CN114441702B - Method for detecting I, II type rhTNK-tPA content - Google Patents
Method for detecting I, II type rhTNK-tPA content Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/89—Inverse chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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Abstract
The invention provides a method for detecting the content of I, II type rhTNK-tPA, and relates to the technical field of analytical chemistry. Preparing a I, II rhTNK-tPA standard substance into a mixed solution according to different percentage contents, performing sample pretreatment, and then adopting a reversed-phase high-performance liquid chromatography for analysis and detection to establish a standard curve; (2) And (3) carrying out pretreatment on the rhTNK-tPA sample solution to be detected, which is the same as that in the step (1), and adopting the chromatographic method, which is the same as that in the step (1), to carry out analysis and detection, thus obtaining the content of I, II type rhTNK-tPA. The method can completely separate the base line of the I/II type peak of the rhTNK-tPA, and the separation degree can reach more than 2.8; the accuracy of the method can reach 86.52-116.90 percent, and the Relative Standard Deviation (RSD) of repeatability and precision is less than 2 percent.
Description
Technical Field
The invention relates to the technical field of analytical chemistry, in particular to a method for detecting the content of I, II-type rhTNK-tPA.
Background
Tissue plasminogen activator (t-PA) is a serine protease consisting of a 527 amino acid polypeptide chain containing 3 potential sites for N-glycosylation (Asn 117, asn184 and Asn 448), where the Asn184 site is incompletely glycosylated.
Recombinant human TNK tissue type plasminogen activator (rhTNK-tPA, mingfule) for injection is a third generation t-PA product with a molecular weight of 65KD, containing 17 pairs of disulfide bonds, t-PA is mutated from 103 th Thr at the N-terminal to Asn,117 th Asn to Gln,296-299 th Lys-His-Arg-Arg to Ala-Ala-Ala, and the peptide bond between Arg275 and Ile276 of rhTNK-tPA is sensitive to plasmin and can be cleaved into molecules consisting of two chains connected by disulfide bonds, wherein 275 amino acid residues at the N-terminal form a heavy chain and 252 amino acid residues at the C-terminal form a light chain. Heavy chains that are glycosylated at the Asn184 position are referred to as type I a chains, heavy chains that are not glycosylated at the Asn184 position are referred to as type II a chains, and light chains are also referred to as B chains.
The polarity of the A chain of the type I or type II rhTNK-tPA is close, and the separation difficulty is extremely high. At present, a relatively mature method for detecting the type I/II content comprises the steps of carrying out protease digestion treatment, reducing by a reducing agent and terminating reaction by a terminating agent on an SDS-PAGE gel electrophoresis detection rt-PA (first generation recombinant human t-PA) published in pharmacopoeia, finally carrying out enzymolysis on proteins into a plurality of independent polypeptide chains, separating the type I A chain and the type II A chain according to the molecular weight difference of the type I A chain and the type II A chain caused by whether glycosylation occurs at an Asn184 site or not, dyeing and decolorizing the separated type I chain and the type II A chain by electrophoresis, obtaining an electrophoresis band result, carrying out gray analysis on the band, establishing a linear relation between the gray value of the band and the concentration of a sample, and calculating the type I/II rt-PA content of the sample by drawing a standard curve.
In the prior art, the method for monitoring the content of t-PA or TNK-tPA using HPLC analysis is to calculate the percentage content of type I and type II by peak area normalization. And the separation of the I/II type rhTNK-tPA chromatographic peak is 1.5, the separation degree only reaches the lower limit of the complete separation of two substances specified in pharmacopoeia, and the I/II type rhTNK-tPA chromatographic peak does not reach the baseline separation, cannot be accurately quantified, and cannot meet the requirement of medicine quality control. Therefore, there is a need to develop an accurate and safe method for detecting the content of type I/II rhTNK-tPA.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide a novel method for detecting the content of I, II type rhTNK-tPA, which not only improves the safety, but also greatly improves the separation degree of I, II type rhTNK-tPA, and is a more accurate method for detecting the content of I, II type rhTNK-tPA.
