A method for determining the molecular weight of a protein or protein complex based on es-dma-cpc

Abstract

The application discloses a method for determining the molecular weight of protein or protein complex based on ES-DMA-CPC, which comprises the following steps: (1) determining the particle size information of protein with known molecular weight by using ES-DMA-CPC; (2) performing polynomial fitting according to the molecular weight information of the known protein and the particle size information measured by ES-DMA-CPC, and establishing a standard curve of protein molecular weight-particle size fitting; (3) determining the molecular weight of unknown protein or protein complex: analyzing the protein sample to be measured by using ES-DMA-CPC to obtain the particle size information of the sample to be measured, and calculating the molecular weight of the protein or protein complex to be measured according to the established standard curve of protein molecular weight-particle size. The method can be used for determining the molecular weight of protein with complex structure, and can also maintain the stability of the structure of biological complex, and realizes the determination of the molecular weight of protein complex.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of protein molecular weight characterization, and particularly relates to a method for determining the molecular weight of a protein or protein complex based on ES-DMA-CPC. BACKGROUND

[0002] Proteins are important executors and regulators of cell life activities, and are involved in the recognition and transmission of signals in the body, material transport, immune response and other reactions. Among them, the molecular weight of the protein is very important for understanding the structure and function of the protein, protein purification, quality control and disease research. First, by determining the molecular weight of the protein, the purity of the protein can be preliminarily identified; second, the change of the molecular weight indicates the degree of protein degradation and aggregation, and by characterizing the molecular weight of the protein aggregate, the product quality of the drug can be controlled to ensure the stability and uniformity of the quality; in addition, the non-covalent interaction of the protein plays an important role in biology and medicine, such as blood clotting, gene expression, immune recognition, formation of lipid granules and viruses, and by characterizing the molecular weight of the protein complex, the formation process of the complex, protein assembly, stoichiometry and other characteristics can be further understood, which is beneficial to the screening and optimization of drug candidate molecules, accelerates the research and development of drugs, and realizes the targeted treatment of drugs.

[0003] The existing methods for characterizing the molecular weight of proteins and their complexes include mass spectrometry, multi-angle light scattering and mass photometry. Mass spectrometry is a relatively accurate method for molecular weight characterization, but this method has high requirements for the purity of the sample, the range of molecular weight determination is limited, and the non-covalent interaction of the protein may be destroyed in mass spectrometry analysis, making it difficult to determine the complete molecular weight of the complex. Multi-angle light scattering is a method for absolute quantification of protein molecular weight, which can effectively identify aggregates and characterize the molecular weight of biological macromolecules, but multi-angle light scattering is not suitable for determining samples that are difficult to separate and elute on a chromatographic column and have complex components. In addition, mass photometry is a new single-molecule technology that can quantify the mass of a molecule by detecting the interference scattered light of a single particle with an optical detector, and weighing the protein. It plays an important role in the study of antibodies and immune complexes, but this method is not suitable for high-concentration samples containing macromolecular impurities, and is easily disturbed by background signals, and is not suitable for small-molecule protein levels.

[0004] Based on the limitations of the above-mentioned existing methods for characterizing the molecular weight of proteins and protein complexes, it is urgent to develop a high-efficiency and rapid method for characterizing the molecular weight of macromolecular proteins or protein complexes with complex structures. SUMMARY

[0005] The present application aims to provide a method for determining the molecular weight of protein or protein complex based on ES-DMA-CPC. The method uses the electric spray-differential electric mobility-particle counting technology to measure the particle size distribution of a series of protein standard substances with known molecular weight, and obtains the protein molecular weight-particle size fitting curve through the measurement of the standard substances, so as to realize the determination of the molecular weight of unknown protein.

[0006] Specifically, the technical scheme adopted by the present application is as follows:

[0007] A method for determining the molecular weight of protein or protein complex based on ES-DMA-CPC, comprising the following steps:

[0008] (1) Determination of the relationship between protein molecular weight and particle size

[0009] The present application adopts the analysis technology based on electric spray-differential electric mobility-particle counting (ES-DMA-CPC) to measure the particle size of protein standard substances with known molecular weight or high-purity (purity > 90%) protein samples. The electric spray-differential electric mobility-particle counting analysis technology (ES-DMA-CPC) is mainly completed by three parts of instruments, including an electric spray aerosol generator (ES), a differential electric mobility analyzer (DMA) and a condensation nucleus particle counter (CPC). In addition, a charge neutralizer is usually added between the electric spray aerosol generator and the differential electric mobility analyzer to balance the charge distribution of the particles, so that more than 90% of the droplets carry only one single charge, so that the particle migration in the DMA is only related to the size of the particle.

