CN115808484B - Method for detecting residual lysyl endonuclease in insulin or insulin analogue - Google Patents
Method for detecting residual lysyl endonuclease in insulin or insulin analogue Download PDFInfo
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- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 title claims abstract description 54
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- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 13
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to the technical field of biological detection, in particular to a method for detecting residual lysyl endonuclease in insulin or an analogue thereof. The method can adjust the concentration limit of Lys-C enzyme in the reference sample according to the requirement, and further simply, stably and accurately judge whether the residue of lysyl endonuclease in the sample to be detected is in a limited range. The detection method has the advantages of high sensitivity, high stability, simplicity and easiness in implementation.
Description
Technical Field
The invention relates to the technical field of biological detection, in particular to a method for detecting residual lysyl endonuclease in insulin or an analogue thereof.
Background
Lysyl-specific endonuclease (Lysyl endopeptidase, ec 3.4.21.50), also called Lysyl endopeptidase, achromobacter protease I, lysine endopeptidase, lysyl endopeptidase, lysine C-terminal endopeptidase, lys-C endopeptidase, is a serine protease, and was originally found and isolated from soil bacteria by Masaki et al. Lysyl endopeptidases are highly specific and specifically cleave peptide bonds at the carboxyl terminus of lysine residues and S-aminoethylcysteine residues in peptide chains.
According to the characteristics that the 29 th site of amino acid of recombinant human insulin, insulin degluded and insulin aspart is Lys, the advantages of high specificity and low cost of Lys-C enzyme are fully utilized, and recombinant constructs containing Lys-C enzyme digestion sites are respectively designed aiming at the recombinant insulin and the insulin analogue. After the construct is purified, insulin or insulin analogues are finally obtained through a series of reactions such as Lys-C enzyme digestion and the like. Therefore, in the insulin or insulin analog prepared by the above method, a certain amount of Lys-C enzyme remains, and therefore it is necessary to detect the residual concentration thereof and to ensure that the amount of Lys-C enzyme remains within a reasonable range. The purified insulin or insulin analogue has very low Lys-C enzyme content. How to accurately detect Lys-C enzyme residues in a sample within a specified limit is a problem which needs to be solved urgently.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for detecting residual lysyl endonuclease in insulin or an analogue thereof through a great deal of research.
The invention provides a method for detecting residual lysyl endonuclease in insulin or an analogue thereof, which comprises the steps of carrying out enzyme digestion reaction on a lysyl endonuclease substrate, a sample to be detected and a positive reference substance respectively, and judging the content of the residual lysyl endonuclease in the sample to be detected by comparing the peak areas of enzyme digestion peptide sections of two enzyme digestion products in high performance liquid chromatography.
The purified sample of insulin or its analogs has a relatively low lysyl endonuclease content. The conventional lysyl endonuclease substrate Lys-PNA determines enzyme activity by detecting enzyme substrate absorbance, and the enzyme substrate absorbance after enzyme digestion can be proportional to the enzyme activity only within a certain enzyme concentration range, has limited sensitivity, and cannot be applied to the detection of Lys-C enzyme residue under a low concentration condition. Therefore, after extensive screening and intensive research, the applicant of the invention finds that HGEGTFTSDLSKX is particularly suitable to be used as a substrate peptide segment, and the enzyme-digested peptide segment HGEGTFTSDLSK (short for "peptide segment H") obtained by enzyme digestion of Lys-C is analyzed and detected at the chromatographic wavelength of 214 nm by high performance liquid, so that the peptide segment has obviously stronger ultraviolet compared with other peptide segments. The peptide fragment is the initial peptide fragment of the exenatide, so that the applicant chooses to use the exenatide as a substrate. Based on further application research of the substrate discovery, the applicant of the invention develops a method for detecting the content of residual Lys-C enzyme in insulin or an analogue thereof. Experiments also show that when the enzyme digestion ratio of the detection method of the invention which selects exenatide as the substrate is 1:50000 hours later, the enzyme digestion peptide still has strong absorption at 214 nm, and the peak shape is good and stable, and can stably and accurately present the detection result.
Exenatide is a novel diabetes treatment drug, is a hypoglycemic analogue developed by American etiquette, is a first member of an incretin analogue family, is a polypeptide compound artificially synthesized, and has a definite and stable structure and wide sources.
In particular, the method of the present invention is applicable to all insulin or insulin analogs, including but not limited to recombinant human insulin, insulin deglutamide or insulin aspart, and the like.
