Method for detecting moisture content of freeze-drying preparation and application of freeze-drying preparation

Abstract

The invention relates to a method for detecting the moisture content of a freeze-drying preparation and application of the freeze-drying preparation. The method comprises the following steps: freeze-drying a dilution buffer agent used by the freeze-drying preparation; placing the obtained sample in a constant-temperature and constant-humidity environment for different times, taking out the sample, and sealing to obtain samples with different water contents; and detecting semaphores of the samples with different water contents by using LF-NMR, calculating the water contents of the samples by using a Karl Fischer water detection method, and drawing a standard curve of the signal values and the water contents. The method is a non-invasive method, does not need sample pretreatment or sample consumption, saves the detection time, and is sensitive in detection.

Description

Technical Field

[0001] The invention belongs to the technical field of analysis and testing, and relates to a method for detecting the moisture content of a freeze-dried preparation and an application thereof. Background Art

[0002] Lipid nanoparticles (LNP) are a type of nanoparticles formed by lipids. In the past, researchers usually used lipid nanoparticles to directly encapsulate chemical drugs. In the field of gene therapy, researchers began to use lipid nanoparticles to encapsulate nucleic acids, such as mRNA, siRNA, pDNA, etc., which are called nucleic acid lipid nanoparticles. Due to the instability of RNA, making it into a freeze-dried preparation can greatly improve its stability in storage and transportation.

[0003] The detection and control of the moisture content of freeze-dried preparations is a huge challenge in the research and development of freeze-dried preparations. The residual moisture content of freeze-dried preparations will directly affect the quality and stability of the product. Generally speaking, the raw materials of freeze-dried preparations are unstable and easily degraded when exposed to moisture and heat. It is precisely because of the unstable characteristics of raw materials when exposed to moisture and heat that such raw materials or drug preparations are developed into freeze-dried preparations. The lower the moisture content in the finished freeze-dried preparation, the more stable the drug can be stored for a long time, so product moisture control is particularly critical during the preparation of freeze-dried preparations.

[0004] As one of the most important factors affecting the stability of freeze-dried preparations, it is necessary to analyze and control the water content of freeze-dried preparations. The traditional method of testing water content is to use a Karl Fischer instrument for determination. The principle is that when the Karl Fischer reagent in the electrolytic cell of the instrument reaches equilibrium, a water-containing sample is injected. Water participates in the redox reaction of iodine and sulfur dioxide in the presence of pyridine and methanol until all the water is consumed. The water content is calculated according to Faraday's law of electrolysis. However, this method has certain limitations: 1. The Karl Fischer instrument needs to be in a well-sealed room and needs to be equipped with a dehumidifier. The instrument needs to be turned on for a long time to balance before use, which takes a long time; 2. The Karl Fischer determination of samples requires opening the lid to take out a sample larger than 0.1g, which is a destructive detection method for the sample. For precious LNP samples, the loss is too large, and the sample powder after opening the lid may float into the air, posing a potential health hazard to the operator; 3. The Karl Fischer reagent used in the Karl Fischer moisture determination method contains methanol, pyridine, iodine, and sulfur dioxide. These substances are toxic. The operation of transferring reagents needs to be carried out in a fume hood. Operators must protect themselves to avoid inhaling toxic substances; 4. The Karl Fischer method is a destructive detection method and cannot be used to continuously detect the same sample. There will also be individual differences in the moisture content between the same batch of freeze-dried products. The Karl Fischer method for detecting the moisture content of samples at different time points may introduce errors caused by the different initial moisture contents of different samples.

[0005] Therefore, an efficient, rapid, non-invasive and highly sensitive moisture content detection technology urgently needs to be developed to support the moisture determination process of LNP freeze-dried preparations. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for detecting the moisture content of a freeze-dried preparation and its application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a method for detecting the moisture content of a lyophilized preparation, the method comprising:

[0009] (1) Freeze-drying the dilution buffer used in the lyophilized preparation;

[0010] (2) placing the samples obtained in step (1) in a constant temperature and humidity environment for different periods of time, taking them out, and sealing them to obtain samples with different water contents;

[0011] (3) The samples with different water contents were detected by LF-NMR, and the water content of the samples was calculated by Karl Fischer water content detection method, and a standard curve of signal value and water content was drawn.

