Method for analyzing purity of protein polymer

Abstract

The invention belongs to the field of biological medicines, and discloses a protein polymer purity analysis method, which comprises: S1, pretreatment of a protein polymer; s2, measuring a sample with a preset sample size, feeding the sample into a capillary electrophoresis apparatus for capillary electrophoresis detection, and setting adaptive detection conditions to obtain a chromatogram; and S3, analyzing the signal peaks in the chromatogram, screening the signal peaks above a predetermined scale as main peaks, taking the main peak with the largest area as a protein peak, and analyzing to obtain the purity information of the protein polymer. According to the purity analysis method of some examples, a chromatogram with good consistency is obtained through pretreatment of the protein polymer and adaptive setting of detection conditions of the capillary electrophoresis apparatus, a detection result with higher credibility is achieved, establishment of quality control standards of the protein polymer is facilitated, and the method is suitable for popularization and application. The systematic qualitative and quantitative analysis of the protein polymer is assisted.

Description

Technical Field

[0001] The invention belongs to the field of biological medicines, and in particular relates to a purity analysis method for protein polymers. Background Art

[0002] Mesenchymal stem cells (MSCs) have the potential for self-replication and multidirectional differentiation. They are widely present in tissues such as bone marrow, fat, synovium, dental pulp, amniotic fluid, placenta, umbilical cord, embryo, umbilical cord blood, amnion, peripheral blood, muscle, and urine. They have the characteristics of wide source, no need for matching, low infection rate, strong differentiation potential, strong proliferation ability, and easy collection. They can produce active factors such as stem cell growth factor (SCF), nerve growth factor (NGF), interleukin-6 (IL-6), interleukin-7 (IL-7), tumor necrosis factor (TNF), interferon (IFN), etc., and participate in regulating cell growth, cell apoptosis, cell differentiation, antiviral, immune maturation and other processes. They can be used for immune regulation, tissue repair and treatment of diseases such as acute lung injury, severe pneumonia, and acute respiratory distress syndrome.

[0003] The inventors have discovered that by stimulating MSCs and then lysing them, a protein polymer with nerve repair activity can be separated and purified from intracellular proteins. This protein polymer has achieved significant results in completed animal and clinical trials, especially for ALS, and has a clear potential for drug development, and has now entered the clinical trial stage.

[0004] At present, protein polymers are obtained by stimulating MSCs expression under specific stress conditions and separating and purifying the extracted bioactive substances. During the experiment, the means of intervention and control of selective gene expression of cells are relatively limited, resulting in relatively difficult quality control of the obtained protein polymers. Therefore, developing a purity analysis method for protein polymers is of great significance for analyzing the purity of protein polymers and stabilizing the quality of protein polymers. Summary of the invention

[0005] The object of the present invention is to overcome at least one disadvantage of the prior art and provide a method for analyzing the purity of protein polymers.

[0006] The technical solution adopted by the present invention is:

[0007] A method for analyzing the purity of a protein polymer, comprising:

[0008] S1: Pre-treat the protein polymer to prepare a sample with a predetermined protein concentration;

[0009] S2: taking a predetermined injection amount of the sample, sending it into a capillary electrophoresis instrument for capillary electrophoresis detection, setting the injection voltage to 3-9 KV, the separation voltage to 10-20 KV, the injection volume to 25-100 μl, the capillary temperature to 10-40° C., the mobile phase to: CE-SDS Gel buffer, the elution pressure to 10-40 psi, and the peak width to 1-8 seconds to obtain a chromatogram; and

[0010] S3: Analyze the signal peaks in the chromatogram, screen the signal peaks above a predetermined scale as main peaks, wherein the main peak with the largest area is the protein peak, and analyze to obtain the purity information of the protein polymer.

