CN115406885B - Method for detecting residual cross-linking agent in disulfide bond cross-linked hyaluronic acid gel - Google Patents
Method for detecting residual cross-linking agent in disulfide bond cross-linked hyaluronic acid gel Download PDFInfo
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Abstract
The invention discloses a method for detecting a residual cross-linking agent in disulfide bond cross-linked hyaluronic acid gel, which comprises the following steps: step (1) standard solution of cross-linking agent; degrading a sample to be detected; ultrafiltration separation of the degradation products in the step (3); step (4), NTSB reagent color reaction; step (5) establishing a standard curve; and (6) measuring the residual of the crosslinking agent. The lowest detection limit of the residual cross-linking agent (taking cystamine dihydrochloride as an example) can reach 0.0539 ppm, the quantitative limit can reach 0.1634 ppm, and the linear correlation coefficient (R) of 1-5 ppm 2 ) 0.9999, and is a color development detection method with high precision, simple and easy operation method, wide linear range and low cost, which can meet the actual requirements and contains a small amount of residual cross-linking agent in the disulfide bond cross-linked hyaluronic acid gel.
Description
Technical Field
The invention relates to the field of disulfide bond cross-linked hyaluronic acid gel, in particular to a method for detecting a residual cross-linking agent in disulfide bond cross-linked hyaluronic acid gel.
Background
Disulfide-bond cross-linked hyaluronic acid gels, commonly used cross-linking agents are disulfide-bond diamines (e.g., cystamine, etc.) or disulfide-bond dihydrazides (e.g., 3' -dithiobis (propionylhydrazine), etc.); the side chain carboxyl of hyaluronic acid can generate coupling reaction with the amino or hydrazide group of the cross-linking agent under the catalytic activity of carbodiimide and the like to form an amido bond cross-linking structure. Amide bond crosslinking, while relatively stable, may present hydrolytic degradation reactions, the hydrolysis products of which are disulfide bond crosslinkers.
The disulfide cross-linking agent (disulfide diamine or disulfide dihydrazide) has amino group, and the content of amino group can be detected by color reaction. For example, the alpha-amino group of an amino acid can be hydratedThe triton reacts to generate a violet compound, a characteristic absorption peak is formed at the position of 570nm, the content of the amino acid can be quantitatively detected through the change of the absorption value, the method is the most common detection method at present, and the method has the advantages of simple and easy operation method, low cost, no need of expensive equipment and the like. For example, a study of ninhydrin colorimetric determination of glutamic acid content (chinese seasoning 2015, 8 2 Only 0.9964). However, the accuracy of the trithione color development method is poor, and the requirement for detecting trace residue of disulfide bond cross-linking agent cannot be met.
Disulfide bonds are functional groups characteristic of disulfide bonds for crosslinking crosslinkers in hyaluronic acid. Disulfide bonds generally exhibit weak absorption in the 245nm region, and the absorption peak is hardly visible at a low concentration, and thus the requirement for detection of trace residues of disulfide bond crosslinkers cannot be met.
The residue of the cross-linking agent may cause potential safety hazard, and the quantitative detection of the residue of the disulfide cross-linking agent in the disulfide cross-linked hyaluronic acid becomes a problem to be solved urgently. In ICH (The International Conference on harnessing of Technical Requirements for Registration of Pharmaceuticals for Human Use), < Q3B (R2): impurities in New drug formulations strict regulations are imposed on degradation product impurities in drugs at the maximum daily dose, e.g. a maximum daily dose of more than 2g, with an identification limit of 0.1%. In disulfide-bond cross-linked hyaluronic acid, hydrolysis of the cross-linking bond is the main pathway of its degradation, and disulfide-bond cross-linker is the main degradation impurity residue. At present, a detection method for trace residues of a cross-linking agent in disulfide bond cross-linked hyaluronic acid gel does not exist, which brings limitation to the wide application of the disulfide bond cross-linked hyaluronic acid gel in medicines or medical instruments, and therefore, the establishment of an effective detection method for trace residues of the cross-linking agent in disulfide bond cross-linked hyaluronic acid gel has important significance.
Disclosure of Invention
The invention aims to provide a developing detection method for trace residue of a cross-linking agent in a disulfide bond cross-linked hyaluronic acid gel, which has the advantages of high accuracy, simple and easy operation method, wide linear range and low cost and can meet the actual requirement.
