CN114875114A - Soluble fms-like tyrosine kinase-1 quality control product and preparation method thereof - Google Patents
Soluble fms-like tyrosine kinase-1 quality control product and preparation method thereof Download PDFInfo
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Abstract
The invention provides a soluble fms-like tyrosine kinase-1 quality control product and a preparation method thereof, relating to the technical field of in vitro diagnosis. The quality control product provided by the invention comprises a quality control product level 1 and a quality control product level 2, and is a freeze-dried product prepared by mixing and freeze-drying recombinant soluble fms-like tyrosine kinase-1 antigen and freeze-dried diluent in different proportions. The quality control product provided by the invention enables the detection result to be more accurate, has the function of quality control, and is suitable for quality control and observation of the soluble fms-like tyrosine kinase-1 determination kit. The invention also provides a preferable freeze-drying diluent formula, the freeze-drying diluent has the advantages of few component additives, simple components and low cost, and the prepared quality control product has good stability and uniformity. The preparation method of the quality control product has the advantages of simple process, simple operation, economy, wide application range and the like.
Description
Technical Field
The invention relates to the field of in-vitro diagnostic reagents, in particular to a soluble fms-like tyrosine kinase-1 quality control product and a preparation method thereof.
Background
Preeclampsia is a kind of clinical special diseases with high morbidity in the gestation period, related complications such as thromboembolism, bleeding and the like easily appear in preeclampsia patients, and if the preeclampsia patients cannot be effectively treated in time, the occurrence and even death of cardiovascular diseases can be further aggravated along with the development of the conditions, and premature delivery of fetuses, kidney damage and the like are caused at the same time. Women with pre-eclampsia have levels of soluble fms-like tyrosine kinase-1 (sFlt-1) that are higher than normal pregnancy levels, while levels of PLGF are lower than normal pregnancy levels. The soluble fms-like tyrosine kinase-1 test kit detects the level of sFlt-1 in blood circulation, and identifies normal pregnancy and preeclampsia before clinical symptoms appear, so as to realize early diagnosis and early treatment of patients with hypertensive disorders of pregnancy.
Clinical trials to obtain reliable assay results require the establishment of a comprehensive quality management system. Among them, quality control in a laboratory is an important link, and quality control products are important material bases for ensuring quality control work. The quality control material is a quality control material for in vitro diagnosis, a reference value analyzed by a manufacturer using an appropriate analysis method or process, and a reference range designated, and is a material, article or apparatus intended for use in a test system for medical use, for the purpose of evaluating or verifying measurement precision, analysis deviation which may occur in the test system due to a change in a reagent or an analysis instrument, and the like. Therefore, the development of a soluble fms-like tyrosine kinase-1 quality control product is very necessary.
Disclosure of Invention
The invention aims to provide a preparation method of a soluble fms-like tyrosine kinase-1 quality control product, which has the advantages of simple process, simple operation, economy, wide application range and the like.
The invention also aims to provide a quality control product prepared by the preparation method of the soluble fms-like tyrosine kinase-1 quality control product. The quality control product is a freeze-dried product prepared by mixing recombinant soluble fms-like tyrosine kinase-1 antigen and freeze-dried diluent in different proportions and freeze-drying the mixture. The quality control product provided by the invention can eliminate matrix effect, the freeze-dried diluent has few component additives, simple components and low cost, and the prepared quality control product has good stability and uniformity.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
In one aspect, the present application provides a method for preparing a quality control product of soluble fms-like tyrosine kinase-1, comprising the following steps: mixing the freeze-dried diluent with a soluble fms-like tyrosine kinase-1 antigen, and stirring and freeze-drying to obtain the quality control product; the freeze-dried diluent comprises a buffer solution, sucrose, bovine serum albumin and a preservative; the buffer solution is phosphate buffer solution or citrate buffer solution.
In another aspect, the present disclosure provides a quality control product prepared by the method for preparing a soluble fms-like tyrosine kinase-1 quality control product.
