Determination of protein adsorption rate in pneumococcal polysaccharide-protein conjugate vaccine

Abstract

The present application improves the precipitation degree of the protein to be tested by changing the concentration of the sodium deoxycholate solution, thereby improving the accuracy of the determination of the protein adsorption rate in the pneumococcal polysaccharide protein conjugate vaccine. At the same time, the present application determines that the protein content is within the range of 60μg / ml to 90μg / ml, which can minimize the impact of the protein content determination diluent on the protein content determination, and make the protein recovery rate between 95% and 105%. The method for determining the protein content in the pneumococcal polysaccharide protein conjugate of the present application has the characteristics of high accuracy and high stability. Compared with the traditional Lowry method, the results are more reliable and can be widely used in the determination of the protein adsorption rate in the pneumococcal polysaccharide protein conjugate vaccine.

Description

Technical Field

[0001] The present application belongs to the technical field of vaccine detection, and relates to a method for determining the protein adsorption rate in a pneumococcal polysaccharide-protein conjugate vaccine. Background Art

[0002] Streptococcus pneumoniae is a type of Gram-positive bacteria, whose pathogenic substance is capsular polysaccharide. According to the different antigenicity of capsular polysaccharide, Streptococcus pneumoniae is divided into 84 serotypes, which mainly cause lobar pneumonia in humans. Pneumococcal polysaccharide protein conjugate vaccine is a polysaccharide protein conjugate formed by covalently binding purified pneumococcal polysaccharide with tetanus toxoid. This polysaccharide protein conjugate can induce thymus-dependent immune response in the body. Its successful development provides an important tool for preventing invasive diseases such as meningitis and pneumonia.

[0003] Adjuvants can non-specifically change or enhance the body's specific immune response to antigens and play an auxiliary role. They can induce the body to produce long-term, efficient specific immune responses and improve the body's protective ability. During the production of pneumococcal polysaccharide protein conjugate vaccines, aluminum phosphate adjuvants are added to the conjugate stock solution to adsorb the conjugate stock solution, thereby enhancing the immunogenicity of the vaccine. Therefore, the ability of the adjuvant to adsorb the conjugate stock solution is an important quality control indicator in the detection of finished products. The adsorption capacity of the adjuvant is generally evaluated by testing the adjuvant's ability to adsorb protein.

[0004] Traditional protein detection methods include the Lowry method, Kjeldahl method, biuret method, etc. Since the sensitivity of the Kjeldahl method and biuret method cannot meet the protein content level requirements in vaccines, the Lowry method is usually used in vaccine protein content detection. However, the existing Lowry method has the problem of inaccurate test results. Summary of the invention

[0005] Based on this, the present application provides a method for accurately and efficiently detecting the protein adsorption rate in pneumococcal polysaccharide protein conjugate vaccines, comprising the following steps:

[0006] Step S1, using the pneumococcal polysaccharide protein conjugate vaccine solution to be tested to prepare adsorbed protein and total protein respectively, wherein the content of total protein in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is controlled at 60 μg / mL to 90 μg / mL.

[0007] The preparation process of the adsorbed protein comprises: taking the pneumococcal polysaccharide protein conjugate vaccine solution to be tested, centrifuging, removing the supernatant, and preparing the adsorbed protein.

[0008] The total protein preparation process comprises: taking the pneumococcal polysaccharide protein conjugate vaccine solution to be tested, adding a first protein precipitant, mixing, standing at room temperature, then adding a second protein precipitant, mixing, standing at room temperature, centrifuging, removing the supernatant, and preparing the total protein.

[0009] Step S2, respectively measuring the adsorbed protein content and the total protein content, and determining the adsorption rate of the protein in the pneumococcal polysaccharide-protein conjugate vaccine to be tested according to the adsorbed protein content and the total protein content.

[0010] In one embodiment, in step S2, the first protein precipitant is sodium deoxycholate solution, and the second protein precipitant is trichloroacetic acid solution.

[0011] In one embodiment, the volume concentration of the sodium deoxycholate solution is 0.8% to 1.2%, and the volume ratio of the sodium deoxycholate solution to the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 1:(8-12).

[0012] The volume concentration of the trichloroacetic acid solution is 70% to 75%, and the volume ratio of the trichloroacetic acid solution to the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 1:(8 to 12).

