Method for determining molecular weight of protein or protein complex based on ES-DMA-CPC

Abstract

The invention discloses a method for determining the molecular weight of a protein or a protein complex based on ES-DMA-CPC. The method comprises the following steps: (1) determining the particle size information of the protein with the known molecular weight by adopting the ES-DMA-CPC; (2) performing polynomial fitting according to known protein molecular weight information and particle size information measured by ES-DMA-CPC, and establishing a protein molecular weight-particle size fitting standard curve; and (3) determination of molecular weight of unknown protein or protein complex: analyzing a to-be-detected protein sample through ES-DMA-CPC to obtain particle size information of the to-be-detected sample, and calculating the molecular weight of the to-be-detected protein or protein complex according to the established protein molecular weight-particle size standard curve. By adopting the method disclosed by the invention, the molecular weight of protein with a complex structure can be measured, the structural stability of a biological compound can be maintained, and the molecular weight of the protein complex can be measured.

Description

Technical Field

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

[0002] Proteins are important executors and regulators of cellular life activities, and are involved in reactions such as signal recognition and transmission, material transport, and immune response in organisms. Among them, the protein molecular weight is very important for understanding the structure and function of proteins, protein purification, quality control, and disease research. First, by measuring the protein molecular weight, the protein purity can be preliminarily identified; secondly, the change in molecular weight indicates the degree of protein degradation and aggregation. By characterizing the molecular weight of protein aggregates, the product quality of drugs can be controlled to ensure the stability and uniformity of quality; in addition, non-covalent interactions of proteins play an important role in biology and medicine, such as blood coagulation, gene expression, immune recognition, the formation of lipid particles and viruses. By characterizing the molecular weight of protein complexes, the formation process, protein assembly, stoichiometry and other characteristics of the complexes can be further understood, which is beneficial to screening and optimizing drug candidate molecules, accelerating drug research and development, and realizing drug targeted therapy.

[0003] Existing methods for characterizing the molecular weight of proteins and their complexes include mass spectrometry, multi-angle light scattering method, and mass photometry method, etc. Mass spectrometry is a relatively accurate method for molecular weight characterization, but this method has high requirements for the purity of samples, limited molecular weight measurement range, and non-covalent interactions of proteins may be destroyed in mass spectrometry analysis, making it difficult to measure the complete molecular weight of complexes. The multi-angle light scattering method is a method for absolute quantification of protein molecular weight, which can effectively identify aggregates and characterize the molecular weight of biological macromolecules. However, the multi-angle light scattering method is not suitable for measuring samples that are difficult to separate and elute on a chromatographic column and have complex components. In addition, as a new type of single molecule technology, mass photometry can quantify the mass of molecules by the interference scattered light of single particles through an optical detector to weigh proteins. It plays an important role in the study of antibodies and their immune complexes, but this method is not suitable for samples with high concentrations and containing macromolecular impurities, and is easily interfered by background signals and is not applicable to the small molecule protein level.

[0004] Based on the limitations of the existing methods for characterizing the molecular weight of proteins and protein complexes, it is urgent to develop a highly efficient and rapid method for characterizing the molecular weight of proteins or protein complexes with large molecular weights and complex structures. Summary of the Invention

[0005] The object of the present invention is to provide a method for determining the molecular weight of proteins or protein complexes based on ES-DMA-CPC. This method uses electrospray-differential mobility-particle counting technology to measure the particle size distribution of a series of protein standard substances with known molecular weights, and obtains a protein molecular weight-particle size fitting curve through the measurement of the standard substances, thereby enabling the determination of the molecular weight of unknown proteins.

[0006] Specifically, the technical solution adopted by the present invention is as follows:

[0007] A method for determining the molecular weight of proteins or protein complexes 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 invention uses an analytical technique based on electrospray-differential mobility-particle counting (ES-DMA-CPC) to measure the particle size of protein standard substances with known molecular weights or high-purity (purity > 90%) protein samples. The electrospray-differential mobility-particle counting analysis technique (ES-DMA-CPC) is mainly completed by three parts of instruments, including an electrospray aerosol generator (ES), a differential mobility analyzer (DMA), and a condensation particle counter (CPC). In addition, a charge neutralizer is usually added between the electrospray aerosol generator and the differential mobility analyzer to balance the charge distribution of the particles, so that more than 90% of the droplets carry only a single charge, so that when the particles migrate in the DMA, it is only related to the size of the particles.

