CN108226263B - Capillary isoelectric focusing detection method of urate oxidase - Google Patents
Capillary isoelectric focusing detection method of urate oxidase Download PDFInfo
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- CN108226263B CN108226263B CN201611151762.7A CN201611151762A CN108226263B CN 108226263 B CN108226263 B CN 108226263B CN 201611151762 A CN201611151762 A CN 201611151762A CN 108226263 B CN108226263 B CN 108226263B
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Abstract
The invention relates to a capillary isoelectric focusing method for detecting urate oxidase, which specifically comprises the steps of mixing an amphoteric carrier, capillary isoelectric focusing gel, urea, an anode stabilizer, a cathode stabilizer, an isoelectric point marker and a urate oxidase sample according to a certain concentration proportion, loading the mixture into a coated capillary, and detecting by a capillary electrophoresis apparatus at a proper temperature, a proper voltage and a proper focusing time to realize effective separation of components in a protein sample to be analyzed. The method has good repeatability, and can be used for quality control of uricoxidase in production process.
Description
Technical Field
The invention relates to a capillary isoelectric focusing detection method for determining urate oxidase charge heterogeneity, which can perform good quality control analysis on urate oxidase drugs.
Background
Uric acid is an end product of purine metabolism in birds, reptiles, and primates, including humans, because of the lack of urate oxidase in these animals, which catalyzes the further breakdown of uric acid to allantoin, carbon dioxide, and hydrogen peroxide using molecular oxygen as a receptor. Uric acid is excreted by the kidney as an end product of purine metabolism in the human body, and when uric acid production exceeds the metabolic capability of the kidney or when the kidney is in a pathological state, plasma uric acid is remarkably increased, and hyperuricemia is formed. Hyperuricemia can cause or exacerbate a variety of diseases due to the low solubility and easy deposition of urea and its salts in the blood, such as: acute inflammation and pain caused by deposition of uric acid crystals in peripheral joints and synovium during hyperuricemia are the main causes of gout; uric acid can not only stimulate the proliferation of vascular smooth muscle cells, but also cause endothelial cell dysfunction; plasma hyperuricemia is also an important risk factor for cardiovascular diseases such as atherosclerosis; uric acid is deposited in kidney tissues and is the main cause of acute renal failure, renal tubular injury and IgA nephritis, so hyperuricemia brings many risks to self health.
The classic scheme for reducing uric acid is to reduce the production of uric acid by using xanthine oxidase inhibitors such as allopurinol and the like, or to promote the excretion of uric acid by using probenecid, benzbromarone and the like. The water solubility of xanthine is lower than that of uric acid, the xanthine has potential danger when accumulated, the xanthine oxidase inhibitor has poor curative effect when the initial uric acid concentration is too high, and can also cause hypersensitivity syndrome which is manifested by fever, toxic epithelial cell necrosis and lysis, hepatitis and eosinophilic cell increase, the death rate reaches 20 percent, and in addition, the xanthine and uric acid excretion promoting medicines such as probenecid, benzbromarone and the like have obvious hepatotoxicity and kidney toxicity. Therefore, the classical uric acid lowering treatment strategies are not safe enough and the curative effect is not ideal enough. The excellent water solubility of allantoin and the efficient excretion capacity of the kidney to allantoin make urate oxidase an ideal drug for treating hyperuricemia and secondary diseases thereof: clinical research shows that the urate oxidase can quickly and efficiently reduce serum uric acid, and has almost no toxic or side effect; urate oxidase is safer and more effective than allopurinol in treating tumor lysis syndrome; can be used for treating gout, and can rapidly decompose uric acid deposited on joint to eliminate inflammation and skin injury.
The urate oxidase is used as a novel medicine for reducing uric acid, wherein a recombinant aspergillus flavus urate oxidase (trade name: labyrinase) is approved by Europe and America to be used for clinically preventing and treating tumor lysis syndrome, so the urate oxidase has good drug-forming property. Of course, as protein biological products, good quality control method is one of the important factors for the drug formation. The present invention is to provide an analytical method with good and stable quality control of urate oxidase.