[0011] By pre-treating the protein polymer, a sample adapted to the capillary electrophoresis instrument is obtained, and the corresponding detection conditions are set. The sample is driven by high voltage in the capillary, and the molecules of different molecular weights are separated from each other due to different electrophoretic speeds, and corresponding signal peaks are generated at different time points to form a chromatogram. The signal peaks above the predetermined scale in the chromatogram are screened as the main peaks, and the main peak with the largest area is defined as the protein peak, and the area ratio of each main peak is calculated to obtain the purity information of the protein polymer. The fewer the number of main peaks screened and the higher the area ratio of the protein peak, the higher the purity of the protein polymer. The area ratio of the protein peak reflects the purity of the protein in the protein polymer. After repeated experiments, the detection results obtained by this purity analysis method have good consistency, and the confidence of the purity analysis results is high. It can be used to establish the quality control standard of the protein polymer, and can also assist in the systematic qualitative and quantitative analysis of the protein polymer. It can be understood that the internal standard in the sample will form an internal standard peak in the chromatogram, and the above-mentioned signal peak does not contain the internal standard peak.

[0012] In some embodiments, the capillary electrophoresis instrument is a high-throughput capillary electrophoresis instrument of model Biophase 8800 produced by AB SCIEX, the sample buffer is Low pH SDS-Sample Buffer of product number C57805 produced by AB SCIEX, the capillary length in the capillary electrophoresis instrument is 30 cm, and an internal standard of a predetermined molecular weight is added to the sample. Using the above instrument reagents and under the preferred detection conditions, a signal peak with a better peak shape is obtained.

[0013] In some embodiments, the pre-treatment of the protein polymer also includes non-reducing treatment or reducing treatment of the protein polymer, wherein the non-reducing treatment is to add a predetermined amount of iodoacetamide to a predetermined amount of the protein polymer; and the reducing treatment is to add a predetermined amount of 2-mercaptoethanol to a predetermined amount of the protein polymer.

[0014] In some embodiments, the separation conditions of the sample obtained by the non-reduction treatment in the capillary electrophoresis instrument are: duration of 10-60 minutes, voltage rise time of 1-5 minutes, and automatic zeroing time is set to 0.1-0.9 minutes.

[0015] In some embodiments, the separation conditions of the sample obtained by the reduction treatment in the capillary electrophoresis instrument are: duration of 10-60 minutes, voltage rise time of 0.5-3 minutes, and automatic zeroing time is set to 2-8 minutes.

[0016] In some embodiments, the protein concentration in the sample ranges from 0.1 mg / ml to 5 mg / ml.

[0017] In some embodiments, the protein polymer is further subjected to ultrafiltration and liquid exchange treatment before pre-treatment, and the ultrafiltration and liquid exchange treatment includes:

[0018] S01: measuring a predetermined volume of the sample and adding it into an ultrafiltration tube;

[0019] S02: sending the ultrafiltration tube into a centrifuge and centrifuging for 10 minutes;

[0020] S03: Add CE Water, mix well, and continue centrifuging in the centrifuge for 10 minutes;

[0021] S04: Repeat the above step S03 three times, take the supernatant after the last centrifugation, and concentrate the supernatant to a predetermined protein concentration.

[0022] In some embodiments, the sample buffer is a low pH sample buffer having a pH value less than 7.

[0023] In some embodiments, the sample needs to be subjected to denaturation culture before being sent to the capillary electrophoresis instrument, and the denaturation culture includes:

[0024] S11: transferring the sample to a centrifuge and centrifuging for 1 minute;

[0025] S12: placing the centrifuged sample in a water bath at 30-100° C. for 5-15 minutes;

[0026] S13: placing the sample after the water bath at room temperature away from light for 3-10 minutes;

[0027] S14: The sample is transferred to the centrifuge again and centrifuged for 1 minute. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the chromatogram obtained after the protein polymer of the present application is treated by a non-reducing reaction.

[0029] Figure 2 It is the chromatogram obtained after the protein polymer of the present application is treated by reduction reaction.

[0030] Figure 3 This is the detection result of the protein polymer of the present application using the SDS-PAGE method.

[0031] Figure 4 It is the test result of concentration screening of the protein polymer purity analysis method of the present application.

[0032] Figure 5 It is the test result of matrix screening of the protein polymer purity analysis method of the present application.

[0033] Figure 6 It is the test result of the sample buffer pH screening of the protein polymer purity analysis method of the present application.

[0034] Figure 7 It is the test result of screening the denaturation reaction conditions of the protein polymer purity analysis method of the present application.