In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:
in one aspect, the invention provides a method of detecting the content of I, II-type rhTNK-tPA, the method comprising:
(1) Preparing a I, II rhTNK-tPA standard substance into mixed solutions according to different percentage contents, performing sample pretreatment, and then adopting a reversed-phase high performance liquid chromatography (RP-HPLC) for analysis and detection to establish a standard curve;
(2) Carrying out pretreatment on the rhTNK-tPA sample solution to be detected, which is the same as that of the step (1), and adopting the chromatographic method, which is the same as that of the step (1), to carry out analysis and detection so as to obtain the contents of the I-type and II-type rhTNK-tPA;
wherein, the reversed-phase high-performance liquid chromatography adopts a Sino Chrom C8 or Zorbax 300SB-C8 reversed-phase chromatographic column; taking 0.1% -0.3% trifluoroacetic acid/water solution as a mobile phase A, and taking 0.1% -0.3% trifluoroacetic acid/acetonitrile solution as a mobile phase B for linear elution;
the sample pretreatment includes:
(a) Dilution and enzymolysis: diluting a sample to a concentration of 1.0-1.5 mg/mL by using a sample diluent; adding 1% -10% (w/w) of plasmin, and performing enzymolysis for 30-90 min at 35-38 ℃; the diluent comprises 54-56 g/L arginine and 0.03-0.05% (v/v) Tween 80;
(b) Denaturation and reduction: diluting the enzymolyzed sample with a denaturation buffer solution to a concentration of 0.45-0.75 mg/mL in a ratio of 1:1 (v/v), and then adding dithiothreitol for reduction; the denaturation buffer solution comprises 475-485 g/L urea, 60.55-60.65 g/L Tris and 1.15-1.25 g/L Na 2 EDTA。
In one embodiment, the pH of the sample diluent is 7.2-7.6.
In one embodiment, the pH of the denaturing buffer is 8.2 to 8.6.
In one embodiment, the reduction is performed by adding dithiothreitol to a final concentration of 15 to 25 mM, preferably 20 mM, and reacting at 35 to 38℃for 30 to 90 minutes.
In one embodiment, the chromatographic column is equilibrated for 15-30 min before sample injection, equilibrated for another 10-15 min after sample injection, and then subjected to linear elution.
In one embodiment, the linear elution conditions are: 0-70 min, 65-75% of mobile phase A and 25-35% of mobile phase B; 70-120 min,50% of mobile phase A and 50% of mobile phase B; the column was then regenerated with 100% mobile phase B.
In one embodiment, the sample injection amount of the reversed-phase high-performance liquid chromatography is 50-100 mu L.
In some specific embodiments, the sample loading in the reverse phase high performance liquid chromatography includes, but is not limited to: 50. 60, 70, 80, 90, 100 μl.
In one embodiment, the column temperature of the chromatographic column is 38-42 ℃, preferably 40 ℃.
In one embodiment, the filler particle size of the chromatographic column is 5-10 μm and the pore size is 200-350 a.
In one embodiment, the flow rate of the reversed phase high performance liquid chromatography mobile phase is 0.5-1.0 mL/min, preferably 0.8 mL/min.
In one embodiment an ultraviolet detector or diode array detector is used for detection, preferably an ultraviolet detector.
In one embodiment, the ultraviolet detection wavelength of the reverse phase high performance liquid chromatography is 214 nm or 280 nm.
In one embodiment, the standard curve is plotted with the ratio of the amount of rhTNK-tPA form I to the amount of rhTNK-tPA form II as the abscissa and the ratio of the peak area of the standard form I rhTNK-tPA form II to the peak area of the standard form II rhTNK-tPA form as the ordinate, and a linear regression equation is established.
Specifically, the ratio of the peak area of the type I rhTNK-tPA to the peak area of the type II rhTNK-tPA of the detection sample is substituted into a regression equation to obtain the content ratio of the type I/II rhTNK-tPA, so that the percentage contents of the type I rhTNK-tPA and the type II rhTNK-tPA are calculated.
In a specific embodiment, the method comprises:
(1) Preparing a mobile phase, filtering and degassing; adjusting the conditions of the high performance liquid chromatograph, and starting up the balance chromatographic column;
(2) Preparing a standard substance solution: mixing I, II type rhTNK-tPA standard products according to different percentage contents;
(3) Pretreating a standard substance solution, injecting the pretreated standard substance solution into a high performance liquid chromatograph, recording a chromatogram and a peak area, and manufacturing a standard curve to obtain a linear regression equation;
(4) Pretreating the solution to be detected, injecting the pretreated solution into a high performance liquid chromatograph, recording a chromatogram and a peak area, and calculating the content by adopting an external standard method.
In a specific embodiment, the chromatographic conditions comprise: equilibrate for 15min before sample injection, equilibrate for 10min after sample injection, then linearly elute from 70% mobile phase a to 50% mobile phase a with 120min, and finally regenerate the column with 100% mobile phase B. Detection wavelength: 214 nm, flow rate 0.8 mL/min, column temperature: 40 ℃.