The urate oxidase adopted by the invention is an escherichia coli engineering strain capable of efficiently expressing urate oxidase, which is obtained by extracting genome DNA from candida utilis, amplifying by PCR, designing a specific primer, separating urate oxidase genes from candida utilis genomes, inserting the urate oxidase genes into escherichia coli expression plasmids to obtain recombinant candida utilis urate oxidase transformants, and transforming the recombinant candida utilis urate oxidase transformants into escherichia coli. And then the thalli expressing the recombinant candida utilis urate oxidase is obtained through fermentation culture, and the purified recombinant candida utilis urate oxidase is obtained through thalli cracking and column chromatography purification (see patent ZL02819387.3 for details). Different types of urate oxidases have the same action mode and similar physicochemical properties for the action of uric acid, so that the detection method of the invention is also applicable to other urate oxidases.
Disclosure of Invention
The invention aims to provide a novel method for analyzing urate oxidase charge heterogeneity based on capillary electrophoresis technology, which is suitable for quality control analysis of urate oxidase drugs.
The capillary isoelectric focusing detection method comprises the following steps:
(1) mixing an amphoteric carrier, capillary isoelectric focusing gel, urea, an anode stabilizer, a cathode stabilizer, an isoelectric point marker and a urate oxidase sample to be analyzed to form a mixed solution;
(2) loading the mixed solution obtained in the step (1) into a coating capillary, and detecting the mixed solution by a capillary electrophoresis apparatus at a proper temperature, voltage and focusing time to obtain an isoelectric focusing spectrogram;
(3) obtaining a standard curve according to the migration time and the pI value of the isoelectric point marker in the isoelectric focusing map in the step (2), and determining the pI value of each component of the protein sample to be analyzed;
(4) and (3) determining the content of each component of the protein sample to be analyzed according to the isoelectric focusing map information in the step (2).
In the step (1), the amphoteric carrier comprises: one or more of Pharmalyte3-10, Pharmalyte5-8, Pharmalyte6.7-7.7, Pharmalyte8-10.5, Servalyt4-9, Servalyt5-7, Servalyt6-9 and Servalyt3-10, wherein the volume range of the mixed solution in the ampholytic vector is 1% -5%;
the concentration of the urea in the mixed solution is 0-4.86 mol/L;
the anode stabilizer is an iminodiacetic acid solution, and the concentration of the iminodiacetic acid solution in the mixed solution is 2.97mmol/L-5.11 mmol/L;
the cathode stabilizer is a levo-arginine solution, and the concentration of the cathode stabilizer in the mixed solution is 74.35mmol/L-127.80 mmol/L;
isoelectric markers including small molecule isoelectric markers available from Beckman corporation; the small molecule isoelectric point markers provided by Beckman include: pI4.1, pI5.5, pI9.5 and pI 10.0;
the concentration of the protein sample to be analyzed in the mixed solution ranges from 0.1mg/mL to 1 mg/mL.
In the step (2), the coated capillary used comprises: FC coated capillaries from Agilent, neutral coated capillaries from Beckman or N-CHO coated capillaries, or other similar non-covalently bonded or covalently bonded coated capillaries;
in the step (2), the temperature of the capillary column is 10-40 ℃, preferably 10 ℃, 25 ℃ and 30 ℃;
in the step (2), the capillary voltage used is 15-50kv, preferably 15kv, 25kv, 30kv, 40 kv;
in the step (2), the focusing time used is 10 to 30 minutes.
The invention can realize the following contents:
1. by selecting proper experimental conditions, each component in the protein sample to be analyzed is effectively separated, and the corresponding isoelectric point of each component in the map is determined.
2. The relative contents of the components in the map can be determined.
3. The method has good repeatability.
Drawings
The abscissa of the figure used is the time unit Minute, the ordinate is UV and the absorption intensity unit AU.
FIG. 1 shows the uricase CE-SDS electrophoretically pure map.