[0013] In one embodiment, the total protein content in the pneumococcal polysaccharide protein conjugate vaccine solution is controlled at 60 μg / mL to 90 μg / mL by adding a protein content determination diluent.

[0014] In one embodiment, the protein content determination diluent contains an adjuvant, a pH adjuster and a dispersant.

[0015] In one embodiment, the adjuvant is aluminum phosphate, the pH adjuster is succinic acid, and the dispersant is Tween 80.

[0016] Optionally, the final concentration range of aluminum phosphate in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 0.20 mg / mL to 0.30 mg / mL.

[0017] Optionally, the final volume concentration of Tween 80 in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is in the range of 0.18% to 0.22%.

[0018] Optionally, the final concentration range of succinic acid in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 3.7 mmol / L to 5.7 mmol / L.

[0019] In one embodiment, step S1 further comprises the step of re-dissolving the collected material with a re-solvent after centrifugation to remove the supernatant.

[0020] Optionally, the complexing solvent includes sodium hydroxide, sodium carbonate, disodium tartrate dihydrate and copper sulfate pentahydrate.

[0021] In one embodiment, the centrifugation condition in the process of preparing the adsorbed protein in step S1 is 6000xg to 10000xg for 8min to 12min.

[0022] In one embodiment, the centrifugation condition during the total protein preparation process in step S1 is 3000×g to 7000×g for 25 min to 35 min.

[0023] In one embodiment, the pneumococcal polysaccharide-protein conjugate vaccine includes any one of the following pneumococcal polysaccharide-protein conjugates: type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 18C, type 19A, type 19F, type 23F, type 8, type 10A, type 11A, type 12F, type 15B, type 22F, and type 33F.

[0024] The present application improves the precipitation degree of the protein to be tested by changing the concentration of the sodium deoxycholate solution, thereby improving the accuracy of the determination of the protein adsorption rate in the pneumococcal polysaccharide protein conjugate vaccine. At the same time, the present application determines that the protein content is within the range of 60μg / mL to 90μg / mL, which can minimize the impact of the protein content determination diluent on the protein content determination, and make the protein recovery rate between 95% and 105%. The method for determining the protein content in the pneumococcal polysaccharide protein conjugate of the present application has the characteristics of high accuracy and high stability. Compared with the traditional Lowry method, the results are more reliable and can be widely used in the determination of the protein adsorption rate in the pneumococcal polysaccharide protein conjugate vaccine. DETAILED DESCRIPTION

[0025] Below in conjunction with embodiment and example, the application is further described in detail.It should be understood that these embodiments and examples are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiments and examples is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms, is not limited to the embodiment and example described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the application, and it should be understood that the application can be implemented without one or more of these details.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0027] the term

[0028] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0029] The terms "combination thereof", "any combination thereof", "any combination thereof" and the like used in this application include all suitable combinations of any two or more of the listed items.

[0030] In this application, the "suitable" mentioned in "suitable combination", "suitable method", "any suitable method", etc. is based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0031] In the present application, "preferred", "better", "more preferred" and "suitable" are only used to describe implementation methods or examples with better effects. It should be understood that they do not constitute limitations on the scope of protection of the present application.

[0032] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0033] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0034] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0035] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0036] Pneumococcal polysaccharide-protein conjugate vaccine: A polysaccharide-protein conjugate formed by covalently bonding purified pneumococcal capsular polysaccharide with tetanus toxoid. This polysaccharide-protein conjugate can induce a thymus-dependent immune response in the body. Its successful development provides an important tool for preventing invasive diseases such as meningitis and pneumonia.

[0037] Protein precipitation: Protein precipitation can be done by adding salt, polyethylene glycol or organic solvents. As a precipitant, salt has a dual role, namely salting out and salting out. Salt ions shield the surface charges of protein molecules, thereby weakening the repulsive forces between molecules.

[0038] Lowry method: It is a combination and development of the biuret method and the folin-phenol method. Its principle is: the protein solution is treated with an alkaline copper solution to form a copper-protein complex salt. After adding the phenol reagent, in addition to the color development of tyrosine, tryptophan and cysteine ​​in the peptide chain, the color development effect of the peptide bonds and alkaline copper in the biuret method is also made stronger.

[0039] Sodium deoxycholate: It is a water-soluble bile acid anionic detergent that can effectively destroy the interactions between many proteins. It forms micelles under acidic conditions and can precipitate protein substances, thereby precipitating polysaccharide-protein conjugates in vaccines.