[0035] Figure 8 It is the test result of capillary temperature screening by the protein polymer purity analysis method of the present application.

[0036] Fig. 9 This is the test result of repeatability experiment of the protein polymer purity analysis method of this application.

[0037] Figure 10-15 It is a statistical chart of linear regression analysis of the test results obtained by the protein polymer purity analysis method of the present application.

[0038] Fig.16 It is the test result of the protein polymer purity analysis method of the present application using molecular weight standards for data calibration.

[0039] Fig.17 Yes Fig.16 Statistical graph of linear regression of the detection results. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further illustrated below in conjunction with experiments.

[0041] The production process of the protein polymer samples used in the following experiments includes:

[0042] Cultivating mesenchymal stem cells and using ultraviolet irradiation to create a stress environment;

[0043] The mesenchymal stem cells are lysed and separated and purified using size exclusion chromatography to obtain protein polymers; wherein, under the size exclusion chromatography condition of a flow rate of 0.2 ml / min, the elution volume of the first component peak is 12-13.2 ml, the elution volume of the second component peak is 15.2-17 ml, the elution volume of the third component peak is 17-20 ml, the elution volume of the fourth component peak is 29-31 ml, and the elution volume of the fifth component peak is 31-34 ml.

[0044] Purity analysis of protein polymer samples by capillary electrophoresis

[0045] A method for analyzing the purity of a protein polymer, comprising:

[0046] S1: Pre-treat the protein polymer to prepare a sample with a predetermined protein concentration;

[0047] S2: taking a predetermined injection amount of the sample, and sending it into a capillary electrophoresis instrument for capillary electrophoresis detection to obtain a chromatogram; and

[0048] S3: Analyze the signal peaks in the chromatogram, screen the signal peaks above a predetermined scale as main peaks, wherein the main peak with the largest area is the protein peak, and analyze to obtain the purity information of the protein polymer.

[0049] Among them, the main instruments and equipment

[0050]

[0051] Main reagents and consumables

[0052]

[0053] In addition, it should be noted that the above-mentioned Biophase CE-SDS Protein Analysis Kit is a product for use with a capillary electrophoresis instrument, and its components include: Basic Wash, Acid Wash, SDS-MW Sample Buffer (pH 6.8), SDS-MW Gel Buffer, 10KD Internal Standard, MW molecular weight standards and Uncoated Capillary.

[0054] The above-mentioned SDS-MW Analysis Kit is a product for use with a capillary electrophoresis instrument, and its components include: Basic Wash, Acid Wash, SDS-MW Sample Buffer (pH 9.5), SDS-MW Gel Buffer, 10KD Internal Standard, MW molecular weight standards and Uncoated Capillary.

[0055] Sample processing and testing conditions:

[0056] The protein concentration of the protein polymer stock solution was measured by a micro-spectrophotometer and was 5 mg / ml;

[0057] Prepare samples with a predetermined protein concentration of 0.25 mg / ml after non-reducing treatment and reducing treatment according to the following table:

[0058]

[0059] The reagent raw materials of the above two groups of treatment systems are mixed respectively and allowed to stand to obtain samples.

[0060] The protein polymer samples treated by the above non-reduction reaction were sent to the machine for testing:

[0061] The detection conditions were set as follows: injection voltage 3-9KV, separation voltage 10-20KV, injection volume 25-100μl, capillary temperature 10-40℃, mobile phase CE-SDS Gel buffer, elution pressure 10-40psi, peak width set to 1-8 seconds; separation conditions were set as follows: duration 10-60 minutes, voltage rise time 1-5 minutes, automatic zeroing time set to 0.1-0.9 minutes;

[0062] The protein polymer samples treated by the above reduction reaction were sent to the machine for testing:

[0063] The detection conditions were set as follows: injection voltage 3-9KV, separation voltage 10-20KV, injection volume 25-100μl, capillary temperature 10-40℃, mobile phase CE-SDS Gel buffer, elution pressure 10-40psi, and peak width set to 1-8 seconds; the separation conditions were set as follows: duration 10-60 minutes, voltage rise time 0.5-3 minutes, and automatic zeroing time set to 2-8 minutes.