Compared with the prior art, the embodiment of the invention has at least the following advantages or beneficial effects:
1. the preparation method of the soluble fms-like tyrosine kinase-1 quality control product provided by the invention comprises the steps of mixing the recombinant soluble fms-like tyrosine kinase-1 antigens with different proportions with freeze-drying diluent, and then carrying out freeze drying on the mixture. The preparation method has the advantages of simple process, simple operation, economy, wide application range and the like.
2. The soluble fms-like tyrosine kinase-1 quality control product provided by the invention is a freeze-dried product, the quality control product can eliminate matrix effect, the freeze-dried diluent has few component additives, the components are simple, the cost is low, and the prepared quality control product has good stability and uniformity.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 shows the results of the long-term stability measurement of quality control level 1 in Experimental example 2 of the present invention;
FIG. 2 shows the results of the long-term stability measurement of quality control level 2 in Experimental example 2 of the present invention;
FIG. 3 shows the results of the long-term stability measurement of quality control level 1 in Experimental example 5 of the present invention;
FIG. 4 shows the results of the long-term stability measurement of quality control level 2 in Experimental example 5 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.
A preparation method of a soluble fms-like tyrosine kinase-1 quality control product is characterized by comprising the following steps: mixing the freeze-dried diluent with a soluble fms-like tyrosine kinase-1 antigen, and stirring to obtain the quality control product; the freeze-dried diluent comprises a buffer solution, sucrose, bovine serum albumin and a preservative; the buffer solution is phosphate buffer solution or citrate buffer solution.
The preparation method of the soluble fms-like tyrosine kinase-1 quality control product provided by the invention comprises the steps of mixing the recombinant soluble fms-like tyrosine kinase-1 antigens with different proportions with freeze-drying diluent, and then carrying out freeze drying on the mixture. The preparation method has the advantages of simple process, simple operation, economy, wide application range and the like.
In some embodiments of the invention, the phosphate is a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate. If the pH value in blood is too high, redundant alkali can be combined with sodium dihydrogen phosphate to generate disodium hydrogen phosphate, otherwise, the redundant alkali can be reacted with the disodium hydrogen phosphate to generate the sodium dihydrogen phosphate, phosphate buffer solutions prepared by the two medicines have secondary dissociation, the buffer pH value range is wide, and the buffer solution has excellent retarding performance and stability.
In some embodiments of the invention, the citrate salt is a mixture of citric acid and sodium citrate. The sodium citrate is a weak acid strong alkali salt, and can be matched with citric acid to form a strong pH buffer solution. In addition, the sodium citrate has excellent solubility, excellent retarding performance and stability, and in the synthesis reaction of the soluble fms-like tyrosine kinase-1 quality control product, the buffer solution can simulate and stabilize the pH environment of a real blood sample, so that the buffer solution has an important effect on the synthesis reaction of the quality control product.
In some embodiments of the invention, the total concentration of phosphate ions in the phosphate is 20 to 80 mM. 20-80mM is isotonic with human blood, the buffer solution with pH value lower than 20mM is obviously reduced, and the osmotic pressure of too small salt ions is reduced; the buffer solution with the pH value higher than 80mM has an increased buffering effect on the pH value, but the overlarge osmotic pressure of salt ions is increased, so that the real blood sample environment is difficult to effectively simulate and the solubility and the stability of active protein are not facilitated.
In some embodiments of the invention, the total citrate ion concentration in the citrate salt is 20 to 80 mM. 20-80mM is isotonic with human blood, the buffer solution with pH value lower than 20mM is obviously reduced, and the osmotic pressure of too small salt ions is reduced; the buffer solution with the pH value higher than 80mM has an increased buffering effect on the pH value, but the excessive osmotic pressure of salt ions is increased, so that the environment of a real blood sample is difficult to effectively simulate and the solubility and the stability of active proteins are not facilitated.
In some embodiments of the present invention, the pH of the phosphate buffer or citrate buffer is in the range of 7.3 to 7.5. Normal blood is weakly alkaline and the pH value of the blood is kept relatively constant, with the pH value of 7.35-7.45. The pH value of the blood is an index which represents the concentration of hydrogen ions in the blood and reflects the strength of the acidity and the alkalinity of the blood, and the pH value of the normal human blood is 7.35-7.45, and the numerical range ensures various biochemical reactions in the blood. The product is just a human serum and plasma sample, and the specificity of a quality control product is reduced when the pH is less than 7.3; at pH > 7.5, the binding capacity of the control to the test agent is reduced and the pH of the control, whether < 7.3 or > 7.5, loses its significance in simulating human blood samples.