FIG. 2 shows the capillary isoelectric focusing pattern of urate oxidase under the conditions of example 2.
FIG. 3 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 3 (2. mu.l of 200mmol/L iminodiacetic acid solution therein).
FIG. 4 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 3 (wherein 200mmol/L iminodiacetic acid solution is 10. mu.l).
FIG. 5 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 3 (wherein 200mmol/L iminodiacetic acid solution is 20. mu.l).
FIG. 6 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 3 (wherein 200mmol/L iminodiacetic acid solution is 50. mu.l).
FIG. 7 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 3 (wherein 200mmol/L iminodiacetic acid solution is 100. mu.l).
FIG. 8 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 4 (wherein 500 mmol/L-arginine solution is 10. mu.l).
FIG. 9 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 4 (wherein the concentration of L-arginine is 500mmol/L in 20. mu.l).
FIG. 10 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 4 (wherein the concentration of L-arginine is 500mmol/L in 30. mu.l).
FIG. 11 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 5 (20. mu.l L-arginine solution 500mmol/L, 2. mu.l iminodiacetic acid solution 200 mmol/L).
FIG. 12 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 5 (40. mu.l of 500 mmol/L-arginine solution, 4. mu.l of 200mmol/L iminodiacetic acid solution).
FIG. 13 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 5 (wherein 500 mmol/L-arginine solution is 60. mu.l, 200mmol/L iminodiacetic acid solution is 6. mu.l).
FIG. 14 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 5 (wherein 500 mmol/L-arginine solution is 80. mu.l, and 200mmol/L iminodiacetic acid solution is 8. mu.l).
FIG. 15 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 5 (wherein 500 mmol/L-arginine solution is 100. mu.l, 200mmol/L iminodiacetic acid solution is 10. mu.l).
FIG. 16 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 6 (in which 30kV is focused for 15 minutes).
FIG. 17 shows capillary isoelectric focusing profiles of urate oxidase under the conditions of example 6 (with 30kV focusing for 17.5 min).
FIG. 18 shows capillary isoelectric focusing profiles of urate oxidase under the conditions of example 6 (in which 30kV is focused for 20 minutes).
FIG. 19 shows capillary isoelectric focusing profiles of urate oxidase under the conditions of example 6 (with 30kV focusing for 22.5 min).
FIG. 20 shows capillary isoelectric focusing profiles of urate oxidase under the conditions of example 6 (with 30kV focusing for 25 min).
FIG. 21 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 7 (wherein 0mol/L urea-cIEF gel solution is 200. mu.l).
FIG. 22 shows the capillary isoelectric focusing pattern of urate oxidase under the conditions of example 7 (wherein 1mol/L urea-cIEF gel solution is 200. mu.l).
FIG. 23 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 7 (wherein 2mol/L urea-cIEF gel solution is 200. mu.l).
FIG. 24 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 7 (wherein 3mol/L urea-cIEF gel solution is 200. mu.l).
FIG. 25 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 7 (where 4mol/L urea-cIEF gel solution is 200. mu.l).
FIG. 26 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 7 (wherein 6mol/L urea-cIEF gel solution is 200. mu.l).
FIG. 27 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 8 (wherein 3mol/L urea-cIEF gel solution is 200. mu.l).
FIG. 28 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 8 (wherein 6mol/L urea-cIEF gel solution is 200. mu.l).
FIG. 29 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 9 (20. mu.l of 500 mmol/L-arginine solution, 2. mu.l of 200mmol/L iminodiacetic acid solution).
FIG. 30 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 9 (40. mu.l of 500 mmol/L-arginine solution, 4. mu.l of 200mmol/L iminodiacetic acid solution).
FIG. 31 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 9 (wherein 500 mmol/L-arginine solution is 60. mu.l, 200mmol/L iminodiacetic acid solution is 6. mu.l).
FIG. 32 shows capillary isoelectric focusing spectra of urate oxidase under the conditions of example 9 (wherein 500 mmol/L-arginine solution is 80. mu.l, and 200mmol/L iminodiacetic acid solution is 8. mu.l).