[0064] By pre-treating the protein polymer, a sample adapted to the capillary electrophoresis instrument is obtained. The sample is driven by high voltage in the capillary, and molecules of different molecular weights are separated from each other due to different electrophoretic speeds, and corresponding signal peaks are generated at different time points to form a chromatogram. The signal peaks above a predetermined scale in the chromatogram are screened as the main peaks, and the main peak with the largest area is defined as the protein peak. The area ratio of each main peak is calculated to obtain the purity information of the protein polymer. The fewer the number of main peaks screened and the higher the area ratio of the protein peak, the higher the purity of the protein polymer. The value of the area ratio of the protein peak reflects the purity of the protein in the protein polymer. It is understandable that the internal standard in the sample will form an internal standard peak in the chromatogram, and the above-mentioned signal peak does not include the internal standard peak.

[0065] On the other hand, SDS-PAGE was used as a control method to analyze the purity of the above samples and obtain an electrophoresis spectrum (see Figure 3 ).

[0066] See also Figure 1 and Figure 2 , we can know that the protein purity in the sample has reached more than 95%, but Figure 3 The protein purity was only 90.1% as determined by SDS-PAGE. Figure 1 and Figure 2 Judging from the sensitivity of the instrument detector, the protein concentration of the sample is too low, so the test result is biased high, but overall, the protein peak area of ​​the sample treated with non-reduction reaction accounts for 98.78%, and the protein peak area of ​​the sample treated with reduction reaction accounts for 97.44%, reflecting that the purity of the protein polymer is relatively high.

[0067] Screen samples for protein concentration:

[0068] In some embodiments, in order to obtain a protein peak with a better peak shape, the protein concentration of the sample needs to be screened to obtain the optimal injection protein concentration with the best detection result accuracy.

[0069] 1. Sample preparation:

[0070] Take the protein polymer stock solution and prepare the sample according to the following table:

[0071]

[0072] The reagents and raw materials contained in each group of samples are fully mixed and allowed to stand to obtain samples.

[0073] 2. On-machine testing

[0074] Each time, 100 μl of the supernatant of the sample was measured and injected into the injection port of the capillary electrophoresis instrument. The detection conditions were the same as those of the above-mentioned reduction-treated sample to obtain a chromatogram.

[0075] See also Figure 4 The higher the protein concentration of the sample, the worse the separation between the signal peaks, and the worse the accuracy of the sample purity analysis. Therefore, the protein concentration of the sample is preferably 1 mg / ml-3 mg / ml.

[0076] Preparation of samples for matrix screening:

[0077] In some embodiments, the protein polymer stock solution is also subjected to ultrafiltration to exchange the liquid in order to obtain a better peak shape of the protein peak. During the detection process, the capillary electrophoresis instrument responds more sensitively to the protein molecules.

[0078] 1. Ultrafiltration liquid replacement treatment:

[0079] Take the sample protein polymer stock solution, and measure the protein concentration of 24 mg / ml by spectrophotometer. Take 100 μl of the stock solution and add it to the ultrafiltration tube. Place the ultrafiltration tube in a centrifuge and centrifuge for 10 minutes. Then add 100 μl CE Water, mix well, and centrifuge for 10 minutes. Repeat the addition of CE Water and centrifugation for 3 times. Finally, concentrate the sample mixture to a protein concentration of 23.853 mg / ml, and the ultrafiltration solution change is completed.

[0080] 2. Sample preparation:

[0081] Prepare the samples according to the following table:

[0082]

[0083] The reagents and raw materials contained in each group of samples are fully mixed and allowed to stand to obtain samples.

[0084] 3. On-machine testing

[0085] Each time, 100 μl of the supernatant of the sample was measured and injected into the injection port of the capillary electrophoresis instrument. The detection conditions were the same as those of the above-mentioned reduction-treated sample to obtain a chromatogram.

[0086] See also Figure 5 The difference in salt concentration between the protein polymer stock solution after ultrafiltration and the protein polymer stock solution has little effect on the instrument response sensitivity. However, considering that when a low protein concentration stock solution is used to prepare a high protein concentration sample, the low protein concentration stock solution needs to be concentrated, and the salt concentration in the concentrated sample will further increase, which may not necessarily avoid the increase in the salt concentration of the sample. The impact on subsequent detection results, therefore, it is preferred to prepare the sample with a protein polymer stock solution that has been replaced by ultrafiltration.