[0012] The present invention has discovered for the first time the application of low-field nuclear magnetic resonance (LF-NMR) technology in detecting the moisture content of LNP freeze-dried preparations. First, the researchers calculated the mass content and determined that the LNP content accounted for less than 0.1% of the total mass of the freeze-dried powder cake, and the contribution to the sample LF-NMR signal value M was negligible, so five common LNP freeze-dried preparations were prepared as dilution buffers to simulate LNP preparations, and then the five dilution buffers were freeze-dried and placed in a constant temperature and humidity chamber with the lid opened for different times. By controlling the sample absorption time, a series of samples with gradient changes in water content were obtained. The present invention uses LF-NMR to perform non-destructive testing on a series of freeze-dried products. The results show that the signal amount M has a relatively high sensitivity to changes in the water content of freeze-dried preparations. Subsequently, the researchers tested the water content of the same series of products by the traditional Karl Fischer water determination method, quantitatively analyzed the water content X and the nuclear magnetic signal amount M, and established a good positive correlation between M and the water content X. On this basis, the present invention uses LF-NMR technology to detect the moisture content in the development process of LNP freeze-dried preparations, helping to evaluate the quality and stability of freeze-dried products of different LNP prescriptions. As a non-invasive method, LF-NMR technology does not require sample processing or sample consumption, and a single test takes only 44 seconds, saving a lot of manpower, material resources and test time, and also greatly saving precious LNP samples.

[0013] Preferably, the lyophilized preparation comprises a drug and a dilution buffer.

[0014] Preferably, the mass percentage of the drug in the lyophilized preparation does not exceed 0.1%, for example 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc. Other specific point values ​​within the above numerical range can be selected and will not be described one by one here.

[0015] Preferably, the lyophilized preparation is a LNP lyophilized preparation.

[0016] Since the LNP content accounts for less than 0.1% of the total mass of the freeze-dried powder, its contribution to the sample LF-NMR signal value M is negligible. Therefore, we prepared lyophilized products with dilution buffer to simulate LNP freeze-dried preparation samples encapsulating different biomacromolecules to evaluate the quantitative relationship between the LF-NMR signal value M of the LNP freeze-dried preparation in different formulations and the moisture content X of the LNP freeze-dried preparation.

[0017] Preferably, the dilution buffer comprises sucrose buffer and trehalose buffer.

[0018] Preferably, the temperature of the constant temperature and humidity environment in step (2) is 23-27° C., and the humidity is 55-65%.

[0019] The temperature can be selected as 23℃, 24℃, 25℃, 26℃, 27℃, etc., and the humidity can be selected as 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0020] Preferably, the number of samples is no less than 6.

[0021] Preferably, the sealing process also includes balancing.

[0022] Preferably, the equilibrium temperature is 20-25°C and the equilibrium time is 30-50 min.

[0023] The temperature can be selected as 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, etc. The time can be selected as 30min, 32min, 34min, 36min, 38min, 40min, 42min, 44min, 46min, 48min, 50min, etc. Other specific point values ​​within the above numerical range can be selected, so they will not be elaborated here.

[0024] Preferably, the time interval between two adjacent sample removals in step (2) is not less than 10 minutes, for example 10 minutes, 12 minutes, 15 minutes, 18 minutes, 20 minutes, 25 minutes, etc. Other specific point values ​​within the above numerical range can be selected and will not be described here one by one.

[0025] Preferably, the method of calculating the water content of a sample using the Karl Fischer water content detection method specifically includes:

[0026] (1) Determine the mass of the sealed vial containing the lyophilized preparation, denoted as m, and measure the water content of the sample by the Karl Fischer method, denoted as X1;

[0027] (2) Determine the mass of the blank vial and stopper, denoted as q;

[0028] (3) Calculate the moisture content of the sample according to the formula: X% = X1 (mq) × 100%.