FIG. 33 shows the capillary isoelectric focusing pattern of urate oxidase under the conditions of example 10.
FIG. 34 shows a superposition spectrum of capillary isoelectric focusing of three consecutive batches of urate oxidase.
Detailed Description
The instruments and reagents used in the following examples were all conventionally purchased commercially, except as otherwise indicated.
Example 1
Preparation of urate oxidase stock solution
According to the invention patent ZL02819387.3, a urate oxidase engineering strain is prepared.
Recovering the uricase engineering bacteria, and culturing the bacteria on an inclined plane at 37 ℃ for 17-24 hours. Then inoculating the strain into a shake flask, and performing shake culture at 37 ℃ and 200-300 rpm. OD of bacterial liquid600When the concentration is 2.0-5.0, inoculating to a fermentation tank for culture. Culturing in a fermentation tank at 450-700 rpm and pH7.0, and keeping dissolved oxygen not less than 50%, OD600IPTG is added for induction expression for 2 hours for 7.5-9.5. And (3) centrifugally collecting thalli, re-suspending the thalli by using a lysate, then treating the suspension by using a homogenizer, centrifugally treating the suspension, and collecting a supernatant. Obtaining pure urate oxidase stock solution by hydrophobic chromatography and ion exchange.
Secondly, detecting the purity of the stock solution
The urate oxidase was subjected to CE-SDS analysis, and the purity of the sample was determined, and the details of the detection results are shown in FIG. 1.
Example 2
First, experimental conditions
Capillary isoelectric focusing sample loading mixtures were prepared according to table 1, placed in an autosampler of a Beckman PA800plus capillary electrophoresis apparatus, set at a column temperature of 30 ℃, analyzed using N-CHO coated capillary (Beckman), focused at 15kV for 15 minutes, analyzed at 25kV for 30 minutes, and detected under a UV detector at a wavelength of 280 nm.
TABLE 1 capillary isoelectric focusing sample loading liquid ratio
| Name of reagent | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solution | 200 | 2.43mol/L |
| 3-10 ampholyte carrier (Pharmalyte3-10) | 12 | |
| 500 mmol/L-arginine cathode |
20 | 40.49mmol/L |
| 200mmol/L Iminodiacetic acid anode |
2 | 1.62mmol/L |
| pI10.0 protein marker | 1 | - |
| pI5.5 protein marker | 1 | - |
| pI4.1 protein marker | 1 | - |
| 10mg/ |
10 | 0.40mg/mL |
Second, data analysis
As shown in FIG. 2, the capillary isoelectric focusing spectrum shows that many heterogeneous peaks are found by analyzing the urate oxidase stock solution, and the urate oxidase stock solution cannot be subjected to effective quality control analysis.
Example 3
First, experimental conditions
Preparing capillary isoelectric focusing sample loading mixed liquor according to the table 2, respectively adding anode stabilizers with different volumes according to the table 3, uniformly mixing, placing in an automatic sample injector of a Beckman company PA800plus capillary electrophoresis apparatus, setting the column temperature to be 30 ℃, analyzing by adopting an N-CHO coating capillary (Beckman company), focusing for 15 minutes at 15kV, analyzing for 30 minutes at 25kV, and detecting under a UV detector with the wavelength of 280 nm.
TABLE 2 capillary isoelectric focusing sample loading liquid ratio
| Name of reagent | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solution | 200 | 1.74-2.43mol/L |
| 3-10 ampholyte carrier (Pharmalyte3-10) | 12 | - |
| 500 mmol/L-arginine cathode |
20 | 28.99-40.49mmol/L |
| pI10.0 protein marker | 1 | - |
| pI5.5 protein marker | 1 | - |
| pI4.1 protein marker | 1 | - |
| 10mg/ |
10 | 0.29-0.40mg/mL |
TABLE 3 Anode stabilizer ratio
Second, data analysis
Capillary isoelectric focusing patterns are shown in fig. 3 to 7, and the volumes of the anode stabilizers are sequentially increased. As shown, no sample peak was detected when the volume of the anode stabilizer was increased to 10. mu.l. Concentration gradient experiments with 200mmol/L anode stabilizer were found to have no good results.
