Quantitative detection method for monoclonal antibody drug low-abundance host cell residual protein

Abstract

The invention belongs to the technical field of protein detection, and particularly relates to a quantitative detection method for monoclonal antibody drug low-abundance host cell residual protein. The method is based on a non-labeled quantitative technology of proteomics, namely, the ratio of the sum of responses of three peptide fragments (Hi3Peptides) with the highest protein response is in direct proportion to the amount of substance, an internal standard protein recovery rate correction factor is introduced, and the host residual protein in a monoclonal antibody sample is relatively quantified by utilizing internal standard protein. The low-abundance host cell residual protein in the monoclonal antibody medicine can be more comprehensively recognized, the accuracy of the relative quantification method of the low-abundance host cell residual protein in the monoclonal antibody medicine based on the proteomics technology is improved, and technical support is provided for biological product quality control. According to the quantitative detection method, the mass concentration of the residual protein of the exogenous host cell with the lowest content can be identified to be about 0.3 ppm, the lower detection limit is low, and the detection is efficient, accurate and high in precision by combining a mass spectrometry method.

Description

Technical Field

[0001] The invention belongs to the technical field of protein detection, and particularly relates to a method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs. Background Art

[0002] Biological products, such as antibody drugs and recombinant protein drugs, are synthesized through biological systems. Although various impurities can be effectively removed through various purification processes, trace amounts of host cell proteins (HCPs) may still remain during the production process. HCPs remaining in monoclonal antibody drugs are one of the key process-related impurities in the production of biological products. As exogenous proteins, HCPs may trigger the body's immune response to varying degrees, leading to allergies or other adverse reactions, and may also cause the degradation of excipient components in the product, affecting the stability of the drug. Therefore, the qualitative and quantitative detection of HCPs in antibody drugs is particularly important.

[0003] Enzyme Linked Immunosorbent Assay (ELISA) has been the most commonly used method for detecting HCPs in the development and quality control of biological products, but its coverage and specific identification information of HCPs are still limited. The polyclonal antibodies used in this method cannot cover HCPs with no immunogenicity or weak immunogenicity. As a high-precision and high-accuracy detection method, high-resolution mass spectrometry has good specificity for the detection of HCPs and can be applied to the monitoring of HCPs in the production process of monoclonal antibody drugs. Summary of the invention

[0004] The present invention aims to provide a method for quantitatively detecting low-abundance host cell residual proteins in monoclonal antibody drugs, which method helps to more comprehensively identify low-abundance host cell residual proteins in monoclonal antibody drugs and can efficiently and accurately determine the residual amount of host cell proteins in monoclonal antibody drugs.

[0005] To achieve the above-mentioned purpose, the present invention is implemented by the following technical solutions: A method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs comprises the following steps: (S.1) Weigh each internal standard protein respectively and prepare and dilute with a buffer to obtain a working solution of each internal standard protein; (S.2) adding ultrapure water to the ultrafiltration tube and centrifuging to remove glycerol, then adding the monoclonal antibody sample and centrifuging, adding a buffer solution for substitution, obtaining the monoclonal antibody sample solution after substitution and determining the total protein content of the monoclonal antibody sample solution; (S.3) adding the replaced monoclonal antibody sample solution obtained in step (S.2) to the pre-saturated centrifuge tube, adding each internal standard protein working solution obtained in step (S.1), and then adding a buffer solution to mix well to obtain a mixed solution; the mixed solution is subjected to non-denaturing enzymatic hydrolysis and precipitation to remove the main monoclonal antibody protein, and sodium deoxycholate is used for denaturing enzymatic hydrolysis to obtain enzymatic peptides; then formic acid is added to the enzymatic peptides to remove the generated sodium deoxycholate precipitate, and then the enzymatic peptides are desalted and redissolved, centrifuged, and the supernatant is taken to obtain a mixed peptide solution containing peptides of the host residual protein and peptides of each internal standard protein, and the total peptide content is determined, and the mixed peptide solution is transferred to an injection bottle to obtain an injection solution to be tested; (S.4) The test injection solution obtained in step (S.3) is detected and data is collected using nano-liquid chromatography-tandem high-resolution mass spectrometry, and the proteomics data processing software Proteome Discoverer 3.0 is used for data analysis. The response values ​​of the internal standard protein in the samples with and without the monoclonal antibody matrix are screened and calculated, respectively, so as to calculate the internal standard protein recovery correction factor, and the response values ​​of the host residual protein are screened and calculated, and the content of the host residual protein in the monoclonal antibody sample is further calculated.

