Method for simultaneously and quantitatively detecting active and inactive components in human immunoglobulin for intravenous injection

Abstract

The invention relates to a method for simultaneously and quantitatively detecting active ingredients and inactive ingredients in an intravenous injection human immune globulin product, the active ingredients comprise one or more immune globulins in IgG, IgD, IgA and IgM, and the inactive ingredients comprise albumin ALB. The method comprises the following steps: carrying out proteolysis to obtain a peptide fragment mixed solution containing characteristic peptides corresponding to the active component and the inactive component, the active component is IgA, and the sequence of the corresponding characteristic peptide is shown as SEQ ID NO.1; the active component is IgD, and the sequence of the corresponding characteristic peptide is shown as SEQ ID NO.2; the active component is IgG, and the sequence of the corresponding characteristic peptide is shown as SEQ ID NO.3; the active component is IgM, and the sequence of the corresponding characteristic peptide is shown as SEQ ID NO.4; the inactive component is albumin ALB, and the sequence of the corresponding characteristic peptide is as shown in SEQ ID NO.5; detecting by adopting a high performance liquid chromatography-mass spectrometer, taking the obtained characteristic peptide as a target peptide fragment, acquiring mass spectrum data based on a PRM, SRM or MRM mode, and carrying out targeted detection on the target peptide fragment, so as to realize quantitative analysis on active components and inactive components in the intravenous injection human immune globulin product.

Description

Technical Field

[0001] The present invention relates to the field of biological drug technical analysis, and more specifically to a method for simultaneously and quantitatively detecting active and inactive components in intravenous human immunoglobulin. Background Art

[0002] Intravenous human immunoglobulin (pH4) is a human immunoglobulin drug for intravenous injection. It is widely used in the clinical treatment of primary and secondary immunodeficiency diseases, neuromuscular diseases and Kawasaki disease. Its main active ingredients include immunoglobulin G (IgG), immunoglobulin D (IgD), immunoglobulin A (IgA) and immunoglobulin M (IgM), of which IgG is the main one. The Chinese Pharmacopoeia stipulates that the IgG content in the stock solution of intravenous human immunoglobulin (pH4) should not be less than 95%, and the IgG content in the finished product should not be less than 90% of the labeled amount. At present, domestic companies mostly use the low-temperature ethanol method to combine at least 1,000 healthy human plasma, and then use low-temperature ethanol graded precipitation, separation and purification, and ultrafiltration to remove ethanol to obtain it. However, due to the limitations of existing processes, in addition to the main active ingredients, intravenous human immunoglobulin products also contain other inactive ingredients (non-immunoglobulin residues) derived from human plasma, such as complement factors.

[0003] In clinical applications, common adverse reactions to intravenous human immunoglobulin include fever, rash, and itching, while serious adverse reactions may include high fever, chills, thrombosis, and anaphylactic shock. Although the exact causes and mechanisms of these adverse reactions are still inconclusive, most researchers believe that their adverse reactions are closely related to the inactive proteins remaining in the product. For example, residual procoagulants such as human coagulation factor XI may lead to thromboembolic events (TEE, literature 1); residual protein components with anticomplement activity (ACA) may trigger abnormal activation of the complement system, thereby causing allergic reactions such as fever and chills (literature 2). Therefore, quantitative detection of active and inactive ingredients in intravenous human immunoglobulin products is conducive to product quality control and reduction of adverse reactions.

[0004] At present, the quantitative detection method for active protein components of intravenous human immunoglobulin products is mainly the Fc segment activity detection method, such as CN 106501524B, a method for detecting the Fc segment activity of intravenous human immunoglobulin, which uses the cell counting method to visually detect the hemolysis of erythrocytes caused by antigens, thereby obtaining the activity results of the Fc segment. The higher the activity of the Fc segment of intravenous human immunoglobulin, the milder the hemolysis of erythrocytes. Although this method reduces the coefficient of difference CV value to less than 5% compared with the detection method specified in the European Pharmacopoeia and the traditional OD value detection method, this method is still affected by the stability of antigen acquisition, the possibility of large human errors in the cell counting process, and the accuracy of instrument judgment, resulting in limited wide application.

[0005] The existing quantitative detection methods for inactive protein components in biological products are mainly enzyme-linked immunosorbent assay (ELISA) and reversed-phase high-performance liquid chromatography (RP-HPLC). However, the accuracy of ELISA is low and it is easily affected by the stability of various test supplies such as primary antibodies, secondary antibodies, and colorimetric solutions. The detection of low-abundance impurity proteins by reversed-phase high-performance liquid chromatography is prone to problems such as baseline fluctuations of chromatographic peaks, changes in peak elution time, and low-abundance impurity protein peaks being masked by noise peaks. At present, there are few methods for quantitative detection of inactive components of intravenous human immunoglobulin, and it is difficult to quantitatively detect its active components and inactive components at the same time. Therefore, based on the characteristics of intravenous human immunoglobulin, a method that can simultaneously quantitatively detect its active components and inactive components is studied, which is of great significance to the quality supervision of existing intravenous human immunoglobulin products and the research and development of high-quality intravenous human immunoglobulin products.