[0010] The process of ES-DMA-CPC for determining the particle size of protein samples is as follows: the known molecular weight protein standard substance is diluted with ammonium acetate buffer solution, the prepared protein sample is injected into the electric spray aerosol particle generator through the sample pump, the atomized high-charge droplets are generated through a 25 μm inner diameter capillary, after the droplets are evaporated and dried, the charged particles pass through the differential electric mobility analyzer, and the particle size distribution of the protein sample is obtained by the particle counter.

[0011] In order to ensure the accuracy of the detection of protein molecular weight, at least three kinds of known molecular weight protein standard substances which can obviously distinguish monomers are selected. The molecular weight difference of the selected several known proteins is at least greater than 15 KDa. When the molecular weight of the protein is similar, the particle size detected by the instrument is similar, which is not conducive to the establishment of the protein molecular weight-particle size curve.

[0012] According to the molecular weight of the known protein standard substance and the particle size information measured by the instrument, the molecular weight and the particle size are fitted by a polynomial, which can be seen from formula (1):

[0013] y=a0+a1x+a2x 2 +a3x3 (1)

[0014] Wherein, x is the particle size of protein standard substance (nm), y is the molecular weight of protein standard substance (KDa). The molecular weight information of known protein standard substance and the particle size information detected by instrument can be fitted using formula (1) to obtain the protein molecular weight-particle size (MW / EMD) fitting curve.

[0015] (2) When the molecular weight of the protein sample to be measured is determined for the known fitting curve:

[0016] According to step (1), the MW / EMD fitting curve of the protein in the range of 17-340 KDa is obtained from the protein standard substance, and when the protein sample with unknown molecular weight is determined, the protein sample to be measured is analyzed by ES-DMA-CPC to obtain the detected particle size distribution, and the molecular weight of the protein sample to be measured is calculated according to the MW / EMD fitting curve.

[0017] In addition, the approximate particle size distribution behavior of the protein sample with known molecular weight in ES-DMA-CPC can also be calculated through the MW / EMD fitting curve, and then it can be inferred whether the protein sample conforms to the basic law of protein.

[0018] When the protein complex sample with unknown molecular weight is determined:

[0019] Select appropriate concentrations of antigen and antibody proteins, and determine the particle size distribution of antigen and antibody protein standard substances according to step (1); analyze the protein complex sample with unknown molecular weight by ES-DMA-CPC to obtain the particle size distribution of the complex; and calculate the molecular weight of the protein complex sample to be measured according to formula (1) from the protein molecular weight-particle size fitting curve.

[0020] In the step (1), the preparation method of the ammonium acetate buffer solution is to take an appropriate amount of ammonium acetate to prepare a 20 mmol / L buffer solution, and adjust the pH of the solution to 8 with ammonia water. The protein sample is diluted with the prepared ammonium acetate buffer solution, and the ammonium acetate buffer solution is filtered 3 times before dilution, and the diluted sample is mainly able to distinguish the existence of monomer in ES-DMA-CPC analysis.

[0021] The sample pump is set to constant flow mode, and the flow rate is set to 0.20 μL / min; the flow rates of dry air and carbon dioxide are 1.2 slpm and 0.1 slpm respectively; the flow rate of DMA sheath gas is set to 15 L / min, and the particle size scanning range is set to the corresponding scanning range; the sampling flow rate of CPC is set to 0.6 L / min.

[0022] Electrospray-differential mobility particle counting (ES-DMA-CPC) is a soft ionization method, which can not dissociate protein and its complex in the process of electrospray, can measure the particle size of protein and its complex completely, has the advantage of high precision molecular weight measurement which mass spectrometry cannot obtain, and can realize the particle size separation of mixed samples, even for complex samples, which makes up the limitations of multi-angle light scattering and mass photometry. On the basis of overcoming the limitations of existing protein and its complex molecular weight detection technology, the ES-DMA-CPC method can realize more accurate protein and its complex molecular weight measurement in a larger range.

