Method for identifying O-glycosylation of hinge region of IgG3 antibody

Abstract

The invention discloses a method for identifying the O-glycosylation of an IgG3 antibody hinge region, which is characterized in that chymotrypsin is used for hydrolyzing an IgG3 antibody, so that the overall O-glycosylation condition of the IgG3 antibody hinge region can be obtained, and the mutual relation information of O-glycosylation sites can be obtained; the method is helpful to discover abnormal conditions of O-glycosylation of the IgG3 antibody of patients with diseases such as rheumatoid arthritis and systemic lupus erythematosus, meanwhile, the analysis efficiency of O-glycosylation of an antibody drug based on IgG3 can be improved, meanwhile, according to the method, domestic gel is loaded into a syringe cavity to form a simple gel column, the purposes of enrichment and purification are achieved, and the method is suitable for large-scale popularization and application. The method is simple and convenient to operate, lower in cost and easy to popularize and use, and has important significance on detection of O-glycosylation of the hinge region of the IgG3 antibody.

Description

Technical Field

[0001] The present invention relates to the technical field of biochemistry, and in particular to a method for identifying O-glycosylation of the hinge region of an IgG3 antibody. Background Art

[0002] Immunoglobulin G (IgG) antibodies are key immunoglobulins in the human immune system, with the IgG3 subtype playing a unique and crucial role in immune responses. IgG3 antibodies differ from other human immunoglobulin subtypes primarily in their presence of an O-glycosylated long hinge region, which significantly increases the structural flexibility of the antibody's Fab-Fab and Fab-Fc structures. O-glycosylation plays a crucial role in the conformational stability, functional activity, and interactions of IgG3 antibodies with other immune molecules.

[0003] Abnormal O-glycosylation in the hinge region of IgG3 antibodies is closely associated with autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. Altered O-glycosylation patterns may lead to the production or clearance of pathological autoantibodies. The anti-tumor activity and immunogenicity of IgG3-based therapeutic antibodies are also closely related to hinge region O-glycosylation. Therefore, analysis of IgG3 antibody hinge region O-glycosylation is crucial for disease detection and quality control of therapeutic antibodies.

[0004] Currently, O-glycosylation in the hinge region of IgG3 antibodies is identified using a conventional bottom-up mass spectrometry approach, which involves proteolytic cleavage of the intact protein into peptide fragments, followed by ultra-high performance liquid chromatography (ULHPLC) separation and subsequent mass spectrometry to collect peptide mass spectrometry information. This method only provides information on individual glycosylation sites, but cannot determine the interaction between individual sites. Furthermore, glycosylation signals may be lost.

[0005] To this end, the present invention provides a method for identifying O-glycosylation of the hinge region of an IgG3 antibody. The IgG3 antibody is hydrolyzed using chymotrypsin, and after ultrafiltration purification, the complete hinge region protein information is collected by mass spectrometry to obtain the overall O-glycosylation status of the hinge region of the IgG3 antibody. Summary of the Invention

[0006] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention provides a method for identifying O-glycosylation of the hinge region of an IgG3 antibody. The IgG3 antibody is hydrolyzed using chymotrypsin, so that the hinge region can remain in the form of a small protein of approximately 8 kD, while the remaining sequences outside the hinge region are enzymatically cleaved into small peptides of 1-2 kD. Further centrifugation through a 10 kD ultrafiltration tube can obtain a high-purity hinge region protein. Mass spectrometry is used to collect information on the complete hinge region protein to obtain the overall O-glycosylation status of the hinge region of the IgG3 antibody.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a method for identifying O-glycosylation of the hinge region of an IgG3 antibody, comprising the following steps: enzymatically cleaving the IgG3 antibody using chymotrypsin, enriching the fragments using an ultrafiltration tube, and detecting the resulting cleaved fragments.

[0009] In some embodiments of the present invention, the mass ratio of the IgG3 antibody to chymotrypsin is 20:1 to 70:1.

[0010] In some embodiments of the present invention, the mass ratio of the IgG3 antibody to chymotrypsin is 40:1 to 60:1.

[0011] In some embodiments of the present invention, the mass ratio of the IgG3 antibody to chymotrypsin is 50:1.

[0012] In some embodiments of the present invention, the chymotrypsin enzymatic digestion is performed overnight.

[0013] In some embodiments of the present invention, the overnight enzymatic digestion time comprises 8-16 hours.