[0006] Source:

[0007] Reference 1: Chen Tao, Sun Ziqiang. Research progress on coagulation factor Ⅺ and venous thromboembolism[J]. Chinese Contemporary Medicine, 2023, 30(11):42-46.

[0008] Reference 2: Xu Han, Zhang Yunyi, Zhang Jianwen, et al. Anti-complement active ingredients in natural products [J]. Chinese Journal of Natural Medicine, 2007, 5(5): 322-332. Summary of the invention

[0009] In view of the above defects or improvement needs of the prior art, the present invention provides a method for simultaneously quantitatively detecting active ingredients and inactive ingredients in intravenous human immunoglobulin products, the purpose of which is to use a high performance liquid chromatography-mass spectrometer to detect intravenous human immunoglobulin, collect data based on the DIA mode, search and analyze the library to screen out characteristic peptides corresponding to 4 immunoglobulins and 12 non-immunoglobulins, take one or more of the characteristic peptides as target peptide segments, use a high performance liquid chromatography-mass spectrometer to detect, and collect mass spectrometry data based on the PRM, SRM or MRM mode to perform targeted detection of the target peptide segments, thereby achieving quantitative detection of active ingredients and inactive ingredients in intravenous human immunoglobulin products, thereby solving the technical problems of poor accuracy and stability of existing quantitative detection of intravenous human immunoglobulin and difficulty in simultaneously detecting its active ingredients and inactive ingredients.

[0010] To achieve the above object, according to one aspect of the present invention, a method for simultaneously and quantitatively detecting active ingredients and inactive ingredients in an intravenous human immunoglobulin product is provided, wherein the active ingredients include one or more immunoglobulins of IgG, IgD, IgA and IgM, and the inactive ingredients include albumin ALB, and the method comprises the following steps:

[0011] (1) Proteolysis: The intravenous human immunoglobulin to be tested is enzymatically hydrolyzed by a protease that uses lysine and arginine as enzymatic sites to obtain a peptide segment mixture containing characteristic peptides corresponding to the active ingredient and the inactive ingredient;

[0012] The active ingredient is IgA, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.1; the active ingredient is IgD, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.2;

[0013] The active ingredient is IgG, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.3;

[0014] The active ingredient is IgM, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.4;

[0015] The inactive ingredient is albumin ALB, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.5;

[0016] (2) Targeted detection: HPLC-MS / MS is used to detect the characteristic peptides as target peptides. Mass spectrometry data is collected based on PRM, SRM or MRM mode, and the target peptides are targeted for detection, thereby achieving quantitative analysis of the active and inactive ingredients in intravenous human immunoglobulin products.

[0017] Preferably, in the method, the active ingredient comprises IgG, and any one or a combination of IgM, IgA and IgD.

[0018] Preferably, in the method, the inactive ingredients also include complement factor CFB and / or complement factor C1QB, wherein the inactive ingredient is complement factor CFB, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.8; the inactive ingredient is complement factor C1QB, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.14.

[0019] Preferably, in the method, the inactive ingredients further include one or more of transferrin TF, gelsolin GSN, and heme-binding protein HPX, wherein the inactive ingredient is transferrin TF, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.7; the inactive ingredient is gelsolin GSN, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.10; the inactive ingredient is heme-binding protein HPX, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.11.

[0020] Preferably, in the method, the inactive ingredient further includes apolipoprotein APOA1 and / or apolipoprotein APOH, wherein the inactive ingredient is apolipoprotein APOA1, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.6; the inactive ingredient is apolipoprotein APOH, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.9.

[0021] Preferably, in the method, the inactive ingredients further include one or more of transferrin TF, gelsolin GSN, and heme-binding protein HPX, wherein the inactive ingredient is transferrin TF, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.7; the inactive ingredient is gelsolin GSN, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.10; the inactive ingredient is heme-binding protein HPX, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.11.

[0022] Preferably, in the method, the inactive ingredients further include one or more of fibrinogen FGA, fibrin FCN3, antithrombin SERPINC1, and antitrypsin SERPINA1, wherein the inactive ingredient is fibrinogen FGA, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.12; the inactive ingredient is fibrin FCN3, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.13; the inactive ingredient is antithrombin SERPINC1, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.15; the inactive ingredient is antitrypsin SERPINA1, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.16.

[0023] Preferably, in the method, the protease with lysine and arginine as enzymatic hydrolysis sites is trypsin, and the protein reaction solution is added at a volume ratio of 1:1 between the protein amount and 1% protein reaction solution to denature and reductively alkylate the protein. After completion, water is added to dilute the protein reaction solution to reduce the concentration to below 0.5%, and trypsin is added for enzymatic hydrolysis.

[0024] Preferably, in the method, the 1% protein reaction solution comprises 1% SDC, 50-150 mM Tris-HCl, 10-50 mM TCEP and 10-50 mM CAA.

[0025] Preferably, in the method, the 1% protein reaction solution further comprises 10-50 mM DTT and / or 10-50 mM IAA.

[0026] Preferably, in the method, the peptide mixture is desalted and then detected using a high performance liquid chromatography-mass spectrometer, wherein mobile phase A comprises 0.1% formic acid and 3% DMSO, and mobile phase B comprises 0.1% formic acid, 3% DMSO and 80% acetonitrile; and mass spectrometry data is collected using the PRM mode.