[0023] Compared with the prior art, the outstanding effect of the present application is that:

[0024] (1) The present application provides a new protein molecular weight characterization method based on single particle analysis, which can completely measure the particle size of protein and its complex, and can also analyze and measure complex samples.

[0025] (2) The protein molecular weight in the solution can be indirectly measured according to the particle size information of the protein sample by using the method of the present application, and the obtained particle size spectrum can distinguish the slight mass difference of the protein.

[0026] (3) The method of the present application uses a soft ionization method to analyze protein analyte, even if the protein has poor stability, it is not easy to dissociate in the ionization process, and it is very suitable for studying the antigen-antibody interaction, and can realize the molecular weight characterization of a larger range of protein samples such as antigen-antibody complex.

[0027] The method for measuring the molecular weight of protein or protein complex based on ES-DMA-CPC described in the present application will be further described below in combination with the accompanying drawings and specific embodiments. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a polynomial fitting graph of the molecular weight and particle size of protein standard substance.

[0029] Figure 2 It is a particle size spectrum graph of TNF-alpha monoclonal antibody (WT) before and after ultrafiltration.

[0030] Figure 3 It is a control graph of TNF-alpha monoclonal antibody (WT) before and after ultrafiltration.

[0031] Figure 4 It is an ES-DMA detection complex particle size spectrum graph.

[0032] Figure 5 It is an MP detection mass distribution graph. DETAILED DESCRIPTION

[0033] Example 1

[0034] The method of determining the molecular weight of protein by ES-DMA-CPC was used to characterize the molecular weight of TNF-a monoclonal antibody, which proved that the method could realize the determination of the molecular weight of unknown protein.

[0035] I. Experimental instruments and reagents:

[0036] FLOW-EZ microfluidic constant flow pump (Fluigent, France); electrospray aerosol particle generator (TSI, USA, model 3482); electrostatic classifier (TSI, USA, model 3082); nano water-based condensed nucleus particle counter (TSI, USA, model 3788); silica capillary (inner diameter 25 μm, length 4.1 cm, TSI, USA); Amicon Ultra protein concentration tube (Merck, Germany); ultramicro high-precision ultraviolet-visible spectrophotometer Nanodrop 2000 (Thermo Fisher Scientific, USA); mass photometer (Refeyn, UK).

[0037] Ammonium acetate (chromatographic grade, purity ≥ 99.0%, Honeywell, Germany); TNF-a monoclonal antibody (China Institute of Metrology); experimental water is ultrapure water.

[0038] II. Sample preparation and standard solution preparation:

[0039] An appropriate amount of ammonium acetate was prepared into a 20 mmol / L buffer solution, filtered and adjusted to pH 8 with ammonia water. The TNF-a monoclonal antibody was diluted to 0.10 mg / mL with the buffer solution. The protein sample was diluted with the prepared ammonium acetate buffer solution, and it was necessary to ensure that the diluted sample existed mainly in the form of monomer in the ES-DMA-CPC analysis.

[0040] III. Experimental conditions and parameters:

[0041] The experimental procedure was as follows: first, the sample was injected into the electrospray aerosol particle generator by the FLOW-EZ microfluidic constant flow pump, and the atomized high-charge droplets were generated through the 25 μm inner diameter capillary. After the droplets evaporated and dried, the charged particles passed through the DMA nanocolumn, and the particle size distribution of the protein sample was obtained by the particle counter.

[0042] The microfluidic constant flow pump was set to constant flow mode with a flow rate of 0.20 μL / min. Dry air and carbon dioxide gas were used as carrier gas with flow rates of 1.2 slpm and 0.1 slpm, respectively. The DMA sheath gas flow rate was set to 15 L / min, and the particle size scanning range was set to the corresponding scanning range (1.96-64.9 nm). The CPC sampling flow rate was set to 0.6 L / min.