In order to solve the technical problem, the invention provides a method for detecting the residue of a cross-linking agent in a disulfide bond cross-linked hyaluronic acid gel, which comprises the following steps: step (1) standard solution of cross-linking agent: accurately weighing a certain amount of cross-linking agent standard, diluting with pure water to prepare a cross-linking agent standard solution with concentration gradient of not less than 4 and concentration range of 1-10 ppm; and (2) degrading a sample to be detected: precisely weighing a certain volume of disulfide bond cross-linked hyaluronic acid gel, and degrading with enzyme, wherein the enzymatic degradation product solution is a uniform transparent solution without obvious particles; and (3) ultrafiltration separation of degradation products: placing the degradation product obtained in the step (2) in an ultrafiltration centrifugal tube, and after centrifugation, allowing the cross-linking agent residue in the degradation product to enter into filtrate; step (4) NTSB reagent color reaction: adjusting the pH of the filtrate obtained in the step (3) to 3-4, mixing the filtrate with the same volume with an NTSB reagent, reacting for 25 minutes at room temperature, and measuring the absorbance at 412nm +/-2 nm; step (5), establishing a standard curve: degrading the standard substance solution obtained in the step (1) according to the method in the step (2), performing ultrafiltration separation in the step (3), determining absorbance after color development in the step (4), and performing linear regression with the concentration of the standard solution of the cross-linking agent to generate a standard curve; step (6) determination of crosslinker residue: substituting the absorbance obtained in the step (4) into the standard curve obtained in the step (5) to obtain the residual content of the cross-linking agent; the absorbance measurement adopts an ultraviolet-visible spectrophotometer.
In particular, the cross-linking agent is cystamine dihydrochloride or 3,3' -dithiobis.
Specifically, the concentration of the disulfide-bond cross-linked hyaluronic acid gel is 3-30 mg/ml.
In particular, the enzyme is hyaluronidase.
Specifically, the hyaluronidase is dissolved by 0.9% sodium chloride, and the enzyme activity is 25-75U/ml.
Specifically, the volume ratio (V/V) of the disulfide-bond cross-linked hyaluronic acid to the hyaluronidase is 1: (1-3).
Specifically, the degradation condition of the hyaluronidase is degradation at a temperature of 37 ℃ for 20 minutes.
Specifically, the centrifugation is performed for 15 minutes at 6000 rpm.
Specifically, the pH adjustment to 3 to 4 can be performed before the centrifugation in the step (3).
Specifically, the concentration gradients of the standard solutions of the crosslinking agents were 1, 2, 3, 4, and 5ppm, respectively.
Specifically, the molecular weight cut-off of the ultrafiltration centrifugal tube is not more than 10KDa.
Specifically, the room temperature is in the range of 10 ℃ to 30 ℃.
Analysis for Disulfide Bonds in Peptides and Proteins (Methods Enzymol 1987, 143.
The disulfide-linked hyaluronic acid gel is first degraded, preferably with hyaluronidase. The activity of the enzyme solution and the time of the enzymatic hydrolysis need to be controlled to improve the accuracy of the detection method of the present invention. When the activity of the adopted hyaluronidase solution is too low, the required degradation time is longer, and the detection efficiency is influenced; when the activity of the enzyme solution is too high, the degradation is too fast, the degradation process is not easy to control, the molecular weight of the degradation product is low, the centrifugal ultrafiltration separation of the degradation product in the subsequent steps is not facilitated, and the detection accuracy is influenced.
The invention has the beneficial effects that:
the method for detecting the trace residue of the disulfide bond cross-linking agent in the disulfide bond cross-linked hyaluronic acid gel provided by the invention has the advantages of simple and easy operation method, low cost, high accuracy and good accuracy, can detect microgram level and can meet the requirement in actual detection.
Drawings
In order to more clearly illustrate the technical solution of the present invention, the drawings required for the present invention are briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a characteristic absorption spectrum of cystamine dihydrochloride after NTSB development;
FIG. 2 is a standard curve for disulfide cross-linkers.
Detailed Description
The technical solutions in the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The device information in the following examples is as follows: UV-visible spectrophotometer (Shimadzu instruments, inc., model: UV-1900), room temperature detection.
Example 1 determination of test conditions
1.1 NTSB reagent
The NTSB reagent was prepared according to the method disclosed by Analysis for Disulfide Bonds in Peptides and Proteins (Methods Enzymol 1987, 143.
1.2 Standard solution of disulfide cross-linker
Weighing 0.01g of disulfide bond cross-linking agent (cystamine dihydrochloride) into a volumetric flask with 100ml, fixing the volume to 100ml, and diluting to different concentration ranges for later use.