[0010] The process of measuring the particle size of protein samples by ES-DMA-CPC is as follows: Dilute the protein standard substance with known molecular weight with ammonium acetate buffer solution, inject the prepared protein sample into the electrospray aerosol particle generator through an injection pump, generate atomized highly charged droplets through a capillary with an inner diameter of 25 μm, and after the droplets evaporate and dry, the charged particles are analyzed by differential mobility, and the particle size distribution of the protein sample is obtained by a particle counter.

[0011] To ensure the accuracy of protein molecular weight detection, at least three or more protein standard substances with known molecular weights that can significantly distinguish monomers are selected in the experiment. And the molecular weight difference of the selected several known proteins is at least greater than 15 KDa. When the protein molecular weights are similar, the particle sizes detected by the instrument are similar, which is not conducive to establishing a protein molecular weight-particle size curve.

[0012] According to the corresponding information of the molecular weight of the protein standard substance and the particle size measured by the instrument, the molecular weight and the particle size are polynomially fitted. Specifically, see formula (1):

[0013] y = a 0 + a 1 x + a2 x 2 +a 3 x 3 (1)

[0014] Among them, x is the particle size (nm) of the protein standard substance, and y is the molecular weight (KDa) of the protein standard substance. Given the molecular weight information of the protein standard substance and the particle size information obtained by instrument detection, they can be fitted using formula (1) to obtain the protein molecular weight-particle size (MW / EMD) fitting curve.

[0015] (2) When measuring the molecular weight of a protein sample to be tested with a known fitting curve:

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

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

[0018] When measuring a protein complex sample with unknown molecular weight:

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

[0020] Among them, in the said 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. The ammonium acetate buffer solution is filtered 3 times before dilution, and it is necessary to ensure that the diluted sample can be clearly distinguished as monomers in the ES-DMA-CPC analysis.

[0021] The injection pump is set to the constant flow mode, and the flow rate is set to 0.20 μL / min; the gas 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 its particle size scanning range is set to the corresponding scanning range; the CPC sampling flow rate is set to 0.6 L / min.

[0022] The electrospray-differential mobility-particle counting technique (ES-DMA-CPC) uses soft ionization, and proteins and their complexes are not easily dissociated during the electrospray process, enabling the complete determination of the particle sizes of proteins and their complexes. It has the advantage of high-precision molecular weight determination that cannot be obtained by mass spectrometry. Moreover, ES-DMA-CPC can achieve particle size separation in mixed samples and can analyze and measure even complex samples, making up for the limitations of multi-angle light scattering and mass photometry. On the basis of overcoming the limitations of existing protein and their complex molecular weight detection techniques, the present invention can achieve a wider range and more accurate determination of the molecular weights of proteins and their complexes through the ES-DMA-CPC method.

[0023] Compared with the prior art, the outstanding effects of the present invention are as follows:

[0024] (1) The present invention provides a brand-new single-particle analysis-based protein molecular weight characterization method, which can completely determine the particle sizes of proteins and their complexes and can also analyze and measure complex samples.

[0025] (2) By using the method of the present invention, the protein molecular weight in solution can be indirectly determined based on the particle size information of the protein sample, and the obtained particle size map can resolve the minute mass differences of the protein.

[0026] (3) The method of the present invention uses soft ionization to analyze protein analytes. Even proteins with poor stability are not easily dissociated during ionization, which is very suitable for studying antigen-antibody interactions and can achieve the molecular weight characterization of a wider range of protein samples such as antigen-antibody complexes.

[0027] The following further describes the method for determining the molecular weight of proteins or protein complexes based on ES-DMA-CPC of the present invention in conjunction with the accompanying drawings and specific examples. Description of the Drawings

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

[0029] Figure 2 It is the particle size spectrum diagram before and after ultrafiltration of TNF-α monoclonal antibody (WT).

[0030] Figure 3 It is the comparison diagram before and after ultrafiltration of TNF-α monoclonal antibody (WT).

[0031] Figure 4 It is the particle size spectrum diagram of the ES-DMA detected complex.