[0043] IV. Determination of the molecular weight of the sample protein:

[0044] (1) Determination of the molecular weight and particle size of the protein standard:

[0045] The particle size distribution information was measured in the ES-DMA-CPC by using the known molecular weight protein standards of 0.05 mg / mL horse heart myoglobin, 0.05 mg / mL bovine serum albumin, 0.05 mg / mL anti-CD20 monoclonal antibody, 0.1 mg / mL fibrinogen, and 0.1 mg / mL NIST 8671 monoclonal antibody. The molecular weight and particle size information of the five protein standards is shown in Table 1.

[0046] Table 1. Particle size determination results of the five protein standards

[0047]

[0048] According to the molecular weight and particle size information of the five protein standards, the molecular weight and particle size were fitted by a polynomial to obtain the fitting equation, which can be seen in Equation (2):

[0049] y = 0.2961x 3 - 3.1595x 2 + 24.096x - 52.428 (2)

[0050] where x is the particle size of the protein standard (nm), and y is the molecular weight of the protein standard (KDa). The equation R 2 is 0.9924, and the fitting degree is good.

[0051] (2) Determination of the molecular weight of the sample:

[0052] In the actual determination, the TNF-α wild type monoclonal antibody cannot distinguish the existence of monomer and oligomer by direct dilution with ammonium acetate buffer, the particle size determination shows obvious peak broadening, deviating from the protein molecular weight-particle size fitting curve; it is suspected that the buffer in the protein solution interferes with the determination of the protein particle size, the sample is treated by ultrafiltration, and the result of the second determination shows that the treated sample can distinguish the existence of monomer and oligomer, and the molecular weight information of the TNF-α wild type monoclonal antibody is calculated according to the protein molecular weight-fitting curve, which can be seen in Table 2, and it is confirmed that the particle size determination result conforms to the basic law of protein. The particle size spectrum and molecular weight corresponding relationship before and after ultrafiltration can be seen in Figures 2-3 .

[0053] Table 2 TNF-α monoclonal antibody molecular weight determination result

[0054]

[0055]

[0056] The determination value of the TNF-α wild type monoclonal antibody by mass photometry (MP) is 114 KDa, and the measurement result of the protein molecular weight characterization based on the ES-DMA-CPC method of the present application is 106.88 KDa, and the RSD of the two methods is less than 5%, which proves that the method can accurately characterize the protein molecular weight.

[0057] Example 2

[0058] The method for determining unknown molecular weight protein based on the ES-DMA-CPC method is used to determine the fusion protein TNF-α antigen, which proves that the method can be used to characterize the protein sample with unknown molecular weight.

[0059] All experimental conditions and parameters are kept consistent with Example 1, and the monomer and dimer particle sizes of the TNF-α antigen with a concentration of 0.06 mg / mL are determined, and the molecular weight information corresponding to the monomer and dimer particle sizes is calculated based on the molecular weight-particle size fitting curve measured in Example 1, and the molecular weight detection result is shown in Table 3.

[0060] Table 3 TNF-α antigen detection result

[0061]

[0062] The theoretical molecular weight of the TNF-α antigen standard substance used is 52.5 KDa, and the measurement result of the method of the present application is 49.59 KDa, and the RSD deviation is 4.03%, which is less than 5%, which proves that the method of the present application can realize the determination of the molecular weight of the fusion protein.

[0063] Example 3

[0064] The method for determining the molecular weight of the protein based on the ES-DMA-CPC method determines the molecular weight of the CD19-FC antigen antibody complex, and proves that the method can accurately characterize the molecular weight of the protein complex.

[0065] The TNF-α wild type monoclonal antibody in Example 1 is replaced by the CD19-FC antigen antibody complex (China Institute of Metrology), and the CD19-FC antigen and the CD19 single-chain antibody are diluted to 0.23 mg / mL and 0.05 mg / mL respectively after ultrafiltration, and then mixed in equal volumes to prepare a mixed solution with a 1:1 molar ratio, and incubated overnight in a 4°C refrigerator, and the other experimental conditions are the same as in Example 1.

[0066] The particle size distribution of the antigen and the antibody is determined by ES-DMA-CPC, and it is determined that it conforms to the basic law of the protein, and then the particle size distribution of the antigen antibody complex is determined, and the specific measurement results are shown in Table 4.