Example 4
First, experimental conditions
Preparing capillary isoelectric focusing sample loading mixed liquor according to table 4, respectively adding cathode stabilizers with different volumes according to table 5, uniformly mixing, placing in an autosampler of a Beckman company PA800plus capillary electrophoresis apparatus, setting the column temperature to be 30 ℃, analyzing by adopting an N-CHO coating capillary (Beckman company), focusing at 30kV for 15 minutes, analyzing at 30kV for 30 minutes, and detecting under a UV detector with the wavelength of 280 nm.
TABLE 4 capillary isoelectric focusing sample loading liquid ratio
| Name of reagent | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solution | 200 | 2.33-2.53mol/L |
| 3-10 ampholyte carrier (Pharmalyte3-10) | 12 | - |
| 200mmol/L Iminodiacetic acid anode |
2 | 1.56-1.69mmol/L |
| pI10.0 protein marker | 1 | - |
| pI5.5 protein marker | 1 | - |
| pI4.1 protein marker | 1 | - |
| 10mg/ |
10 | 0.39-0.42mg/mL |
TABLE 5 Anode stabilizer ratio
Second, data analysis
Capillary isoelectric focusing spectra are shown in FIGS. 8 to 10, and only 500mmol/L of cathode stabilizer is subjected to concentration gradient investigation, which does not produce good effect.
Example 5
First, experimental conditions
Preparing a capillary isoelectric focusing sample loading mixed solution according to table 6, adding anode stabilizers and cathode stabilizers with different volumes according to table 7 respectively, mixing uniformly, placing in an autosampler of a Beckman PA800plus capillary electrophoresis apparatus, setting the column temperature to 10 ℃, analyzing by adopting an N-CHO coated capillary (Beckman), focusing at 30kV for 15 minutes, analyzing at 30kV for 30 minutes, and detecting at 280 nm.
TABLE 6 capillary isoelectric focusing sample loading liquid ratio
| Name of reagent | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solution | 200 | 1.79-2.43mol/L |
| 3-10 ampholyte carrier (Pharmalyte3-10) | 12 | - |
| pI10.0 protein marker | 1 | - |
| pI5.5 protein marker | 1 | - |
| pI4.1 protein marker | 1 | - |
| 10mg/ |
10 | 0.30-0.40mg/mL |
TABLE 7 cathode/anode stabilizer ratio
Second, data analysis
Capillary isoelectric focusing patterns are shown in fig. 11-15, which simultaneously multiply increase the volume of the cathode and anode stabilizers. As shown, the sample heterogeneous peak decreased with increasing stabilizer concentration, and no sample peak was detected at a stabilizer volume of 5N. The volume of the stabilizer is 2-4N (namely the volume of the anolyte is 2.97-5.11 mmol/L, and the volume of the catholyte is 74.35-127.80 mmol/L), so that a better experimental effect can be achieved, and the final concentration range of 3mol/L urea in the system corresponding to the concentration range is 1.92-2.23 mol/L. The concentrations of the stabilizers, 1N and 5N, were not suitable for the detection of urate oxidase samples.
We also experimentally confirmed that stabilizer concentrations below 1N would detect more signal peaks, making urate oxidase difficult to quality control. Concentrations of the stabilizer above 5N resulted in no detectable signal from the sample.
We also generated similar experimental results to this example using different concentration configurations of the same final concentration of cathode and anode stabilizers. It is important to note that the choice of the final concentration of the cathode/anode stabilizer formulation is the key to the practice of the present invention.
Example 6
First, experimental conditions
Capillary isoelectric focusing sample loading mixtures were prepared according to table 8, placed in an autosampler of a Beckman PA800plus capillary electrophoresis apparatus, set at a column temperature of 25 ℃, and analyzed using N-CHO coated capillary (Beckman) at 30kV focused for 15 minutes, 17.5 minutes, 20 minutes, 22.5 minutes, and 25 minutes, respectively, and 30kV analyzed for 30 minutes, and detected at 280 nm.