[0087] The buffer system was screened using samples with a protein concentration of 2 mg / ml after reduction reaction:

[0088] 1. Sample preparation:

[0089] Take the protein polymer stock solution after ultrafiltration and liquid exchange treatment, and prepare the sample according to the following table:

[0090]

[0091]

[0092] The reagents and raw materials contained in each group of samples are fully mixed and allowed to stand to obtain samples.

[0093] 2. On-machine testing

[0094] Each time, 100 μl of the supernatant of the sample was measured and injected into the injection port of the capillary electrophoresis instrument. The detection conditions were the same as those of the above-mentioned reduction-treated sample to obtain a chromatogram.

[0095] See also Figure 6 Buffer systems with different pH values ​​have little effect on the instrument response sensitivity, and the test results are relatively consistent. However, considering that the relevant detection method introduced in the "Chinese Pharmacopoeia (General Chapter 0542) Capillary Electrophoresis" uses a low pH buffer, it is preferred to use a low pH buffer to prepare samples.

[0096] Screening of denaturing conditions for sample preparation:

[0097] In some embodiments, in order to obtain a protein peak with a better peak shape, it is also necessary to screen the denaturation conditions during the sample preparation process, aiming to obtain the optimal denaturation conditions with the best detection result accuracy.

[0098] 1. Sample preparation:

[0099] Take the protein polymer stock solution and prepare the sample according to the following table:

[0100]

[0101] The reagents and raw materials contained in the sample were mixed thoroughly and centrifuged for 1 minute.

[0102] 2. Denaturation reaction

[0103] The sample was divided into 4 parts and denatured in a water bath at 70°C for 15 min, 70°C for 5 min, 70°C for 10 min and 100°C for 5 min.

[0104] After the denaturation reaction is completed, each group of samples is placed at room temperature and protected from light for 10 minutes to obtain the sample.

[0105] 3. On-machine testing

[0106] Each time, 100 μl of the supernatant of the sample was measured and injected into the injection port of the capillary electrophoresis instrument. The detection conditions were the same as those of the above-mentioned reduction-treated sample to obtain a chromatogram.

[0107] See also Figure 7 The samples denatured in a 70°C water bath for 5 or 10 minutes showed poor peak shape in the chromatogram, which may mean that the denaturation reaction was not complete. Based on the denaturation conditions mentioned in the "Capillary Electrophoresis Method of the Chinese Pharmacopoeia (General Chapter 0542)", the denaturation conditions are preferably 70°C water bath for 15 minutes.

[0108] Screening of capillary temperature during sample testing:

[0109] In some embodiments, in order to obtain a protein peak with a better peak shape, the capillary temperature of the capillary electrophoresis instrument needs to be screened to obtain the optimal capillary temperature with the best detection result accuracy.

[0110] 1. Sample preparation:

[0111] Take the protein polymer stock solution after ultrafiltration and prepare the sample according to the following table:

[0112]

[0113] The reagents and raw materials contained in each group of samples are fully mixed and allowed to stand to obtain samples.

[0114] 2. On-machine testing

[0115] The above sample was divided into three parts and tested at capillary temperatures of 20°C, 25°C and 30°C respectively.

[0116] Each time, 100 μl of the supernatant of the sample was measured and injected into the injection port of the capillary electrophoresis instrument. The detection conditions were the same as those of the above-mentioned reduction-treated sample to obtain a chromatogram.

[0117] See also Figure 8 It can be known that the higher the capillary temperature, the shorter the migration time of the protein molecules. However, no change in the resolution of the chromatogram was found in the samples of this application at the above three experimental temperatures. Therefore, a capillary temperature of 25°C is preferably adopted to avoid re-denaturation of sensitive proteins due to excessively high temperature, resulting in result errors.