[0040] Aluminum phosphate adjuvant: Aluminum phosphate adjuvant is not a single substance, but a hydroxyaluminum phosphate complex. The degree of replacement of the phosphate group for the hydroxyl group depends on the reactants and precipitation conditions and their isoelectric point. Generally speaking, the isoelectric point of aluminum phosphate adjuvant is 5.0. It exists in anionic form in a solution with a pH of 7.4 and is a good adsorbent for cationic antigens.

[0041] UV-Visible Spectrophotometry: UV-Visible Spectrophotometry is a method for determining the absorbance of a substance in the wavelength range of 190 to 800 nm, and is used for identification, impurity inspection, and quantitative determination. When light passes through the solution of the substance being measured, the degree of absorption of light by the substance varies with the wavelength of the light. Therefore, by measuring the absorbance of the substance at different wavelengths and plotting the relationship between its absorbance and wavelength, the absorption spectrum of the substance being measured can be obtained.

[0042] BSA: Bovine serum albumin (BSA) is a globulin in bovine serum, containing 607 amino acid residues, with a molecular weight of 66.446KDa and an isoelectric point of 4.7. Bovine serum albumin is widely used in biochemical experiments.

[0043] The present application provides a method for determining the protein adsorption rate in a pneumococcal polysaccharide-protein conjugate vaccine, comprising the following steps:

[0044] Step S1: using the pneumococcal polysaccharide protein conjugate vaccine solution to be tested to prepare adsorbed protein and total protein respectively, and the content of total protein in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is controlled at 60 μg / mL to 90 μg / mL.

[0045] Adsorbed protein refers to the protein that can be adsorbed by the adjuvant in the vaccine. Total protein includes the protein that is not adsorbed by the adjuvant and the protein that is adsorbed by the adjuvant.

[0046] The preparation process of the adsorbed protein comprises: taking the pneumococcal polysaccharide protein conjugate vaccine solution to be tested, centrifuging, removing the supernatant, and preparing the adsorbed protein.

[0047] The preparation process of total protein includes: taking the pneumococcal polysaccharide protein conjugate vaccine solution to be tested, adding a first protein precipitant, mixing, standing at room temperature, then adding a second protein precipitant, mixing, standing at room temperature, centrifuging, removing the supernatant, and preparing total protein.

[0048] Step S2, respectively measuring the adsorbed protein content and the total protein content, and determining the adsorption rate of the protein in the pneumococcal polysaccharide protein conjugate vaccine to be tested according to the adsorbed protein content and the total protein content. The specific calculation method can be based on the adsorbed protein content / total protein content×100%.

[0049] In a specific example, in step S2, the first protein precipitant is sodium deoxycholate solution, and the second protein precipitant is trichloroacetic acid solution.

[0050] In a specific example, the volume concentration of the sodium deoxycholate solution is 0.8% to 1.2%, and the volume ratio of the sodium deoxycholate solution to the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 1: (8 to 12). For example, the volume concentration of the sodium deoxycholate solution is 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, and the volume ratio of the sodium deoxycholate solution to the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 1: 8, 1: 9, 1: 10, 1: 11, 1: 12.

[0051] Optionally, the volume concentration of the trichloroacetic acid solution is 70% to 75%, and the volume ratio of the trichloroacetic acid solution to the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 1:(8-12). For example, the volume concentration of the trichloroacetic acid solution is 70%, 71%, 72%, 73%, 74%, 75%, and the volume ratio of the trichloroacetic acid solution to the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 1:8, 1:9, 1:10, 1:11, 1:12.

[0052] Furthermore, the total protein content in the pneumococcal polysaccharide protein conjugate vaccine solution is controlled to be 60 μg / mL to 90 μg / mL by adding a protein content determination diluent, for example 60 μg / mL, 65 μg / mL, 70 μg / mL, 75 μg / mL, 80 μg / mL, 85 μg / mL, 90 μg / mL.

[0053] In a specific example, the protein content determination diluent contains an adjuvant, a pH regulator and a dispersant. Specifically, the adjuvant is aluminum phosphate, the pH regulator is succinic acid, and the dispersant is Tween 80.

[0054] Optionally, the final concentration of aluminum phosphate in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested ranges from 0.20 mg / mL to 0.30 mg / mL, for example, 0.20 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.30 mg / mL.

