Construction method of protein mass calculation model based on cation mass difference

Abstract

The invention belongs to the field of physical testing, and particularly relates to a construction method of a protein mass calculation model based on cation mass difference. The method comprises the following steps: acquiring a cation mass difference measured by a target protein in at least two different cation solutions with equal molar concentrations; based on the at least two molar concentrations, respectively extracting double-electric-ball concentration factors of the target protein; fitting a mathematical regression model of the double-electric-ball concentration factor and the cation mass difference based on the at least two molar concentrations and the corresponding cation mass difference, and solving to obtain a slope parameter and an intercept parameter of the mathematical model; and calculating the mass of the target protein based on the slope parameter and the intercept parameter. The construction method of the protein mass calculation model based on the cation mass difference, provided by the invention, is realized by codes, so that convenience and rapidness in calculation are realized; and the result obtained by calculation is highly consistent with the result of the protein content obtained by using a conventional protein content calculation method.

Description

Technical Field

[0001] The present invention relates to the field of physical testing, and tests or analyzes materials by measuring the chemical or physical properties of the materials; more specifically, it relates to a method, device, medium and program product for constructing a protein mass calculation model based on the cation mass difference. Background Art

[0002] Protein content measurement has always been an important technology in the fields of biology, medicine and chemistry. Currently, the commonly used methods for detecting protein content in the field include: Kjeldahl method, Lowry method, BCA method, HPLC method, amino acid composition method. Among them, the basic steps of the Lowery method and the BCA method are to add a solution (such as sodium chloride solution) to a standard substance and then develop a color corresponding to a value A; the same solution is also added to a drug sample and then develop a color corresponding to a value B, and the content of the drug protein is calculated by substituting this value B into the curve; however, the problem with these two methods is that a standard substance must be used as a control; but due to the difficulty in obtaining the standard substance, the error caused by the standard substance cannot be eliminated from the subsequent measurement calculations, which causes serious problems for the accurate detection of drug protein content.

[0003] Although with the replacement of technology, high performance liquid chromatography (HPLC) has been gradually widely used in protein content measurement. The HPLC method is based on the principle of chromatography, and uses a high-pressure infusion pump to separate and purify samples by passing the mobile phase (usually a liquid) through the stationary phase (usually a solid or liquid particle). The distribution coefficients of the components in the sample solution in the mobile phase and the stationary phase are different. When they move relatively in the two phases, through continuous adsorption-desorption distribution processes, the mixed components gradually separate from each other, and finally flow out of the column in turn as individual separated components. A highly sensitive detector is used to convert the sample concentration into an electrical signal and transmit it to a recorder, and the sample data is printed in the form of a chromatogram, so as to realize the analysis and separation of the sample. SEC-HPLC (size / volume exclusion high performance liquid chromatography), as a special type of HPLC (high performance liquid chromatography), uses stationary phases (packings) with different pore sizes to separate the molecules in the sample. When the sample solution passes through the chromatographic column, macromolecular substances cannot enter the small pore diameters inside the packing and can only pass through the gaps between the packing particles, so their paths are shorter and the retention times are also shorter; while small molecular substances can enter the small pore diameters inside the packing, with longer paths and longer retention times. In this way, molecules of different sizes can be separated in the chromatographic column.

[0004] The steps for purity detection using SEC-HPLC basically include:

[0005] 1. SEC-HPLC Detection: (1) System Equilibration: Buffer Selection: Select an eluent that matches the sample buffer to ensure the stability of the sample during analysis; System Preheating: Preheat the SEC-HPLC system to the set temperature to ensure temperature consistency during analysis; Equilibrate the chromatographic column: Pass the eluent through the chromatographic column until the baseline is stable, ensuring that the packing material in the column is fully wetted and reaches an equilibrium state. (2) Sample Preparation and Loading: Sample Amount: Calculate the appropriate sample loading amount based on the capacity of the chromatographic column and the concentration of the sample to avoid overloading. Injection Method: An autosampler can be used for injection, or manual injection can be performed to ensure that the sample enters the chromatographic system. (3) Elution and Detection: Elution Program: Elute through the chromatographic column at a constant flow rate, usually using isocratic elution. Detector Settings: Set the appropriate detection wavelength according to the properties of the protein. Usually, an ultraviolet absorption detector is used to detect proteins at 280 nm. Record the Elution Profile: Record the signal changes during elution to generate an elution profile. (4) Post-Elution Processing: Column Cleaning: After analysis, clean the chromatographic column with the eluent to remove residual proteins and impurities. System Shutdown: Shut down the HPLC system and properly store the chromatographic data.

[0006] 2. Data Analysis of SEC-HPLC Purity Detection: (1) Peak Identification and Molecular Weight Estimation: Peak Identification: In the elution profile, identify the main protein peaks and possible impurity peaks based on the elution volume (or elution time). The main peak usually corresponds to the target protein, while smaller peaks may represent protein aggregates, degradation products, or other impurities. Molecular Weight Estimation: By comparing the elution volume of the sample with that of a standard substance of known molecular weight, the molecular weight of the protein can be estimated. This is usually achieved by plotting a standard curve (a graph of molecular weight versus elution volume), and the standard curve is usually plotted using linear regression methods. By fitting the relationship between the elution volume and molecular weight of the standard substance of known molecular weight, a linear equation can be established to estimate the molecular weight of the unknown sample. (2) Quantitative Analysis: Peak Area Integration: Integrate the area of each peak in the elution profile to quantitatively analyze the relative content of each component. Purity Calculation: Calculate the ratio of the area of the main protein peak to the total area (including all detected peaks) to estimate the purity of the sample. Purity = (Area of Main Protein Peak / Total Area) × 100%.

[0007] Thus, even the more advanced technology of SEC-HPLC has not overcome the limitation of the Lowery method and the BCA method that require standard substances. The molecular weight of SEC-HPLC is still estimated by comparing the elution volume of the sample with that of a standard substance of known molecular weight. However, it is difficult to obtain an accurate standard substance, and the resulting error in molecular weight estimation cannot be eliminated in current technologies. Summary of the Invention

[0008] In view of the problems caused by the difficulty in obtaining reference materials, the present invention first proposes not to use reference materials, but instead to calculate the protein content by using the difference between two cations measured at equimolar concentrations. However, in the process of solving for the protein content based on the measured cation mass difference, a series of technical problems need to be solved. This application provides a method for constructing a protein mass calculation model based on the cation mass difference, which solves a series of technical problems in calculating the protein content using the cation mass difference instead of reference materials.

[0009] This application (in the first aspect) discloses a method for constructing a protein mass calculation model based on the cation mass difference.

[0010] Obtain the cation mass difference of the target protein measured in at least two different cation solutions with equimolar concentrations.

[0011] Based on the at least two molar concentrations, extract the double-electron sphere concentration factor of the target protein, which is determined by the molar concentration, the average charge constant of the protein, the ion displacement constant of the protein, and the applicable type constant.

[0012] Based on the at least two molar concentrations and their corresponding cation mass differences, fit a mathematical regression model of the double-electron sphere concentration factor and the cation mass difference to solve for the slope parameter and intercept parameter of the mathematical model. The mathematical regression model includes a slope parameter and an intercept parameter, where the intercept parameter represents the cation mass difference when the molar concentration is equal to the average charge constant of the target protein, and the slope parameter represents: the change in the cation mass difference when the molar concentration is twice the multiple of the average charge constant of the target protein compared to the cation mass difference at equality.

[0013] Calculate the mass of the target protein based on the slope parameter and the intercept parameter. The calculation basis is: the product of the applicable constant power of the intercept parameter and the slope parameter, and the dimension is reduced to the mass unit by taking the square root of the product.