In some embodiments of the invention, the control comprises a control level 1 and a control level 2, the concentration of the soluble fms-like tyrosine kinase-1 antigen in the control level 1 is 7000pg/mL, and the concentration of the soluble fms-like tyrosine kinase-1 antigen in the control level 2 is 700 pg/mL. Studies have shown that the concentration of sFlt-1 in blood samples is closely related to the development and progression of pre-eclampsia, and that the level of sFlt-1 in the blood circulation can be used to distinguish between normal pregnancy and pre-eclampsia before clinical symptoms appear. The content of soluble fms-like tyrosine kinase-1 in normal women is 900pg/mL in 1-4 weeks of pregnancy, and the content of soluble fms-like tyrosine kinase-1 in 34-34 weeks of pregnancy is 8000pg/mL in 6000, so the intermediate concentration of the quality control product is selected as the quality control product, namely the concentration of the soluble fms-like tyrosine kinase-1 antigen in the quality control product level 1 is 7000pg/mL, and the concentration of the soluble fms-like tyrosine kinase-1 antigen in the quality control product level 2 is 700 pg/mL.
In some embodiments of the invention, the bovine serum albumin has a mass fraction of 0.5% to 1%. Bovine Serum Albumin (BSA), referred to as BSA for short, mainly plays a role in maintaining osmotic pressure and providing pH buffering and a carrier, the BSA hardly plays a role in pH buffering and carrier when the concentration of the BSA is lower than 0.5%, and the BSA with the concentration higher than 1% makes the osmotic pressure too large to simulate the real blood sample environment of a human body, so that the synthetic environment of the soluble fms-like tyrosine kinase-1 quality control product in the invention is not stable.
In some embodiments of the invention, the preservative is PC-300. PC-300 is a broad-spectrum bacteriostatic preservative, and the main active ingredients are a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (5-chloro-2-methyl-4-isothiazolin-3-one, CMIT) and 2-methyl-4-isothiazolin-3-one (2-methyl-4-isothiazolin-3-one, MIT), which can inhibit the growth of microorganisms and promote the death of microorganisms. The principle is that within minutes after contact of CMIT/MIT with the cell membrane and after contact with the microorganism, the active components can permeate through the cell wall and thereby inhibit the activity of certain specific enzymes in the Krebs cycle. The growth of microorganisms, the synthesis of macromolecules and respiratory chain reaction are effectively inhibited, and finally, the growth is inhibited or the cells die because of the collapse of an energy system and the failure of normal anabolism of the cells. The bacteria and fungi have all or part of tricarboxylic acid cycle, so that the product is suitable for both bacteria and fungi, has very broad antibacterial spectrum, and has several dehydrogenase acting sites, so that the medicine resistance of microbes caused by variation is greatly reduced.
A quality control product prepared by a preparation method of a soluble fms-like tyrosine kinase-1 quality control product.
The soluble fms-like tyrosine kinase-1 quality control product provided by the invention is a freeze-dried product, the quality control product can eliminate matrix effect, the freeze-dried diluent has few component additives, the components are simple, the cost is low, and the prepared quality control product has good stability and uniformity.
Example 1
A method for preparing a soluble fms-like tyrosine kinase-1 quality control product comprises the following steps:
preparation of 50mM phosphate (disodium hydrogen phosphate-sodium dihydrogen phosphate) lyophilized dilution (to formulate 500mL as an example): 8.956g of disodium hydrogen phosphate dodecahydrate and 3.900 g of sodium dihydrogen phosphate dihydrate were accurately weighed on an analytical balance and dissolved in 450mL of purified water, and then the pH was adjusted to 7.4 with 1M NaOH/HCl solution, and the volume was adjusted to 500mL with purified water.