[0006] Due to the influence of drug matrix in monoclonal antibody drugs, the present invention is based on the non-labeled quantitative technology of proteomics, that is, the ratio of the sum of the responses of the three peptides (Hi3 Peptides) with the highest protein response is proportional to the amount of its substance, and the host residual protein in the monoclonal antibody sample is relatively quantified by introducing the internal standard protein recovery correction factor. The quantitative detection method of the present invention helps to more comprehensively identify low-abundance host cell residual proteins in monoclonal antibody drugs, improves the accuracy of the relative quantitative method of low-abundance host cell residual proteins in monoclonal antibody drugs based on proteomics technology, can be used to evaluate the residual amount of host cell proteins in monoclonal antibody drugs, and provides technical support for the quality control of biological products. The mass concentration of the exogenous host cell residual protein with the lowest content that can be identified by the quantitative detection method of the present invention is about 0.3ppm, and the detection limit is low. Combined with mass spectrometry, the detection is efficient, accurate, and has high precision.

[0007] The present invention is based on non-targeted proteomics, and comprehensively analyzes the proteins contained in the monoclonal antibody to obtain qualitative information of residual host proteins. At the same time, the present invention introduces an internal standard protein recovery correction factor during calculation by adding an internal standard protein, and further obtains the relative quantitative results of all residual host proteins in the monoclonal antibody sample.

[0008] Preferably, the internal standard protein in step (S.1) is any one or more combinations of cytochrome c, α-lactoprotein, C-reactive protein, thioredoxin 1, myoglobin, and hemoglobin S.

[0009] Preferably, the specific method for pre-saturating the centrifuge tube in step (S.3) is as follows: Pre-saturate the centrifuge tube with bovine serum albumin and discard the solution in the centrifuge tube before use.

[0010] Preferably, in step (S.3), 10 ppm of thioredoxin 1, 15 ppm of myoglobin, 20 ppm of cytochrome c, 40 ppm of hemoglobin S, 50 ppm of α-lactoprotein and 50 ppm of C-reactive protein are added to the monoclonal antibody sample solution after replacement for every 500 μg of monoclonal antibody protein contained therein.

[0011] Preferably, the specific process of obtaining the enzymatic peptide fragments in step (S.3) is: Trypsin was added to the mixture at an enzyme:protein ratio of 1:400 (w / w), and digested at 37°C for 2h; dithiothreitol was added to make the final concentration 5mM, denatured at 95°C for 10min, centrifuged at 14000g for 15min, and the supernatant was taken; 10% sodium deoxycholate was added to make the final concentration 1%; dithiothreitol was added to make the final concentration 5mM, reacted at 95°C for 10min, and cooled to room temperature; iodoacetamide was added to make the final concentration 10mM, and reacted in the dark for 30min; trypsin was added, and digested at 37°C overnight; 10% formic acid was added to make the final concentration 1%, mixed, centrifuged at 14000g for 15min, and the supernatant was taken.

[0012] Preferably, the specific process of desalting and re-dissolving in step (S.3) is: After desalting the enzymatic peptides using a C18 desalting column, the solvent was removed and the peptides were reconstituted with formic acid-water solution.

