Method for detecting protein oxidation through compound mode volume exclusion chromatography

Abstract

The invention belongs to the field of biological pharmacy, and particularly relates to a method for detecting protein oxidation through compound mode volume exclusion chromatography. The method comprises the following steps: properly diluting a protein solution, injecting the diluted protein solution into a high performance liquid chromatography, and separating the oxidized variant from other components according to different retention times of a sample in a composite mode chromatographic column, so as to calculate the proportion of the oxidized variant. The method has the characteristics of simplicity and high efficiency, and can provide powerful support for research and production in the field of biological pharmacy.

Description

Technical Field

[0001] The invention relates to the field of biopharmaceuticals, and in particular to a method for detecting protein oxidation by using composite mode size exclusion chromatography. Background Art

[0002] In recent years, biopharmaceuticals have shined in the global pharmaceutical market with their high activity, strong specificity, and good effects. Among them, protein drugs have shown clinical advantages that are significantly superior to traditional drugs and have become the driving force for the development of the biopharmaceutical industry. However, protein drugs undergo multiple post-translational modifications and degradation during the production process, and their biochemical and physicochemical properties are heterogeneous. These heterogeneities can lead to changes in molecular weight, hydrophobicity, and structure, thereby affecting efficacy, half-life, and safety. Therefore, the key quality attributes of proteins require special attention. According to the ICH Guideline M4Q, applicants need to provide regulatory agencies with detailed structural characterization data of the API and use a large number of analytical methods to elucidate the structure of the API from different aspects.

[0003] Protein oxidation commonly occurs in various processes such as drug production and storage, and is a major obstacle to safety and efficacy. Therefore, oxidation is usually monitored throughout the production process of protein drugs. The main method is liquid chromatography-mass spectrometry. This method requires the sample to be reduced to open the disulfide bonds, and then the free sulfhydryl groups and other groups are alkylated with an alkylating agent. After desalting, enzymatic hydrolysis is performed, and then the sample is separated by liquid phase and analyzed by a mass spectrometer to calculate the proportion of protein oxidation (see Yanpeng Xu et al., (2019): Physicochemical and functional assessments demonstrating analytical similarity between rituximab biosimilar HLX01 and the mAbs,DOI:

[0004] 10.1080 / 19420862.2019.1578147). This method is relatively complicated, expensive, and requires a high level of knowledge in biology and chemistry. Therefore, there is an urgent need for a method with a relatively low threshold, such as simple operation, low cost, and high throughput, to detect protein oxidation.

[0005] Composite mode size exclusion chromatography is a new detection method that has emerged in recent years. The filler of the composite mode chromatographic column is a uniform, hydrophilic, neutral nanofilm chemically bonded to a high-purity silica gel with enhanced mechanical stability. This special surface technology allows the separation of components by characteristics such as molecular size and hydrophobicity, which is different from ordinary size exclusion chromatography. The detection of protein oxidation using composite mode size exclusion chromatography only requires diluting the sample and then performing liquid phase separation to simply and quickly complete the detection of protein oxidation. Therefore, the present invention is simple to operate and low in cost, and can also achieve high-throughput detection of protein oxidation. Both the production and quality control of protein drugs will benefit from this new detection method. Summary of the invention

[0006] In order to solve the problem of rapid and simple detection of protein oxidation variants, the present invention provides a method for detecting protein oxidation by composite mode size exclusion chromatography with simple operation and low cost.

[0007] One aspect of the present invention provides a method for detecting protein oxidation, comprising the following steps: diluting a protein sample to obtain a diluted protein sample; detecting and analyzing the diluted protein sample using a high performance liquid chromatograph; and calculating the proportion of oxidized variants in the protein sample using an area normalization algorithm, wherein the high performance liquid chromatograph uses a composite mode size exclusion chromatography column.

[0008] In some embodiments, the protein or protein sample is selected from a monoclonal antibody, a bispecific antibody, a multispecific antibody, a nanobody, an antibody fragment, an antigen-binding fragment, an antibody-drug conjugate, or a fusion protein.