[0014] Further, calculate the mass of the target protein based on the slope parameter, the intercept parameter, and the constant calculated from the applicable type.

[0015] Optionally, the mathematical regression model is expressed as:

[0016] m i =k·Factor + b

[0017] where m i represents the cation mass difference at the i-th molar concentration, Factor represents the double-electron sphere concentration factor, k is the slope parameter, and b is the intercept parameter.

[0018] Further, the double electric sphere concentration factor is the exponent of the part where the difference between the larger value and the smaller value of the molar concentration and the protein average charge constant is higher than 1, and the exponential term is determined by the applicable type constant and the protein ion-excluding constant;

[0019] Optionally, the double electric sphere concentration factor is expressed as:

[0020]

[0021] where Factor represents the double electric sphere concentration factor, C i represents the molar concentration, a represents the protein average charge constant, n represents the protein ion-excluding constant, p represents the applicable type constant, and the value is 2 or 3; C i > a? C i : a is a ternary operation expression, indicating that if the expression on the left side of the question mark holds, the value or symbol on the left side of the colon is taken, otherwise the value or symbol on the right side is taken; 5 - p(C i > a? + : - )n means that if C i > a holds, the exponent is 5 - p + n, otherwise it is 5 - p - n;

[0022] Optionally, if the molar concentration is greater than the protein average charge constant, the double electric sphere concentration factor is the exponent of the ratio of the difference between the molar concentration and the protein average charge constant to the protein average charge constant, and the exponential term is the difference between the applicable type constant and the protein ion-excluding constant;

[0023] Optionally, if the molar concentration is less than the protein average charge constant, the double electric sphere concentration factor is the exponent of the ratio of the difference between the molar concentration and the protein average charge constant to the molar concentration, and the exponential term is the sum of the applicable type constant and the protein ion-excluding constant;

[0024] Optionally, the protein ion-excluding constant and the average charge constant are obtained by looking up the table according to the data recorded in previous experiments;

[0025] Optionally, at least four molar concentrations and their corresponding cation mass differences are obtained, at least four double electric sphere concentration factors are extracted, and the slope parameter, intercept parameter, the protein ion-excluding constant, and the average charge constant are solved based on the cation differences and double electric sphere concentration factors of at least four molar concentrations.

[0026] Further, the way the calculation output layer calculates the protein mass is expressed as: where k is the slope parameter, b is the intercept parameter, and p represents the applicable type constant;

[0027] Optionally, the applicable type constant is obtained based on the structural analysis of the protein;

[0028] Optionally, preset the applicable type constant as 3, solve to obtain the slope parameter k. If k > 0, confirm that the applicable type constant is 3; otherwise, modify the applicable type constant to 2 and re-solve to obtain the slope parameter and the intercept parameter.

[0029] Optionally, if the applicable type constant is 3, the protein calculation formula is expressed as: where b represents the intercept parameter and k represents the slope parameter.

[0030] Optionally, if the applicable type constant is 2, the protein calculation formula is expressed as:

[0031] where b represents the intercept parameter and k represents the slope parameter.

[0032] Further, the specific steps for obtaining the cation mass difference measured for the target protein in at least two different cation solutions with equimolar concentrations include:

[0033] Step 1: Obtain the protein solution of the sample to be measured, and the congeners of the first cation and the second cation.

[0034] Step 2: Set the first molar concentration, and use the congeners of the first cation and the second cation to prepare a first solution and a second solution with the first concentration as the first mobile phase and the second mobile phase with equimolar concentrations respectively.

[0035] Step 3: After respectively injecting equal amounts of the said protein solution into the SEC-HPLC system of the first mobile phase and the SEC-HPLC system of the second mobile phase, collect the effluent during the target protein peak period and weigh it to obtain the mass of the first protein solution and the mass of the second protein solution; without injection, turn on the SEC-HPLC systems of the first mobile phase and the second mobile phase, collect the effluent during the target protein peak period and weigh it to obtain the mass of the first solution and the mass of the second solution.

[0036] Step 4: Obtain the mass difference of the first cation caused by the protein based on the difference between the mass of the first solution and the mass of the first protein solution; obtain the mass difference of the second cation caused by the protein based on the difference between the mass of the second solution and the mass of the second protein solution; obtain the mass difference of the cations at the first molar concentration based on the difference between the mass difference of the first cation and the mass difference of the second cation.

[0037] Step 5: Change the first molar concentration in Step 2 to different molar concentrations, and repeat the steps of Steps 2 to 4 to obtain the mass differences of the cations at different molar concentrations.

[0038] A method for calculating the protein mass based on a protein mass calculation model, the method comprising:

[0039] Obtain at least two molar concentrations and their corresponding cation mass differences;

[0040] The at least two molar concentrations and their corresponding cation mass differences are input into a protein mass calculation model to obtain the protein mass, and the protein mass calculation model is constructed based on the method described above.

[0041] A method for calculating protein content based on a protein mass calculation model, the method comprising:

[0042] Obtain a sample to be tested;

[0043] Use the method for calculating protein mass based on the protein mass calculation model to obtain the protein mass in the sample to be tested;

[0044] Obtain the protein content based on the ratio of the protein mass to the mass of the sample to be tested.

[0045] The second aspect of the present application discloses a construction system for a protein mass calculation model based on cation mass difference, comprising:

[0046] Acquisition module 201: Obtain the cation mass difference measured for the target protein in different cation solutions with at least two equimolar concentrations;

[0047] Factor extraction module 202: Extract the double electric sphere concentration factor of the target protein based on the at least two molar concentrations, and the double electric sphere concentration factor is determined by the molar concentration, the protein average charge constant, the protein ion displacement constant, and the applicable type constant;

[0048] Solution module 203: Solve the slope parameter and intercept parameter of the mathematical regression model of the double electric sphere concentration factor and the cation mass difference by fitting based on at least two molar concentrations and their corresponding cation mass differences, wherein the intercept parameter represents the cation mass difference when the molar concentration is equal to the average charge constant of the target protein, and the slope parameter represents: the change in the cation mass difference when the molar concentration is twice the multiple of the average charge constant of the target protein compared to the equal cation mass difference;

[0049] Calculation and output module 204: Calculate the mass of the target protein based on the slope parameter and the intercept parameter, and the calculation basis is: the product of the applicable constant power of the intercept parameter and the slope parameter, and the dimension is reduced to the mass unit by taking the square root of the product.

[0050] The third aspect of the present application discloses a computer device, the device comprising: a memory and a processor; the memory is used to store program instructions; the processor is used to call the program instructions, and when the program instructions are executed, it is used to execute the steps of the method described above.

[0051] A fourth aspect of the present application discloses a computer-readable storage medium, on which a computer program is stored, and when the computer program is executed by a processor, the steps of the above method are implemented.

[0052] A fifth aspect of the present application discloses a computer program product, including a computer program, and when the computer program is executed by a processor, the steps of the above method are implemented.