[0032] Figure 5 It is the mass distribution diagram detected by MP. Detailed Embodiments

[0033] Example 1

[0034] The molecular weight of TNF-α monoclonal antibody was characterized by the ES-DMA-CPC method for determining the molecular weight of proteins, which proved that this method can achieve the determination of the molecular weight of unknown proteins.

[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-aqueous condensation particle counter (TSI, USA, model 3788); silica capillary (inner diameter 25μm, length 4.1 cm, TSI, USA); Amicon Ultra protein concentration tube (Merck, Germany); ultra-micro high-precision ultraviolet-visible spectrophotometer Nanodrop 2000 (ThermoFisher Scientific, USA); mass photometer (Refeyn, UK).

[0037] Ammonium acetate (chromatographic grade, purity ≥99.0%, Honeywell, Germany); TNF-α monoclonal antibody (National Institute of Metrology, China); the experimental water used was 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 the pH of the solution was adjusted to 8 with ammonia water. The TNF-α 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 mainly existed in the monomer form in the ES-DMA-CPC analysis.

[0040] III. Experimental conditions and parameters:

[0041] The experimental process was as follows: First, the sample was injected into the electrospray aerosol particle generator through the FLOW-EZ microfluidic constant flow pump, and atomized highly charged droplets were generated through a capillary with an inner diameter of 25μm. After the droplets evaporated and dried, the charged particles passed through the DMA nano-column, and the particle size distribution of the protein sample was obtained by the particle counter.

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

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

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

[0045] By using protein standard substances with known molecular weights 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 particle size distribution information was measured in ES - DMA - CPC. The molecular weight and particle size information of the five protein standard substances are shown in Table 1 specifically.

[0046] Table 1 Particle size determination results of five protein standard substances

[0047]

[0048] According to the corresponding information of the molecular weight and particle size of the five protein standard substances, the molecular weight and particle size were polynomially fitted to obtain the fitting equation, as shown in formula (2) specifically:

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

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

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

[0052] In actual measurements, the TNF-α wild-type monoclonal antibody could not distinguish the presence of monomers and oligomers by direct dilution with ammonium acetate buffer. There was an obvious peak broadening in the measurement of its particle size, deviating from the protein molecular weight-particle size fitting curve. It was suspected that the buffer in the protein solution interfered with the measurement of the protein particle size. The sample was processed by ultrafiltration, and the results of the re-measurement showed that the processed sample could distinguish the presence of monomers and oligomers. The molecular weight information of the TNF-α wild-type monoclonal antibody was calculated according to the protein molecular weight-fitting curve. Specifically, see Table 2, which confirmed that the results of its particle size measurement conform to the basic laws of proteins. The particle size spectra and the corresponding relationships of molecular weights before and after ultrafiltration are specifically shown in Figure 2 - Figure 3 。

[0053] Table 2 Measurement results of the molecular weight of TNF-α monoclonal antibody

[0054]

[0055]

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

[0057] Example 2

[0058] The fusion protein TNF-α antigen was measured by the method for measuring proteins with unknown molecular weights based on the ES-DMA-CPC method, proving that this method can be used to characterize protein samples with unknown molecular weights.

[0059] Keeping all experimental conditions and parameters the same as in Example 1, the particle sizes of the monomers and dimers of the TNF-α antigen at a concentration of 0.06 mg / mL were measured. Using the molecular weight-particle size fitting curve measured in Example 1, the molecular weight information corresponding to the particle sizes of the monomers and dimers was calculated. The molecular weight detection results are shown in Table 3.

[0060] Table 3 Detection results of TNF-α antigen

[0061]

[0062] The theoretical molecular weight of the TNF-α antigen standard substance used was 52.5 KDa, while the measurement result by the method of the present invention was 49.59 KDa. The RSD deviation between the two was 4.03%, less than 5%, proving that the method of the present invention can achieve the measurement of the molecular weight of the fusion protein.

[0063] Example 3

[0064] The molecular weight of the CD19-Fc antigen-antibody complex was determined by the method for determining the molecular weight of proteins based on the ES-DMA-CPC method, demonstrating that this method can accurately characterize the molecular weight of protein complexes.