TABLE 8 capillary isoelectric focusing sample loading liquid ratio
Second, data analysis
The capillary isoelectric focusing spectra are shown in the graphs from 16 to 20, the focusing time is sequentially prolonged, the urate oxidase peak shape is not obviously changed, and the quality control analysis of the urate oxidase sample is not influenced by the change of the focusing time. But our subsequent experiments confirmed that 30kV focusing times below 10 minutes will affect the experimental effect. Therefore, it is recommended that the focusing time used be 10-30 minutes.
Related experiments are carried out on the selection of the capillary tube voltage, and the focusing time can be adjusted sequentially by adopting different voltages of 15-50kV, so that the result similar to that of the embodiment can be achieved. Voltages below 15kV will result in too long focusing times and thus affect the efficiency of the operation. A capillary tube with a voltage higher than 50kV will be easily damaged, increasing the cost of the experiment.
Example 7
First, experimental conditions
Preparing a capillary isoelectric focusing sample loading mixed solution according to the table 9, respectively adding urea solutions with different concentrations according to the table 10, uniformly mixing, placing in an automatic sample injector of a Beckman company PA800plus capillary electrophoresis apparatus, setting the column temperature to be 30 ℃, analyzing by adopting an N-CHO coated capillary (Beckman company), focusing at 30kV for 10 minutes, analyzing at 30kV for 30 minutes, and detecting at 280 nm.
TABLE 9 capillary isoelectric focusing sample loading liquid ratio
TABLE 10 Urea-cIEF gel solutions
| Name of reagent | Reagent volume (μ l) | Final concentration |
| 0mol/L urea-cIEF gel solution | 200 | 0mol/L |
| 1mol/L urea-cIEF gel solution | 200 | 0.81mol/L |
| 2mol/L urea-cIEF gel solution | 200 | 1.62mol/L |
| 3mol/L urea-cIEF gel solution | 200 | 2.43mol/L |
| 4mol/L urea-cIEF gel solution | 200 | 3.24mol/L |
| 6mol/L urea-cIEF gel solution | 200 | 4.86mol/L |
Second, data analysis
The capillary isoelectric focusing patterns are shown in FIGS. 21-26, where the uricase heterogeneous peak decreases with increasing urea concentration. The foregoing examples have demonstrated that appropriate increases in cathode and anode stabilizer concentrations are effective in reducing uricase heterogeneity, but the stabilizers are expensive. This example demonstrates that increasing the concentration of urea is also effective in reducing urease heterogeneity. The absorption peak of the sample can be increased without adding urea, and the difficulty of liquid preparation is increased by using urea with the concentration of more than 6 mol/L.
Example 8
First, experimental conditions
Capillary isoelectric focusing sample loading mixed solution is prepared according to the table 11, urea solutions with different concentrations are respectively added according to the table 12, the mixed solution is placed in an automatic sample injector of a Beckman company PA800plus capillary electrophoresis apparatus after being mixed uniformly, the column temperature is set to be 25 ℃, neutral coating capillary (Beckman company) is adopted for analysis, 40kV focusing is carried out for 10 minutes, 40kV analysis is carried out for 30 minutes, and detection is carried out under 280 nm.
TABLE 11 capillary isoelectric focusing sample loading liquid ratio
TABLE 12 Urea-cIEF gel solutions
| Name of reagent | Reagent volume (μ l) | Final concentration |
| 3mol/L urea-cIEF gel solution | 200 | 2.43mol/L |
| 6mol/L urea-cIEF gel solution | 200 | 4.86mol/L |
Second, data analysis
Capillary isoelectric focusing profiles as shown in figures 27 to 28, the heterogeneous peaks of uricase decreased significantly with increasing urea concentration.