In a specific embodiment, the sample enzymatic hydrolysis comprises: sample dilutions were prepared according to the following formulation: 55 g/L arginine, 0.04% Tween 80, pH 7.4; samples containing rhTNK-tPA were diluted to 1.5. 1.5 mg/mL with sample dilution, and 2% (w/w) plasmin was added for enzymatic hydrolysis at 37℃for 90 min.
In a specific embodiment, sample denaturation and reduction comprises: the denaturation buffer was formulated as follows: 480 g/L urea, 60.60 g/L Tris and 1.2 g/L Na 2 EDTA, pH adjusted to 8.4; the protease-treated samples were diluted with denaturation buffer, dithiothreitol (DTT) was added to a final concentration of 20 mM to reduce disulfide bonds in the samples, and reacted at 37 ℃ for 60 min.
Compared with the prior art, the invention has the beneficial effects that:
after the Asn184 amino acid of the type I A chain in rhTNK-tPA is glycosylated, the polarity is increased, the type II A chain is not glycosylated, and the polarity is slightly smaller than that of the type I A chain. In reverse phase HPLC systems, more polar species peak first and less polar species remain longer. Thus, the type I A chain and the type II A chain can be theoretically separated by reversed phase HPLC, but it is difficult to separate the two peaks to the extent of baseline separation. The method determines the optimal condition for measuring the content of I, II type rhTNK-tPA by using a reversed-phase high-performance liquid chromatography, and can separate two substances well. The chromatographic condition of the invention can completely separate the base line of I/II type peak of rhTNK-tPA, and the separation degree can be greatly improved and can reach more than 2.8.
The quantification method in the prior art is an area normalization method, a standard curve is not drawn by using a standard substance, and the quantification accuracy is poor. The invention adopts the high performance liquid chromatography external standard method, can rapidly and accurately measure the I, II rhTNK-tPA content, has good linear relation between the sample concentration and the peak area, high accuracy and good reproducibility and stability. The method adopts I, II rhTNK-tPA standard substance to establish standard curve and perform quantitative detection, thus being a method with higher accuracy; the accuracy can reach 86.52% -116.90%, and the Relative Standard Deviation (RSD) of the repeatability and the precision of the method is less than 2%.
According to the method disclosed by the invention, sodium azide is not required for sample treatment, so that the safety is greatly improved; the method has simple operation and low cost, and can be used for large-scale production.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a chromatogram of the invention for detecting I, II type content in a rhTNK-tPA sample to be detected by reverse phase high performance liquid chromatography under the condition of example 1;
FIG. 2 is a standard graph of a I, II type rhTNK-tPA standard under the conditions of example 1 of the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1:
a method for detecting the type I and type II contents in rhTNK-tPA by using an RP-HPLC method comprises the following steps:
step (a): preparing a mobile phase;
step (b): preparing I-type and II-type standard mixed solutions with different proportions;
carrying out sample pretreatment on the standard mixed solution, carrying out high performance liquid chromatography on the I, II type standard mixed solution by adopting a high performance liquid chromatograph, preparing a standard curve, and drawing an I/II type proportion-peak area standard curve to obtain a standard curve regression equation;
step (c): preprocessing the rhTNK-tPA sample solution to be detected, carrying out high performance liquid chromatography analysis by adopting the chromatographic conditions which are completely the same as those of the step (b), and calculating the I/II type content in the rhTNK-tPA by using an external standard method according to the standard curve regression equation of the step (b) according to the peak area.
The method comprises the following specific steps:
s1, preparing a mobile phase, filtering, degassing, adjusting an instrument and balancing a chromatographic column.
Mobile phase: the mobile phase A is 0.1-0.3% of trifluoroacetic acid (TFA)/water solution by mass, and the mobile phase B is 0.1-0.3% of trifluoroacetic acid (TFA)/acetonitrile solution by mass.
S2, sample pretreatment:
A1. sample enzymolysis:
sample dilutions were prepared according to the following formulation: 54-56 g/L arginine, 0.03-0.05% tween 80, and pH 7.2-7.6; and diluting the sample containing rhTNK-tPA to 1.0-1.5 mg/mL by using a sample diluent, adding 1-10% (w/w) plasmin, and carrying out enzymolysis for 30-90 min at 35-38 ℃.
