), the gel permeation chromatography analysis is performed as follows: adding a chloroform-methanol solution into the sample, wherein the volume ratio of chloroform to methanol is (93-97): (7-3), slightly shaking to dissolve, and sampling the dissolved solution to analyze according to the following chromatographic conditions:

9. use of a method according to claims 1-8 for determining the mixing time of the first oil phase solution with the second oil phase solution during the preparation of microspheres.

Technical Field

The invention relates to the field of drug analysis, in particular to a method for accurately monitoring the change of the molecular weight of PLG in the preparation process of microspheres.

Background

In the preparation process of the sustained-release preparation microsphere, a high molecular compound PLG (glycolide-lactide copolymer) is often used as an auxiliary material, and the sustained-release and controlled-release of the active drug are realized through the chemical reaction of the PLG, such as ester bond hydrolysis and enzymatic degradation chain scission. Researches prove that the molecular weight of PLG in the microspheres has obvious influence on the in vivo slow release effect of the medicament. However, the whole preparation process of the microspheres is complex and long, and the PLG is often degraded in the preparation process, so that the molecular weight of the PLG in the final product of the microspheres cannot be expected, and the in vivo release effect of the microspheres is influenced. How to strictly control the molecular weight of PLG in the preparation process of microspheres and keep the molecular weight of PLG in the final product microspheres at an ideal size is a technical problem which is always faced by developers of microsphere preparations.

It is disclosed in EP1925297a1 that microspheres of PLG of different molecular weights can be obtained by adding a nucleophilic compound to PLG and controlling the reaction time and reaction temperature of both. When the technical content of the patent document is repeated by taking nucleophilic compound risperidone as a raw material, the risperidone microsphere end product with ideal PLG molecular weight cannot be obtained. In fact, we have found that PLG degrades very rapidly in molecular weight during the preparation of microspheres, often to a large extent within a few minutes. Therefore, in order to obtain a microsphere preparation with an ideal release profile, the researchers must find the degradation law of PLG in the whole microsphere preparation process, so that the objective of controlling the molecular weight of PLG is expected to be achieved by selecting PLG with a proper starting molecular weight and strictly controlling the mixing reaction time of PLG and active drug (nucleophile), and the above patent documents obviously do not fully disclose the degradation law of PLG in the process.

At present, PLG is mainly precipitated from a reaction mixture solution by using a large amount of aqueous methanol solution, and the precipitate is lyophilized for a long time to remove residual solvent, and finally subjected to Gel Permeation Chromatography (GPC) analysis, which is also adopted in EP1925297a1, for monitoring and analyzing PLG molecular weight during microsphere preparation. However, probably due to the trade secrets of the core process and the manufacturer, the related analysis methods that have been disclosed so far are not fully disclosed for the specific parameters of the analysis method and the key techniques of sample processing, or even if disclosed in practice, the results consistent with the reports cannot be repeated. And these reported methods all undergo a long drying and waiting period during which risperidone and benzyl alcohol molecules are still entrapped in the PLG, with the risk of degradation of the PLG again, which would cause the test result not to truly reflect the actual molecular weight of PLGA at the sampling point.

Disclosure of Invention

In order to solve the technical problems, the invention provides a method for accurately monitoring the change of the molecular weight of the PLG in the preparation process of the microsphere.

The specific technical scheme of the invention is as follows: a method for accurately monitoring the change of the molecular weight of PLG in the preparation process of microspheres comprises the following steps:

1) preparation of a first oil phase solution, i.e. a PLG solution.

2) Preparing the second oil phase solution, namely the raw material medicine solution.

3) And under the conditions of stirring and water bath heating, quickly adding the second oil phase solution into the first oil phase solution to obtain a mixed solution of the two.

4) Sampling time points were set, timing was started after the solutions were mixed, and the mixed solution was sampled at each set time point and directly subjected to gel permeation chromatography analysis to detect the weight average molecular weight of PLG.

It is generally accepted in the art that PLG must undergo a complex series of precipitation, drying, etc. (e.g. EP1925297a1) to be able to accurately measure its molecular weight, and therefore this series of operations is essential. In the process of research, the inventor unexpectedly finds that the change rule of the molecular weight of the PLG in the preparation process of the microsphere can be rapidly and accurately monitored by changing the sample processing mode for GPC analysis. Specifically, GPC analysis was performed using the reaction mixture solution as a sample.