[0014] In some embodiments of the present invention, the temperature of the chymotrypsin cleavage is 25°C to 50°C.

[0015] In some embodiments of the present invention, the temperature of the chymotrypsin cleavage is 25°C to 30°C.

[0016] In some embodiments of the present invention, the temperature of the chymotrypsin cleavage is 30°C.

[0017] In some embodiments of the present invention, the source of the IgG3 antibody includes serum or cell culture supernatant.

[0018] In some embodiments of the present invention, the serum or cell culture supernatant is purified by gel column to obtain IgG3 antibodies.

[0019] In some embodiments of the present invention, the gel column comprises a gel column prepared by adding Protein G and / or Protein A into a syringe.

[0020] In some embodiments of the present invention, the step of purifying using a gel column comprises taking a sample, diluting it with PBS, passing it through a Protein G gel column, and then eluting it with a glycine solution to obtain an immunoglobulin mixed solution. The immunoglobulin mixed solution is passed through a Protein A gel column to obtain purified IgG3 antibodies.

[0021] In some embodiments of the present invention, the pore size of the ultrafiltration tube is 5-10 kD.

[0022] In some embodiments of the present invention, the pore size of the ultrafiltration tube is 10 kD.

[0023] In some embodiments of the present invention, the enzyme-digested fragments obtained after enrichment by ultrafiltration tube include fragments with a size of 4 to 9 kD.

[0024] In some embodiments of the present invention, the enzyme-digested fragments obtained after enrichment by ultrafiltration tube include fragments with a size of 8 kD.

[0025] The second aspect of the present invention provides the use of chymotrypsin in preparing a product for detecting O-glycosylation of the hinge region of an IgG3 antibody.

[0026] The third aspect of the present invention provides the use of chymotrypsin in the preparation of a product for detecting diseases associated with abnormal O-glycosylation of the hinge region of an IgG3 antibody.

[0027] In some embodiments of the present invention, the disease associated with abnormal O-glycosylation in the hinge region of an IgG3 antibody includes an autoimmune disease.

[0028] In some embodiments of the invention, the autoimmune disease includes rheumatoid arthritis and systemic lupus erythematosus.

[0029] A fourth aspect of the present invention provides the use of chymotrypsin in the detection and analysis of IgG3 antibody drug quality, wherein the use is used for non-disease diagnosis and treatment.

[0030] The beneficial effects of the present invention are:

[0031] The present invention provides a method for identifying O-glycosylation of the hinge region of an IgG3 antibody. The method uses chymotrypsin to hydrolyze the IgG3 antibody, and then performs ultrafiltration purification to obtain the overall O-glycosylation status of the hinge region of the IgG3 antibody, which is conducive to obtaining information on the relationship between O-glycosylation sites. On the one hand, it helps to discover abnormal O-glycosylation of IgG3 antibodies in patients with diseases such as rheumatoid arthritis and systemic lupus erythematosus; on the other hand, it can improve the efficiency of O-glycosylation analysis of IgG3-based antibody drugs. At the same time, the method of the present invention loads domestically produced gel into a syringe cavity to form a simple gel column to achieve the purpose of enrichment and purification. The method is simple to operate, has lower cost, and is easy to promote and use. It is of great significance for the detection of O-glycosylation of the hinge region of IgG3 antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Comparison of IgG3 antibodies with other IgG subtypes and schematic diagram of O-glycosylation.

[0033] Figure 2 Schematic diagram of the differences in mass spectrometry sample pretreatment between the technical solution of the present invention and conventional identification methods.

[0034] Figure 3 Schematic diagram of the differences between the plasma IgG3 antibody purification steps of the technical solution of the present invention and the conventional identification method.

[0035] Figure 4 The IgG3 antibody purified in Example 1 of the present invention and Comparative Example 1 is shown in the staining diagram.

[0036] Figure 5 The results of mass spectrometry identification of O-glycosylation of the hinge region of the IgG3 antibody of Example 1 of the present invention are shown; A is the original protein nuclear ratio map; B is the protein precursor ion molecular weight map after deconvolution.

[0037] Figure 6 These are the mass spectrometry identification results of the O-glycosylated peptide segments of the IgG3 antibody of Comparative Example 1 of the present invention; A is the total ion current; B is the signal peak of the extracted non-glycosylated peptide segment; C is the signal peak of the O-glycosylated peptide segment with a signal intensity of 4.91E5; and D is the signal peak of the O-glycosylated peptide segment with a signal intensity of 1.04E6. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0039] Example 1

[0040] This example provides a method for identifying O-glycosylation in the hinge region of an IgG3 antibody. IgG3 antibodies are compared with other IgG subtypes and O-glycosylation is Figure 1 The specific experimental steps are as follows.