[0053] The present application has the following beneficial effects:

[0054] (1) The present application first proposes to prepare a mobile phase by using two different ionic solutions, measure the cation mass difference, and then input the cation mass difference into a system of equations constructed based on a mathematical model of the cation mass difference and the double electric sphere concentration factor to solve for protein-related parameters, and further solve for the protein mass and protein content;

[0055] (2) Implementing the method for constructing a protein mass calculation model based on the cation mass difference proposed in the present application in code can quickly calculate the protein mass from the measured cation mass difference, achieving convenient, fast, and highly available calculation; and the results obtained by calculation are highly consistent with the results of protein content obtained by using conventional protein content calculation methods, proving the effectiveness and practicality of the method of the present application;

[0056] (3) Based on the method of the mathematical model proposed in the present application, compared with the problem that it is difficult to eliminate the error of the reference material, the more experimental groups with different molar concentrations in the present application, the more accurate the parameters obtained by optimization after fitting, making the method of the present application more accurate and effective when overcoming the problem that it is difficult to eliminate the error of the reference material;

[0057] (4) The present application proposes a method that is completely different from the technical concept of the prior art from the source. Generally speaking, the present application solves an entire technical problem of protein content measurement. However, in the process of solving this entire technical problem, the inventor overcomes technical obstacles in multiple steps and finally forms an overall technical solution. Among them, the novelty of each step has its independence. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those skilled in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0059] Figure 1 It is a schematic flowchart of the method provided in the first aspect of the embodiment of the present invention;

[0060] Figure 2 It is a schematic diagram of a program product provided in the second aspect of the embodiments of the present invention;

[0061] Figure 3 It is a schematic diagram of a computer device provided in the embodiments of the present invention;

[0062] Figure 4 It is a schematic diagram of the architecture of an exemplary computing device provided in the embodiments of the present invention;

[0063] Figure 5 It is a schematic diagram of a storage medium provided in the embodiments of the present invention;

[0064] Figure 6 It is the protein peak appearance time of two different protein solutions measured by SEC-HPLC provided in the embodiments of the present invention;

[0065] Figure 7 It is a schematic diagram of the hydration layer and double electric layer structure of a protein solution provided in the embodiments of the present invention;

[0066] Figure 8 It is a schematic diagram of the double electric layer structure of another protein solution provided in the embodiments of the present invention;

[0067] Figure 9 It is a schematic diagram of calculating the protein content provided in the embodiments of the present invention, where P in the figure represents protein; Figure 10 It is a schematic diagram of the compliance of the measured cation mass difference with the salt molar concentration, the salt molar concentration with the mathematical model, and the cation mass difference with the mathematical model provided in the embodiments of the present invention;

[0068] Figure 11 It is a schematic diagram of deriving the conversion coefficient f provided in the embodiments of the present invention;

[0069] Figure 12 It is an application schematic diagram of a method for calculating protein mass based on a protein mass calculation model provided in the embodiments of the present invention;

[0070] Figure 13 It is the protein peak appearance time of two different protein solutions measured by another SEC-HPLC provided in the embodiments of the present invention;

[0071] Figure 14 It is a schematic diagram of the compliance of the measured cation mass difference with the salt molar concentration, the salt molar concentration with the mathematical model, and the cation mass difference with the mathematical model of another measurement provided in the embodiments of the present invention;

[0072] Figure 15 It is an application schematic diagram of another method for calculating protein mass based on a protein mass calculation model provided in the embodiments of the present invention. Detailed implementation manners

[0073] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0074] In some processes described in the specification, claims and above-mentioned accompanying drawings of the present invention, a plurality of operations appear in a specific order. However, it should be clearly understood that these operations may not be executed in the order in which they appear herein or may be executed in parallel. The serial numbers of the operations, such as S101, S102, etc., are only used to distinguish different operations, and the serial numbers themselves do not represent any execution order. In addition, these processes may include more or fewer operations, and these operations may be executed in sequence or in parallel. It should be noted that the descriptions such as "first" and "second" in this article are used to distinguish different messages, devices, modules, etc., and do not represent a sequence, nor do they limit that "first" and "second" are of different types.

[0075] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work belong to the scope of protection of the present invention.

[0076] Figure 1 It is a schematic diagram of a method for constructing a protein mass calculation model based on the cation mass difference provided by an embodiment of the present invention. Specifically, the method includes the following steps:

[0077] S101: Obtain the cation mass difference measured for the target protein in different cation solutions with at least two equimolar concentrations;

[0078] S102: Extract the double electric sphere concentration factor of the target protein based on the at least two molar concentrations, and the double electric sphere concentration factor is determined by the molar concentration, the protein average charge constant, the protein ion exclusion constant, and the applicable type constant;

[0079] S103: Fit a mathematical regression model of the double electric sphere concentration factor and the cation mass difference based on at least two molar concentrations and their corresponding cation mass differences, and solve for the slope parameter and intercept parameter of the mathematical model. The mathematical regression model includes a slope parameter and an intercept parameter;

[0080] S104: Calculate the protein mass based on the slope parameter and the intercept parameter.

[0081] The main steps of the technical solution provided by this application include:

[0082] I. Determine the time period during which the target protein peak appears through preliminary experiments

[0083] In some embodiments, a sodium sulfate solution is prepared as the mobile phase. After pretreating the sample, it is loaded into the SEC-HPLC instrument, and the start and end times for collecting the effluent during the subsequent experiment are determined based on the time when the target protein peak appears in the chromatogram.

[0084] In some embodiments, a potassium sulfate solution is prepared as the mobile phase. After pretreating the sample, it is loaded into the SEC-HPLC instrument, and the start and end times for collecting the effluent during the subsequent experiment are determined based on the time when the target protein peak appears in the chromatogram.

[0085] As Figure 6 shown, the collection period for all proteins determined through preliminary experiments is a 6-minute interval from the 5th minute to the 11th minute.

[0086] In some embodiments, if it is desired to measure the start and end times for collecting the target protein, as Figure 6 shown, if it is desired to measure the content of the main peak protein, the collection period is determined to be from the 8.5th minute to the 11th minute.

[0087] II. Experimentally collect the effluent, weigh it, and calculate (using a high-precision balance for weighing)

[0088] 2.1 Prepare the sample to be measured, sodium salts, and potassium salts;

[0089] 2.2 Prepare the mobile phase: Use sodium sulfate solution and potassium sulfate solution with equal molar concentrations to prepare the mobile phase respectively. For example, first prepare sodium sulfate solution and potassium sulfate solution with a molar concentration of 0.2 M;

[0090] 2.3 Prepare the sample for loading

[0091] If the sample to be measured is solid, it is diluted with physiological saline, or it can also be diluted with water or a solution of the mobile phase to obtain a liquid sample of the sample;

[0092] If the sample is liquid, the buffer system can be replaced or other pretreatment operations can be performed. For example, the protein solution of the sample to be measured is diluted with physiological saline to approximately 20 mg / ml.

[0093] This step (2.3) can adopt the sample processing methods of existing technologies in the field.

[0094] Only for the convenience of finally obtaining the corresponding protein content in the sample based on the calculated protein mass, it is necessary to record the mass of the initial sample to be measured.

[0095] 2.4 Collect and weigh using the SEC-HPLC system (core step)

[0096] 2.4.1 Collection of the first group

[0097] Load the sample into the SEC-HPLC system with a sodium sulfate solution mobile phase for detection and collect the effluent from the 5th minute to the 11th minute to obtain the effluent of the sodium salt protein solution; pay attention to the treatment of the liquid droplets at the tube head during collection;

[0098] Specific step example: Inject 100 μL, collect all protein peaks, and precisely collect the protein effluent (all) in the 6-minute period;

[0099] Change the mobile phase to a precisely prepared 0.2 M K 2 SO4 solution (the salt needs to be of constant weight), and conduct another experiment to collect the effluent of the potassium salt protein solution; Note: Repeat the above steps of sample loading - collection when changing the buffer system;

[0100] Meanwhile, it is necessary to collect the effluent of the sodium salt solution in the 6-minute period of the SEC-HPLC system with a sodium sulfate solution mobile phase without sample loading;

[0101] Collect the effluent of the potassium salt solution in the 6-minute period of the SEC-HPLC system with a sodium sulfate solution mobile phase without sample loading;

[0102] Through the above steps, we have collected the effluent of the sodium salt protein solution, potassium salt protein solution, sodium salt solution, and potassium salt solution at a molar concentration of 0.2 M;

[0103] Use a high-precision balance to weigh and obtain the masses of the effluent of the sodium salt protein solution, potassium salt protein solution, sodium salt solution, and potassium salt solution respectively.