[0009] In some embodiments, the protein or protein sample includes but is not limited to abatacept, teriparatide, eculizumab, emicizumab, pegfilgrastim, semaglutide, dulaglutide, sargramostim, ustekinumab, secukinumab, tocilizumab, Vedolizumab, natalizumab, interferon beta-1a, denosumab, alirocumab, evolocumab, adalimumab, etanercept, golimumab, trastuzumab, pembrolizumab, pertuzumab, ARO-HBV, glatiramer acetate acetate), LY-3321367, cetuximab, ipilimumab, daratumumab, albumin-bound paclitaxel, tanezumab, LY-2510924, LCAR-B38M, PF-004518600, TAK-079, PF-06730512, LY-3076226, NOV-13, FAZ-053, LY-3375880, PF-06823859, CNGB3 gene therapy, mosune tuzumab), RG-6147, ranibizumab, cofetuzumab bpelidotin, SHR-A1201, TAK-671, NG-HER2 antibody drug conjugate, TAK-164, RG-7861, JNJ-61186372, PF-05206388, NJH-395, PF-05230907, BIIB-059, PF-06688992, ianalumab, TAK-573, PF-06755347, CD200RmAb agonists, cetrelimab, ligelizumab, PF-06801591, JNJ-64407564, polatuzumab vedotin, PF-06817024, NOV-12, BIIB-054, CTL-119, JNJ-61178104, spartalizumab, RNA CART123, LY-3300054, PD-1mAb agonist, CART-EGFRvIII, NOV-10, TQJ-230, PF-06863135, PCA-062, JNJ-64041757, CNTO-2476, tiragolumab, PF-06946860, elgemtumab, LY-3415244, LKA-651, RG-6109, ECF-843, JNJ-61610588, AAV8-RLBP1 gene therapy, LAG-525, MOR-106, BTLA agonist mAb, AMV-564, JNJ-64041809, MBG-453, CGF-166, brolucizumab, NOV-9, CJM-112, tesidolumab, NIZ-985, MCS-110, BHQ-880, NOV-8, CLR-325, XmAb-13676, huMesoCART, NZV-930, CGM-097, NOV-7, Ociperlimab, tislelizumab and certolizumab pegol; and biosimilars thereof; and glycosylation variants thereof.

[0010] In some embodiments, the antibody comprises three complementary determining regions HCDR of the heavy chain variable region, and three complementary determining regions LCDR of the light chain variable region, wherein (i) HCDR1 consists of the amino acid sequence shown in SEQ ID NO:1, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:2, HCDR3 consists of the amino acid sequence shown in SEQ ID NO:3, LCDR1 consists of the amino acid sequence shown in SEQ ID NO:4, LCDR2 consists of the amino acid sequence shown in SEQ ID NO:5, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:6; or (ii) HCDR1 consists of the amino acid sequence shown in SEQ ID NO:12, HCDR2 consists of the amino acid sequence shown in SEQ ID NO:13, HCDR3 consists of the amino acid sequence shown in SEQ ID NO:14, LCDR1 consists of the amino acid sequence shown in SEQ ID NO:15, LCDR2 consists of the amino acid sequence shown in SEQ ID NO:16, and LCDR3 consists of the amino acid sequence shown in SEQ ID NO:17.

[0011] In some embodiments, the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NO:7 or 18, or consists of said sequence; and / or, the antibody comprises a light chain variable region, wherein the light chain variable region comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from SEQ ID NO:8 or 19, or consists of said sequence.

[0012] In some embodiments, the antibody is a monoclonal antibody, preferably Ociperlimab or Tislelizumab.

[0013] In some embodiments, the filler of the composite mode size exclusion chromatography column is formed by chemical bonding of a uniform, hydrophilic, neutral nanomembrane on a high-purity and mechanically stable silica gel, and the sample can be separated by molecular size and hydrophobicity; preferably, the composite mode size exclusion chromatography column is a Sepax BioMix-300 SEC column (specifications 7.8×300 mm, particle size 3 μm, pore size )(See https: / / www.sepax-tech.com / usermanual / UserManual_BioMix.pdf , the entire contents of which are incorporated herein by reference).

[0014] In some embodiments, the concentration of the protein sample after dilution is 0.1-5 mg / mL, preferably 1 mg / mL.

[0015] In some embodiments, the mobile phase in the chromatographic conditions of the detection method is a mixed aqueous solution of phosphate, potassium chloride and sodium chloride; wherein the phosphate is selected from sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, potassium hydrogen phosphate and mixtures thereof; preferably, the phosphate concentration is 5-50 mM, the potassium chloride concentration is 1-50 mM and the sodium chloride concentration is 50-150 mM.