1.3 Hyaluronidase solutions
Weighing 1mg of hyaluronidase, adding a proper amount of 0.9% sodium chloride solution, and the active concentration of the enzyme is 10-100U/ml.
1.4 Degradation of disulfide-bond cross-linked hyaluronic acid
1ml of each of two disulfide-bond cross-linked sodium hyaluronate gels (cross-linking agent: cystamine dihydrochloride) having concentrations of (5 mg/ml and 10mg/ml, respectively) was taken, placed in a 50ml centrifuge tube, 1.92ml of hyaluronidase solution was added, and the mixture was shaken for 10 to 20min in a 37 ℃ constant temperature shaker.
When the activity of the adopted hyaluronidase solution is too low, the required degradation time is longer, and the detection efficiency is influenced; when the activity of the enzyme solution is too high, the degradation is too fast, the degradation process is not easy to control, the molecular weight of the degradation product is low, the centrifugal ultrafiltration separation of the degradation product in the subsequent steps is not facilitated, and the detection accuracy is influenced. The active concentration of the enzyme is preferably 25 to 75U/ml, more preferably 50U/ml. When the active concentration of the enzyme is 50U/ml, samples with different enzymolysis time (10 min, 15min and 20 min) are respectively observed, and when the enzymolysis is carried out for 10min and 15min, the disulfide bond cross-linked sodium hyaluronate gel with two concentrations is not completely degraded, and obvious particles can be observed; when enzymolysis is carried out for 20min, the particles are completely degraded and disappear, and the degradation products of the disulfide bond cross-linked sodium hyaluronate gel with two concentrations are uniform and transparent solutions. Therefore, in the step (1) of the present invention, when the activity of the hyaluronidase solution is preferably 50U/ml, the time for the enzymatic hydrolysis is preferably 20min.
1.5 Ultrafiltration centrifugation of the degradation product solution
And (3) placing the enzymatic degradation product solution into an ultrafiltration centrifugal tube, and allowing small molecules with molecular weight lower than the molecular weight cut-off to enter the filtrate under the action of centrifugal force according to the molecular weight cut-off of the ultrafiltration centrifugal tube, thereby realizing separation. The molecular weight of the disulfide cross-linking agent is typically between 100 and 1000Da (e.g., 225 Da for cystamine dihydrochloride and 238 Da for 3,3' -dithiobis (propionohydrazide)), so the molecular weight cut-off of the eppendorf tube is preferably 10kDa or less. And (4) centrifuging for 15 minutes at 6000 rpm, and allowing disulfide bond cross-linking agent residues in the degradation product to enter filtrate obtained by ultrafiltration and centrifugation.
1.6 Determination of detection wavelength
The characteristic absorption peak of the disulfide bond cross-linking agent after NTSB development has good specificity in the 412nm region, and the characteristic absorption spectrum of cystamine dihydrochloride after NTSB development is shown in figure 1. The maximum absorption wavelength of the characteristic absorption peak is 410-414 nm, preferably 412nm.
Agents such as hyaluronidase and disulfide-linked hyaluronic acid gel and its degradation products themselves are not absorbed at the region of 412nm and do not interfere. In addition, degradation parameters are optimized, degradation products of the disulfide bond cross-linked hyaluronic acid gel are intercepted by ultrafiltration centrifugation and do not enter filtrate, so that the color development reaction of the NTSB reagent is not interfered, and only disulfide bond cross-linking agents enter the filtrate of the ultrafiltration centrifugation and react with the NTSB reagent for color development.
1.7 Adjustment of pH of degradation product solution
The pH adjustment of the degradation product solution has an important influence on the detection precision, and the sample adding recovery rate is obviously influenced.
The influence of the pH value of the degradation product solution on the sample recovery rate is examined according to the following process: adding 0.08ml of 1mol/l hydrochloric acid (pH 3.2) (pure water is used as a control) into the enzyme degradation product solution obtained in the step 1.5, then adding 1ml of standard solutions of three disulfide bond cross-linking agents (cystamine dihydrochloride) with different concentrations (6.4, 8.0 and 9.6 ppm), pouring the uniformly shaken sample into an ultrafiltration centrifugal tube, and carrying out centrifugal filtration (6000 r/min and 15 min); 2ml of the filtrate was removed, 2ml of NTSB reagent was added, the reaction was carried out at room temperature for 25 minutes, and the absorbance was measured at a wavelength of 412nm. The recovery results for each group are summarized in table 1 below, with 3 replicates:
TABLE 1
The added 0.08ml hydrochloric acid (1 mol/l) has important influence on the precision of the detection method, and the sample adding recovery rate is obviously improved. When the concentration of the added hydrochloric acid is 0 mol/l (pure water control), the sample recovery rate is only 67.5-73.2%, and the requirement (80-120%) on the sample recovery rate in the detection method cannot be met. The invention carries out various attempts, finally finds that proper hydrochloric acid is added into the enzymatic degradation product solution to adjust the pH value to be acidic (3-4), the sample adding recovery rate is obviously improved (99.7-104.3%), and the requirement of 80-120% sample adding recovery rate can be well met. Therefore, in the present invention, an appropriate amount of acid is added to the degradation product solution to adjust the pH value to acidity, the acid used is usually dilute hydrochloric acid, and the acidic pH value is preferably 3 to 4.