[0104] In some embodiments, the high-precision balance used is the Sartorius MSA125P balance (one in a hundred thousand) from Sartorius, Germany.

[0105] 2.4.2 Collection of the above four solutions at different molar concentrations and weighing

[0106] Change the mobile phase to solutions of other precisely prepared molar concentrations, such as 0.4 M, 0.3 M, 0.1 M, and 0.05 M Na 2 SO4 solutions (the salts need to be of constant weight) and K 2 SO4 solutions, and repeat the experiment.

[0107] The usage specifications of the SEC-HPLC system in the above collection process are consistent with the conventional operations. Only a brief description is provided here. For the parts not involved, please refer to the conventional operations in the field.

[0108] In some embodiments, the SEC-HPLC system conditions guarantee for scheme detection include:

[0109] ① Conduct HPLC calibration: Ensure that the flow rate: RSD < 2%; injection volume: RSD < 2%;

[0110] ② Calibrate the high-precision balance used for weighing; Standard operating procedures are required during weighing;

[0111] ③ The chromatographic column used in the liquid phase method is a TSK-G3000SW gel chromatographic column; The detection wavelengths used for the sodium sulfate solution and potassium sulfate solution are 280 nm and 214 nm respectively (as Figure 6 shown);

[0112] ④ Flow rate 1 mL / min; Column temperature: 25 °C; Sample cell temperature: 10 °C; Injection volume: 100 μL.

[0113] In some embodiments, in order to reduce the weighing error, conduct the above experimental weighings multiple times, and use the average value of the multiple collected weighings as the mass of the effluent of the sodium salt protein solution, the mass of the effluent of the potassium salt protein solution, the mass of the effluent of the sodium salt solution, and the mass of the effluent of the potassium salt solution (the total mass of the collected liquid in Table 1). In a specific embodiment, the data obtained by weighing the finally collected liquid in the experiment are shown in Table 1:

[0114] Table 1 Experimental data collection of sodium-potassium solutions with equimolar concentration of Sample A

[0115]

[0116]

[0117] Table note: The total weight (mg) of the collected liquid is the mass (or average mass) of the effluent of the four solutions; P_na represents the total mass of the liquid collected within the protein peak appearance time after adding the test sample to the SEC-HPLC system with Na 2 SO 4 as the mobile phase, that is, the mass of the effluent of the sodium salt protein solution; P_K represents the total mass of the liquid collected within the protein peak appearance time after adding the test sample to the SEC-HPLC system with K 2 SO 4 as the mobile phase, the mass of the effluent of the potassium salt protein solution; Na2SO 4 represents the total mass of the liquid collected within the protein peak appearance time of the SEC-HPLC system with Na 2 SO 4 as the mobile phase without adding a sample, that is, the mass of the effluent of the sodium salt solution; K 2 SO 4 represents the total mass of the liquid collected within the protein peak appearance time of the SEC-HPLC system with K 2 SO 4The total mass of the liquid collected by the mobile phase SEC-HPLC system during the protein peak appearance time, that is, the mass of the potassium salt solution effluent, and the differences in the table are all absolute values of the differences.

[0118] III. Ion mass difference calculation (core principle)

[0119] Figure 9 The steps of ion mass difference calculation are shown and outlined as follows: Figure 9 The difference between A and Figure 9 C gives Figure 9 E; Figure 9 The difference between B and Figure 9 D gives Figure 9 F; Figure 9 The difference between E and Figure 9 F gives Figure 9 G. The principle of this calculation step involves the physicochemical structure of proteins, which is the creative labor result of the applicant. To enable those in the field to understand Figure 9 the basis of the calculation process shown, this part makes the following explanations of Figure 9 the calculation process with specific examples:

[0120] First of all, those in the field can know that proteins have a hydration layer / hydration sphere and a double electric layer / double electric sphere structure. Figure 7Schematic diagram of the hydration layer (Bulk Water) and double - layer structure of a protein solution provided by an embodiment of the present invention; most globular proteins have many polar groups on their surfaces, which are easy to adsorb water molecules to form a hydration layer. It not only makes the protein soluble in water but also isolates the protein, making it not easy to precipitate. Generally, each gram of protein can adsorb 0.3 to 0.5 grams of water. The hydration layer is closely related to the biological function of proteins. In the absence of water, almost all biological macromolecules are inactive. The hydration layer is particularly important for the stability of protein structure and the function of specific sites. Research shows that the water molecules in the hydration layer are ordered and also dynamically changing. Rigid water molecules hinder the function of proteins, while mobile and less rigid water structures are beneficial to their activity; proteins are amphoteric electrolytes, and the dissociable groups in the molecule are mainly side - chain groups, including terminal amino and carboxyl groups. Proteins also have an isoelectric point, that is, the pH value at which the net charge carried is zero. The isoelectric points of most proteins are slightly acidic to neutral, about 5. The isoelectric point of a protein is mainly determined by the side - chain groups in the peptide chain. Most dissociable groups in a protein are distributed on the molecular surface, but there can also be a small number of groups buried inside the molecule, which do not contribute to the isoelectric point. The pK values of dissociable groups in proteins are close to those of the corresponding amino acids but are not exactly the same. When a protein is at its isoelectric point, the net charge is zero, so there is no repulsion between like charges, and thus it is unstable, has the lowest solubility, and is easy to aggregate and precipitate. The dissociable groups in a protein can combine with ions in the solution, so the isoelectric point is actually related to the ionic composition of the solution. In the absence of other salts, the pH at which the number of protons dissociated by the proton donor of the protein is equal to the number of protons combined by the proton acceptor is called the plasma point. Strictly speaking, the plasma point of a protein is a constant; when the dissociable groups on the protein molecular surface carry the same charge, they can form a stable double - layer ( Figure 7 The + - shown represents charge), increasing the stability of the protein. This is also one of the reasons why proteins can form stable colloids. Due to the existence of the hydration layer and double - layer of proteins, the hydration sphere (inside the hydration layer), with a density similar to that of the surrounding water, including the volume of the protein within the double - layer changes with the change of the external salt - ion concentration; in the double - layer, the salt - ion concentration changes with the change of the external salt - ion concentration and the change of the interaction with the protein charge.

[0121] As Figure 9 shown, first, the mass difference of the sodium - salt solution caused by the protein (including the mass difference of partial sodium ions, anions, and the water displaced by the ions) is calculated based on the mass difference between the mobile phase of the sodium - sulfate solution of the sample and the effluent of the sodium - sulfate solution, and the mass difference of the potassium - salt solution caused by the protein (including the mass difference of partial potassium ions, anions, and the water displaced by the ions) is calculated based on the difference between the mobile phase of the potassium - sulfate solution of the sample and the potassium - sulfate solution, that is Figure 9 The difference between A and Figure 9 C is Figure 9E: This is because when protein is added to a sodium sulfate solution, compared with the sodium sulfate solution, the volume of the protein displaces the sodium sulfate solution. For example, when an iron ball is placed in a basin full of water, the volume of the iron ball displaces an equal volume of water molecules, causing the water to overflow. However, this is because the iron ball replaces the original volume of water, and the weight of the basin will change now. The volume of the protein displacing the sodium sulfate solution in this application is similar; the protein displaces the original volume of the sodium sulfate solution in terms of volume. At the same time, due to the existence of the double electric layer, free sodium ions and sulfate ions in the sodium sulfate solution will be adsorbed on the double electric layer of the protein, which is equivalent to displacing the water molecules that should have been in this position, as Figure 9 shown in E, the anions on the outer side of the double electric layer adsorb sodium ions and displace water molecules and the sulfate ions corresponding to the free sodium ions (for simplicity, the sulfate ions are not marked on the figure). The anions on the inner side of the double electric layer displace sodium ions (charge repulsion, for simplicity, to represent sulfate ions), and adsorb water molecules (hydration layer); similarly, for the potassium sulfate solution, Figure 9 the difference between B and Figure 9 D is Figure 9 F.