[0013] Preferably, the calculation formula for the internal standard protein recovery correction factor calculated in step (S.4) is as follows:

[0014] Preferably, in step (S.4), the method for respectively screening and calculating the response value of the internal standard protein in the samples with and without the monoclonal antibody matrix is ​​as follows: Proteomics data processing software Proteome Discoverer 3.0 was used to analyze the data to obtain a data table. The three peptides with the highest response were screened out from the data table, and the sum of the response values ​​of the three peptides with the highest response was calculated and used as the response value of the internal standard protein.

[0015] Preferably, the calculation formula for calculating the content of host residual protein in the monoclonal antibody sample in step (S.4) is as follows: Where: i represents the internal standard protein, and n represents the number of internal standard proteins.

[0016] Preferably, the method for screening and calculating the response value of the host residual protein in step (S.4) is as follows: Proteomics data processing software Proteome Discoverer 3.0 was used to analyze the data to obtain a data table. Target proteins with no less than 2 characteristic peptides were screened from the data table, and peptides were selected from the peptides corresponding to the target proteins. The sum of the response values ​​of the selected peptides was calculated and used as the response value of the host residual protein.

[0017] Therefore, the present invention has the following beneficial effects: (1) The present invention is based on a non-labeled quantitative technology of proteomics, that is, the ratio of the sum of the responses of the three peptides (Hi3Peptides) with the highest protein response is proportional to the amount of the substance, and the internal standard protein is introduced to perform relative quantification of the host residual protein in the monoclonal antibody sample by using the internal standard protein; (2) The quantitative detection method of the present invention helps to more comprehensively identify low-abundance host cell residual proteins in monoclonal antibody drugs, improves the accuracy of the relative quantitative method of low-abundance host cell residual proteins in monoclonal antibody drugs based on proteomics technology, and can be used to evaluate the residual amount of host cell proteins in monoclonal antibody drugs, providing technical support for the quality control of biological products; (3) The quantitative detection method of the present invention can identify the lowest content of exogenous host cell residual protein with a mass concentration of about 0.3 ppm, with a low detection limit. Combined with the mass spectrometry analysis method, the detection is efficient, accurate and highly precise. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the TIC diagram of non-denaturing enzymatic hydrolysis of monoclonal antibody injection combined with SDC denaturing enzymatic hydrolysis.

[0019] Figure 2 Peptide retention time distribution diagram.

[0020] Figure 3 Peptide mass deviation diagram.

[0021] Figure 4 Figure A is a Venn diagram of the number of HCPs proteins identified; Figure 4 Figure B is a Venn diagram of the number of HCPs peptides identified.

[0022] Figure 5 This is the lower limit of protein detection.

[0023] Figure 6 is the recovery correction factor of each internal standard protein under the influence of the matrix.

[0024] Figure 7Figure A in the figure is the internal standard protein quantitative heat map when there is no recovery correction factor correction; Figure 7 Figure B is the quantitative heat map of the internal standard protein when corrected with the recovery correction factor. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments of the specification. Those of ordinary skill in the art will be able to implement the present invention based on these descriptions. In addition, the embodiments of the present invention involved in the following description are generally only embodiments of a part of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work should fall within the scope of protection of the present invention.

[0026] Example 1 1.1 Drugs and reagents: 1M Tris buffer, pH = 8.0 (Invitrogen, batch number: 91221415); mass spectrometry grade trypsin (Trypsin, Beijing Shengxia, batch number: 20230808C); dithiothreitol (Dithiothreitol, DDT, Sigma, batch number: SLCM6245); iodoacetamide (Iodoacetamide, IAA, Sigma, batch number: SLCL4705); sodium deoxycholate (Sodium deoxycholate, SDC, Sigma, batch number: BCCK0642); mass spectrometry grade methanol (Fisher, batch number: 221157); mass spectrometry grade acetonitrile (Fisher, batch number: 226101); mass spectrometry grade formic acid (Fisher, batch number: 220622); trifluoroacetic acid (Trifluoroacetic acid, TFA, Sigma, batch number: STBK6505).