[0029] In a second aspect, the present invention provides an application of the method for detecting the moisture content of a freeze-dried preparation according to the first aspect in monitoring moisture content changes and detecting the stability of freeze-dried products.

[0030] Based on the excellent performance of LF-NMR in moisture detection of LNP freeze-dried preparations, the present invention is expected to realize real-time online monitoring of moisture content on the LNP freeze-dried preparation production line in the near future, timely remove unqualified samples, and ensure the sample qualification rate.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention utilizes the characteristics of LF-NMR, which is fast, efficient, highly sensitive and non-invasive, to achieve non-destructive and simple determination of the water content of freeze-dried preparations. First, the present invention utilizes LF-NMR to perform non-destructive detection on freeze-dried products, obtains a signal amount M and compares it with the water content X detected by the traditional Karl Fischer water determination method, and finds that the signal amount M has a good linear relationship with the sample water content X. Secondly, the present invention further establishes a standard curve of the LF-NMR detection signal amount M and the water content X of a common freeze-dried preparation prescription. On this basis, the present invention uses LF-NMR technology for water content detection in the development process of freeze-dried preparations to help evaluate the quality and stability of freeze-dried products of different prescriptions. As a non-invasive method, LF-NMR technology does not require sample processing or sample consumption, and a single detection only takes 44 seconds, saving a lot of manpower, material resources and detection time, and also greatly saving precious samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is the experimental flow chart.

[0034] Figure 2It is the standard curve of water content X and NMR signal quantity of samples 1-6.

[0035] Figure 3 It is the standard curve of water content X and NMR signal quantity of samples 7-12.

[0036] Figure 4 It is the standard curve of water content X and NMR signal quantity of samples 13-18.

[0037] Figure 5 It is the standard curve of water content X and NMR signal quantity of samples 19-24.

[0038] Figure 6 It is the standard curve of water content X and NMR signal quantity of samples 25-30.

[0039] Figure 7 It is the test result of the water content of the sample by LF-NMR after the sample is placed under different temperature conditions for different time periods.

[0040] Figure 8 It is a comparison of the water content test results of LF-NMR and Karl Fischer water determination methods after the samples were placed at different temperatures for different periods of time.

[0041] Fig. 9 It is the evaluation result of the change of moisture content between different batches of LNP freeze-dried preparations, where Figure A is the signal value of different batches of LNP freeze-dried preparations with the same prescription detected by LF-NMR, and Figure B is the result of testing the moisture content using the Karl Fischer water determination method. DETAILED DESCRIPTION

[0042] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0043] Example 1

[0044] This example provides the preparation of five common LNP lyophilized preparations using dilution buffers. The sources of the preparation raw materials are shown in Table 1.

[0045] Table 1

[0046] name grade supplier MW(g / mol) Part Number sucrose NF / EP / JP / ChP Pfanstiehl 342.30 S-124-2-MC Trehalose NF / EP / JP / ChP Pfanstiehl 378.33 T-104-4 Mannitol USP / EP / JP / ChP Pfanstiehl 182.17 M-109-7 Tris USP / EP / BP / JPC Avantor 121.14 4102 hydrochloric acid PhEur / BP / JP / NF Merck 36.46 1.00314

[0047] Table 2

[0048]

[0049] The dilution buffers of five common LNP freeze-dried preparations were prepared according to Table 2. The prepared samples were filtered through a 0.22 μm polyvinylidene fluoride (PVDF) filter and filled into 2 mL glass vials, 1.0 mL per vial, and then half-capped and labeled. All sample preparation procedures were performed in a biosafety cabinet at room temperature. The samples were transferred to the freeze dryer and the parameters were set. The freeze drying procedure is shown in Table 3. After the freeze drying procedure of the freeze dryer was completed, the samples were taken out and fully plugged and capped in the biosafety cabinet.