[0027] In general, compared with the prior art, the above technical scheme conceived by the present invention can achieve the following beneficial effects by screening out characteristic peptides corresponding to 4 immunoglobulins and 12 non-immunoglobulins from enzymatic peptides of intravenous human immunoglobulin enzymes, using one or more of the screened characteristic peptides as target peptides, and performing targeted detection of the target peptides based on PRM, SRM or MRM technology:

[0028] The present invention provides a method for simultaneously quantitatively detecting active ingredients and inactive ingredients in an intravenous human immunoglobulin product. The active ingredients include one or more immunoglobulins of IgG, IgD, IgA and IgM, and the inactive ingredients include albumin ALB, wherein the active ingredient is IgA, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.1; the active ingredient is IgD, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.2; the active ingredient is IgG, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.3; the active ingredient is IgM, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.4; the inactive ingredient is albumin ALB, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.5; high performance liquid chromatography-mass spectrometry is used for detection, the obtained characteristic peptide is used as the target peptide segment, mass spectrum data is collected based on PRM, SRM or MRM mode, and targeted detection is performed on the target peptide segment, thereby realizing quantitative analysis of the active ingredients and inactive ingredients in the intravenous human immunoglobulin product. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1This is an overview of protein identification results, where orange represents non-immunoglobulins and blue represents immunoglobulins;

[0030] Figure 2 This is an overview of the protein detection signal results, where orange represents non-immunoglobulin and blue represents immunoglobulin;

[0031] Figure 3 This is the distribution result of the coefficient of variation of the target protein of the intravenous human immunoglobulin product of manufacturer 1 in 9 PRM tests;

[0032] Figure 4 is the quantitative value of the target protein of different batches of products from manufacturer 1;

[0033] Figure 5 It is the ratio of the signal intensity of different types of target proteins to the total protein signal intensity in the intravenous human immunoglobulin product of manufacturer 1;

[0034] Figure 6 This is the distribution result of the coefficient of variation of the target protein of the intravenous human immunoglobulin product of manufacturer 2 in 9 PRM tests;

[0035] Figure 7 is the quantitative value of the target protein of different batches of products from manufacturer 2;

[0036] Figure 8 It is the ratio of the signal intensity of different types of target proteins to the total protein signal intensity in the intravenous human immunoglobulin product of manufacturer 2;

[0037] Fig. 9 This is the distribution result of the coefficient of variation of the target protein of the intravenous human immunoglobulin product of manufacturer 3 in 9 PRM tests;

[0038] Fig.10 is the quantitative value of the target protein of different batches of products from manufacturer 3;

[0039] Fig.11 It is the ratio of the signal intensity of different types of target proteins to the total protein signal intensity in the intravenous human immunoglobulin product of manufacturer 3;

[0040] Fig.12 It is the PCA principal component analysis diagram;

[0041] Fig.13 This is a quantitative bar chart of target proteins of intravenous human immunoglobulin products from three manufacturers. DETAILED DESCRIPTION

[0042] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation mode, structure, characteristics and effects of the present invention are described in detail below in combination with the accompanying drawings and preferred embodiments.

[0043] The present invention is based on intravenous human immunoglobulin samples from different batches of different manufacturers, adopts high performance liquid chromatography-mass spectrometry for detection, and based on DIA mode data collection, analyzes and screens out characteristic peptides of 4 immunoglobulins and 12 non-immunoglobulins; based on the screened characteristic peptides of the target protein, adopts high performance liquid chromatography-mass spectrometry for detection, and uses PRM, SRM or MRM mode to collect data to perform targeted detection of the target peptide segment, which can realize the quantitative analysis of the active ingredients and inactive ingredients in the intravenous human immunoglobulin product at the same time.

[0044] Based on this, the present invention provides a method for simultaneously and quantitatively detecting active ingredients and inactive ingredients in an intravenous human immunoglobulin product, wherein the active ingredients include one or more immunoglobulins of IgG, IgD, IgA and IgM, and the inactive ingredients include albumin ALB, specifically comprising the following steps:

[0045] (1) Proteolysis: The intravenous human immunoglobulin to be tested is enzymatically hydrolyzed by a protease that uses lysine and arginine as enzymatic sites to obtain a peptide segment mixture containing characteristic peptides corresponding to the active ingredient and the inactive ingredient;

[0046] The active ingredient is IgA, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.1;

[0047] The active ingredient is IgD, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.2;

[0048] The active ingredient is IgG, and the sequence of its corresponding characteristic peptide is the amino acid sequence shown in SEQ ID NO.3 (DTLMISR);

[0049] The active ingredient is IgM, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.4;

[0050] The inactive ingredient is albumin ALB, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.5.

[0051] (2) Targeted detection: HPLC-MS / MS is used to detect the characteristic peptides as target peptides. Mass spectrometry data is collected based on PRM, SRM or MRM mode to perform targeted detection on the target peptides, thereby achieving quantitative detection of the active ingredients and inactive ingredients.