[0065] Replace the TNF-α wild-type monoclonal antibody in Example 1 with the CD19-Fc antigen-antibody complex (National Institute of Metrology, China). After ultrafiltration of its CD19-Fc antigen and CD19 single-chain antibody, they were diluted to 0.23 mg / mL and 0.05 mg / mL with ammonium acetate buffer respectively, and then an equal volume of them was mixed to prepare a mixed solution with a molar ratio of 1:1, and incubated overnight in a 4°C refrigerator. Other experimental conditions were the same as those in Example 1.

[0066] The particle size distributions of the antigen and antibody were detected by ES-DMA-CPC respectively to determine that they conform to the basic laws of proteins, and then the particle size distribution of the antigen-antibody complex was measured. The specific measurement results are shown in Table 4.

[0067] Table 4 CD19 antigen-antibody particle size measurement results and corresponding molecular weights

[0068]

[0069]

[0070] The molecular weight of the first peak of the antigen-antibody complex was obtained by formula (1) as MW = 159.91 kDa, which is the peak of 1:1 binding of the antigen and antibody; the molecular weight of the second peak is MW = 308.96 kDa, which may be the peak of 2:1 binding of the antigen and antibody. The specific ES-DMA-CPC measurement results can be seen Figure 4 . At the same time, the molecular weight of the antigen-antibody complex was analyzed and verified by mass photometry (MP). The specific mass photometry measurement results can be seen Figure 5 .

[0071] In view of the fact that the mass photometry did not detect the peak of 2:1 binding of the antigen and antibody, the peak of 1:1 binding of the antigen and antibody was mainly compared and analyzed. The mass photometry measurement value of the 1:1 binding peak of the antigen-antibody complex standard substance used was 163 kDa, and the measurement result of the method of the present invention was 159.91 kDa. The RSD deviation from the mass photometry measurement result was 1.35%, demonstrating that this method can accurately determine the molecular weight of the protein antigen-antibody complex.

[0072] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention should fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for determining the molecular weight of a protein or protein complex based on ES-DMA-CPC, characterized in that: The following steps are involved: (1) Determination of protein molecular weight-particle size fitting standard curve First, ES-DMA-CPC is used to determine the particle size of a protein standard substance with a known molecular weight or a high-purity protein sample; the protein standard substance with a known molecular weight or a 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 an injection pump, and atomized high-charge droplets are generated through a capillary with an inner diameter of 25 μm. After the droplets are evaporated and dried, the charged particles are analyzed by differential mobility and a particle counter to obtain the particle size distribution of the protein sample; According to the molecular weights of at least three or more protein standard substances or high-purity protein samples and the protein particle size information determined by ES-DMA-CPC, a polynomial fitting of the molecular weight and particle size is performed to obtain a standard curve of protein molecular weight-particle size fitting; (2) Determination of proteins or protein complexes of unknown molecular weight According to the method for determining protein particle size by ES-DMA-CPC in step (1), the protein sample is analyzed by ES-DMA-CPC to obtain the particle size distribution of the protein sample of unknown molecular weight, and the molecular weight of the protein sample to be tested is calculated based on the standard curve of protein molecular weight-particle size obtained in step (1) and the polynomial fitted in step (1).

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 the step (1), at least three or more proteins of known molecular weight that can clearly distinguish monomers are selected as particle size measurement standards, and the difference in molecular weight between the selected several known protein standard substances is at least greater than 15KDa.

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: In the step (1), the purity of the protein standard substance and the high-purity protein sample is at least greater than 90%.

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

5. The method for determining the molecular weight of a protein or protein complex based on ES-DMA-CPC according to claim 4, characterized in that: In the step (1), 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 clearly distinguish the presence of monomers in the ES-DMA-CPC analysis; For samples where the presence of monomers cannot be distinguished, ultrafiltration is used for pretreatment.

6. The method for determining the molecular weight of a protein or protein complex based on ES-DMA-CPC according to claim 5, characterized in that: In the step (1), the injection pump is set to a constant flow mode, and the flow rate is set to 0.20 μL / min; the air 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 its particle size scanning range is set to the corresponding scanning range; the CPC sampling flow rate is set to 0.6 L / min.

7. 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 protein samples with known molecular weight in ES-DMA-CPC, and then infer whether the protein sample conforms to the basic laws of protein.

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