[0016] In some embodiments, the mobile phase is an aqueous solution of 7-10 mM sodium phosphate, 0.5-3 mM potassium phosphate, 1-4 mM potassium chloride and 120-150 mM sodium chloride; preferably, the mobile phase is an aqueous solution of 8.1 mM sodium phosphate, 1.5 mM potassium phosphate, 2.7 mM potassium chloride and 137 mM sodium chloride. In some embodiments, the dilution is performed using a mobile phase.

[0017] In some embodiments, the pH range of the chromatographic conditions of the detection method is 6.0-9.0; the pH is preferably 7.4.

[0018] In some embodiments, the detection wavelength is 210-280 nm, the flow rate is 0.1-5 mL / min, the column temperature is 20-40°C, and the injection volume is 1-100 μL; preferably, the detection wavelength is 214 nm, the flow rate is 1 mL / min, the column temperature is 25°C, and the injection volume is 10 μL.

[0019] In some embodiments, the quantitative method in the detection method is: calculate the peak area of ​​all peaks in the chromatogram except the solvent peak, normalize the peak area, and then calculate the percentage of oxidized variants in the protein. The calculation formula is: percentage of oxidized variants (%) = shoulder peak (oxidized variant peak) peak area / total peak area (excluding solvent peak) × 100%.

[0020] In some embodiments, the present invention provides a method for detecting protein oxidation, comprising the following steps:

[0021] 1) Preparation of mobile phase: prepare an aqueous solution of 8.1 mM sodium phosphate, 1.5 mM potassium phosphate, 2.7 mM potassium chloride and 137 mM sodium chloride, adjust the pH value to 7.4 with an acid-base regulator, and filter with a microporous filter membrane to obtain the mobile phase;

[0022] 2) Dilute the protein sample to 1 mg / mL with mobile phase;

[0023] 3) Chromatographic detection: using a high performance liquid chromatograph, a Sepax BioMix-300 SEC column, using the mobile phase prepared in step 1), a detection wavelength of 214 nm, a flow rate of 1 mL / min, and a column temperature of 25°C; injecting the test sample prepared in step 2) into the high performance liquid chromatograph, with an injection volume of 10 μL, and isocratic elution; using an area normalization algorithm to calculate the proportion of oxidized variants in the protein sample.

[0024] Beneficial Effects

[0025] Compared with the existing LC-MS method, the composite mode size exclusion chromatography method for detecting protein oxidation provided by the present invention has the advantages of simple operation, low cost, easy to achieve high throughput and relatively easy data analysis. This method greatly reduces the threshold of protein oxidation analysis, is of great significance for the monitoring of oxidation in the entire production process of protein drugs, and can also provide important help for the research and development of protein drug processes.

[0026] General Terms and Definitions

[0027] Unless defined otherwise, technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0028] As used herein, "liquid chromatography" refers to the process of selectively retarding one or more components of a fluid solution when the fluid is uniformly filtered through a column of finely divided material or through a capillary channel. The retardation is due to the distribution of the components of the mixture between one or more stationary phases and the main fluid (i.e., mobile phase) when the fluid moves relative to one or more stationary phases. As used herein, the composite mode size exclusion chromatography refers to a high performance liquid chromatograph using a composite mode size exclusion chromatography column. As used herein, a high performance liquid chromatograph (HPLC) is an instrument that applies the principle of high performance liquid chromatography and is mainly used to analyze high boiling point, non-volatile, thermally unstable and large molecular weight organic compounds. It consists of a reservoir, a pump, an injector, a chromatographic column, a detector, a recorder and the like. The mobile phase in the reservoir is pumped into the system by a high-pressure pump, and the sample solution enters the mobile phase through the injector and is carried into the chromatographic column (stationary phase) by the mobile phase. Since the components in the sample solution have different distribution coefficients in the two phases, when they move relative to each other in the two phases, after repeated adsorption-desorption distribution processes, the components have large differences in movement speed and are separated into individual components that flow out of the column in turn. When passing through the detector, the sample concentration is converted into an electrical signal and transmitted to the recorder, and the data is printed out in the form of a graph. The necessary components of common high-performance liquid chromatographs include high-pressure infusion pumps, injectors, chromatographic columns, and detectors. According to special needs, there may be some other accessories, such as degassers, automatic injectors, column ovens, etc.