[0055] Optionally, the final volume concentration of Tween 80 in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is in the range of 0.18% to 0.22%; for example, it can be 0.18%, 0.19%, 0.20%, 0.21%, or 0.22%.

[0056] Optionally, the final concentration of succinic acid in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested ranges from 3.7 mmol / L to 5.7 mmol / L. For example, it can be 3.7 mmol / L, 3.8 mmol / L, 3.9 mmol / L, 4.0 mmol / L, 4.1 mmol / L, 4.2 mmol / L, 4.3 mmol / L, 4.4 mmol / L, 4.5 mmol / L, 4.6 mmol / L, 4.7 mmol / L, 4.8 mmol / L, 4.9 mmol / L, 5.0 mmol / L, 5.1 mmol / L, 5.2 mmol / L, 5.3 mmol / L, 5.4 mmol / L, 5.5 mmol / L, 5.6 mmol / L, 5.7 mmol / L.

[0057] In a specific example, step S1 further includes the step of re-dissolving the collected material with a re-solvent after centrifugation to remove the supernatant.

[0058] Optionally, the resolvent includes sodium hydroxide, sodium carbonate, disodium tartrate dihydrate, and copper sulfate pentahydrate.

[0059] In one embodiment, the centrifugation condition in the process of preparing the adsorbed protein in step S1 is 6000×g to 10000×g for 8 min to 12 min. For example, the centrifugation condition is 6000×g, 7000×g, 8000×g, 9000×g, 10000×g, and centrifugation is 8 min, 9 min, 10 min, 11 min, and 12 min.

[0060] In one embodiment, the centrifugation condition during the total protein preparation process in step S1 is 3000×g-7000×g for 25min-35min. For example, the centrifugation condition is 3000×g, 4000×g, 5000×g, 6000×g, 7000×g for 25min, 26min, 27min, 28min, 29min, 30min, 31min, 32min, 33min, 34min, 35min.

[0061] In one embodiment, the pneumococcal polysaccharide-protein conjugate vaccine includes any one of the following pneumococcal polysaccharide-protein conjugates: type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 18C, type 19A, type 19F, type 23F, type 8, type 10A, type 11A, type 12F, type 15B, type 22F, and type 33F.

[0062] Example 1: Preparation of vaccine protein samples to be tested

[0063] 1. Preparation of adsorbed protein

[0064] Take 1.0mL of the 13-valent pneumococcal polysaccharide protein conjugate vaccine to be tested in a centrifuge tube (if the protein concentration is higher than 90, the vaccine needs to be diluted with adjuvants and auxiliary materials to a protein content of 60μg / mL to 90μg / mL), centrifuge at 8000xg for 10 minutes, discard the supernatant, and keep the precipitate for the next step of the experiment.

[0065] 2. Total Protein Preparation

[0066] Take 1.0mL of 13-valent pneumococcal polysaccharide protein conjugate vaccine to be tested in a centrifuge tube, add 0.1mL of 1.0% sodium deoxycholate solution, place at room temperature for 10 minutes, add 0.1mL of 72% trichloroacetic acid solution, mix well, centrifuge at 5000×g for 30min, discard the supernatant, and keep the precipitate to continue the next experiment.

[0067] 3. Determination of adsorbed protein

[0068] Dissolve the prepared adsorbed protein with 1.0 ml of test solution C, transfer to a 15 ml plastic centrifuge tube, add 5 ml of test solution C, and mix. Place at room temperature for 10 min, add 0.5 ml of Folin phenol test solution (1 mol / L), mix immediately, and place at room temperature for 30 min. Use the 0.0 ml tube as a blank and detect the absorbance with a UV-visible spectrophotometer at a wavelength of 750 nm.

[0069] 4. Determination of total protein

[0070] Dissolve the prepared total protein with 1.0 mL of test solution C, transfer to a 15 mL plastic centrifuge tube, add 5 mL of test solution C, and mix. Place at room temperature for 10 min, add 0.5 mL of Folin phenol test solution (1 mol / L), mix immediately, and place at room temperature for 30 min. Use the 0.0 mL tube as a blank and detect the absorbance value with a UV-visible spectrophotometer at a detection wavelength of 750 nm.