Through detection, the molecular weight degradation rule of the PLG detected by the method is closer to the objective reality. Taking risperidone microspheres as an example, since chemical reagents such as benzyl alcohol and ethyl acetate are used in the preparation process of the risperidone microspheres, and risperidone and benzyl alcohol are used as nucleophilic reagents, once they are mixed and contacted with PLG, chemical reaction inevitably occurs, which induces PLG molecule degradation, thereby causing PLG molecular weight reduction. And timely sampling detection enables the PLG in the sample to be quickly separated from the nucleophilic environment, meanwhile, the uncontrollable external environment interference is reduced, and the possible large-scale degradation phenomenon is avoided.

Preferably, the microspheres are risperidone microspheres.

Preferably, the specific process of the step 1) is as follows: weighing the PLG and the ethyl acetate, mixing, heating in a water bath at 20-30 ℃ in a dark condition, stirring for 2-4h until dissolving to obtain a first oil phase solution with the PLG concentration of 15-18.5%.

Preferably, in the step 1), the model of the PLG is 7525, and the weight average molecular weight is 7000-170000 Da.

Preferably, the specific process of the step 2) is as follows: weighing risperidone and benzyl alcohol, mixing, heating in water bath at 20-30 ℃, stirring and dissolving to obtain a second oil phase solution with the concentration of 21.5-26.5%.

Preferably, in step 3), the water bath heating temperature is 20-30 ℃.

Preferably, the sampling is set once at 0min in the step 4), and the sampling time point is set again within a time not longer than 30 min.

In the step 4): gel permeation chromatography analysis was performed as follows: adding chloroform-methanol solution into the sample, wherein the volume ratio of chloroform to methanol is (93-97) to (7-3), slightly shaking to dissolve, and sampling the dissolved solution to analyze according to the following chromatographic conditions:

compared with the prior art, the invention has the beneficial effects that: the whole process of the invention is simple and convenient to operate, samples are taken and measured at any time, a methanol-water system is not needed for precipitation, a drying step is not needed, the efficiency is high, and the risk of degrading the PLG again is thoroughly solved. By adopting the method, the conclusion can be rapidly and efficiently obtained, and the next process is guided; meanwhile, the use amount of methanol which is a toxic substance is reduced, so that the method is more friendly to the environment and operators.

Drawings

FIG. 1 shows the molecular weight change tendency of PLG obtained by the method of example 1;

FIG. 2 shows the molecular weight change tendency of PLG obtained by the method of comparative example 1.

Detailed Description

The present invention will be further described with reference to the following examples.

General examples

A method for accurately monitoring the change of the molecular weight of PLG in the preparation process of microspheres comprises the following steps:

1) preparation of a first oil phase solution, i.e. a PLG solution.

2) Preparing the second oil phase solution, namely the raw material medicine solution.

3) And under the conditions of stirring and water bath heating, quickly adding the second oil phase solution into the first oil phase solution to obtain a mixed solution of the two.

4) Sampling time points were set, timing was started after the solutions were mixed, and the mixed solution was sampled at each set time point and directly subjected to gel permeation chromatography analysis to detect the weight average molecular weight of PLG.

Preferably, the microspheres are risperidone microspheres.

Preferably, the specific process of the step 1) is as follows: weighing the PLG and the ethyl acetate, mixing, heating in a water bath at 20-30 ℃ in a dark condition, stirring for 2-4h until dissolving to obtain a first oil phase solution with the PLG concentration of 15-18.5%.

Preferably, in the step 1), the model of the PLG is 7525, and the weight average molecular weight is 7000-170000 Da.

Preferably, the specific process of the step 2) is as follows: weighing risperidone and benzyl alcohol, mixing, heating in water bath at 20-30 ℃, stirring and dissolving to obtain a second oil phase solution with the concentration of 21.5-26.5%.

Preferably, in step 3), the water bath heating temperature is 20-30 ℃.

Preferably, the sampling is set once at 0min in the step 4), and the sampling time point is set again within a time not longer than 30 min.

In the step 4): gel permeation chromatography analysis was performed as follows: adding a chloroform-methanol solution into the sample, wherein the volume ratio of chloroform to methanol is (93-97): (7-3), slightly shaking to dissolve, and sampling the dissolved solution to analyze according to the following chromatographic conditions:

example 1 monitoring of PLG molecular weight during preparation of Risperidone microspheres

(1) First oil phase solution preparation (PLG solution)

About 12.07g of PLG (7525, 8E, intrinsic viscosity 0.70-0.90dl/g) was weighed into a 200mL reagent bottle, about 60.00g of ethyl acetate was added, the solution was wrapped with tinfoil and protected from light, heated in a water bath at a temperature of 25 ℃ and dissolved by magnetic force (model: RH B1S25, manufacturer: IKA) for about 3 hours to obtain a first phase solution.

(2) Second oil phase preparation (bulk drug solution)

About 8.03g of risperidone was weighed into a 100mL beaker, about 25.41g of benzyl alcohol was added, the temperature was controlled to 25 ℃ in a water bath, and the solution was dissolved with stirring to obtain a second phase solution.