Specifically, the detection method at least comprises the following steps:
s1, respectively preparing a substrate solution, a Lys-C enzyme positive reference solution, a diluent and a sample solution to be detected;
s2, mixing a Lys-C enzyme positive reference substance solution, a substrate solution and a diluent in proportion, carrying out enzyme digestion reaction, and terminating the reaction after the reaction is finished to obtain a positive reference substance reaction solution;
s3, mixing ultrapure water, a substrate solution and a sample solution to be detected, carrying out enzyme digestion reaction under the same condition as the S2, and terminating the reaction after the reaction is finished to obtain a sample reaction solution to be detected;
wherein, the volume of the ultrapure water is equal to the volume of the Lys-C enzyme positive reference substance solution in the S2, and the volume of the sample solution to be detected is equal to the volume of the diluent in the S2; the dosage of the substrate solution in the S2 and the S3 is the same;
and S4, respectively carrying out high performance liquid chromatography detection on the reaction liquid of the positive control substance and the reaction liquid of the sample to be detected, comparing peak areas of enzyme digestion peptide fragments, and judging whether the lysyl endonuclease residue in the sample to be detected exceeds the concentration of the solution of the positive control substance.
In S3, the same conditions including the volume of the reaction solution, the time and the temperature of the enzyme digestion are all the same.
Preferably, in S2 and S3, the enzyme digestion reaction is carried out for 5-15 hours at 35-40 ℃, further for 5-10 hours at 36-38 ℃, and more preferably for 6 hours at 37 ℃ in a water bath.
Preferably, in S2, the mass ratio of the lysyl endonuclease standard substance to the substrate in the Lys-C enzyme positive control solution is 40000-55000, and preferably 1:50000.
in a preferred embodiment of the present invention, the concentration of the substrate solution in S2 is 0.5 to 2 mg/mL, preferably 0.75 to 1.5 mg/mL, and more preferably 1 mg/mL.
In a preferred embodiment of the present invention, the concentration of Lys-C enzyme in the positive control solution of Lys-C enzyme in S2 is 0.2-1 μ g/mL, preferably 0.25-0.75 μ g/mL, and more preferably 0.5 μ g/mL.
As a preferable technical scheme of the invention, in S3, the concentration of insulin or the like in the sample solution to be detected is 10-30 mg/mL, preferably 15-25 mg/mL, and more preferably 20 mg/mL.
In a preferred embodiment of the present invention, in S2, the volume ratio of the positive control solution, the substrate solution and the diluent is 1:25:25.
as a preferable technical scheme of the invention, in S3, the volume ratio of Lys-C enzyme ultrapure water, substrate solution and sample solution to be detected is 1:25:25.
as a preferable technical scheme of the invention, in S2, the volumes of the Lys-C enzyme positive control solution, the substrate solution and the diluent are respectively 10 mu L, 250 mu L and 250 mu L.
As a preferable technical scheme of the invention, in S3, the volumes of the ultrapure water, the substrate solution and the sample solution to be tested are respectively 10 mu L, 250 mu L and 250 mu L.
As a preferred embodiment of the present invention, the reaction is terminated in both S2 and S3 by adding 10. Mu.L of 1.5mol/L acetic acid solution.
As a preferable technical scheme of the invention, the substrate solution and the sample solution to be detected are both prepared by adopting diluent.
As a preferable technical scheme, the diluent adopted in the invention is 0.05 mol/L of Tirs-HCl buffer solution, and the pH value is 8.0.
As a preferable embodiment of the present invention, the Lys-C enzyme positive control solution has a Lys-C enzyme concentration of A and a Lys-C enzyme dosage of V in S2 and S3, respectively a The concentration of insulin or its analogue in the sample to be tested is B, and the amounts thereof in S2 and S3 are V respectively b Then, in S4, according to the comparison of the peak areas of the enzyme digestion peptide fragments, whether the content of Lys-C enzyme in the sample to be detected exceeds the detection limit (A x V) a ）/（B×V b ）。
As a preferred technical scheme of the invention, the conditions of the high performance liquid detection are as follows:
the chromatographic column is selected from C18 reverse phase chromatographic column; the specific optional Welch Ultimate ® An XB-C18 (4.6X 250 mm,5 μm, 300) reverse phase chromatography column or equivalent chromatography column;
mobile phase: mobile phase a was 0.1% tfa in water, mobile phase B was acetonitrile containing 0.1% tfa;
flow rate: 1.0 mL/min;
column temperature: 50 ℃;
detection wavelength: 214 And (5) nm.
The technical scheme of the invention at least has the following technical effects:
the detection method has high sensitivity, and the mass ratio of lysyl endonuclease to substrate is 1:50000 can also be detected. The detection method has high stability, and the method is simple and easy to implement.
Drawings
Fig. 1 is a high performance liquid chromatography obtained after the detection of the insulin aspart sample solution in example 1, wherein the abscissa (not shown) represents retention time, and the ordinate (not shown) represents intensity.