[0029] The chromatographic column usually includes a medium (i.e., a filler) to promote the separation of chemical parts (i.e., fractional separation). The medium may include tiny particles. The particles may include a bonding surface that interacts with various chemical parts to promote the separation of chemical parts. The chromatographic column may include an inlet for receiving a sample and an outlet for discharging an effluent containing the fractionated sample. In this method, the sample (or a pre-purified sample) may be applied to the column at the inlet, eluted with a solvent or a solvent mixture, and discharged at the outlet. Different solvent modes may be selected to elute different analytes of interest. For example, liquid chromatography may be performed using a gradient mode, an isocratic mode, or a polymorphic (i.e., mixed) mode.

[0030] [k1] For example, the terms "comprises," "comprising," or grammatical variations thereof used in the present invention mean including the listed elements but not excluding others.

[0031] Unless the context indicates otherwise, the word "comprise" and variations such as "include" and "comprising" will be understood to imply the inclusion of the stated amino acid sequence, DNA sequence, step or group thereof, but not the exclusion of any other amino acid sequence, DNA sequence, step. When used herein, the term "comprise" may be replaced with the term "comprising", "including" or sometimes "having".

[0032] Unless explicitly indicated to the contrary, all ranges cited herein are inclusive; that is, the range includes the values ​​of the upper and lower limits of the range and all values ​​in between. For example, the temperature ranges, percentages, equivalent ranges, etc. described herein include the upper and lower limits of the range and any values ​​of the continuous intervals therebetween. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the chromatographic detection spectrum of Protein A in Example 1.

[0034] Figure 2 This is an overlay of the chromatographic detection of sample A-F1 collected for protein A and shoulder peak in Example 1.

[0035] Figure 3 This is the chromatographic detection spectrum of Protein A in Comparative Example 1 using conventional size exclusion chromatography.

[0036] Figure 4A For the chromatographic detection spectrum of protein B in Example 2, protein B samples containing 1 ppm, 20 ppm, 50 ppm, 100 ppm, 500 ppm, and 1000 ppm of hydrogen peroxide were prepared, respectively, and recorded as B-40C2W-1, B-40C2W-20, B-40C2W-50, B-40C2W-100, B-40C2W-500, and B-40C2W-1000; Figure 4B for Figure 4A A partial enlarged view of .

[0037] Figure 5A The chromatographic detection spectrum of protein B in Example 3, the initial sample of protein B is recorded as B-T0, and the sample after being placed at 40°C for 2 weeks is recorded as B-40C2W; Figure 5B for Figure 5A A partial enlarged view of .

[0038] Fig. 6A The chromatographic detection spectrum of protein B in Example 4, the initial sample of protein B is recorded as B-T0, and the sample after being placed under light conditions for 2 weeks is recorded as B-pho2W; Figure 6B for Fig. 6A A partial enlarged view of . DETAILED DESCRIPTION

[0039] The experimental methods in the following examples are conventional methods unless otherwise specified. The chemical raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.

[0040] Abbreviations and their meanings

[0041] abbreviation meaning abbreviation meaning DPBS Dulbecco's Phosphate Buffered Saline Lys_loss Lysine deficiency Lys-C Lysyl endo-enzyme Oxidation Oxidation Trypsin Trypsin Deamidation Deamidation ppm One in a million Glycation Saccharification Pyro-Glu Pyroglutamate cyclization

[0042] Example 1

[0043] Sample protein A (Ociperlimab, BGB-A1217) was selected for the experiment. Protein A (Ociperlimab, BGB-A1217) is an anti-TIGIT antibody disclosed in PCT patent number WO2019 / 129261, and its sequence is shown in Table 1 below.

[0044]

[0045]

[0046]

[0047] Chromatographic conditions: Waters high performance liquid chromatograph, column Sepax BioMix-300 (7.8×300 mm, 3 μm), mobile phase: 8.1 mM Na 3 PO 4 , 1.5 mM K 3 PO 4 , 2.7mM KCl, 137mM NaCl, pH7.4; flow rate 1mL / min, sample diluted to 1mg / mL with mobile phase before injection, injection volume 10μL, detection wavelength 214nm, column temperature 30℃. The chromatogram is shown in Figure 1As shown in the figure, a shoulder peak is found before the main peak. According to the principle of multimodal chromatographic columns, the shoulder peak may be caused by a molecular weight larger than the main peak or a weaker hydrophobicity, or both a large molecular weight and a weaker hydrophobicity.