EXAMPLE 2 establishment of Standard Curve
Taking 1ml of pure water, placing the pure water into a 50ml centrifuge tube, adding 1.92ml of 50U/ml enzyme solution, oscillating the pure water in a constant temperature oscillator at 37 ℃ for 20min, adding 0.08ml of 1mol/L hydrochloric acid solution (adjusting the pH value to 3-4), shaking the pure water evenly, adding 1ml of disulfide bond cross-linking agent (cystamine dihydrochloride) labeling solution with different concentrations, pouring the evenly-shaken sample into an ultrafiltration centrifuge tube, carrying out centrifugal filtration (6000 r/min, centrifuging for 15 min), removing the lower solution 2ml after centrifugation and 2ml of NTSB analysis solution, reacting the solution for 25 min at room temperature, and measuring the absorbance at the wavelength of 412nm.
The results of the absorbance measurements are given in Table 2 below (with no NTSB development and a weak characteristic absorption at 245nm of the disulfide bond as a control):
TABLE 2
As can be seen from the standard curve (FIG. 2), the disulfide cross-linker has a good linear range in the range of 1-5 ppm, and its linear correlation coefficient (R) 2 ) 0.9999, satisfies the detection method with respect to the linear correlation coefficient (R) 2 ) Greater than 0.998.
Disulfide bonds usually show weak absorption in the 245nm region, and the absorption peak is hardly visible at a low concentration, so that the detection requirement of trace residues of disulfide bond crosslinking agents cannot be met.
Example 3 detection and quantitation limits
The detection limit and the quantification limit were determined according to the guidelines of the pharmacopoeia of the people's republic of China (2020 edition four) 9101 "guidelines for validation of analytical methods".
Limit of detection LOD =3.3 delta/S
Limit of quantitation LOD =10 δ/S
Where δ is the standard deviation of the 10 blank values and S is the slope of the standard curve.
The test results are shown in the following table 3:
TABLE 3
Example 4 precision
6 samples (concentration 2.5 ppm) of disulfide bond cross-linker (cystamine hydrochloride) were prepared, measured 6 times, absorbance recorded, and relative standard deviation calculated, as determined according to the procedure of example 2. The test results are shown in the following table 4:
TABLE 4
The RSD is about 0.6 percent, has good precision and meets the target requirement that the RSD is less than or equal to 2 percent.
Example 5 accuracy
See example 1, section 1.7. By adopting a sample-adding recovery test method, three disulfide bond cross-linking agent (cystamine dihydrochloride) standard solutions (6.4 ppm, 8.0ppm and 9.6 ppm) with different concentrations are added into the disulfide bond cross-linked hyaluronic acid gel, the determination recovery rate is 99.7-104.3% (RSD 2.2%), and the requirement of 80-120% sample-adding recovery rate can be well met.
EXAMPLE 6 durability
As measured according to the procedure of example 2, 9 parts of a disulfide bond crosslinking agent (cystamine dihydrochloride) standard solution (2 ppm) was prepared, absorption was measured at 412. + -.2 nm, and the relative standard deviation was calculated. The test results are shown in the following table 5:
TABLE 5
The RSD is 6.95 percent, has good durability and meets the target requirement that the RSD is less than or equal to 12 percent.
EXAMPLE 7 application of the method
Taking 1ml (concentration is 10 mg/ml) of 3 batches of disulfide bond cross-linked hyaluronic acid gel (cross-linking agent: cystamine dihydrochloride), adding 1.92ml of 50U/ml enzyme solution into a 50ml centrifuge tube, oscillating for 20min in a constant-temperature oscillator at 37 ℃, adding 0.08ml of 1mol/L hydrochloric acid solution, shaking uniformly, adding 1ml of pure water, pouring the uniformly shaken sample into an ultrafiltration centrifuge tube, carrying out centrifugal filtration (6000 r/min, centrifuging for 15 min), transferring the solution 2ml below after centrifugation and 2ml of NTSB analysis solution, reacting for 25 min at room temperature, and measuring absorbance at the wavelength of 412nm. The trace residue of the disulfide cross-linker was calculated according to the standard curve established in example 2.