[0052] Among them, PRM targeted detection is specific:

[0053] In the PRM mass spectrometry acquisition mode, according to the target peptide precursor ion (Precursor) parameters set for acquisition, the precursor ions of the target peptide are selectively screened into the collision cell HCD for fragmentation to generate corresponding fragment ions (Fragment); then, the Orbitrap or TOF mass analyzer detects the fragment ions to obtain a high-resolution mass spectrum of the fragment ions, i.e., the secondary mass spectrum MS2; finally, the target peptide is quantitatively analyzed based on MS2.

[0054] In some embodiments, the active ingredient includes IgG, and any one or combination of IgM, IgA and IgD, preferably IgG and IgD; wherein the active ingredient is IgD, and the sequence of its corresponding characteristic peptide is such as the amino acid sequence (VTISVDTSR) shown in SEQ ID NO.2; the active ingredient is IgM, and the sequence of its corresponding characteristic peptide is such as the amino acid sequence (DVMQGTDEHVVCK) shown in SEQ ID NO.4; the active ingredient is IgA, and the sequence of its corresponding characteristic peptide is such as the amino acid sequence (WLQGSQELPR) shown in SEQ ID NO.1.

[0055] In some embodiments, the inactive ingredients further include one or more non-immunoglobulins selected from apolipoprotein APOA1, transferrin TF, complement factor CFB, apolipoprotein APOH, gelsolin GSN, hemoglobin HPX, fibrinogen FGA, fibrin FCN3, complement factor C1QB, antithrombin SERPINC1, and antitrypsin SERPINA1;

[0056] The inactive ingredient is albumin ALB, and the sequence of its corresponding characteristic peptide is the amino acid sequence (VFDEFKPLVEEPQNLIK) shown in SEQ ID NO.5.

[0057] The inactive ingredient is apolipoprotein APOA1, and the sequence of its corresponding characteristic peptide is the amino acid sequence (THLAPYSDELR) shown in SEQ ID NO.6.

[0058] The inactive ingredient is transferrin TF, and the sequence of the corresponding characteristic peptide is the amino acid sequence (EGYYGYTGAFR) shown in SEQ ID NO.7.

[0059] The inactive ingredient is complement factor CFB, and the sequence of its corresponding characteristic peptide is the amino acid sequence (DAQYAPGYDK) shown in SEQ ID NO.8.

[0060] The inactive ingredient is apolipoprotein APOH, and the sequence of its corresponding characteristic peptide is the amino acid sequence (FICPLTGLWPINTLK) shown in SEQ ID NO.9.

[0061] The inactive ingredient is gelsolin GSN, and the sequence of its corresponding characteristic peptide is the amino acid sequence (HVVPNEVVVQR) shown in SEQ ID NO.10.

[0062] The inactive ingredient is heme-binding protein HPX, and the sequence of its corresponding characteristic peptide is the amino acid sequence (NFPSPVDAAFR) shown in SEQ ID NO.11.

[0063] The inactive ingredient is fibrinogen FGA, and the corresponding characteristic peptide sequence is the amino acid sequence (DSHSLTTNIMEILR) shown in SEQ ID NO. 12.

[0064] The inactive ingredient is fibrin FCN3, and the sequence of its corresponding characteristic peptide is the amino acid sequence (YGIDWASGR) shown in SEQ ID NO. 13.

[0065] The inactive ingredient is complement factor C1QB, and the sequence of its corresponding characteristic peptide is the amino acid sequence (FDHVITNMNNNYEPR) shown in SEQ ID NO.14.

[0066] The inactive ingredient is antithrombin SERPINC1, and the sequence of its corresponding characteristic peptide is the amino acid sequence (VAEGTQVLELPFK) shown in SEQ ID NO.15.

[0067] The inactive ingredient is antitrypsin SERPINA1, and the sequence of the corresponding characteristic peptide is the amino acid sequence (SVLGQLGITK) shown in SEQ ID NO.16.

[0068] In some embodiments, the protease with lysine and arginine as enzymatic sites is trypsin, and the protein reaction solution is added at a volume ratio of 1:1 between the protein amount and the 1% protein reaction solution to denature and reduce the alkylation of the protein. After completion, water is added to dilute the protein reaction solution to reduce the concentration of the protein reaction solution to less than 0.5%, and trypsin is added for enzymatic hydrolysis; the 1% protein reaction solution includes SDC, TCEP, CAA and Tris-HCl, and preferably also includes DTT and / or IAA, wherein SDC is sodium deoxycholate, TCEP is the abbreviation of tris (2-carboxyethyl) phosphine hydrochloride, CAA is chloroacetic acid, DTT is the abbreviation of dithiothreitol, and IAA is the abbreviation of indole-3-acetic acid.

[0069] In some embodiments, the 1% protein reaction solution includes 1% SDC, 50-150 mM Tris-HCl, 10-50 mM TCEP and 10-50 mM CAA, and preferably also includes 10-50 mM DTT and / or 10-50 mM IAA.

[0070] The peptide mixture is desalted and detected by high performance liquid chromatography-mass spectrometry, wherein mobile phase A comprises 0.1% formic acid and 3% DMSO, and mobile phase B comprises 0.1% formic acid, 3% DMSO and 80% acetonitrile; preferably, PRM mode is used for mass spectrometry data acquisition.