A2. Sample denaturation and reduction:
the denaturation buffer was formulated as follows: 475-485 g/L urea, 60.55-60.65 g/L Tris and 1.15-1.25 g/L Na 2 EDTA, adjusting the pH to 8.2-8.6; diluting the protease-treated sample with a denaturation buffer solution, adding Dithiothreitol (DTT) to a final concentration of 15-25 mM to reduce disulfide bonds in the sample, and reacting for 30-90 min at 35-38 ℃;
s3, chromatographic conditions:
before sample injection, balancing the chromatographic column for 15-30 min, balancing for 10-15 min after injection, wherein the proportion of an initial organic phase solvent (mobile phase B) is 25% -35%, the proportion of an aqueous phase solvent (mobile phase A) is 65% -75%, changing into 50% through a linear gradient for 70-120 min, and then regenerating the chromatographic column by using 100% of the organic phase solvent.
Mobile phase: mobile phase A is 0.1% trifluoroacetic acid (TFA)/water solution, mobile phase B is 0.1% trifluoroacetic acid (TFA)/acetonitrile solution;
sample injection amount: 50-100 mu L;
flow rate: 0.5-1.0 mL/min;
ultraviolet detection wavelength: 214 nm or 280 nm, the separation degree can reach 2.8-3.0.
Chromatographic column: the filler diameter is preferably 5-10 mu m, and the pore size is preferably 200-350A.
Chromatographic column temperature: 38-42 ℃.
S4, data processing:
according to the invention, I, II type rhTNK-tPA standard substances are mixed according to different percentages, pretreatment is carried out, detection is carried out, the ratio of the content of I type rhTNK-tPA to the content of II type rhTNK-tPA is taken as an abscissa, the ratio of the peak area of the I type rhTNK-tPA standard substance to the peak area of the II type rhTNK-tPA standard substance is taken as an ordinate, a standard curve is established, and the ratio of the peak area of the I type rhTNK-tPA to the peak area of the II type rhTNK-tPA of a detected sample is substituted into a regression equation to obtain the content ratio of I/II type rhTNK-tPA, so that the percentage content of I type rhTNK-tPA is calculated.
In a specific operation:
preparing mixed liquor of I, II type rhTNK-tPA standard products (I, II type rhTNK-tPA standard products are prepared by Guangzhou Ming kang biological engineering Co., ltd.) in different proportions: mixing I, II type rhTNK-tPA standard products according to the proportion of 10:90, 20:80, 30:70, 40:60 and 50:50 respectively;
carrying out sample pretreatment on the standard mixed solution, carrying out high performance liquid chromatography on the I, II type standard mixed solution by adopting a high performance liquid chromatograph, preparing a standard curve, and drawing an I/II type proportion-peak area standard curve to obtain a standard curve regression equation; preprocessing the rhTNK-tPA sample solution to be detected, carrying out high performance liquid chromatography analysis by adopting the chromatographic conditions which are completely the same as those of the step (b), and calculating the I/II type content in the rhTNK-tPA by using an external standard method according to the standard curve regression equation of the step (b) according to the peak area.
Specifically, the steps are as follows:
s1, preparing a mobile phase, filtering, degassing, adjusting an instrument and balancing a chromatographic column.
Mobile phase: mobile phase a was 0.1% trifluoroacetic acid (TFA)/aqueous solution by mass and mobile phase B was 0.1% trifluoroacetic acid (TFA)/acetonitrile solution by mass.
S2, sample pretreatment:
sample enzymolysis: sample dilutions were prepared according to the following formulation: 55 g/L arginine, 0.04% Tween 80, pH 7.4; samples containing rhTNK-tPA were diluted to 1.5. 1.5 mg/mL with sample dilution, and 2% (w/w) plasmin was added for enzymatic hydrolysis at 37℃for 90 min.
Sample denaturation and reduction: denaturation buffer 480 g/L Urea, 60.60 g/L Tris and 1.2 g/L Na was formulated as follows 2 EDTA, pH adjusted to 8.4; diluting the protease-treated sample with a denaturation buffer at 1:1 (v/v) to a concentration of 0.728 mg/mL, adding Dithiothreitol (DTT) to a final concentration of 20 mM to reduce disulfide bonds in the sample, and reacting at 37℃for 60 min;
s3, chromatographic conditions: equilibrate for 15min before sample injection, equilibrate for 10min after sample injection, then linearly elute from 70% mobile phase a to 50% mobile phase a with 120min, and finally regenerate the column with 100% mobile phase B.