[0048] The shoulder peak was collected using a Waters WFCⅢ component collector, and the collected samples were pooled together and replaced with DPBS using a Merck 10kD ultrafiltration centrifuge tube, named A-F1, and then detected using the aforementioned method. The superimposed chromatograms of samples A and A-F1 are shown in Figure 2 As shown, the peak areas of all peaks except the solvent peak in the chromatogram are calculated. After normalizing the peak areas, the percentage of oxidized variants in the protein is calculated. From the calculation, it can be seen that the proportion of the shoulder peak in sample A-F1 can reach 90.13%, so to a certain extent, A-F1 can represent the shoulder peak.

[0049] Mass spectrometry verification

[0050] Take an appropriate amount of A-F1 test sample in guanidine hydrochloride buffer, reduce and alkylate with dithiothreitol and iodoacetic acid, add Lys-C enzyme for pre-cutting at 37℃ for 2h, add Trypsin for enzymatic digestion at 37℃ for 4h, and add formic acid to terminate the reaction. The enzymatic hydrolysis sample is separated by ultra-high performance liquid chromatography system. The enzymatic peptide fragments are separated by ultra-high performance liquid chromatography and analyzed by mass spectrometry using Thermo Q-ExactivePlus mass spectrometer.

[0051] 1) Specific chromatographic conditions: Phase A is 0.1% formic acid aqueous solution, and phase B is 0.1% formic acid acetonitrile aqueous solution. Gradient elution is adopted, and the mobile phase changes evenly according to the gradient retention time in the following table:

[0052] Mobile phase gradient elution parameters:

[0053]

[0054]

[0055] 2) Specific mass spectrometry conditions: analysis time is 75 min, detection mode is positive ion, parent ion scanning range: 300-1800 m / z, primary mass spectrometry resolution is 70000 at m / z 200, secondary mass spectrometry resolution is 17500 at m / z 200. Mass spectrometry data were processed using Biopharma Finder 3.2 software. The results are shown in the following table:

[0056]

[0057] Note: 1. ~ indicates that the modification site is unclear because the identified peptide contains multiple modification sites.

[0058] 2.N / A means not applicable, i.e. not detected.

[0059] As can be seen from the table above, the main difference between sample A and sample A-F1 is the oxidation ratio of M34, M106, M254, M360, M430, and W50. The oxidation ratio of sample A-F1 at M34, M106, M254, M360, M430, and W50 is much higher than that of sample A. Since oxidation can lead to the production of more acidic isomers with weaker hydrophobicity, they will be eluted before the main peak in the composite mode size exclusion chromatography. It can be seen that the shoulder peak is caused by oxidation, so protein oxidation can be detected by measuring the proportion of the shoulder peak in the sample.

[0060] Comparative Example 1

[0061] Sample protein A was selected for the experiment. Chromatographic conditions: Waters high performance liquid chromatograph was used, chromatographic column: TOSOHTSKgel SuperSW mAB HR chromatographic column (7.8×300mm, 4μm), and the experiment was carried out according to the molecular exclusion chromatography method in Part III 0514 of the Pharmacopoeia of the People's Republic of China (2020 edition), where the mobile phase was 8.1mM Na 3 PO 4 , 1.5 mM K 3 PO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.4; flow rate 1 mL / min, the sample was diluted to 1 mg / mL with mobile phase before injection, injection volume 10 μL, detection wavelength 214 nm, column temperature 30 °C.

[0062] The chromatogram obtained from the experiment is as follows Figure 3 As shown, when the protein A sample was analyzed using the conventional method, no shoulder peak was found, indicating that the conventional method is not suitable for the detection of protein oxidation.