Preparation of soluble fms-like tyrosine kinase-1 quality control (the buffer is phosphate buffer):
the soluble fms-like tyrosine kinase-1 quality control product (the buffer solution is phosphate buffer solution) provided by the invention comprises a quality control product level 1 and a level 2, wherein the component composition and the proportion of the quality control product level 1 are shown in table 1, and the component composition and the proportion of the quality control product level 2 are shown in table 2.
TABLE 1 ingredient composition and ratio of quality control level 1
| sFlt-1 antigen | Phosphate buffer | Sucrose | Bovine serum albumin | PC-300 |
| 7000pg/mL | 50mM | 5％ | 0.5％ | 0.1％ |
TABLE 2 ingredient composition and ratio of quality control level 2
| sFlt-1 antigen | Phosphate buffer | Sucrose | Bovine serum albumin | PC-300 |
| 700pg/mL | 50mM | 5％ | 0.5％ | 0.1％ |
The preparation method of the quality control product 1 in this embodiment is as follows: 5g of sucrose and 0.5g of bovine serum albumin are sequentially weighed on an analytical balance and added into 100mL of 50mM phosphate buffer (pH 7.4), 0.1mL of PC-300 is added after the sucrose and the bovine serum albumin are fully dissolved, then the sFlt-1 antigen is added to 7000pg/mL, and after the mixture is fully mixed, the content of sFlt-1 in the intermediate solution is detected, and the measured value is controlled within 10% of the theoretical value of sFlt-1. And subpackaging according to the specification requirement, freeze drying, and performing gland assignment to obtain the quality control product level 1.
The preparation method of the quality control material 2 in this embodiment is as follows: 5g of sucrose and 0.5g of bovine serum albumin are sequentially weighed on an analytical balance and added into 100mL of 50mM phosphate buffer (pH 7.4), 0.1mL of PC-300 is added after the sucrose and the bovine serum albumin are fully dissolved, then the sFlt-1 antigen is added to 700pg/mL, the intermediate solution is fully mixed, the content of the sFlt-1 in the intermediate solution is detected, and the measured value is controlled within 10% of the theoretical value of the sFlt-1. And subpackaging according to the specification requirement, freeze-drying, and assigning a gland to obtain the quality control product level 2.
The method for preparing quality control 1 and quality control 2 according to this example was carried out according to the above procedure, each reagent was completely dissolved and the next reagent was added, the precision of the balance used was of the order of hundred thousand, and the pH was adjusted to 7.4 using hydrochloric acid or sodium hydroxide of appropriate concentration.
Example 2
A method for preparing a soluble fms-like tyrosine kinase-1 quality control product comprises the following steps:
preparation of 50mM citrate (citric acid-sodium citrate) lyophilized dilution (as an example to make 500 mL): 5.254g of citric acid monohydrate and 7.353g of trisodium citrate dihydrate were weighed out accurately on an analytical balance, dissolved thoroughly in 450mL of purified water, the pH was adjusted to 7.4 with 1M NaOH/HCl solution, and the volume was made up to 500mL with purified water.
Preparation of soluble fms-like tyrosine kinase-1 quality control (the buffer is citric acid buffer):
the soluble fms-like tyrosine kinase-1 quality control product (the buffer solution is citrate buffer solution) provided by the invention comprises a quality control product level 1 and a level 2, wherein the component composition and the proportion of the quality control product level 1 are shown in a table 3, and the component composition and the proportion of the quality control product level 2 are shown in a table 4.
TABLE 3 ingredient composition and ratio of quality control level 1
| sFlt-1 antigen | Citrate buffer | Sucrose | Bovine serum albumin | PC-300 |
| 7000pg/mL | 50mM | 5％ | 0.5％ | 0.1％ |
TABLE 4 composition and ratio of quality control level 2
| sFlt-1 antigen | Citrate buffer | Sucrose | Bovine serum albumin | PC-300 |
| 700pg/mL | 50mM | 5％ | 0.5％ | 0.1％ |
The preparation method of the quality control product 1 in this embodiment is as follows: weighing 5g of sucrose and 0.5g of bovine serum albumin in turn on an analytical balance, adding the sucrose and the bovine serum albumin into 100mL of 50mM citrate buffer solution (pH 7.4), adding 0.1mL of PC-300 after fully dissolving, then adding the sFlt-1 antigen to 7000pg/mL, fully mixing uniformly, detecting the content of sFlt-1 in an intermediate solution, and controlling the measured value within 10% of the theoretical value of sFlt-1. And subpackaging according to the specification requirement, freeze drying, and assigning a gland to obtain the quality control product level 1.