[0050] Table 3

[0051]

[0052]

[0053] Example 2

[0054] Preparation of a series of samples with gradient changes in the moisture content of freeze-dried products of five common LNP dilution buffers.

[0055] Preparation process Figure 1 As shown, the five groups of samples obtained after freeze-drying in Example 1 were opened and placed in a constant temperature and humidity chamber at 25°C for 0 min, 15 min, 30 min, 45 min, 1 h and 2 h to obtain a series of samples with a gradient change in moisture content. The samples were collected at the corresponding time points and immediately stoppered and capped, and then equilibrated at room temperature for 30 min to ensure that the moisture absorbed by the freeze-dried powder cake was completely and evenly dispersed.

[0056] Among them, the series of samples with a prescription of 20mM Tris, 15% sucrose, and placed in a constant temperature and humidity chamber at 25°C with the lid opened for 0min, 15min, 30min, 45min, 1h and 2h are marked as samples 1-6; the series of samples with a prescription of 20mM Tris, 20% sucrose, and placed in a constant temperature and humidity chamber at 25°C with the lid opened for 0min, 15min, 30min, 45min, 1h and 2h are marked as samples 7-12; the series of samples with a prescription of 20mM Tris, 20% trehalose, and placed in a constant temperature and humidity chamber at 25°C with the lid opened for 0min, 15min, 30min, 45min, 1h and 2h are marked as samples 13-18; the series of samples with a prescription of 20mM Tris, 20% trehalose, and placed in a constant temperature and humidity chamber at 25°C with the lid opened for 0min, 15min, 30min, 45min, 1h and 2h are marked as samples 14-15 A series of samples with a prescription of Tris, 10% sucrose, and 10% mannitol, which were opened and placed in a constant temperature and humidity chamber at 25°C for 0 min, 15 min, 30 min, 45 min, 1 h, and 2 h, were labeled as samples 19-24; a series of samples with a prescription of 20 mM Tris, 10% sucrose, and 10% trehalose, which were opened and placed in a constant temperature and humidity chamber at 25°C for 0 min, 15 min, 30 min, 45 min, 1 h, and 2 h, were labeled as samples 25-30.

[0057] Example 3

[0058] Samples 1-30 prepared in Example 2 were equilibrated in a constant temperature and humidity chamber at 25° C. for 30 min, and then the signal amounts of the samples 1-30 were measured using a LF-NMR analyzer (Suzhou Newmai Analytical Instruments Co., Ltd.) at a magnetic field of 0.47 T and a resonance frequency of 19.5 MHz.

[0059] Place the 2 mL glass vial directly into the 22 mm glass injection tube for the instrument. In order to stabilize the operation of the LF-NMR analyzer, the temperature in the constant temperature chamber is set to 32 ° C (± 0.5 ° C). The first point signal quantity M is measured using the MSE_CPMG pulse sequence. The total measurement time for one sample is about 44 s.

[0060] The LF-NMR results showed that the signal quantity M values ​​of samples 1-30 ranged from approximately 20844 to 43895. The results showed that for samples with the same prescription, the signal quantity M values ​​increased gradually with the extension of the incubation time for water absorption.

[0061] LF-NMR analyzer sequence parameters:

[0062] Sequence: MSE_CPMG

[0063] Sampling frequency: 250(kHz)

[0064] Main frequency: 20(MHz)

[0065] RF delay (ms): 0.02000

[0066] 90 degree pulse width (us): 12.60

[0067] Example 4

[0068] Determination of water content of five lyophilized dilution buffers by Karl Fischer method

[0069] The sample for which the nuclear magnetic resonance signal M has been detected in Example 3 is further tested for water content by the Karl Fischer method.