[0122] Furthermore, Figure 9 the difference between E and Figure 9 F is Figure 9 G: Since Figure 9 the anion concentrations in E and Figure 9 F are the same (for potassium sulfate solution and sodium sulfate solution with equal molar concentrations, based on the same structure of the protein double electric layer, the concentration of free anions is the same), so after subtraction, the mass of the protein and the mass of the anions (sulfate ions) are eliminated. The resulting difference is essentially the mass difference of the cations and the mass difference of the water. For simplicity, it is expressed as the mass difference of the cations. The essence of this mass difference is the difference in the adsorbed or displaced sodium ions and potassium ions determined by the structure and properties of the protein: on the outer side of the double electric sphere, the mass difference is the mass difference of the adsorbed sodium ions and potassium ions (including the mass difference of the displaced water), and on the inner side of the double electric sphere, the mass difference is the mass difference of the displaced sodium ions and potassium ions. In this way, the double-ion mass difference we measured is essentially the mass difference of the cations (sodium and potassium ions) caused by equal amounts of protein (including the mass difference of the ions displacing water), and from Figure 9 G, it can be obtained that this difference is twice the mass difference of the sodium and potassium ions, and the directions on the inner and outer sides of the double electric layer are opposite;

[0123] when the salt molar concentration changes, the salts (sodium salts, potassium salts) adsorbed and displaced inside and outside the double electric layer are different Figure 10 As shown in A, the relationship curve between the salt molar concentration and the sodium-potassium mass difference obtained by the final weighing is similar to a parabola,

[0124] By measuring based on different molar concentrations, the protein mass in the sample can be inversely deduced from the ion mass difference determined by the protein properties and structure.

[0125] Taking the case when the molar concentration of TEST1 in Table 1 is 0.4M as an example:

[0126] Na 2 SO 4 The mass of the collected liquid is 6.28804mg, the mass of the P_na collected liquid is 6.28131mg, and the mass difference is 6.28804 - 6.28131 = 0.00673mg.

[0127] The mass of the K2SO4 collected liquid is 6.31077mg, the mass of the P_K collected liquid is 6.29111mg, and the mass difference is 6.31077 - 6.29111 = 0.01966mg.

[0128] Finally, by further taking the difference, the mass difference between sodium and potassium ions and the mass difference of water molecules are 0.01966 - 0.00673 = 0.1293mg.

[0129] Taking Figure 1 the mass differences under 5 different equimolar concentrations in

[0130] Table 2 is obtained as follows:

[0131]

[0132]

[0133] IV. Parameter Solving

[0134] The mass difference between sodium and potassium ions (Na + -K + +△mH 2 O) finally calculated in Table 2 conforms to the following mathematical model ( Figure 10 shown in A):

[0135]

[0136] where a≠0, C i ≠0, n∈[-11,11]; C iIt represents the salt molar concentration of the solution, a represents the average charge concentration constant of the protein, and this constant represents the ability of the protein to adsorb Na ions on the double electric layer as described above; n represents the relationship between the ions on the double electric layer of the protein and the salt ions displaced by the corresponding space of the double electric layer; a and n can be determined for a specific target protein because these two values are related to the function and structure of the protein itself. For example, for protein HAS: a = 0.167; n = 0.398.

[0137] Among them, the average charge concentration constant a can be understood as the interaction between the protein and the surrounding charge concentration, and this value is related to the protein structure. For example, if a protein has a charge at a certain point, the charge intensity is fixed. That is to say, for a determined protein, that is, the spatial structure of the protein is determined, then the charge intensity on the protein surface is fixed, and a measures the average number of charges on the protein surface.

[0138] In a sense, the molar concentration characterizes the distance between charges; by changing the molar concentration, the values of a and n can be calculated.

[0139] The value of b represents the difference in cation mass and water mass corresponding to 1-fold protein mass (concentration), and k represents the change in cation mass and water mass caused by 1-fold protein mass (concentration). For the sake of simplicity, it is expressed that k represents the change in cation mass caused by 1-fold protein mass, and b represents the difference in cation mass corresponding to 1-fold protein mass;

[0140] Since the available data for a and n in the prior art in this application is missing, this application maintains a lookup table for a and n of proteins (shown in Table 3). When initially measuring a new protein, at least four different molar concentrations are required to obtain the four parameters k, b, a, and n in formula (1); however, since a and n are determined by the properties of a specific target protein and are not affected by the protein content in the sample, after their values are obtained, in subsequent measurements of the protein content of the same protein or the protein component content of the same drug, it is not necessary to repeat the solution of a and n. The protein mass of the sample can be measured and calculated more quickly through the difference in cation mass at two different molar concentrations;

[0141] Table 3 Protein Constant Table

[0142] Protein Name a n HAS 0.167 0.398 G-HAS 0.203 1.641

[0143] For the a and n tables of the proteins not yet disclosed in this application; when measuring for the first time, at least four sets of equimolar concentrations are required to weigh the mass difference between two cations to obtain four unknown parameters; when measuring again, the weighing steps can be simplified according to the already calculated a and n values, and only two sets need to be weighed for parameter solving, and only k and b are solved. Although when measuring less than four sets, the values of k and b can be obtained by eliminating a and n during the solving process, or by iterative optimization, the accuracy at this time is not high. cv

[0144] Figure 10 As shown in B, the relationship between different salt molar concentrations and Factor is shown. Factor is the factor after k in formula (1) and is expressed as:

[0145]

[0146] Since the values of a and n are determined for a specific protein, it can be seen that Figure 10 B and Figure 10 the trend of the distribution of the measurement points in A is consistent;

[0147] Figure 10 C shows the relationship between Factor and the cation mass difference; that is, formula (1) is converted into a regression line for Factor, expressed as formula (3). It can be seen that the measured mass difference and Factor conform to a linear relationship; based on this linear relationship, the parameters k and b can be solved;

[0148] |m i | = k·Factor + b (3)

[0149] The value of k represents the slope of the conformity between the mass difference |m i | and the mathematical model (formula (3)), and the value of b represents the intercept of the conformity between the mass difference |m i | and the mathematical model (formula (3)).

[0150] In formula (1), |m i | is the mass difference between sodium and potassium ions and the mass difference of water molecules at different molar concentrations calculated through experimental measurements in Table 2,

[0151] As mentioned above, when a and n are unknown, at least four different molar concentrations C i and m i are required to solve the parameters k, b, a, n; as Figure 10 shown, based on the measured mass difference m i (where i = 1, 2,..., 5) represents the mass difference m measured at 5 different molar concentrations i, after substituting into the mathematical model and solving, we get: k = 0.000334, b = 0.000335; (when the number of measurement groups is more than the number of parameters, the optimal solution is obtained through model fitting optimization, and the commonly used optimization method is least squares optimization).

[0152] V. Calculate the protein mass in the sample according to the obtained parameters

[0153] Regarding the calculation result of Table 2 (Na + -K + +△m H2O ) as a virtual ion "∑△", the protein is suspended in the solution composed of this virtual ion with equal density, and the mass of the virtual ion displaced by the protein and the mass difference of the virtual ion within the double electric layer ( Figure 9 shown in G) is twice the mass of the virtual ion "∑△".