[0041] (1) Gel column preparation: Plug a small amount of cotton into the outlet of a 2 mL syringe and add Protein G and Protein A gel solutions to prepare Protein G gel columns and Protein A gel columns, respectively. Keep the gel moist and use them as gel columns for later use.

[0042] (2) Acquisition and purification of human immunoglobulins: 10 mL of human plasma was taken and diluted to 100 mL by adding 90 mL of PBS; the dilution solution was passed through a gel column containing 200 μL of Protein G three times; the gel column was washed three times with 400 μL of PBS; and a mixture of human immunoglobulins IgG1, IgG2, IgG3, and IgG4 was obtained by adding 800 μL of 50 mM glycine solution (pH 2.7) for elution.

[0043] (3) Purification of IgG3 antibody: 80 μL of 1M Tris-HCl (pH 8.8) was added to 800 μL of the human immunoglobulin mixture obtained in step (2) for neutralization, and PBS was added to dilute to about 50 mL; the dilution solution was passed through a gel column containing 200 μL of Protein A three times to achieve the purpose of adsorbing IgG1, IgG2 and IgG4; the effluent was further passed through a gel column containing 200 μL of Protein G three times to achieve the purpose of enriching IgG3; the gel column was washed three times with 400 μL of PBS; 200 μL of 50 mM glycine solution (pH 2.7) was added to elute the IgG3 antibody, and 20 μL of 1M Tris-HCl (pH 8.8) was added for neutralization and then used for later use.

[0044] (4) Enzymatic hydrolysis of IgG3 antibody: 220 μL of the solution obtained in (3) was added to a 30 kD ultrafiltration tube, centrifuged at 6000 g for 5 min, 400 μL of 100 mM TrisHCl, 2 mM CaCl2 was added, and centrifuged at 6000 g for 5 min, repeated three times; the protein concentration was measured using a nano-micro spectrophotometer, and chymotrypsin (Thermo Fisher Scientific) was added at a protein to enzyme mass ratio of 50:1, and the enzyme digestion was carried out at 30°C overnight.

[0045] (5) Purification of the IgG3 hinge region: The mixture after overnight enzymatic digestion was added to a 10 kD ultrafiltration tube and centrifuged at 12,000 g for 5 min. Ultrapure water was added and centrifuged at 12,000 g for 5 min. This process was repeated three times until the final volume of the solution was approximately 50 μL. Desalting was performed using conventional mass spectrometry pretreatment procedures.

[0046] (6) IgG3 antibody hinge region small protein spectrum data acquisition: UPLC-MS was used for mass spectrometry data acquisition, and the separation column used was a Waters ACQUITY UPLC BEH C18 1.7μm column, and the Orbitrap Exploris TM Data were collected using a 240 mass spectrometer. The injection volume was 5 μg; the liquid phase consisted of 0.1% formic acid in acetonitrile and 0.1% formic acid in water; the separation time was 120 min, the gradient was 8-35, and the mass spectrometry data acquisition range was 600-3000.

[0047] Comparative Example 1

[0048] This comparative example 1 is a conventional method for detecting IgG3 antibodies. The specific experimental steps are as follows.

[0049] (1) Purification of human immunoglobulins from human plasma: Using Cytiva's HiTrap Protein G HP product, follow the instructions to obtain a mixture of human immunoglobulins IgG1, IgG2, IgG3, and IgG4.

[0050] (2) Purification of IgG3 antibodies from human immunoglobulin mixture: Cytiva HiTrap Protein A HP product was used to remove IgG1, IgG2, and IgG4 antibodies according to the instructions, and IgG3 antibodies were concentrated using an ultrafiltration tube. The replacement solution was 50 mM NH4HCO3.

[0051] (3) Enzymatic digestion of IgG3 antibody: Trypsin was added at a protein to enzyme ratio of 50:1 and digested at 37°C overnight. Desalting was performed according to conventional mass spectrometry pretreatment procedures.