[0070] How to use Karl Fischer moisture analyzer:

[0071] 1) Turn on the instrument power and instrument balance, and calibrate the balance to ensure that the sample mass can be accurately measured;

[0072] 2) Prepare Karl Fischer test solution and ensure its quality;

[0073] 3) Use a clean weighing spoon to stir the freeze-dried powder into powder, add the powder into the titration container, and determine the mass of the solid added into the titration container by weight loss method and ensure that the amount of sample added is not less than 0.1g;

[0074] 4) Input the mass of added powder into the instrument calculation program;

[0075] 5) Obtain the moisture content of the sample and print the record;

[0076] 6) After the instrument is balanced, test the moisture content of the next sample.

[0077] Calculation method of sample water content:

[0078] 1) firstly measure the mass m1-m30 of the unopened samples 1-30, and then determine the moisture content x1%-x30% of the samples by Karl Fischer method;

[0079] 2) Pour out all the remaining powder of the sample to obtain an empty vial, clean the empty vial and its corresponding rubber stopper with ultrapure water, and then put them in an oven to dry;

[0080] 3) Determine the mass of the vial and the rubber stopper after drying, q1-q30, and calculate the mass of the powder cake of 30 samples as mq;

[0081] 4) Sample moisture content calculation formula: X% = x% (mq).

[0082] The moisture content results of the five common LNP dilution buffer freeze-dried products with gradient changes obtained in Example 2 are shown in Table 2.

[0083] Table 4

[0084]

[0085] Example 5

[0086] Establishment of the linear relationship between the LF-NMR signal value M and the water content of the LNP freeze-dried preparation

[0087] Each group of freeze-dried samples was tested three times by LF-NMR to obtain three groups of signal quantities M1, M2, and M3, and the average value Mave was calculated. The moisture content of the sample was measured by the Karl Fischer method, and the moisture content X of the sample was calculated according to the method in Example 4. The average value Mave of the nuclear magnetic signal quantity of each sample was matched with its moisture content X one by one, and the data table was imported into an Excel table to draw a scatter plot.

[0088] The methods for drawing scatter plots include:

[0089] 1) Select the data area, click "Insert" and select the scatter chart;

[0090] 2) Click any point on the scatter plot, right-click and select "Add Trend Line";

[0091] 3) Check "Show formula" and "Show R square value" in the trend line options.

[0092] The standard curves of NMR signal M-water content X for five groups of freeze-dried products were obtained. Figure 2-6 shown.

[0093] Example 6

[0094] Verification of the reproducibility of the LF-NMR method

[0095] The samples with water content gradient changes of the five prescriptions prepared in Example 3 were taken, numbered 1-30, and tested three times a day by LF-NMR for three days to obtain nine groups of sample signal quantities. The average value, standard deviation and range of the nine groups of values ​​were calculated. The results are shown in Table 5. The results can characterize the measurement repeatability from two perspectives. The standard deviation is distributed between 188.2 and 798.4, and the repeatability is good.

[0096] Table 5

[0097]

[0098]

[0099] Example 7

[0100] The water content of the LNP freeze-dried preparation was detected by measuring the nuclear magnetic resonance signal M.

[0101] The LNP formulation was filtered with a 0.22 μm PVDF filter and filled into 2 mL glass bottles (1.0 mL per bottle), then half-stoppered and labeled. All sample filtering and filling steps were performed aseptically in a biosafety cabinet. The samples were then transferred to a freeze dryer, the freeze drying parameters were set, and after the freeze dryer freeze drying process was completed, the samples were taken out and fully stoppered and capped in a biosafety cabinet.

[0102] The samples were incubated at 5°C, 25°C and 40°C for 4 weeks, and samples were taken at T0, 4 weeks, and the NMR signal of the samples was detected. After the LF-NMR detection was completed, the water content of the samples was determined using the Karl Fischer method. The results are shown in Figure 7-8 As shown, the results show that the error between the LF-NMR method and the Karl Fischer method is within 5%.

[0103] Table 6

[0104]

[0105]

[0106] X represents the moisture content detected by Karl Fischer method and LF-NMR method respectively.