Example 9
First, experimental conditions
Preparing a capillary isoelectric focusing sample loading mixed solution according to table 13, adding anode stabilizers and cathode stabilizers with different volumes respectively according to table 14, mixing uniformly, placing in an autosampler of a Beckman PA800plus capillary electrophoresis apparatus, setting the column temperature to 25 ℃, analyzing by using a neutral coating capillary (Beckman), focusing at 25kV for 10 minutes, analyzing at 25kV for 30 minutes, and detecting at 280 nm.
TABLE 13 capillary isoelectric focusing sample loading liquid ratio
| Name of reagent | Reagent volume (μ l) | Final concentration |
| 6mol/L urea-cIEF gel solution | 200 | 3.83-4.86mol/L |
| 3-10 ampholyte carrier (Pharmalyte3-10) | 12 | - |
| pI10.0 protein marker | 1 | - |
| pI5.5 protein marker | 1 | - |
| pI4.1 protein marker | 1 | - |
| 10mg/ |
10 | 0.32-0.40mg/mL |
TABLE 14 cathode/anode stabilizer ratio
Second, data analysis
Capillary isoelectric focusing spectra are shown in FIGS. 29 to 32, using a similar protocol to that of example 5. The concentration of the urea is increased to 6mol/L, the final concentration of the stabilizer is in a lower concentration range of 2N-4N, the corresponding anode stabilizer is 2.97-5.11 mmol/L, the final concentration of the cathode stabilizer is 74.3-127.80 mmol/L, still better experimental results can be obtained, and the final concentration range of the 6mol/L urea corresponding to the concentration range in the system is 3.83-4.46 mol/L.
The isoelectric point marker selects pI9.5, and the similar effect to pI10 can be achieved. Isoelectric point markers from different manufacturers can achieve the same effect.
Example 10
First, experimental conditions
Capillary isoelectric focusing sample loading mixtures were prepared according to table 15, placed in an autosampler of a Beckman PA800plus capillary electrophoresis apparatus, set at a column temperature of 30 ℃, analyzed using N-CHO coated capillary (Beckman), focused at 15kV for 15 minutes, analyzed at 20kV for 30 minutes, and detected at 280 nm.
TABLE 15 capillary isoelectric focusing sample loading liquid ratio
Second, data analysis
Capillary isoelectric focusing spectra As shown in FIG. 33, urate oxidase achieved complete baseline separation, yielding a total of 4 component peaks. The isoelectric points and percentage contents of all components of the urate oxidase can be detected by the optimized method (see table 16), and further, effective quality control analysis is carried out on urate oxidase medicaments.
TABLE 16 isoelectric points and percentages of the various components of urate oxidase
| Target peak | Migration time | Peak area | Percentage of | |
| 1 | 39.017 | 435618 | 33.67 | 7.26 |
| 2 | 39.208 | 470038 | 36.33 | 7.14 |
| 3 | 39.425 | 251615 | 19.45 | 7.00 |
| 4 | 39.733 | 136366 | 10.54 | 6.80 |
Under the same conditions, looking again at capillary manufacturers, the coated capillaries produced by Agilent and Beckman produced similar experimental results and could be used. Theoretically, as long as the coating capillary capable of effectively inhibiting electroosmotic flow is suitable for the experiment, electroosmotic flow can generate larger interference to the experiment.
We also investigated the manufacturers and ranges of the amphoteric carriers, the amphoteric carriers in the same range of GE and SERVA companies can produce similar experimental results, the mixing of the amphoteric carriers in the range of 3-10 with carriers in other ranges can change the retention time or peak capacity of the peak, and the related experimental results can also effectively perform quality control analysis on urate oxidase drugs.
The urate oxidase can be accepted after mixing at 0.1-1 mg/ml, the corresponding value of the urate oxidase lower than 0.1mg/ml is lower, the concentration multiple of the urate oxidase higher than 1mg/ml is increased, and the sample is viscous, so that accurate transfer is difficult.