[0063] Example 2: Effect of hydrogen peroxide on protein oxidation

[0064] Hydrogen peroxide is widely used in protein oxidation research. The higher the hydrogen peroxide concentration in the sample, the higher the protein oxidation ratio (see Neeraj J. Agrawal et al., (2018): Prediction of the Hydrogen Peroxide-Induced Methionine Oxidation Propensity in Monoclonal Antibodies, Journal of Pharmaceutical Sciences). The sample protein B (Tislelizumab, BGB-A317) was selected for the experiment. Protein B (Tislelizumab, BGB-A317) is an anti-PD1 antibody disclosed in U.S. Patent 8,735,553, and its sequence is as follows.

[0065]

[0066]

[0067] 30% hydrogen peroxide was mixed with sample B to prepare samples containing 1ppm, 20ppm, 50ppm, 100ppm, 500ppm, and 1000ppm hydrogen peroxide, respectively. The samples were then placed at 40°C / 75%RH for 2 weeks and were recorded as B-40C2W-1, B-40C2W-20, B-40C2W-50, B-40C2W-100, B-40C2W-500, and B-40C2W-1000. Chromatographic conditions: Waters high performance liquid chromatograph, chromatographic column Sepax BioMix-300 (7.8×300mm, 3μm), mobile phase: 8.1mM Na 3 PO 4 , 1.5 mM K 3 PO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.4; flow rate 1 mL / min, the sample was diluted to 1 mg / mL with mobile phase before injection, injection volume 10 μL, detection wavelength 214 nm, column temperature 30 °C.

[0068] Chromatographic overlay Figure 4A and Figure 4B As shown, the peak areas of all peaks except the solvent peak in the chromatogram were calculated. After normalizing the peak areas, the percentage of oxidized variants in the protein was calculated. The calculated proportions of oxidized variants are shown in the table below.

[0069] Sample name Final concentration of hydrogen peroxide Oxidation variant content percentage (%) B-40C2W-1 1ppm 10.21 B-40C2W-20 20ppm 10.23 B-40C2W-50 50ppm 10.53 B-40C2W-100 100ppm 11.36 B-40C2W-500 500ppm 12.76 B-40C2W-1000 1000ppm 17.47

[0070] The results showed that as the concentration of hydrogen peroxide in the sample increased, the content of oxidative variants also increased accordingly, and the concentration of hydrogen peroxide was positively correlated with the proportion of oxidative variants. Therefore, the method of the present invention can detect the changes in the content of oxidative variants caused by hydrogen peroxide, which further verifies that the method of the present invention is scientific and feasible, and can provide strong support for the quality control and process development of protein drugs.

[0071] Example 3: Effect of high temperature on protein oxidation

[0072] In the field of biopharmaceuticals, high temperature (such as 40°C) conditions are often used to study protein oxidation. Proteins will produce more oxidative variants under high temperature. Sample protein B (tislelizumab, BGB-A317) was selected for the experiment. The sample was placed in 40°C / 75% RH conditions for 2 weeks, recorded as B-40C2W, and the original sample was recorded as B-T0. Chromatographic conditions: Use Waters high-performance liquid chromatograph, chromatographic column Sepax BioMix-300 (7.8×300mm, 3μm), mobile phase: 8.1mM Na 3 PO 4 , 1.5mMK 3 PO 4 , 2.7 mM KCl, 137 mM NaCl, pH 7.4; flow rate 1 mL / min, the sample was diluted to 1 mg / mL with mobile phase before injection, injection volume 10 μL, detection wavelength 214 nm, column temperature 30 °C.

[0073] Chromatographic overlay Figure 5A and 5B As shown, the peak areas of all peaks except the solvent peak in the chromatogram are calculated, and after the peak areas are normalized, the percentage of oxidized variants in the protein is calculated. The results show that the proportion of oxidized variants in the initial sample B-T0 is 5.25%, and after being placed at high temperature, the proportion of oxidized variants in the sample B-40C2W is 10.17%, and the proportion of oxidized variants is significantly increased. It can be seen that the method of the present invention can detect the changes in the content of oxidized variants caused by high temperature.

[0074] Example 4: Effect of light on protein oxidation

[0075] In the field of biopharmaceuticals, light conditions are often used to study the photooxidation of proteins. Sample protein B (tislelizumab, BGB-A317) was selected for the experiment. The sample was placed under visible light 6230Lux and ultraviolet light 344μW / cm for 2 weeks, recorded as B-pho2W, and the original sample was recorded as B-T0. Chromatographic conditions: Waters high-performance liquid chromatograph, chromatographic column SepaxBioMix-300 (7.8×300mm, 3μm), mobile phase: 8.1mM Na3 PO 4 , 1.5 mM K 3 PO 4 , 2.7mM KCl, 137mMNaCl, pH 7.4; flow rate 1mL / min, the sample was diluted to 1mg / mL with mobile phase before injection, injection volume 10μL, detection wavelength 214nm, column temperature 30℃.