The preparation method of the quality control product 2 in this embodiment is as follows: weighing 5g of sucrose and 0.5g of bovine serum albumin in turn on an analytical balance, adding the sucrose and the bovine serum albumin into 100mL of 50mM citrate buffer solution (pH 7.4), adding 0.1mL of PC-300 after fully dissolving, then adding the sFlt-1 antigen to 700pg/mL, fully mixing uniformly, detecting the content of sFlt-1 in an intermediate solution, and controlling the measured value within 10% of the theoretical value of sFlt-1. And subpackaging according to the specification requirement, freeze-drying, and assigning a gland to obtain a quality control product level 2.
The method for preparing quality control 1 and quality control 2 according to this example was carried out according to the above procedure, each reagent was completely dissolved and the next reagent was added, the precision of the balance used was of the order of hundred thousand, and the pH was adjusted to 7.4 using hydrochloric acid or sodium hydroxide of appropriate concentration.
Example 3
This example is prepared essentially identically to example 1, except that: 1. the concentration of the freeze-dried diluted release solution (the buffer solution is phosphate buffer solution) is 20 mM; 2. the mass fraction of the bovine serum albumin is 1%; 3. the pH of the phosphate buffer was 7.3.
Example 4
This example is prepared essentially identically to example 1, except that: 1. the concentration of the freeze-dried diluted release solution (the buffer solution is phosphate buffer solution) is 80 mM; 2. the mass fraction of the bovine serum albumin is 0.7 percent; 3. the pH of the phosphate buffer was 7.5.
Example 5
This example is prepared essentially identically to example 2, except that: 1. the concentration of the freeze-dried diluted release solution (citrate buffer solution) used was 20 mM; 2. the mass fraction of the bovine serum albumin is 1 percent; 3. the pH of the phosphate buffer was 7.5.
Example 6
This example is prepared essentially identically to example 2, except that: 1. the concentration of the freeze-dried diluted release solution (citrate buffer solution) used is 80 mM; 2. the mass fraction of the bovine serum albumin is 0.7 percent; 3. the pH of the phosphate buffer was 7.3.
Experimental example 1
This example is a uniformity measurement of a soluble fms-like tyrosine kinase-1 quality control substance (the buffer solution is a phosphate buffer solution), and the quality control substances used in this example are the quality control substance level 1 and the quality control substance level 2 prepared in example 1.
To examine the homogeneity of a quality control of soluble fms-like tyrosine kinase-1 as provided in example 1, the following experiment was performed: 10 samples of each of the quality control substance levels 1 and 2 prepared in example 1 were randomly extracted, and after redissolving with distilled water, the in-bottle and inter-bottle uniformity of the quality control substance levels 1 and 2 were measured on a fluorescence immunoassay analyzer (Tigsun-600) manufactured by limited Biotech, Beijing Tegaku, and on a soluble fms-like tyrosine kinase-1 measurement kit, respectively. The results of in-vial and inter-vial uniformity measurements for quality control level 1 are shown in Table 5, and the results of in-vial and inter-vial uniformity measurements for quality control level 2 are shown in Table 6.
It should be noted that the Coefficient of Variation (CV) of repeatability should be no more than 10%, the target value range of 1 level of quality control is 5529.67-8470.33pg/mL, and the target value range of 2 level of quality control is 550.59-849.41 pg/mL.
TABLE 5 homogeneity determination results of quality control level 1
TABLE 6 homogeneity determination results of quality control level 2
As can be seen from the measurement results in tables 5 and 6, the intra-vial and inter-vial CV values of the level 1 and the level 2 of the quality control product are all within 10% and within the target value range, and the experimental result shows that the soluble fms-like tyrosine kinase-1 quality control product provided by the invention has better uniformity and can meet the clinical requirements.