[0107] Example 8

[0108] Evaluation of inter-batch moisture content variation of LNP freeze-dried preparations by measuring the NMR signal M

[0109] The samples of different batches were equilibrated at 25°C for more than 30 min. LF-NMR was used to detect the nuclear magnetic signal M, and the analyzer sequence parameters were as follows:

[0110] Sequence: MSE_CPMG; Sampling frequency: 250 (kHz); Main frequency: 20 (MHz); RF delay (ms): 0.02000; 90 degree pulse width (us): 12.60.

[0111] The moisture content of the samples was tested by the Karl Fischer method.

[0112] The results are as follows Fig. 9 As shown in the figure, during the freeze-drying parameter optimization process, different batches of LNP freeze-dried preparations can be obtained by adjusting the freeze-drying process parameters. The formulations of different batches of preparations are consistent and the loading volume is consistent. However, due to the adjustment of freeze-drying parameters, the water content of samples between batches may change. We use the LF-NMR analysis method to measure the signal quantity M of each sample in each batch, calculate the mean value of the signal quantity M of each batch of samples, and compare the mean values ​​of M of each batch. It can be analyzed which batch has a low water content, and the freeze-drying process parameters used for the freeze-dried products in this batch are more appropriate. LF-NMR has unique advantages in evaluating the changes in the water content of LNP freeze-dried preparations between batches. The operation process is simple, the screening speed is faster, and no pretreatment procedures are required.

[0113] The applicant declares that the present invention illustrates a method for detecting the moisture content of a freeze-dried preparation and its application through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0114] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0115] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A method for detecting the moisture content of a freeze-dried preparation, characterized in that: The method comprises: (1) Freeze-drying the dilution buffer used in the lyophilized preparation; (2) placing the samples obtained in step (1) in a constant temperature and humidity environment for different periods of time, taking them out, and sealing them to obtain samples with different water contents; (3) The samples with different water contents were detected by LF-NMR, and the water content of the samples was calculated by Karl Fischer water content detection method, and a standard curve of signal value and water content was drawn.

2. The method for detecting the moisture content of a freeze-dried preparation according to claim 1, characterized in that: The lyophilized preparation includes a drug and a dilution buffer.

3. The method for detecting the moisture content of a freeze-dried preparation according to claim 2, characterized in that: The mass percentage of the drug in the freeze-dried preparation does not exceed 0.1%.

4. The method for detecting the moisture content of a freeze-dried preparation according to any one of claims 1 to 3, characterized in that: The lyophilized preparation includes a lyophilized LNP preparation.

5. The method for detecting the moisture content of a freeze-dried preparation according to any one of claims 1 to 4, characterized in that: The dilution buffer includes a sucrose buffer and / or a trehalose buffer.

6. The method for detecting the moisture content of a freeze-dried preparation according to any one of claims 1 to 5, characterized in that: The temperature of the constant temperature and humidity environment in step (2) is 23-27° C., and the humidity is 55-65%.

7. The method for detecting the moisture content of a freeze-dried preparation according to any one of claims 1 to 6, characterized in that: The number of the samples is no less than 6.

8. The method for detecting the moisture content of a freeze-dried preparation according to any one of claims 1 to 7, characterized in that: After the sealing process, balancing is also included; Preferably, the equilibrium temperature is 20-25°C and the time is 30-50min; Preferably, the time interval between two consecutive sample removals in step (2) is no less than 10 minutes.

9. The method for detecting the moisture content of a freeze-dried preparation according to any one of claims 1 to 8, characterized in that: Calculating the water content of a sample using the Karl Fischer method involves: (1) Determine the mass of the sealed vial containing the lyophilized preparation, denoted as m, and measure the water content of the sample by the Karl Fischer method, denoted as X1; (2) Determine the mass of the blank vial and stopper, denoted as q; (3) Calculate the moisture content of the sample according to the formula: X% = X1 (mq) × 100%.

10. Use of the method for detecting the moisture content of a freeze-dried preparation according to any one of claims 1 to 9 in monitoring the change of moisture content and detecting the stability of freeze-dried products.

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