Mobile phase: mobile phase A is 0.1% trifluoroacetic acid (TFA)/water solution, mobile phase B is 0.1% trifluoroacetic acid (TFA)/acetonitrile solution;
sample injection amount: 50. mu L; flow rate: 0.8 mL/min;
ultraviolet detection wavelength: 214 nm, the separation degree can reach 2.9.
Chromatographic column: zorbax 300SB-C8, preferably 5 μm filler diameter and 300A pore size. Chromatographic column temperature: 40 ℃.
FIG. 1 is a chromatogram of the invention for detecting I, II type content of a rhTNK-tPA sample to be detected by reverse phase high performance liquid chromatography under the condition of example 1; FIG. 2 is a standard graph of a I, II type rhTNK-tPA standard under the conditions of example 1 of the present invention.
Example 2
1. Precision test:
precision refers to the degree of closeness between the results obtained by sampling and measuring the same sample under prescribed conditions, including reproducibility, intermediate precision and reproducibility. The method is subjected to precision test evaluation, the repeatability RSD value of the method is 1.1% -1.5%, the intermediate precision RSD value is 0.5% -1.1%, and the method is proved to have good precision in measuring I, II-type rhTNK-tPA.
2. Linear relationship:
linearity refers to the ability of a linear test result to directly scale with the concentration of the analyte in a sample within a designed range. The linear test result shows that the linear equation y=1.497x-0.0306 and the R value is 0.9993, and the method is proved to have good linear relation.
3. Accuracy test:
accuracy refers to the degree to which the result of a measurement by the established method is close to the true or reference value, typically expressed in terms of recovery (%). The method for measuring I, II-type rhTNK-tPA has good accuracy, and the accuracy is 86.52% -116.90%.
Comparative test
The detection is carried out by adopting the reversed-phase high performance liquid chromatography which is the same as the invention, and the difference is that:
(1) Sample enzymolysis: sample dilutions were prepared according to the following formulation: 125 mM Na 2 HPO 4 200 mM arginine and 0.01% NaN 3 pH 7.5; diluting a sample containing rhTNK-tPA with a sample diluent of 1:3 (v/v), adding 1% (w/w) plasmin, and performing enzymolysis at 37 ℃ for 30 min;
(2) The time for reducing the sample is 37 ℃ and the reaction is carried out for 60 min;
(3) Balancing for at least 15min before sample injection, balancing for 5min after sample injection, linearly eluting from 70% mobile phase A to 50% mobile phase A for 60 min, and regenerating chromatographic column with 100% mobile phase B;
(4) Detection wavelength: excitation wavelength 275 nm, emission wavelength 340 nm, flow rate 1 mL/min.
Compared with a comparison test, the invention has the following advantages:
(a) In the pretreatment of the sample, the invention does not need to use extremely toxic substances, and has higher safety. The invention does not use sodium azide, and the solution is filtered and stored, so that the antibacterial effect of the solution can be achieved.
(b) The invention adjusts and optimizes the equilibrium time and the linear gradient change time after the sample injection. The equilibrium is carried out for 5min after the injection of the comparative test sample, the linear gradient change time is 60 min, the separation degree of the I/II type rhTNK-tPA peak is only 1.5-2.0, and the separation degree of the I type rhTNK-tPA peak and impurities is low; the invention balances for 10-15 min after injection, the linear gradient change time is 120min, and the peak separation degree of the type I rhTNK-tPA and the type II rhTNK-tPA can reach 2.8-3.0.
(c) The invention adopts I, II type standard substance solutions mixed in different proportions to establish a standard curve to quantify the I/II type in rhTNK-tPA, and has higher accuracy (the same sample to be tested, the comparison test result is that the I type content is 42.51%, the II type result is 57.49%, and the invention is detected as 35.68% of the I type content and 64.32% of the II type result).
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.