Example 11
First, experimental conditions
Capillary isoelectric focusing sample loading mixtures were prepared according to table 15, placed in an autosampler of a Beckman PA800plus capillary electrophoresis apparatus, set at a column temperature of 30 ℃, analyzed using N-CHO coated capillary (Beckman), focused at 15kV for 15 minutes, analyzed at 20kV for 30 minutes, and detected at 280 nm.
Second, data analysis
Capillary isoelectric focusing spectra are shown in FIG. 34, and three batches of urate oxidases were analyzed continuously, and complete baseline separation was achieved, yielding 4 component peaks in total. The optimized method is stable, and the isoelectric points and percentage contents of all components of the urate oxidase can be detected by the method, so that the method can be used for quality detection and control in product production.
TABLE 17 isoelectric points and percentages of the three urate oxidase batches
Claims (6)
1. A capillary isoelectric focusing detection method of urate oxidase, comprising the following steps:
(1) mixing an amphoteric carrier, capillary isoelectric focusing gel, urea, an iminodiacetic acid solution, an L-arginine solution, an isoelectric point marker and a urate oxidase sample to be analyzed to form a mixed solution, wherein the final concentration of the urea in the mixed solution is 1.92-2.23 mol/L or 3.83-4.46 mol/L, the final concentration of the iminodiacetic acid solution in the mixed solution is 2.97-5.11 mmol/L, the final concentration of the L-arginine solution in the mixed solution is 74.35-127.80 mmol/L,
(2) loading the mixed solution obtained in the step (1) into a coating capillary, detecting by a capillary electrophoresis apparatus at proper temperature, voltage and focusing time to obtain an isoelectric focusing spectrogram,
(3) obtaining a standard curve according to the migration time and the pI value of the isoelectric point marker in the isoelectric focusing map in the step (2), determining the pI value of each component of the urate oxidase sample,
(4) and (3) determining the content of each component of the protein sample to be analyzed according to the isoelectric focusing map information in the step (2).
2. The method of claim 1, wherein the range of the ampholytic vector in step (1) is 3-10, the range of the ampholytic vector in the mixture is 1% -5% by volume, and the ampholytic vector is selected from one or more of Pharmalyte3-10, Pharmalyte5-8, Pharmalyte6.7-7.7, Servalyt4-9, Servalyt5-7, Servalyt6-9, and Servalyt 3-10.
3. The method of claim 1, wherein the isoelectric marker of step (1) ranges from one or more of pI4.1, pI5.5, pI9.5, and pI 10.0.
4. The method of claim 1, wherein the urate oxidase sample of step (1) is present in the mixture at a final concentration in the range of 0.1mg/mL to 1 mg/mL.
5. The method of claim 1, wherein the capillary column temperature of step (2) is 10-30 ℃, the capillary voltage used is 15-40kv, and the focusing time is 10-30 minutes.
6. A capillary isoelectric focusing detection method of urate oxidase, comprising the following steps:
(1) mixing an ampholyte carrier, a urea-cIEF gel solution, a levorotatory arginine cathode stable solution, an iminodiacetic acid anode stable solution, pI4.1, pI5.5, pI10.0 protein markers and a urate oxidase sample to form a mixed solution, wherein the final concentration of the urea-cIEF gel solution in the mixed solution is 4.12mol/L, the final concentration of the levorotatory arginine cathode stable solution in the mixed solution is 103.09mmol/L, the final concentration of the iminodiacetic acid anode stable solution in the mixed solution is 4.12mmol/L, and the final concentration of the urate oxidase sample in the mixed solution is 0.34mg/mL,
(2) loading the mixed solution obtained in the step (1) into a coating capillary, setting the column temperature at 30 ℃, focusing at 15kV for 15 minutes, analyzing at 20kV for 30 minutes, detecting at 280nm to obtain an isoelectric focusing spectrogram,
(3) obtaining a standard curve according to the migration time and the pI value of the isoelectric point marker in the isoelectric focusing map in the step (2), determining the pI value of each component of the urate oxidase sample,
(4) and (3) determining the content of each component of the protein sample to be analyzed according to the isoelectric focusing map information in the step (2).
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