[0154] As shown in Figure 11, by normalizing the volume of the virtual ion, it can be obtained that the k value is the cube of the virtual ion mass (b) converted from the actual mass of the protein and the conversion coefficient (f); the virtual ion mass here is the mass difference between sodium and potassium ions and the mass difference of water molecules obtained in Table 2; that is, the b value is: the virtual ion mass converted from the actual mass of the drug protein in the mobile phase;

[0155] Figure 11 A indicates k; Figure 11 B indicates b; Figure 11 C indicates m p , that is Figure 11 A represents the composition factors of k under 1-fold protein mass, including the second or third power of b and f, and Figure B represents the corresponding cation mass difference under 1-fold protein mass; Figure 11 C is 1-fold protein mass converted by f in b and k;

[0156] m p = b·f, where m p represents 1-fold protein mass, and f is the conversion coefficient that maps the cation mass difference corresponding to 1-fold protein mass to 1-fold protein mass; among them, the conversion coefficient f can be calculated from k and b:

[0157]

[0158] It can also be expressed as: k = b·f 3 ;

[0159] |m i | and b have the dimension of mass unit gram (g), C i and a have the dimension of molar concentration unit (mol / L), and f and n are conversion and adjustment coefficients without dimension;

[0160] Then the mass m of the target protein p has multiple representations:

[0161] or or m p = b·f or

[0162] Thus, the mass m of the target protein in the drug of the preparation sample is obtained p , so the protein content is obtained by multiplying the ratio of the mass of the target protein to the mass or volume of the drug by 100%. Figure 12 shows the protein content results measured based on five different molar concentrations. It must be stated here that Figure 12 the code of the shown calculation software is the code implementation of the construction method of the protein mass calculation model based on the cation mass difference of this application.

[0163] In some embodiments, the protein content obtained by comparing with a reference substance using the current method is 0.002 g, and the protein content calculated by the method of this application is 0.000335 g / mL × 6 mL( Figure 12 ), that is, the protein content is 0.002008 g (the decimal places of this application retain multiple decimal places of weighing, and the weighing error is reduced by taking the average value through multiple measurements).

[0164] It can be seen from this that this method is practical and can obtain good results.

[0165] After arranging the relationship shown in Figure 11 and the expression of the target protein content, the following conversion formula is obtained:

[0166]

[0167] where m i represents the ion mass difference at the i-th molar concentration, i = 1, 2,..., T; T represents the experimental results of a total of T groups of different molar concentrations;

[0168] a ≠ 0, Ci ≠ 0, k ≠ 0, n ∈ [-11, 11]. If Ci = a, |Ci - a| / (Ci - a) = 1

[0169] After arrangement, the relationship between the measured m i and the target protein mass m p is expressed as follows:

[0170]

[0171] After conversion, it is:

[0172]

[0173] a ≠ 0, Ci ≠ 0, k ≠ 0, n ∈ [-11, 11]. If Ci = a, |Ci - a| / (Ci - a) = 1

[0174] VI. Experimental Calibration

[0175] The above method of this application has undergone rigorous theoretical derivation, and its measurement effect has been verified by experimental results. The inventors have conducted multiple repeated tests. The theoretical value of k is greater than 0. However, in some embodiments, the k calculated by the above steps is less than 0, resulting in the inability to obtain the accurate protein mass by the above steps. After analyzing the physicochemical structures of these special proteins, the inventors believe that the proteins with double electric spheres applicable to the above calculation process have a relatively large volume, which conforms to the theory of protein displacing water, and it is a situation dominated by the three-dimensional volume conversion coefficient. Therefore, the conversion coefficient is obtained through volume normalization, and the conversion coefficient has a cubic relationship: k = b·d 3 ;

[0176] When the structure of the protein is relatively loose, the conversion coefficient is dominated by the square at this time, and the mathematical model at this time is actually an area mathematical model; the above-mentioned is a volume mathematical model:

[0177] When k < 0 calculated based on the volume mathematical model, the mathematical model for updating parameters is an area mathematical model:

[0178]

[0179] The calculation method of protein mass is:

[0180]

[0181] The experiment verifies the effectiveness of the calculation in this calibrated case.

[0182] If the protein structure is relatively tight, it occupies a relatively small space. Then the distance between the double electric spheres of the protein and the surface layer of the protein structure is relatively far, which can be understood as a structure with a small core and relatively large double electric spheres. In this case, due to the relatively large double electric spheres, using the volume mathematical model conforms to the theoretical assumption, and this theoretical assumption has been verified by experimental data.

[0183] However, the situation where k < 0 obtained based on the volume mathematical model is as follows: the structure of the protein is relatively loose, and its double electric layer is very close to the protein itself, that is to say, almost all of the double electric spheres are a relatively large protein core. Therefore, in the above calculation process, it is no longer the large double electric spheres that play a dominant role. After analyzing the structure of this loose protein and its double electric sphere structure, we have corrected and obtained the area mathematical model of the loose protein.

[0184] That is to say, when k calculated based on the volume model is positive, the calculation is based on the volume displacement; the conversion coefficient is also converted based on the volume;

[0185] When k calculated based on the volume model is positive and K is negative, the displacement is calculated based on the square.

[0186] Since the k value currently adopted in this application first calculates the k value using the volume mathematical model, if the obtained k value is less than 0, then the area mathematical model is used to recalculate.

[0187] In some embodiments, this application realizes the classification judgment of the applicable area model or volume model for protein content determination according to the protein structure by collecting protein data and combining protein performance structures for modeling analysis and prediction.

[0188] So far, this application has constructed a complete method for solving protein content based on cation mass; and its accuracy and effectiveness have been repeatedly verified by experiments.

[0189] In another embodiment, the collection period of the protein peak obtained through preliminary experiments is: 5 - 12 min ( Figure 13 as shown); the measured mass is shown in Table 4:

[0190] Table 4 Experimental data collection of sodium-potassium solution with equimolar concentration of sample B

[0191]

[0192]

[0193] The relationships between the cation mass difference calculated based on the mass in Table 4, the salt molar concentration and the mathematical model, and the factor and the mass difference are as Figure 14 shown:

[0194] The relevant constants of protein G-HAS are: a = 0.203; n = 1.641;

[0195] The calculated k and b values are as Figure 15 shown, which are 0.001277 and 0.000130 respectively;

[0196] Figure 15 The calculation results of the software product of the calculation method are shown as follows: the protein mass is 0.001951 g; the protein concentration is: 0.0002779 g / mL, which is close to the protein mass of 0.002 g measured by other methods, and the decimal places of this application are more accurate.

[0197] In one embodiment, the target protein measured in this application has been measured before, so the parameters a, n, and p are all known. After solving for k and b by measuring the cation mass difference at two different equimolar concentrations in this application, the protein content is successfully obtained.

[0198] In one embodiment, the target protein measured in this application has not been measured before, and the parameters a, n, and p are all unknown. By measuring the cation mass difference at four different equimolar concentrations in this application, first, a, n, k, and b are solved based on the volume mathematical model, and k > 0. Therefore, it is determined that p = 3, and the protein content is obtained by substituting it into the calculation formula.

[0199] In one embodiment, the target protein measured in this application has not been measured before, and the parameters a, n, and p are all unknown. By measuring the cation mass difference at four different equimolar concentrations in this application, first, a, n, k, and b are solved based on the volume mathematical model, and k < 0. Therefore, it is determined that p = 2, and the protein content is obtained by substituting it into the calculation formula based on the area mathematical model.