[0067] Table 4: CD19 antigen antibody particle size determination results and corresponding molecular weight

[0068]

[0069]

[0070] The molecular weight MW of the first peak of the antigen antibody complex is 159.91 kDa, which is the peak of the 1:1 combination of the antigen and the antibody, and the molecular weight MW of the second peak is 308.96 kDa, which is the peak of the 2:1 combination of the antigen and the antibody, and the ES-DMA-CPC measurement results are shown in Table 4. Figure 4 At the same time, the mass photometry (MP) is used to analyze and verify the molecular weight of the antigen antibody complex, and the mass photometry determination results are shown in Table 4. Figure 5 .

[0071] Since the mass photometry does not detect the peak of the 2:1 combination of the antigen and the antibody, the peak of the 1:1 combination of the antigen and the antibody is mainly compared and analyzed. The mass photometry determination value of the 1:1 combination peak of the antigen antibody complex standard substance is 163 KDa, the measurement result of the method is 159.91 KDa, and the RSD deviation of the mass photometry determination result is 1.35%, which proves that the method can accurately determine the molecular weight of the protein antigen antibody complex.

[0072] The above examples only describe the preferred embodiments of the present application, and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements of the technical solutions of the present application made by those skilled in the art shall fall within the protection scope determined by the claims of the present application.

Claims

1. A method for determining the molecular weight of a protein or protein complex based on ES-DMA-CPC, characterized in that, The method comprises the following steps: (1) Determination of protein molecular weight-particle size fitting standard curve First, the particle size of a known molecular weight protein standard or a high-purity protein sample is determined by ES-DMA-CPC. The known molecular weight protein standard or the high-purity protein sample is diluted to a suitable measurement concentration with an ammonium acetate buffer solution. The prepared protein sample is injected into an electrospray aerosol particle generator through a sample pump, and atomized high-charge droplets are generated through a 25-μm inner diameter capillary. After the droplets are evaporated and dried, the charged particles are analyzed by a differential mobility analyzer and a particle counter to obtain the particle size distribution of the protein sample. At least three known molecular weight proteins that can significantly distinguish monomers are selected as particle size measurement standards, and the molecular weights of the selected several known protein standards differ by at least 15 Kda. The protein sample is diluted with the prepared ammonium acetate buffer solution, and the concentration of the protein analyte is selected to ensure that the diluted sample can significantly distinguish the presence of monomers in the ES-DMA-CPC analysis. For samples that cannot distinguish the presence of monomers, an ultrafiltration pretreatment is used. According to the molecular weights of at least three known molecular weight proteins or high-purity protein samples and the protein particle size information determined by ES-DMA-CPC, a polynomial fitting is performed on the molecular weight and the particle size to obtain a protein molecular weight-particle size fitting standard curve. (2) Determination of unknown molecular weight protein or protein complex According to the method of step (1), the protein sample is analyzed by ES-DMA-CPC to obtain the particle size distribution of the unknown molecular weight protein sample. According to the protein molecular weight-particle size fitting standard curve obtained in step (1) and the polynomial fitting in step (1), the molecular weight of the protein sample to be measured is calculated.

2. The method for determining the molecular weight of a protein or protein complex based on ES-DMA-CPC according to claim 1, characterized in that: In step (1), the purity of the protein standard and the high-purity protein sample is at least 90%.

3. The method for determining the molecular weight of a protein or protein complex based on ES-DMA-CPC according to claim 2, characterized in that: The soft ionization mode of ES does not destroy the non-covalent bonds in the antigen-antibody complex, and the particle size of the entire complex can be completely measured.

4. The method for determining the molecular weight of a protein or protein complex based on ES-DMA-CPC according to claim 1, characterized in that: In step (1), the sample pump is set to a constant flow mode, the flow rate is set to 0.20 μL / min, the flow rates of dry air and carbon dioxide are 1.2 slpm and 0.1 slpm respectively, the DMA sheath gas flow rate is set to 15 L / min, and the particle size scanning range is set to the corresponding scanning range. The sampling flow rate of CPC is set to 0.6 L / min.

5. The method for determining the molecular weight of a protein or protein complex based on ES-DMA-CPC according to claim 1, characterized in that: The molecular weight / particle size fitting curve can also be used to calculate the approximate particle size distribution behavior of a known molecular weight protein sample in ES-DMA-CPC, and to infer whether the protein sample meets the basic rules of proteins.

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