Claims (10)
1. A method for detecting the content of I, II-type rhTNK-tPA, comprising:
(1) Preparing a I, II rhTNK-tPA standard substance into a mixed solution according to different percentage contents, performing sample pretreatment, and then adopting a reversed-phase high-performance liquid chromatography for analysis and detection to establish a standard curve;
(2) Carrying out pretreatment on the rhTNK-tPA sample solution to be detected, which is the same as that of the step (1), and adopting the chromatographic method, which is the same as that of the step (1), to carry out analysis and detection so as to obtain the contents of the I-type and II-type rhTNK-tPA;
wherein, the reversed-phase high-performance liquid chromatography adopts a Sino Chrom C8 or Zorbax 300SB-C8 reversed-phase chromatographic column; taking 0.1% -0.3% of trifluoroacetic acid and an aqueous solution as a mobile phase A, and taking 0.1% -0.3% of trifluoroacetic acid and an acetonitrile solution as a mobile phase B for linear elution;
the sample pretreatment includes:
(a) Dilution and enzymolysis: diluting a sample to a concentration of 1.0-1.5 mg/mL by using a sample diluent; adding 1% -10% w/w of plasmin, and performing enzymolysis for 30-90 min at 35-38 ℃; the diluent comprises 54-56 g/L arginine and 0.03-0.05% tween 80;
(b) Denaturation and reduction: diluting the enzymolyzed sample with a denaturation buffer solution at a concentration of 1:1v/v to 0.45-0.75 mg/mL, and then adding dithiothreitol for reduction; the denaturation buffer solution comprises 475-485 g/L urea, 60.55-60.65 g/L Tris and 1.15-1.25 g/L Na 2 EDTA；
The linear elution conditions were: 0-70 min, 65-75% of mobile phase A and 25-35% of mobile phase B; 70-120 min,50% of mobile phase A and 50% of mobile phase B; regenerating the column with 100% mobile phase B;
the standard curve is formed by plotting the ratio of the content of the type I rhTNK-tPA and the content of the type II rhTNK-tPA as an abscissa and the ratio of the peak area of the type I rhTNK-tPA standard substance to the peak area of the type II rhTNK-tPA standard substance as an ordinate, and establishing a linear regression equation.
2. The method of claim 1, wherein the reducing is performed by adding dithiothreitol to a final concentration of 15-25 mM and reacting at 35-38 ℃ for 30-90 min.
3. The method according to claim 1, wherein the chromatographic column is equilibrated for 15-30 min before sample introduction, equilibrated for another 10-15 min after sample introduction, and then subjected to linear elution.
4. The method of claim 1, wherein the sample injection amount of the reverse phase high performance liquid chromatography is 50-100 μl.
5. The method of claim 1, wherein the column temperature of the chromatographic column is 38-42 ℃.
6. The method of claim 1, wherein the column temperature of the chromatographic column is 40 ℃.
7. The method of claim 1, wherein the chromatographic column has a filler particle size of 5-10 μm and a pore size of 200-350 a.
8. The method of claim 1, wherein the flow rate of the mobile phase of the reverse phase high performance liquid chromatography is 0.5-1.0 mL/min.
9. The method of claim 1, wherein the mobile phase of reverse phase high performance liquid chromatography has a flow rate of 0.8 mL/min.
10. The method of claim 1, wherein the reverse phase high performance liquid chromatography has an ultraviolet detection wavelength of 214 nm or 280 nm.
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| CN108333267A (en) * | 2018-01-23 | 2018-07-27 | 广州铭康生物工程有限公司 | The method for measuring arginine content in the multiple pleasure of inscription using high performance liquid chromatography |
| CN109459514A (en) * | 2018-12-18 | 2019-03-12 | 湖北吉和昌化工科技有限公司 | A kind of HPLC analytical method of dibenzenesulfonimide |
| CN110658286A (en) * | 2019-11-19 | 2020-01-07 | 浙江树人学院（浙江树人大学） | Method for simultaneously and rapidly detecting contents of phenyllactic acid and 4-hydroxy phenyllactic acid by RP-HPLC (reverse phase-high performance liquid chromatography) |
| CN113337490A (en) * | 2021-06-18 | 2021-09-03 | 广州铭康生物工程有限公司 | Method for large-scale rapid separation and purification of rhTNK-tPAI/II type |
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| CN108333267A (en) * | 2018-01-23 | 2018-07-27 | 广州铭康生物工程有限公司 | The method for measuring arginine content in the multiple pleasure of inscription using high performance liquid chromatography |
| CN109459514A (en) * | 2018-12-18 | 2019-03-12 | 湖北吉和昌化工科技有限公司 | A kind of HPLC analytical method of dibenzenesulfonimide |
| CN110658286A (en) * | 2019-11-19 | 2020-01-07 | 浙江树人学院（浙江树人大学） | Method for simultaneously and rapidly detecting contents of phenyllactic acid and 4-hydroxy phenyllactic acid by RP-HPLC (reverse phase-high performance liquid chromatography) |
| CN113337490A (en) * | 2021-06-18 | 2021-09-03 | 广州铭康生物工程有限公司 | Method for large-scale rapid separation and purification of rhTNK-tPAI/II type |
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