[0052] (5) Mass spectrometry data acquisition: UPLC-MS was used for mass spectrometry data acquisition. The separation column used was a Waters ACQUITY UPLC BEH C18 1.7 μm column and a Thermo Fisher Scientific Orbitrap Exploris TM Data were collected using a 240 mass spectrometer. The injection volume was 5 μg; the liquid phase consisted of 0.1% formic acid in acetonitrile and 0.1% formic acid in water; the separation time was 20 min, the gradient was 8-35, and the mass spectrometry data acquisition range was 300-2000.

[0053] Effect Example 1

[0054] This effect example provides a comparison of the detection methods of Example 1 and Comparative Example 1. The differences in the mass spectrometry sample pretreatment between Example 1 and Comparative Example 1 are as follows: Figure 2 As shown, the plasma IgG3 antibody purification steps differ as follows Figure 3 shown.

[0055] Specific experimental procedures: IgG3 antibodies were detected according to the experimental procedures of Example 1 and Comparative Example 1. The human immunoglobulin IgG1, IgG2, IgG3, and IgG4 mixed solutions and the purified IgG3 antibodies obtained in Example 1 and Comparative Example 1 were subjected to SDS-PAGE electrophoresis and then stained with Coomassie Brilliant Blue (Coomassie staining). At the same time, the fragments obtained by enzyme digestion of the purified IgG3 antibodies obtained in Example 1 and Comparative Example 1 were sequenced using mass spectrometry. The amino acid sequence of the hinge region of IgG3 after enzyme digestion is shown in Table 1 below.

[0056] Table 1 Amino acid sequence of hinge region after IgG3 enzyme cleavage

[0057]

[0058] The experimental results are as follows Figure 4-6 As shown, Figure 4 This is a staining diagram of the purified IgG3 antibody of Example 1 and Comparative Example 1. Figure 5 The results of mass spectrometry identification of O-glycosylation of the hinge region of the IgG3 antibody in Example 1 are shown. Figure 6 The mass spectrometry results of the O-glycosylated peptide fragments of the IgG3 antibody of Comparative Example 1 are shown. The experimental results show that after enzymatic digestion of the purified IgG3 antibodies of Example 1 and Comparative Example 1, Example 1 retains the complete hinge region, while Comparative Example 1 cuts the hinge region into smaller fragments. At the same time, the detection results of Example 1 show the O-glycosylated protein in the hinge region. Figure 5 A in the middle is the original protein core ratio, Figure 5 B in the middle is the molecular weight of the protein parent ion after deconvolution, which clearly shows that the O-glycosylation structure is the Core1 form with two sialic acids. Figure 6 The test results for Comparative Example 1 are as follows: Figure 6 A in the figure is the total ion current diagram, Figure 6 B in the middle is the signal peak of the extracted non-glycosylated peptide segment, with an intensity of 1.11E9. Figure 6 C and D in the middle are the signal peaks of the corresponding O-glycosylated peptides, and there are only impurity signals with signal intensities of 4.91E5 and 1.04E6.

[0059] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for identifying O-glycosylation of the hinge region of an IgG3 antibody, characterized in that: The method comprises the following steps: using chymotrypsin to enzymatically digest IgG3 antibodies, enriching them through an ultrafiltration tube, and then detecting the obtained enzymatically digested fragments.

2. The method according to claim 1, characterized in that The mass ratio of the IgG3 antibody to chymotrypsin is 40:1 to 60:

1.

3. The method according to claim 1, characterized in that The chymotrypsin enzymatic digestion time is 8 to 16 hours.

4. The method according to claim 1, wherein The temperature of the chymotrypsin enzymatic digestion is 25°C to 30°C.

5. The method according to claim 1, wherein The pore size of the ultrafiltration tube is 5-10 kD.

6. The method according to claim 1, characterized in that The IgG3 antibody source includes serum or cell culture supernatant.

7. The method according to claim 1, characterized in that The enzyme-digested fragments obtained after enrichment in the ultrafiltration tube include fragments with a size of 4 to 9 kD.

8. Application of chymotrypsin in the preparation of products for detecting O-glycosylation of the hinge region of IgG3 antibodies.

9. Use of chymotrypsin in the preparation of a product for detecting diseases associated with abnormal O-glycosylation of the hinge region of an IgG3 antibody, characterized in that: Diseases associated with abnormal O-glycosylation in the hinge region of IgG3 antibodies include autoimmune diseases; and the autoimmune diseases include rheumatoid arthritis and systemic lupus erythematosus.

10. Application of chymotrypsin in the quality detection and analysis of IgG3 antibody drugs.

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