[0118] In a second aspect, in some embodiments, the present application also provides a repeatability verification method for verifying the repeatability of the above purity analysis method:

[0119] The repeatability verification method comprises:

[0120] S41: preparing several groups of samples with different protein concentrations;

[0121] S42: measuring a predetermined injection amount of several groups of the samples, and sending them into a capillary electrophoresis instrument for capillary electrophoresis detection to obtain several groups of chromatograms;

[0122] S43: Repeat the above step S2 three times, perform capillary electrophoresis detection three times in parallel for each group of samples, select three main peaks in the chromatogram, count the area proportions of each main peak in each chromatogram and perform data analysis.

[0123] By verifying the repeatability of the purity analysis method of the above-mentioned protein polymer, calculating the relative standard deviation of the peak area ratios of parallel experiments at the same protein concentration, and the relative standard deviation of the main peak at the same peak time at different protein concentrations, the accuracy of the purity analysis method proposed in this application is evaluated, and the credibility of each indicator in the test results of the purity analysis method proposed in this application is predicted. Specific implementation method:

[0125] 1. Sample preparation:

[0126] Take the protein polymer stock solution after ultrafiltration and prepare the sample according to the following table:

[0127]

[0128] The reagents and raw materials contained in each group of samples were fully mixed and allowed to stand to obtain the samples. Three copies of each target protein concentration were prepared for parallel experiments with the same protein concentration.

[0129] 2. On-machine testing

[0130] Each time, 100 μl of the supernatant of the sample was measured and injected into the injection port of the capillary electrophoresis instrument. The detection conditions were the same as those of the above-mentioned reduction-treated sample to obtain a chromatogram.

[0131] See also Fig. 9 , the peak area percentages of the three signal peaks with the largest peak areas in each chromatogram are counted, among which the largest signal peak is the protein peak, and the following table is obtained:

[0132]

[0133]

[0134] It can be seen from the above table that the peak shape and peak area ratio of the protein peaks measured in parallel experiments for samples with the same protein concentration and samples with different protein concentrations are basically the same, and the relative standard deviation RSD is less than 2.0%, which fully proves that the protein purity analysis method proposed in this application is repeatable. At the same time, it also indirectly confirms that the protein purity in the samples of this application is very high, and there is almost no interference from miscellaneous peaks.

[0135] In a third aspect, in some embodiments, the present application further provides a linear verification method for verifying the linear relationship between the protein concentration of the sample and the peak area of ​​the peak in the chromatogram in the above-mentioned protein polymer purity analysis method:

[0136] The linear relationship verification method comprises:

[0137] S51: preparing several groups of samples with different protein concentrations;

[0138] S52: measuring a predetermined injection amount of several groups of the samples, and sending them into a capillary electrophoresis instrument for capillary electrophoresis detection to obtain several groups of chromatograms;

[0139] S53: Select three main peaks in the chromatogram, count the areas of the main peaks in the chromatograms and perform data analysis.

[0140] By verifying the linear relationship between the protein concentration of the sample in the purity analysis method of the above-mentioned protein polymer and the peak area of ​​the main peak in the chromatogram, a regression curve with protein concentration as - peak area is obtained, the optimal detection protein concentration of the purity analysis method of the protein polymer proposed in this application is determined, and the protein concentration of the sample is adjusted to improve the accuracy of the detection result. Specific implementation method:

[0142] 1. Sample preparation:

[0143] Take the protein polymer stock solution after ultrafiltration and prepare the sample according to the following table:

[0144]

[0145]

[0146] The reagents and raw materials contained in each group of samples are fully mixed and allowed to stand to obtain the samples. The protein concentration of the protein polymer stock solution is obtained by adjusting the addition ratio of CE Water when the protein polymer raw material is ultrafiltered to obtain a stock solution with a target protein concentration.

[0147] 2. On-machine testing

[0148] Each time, 100 μl of the supernatant of the sample was measured and injected into the injection port of the capillary electrophoresis instrument. The detection conditions were the same as those of the above-mentioned reduction-treated sample to obtain a chromatogram.