[0071] The following are examples

[0072] Example 1 Screening of characteristic peptides of immunoglobulins and non-immunoglobulins in intravenous human immunoglobulin products

[0073] In this example, three different batches of intravenous human immunoglobulin products from three different manufacturers were selected for protein extraction and enzymatic hydrolysis and peptide desalting treatment, and high-performance liquid chromatography-mass spectrometry was used for detection and DIA data independent acquisition. The DIA raw data was searched using DIA-NN (version 1.8) software. The search refers to downloading the human protein database from the Uniprot official website. The search software uses an algorithm to construct a theoretical spectrum library for the protein database, that is, the theoretical spectrum corresponding to the peptide segment after the protein theoretical enzyme cleavage, and then the actual detected spectrum is matched and scored with the theoretical spectrum to determine which protein the detected peptide segment belongs to. The human protein database used is: Uniprot.Swissprot.Human.20230619.fasta. The characteristic peptides corresponding to the protein can be screened out based on the DIA data acquisition mode.

[0074] The experimental process includes enzymatic hydrolysis of intravenous human immunoglobulin samples, peptide desalting, high performance liquid chromatography fractionation, and mass spectrometry detection and screening of peptides. The specific methods are as follows:

[0075] 1. Select intravenous human immunoglobulin products from 3 different manufacturers (Nanyue Biopharmaceutical Co., Ltd., Tonglu Biopharmaceutical Co., Ltd., and Sinopharm Wuhan Blood Products Co., Ltd.), each of which needs to have 3 different batches of products, for a total of 9 protein samples. Prepare 9 1.5mL EP tubes, labeled NY10, NY12, NY18, TL36, TL30, TL43, WS08, WS11, and WS24.

[0076] 2. After thoroughly mixing each intravenous human immunoglobulin sample, add 1 μL of protein solution (50 mg / mL) to each 1.5 mL EP tube, i.e., 50 μg of starting protein.

[0077] 3. Add 50 μL of 1% protein reaction solution, mix thoroughly, and incubate at 60°C with shaking for 30 min to perform protein denaturation and reductive alkylation reaction.

[0078] The 1% protein reaction solution was obtained by mixing the mother solution 10% SDC: 100mM TCEP: 400mM CAA: 1M Tris-HCl in a volume ratio of 1:1:1:1, wherein the 10% SDC mother solution was prepared by dissolving 10g of sodium deoxycholate (SDC) in 100mL of water.

[0079] 4. After the sample has cooled naturally, add ddH2O to dilute it so that the SDC volume concentration drops below 0.5%.

[0080] 5. Add 1 μg of Trypsin (lysine and arginine are used as enzymatic sites) and incubate at 37°C with shaking overnight for enzymatic hydrolysis.

[0081] 6. After overnight enzymatic hydrolysis, add TFA to terminate the enzymatic cleavage, take the supernatant and desalt it with SDB-RPS desalting column, vacuum dry it and freeze it at -20℃.

[0082] 7. High performance liquid chromatography-mass spectrometer detection: Use high performance liquid chromatography-mass spectrometer detection, the specific detection conditions are as follows:

[0083] ①Instrument system: Q Exactive HF mass spectrometer connected in series with UltiMate 3000RSLCnano liquid chromatography-mass spectrometry system.

[0084] ②Chromatographic column: 25cm long C18 chromatographic column.

[0085] ③Mobile phase: Mobile phase A: 0.1% formic acid, 3% DMSO; Mobile phase B: 0.1% formic acid, 3% DMSO, 80% acetonitrile, detection time 30-65 min.

[0086] ④Flow rate: 300nL / min.

[0087] ⑤Injection volume: 50μL dissolved sample, injection volume 1μL.

[0088] ⑥Data acquisition mode: Data-independent acquisition (DIA) acquisition mode. The specific DIA mass spectrometry detection parameter settings are shown in Table 2 below.

[0089] The HPLC detection gradient with a detection time of 35 min is shown in Table 1 below:

[0090] Table 1 HPLC detection gradient

[0091]

[0092]

[0093] Table 2 DIA mass spectrometry detection parameter settings

[0094]

[0095] 8. Use DIA-NN (version 1.8) software to search the DIA raw data.

[0096] The search results are summarized as follows Figure 1 and Figure 2As shown in the table, the main protein types in intravenous human immunoglobulin products from different manufacturers are immunoglobulins, and the detection signal intensity of immunoglobulins accounts for more than 99%. By classifying the quantified immunoglobulins, they mainly include IgG, IgD, IgM, and IgA. According to the signal intensity of non-immunoglobulins, the target proteins for subsequent PRM targeted detection of inactive ingredients in intravenous human immunoglobulin products selected 12 non-immunoglobulins with relatively high contents and related to adverse reactions. The specific non-immunoglobulins and their functions are shown in Table 3:

[0097] Table 3 Determination of non-immunoglobulins and their functional information

[0098]

[0099]

[0100] One peptide segment was selected for each protein for subsequent quantitative analysis of the protein, wherein the selected peptide segment sequence only matched the sequence of the protein, namely, the characteristic peptide. The specific peptide segment sequence information of the selected characteristic peptide is shown in Table 4 below:

[0101] Table 4 Characteristic peptides corresponding to the 4 immunoglobulins and 12 non-immunoglobulins screened

[0102]

[0103] Based on the above results, 4 immunoglobulins (IgG, IgD, IgM, IgA) and 12 non-immunoglobulins were selected for subsequent PRM targeted detection. Parallel reaction monitoring (PRM) technology is a targeted proteomics quantitative strategy based on high-resolution mass spectrometry. In the PRM mass spectrometry acquisition mode, according to the target peptide precursor ion (Precursor) parameters set for acquisition, the precursor ions of the target peptide are selectively screened into the collision cell HCD for fragmentation to generate corresponding fragment ions (Fragment); then, the fragment ions are detected by mass analyzers such as Orbitrap or TOF to obtain a high-resolution mass spectrum of the fragment ions, namely the secondary mass spectrometry MS2; finally, the target peptide is quantitatively analyzed based on MS2.

[0104] The present invention uses one or more of the characteristic peptides corresponding to the screened immunoglobulins and non-immunoglobulins as target peptide segments, performs targeted detection of the target peptide segments based on PRM technology, and then realizes quantitative detection of the target protein, so as to quantitatively detect the corresponding active ingredients and inactive ingredients in the intravenous human immunoglobulin product.

[0105] Example 2 Verification of the stability of this method

[0106] Based on the characteristic peptides corresponding to immunoglobulins and non-immunoglobulins screened in Example 1 as target peptides, the PRM technology was used to quantitatively detect the protein components of the intravenous human immunoglobulin product of manufacturer 1. In order to verify the stability of this method, three other batches of samples from manufacturer 1 were selected, and the same sample was tested three times, for a total of 9 mass spectrometry analyses. The sample processing is as follows:

[0107] After each intravenous human immunoglobulin sample was fully mixed, 1 μL of protein solution (50 mg / mL) was added to each 1.5 mL EP tube, i.e., 50 μg of starting protein amount.

[0108] 50 μL of 1% protein reaction solution (containing SDC, TCEP, CAA and Tris-HCl) was added to the protein solution, and after thorough mixing, the solution was incubated at 60° C. with shaking for 30 min to perform protein denaturation and reduction alkylation reaction.

[0109] After the reaction was completed, the sample was cooled naturally and then an equal volume of ddH2O was added to reduce the SDC concentration to 0.5%.

[0110] Add 1 μg of Trypsin, incubate at 37°C with shaking for 6 to 15 hours for enzymatic hydrolysis. After the enzymatic hydrolysis is completed, add TFA to terminate the enzymatic cleavage, and take the supernatant for desalting on a SDB-RPS desalting column.

[0111] Detection by high performance liquid chromatography-mass spectrometry:

[0112] ①Instrument system: Q Exactive HF mass spectrometer connected in series with UltiMate 3000RSLCnano liquid chromatography-mass spectrometry system.

[0113] ②Chromatographic column: 25cm long C18 chromatographic column.

[0114] ③ Mobile phase: Mobile phase A: 0.1% formic acid, 3% DMSO; Mobile phase B: 0.1% formic acid, 3% DMSO, 80% acetonitrile. The specific liquid phase detection conditions are the same as those in Example 1;

[0115] ④Flow rate: 300nL / min.

[0116] ⑤Injection volume: 50μL dissolved sample, injection volume 1μL.

[0117] ⑥Data acquisition mode: PRM, SRM or MRM mode acquisition, where the PRM mode detection parameters are set as shown in the following table.

[0118] The PRM mass spectrometry detection parameter settings are shown in Table 5 below:

[0119] Table 5 PRM mass spectrometry detection parameter settings

[0120]

[0121]

[0122] The HPLC gradient elution conditions and PRM mass spectrometry detection parameters in this embodiment are not intended to limit the detection conditions of this method. For example, SRM or MRM mode may be selected to collect mass spectrometry data. In the present invention, the parameters may be adjusted according to the actual detection situation to achieve quantitative detection of the target protein.

[0123] The raw data were imported into Skyline software (Version 24.1) to extract the fragment ion peaks of the target peptide and perform quantitative analysis to obtain the quantitative detection results of the target protein based on PRM technology, such as Figure 3 and Figure 4 shown.

[0124] Figure 3 This is the distribution diagram of the coefficient of variation of the target protein of the intravenous human immunoglobulin product of manufacturer 1 in 9 PRM tests. The horizontal axis represents the products of different batches of manufacturer 1, and the vertical axis represents the protein quantitative coefficient of variation of 3 repeated tests of the same product. One point represents one protein. Figure 4 The results showed that the coefficient of variation of the protein quantification values ​​of all samples was less than 10%, and more than 90% of the proteins had a coefficient of variation of less than 5%. This indicates that this method is based on the screened characteristic peptides and uses PRM technology to detect intravenous human immunoglobulin products to maintain better stability, and this method has better detection stability.