[0200] In one embodiment, the target protein measured in this application has not been measured before, and the parameters a, n, and p are all unknown. However, through the analysis of the protein structure in this application, it is determined that p = 2. By measuring the cation mass difference at four different equimolar concentrations, a, n, k, and b are solved based on the area mathematical model, and the protein content is obtained by substituting it into the calculation formula based on the area mathematical model.

[0201] In some embodiments, the structure and parameter p of the protein that has been tested in this application are input into the machine learning model to train the applicable constant discrimination model, which is used for the judgment of the protein applicable model and subsequent solution.

[0202] In one embodiment, the target protein measured in this application has been measured before, so the parameters a and n are unknown. By judging the applicable type constant, it is obtained that p = 3. After solving for k and b by measuring the cation mass difference at two different equimolar concentrations in this application, the protein mass is calculated. Since a and n are not solved in the calculation process at this time, the result of the protein mass is not as reliable as that calculated based on four groups.

[0203] Figure 3 is a schematic diagram of a computer device provided by an embodiment of the present invention, as Figure 3 shown, the device may include: one or more processors, and one or more memories; wherein, computer-readable code is stored in the memory, and when the computer-readable code is run by the one or more processors, the method described above can be executed.

[0204] The processor in this embodiment may be an integrated circuit chip with signal processing capabilities. The above-mentioned processor may be a general-purpose processor, a digital signal processor (DSP), an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components. It can implement or execute the various methods, operations, and logic block diagrams disclosed in the embodiments of the present disclosure. The general-purpose processor may be a microprocessor, or the processor may also be any conventional processor, etc., and may be of the X86 architecture or the ARM architecture.

[0205] Generally speaking, the various exemplary embodiments of the present disclosure may be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Some aspects may be implemented in hardware, while other aspects may be implemented in firmware or software that can be executed by a controller, a microprocessor, or other computing devices. When aspects of the embodiments of the present disclosure are illustrated or described as block diagrams, flowcharts, or using some other graphical representation, it will be understood that the blocks, devices, systems, techniques, or methods described herein may be implemented as non-limiting examples in hardware, software, firmware, dedicated circuits or logic, general hardware or a controller or other computing devices, or some combination thereof.

[0206] For example, the method or device according to the embodiments of the present disclosure may also be implemented by means of Figure 4 the architecture of the computing device 3000 shown. As Figure 4 shown, the computing device 3000 may include a bus 3010, one or more CPUs 3020, a read-only memory (ROM) 3030, a random access memory (RAM) 3040, a communication port 3050 connected to a network, an input / output component 3060, a hard disk 3070, etc. The storage device in the computing device 3000, such as the ROM 3030 or the hard disk 3070, may store various data or files used for the processing and / or communication of the method provided by the present disclosure and the program instructions executed by the CPU. The computing device 3000 may also include a user interface 3080. Of course, Figure 4 the architecture shown is only exemplary, and when implementing different devices, one or more components shown in the Figure 4 computing device may be omitted according to actual needs.

[0207] The embodiment of the present invention also provides a computer-readable storage medium, such as Figure 5As shown, it is a schematic diagram of a storage medium provided by an embodiment of the present invention. Computer-readable instructions 4010 are stored on the computer storage medium 4020. When the computer-readable instructions 4010 are run by a processor, the method according to the embodiments of the present disclosure described with reference to the above drawings can be executed. The computer-readable storage medium in the embodiments of the present disclosure can be a volatile memory or a non-volatile memory, or can include both volatile and non-volatile memories. The non-volatile memory can be a read-only memory (ROM), a programmable read-only memory (PROM), an erasable programmable read-only memory (EPROM), an electrically erasable programmable read-only memory (EEPROM), or a flash memory. The volatile memory can be a random access memory (RAM), which is used as an external cache. By way of example but not limitation, many forms of RAM are available, such as static random access memory (SRAM), dynamic random access memory (DRAM), synchronous dynamic random access memory (SDRAM), double data rate synchronous dynamic random access memory (DDR SDRAM), enhanced synchronous dynamic random access memory (ESDRAM), synchronous link dynamic random access memory (SLDRAM), and direct memory bus random access memory (DR RAM). It should be noted that the memories of the methods described herein are intended to include but not be limited to these and any other suitable types of memories. It should be noted that the memories of the methods described herein are intended to include but not be limited to these and any other suitable types of memories.

[0208] The embodiments of the present disclosure also provide a computer program product or a computer program. When the computer program is executed by a processor, the steps of the above method are implemented, as Figure 2 shown. The computer program product or the computer program includes:

[0209] Acquisition module 201: Acquire the cation mass difference measured for a target protein in different cation solutions with at least two equimolar concentrations;

[0210] Factor extraction module 202: Extract the double electric sphere concentration factor of the target protein based on the at least two molar concentrations. The double electric sphere concentration factor is determined by the molar concentration, the protein average charge constant, the protein ion displacement constant, and the applicable type constant;

[0211] Parameter solving module 203: Solve the slope parameter and intercept parameter of the mathematical regression model of the double electric sphere concentration factor and the cation mass difference by fitting the at least two molar concentrations and their corresponding cation mass differences. The intercept parameter represents the cation mass difference when the molar concentration is equal to the average charge constant of the target protein, and the slope parameter represents: the change amplitude of the cation mass difference when the molar concentration has a two-fold multiple relationship with the average charge constant of the target protein compared to the cation mass difference when they are equal;

[0212] Calculation output module 204: Calculate the mass of the target protein based on the slope parameter and the intercept parameter. The basis for the calculation is the product of the applicable constant power of the slope-intercept parameter and the slope parameter, and the dimension is reduced to the mass unit by taking the square root of the product.

[0213] It should be noted that the flowcharts and block diagrams in the accompanying drawings illustrate the possible architectures, functions, and operations of systems, methods, and computer program products according to various embodiments of the present disclosure. In this regard, each block in the flowchart or block diagram may represent a module, a program segment, or a part of code that contains one or more executable instructions for implementing the specified logical function. It should also be noted that in some alternative implementations, the functions marked in the blocks may occur in a different order than marked in the accompanying drawings. For example, two consecutive blocks shown may actually be executed substantially in parallel, and they may sometimes be executed in the reverse order, depending on the functions involved. It should also be noted that each block in the block diagram and / or flowchart, and combinations of blocks in the block diagram and / or flowchart, can be implemented by a dedicated hardware-based system for performing the specified functions or operations, or can be implemented by a combination of dedicated hardware and computer instructions.

[0214] Generally speaking, various example embodiments of the present disclosure can be implemented in hardware or dedicated circuits, software, firmware, logic, or any combination thereof. Some aspects can be implemented in hardware, while other aspects can be implemented in firmware or software that can be executed by a controller, a microprocessor, or other computing devices. When aspects of the embodiments of the present disclosure are illustrated or described as block diagrams, flowcharts, or using some other graphical representation, it will be understood that the blocks, devices, systems, technologies, or methods described herein can be implemented as non-limiting examples in hardware, software, firmware, dedicated circuits or logic, general hardware or controllers or other computing devices, or some combination thereof.

[0215] Those skilled in the art can clearly understand that for the convenience and simplicity of description, the specific working processes of the systems, devices, and units described above can refer to the corresponding processes in the foregoing method embodiments, and will not be elaborated herein.

[0216] In several embodiments provided by the present application, it should be understood that the disclosed systems, devices, and methods can be implemented in other ways. For example, the device embodiments described above are merely illustrative. For example, the division of the units is only a logical function division. In actual implementation, there may be other division methods. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the displayed or discussed coupling, direct coupling, or communication connection between each other can be through some interfaces, and the indirect coupling or communication connection of the devices or units can be in electrical, mechanical, or other forms.