Experimental example 2
This example is a long-term stability measurement of a soluble fms-like tyrosine kinase-1 quality control substance (the buffer is a phosphate buffer), and the quality control substances used in this example are the quality control substance level 1 and the quality control substance level 2 prepared in example 1.
To test the long-term stability of a soluble fms-like tyrosine kinase-1 quality control provided in this example, the following experiments were performed: the quality control substance level 1 and the quality control substance level 2 prepared in example 1 were stored in a refrigerator at 2 to 8 ℃ and tested for the content of sFlt-1 once in each of 0, 3, 6, 9, 12, 15, 18, 21 and 24 months from the start of storage. The results of the long-term stability measurements of the quality control level 1 are shown in Table 7 and FIG. 1, and the results of the long-term stability measurements of the quality control level 2 are shown in Table 8 and FIG. 2.
It should be noted here that the target value range of the quality control level 1 is 5529.67-8470.33pg/mL, the target value range of the quality control level 2 is 550.59-849.41pg/mL, and the CV is all within 10%.
TABLE 7 Long-term stability assay results for quality control level 1
TABLE 8 Long-term stability determination results of quality control level 2
As can be seen from the analysis of Table 7, Table 8, FIG. 1 and FIG. 2, the analyte concentration of the lyophilized soluble fms-like tyrosine kinase-1 quality control product provided in this example is still within the target value range within 24 months of storage at 2-8 deg.C, and the CV is within 10%. The experimental result shows that the soluble fms-like tyrosine kinase-1 quality control product provided by the invention has better long-term stability.
Experimental example 3
This example is a measurement of the reconstitution stability of a soluble fms-like tyrosine kinase-1 quality control substance (the buffer is a phosphate buffer), and the quality control substances used in this example are the quality control substance level 1 and the quality control substance level 2 prepared in example 1.
To examine the post-reconstitution stability of a soluble fms-like tyrosine kinase-1 quality control provided in example 1, the following experiments were performed: after redissolving the quality control material level 1 and the quality control material level 2 prepared in the example 1, the raw bottles are sealed and stored in a refrigerator at the temperature of 2-8 ℃ for 0 day, 3 days, 5 days, 7 days and 9 days respectively to carry out the detection of the content of the sFlt-1 once. The results of measurement of reconstitution stability at quality control level 1 are shown in Table 9, and the results of measurement of reconstitution stability at quality control level 2 are shown in Table 10.
It should be noted here that the target value range of the quality control level 1 is 5529.67-8470.33pg/mL, the target value range of the quality control level 2 is 550.59-849.41pg/mL, and the CV is all within 10%.
TABLE 9 quality control level 1 redissolution stability assay results
TABLE 10 quality control level 2 reconstitution stability assay results
As can be seen from the measurement results in tables 9 and 10, the analyte concentration of the lyophilized soluble fms-like tyrosine kinase-1 quality control provided in this example is still within the target value range after reconstitution within 24 months at 2-8 deg.C, and CV is within 10%. The experimental result shows that the soluble fms-like tyrosine kinase-1 quality control product provided by the invention has better stability after redissolution.
Experimental example 4
This example is a uniformity measurement of a soluble fms-like tyrosine kinase-1 quality control substance (the buffer solution is a citrate buffer solution), and the quality control substances used in this example are the quality control substance level 1 and the quality control substance level 2 prepared in example 2.
To examine the homogeneity of a quality control of soluble fms-like tyrosine kinase-1 as provided in example 2, the following experiment was performed: after randomly extracting 10 samples of each of the level 1 and the level 2 of the quality control material prepared in example 2, redissolving the samples with distilled water, and then respectively measuring the uniformity of the level 1 and the level 2 of the quality control material on a fluorescence immunoassay analyzer (Tigsun-600) and a soluble fms-like tyrosine kinase-1 measuring kit, wherein the CV values are not more than 10%, the target value range of the level 1 of the quality control material is that the quality control material prepared in example 2 is redissolved and then is sealed and stored in an original bottle at 2-8 ℃ for 0 day, 3 days, 5 days, 7 days and 9 days, and then sampling detection is carried out, the uniformity measurement results of the level 1 of the quality control material in the bottle and among the bottles are shown in table 11, and the uniformity measurement results of the level 2 of the quality control material in the bottle and among the bottles are shown in table 12.