Detailed Description
In order that the above objects, features and advantages of the present invention may be more clearly understood, a solution of the present invention will be further described below. It should be noted that the embodiments of the present invention and features of the embodiments may be combined with each other without conflict. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the invention may be practiced otherwise than as described herein; it is to be understood that the embodiments described in this specification are only some embodiments of the invention, and not all embodiments.
The reagents used in the following examples are all commercially available.
Example 1: method for detecting Lys-C enzyme residue in insulin aspart
S1, solution preparation:
(1) Diluent (0.05 mol/L of Tirs-HCl buffer, pH 8.0): weighing 0.606 g of Tris, dissolving in 80 mL of ultrapure water, adjusting the pH to 8.0 +/-0.1 by using hydrochloric acid, metering the volume to 100mL by using the ultrapure water, and uniformly mixing to obtain the finished product.
(2) 1.5mol/L acetic acid solution: measuring 1.0mL of acetic acid, adding 10.7 mL of ultrapure water, and mixing uniformly to obtain the product.
(3) Substrate (exenatide) solution: taking a proper amount of exenatide, dissolving and diluting the exenatide with a diluent to 1mg/mL, and uniformly mixing to obtain the exenatide acetate.
(4) Lys-C enzyme positive control solution (0.5. Mu.g/mL): lys-C enzyme was dissolved in ultrapure water to a concentration of 0.5. Mu.g/mL.
(5) Insulin aspart test sample solution (20 mg/mL): taking a proper amount of insulin aspart samples, precisely weighing, dissolving and diluting to 20 mg/mL by using a diluent, and uniformly mixing to obtain the insulin aspart.
(6) Pure solution of insulin aspart (20 mg/mL): taking a proper amount of pure insulin aspart, precisely weighing, dissolving and diluting to 20 mg/mL by using a diluent, and uniformly mixing to obtain the insulin aspart.
S2, carrying out enzyme digestion reaction on a positive control solution: taking 10 mu L of Lys-C enzyme positive control solution, 250 mu L of substrate (exenatide) solution and 250 mu L of diluent, uniformly mixing, and placing in a water bath at 37 ℃ for reacting for 6 h; after completion of the reaction, 10. Mu.L of an acetic acid solution (1.5 mol/L) was added to terminate the reaction.
S3, carrying out enzyme digestion reaction on a pure product solution adapted to the system: taking 10 mu L of positive control solution, 250 mu L of substrate (exenatide) solution and 250 mu L of pure solution of insulin aspart, mixing uniformly, and placing in a 37 ℃ water bath for reaction for 6 h; after completion of the reaction, 10. Mu.L of an acetic acid solution (1.5 mol/L) was added to terminate the reaction.
S4, respectively carrying out high performance liquid detection on the reaction liquid after the reaction in S2 and S3 is terminated, and detecting the chromatographic peak area of the enzyme digestion peptide fragment H at 214 nm, wherein the chromatographic conditions are as follows:
reversed phase chromatography column with WelchUltimate XB-C18 (4.6X 250 mm,5 μm, 300A); as mobile phase A, an aqueous solution of 0.1% TFA and as mobile phase B, an acetonitrile solution of 0.1% TFA were used, and the flow rate was 1.0mL/min, the column temperature was 50 ℃ and the detection wavelength was 214 nm.
The assay was performed with the system adaptation solution and the positive control solution as described above (three replicates), the feasibility of the method was verified, and the results are shown in table 1:
TABLE 1
As shown in the repeated results in table 1, the areas of the H peaks of the enzymatic peptide fragments in the positive control and the pure solution for system adaptation are substantially the same, and the detection results are stable and reliable; the difference between the two is very small; and repeated for many times, the result is stable. Moreover, the enzymolysis peptide fragment H has good separation degree with substrate, insulin and the like, and no peak mixing condition exists.
S5, enzyme digestion reaction of a reaction solution of a sample to be detected: taking 10 mu L of ultrapure water, 250 mu L of substrate (exenatide) solution and 250 mu L of insulin aspart sample solution to be detected, and placing the ultrapure water, the substrate (exenatide) solution and the insulin aspart sample solution in a water bath at 37 ℃ for reaction for 6 hours; after the reaction was completed, 10. Mu.L of an acetic acid solution (1.5 mol/L) was added to terminate the reaction.
And detecting the purified insulin aspart sample solution to be detected according to the high performance liquid chromatography method in the S4, wherein the detection result is shown in figure 1.
As shown in FIG. 1, no peptide fragment H peak was detected in the samples of insulin aspart purified and prepared by the applicant, indicating that the Lys-C enzyme content was much lower than the limited amount.
The above experiments show that the detection method of the invention can accurately react the residual amount of Lys-C enzyme in a sample. According to the detection method of this example, the area below the control peak indicates that the Lys-C enzyme content in the sample is less than 1. Mu.g per g of sample.