The detection results showed that the residual amount of the disulfide bond cross-linking agent in the 3 batches was 0.5 to 0.8ppm, which was in accordance with the specification.
Root ICH "Q3 AR2: impurities in New pharmaceutical preparation the identification limit for degradation products in drugs at the maximum daily dose (greater than 2 g) was 0.1%, with a signed limit of 10ppm based on disulfide-linked hyaluronic acid active ingredient (10 mg/ml).
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (11)
1. A method for detecting the residue of a cross-linking agent in a disulfide bond cross-linked hyaluronic acid gel is characterized by comprising the following steps:
step (1) standard solution of cross-linking agent: accurately weighing a certain amount of cross-linking agent standard, diluting with pure water to prepare a cross-linking agent standard solution with concentration gradient of not less than 4 and concentration range of 1-10 ppm;
and (2) degrading a sample to be detected: precisely weighing a certain volume of disulfide bond cross-linked hyaluronic acid gel, and degrading the disulfide bond cross-linked hyaluronic acid gel by using an enzyme, wherein the enzymatic degradation product solution is a uniform transparent solution without obvious particles;
and (3) ultrafiltration separation of degradation products: placing the degradation product obtained in the step (2) in an ultrafiltration centrifugal tube, and centrifuging to ensure that the cross-linking agent residue in the degradation product enters filtrate;
step (4) NTSB reagent color reaction: adjusting the pH of the filtrate obtained in the step (3) to 3-4, mixing the filtrate with the same volume with an NTSB reagent, reacting for 25 minutes at room temperature, and measuring the absorbance at 412nm +/-2 nm;
step (5), establishing a standard curve: degrading the standard substance solution obtained in the step (1) according to the method in the step (2), performing ultrafiltration separation in the step (3), determining absorbance after color development in the step (4), and performing linear regression with the concentration of the standard solution of the cross-linking agent to generate a standard curve;
step (6) determination of crosslinker residue: substituting the absorbance obtained in the step (4) into the standard curve obtained in the step (5) to obtain the residual content of the cross-linking agent;
the absorbance measurement adopts an ultraviolet-visible spectrophotometer; the molecular weight cut-off of the ultrafiltration centrifugal tube is not more than 10KDa.
2. The detection method according to claim 1, wherein the cross-linking agent is cystamine dihydrochloride or 3,3' -dithiobis.
3. The method according to claim 1, wherein the concentration of the disulfide-bond cross-linked hyaluronic acid gel is 3 to 30 mg/ml.
4. The method of detecting according to claim 1, wherein the enzyme is hyaluronidase.
5. The detection method according to claim 4, wherein the hyaluronidase is dissolved with 0.9% sodium chloride and has an enzyme activity of 25-75U/ml.
6. The method of detecting according to claim 5, wherein the volume ratio (V/V) of the disulfide-bond cross-linked hyaluronic acid gel to the hyaluronidase is 1: (1-3).
7. The assay of claim 4, wherein the hyaluronidase degradation condition is 20 minutes at a temperature of 37 ℃.
8. The assay of claim 1, wherein the centrifugation conditions are centrifugation at 6000 rpm for 15 minutes.
9. The method of claim 1, wherein the pH adjustment to 3 to 4 is performed before the centrifugation in step (3).
10. The method of claim 1, wherein the standard solution of the cross-linking agent has a concentration gradient of 1, 2, 3, 4, and 5ppm, respectively.
11. The method of claim 1, wherein the room temperature is in the range of 10 ℃ to 30 ℃.
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| "Self-assembling and auto-crosslinkable hyaluronic acid hydrogels with a fibrillar structure";F.S. Palumbo et al;《Acta Biomaterialia》;20100630;第195–204页 * |
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Address after: 213000 block B, No. 117 Xueye Road, Xinbei District, Changzhou City, Jiangsu Province Patentee after: Changzhou Bairuiji Biopharmaceutical Co.,Ltd. Address before: Building B, No. 117 Xueye Road, Xinbei District, Changzhou City, Jiangsu Province, 213125 Patentee before: BIOREGEN BIOMEDICAL (CHANGZHOU) Co.,Ltd. |
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