(3) Analysis of samples

The obtained sample was placed in a 10mL volumetric flask, 8mL of chloroform/methanol bath (volume ratio 95: 5) was added thereto and the solution was gently shaken to dissolve, and after shaking to a constant volume, a sample was taken and subjected to GPC analysis to detect the weight average molecular weight of PLG.

Comparative example 1

(1) First oil phase solution preparation (PLG solution), same as example 1.

(2) The second oil phase was prepared (bulk drug solution) as in example 1.

(3) Sampling process at different time points

(3.1) sample preparation at 3h sampling Point

Weighing 10.03g of the second oil phase solution into a 60ml penicillin bottle, adding 4.61g of the first oil phase solution under the stirring condition, heating in a water bath to control the temperature to be 25 ℃, sampling 3h after mixing, slowly adding the mixture into 200ml methanol by using a syringe, and filtering to obtain a precipitate.

(3.2) sample preparation at 2h sampling Point

Weighing 10.00g of the second oil phase solution into a 60ml penicillin bottle, adding 4.64g of the first oil phase solution under the stirring condition, heating in a water bath to control the temperature to be 25 ℃, sampling 2h after mixing, slowly adding the mixture into 200ml methanol by using a syringe, and filtering to obtain a precipitate.

(3.3)1h sample Point sample preparation

Weighing 10.02g of the second oil phase solution into a 60ml penicillin bottle, adding 4.60g of the first oil phase solution under the stirring condition, heating in a water bath to control the temperature to be 25 ℃, sampling 1h after mixing, slowly adding the mixture into 200ml methanol by using a syringe, and filtering to obtain a precipitate.

(3.4) sample preparation at 15min sample Point

Weighing 10.09g of the second oil phase solution into a 60ml penicillin bottle, adding 4.63g of the first oil phase solution under stirring, heating in a water bath to control the temperature to 25 ℃, slowly adding into 200ml methanol by using a syringe, and filtering to obtain a precipitate.

(3.5) sample preparation at 0min sample Point

Weighing 10.04g of the second oil phase solution into a 60ml penicillin bottle, adding 4.59g of the first oil phase solution under the stirring condition, heating in a water bath to control the temperature to be 25 ℃, slowly adding into 200ml methanol by using a syringe, and filtering to obtain a precipitate.

(4) Sample drying

And (4) uniformly carrying out vacuum drying on the precipitate obtained by sampling treatment at each time point in the step (3) at 25 ℃ for about 24 hours. The weight of the dried product at each time point was weighed as follows:

sampling time (start timing after mixing, unit: hour)	Weight of dried product (unit: mg)
		0	28.95
0.25	28.52
		1	27.12
2	29.72
		3	27.09

Placing the above dried products into 10mL volumetric flasks, adding 8mL chloroform/methanol solution (volume ratio is 95: 5) and shaking slightly to dissolve, storing at room temperature in the dark for 24h, shaking to constant volume, sampling, and performing GPC analysis to detect weight average molecular weight of PLG.

The GPC analysis condition parameters of the above example 1 and comparative example 1 are as shown in the following table:

the results are shown in the following table:

FIG. 1 is a graph showing the change in molecular weight of PLG according to the results of the test in example 1; FIG. 2 is a change curve of the molecular weight of PLG shown in accordance with the results of the test of comparative example 1.

The test results of example 1 show that the PLG is slowly degraded with the increase of the mixing time, and after 3 hours, the weight average molecular weight of the PLG is reduced by about 30000Da, and the degradation speed is in line with the theoretical expectation and the actual situation.

The test results in comparative example 1 show that PLG changes little by little in weight average molecular weight throughout the preparation of the microspheres, does not degrade continuously, and is contrary to theory. The initial weight average molecular weight of PLG (75258E) in the test was 129853Da, whereas the value monitored in comparative example 1 at 0min had been reduced to 76582Da, which is far from the actual value. It can be seen that the detection method in comparative example 1 does not provide the actual change rule of molecular weight of PLG during the microsphere preparation process.

Accordingly, since the molecular weight of PLGA in the finished risperidone microsphere is about 111142Da (see "A protocol for assay of poly (lactic-co-glycolic) in a clinical product", Garner J, et al, Int J pharm.495 (1): 87-92, Aug 28, 2015), we can estimate the time for mixing PLG with reagents such as benzyl alcohol and risperidone to be controlled within 30min according to the change rule of PLG molecular weight monitored in example 1 (see FIG. 1). The developer can perform experimental groping with 15min as an intermediate value to obtain the optimal mixing time.

The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.