[0071] 5. Specific instruments and reagents

[0072] Table 1 Instruments

[0073] Instrument / Equipment Name Specifications / Models factory UV-Vis Spectrophotometer Shimadzu UV-2600 Shimadzu Enterprise Management (China) Co., Ltd. High-speed refrigerated centrifuge ST16R Thermo Electronic balance BSA224S-CW Sartorius Pipette 20~200μL Brand Pipette 100~1000μL Brand Pipette 0.5~5mL Brand

[0074] Table 2 Reagents

[0075] name Manufacturer batch number Anhydrous sodium carbonate Sinopharm Chemical Reagent Co., Ltd. 20191127 Sodium hydroxide Sinopharm Chemical Reagent Co., Ltd. 20201016 Disodium tartrate dihydrate Sinopharm Chemical Reagent Co., Ltd. 20200317 Copper Sulfate Pentahydrate Sinopharm Chemical Reagent Co., Ltd. 20181012 Folin Sinopharm Chemical Reagent Co., Ltd. 20210107 Sodium deoxycholate Sigma SLCC7723 Trichloroacetic acid (TCA) Sinopharm Chemical Reagent Co., Ltd. 20210323 Potassium tartrate (hemihydrate) Sinopharm Chemical Reagent Co., Ltd. 20200309

[0076] Example 2: Effects of adjuvants and excipients on detection of different protein concentrations

[0077] 1. Operation steps

[0078] Take two portions of 13-valent pneumococcal polysaccharide-protein conjugate stock solutions with protein concentrations of approximately 60 μg / mL, 70 μg / mL, 80 μg / mL, and 90 μg / mL, add aluminum phosphate adjuvant, Tween 80 solution, and succinic acid buffer solution to one portion, so that the final concentrations of aluminum phosphate adjuvant, Tween 80 solution, and succinic acid buffer solution are 0.25 mg / mL, 0.02%, and 5 mmol / L, respectively, and set aside; no addition is required to the other portion.

[0079] Take 2 portions of 20-valent pneumococcal polysaccharide-protein conjugate stock solutions with protein concentrations of about 100 μg / mL, 120 μg / mL, 140 μg / mL, 160 μg / mL, and 180 μg / mL, add aluminum phosphate adjuvant, Tween 80 solution, and succinic acid buffer solution to one portion, so that the final concentrations of aluminum phosphate adjuvant, Tween 80 solution, and succinic acid buffer solution are 0.25 mg / mL, 0.02%, and 5 mmol / L, respectively, and dilute it 2 times with the mixed adjuvant for standby use; no addition is required to the other portion.

[0080] Accurately pipette 1.0 mL of the above solution into a test tube and determine the protein content by Lowry method 2. Compare the recovery rates of the original solution of the conjugate and the original solution of the conjugate with adjuvants added (i.e., semi-finished product solution). The recovery rate is required to be between 95% and 105%. The calculation formula is as follows:

[0081]

[0082] 2. The experimental results are shown in Table 3.

[0083] Table 3 Effects of adjuvants and excipients on detection of different protein concentrations

[0084]

[0085] Data analysis: When the protein content is in the range of 60μg / mL to 90μg / mL, the effect of adjuvants on protein content detection can be ignored. Test samples exceeding this range can be diluted with adjuvants to within the range for determination.

[0086] Example 3: Corresponding relationship between protein content and recovery rate (accuracy of protein content detection)

[0087] Add different concentrations of semi-finished product solutions to the BSA solution and calculate the recovery rate of the protein content of the added semi-finished product solution. The recovery rate is between 95% and 105%, indicating that the protein content detection is accurate.

[0088] The formula is as follows:

[0089]

[0090] 1. Operation steps

[0091] Add 13-valent pneumococcal conjugate vaccine semi-finished product to the BSA solution, so that the final protein concentration in the semi-finished product is 50μg / mL, 60μg / mL, 90μg / mL, and 100μg / mL, respectively, to prepare a mixed solution of the semi-finished product and BSA. If the protein content of the solution exceeds 90μg / ml, it needs to be diluted with a diluent to a protein content within 60-90μg / ml for use. Accurately pipette 1.0mL of the above mixed solution, semi-finished solution, and BSA solution into a test tube, and determine the protein content by Lowry method 2; compare the recovery rates.

[0092] 2. The experimental results are shown in Table 4.

[0093] Table 4: Accuracy of protein content detection

[0094]

[0095] Data analysis: Add different concentrations of semi-finished solution to BSA solution, and calculate the recovery rate of protein content of the added semi-finished solution. The recovery rate of protein content of the semi-finished solution in the range of 50μg / mL to 100μg / mL is between 95% and 105%, and the method has good accuracy.