It should be noted here that the target value range of the quality control level 1 is 5529.67-8470.33pg/mL, the target value range of the quality control level 2 is 550.59-849.41pg/mL, and the CV is all within 10%.
TABLE 11 homogeneity determination of quality control level 1
TABLE 12 quality control level 2 homogeneity determination results
As can be seen from the measurement results in tables 11 and 12, the intra-and inter-vial CVs of the quality control substance level 1 and level 2 are all within 10% and within the target value range, and the experimental result shows that the soluble fms-like tyrosine kinase-1 quality control substance provided by the invention has better uniformity and can meet the clinical requirements.
Experimental example 5
This example is a measurement of the long-term stability of a soluble fms-like tyrosine kinase-1 quality control substance (the buffer is citrate buffer), and the quality control substances used in this example are the quality control substance level 1 and the quality control substance level 2 prepared in example 2.
To test the long-term stability of a soluble fms-like tyrosine kinase-1 quality control provided in example 2, the following experiments were performed: the quality control products prepared in example 2 were stored at 2-8 ℃ and tested for sFlt-1 content once in each of 0, 3, 6, 9, 12, 15, 18, 21, and 24 months from the start of storage, with the long-term stability test results for quality control level 1 shown in Table 13 and FIG. 3, and the long-term stability test results for quality control level 2 shown in Table 14 and FIG. 4.
It should be noted here that the target value range of the quality control level 1 is 5499.06-8500.94pg/mL, the target value range of the quality control level 2 is 560.00-840.00pg/mL, and the CV is all within 10%.
TABLE 13 Long-term stability determination results of quality control level 1
TABLE 14 Long-term stability assay results for quality control level 2
As can be seen from the analysis in Table 13, Table 14, FIG. 3 and FIG. 4, the lyophilized product of the soluble fms-like tyrosine kinase-1 quality control provided in this example has an analyte concentration within the target value range within 24 months of storage at 2-8 deg.C, and CV values within 10%. The experimental result shows that the soluble fms-like tyrosine kinase-1 quality control product provided by the invention has better long-term stability.
Experimental example 6
This example is a measurement of the reconstitution stability of a soluble fms-like tyrosine kinase-1 quality control (the buffer is citrate buffer), and the quality control used in this example was the quality control level 1 and the quality control level 2 prepared in example 2.
To examine the post-reconstitution stability of a soluble fms-like tyrosine kinase-1 quality control provided in example 2, the following experiments were performed: the quality control products prepared in the example 2 were re-dissolved and then stored in original bottles at 2-8 ℃ for 0 day, 3 days, 5 days, 7 days and 9 days in a sealed manner, and the results of the re-dissolution stability measurement of the quality control product level 1 are shown in table 15 and the results of the re-dissolution stability measurement of the quality control product level 2 are shown in table 16.
It should be noted here that the target value range of the quality control level 1 is 5499.06-8500.94pg/mL, the target value range of the quality control level 2 is 560.00-840.00pg/mL, and the CV is all within 10%.
TABLE 15 measurement results of reconstitution stability of quality control level 1
TABLE 16 measurement results of reconstitution stability of quality control level 2
As can be seen from the measurement results in tables 15 and 16, the analyte concentration of the lyophilized soluble fms-like tyrosine kinase-1 quality control provided in this example was within the target value range after reconstitution at 2-8 deg.C for 24 months, with CV values within 10%. The experimental result shows that the soluble fms-like tyrosine kinase-1 quality control product provided by the invention has better stability after redissolution.
In summary, the quality control product of the soluble fms-like tyrosine kinase-1 and the preparation method thereof of the embodiment of the invention comprises a quality control level 1 and a quality control level 2, wherein the quality control product is prepared by mixing the recombinant soluble fms-like tyrosine kinase-1 antigen and the freeze-drying diluent in different proportions, and then freeze-drying the mixture to obtain the quality control product. The preparation method has the advantages of simple process, simple operation, economy, wide application range and the like. The invention also provides a freeze-dried diluent formula, the freeze-dried diluent has few component additives, simple components and low cost, and a quality control product prepared by the freeze-dried diluent formula has good stability and uniformity.