[0217] The units described as separate components may or may not be physically separated. The components displayed as units may or may not be physical units, that is, they may be located in one place or distributed to multiple network units. Some or all of the units can be selected according to actual needs to achieve the purpose of the solution of this embodiment.

[0218] In addition, in each embodiment of the present invention, the functional units can be integrated in a processing unit, or each unit can exist physically alone, or two or more units can be integrated in one unit. The above-mentioned integrated units can be implemented in the form of hardware or in the form of software functional units.

[0219] The exemplary embodiments of the present disclosure described in detail above are merely illustrative and not restrictive. Those skilled in the art should understand that various modifications and combinations can be made to these embodiments or their features without departing from the principles and spirit of the present disclosure, and such modifications should fall within the scope of the present disclosure.

Claims

1. A method for constructing a protein mass calculation model based on cation mass difference, characterized in that: Obtaining the cation mass difference of the target protein measured in at least two cation solutions with equimolar concentrations; Extracting double electric sphere concentration factors of target proteins based on at least two molar concentrations, respectively, wherein the double electric sphere concentration factors are determined by the molar concentration, the protein average charge constant, the protein excluded ion constant, and the applicable type constant; A mathematical regression model of the double electric sphere concentration factor and the cation mass difference is fitted based on at least two molar concentrations and their corresponding cation mass differences to obtain the slope parameter and intercept parameter of the mathematical model, wherein the intercept parameter represents the cation mass difference when the molar concentration is equal to the average charge constant of the target protein, and the slope parameter represents: the change in the cation mass difference when the multiple relationship between the molar concentration and the average charge constant of the target protein changes by one time; The mass of the target protein is calculated based on the slope parameter and the intercept parameter. The calculation basis is: the product of the applicable constant power of the intercept parameter and the slope parameter. The mass of the target protein is obtained by taking the root of the product and reducing the dimension to the mass unit.

2. The method for constructing a protein mass calculation model based on cation mass difference according to claim 1, characterized in that: The mass of the target protein is calculated based on the slope parameter, the intercept parameter, and the applicable type generation constant; Optionally, the mathematical regression model is expressed as: m i =k·Factor+b Among them, m i represents the mass difference of the cation with the i-th molar concentration, Factor represents the bi-electrosphere concentration factor, k is the slope parameter, and b is the intercept parameter.

3. The method for constructing a protein mass calculation model based on cation mass difference according to claim 1, characterized in that: The double electric sphere concentration factor is an exponent of the part of the difference between the larger value and the smaller value of the molar concentration and the protein average charge constant that is higher than 1, and the exponential term is determined by the applicable type constant and the protein exclusion ion constant; Optionally, the double electric sphere concentration factor is expressed as: Among them, Factor represents the double electric sphere concentration factor, C i represents the molar concentration, a represents the average charge constant of the protein, n represents the protein expulsion ion constant, and p represents the applicable type constant, which takes a value of 2 or 3; C i >a? C i :a is a ternary operation expression, which means that if the expression on the left side of the question mark is true, then the value or symbol on the left side of the colon is taken, otherwise the value or symbol on the right side is taken; 5-p(C i >a? +:-)n means if C i >a holds, the exponent is 5-p+n, otherwise it is 5-pn; Optionally, if the molar concentration is greater than the protein average charge constant, the double charge sphere concentration factor is the exponential of the ratio of the difference between the molar concentration and the protein average charge constant to the protein average charge constant, with the exponential term being the difference between the applicable type constant and the protein excluded ion constant; Optionally, if the molar concentration is less than the protein average charge constant, the double charge concentration factor is the exponential of the ratio of the difference between the molar concentration and the protein average charge constant to the molar concentration, where the exponential term is the sum of the applicable type constant and the protein excluded ion constant; Optionally, the protein ion exclusion constant and average charge constant are obtained by looking up data from previous experimental records; Optionally, at least four molar concentrations and their corresponding cation mass differences are obtained, at least four double electric sphere concentration factors are extracted, and the slope parameter, intercept parameter, protein ion exclusion constant, and average charge constant are obtained based on the cation differences and double electric sphere concentration factors of at least four molar concentrations.

4. The method for constructing a protein mass calculation model based on cation mass difference according to claim 1, characterized in that: The calculated mass of the target protein is expressed as: Among them, k is the slope parameter, b is the intercept parameter, and p represents the applicable type constant; Optionally, the applicable type constant is obtained based on structural analysis of the protein; Optionally, the applicable type constant is preset to 3, and the slope parameter k is obtained by solving. If k>0, the applicable type constant is confirmed to be 3, otherwise the applicable type constant is modified to 2, and the slope parameter and the intercept parameter are obtained by solving again; Optionally, if the applicable type constant is 3, the protein calculation is expressed as: Among them, b represents the intercept parameter and k represents the slope parameter; Optionally, if the applicable type constant is 2, the protein calculation is expressed as: Among them, b represents the intercept parameter and k represents the slope parameter.

5. The method for constructing a protein mass calculation model based on cation mass difference according to claim 1, characterized in that: The specific steps of obtaining the cation mass difference of the target protein measured in at least two cation solutions with equimolar concentrations include: Step 1: obtaining a protein solution of a sample to be tested, a compound having the same root as the first cation and the second cation; Step 2: setting a first molar concentration, using the compound having the same root as the first cation and the second cation to prepare a first solution and a second solution having the first molar concentration, respectively, as a first mobile phase and a second mobile phase having equimolar concentrations; Step 3: add equal amounts of the protein solution to the SEC-HPLC system of the first mobile phase and the SEC-HPLC system of the second mobile phase, respectively, collect the effluent in the target protein peak period, and weigh them to obtain the mass of the first protein solution and the mass of the second protein solution; start the SEC-HPLC system of the first mobile phase and the SEC-HPLC system of the second mobile phase without adding samples, collect the effluent in the target protein peak period, and weigh them to obtain the mass of the first solution and the mass of the second solution; Step 4: obtaining a first cation mass difference caused by protein based on the difference between the mass of the first solution and the mass of the first protein solution; obtaining a second cation mass difference caused by protein based on the difference between the mass of the second solution and the mass of the second protein solution; obtaining a cation mass difference of the first molar concentration based on the difference between the first cation mass difference and the second cation mass difference; Step 5: Change the first molar concentration in step 2 to a different molar concentration, and repeat steps 2 to 4 to obtain cation mass differences of different molar concentrations.

6. A method for calculating protein mass based on a protein mass calculation model, characterized in that: The method comprises: Obtain at least two molar concentrations and their corresponding cation mass differences; The at least two molar concentrations and their corresponding cation mass differences are input into a protein mass calculation model to obtain protein mass, and the protein mass calculation model is constructed based on the method described in any one of claims 1-5.

7. A method for calculating protein content based on a protein mass calculation model, characterized in that: The method comprises: Obtaining a sample to be tested; Obtaining the protein mass in the sample to be tested using the method of claim 6; The protein content is obtained based on the ratio of the protein mass to the mass of the sample to be tested.

8. A computer device, characterized in that: The device comprises: a memory and a processor; the memory is used to store a computer program; the processor executes the computer program to implement the steps of the method according to any one of claims 1 to 7.

9. A computer-readable storage medium, characterized in that: A computer program is stored thereon, and when the computer program is executed by a processor, the steps of the method according to any one of claims 1 to 7 are implemented.

10. A computer program product, comprising a computer program, characterized in that When the computer program is executed by a processor, the steps of the method described in any one of claims 1 to 7 are implemented.

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