[0149] The peak areas of the three signal peaks with the largest peak areas in each chromatogram are counted, among which the largest signal peak is the protein peak, and the following table is obtained:

[0150]

[0151] 3. Linear Regression

[0152] The peak areas of the three signal peaks detected at different protein concentrations were linearly fitted to obtain Figure 10-12 The five-point fitting statistical graph shown, Figure 10-12 The fitting degree R in the five-point fitting statistical diagram 2 All of them are less than 0.980, indicating a poor linear relationship. Therefore, the highest point with large deviation is removed to produce Figure 13-Figure 15 The four-point fitting graph shown in the figure has a linear fit R 2 They are all greater than 0.980, with good linearity, which is sufficient to prove that the protein concentration of the sample is proportional to the peak area of ​​the protein peak. Therefore, the protein concentration of the sample can be subsequently calculated through the linear regression equation of the protein peak. Specifically, capillary electrophoresis is performed on samples with unknown protein concentration to obtain the peak area value of the protein peak, and then the peak area value is substituted into the linear regression equation to obtain the protein concentration value of the sample.

[0153] At the same time, the deviation calculation is performed on the same signal peak of different concentrations to obtain the following table:

[0154]

[0155] The relative standard deviation RSD (n=5) of the peak area ratio of the same signal peak of samples with different protein concentrations is less than 5.0%. The poor fitting degree in the above five-point fitting statistical graph may be due to the overload of the protein concentration of the sample. Therefore, only samples with a protein concentration between 1mg / ml and 4mg / ml have a linear relationship between protein concentration and the peak area of ​​the signal peak. Therefore, the protein concentration of the sample obtained by the linear regression equation of the protein peak is greater than 4mg / ml, which may not be accurate.

[0156] In a fourth aspect, in some embodiments, the present application further provides a molecular weight calculation method for calculating the molecular weight of the protein molecule represented by each signal peak by referring to the peak time in the chromatogram in the purity analysis method of the protein polymer provided in the present application:

[0157] The molecular weight calculation method comprises:

[0158] S61: preparing a standard sample, wherein the standard sample is obtained by mixing a predetermined amount of a molecular weight standard, a sample buffer and an internal standard, wherein the molecular weight standard comprises calibration molecules of different molecular weights;

[0159] S62: taking a predetermined injection amount of the standard sample, feeding it into a capillary electrophoresis instrument for capillary electrophoresis detection, and obtaining a calibration chromatogram, wherein the molecular weight standards form corresponding calibration peaks on the calibration chromatogram in ascending order of molecular weight;

[0160] S63: Counting the corrected peak time and the corrected molecular weight of each calibration peak in the calibration chromatogram, and performing linear regression.

[0161] By using molecular weight standards to prepare standard samples, a calibration chromatogram is obtained in which corresponding calibration peaks are formed in sequence according to the molecular weight. The corrected peak elution time and the corrected molecular weight of each calibration peak are associated for linear regression to obtain a regression curve of the corrected peak elution time and the corrected molecular weight, which is convenient for subsequently obtaining the molecular weight of the protein molecules in the protein polymer based on the peak elution time of the protein peak in the chromatogram of the purity analysis method of the protein polymer. Specific implementation method:

[0163] 1. Preparation of standard samples:

[0164] Take molecular weight standards and prepare samples according to the following table:

[0165]

[0166] The reagents and raw materials contained in the sample are fully mixed and allowed to stand to obtain the sample. The molecular weight standard contains calibration molecules with molecular weights of 10KD, 20KD, 35KD, 50KD, 100KD, 150KD, and 225KD, respectively.

[0167] 2. On-machine testing

[0168] Each time, 100 μl of the supernatant of the sample was measured and injected into the injection port of the capillary electrophoresis instrument. The detection conditions were the same as those of the above-mentioned reduction-treated sample to obtain a chromatogram (see Fig.16 ).

[0169] The peak time and molecular weight of the signal peak corresponding to the calibration molecule of each molecular weight are counted, and a linear fit is performed to obtain the following: Fig.17 The molecular weight linear plot shown, where the fit R 2 It is 0.9998, with good linearity. Therefore, the molecular weight of the protein molecules in the sample can be calculated through the linear regression equation of the molecular weight. Specifically, the sample with unknown molecular weight of the protein molecule is subjected to capillary electrophoresis detection to obtain the peak time of the protein peak, and then the peak time value is substituted into the linear regression equation to obtain the molecular weight of the protein molecule in the sample.