As can be seen from the results of the analyses in tables 5 to 16, the intra-bottle and inter-bottle CVs of the quality control products of level 1 and level 2 prepared by the preparation method of the soluble fms-like tyrosine kinase-1 quality control product provided by the invention are both within 10% and within the range of target values, and the experimental results show that the soluble fms-like tyrosine kinase-1 quality control product provided by the invention has better uniformity and can meet the clinical requirements. The analyte concentration of the lyophilized soluble fms-like tyrosine kinase-1 quality control product provided in the embodiment is still within a target value range within 24 months of storage at the temperature of 2-8 ℃, and CV is within 10%. The experimental result shows that the soluble fms-like tyrosine kinase-1 quality control product provided by the invention has better long-term stability and can meet the clinical requirement. The analyte concentration of the lyophilized soluble fms-like tyrosine kinase-1 quality control product provided in the embodiment is still within the target value range after being reconstituted within 24 months at the temperature of 2-8 ℃, and CV is within 10%. The experimental result shows that the soluble fms-like tyrosine kinase-1 quality control product provided by the invention has better stability after redissolution and can meet the clinical requirements.
In conclusion, the quality control product prepared by the preparation method of the soluble fms-like tyrosine kinase-1 quality control product provided by the invention has good uniformity, repeatability and stability, and can meet clinical requirements.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. A preparation method of a soluble fms-like tyrosine kinase-1 quality control product is characterized by comprising the following steps: mixing the freeze-dried diluent with soluble fms-like tyrosine kinase-1 antigen, stirring and freeze-drying to obtain the quality control product; the freeze-dried diluent comprises a buffer solution, sucrose, bovine serum albumin and a preservative; the buffer solution is phosphate buffer solution or citrate buffer solution.
2. The method of claim 1, wherein the phosphate is a mixture of disodium hydrogen phosphate and sodium dihydrogen phosphate.
3. The method for preparing the quality control product of the soluble fms-like tyrosine kinase-1 as claimed in claim 1, wherein the citrate is a mixture of citric acid and sodium citrate.
4. The method for preparing the quality control product of the soluble fms-like tyrosine kinase-1 as claimed in claim 1, wherein the concentration of phosphate ions in the phosphate is 20-80 mM.
5. The method for preparing the quality control product of the soluble fms-like tyrosine kinase-1 as claimed in claim 1, wherein the citrate ion concentration in the citrate is 20-80 mM.
6. The method for preparing the quality control product of the soluble fms-like tyrosine kinase-1 as claimed in claim 1, wherein the pH value of the phosphate buffer or citrate buffer is in the range of 7.3-7.5.
7. The method for preparing the quality control product of the soluble fms-like tyrosine kinase-1 according to claim 1, wherein the quality control product comprises the following steps: the quality control product comprises a quality control product level 1 and a quality control product level 2, the concentration of the soluble fms-like tyrosine kinase-1 antigen in the quality control product level 1 is 7000pg/mL, and the concentration of the soluble fms-like tyrosine kinase-1 antigen in the quality control product level 2 is 700 pg/mL.
8. The method for preparing the quality control product of the soluble fms-like tyrosine kinase-1 according to claim 1, wherein the bovine serum albumin has a mass fraction of 0.5% -1%.
9. The method for preparing the quality control product of the soluble fms-like tyrosine kinase-1 as claimed in claim 1, wherein the preservative is PC-300.
10. A quality control product is characterized in that: the quality control product is prepared by the preparation method of the soluble fms-like tyrosine kinase-1 quality control product as claimed in any one of claims 1 to 9.
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| CN115791340A (en) * | 2023-01-17 | 2023-03-14 | 北京水木济衡生物技术有限公司 | Composite quality control product for epilepsy as well as preparation method and application thereof |
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| CN115791340A (en) * | 2023-01-17 | 2023-03-14 | 北京水木济衡生物技术有限公司 | Composite quality control product for epilepsy as well as preparation method and application thereof |
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