[0125] Figure 4 This is a quantitative bar chart of the target protein of the intravenous human immunoglobulin product of manufacturer 1. The horizontal axis represents different target proteins, and the vertical axis represents the logarithm (log10) of the PRM detection signal. Figure 4 The results showed the quantitative values ​​of target proteins in different batches of products from manufacturer 1. Among them, apolipoprotein APOA1, apolipoprotein APOH, fibrinogen FGA, antithrombin SERPIN C1 and antitrypsin SERPINA 1 were not detected in the intravenous human immunoglobulin products of manufacturer 1, which indicates that the quality of the intravenous human immunoglobulin is good. Except for IGM protein, the quantitative values ​​of other target proteins are not statistically significant in different batches, indicating that the quality stability of the intravenous human immunoglobulin products of this manufacturer is good. It also shows that the method used to quantitatively detect target proteins in intravenous human immunoglobulin has good repeatability.

[0126] According to the original signal value of PRM targeted detection, the ratio of the signal intensity of different types of target proteins in the intravenous human immunoglobulin product of manufacturer 1 to the total protein signal intensity was calculated, and the composition ratio of immunoglobulins and non-immunoglobulins in the intravenous human immunoglobulin product of manufacturer 1 was obtained by analysis. The results are as follows: Figure 5 shown.

[0127] Figure 5 The results showed that immunoglobulins accounted for 99.65% of the intravenous human immunoglobulin product of manufacturer 1, of which IgG accounted for 95.2%, while other non-immunoglobulin components such as albumin, transferrin, complement factors, etc. were distributed in trace amounts, that is, this method can accurately detect extremely low levels of non-immunoglobulins. It can be seen that the detection accuracy and sensitivity of this method are both high.

[0128] Example 3

[0129] Based on the characteristic peptides corresponding to the immunoglobulins and non-immunoglobulins screened out in Example 1 as the target peptides, the PRM technology was used to quantitatively detect the protein components of the intravenous human immunoglobulin product of Manufacturer 2. During the mass spectrometry loading test, the same sample was tested 3 times, for a total of 9 mass spectrometry analyses. The sample processing and detection conditions were the same as in Example 2, and the high performance liquid chromatography-mass spectrometer was used for detection, and the mass spectrometry data was collected in PRM mode. Subsequently, the raw data was imported into the Skyline software (Version 24.1), and the fragment ion peaks of the target peptides were extracted and quantitatively analyzed to obtain the quantitative results of the target protein.

[0130] Figure 6 This is the distribution of coefficient of variation of the target protein of the intravenous human immunoglobulin product of manufacturer 2 in 9 PRM tests. The results show that the coefficient of variation of the protein quantification values ​​of all samples is less than 10%, among which more than 85% of the proteins have a coefficient of variation less than 5%. This also indicates that this method has good stability when detecting the intravenous human immunoglobulin product of manufacturer 2.

[0131] Figure 7 These are the quantitative values ​​of target proteins of different batches of intravenous human immunoglobulin products from manufacturer 2. Among them, IgM, apolipoprotein APOA1, fibrinogen FGA, antithrombin SERPIN C1 and antitrypsin SERPINA 1 were not detected in the intravenous human immunoglobulin products from manufacturer 2, which indicates that the quality of this intravenous human immunoglobulin is better. The quantitative values ​​of all other target proteins do not have significant differences in different batches, indicating that the product preparation process of this manufacturer and the detection method have good repeatability.

[0132] According to the original signal value of PRM targeted detection, the ratio of the signal intensity of different types of target proteins to the total protein signal intensity was calculated. The results are as follows: Figure 8 As shown, immunoglobulins accounted for 97.43%, of which IgG accounted for 92.67%, while other non-immunoglobulin components such as albumin and apolipoprotein were distributed in trace amounts.

[0133] Example 4

[0134] Based on the characteristic peptides corresponding to the immunoglobulins and non-immunoglobulins screened out in Example 1 as the target peptides, the PRM technology was used to quantitatively detect the protein components of the intravenous human immunoglobulin product of Manufacturer 3. During the mass spectrometry loading test, the same sample was tested 3 times, for a total of 9 mass spectrometry analyses. The sample processing and detection conditions were the same as in Example 2, and the high performance liquid chromatography-mass spectrometer was used for detection, and the mass spectrometry data was collected in PRM mode. Subsequently, the raw data was imported into the Skyline software (Version 24.1), and the fragment ion peaks of the target peptides were extracted and quantitatively analyzed to obtain the quantitative results of the target protein.

[0135] Fig. 9 This is the distribution of coefficient of variation of the target protein of the intravenous human immunoglobulin product of manufacturer 3 in 9 PRM tests. The results show that the coefficient of variation of the protein quantification values ​​of all samples is less than 10%, among which more than 90% of the proteins have a coefficient of variation less than 5%. This also indicates that this method has good stability when detecting the intravenous human immunoglobulin product of manufacturer 3.

[0136] Fig.10 The quantitative values ​​of target proteins of different batches of products from manufacturer 3 are shown in Table 1. There is no significant difference in the quantitative values ​​of all target proteins in different batches, indicating that the product preparation process of this manufacturer and the detection method have good repeatability. According to the original signal value of PRM targeted detection, the ratio of the signal intensity of different types of target proteins to the total protein signal intensity is calculated. The results are shown in Table 1. Fig.11 As shown, the results showed that immunoglobulins accounted for 85.92%, of which IgG accounted for 76.38%, while other non-immunoglobulin components such as albumin and apolipoprotein were distributed in trace amounts.