[0096] Example 4: Comparison of the test results of the present method and the traditional Lowry method

[0097] The test results are shown in Table 5.

[0098] Table 5: Comparison of the test results of this application method and the traditional Lowry method

[0099]

[0100]

[0101] Data analysis: By comparing the adsorption rates of 13-valent and 20-valent pneumococcal conjugate vaccines measured by the two methods, it can be seen that the adsorption rate measured by the method of the present application is about 10% higher than that of the traditional Lowry method; the adsorption rate of 20-valent pneumococcal conjugate vaccine is about 10% higher than that of the traditional Lowry method.

[0102] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0103] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be based on the attached claims.

Claims

1. A method for determining the protein adsorption rate in a pneumococcal polysaccharide-protein conjugate vaccine. It is characterized in that The following steps are involved: Step S1, using the pneumococcal polysaccharide protein conjugate vaccine solution to be tested to prepare adsorbed protein and total protein respectively, wherein the content of total protein in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is controlled at 60 μg / mL to 90 μg / mL; The preparation process of the adsorbed protein comprises: taking the pneumococcal polysaccharide protein conjugate vaccine solution to be tested, centrifuging, removing the supernatant, and preparing the adsorbed protein; The total protein preparation process comprises: taking the pneumococcal polysaccharide protein conjugate vaccine solution to be tested, adding a first protein precipitant, mixing, leaving at room temperature, then adding a second protein precipitant, mixing, leaving at room temperature, centrifuging, removing the supernatant, and preparing the total protein; Step S2, respectively measuring the adsorbed protein content and the total protein content, and determining the adsorption rate of the protein in the pneumococcal polysaccharide-protein conjugate vaccine to be tested according to the adsorbed protein content and the total protein content; In step S2, the first protein precipitant is a sodium deoxycholate solution, and the second protein precipitant is a trichloroacetic acid solution; The total protein content in the pneumococcal polysaccharide protein conjugate vaccine solution is controlled to be 60 μg / mL to 90 μg / mL by adding a protein content determination diluent; The protein content determination diluent contains an adjuvant, a pH regulator and a dispersant.

2. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to claim 1, It is characterized in that The volume concentration of the sodium deoxycholate solution is 0.8% to 1.2%, and the volume ratio of the sodium deoxycholate solution to the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 1:(8-12); The volume concentration of the trichloroacetic acid solution is 70% to 75%, and the volume ratio of the trichloroacetic acid solution to the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 1:(8 to 12).

3. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to claim 1, It is characterized in that The adjuvant is aluminum phosphate, the pH regulator is succinic acid, and the dispersant is Tween 80.

4. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to claim 3, It is characterized in that The final concentration range of the aluminum phosphate in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 0.20 mg / mL to 0.30 mg / mL.

5. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to claim 3, It is characterized in that The final volume concentration range of Tween 80 in the tested pneumococcal polysaccharide protein conjugate vaccine solution is 0.18% to 0.22%.

6. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to claim 3, It is characterized in that The final concentration range of succinic acid in the pneumococcal polysaccharide protein conjugate vaccine solution to be tested is 3.7 mmol / L to 5.7 mmol / L.

7. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to claim 1, It is characterized in that Step S1 further includes the step of re-dissolving the collected material with a re-solvent after centrifugation to remove the supernatant.

8. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to claim 7, It is characterized in that The complex solvent comprises sodium hydroxide, sodium carbonate, disodium tartrate dihydrate and copper sulfate pentahydrate.

9. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to any one of claims 1 to 8, It is characterized in that The centrifugation condition in the process of preparing the adsorbed protein in step S1 is 6000×g to 10000×g for 8 min to 12 min.

10. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to any one of claims 1 to 8, It is characterized in that The centrifugation condition during the total protein preparation process in step S1 is 3000×g to 7000×g for 25 min to 35 min.

11. The method for determining the protein adsorption rate in the pneumococcal polysaccharide-protein conjugate vaccine according to any one of claims 1 to 8, It is characterized in that The pneumococcal polysaccharide-protein conjugate vaccine includes any one of the following pneumococcal polysaccharide-protein conjugates: type 1, type 3, type 4, type 5, type 6A, type 6B, type 7F, type 9V, type 14, type 18C, type 19A, type 19F, type 23F, type 8, type 10A, type 11A, type 12F, type 15B, type 22F, and type 33F.

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