[0170] Furthermore, for the above linear regression equation, samples of different concentrations were prepared using a standard substance with a molecular weight of 60KD, and the peak time was obtained by machine detection. The molecular weight was obtained by substituting it into the linear regression equation. The specific comparison is shown in the table below:

[0171]

[0172] It can be seen from the above table that the molecular weight range obtained by the regression equation is 57KDa ~ 60KDa, which is somewhat different from the molecular weight of the standard. The prediction accuracy is not high, and there will be deviations at different detection times for the same concentration. However, it has certain reference value in practical applications.

[0173] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A method for analyzing the purity of a protein polymer, characterized in that: The method comprises: S1: pre-treating the protein polymer to prepare a sample with a predetermined protein concentration; S2: taking a predetermined injection amount of the sample, sending it into a capillary electrophoresis instrument for capillary electrophoresis detection, setting the injection voltage to 3-9 KV, the separation voltage to 10-20 KV, the injection volume to 25-100 μl, the capillary temperature to 10-40° C., the mobile phase to: CE-SDS Gelbuffer, the elution pressure to 10-40 psi, and the peak width to 1-8 seconds to obtain a chromatogram; and S3: Analyze the signal peaks in the chromatogram, screen the signal peaks above a predetermined scale as main peaks, wherein the main peak with the largest area is the protein peak, and analyze to obtain the purity information of the protein polymer.

2. The purity analysis method according to claim 1, characterized in that: The capillary electrophoresis instrument is a high-throughput capillary electrophoresis instrument of model Biophase 8800 produced by AB SCIEX, the sample buffer is Low pH SDS-Sample Buffer of product number C57805 produced by AB SCIEX, the length of the capillary in the capillary electrophoresis instrument is 30 cm, and an internal standard of a predetermined molecular weight is also added to the sample.

3. The purity analysis method according to claim 2, characterized in that: The pretreatment of the protein polymer also includes non-reducing treatment or reducing treatment of the protein polymer. The non-reducing treatment is to add a predetermined amount of iodoacetamide to a predetermined amount of the protein polymer; the reducing treatment is to add a predetermined amount of 2-mercaptoethanol to a predetermined amount of the protein polymer.

4. The purity analysis method according to claim 3, characterized in that: The separation conditions of the sample obtained by the non-reduction treatment in the capillary electrophoresis instrument are as follows: duration of 10-60 minutes, voltage rise time of 1-5 minutes, and automatic zero adjustment time of 0.1-0.9 minutes.

5. The purity analysis method according to claim 3, characterized in that: The separation conditions of the sample obtained by the reduction treatment in the capillary electrophoresis instrument are: duration of 10-60 minutes, voltage rise time of 0.5-3 minutes, and automatic zero adjustment time of 2-8 minutes.

6. The purity analysis method according to claim 5, characterized in that: The protein concentration in the sample ranged from 0.1 mg / ml to 5 mg / ml.

7. The purity analysis method according to claim 1, characterized in that: The protein polymer is further subjected to ultrafiltration and liquid exchange treatment before pre-treatment, and the ultrafiltration and liquid exchange treatment includes: S01: measuring a predetermined volume of the sample and adding it into an ultrafiltration tube; S02: sending the ultrafiltration tube into a centrifuge and centrifuging for 10 minutes; S03: Add CE Water, mix well, and continue centrifuging in the centrifuge for 10 minutes; S04: Repeat the above step S03 three times, take the supernatant after the last centrifugation, and concentrate the supernatant to a predetermined protein concentration.

8. The purity analysis method according to claim 1, characterized in that: The sample buffer is a low pH sample buffer with a pH value less than 7.

9. The purity analysis method according to claim 1, characterized in that: The sample needs to be subjected to denaturation culture before being sent to the capillary electrophoresis instrument, and the denaturation culture includes: S11: transferring the sample to a centrifuge and centrifuging for 1 minute; S12: placing the centrifuged sample in a water bath at 30-100° C. for 5-15 minutes; S13: placing the sample after the water bath at room temperature away from light for 3-10 minutes; S14: The sample is transferred to the centrifuge again and centrifuged for 1 minute.

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