[0137] Example 5

[0138] Since different manufacturers have different processes for preparing intravenous human immunoglobulin products, in order to further verify whether this method can be effectively used to distinguish intravenous human immunoglobulin products from different manufacturers, this example combines the quantitative results of Examples 2, 3, and 4 to conduct a comprehensive analysis of the target proteins of intravenous human immunoglobulin products from three manufacturers. The results are as follows: Fig.12 and Fig.13 .

[0139] Fig.12This is a PCA principal component analysis diagram, which shows the data distribution of target proteins of products from three different manufacturers. Through two-dimensional principal component analysis, it can be seen that the target proteins of products from the same manufacturer are all in the same confidence ellipse. Manufacturer 3 (red, WS) and the other two manufacturers (purple and blue, NY and TL, respectively) are clearly separated in the two principal component dimensions, showing smaller intra-group differences and larger inter-group differences. This result preliminarily shows that the production processes of intravenous human immunoglobulin products from different manufacturers may be inconsistent. Under the premise of ensuring the stability and repeatability of the test, the test results based on this method can effectively distinguish intravenous human immunoglobulin products from different manufacturers.

[0140] from Fig.13 It can also be seen that there are significant quantitative differences in most target proteins between the products of manufacturers 1 (NY) and 2 (TL) and those of manufacturer 3 (WS) in the t-test, which once again shows that the test results based on this method can effectively distinguish intravenous human immunoglobulin products from different manufacturers.

[0141] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for simultaneously and quantitatively detecting active ingredients and inactive ingredients in intravenous human immunoglobulin products, characterized in that: The active ingredient comprises one or more immunoglobulins of IgG, IgD, IgA and IgM, the inactive ingredient comprises albumin ALB, and the method comprises the following steps: (1) Proteolysis: The intravenous human immunoglobulin to be tested is enzymatically hydrolyzed by a protease that uses lysine and arginine as enzymatic sites to obtain a peptide segment mixture containing characteristic peptides corresponding to the active ingredient and the inactive ingredient; Where the active ingredient is IgA, the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.1; the active ingredient is IgD, the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.2; the active ingredient is IgG, the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.3; the active ingredient is IgM, the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.4; The inactive ingredient is albumin ALB, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.5; (2) Targeted detection: HPLC-MS / MS is used to detect the characteristic peptides as target peptides. Mass spectrometry data is collected based on PRM, SRM or MRM mode, and the target peptides are targeted for detection, thereby achieving quantitative analysis of the active and inactive ingredients in intravenous human immunoglobulin products.

2. The method according to claim 1, characterized in that The active ingredients include IgG, and any one or more combinations of IgM, IgA and IgD.

3. The method according to claim 1 or 2, characterized in that The inactive ingredients also include complement factor CFB and / or complement factor C1QB, wherein the inactive ingredient is complement factor CFB, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.8; the inactive ingredient is complement factor C1QB, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.

14.

4. The method according to claim 1 or 3, characterized in that The inactive ingredients also include one or more of transferrin TF, gelsolin GSN, and heme-binding protein HPX, wherein the inactive ingredient is transferrin TF, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.7; the inactive ingredient is gelsolin GSN, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.10; the inactive ingredient is heme-binding protein HPX, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.

11.

5. The method according to claim 1 or 4, characterized in that The inactive ingredient also includes apolipoprotein APOA1 and / or apolipoprotein APOH, wherein the inactive ingredient is apolipoprotein APOA1, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.6; the inactive ingredient is apolipoprotein APOH, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.

9.

6. The method according to claim 1 or 5, characterized in that The inactive ingredients also include one or more of fibrinogen FGA, fibrin FCN3, antithrombin SERPINC1, and antitrypsin SERPINA1, wherein the inactive ingredient is fibrinogen FGA, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.12; the inactive ingredient is fibrin FCN3, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.13; the inactive ingredient is antithrombin SERPINC1, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.15; the inactive ingredient is antitrypsin SERPINA1, and the sequence of its corresponding characteristic peptide is shown in SEQ ID NO.

16.

7. The method according to any one of claims 1 to 6, characterized in that: The protease with lysine and arginine as enzymatic sites is trypsin, and the protein reaction solution is added according to the volume ratio of protein mass to 1% protein reaction solution of 1:1 to denature the protein and perform reductive alkylation. After completion, water is added to dilute the protein reaction solution to reduce the concentration to below 0.5%, and trypsin is added for enzymatic hydrolysis.

8. The method according to claim 7, characterized in that The 1% protein reaction solution includes 1% SDC, 50-150mM Tris-HCl, 10-50mM TCEP and 10-50mM CAA.

9. The method according to claim 8, characterized in that The 1% protein reaction solution also includes 10-50 mM DTT and / or 10-50 mM IAA.

10. The method according to claim 9, characterized in that The peptide mixture was desalted and detected by high performance liquid chromatography-mass spectrometry, wherein mobile phase A included 0.1% formic acid and 3% DMSO, and mobile phase B included 0.1% formic acid, 3% DMSO and 80% acetonitrile; and mass spectrometry data were collected in PRM mode.

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