The above description is merely illustrative of particular embodiments of the invention that enable those skilled in the art to understand or practice the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (9)
1. A method for detecting residual lysyl endonuclease in insulin or an analog thereof, comprising at least the steps of:
s1, respectively preparing a substrate solution, a Lys-C enzyme positive reference solution, a diluent and a sample solution to be detected; the substrate is exenatide;
s2, mixing the Lys-C enzyme positive control solution, the substrate solution and the diluent according to a ratio, performing enzyme digestion reaction, and terminating the reaction after the reaction is finished to obtain a positive control reaction solution; the mass ratio of the lysyl endonuclease standard substance to the substrate in the Lys-C enzyme positive control solution is 1: 40000-55000; the concentration of the substrate solution is 0.5-2 mg/mL, and the concentration of Lys-C enzyme in the Lys-C enzyme positive control solution is 0.2-1 mu g/mL;
s3, taking ultrapure water, the substrate solution and the sample solution to be detected, mixing, performing enzyme digestion reaction under the same condition as S2, and terminating the reaction after the reaction is finished to obtain a sample reaction solution to be detected;
wherein, the volume of ultrapure water is equal to the volume of the Lys-C enzyme positive control solution in S2, and the volume of the sample solution to be detected is equal to the volume of the diluent in S2; the dosage of the substrate solution in the S2 and the S3 is the same; the concentration of insulin or insulin analogues in the sample solution to be detected is 10-30 mg/mL;
s4, respectively carrying out high performance liquid chromatography detection on the positive reference substance reaction solution and the reaction solution of the sample to be detected, comparing peak areas of enzyme digestion peptide fragments, and judging whether the lysyl endonuclease residue in the sample to be detected exceeds the concentration of the positive reference substance solution or not;
the detection conditions of the high performance liquid chromatography are as follows:
the chromatographic column is selected from C18 reverse phase chromatographic column;
mobile phase: mobile phase a was 0.1% tfa in water, mobile phase B was acetonitrile containing 0.1% tfa;
flow rate: 1.0 mL/min;
column temperature: 50 ℃;
detection wavelength: 214 And (5) nm.
2. The detection method according to claim 1, wherein the conditions of the enzyme digestion reaction in S2 and S3 are the same, and the reaction is carried out for 5-15 hours at 35-40 ℃.
3. The detection method according to claim 1, wherein in S2, the mass ratio of the lysyl endonuclease standard substance to the substrate in the Lys-C enzyme positive control solution is 1:50000.
4. the detection method according to claim 1, wherein in S2, the concentration of the substrate solution is 1mg/mL, the concentration of Lys-C enzyme in the Lys-C enzyme positive control solution is 0.5. Mu.g/mL,
in S3, the concentration of insulin or the like in the sample solution to be detected is 20 mg/mL.
5. The detection method according to claim 4, wherein in S2, the volume ratio of the Lys-C enzyme positive control solution, the substrate solution and the diluent is 1:25:25;
in S3, the volume ratio of the ultrapure water to the substrate solution to the sample solution to be detected is 1:25:25.
6. the detection method according to claim 5, wherein in S2, the volumes of the Lys-C enzyme positive control solution, the substrate solution, and the diluent are 10. Mu.L, 250. Mu.L, and 250. Mu.L, respectively;
in S3, the volumes of the ultrapure water, the substrate solution and the sample solution to be detected are respectively 10 muL, 250 muL and 250 muL.
7. The detection method according to claim 6, wherein the reaction is terminated in each of S2 and S3 by adding 10. Mu.L of a 1.5mol/L acetic acid solution.
8. The detection method according to claim 1, wherein the substrate solution and the sample solution to be detected are both prepared by using a diluent; the diluent is 0.05 mol/L of Tirs-HCl buffer solution, and the pH value is 8.0.
9. The detection method according to claim 1, wherein the concentration of Lys-C enzyme in the Lys-C enzyme positive control solution is A, and the amounts thereof in S2 and S3 are V, respectively a The concentration of the insulin or the analogue thereof in the sample to be tested is B, and the dosage of the insulin or the analogue thereof in S2 and S3 is V respectively b Then, in S4, according to the comparison of the peak areas of the enzyme digestion peptide fragments, judging whether the content of Lys-C enzyme in the sample to be detected exceeds the detection limit (A x V) a ）/（B×V b ）。
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Denomination of invention: A detection method for residual lysine endonuclease in insulin or its analogues Effective date of registration: 20231013 Granted publication date: 20230414 Pledgee: Jilin Bank Co.,Ltd. Tonghua Meihekou Branch Pledgor: Jilin Huisheng Biopharmaceutical Co.,Ltd. Registration number: Y2023220000102 |
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