[0076] Chromatographic overlay Fig. 6A and 6B As shown, the peak areas of all peaks except the solvent peak in the chromatogram are calculated, and after the peak areas are normalized, the percentage of oxidized variants in the protein is calculated. The results show that the proportion of oxidized variants in the initial sample B-T0 is 5.25%, and after the sample is placed under light, the proportion of oxidized variants in the sample B-pho2W is 8.11%, and the proportion of oxidized variants is significantly increased. It can be seen that the method of the present invention can detect changes in the content of oxidized variants caused by light.

[0077] The present invention has been described in detail above by means of general explanations, specific implementation methods and experiments. Any modifications or improvements made without departing from the spirit of the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A method for detecting protein oxidation, comprising the following steps: diluting the protein sample to obtain a diluted protein sample; The diluted protein sample is detected and analyzed by high performance liquid chromatography. The area normalization algorithm was used to calculate the proportion of oxidative variants in protein samples. Characterized in that the high performance liquid chromatograph adopts a composite mode size exclusion chromatography column.

2. The method of claim 1, wherein the protein is selected from a monoclonal antibody, a bispecific antibody, a multispecific antibody, a nanobody, an antibody fragment, an antigen binding fragment, an antibody-drug conjugate or a fusion protein.

3. The method according to any one of claims 1 to 2, wherein the filler of the composite mode size exclusion chromatography column is formed by chemical bonding of a uniform, hydrophilic, neutral nanomembrane on a high purity and mechanically stable silica gel.

4. The method according to any one of claims 1 to 3, wherein the multimodal size exclusion chromatography column is a Sepax BioMix-300 SEC chromatography column.

5. The method according to any one of claims 1 to 4, wherein the dilution is carried out using a mobile phase in a chromatographic column, and the mobile phase is a mixed aqueous solution of phosphate, potassium chloride and sodium chloride; wherein, The phosphate is selected from sodium phosphate, disodium hydrogen phosphate, sodium dihydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, potassium hydrogen phosphate and mixtures thereof; preferably, the phosphate concentration is 5-50 mM, the potassium chloride concentration is 1-50 mM and the sodium chloride concentration is 50-150 mM.

6. The method of claim 5, wherein the mobile phase is an aqueous solution of 7-10 mM sodium phosphate, 0.5-3 mM potassium phosphate, 1-4 mM potassium chloride and 120-150 mM sodium chloride; preferably, the mobile phase is an aqueous solution of 8.1 mM sodium phosphate, 1.5 mM potassium phosphate, 2.7 mM potassium chloride and 137 mM sodium chloride.

7. The method according to any one of claims 1 to 6, wherein the concentration of the diluted protein sample is 0.1 to 5 mg / mL, preferably 1 mg / mL.

8. The method according to any one of claims 1 to 7, wherein in the area normalization algorithm, the percentage of the oxidized variants in the protein sample is: peak area of ​​the oxidized variant peak / total peak area × 100%, wherein the total peak area does not include the solvent peak.

9. A method for detecting protein oxidation, comprising the following steps: 1) Preparation of mobile phase: prepare an aqueous solution of 8.1 mM sodium phosphate, 1.5 mM potassium phosphate, 2.7 mM potassium chloride and 137 mM sodium chloride, adjust the pH value of the aqueous solution to 7.4 with an acid-base regulator, and filter through a microporous filter membrane to obtain a mobile phase; 2) diluting the protein sample to 1 mg / mL with the mobile phase; 3) Chromatographic detection: using a high performance liquid chromatograph, a Sepax BioMix-300 SEC column, using the mobile phase prepared in step 1), a detection wavelength of 214 nm, a flow rate of 1 mL / min, a column temperature of 25°C, injecting the test sample prepared in step 2) into the high performance liquid chromatograph, an injection volume of 10 μL, and isocratic elution; the area normalization algorithm is used to calculate the proportion of oxidized variants in the protein sample.

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