[0027] 30kD ultrafiltration tube (Millipore, batch number: 0000186797); C18 desalting column (3M, batch number: 340166D); Acclaim PepMap RSLC C18 chromatographic column (Thermo Fisher, batch number: 2765105); Acclaim PepMap 100C18 Trap column (Thermo Fisher, batch number: 2726780); quantitative peptide detection kit (Thermo Fisher, catalog number: 23275); protein detection kit (Thermo Fisher, catalog number: 23236).

[0028] The monoclonal antibody injection was provided by Sichuan Institute of Drug Control, which was a total of 26 batches of samples of the same variety. The E. coli mixed protein came from the E. coli calibrator (batch number: 24030401) of Huzhou Shenke Biotechnology Co., Ltd.

[0029] 1.2 Protein standards: In this example, six protein standards were used as internal standard proteins: cytochrome c (source: horse, UniProt registration number: P00004, batch number: SLCL7912); alpha-lactalbumin (source: human, UniProt registration number: P00709, batch number: SLCP3207); C-reactive protein (source: human, UniProt registration number: P02741, batch number: SLCK3864); Thioredoxin 1 (source: Escherichia coli, UniProt registration number: P0AA25, batch number: 0000222615); myoglobin (source: horse, UniProt registration number: P68082, batch number: SLCL3317); hemoglobin S (Hemoglobin subunit beta, source: human, UniProt accession number: P68871, batch number: SLCC7764) were purchased from Sigma.

[0030] 1.3 Instruments: Nano-liquid chromatography-ultra-high resolution Orbitrap mass spectrometry (Thermo Fisher Scientific), equipped with Vanquish neo nano-liquid chromatography and Q Exactive HF mass spectrometer. SpeedVac drying instrument (Thermo Fisher Scientific), model SPD2030. SpectraMax multi-function microplate reader (Molecular Devices), model M2.

[0031] 1.4 Analysis methods: 1.4.1 Liquid phase method: Nano-LC-ESI-Orbitrap was used for sample detection. Chromatographic analysis column: Acclaim PepMap RSLC C18 Column (2 μm, 75 μm×25 cm); Trap column: Acclaim PepMap100C18 Column (3 μm, 75μm×15cm); mobile phase A: 0.1% formic acid-water; mobile phase B: 0.1% formic acid-acetonitrile; column temperature: 55°C; injection volume: 0.5μg peptide; liquid phase elution gradient is shown in Table 1 below.

[0032] Table 1

[0033] 1.4.2 Mass spectrometry method: Ion source spray voltage: 2.0 kV; capillary temperature: 275 °C. Mass spectrometry scanning acquisition mode: Data Dependent Acquisition (DDA). Primary resolution: 60000 at m / z 200; scanning range m / z: 400-1600; AGC target: 3e6; Max IT: 50 ms. Secondary resolution: 30000 at m / z 200; scanning range m / z: 110 start; AGC target: 2e5; Max IT: 100 ms; dynamic exclusion: 20 s; TopN=20.

[0034] 1.5 Sample preparation: Accurately weigh thioredoxin 1 and prepare it with 25 mM Tris (pH = 8.0) solution to a concentration of 2 mg mL -1 The remaining internal standard proteins (i.e., cytochrome c, α-lactoprotein, C-reactive protein, myoglobin, and hemoglobin S) were prepared in the same manner to a concentration of 5 mg mL -1 When preparing samples, dilute each internal standard protein stock solution to 0.01 mg mL with 25 mM Tris (pH = 8.0) solution. -1 As the corresponding internal standard protein working solution.

[0035] Ultrapure water was added to the 30kD ultrafiltration tube, and the solution was centrifuged at 14000g for 10 min to remove a small amount of glycerol in the ultrafiltration tube. Then, the monoclonal antibody sample was added to the ultrafiltration tube, and the solution was centrifuged at 14000g for 20 min. 25mM Tris (pH=8.0) was added for buffer replacement, and the monoclonal antibody sample solution after replacement was obtained and the total protein content was determined using a protein detection kit.

[0036] Use 0.2mg·mL -1Bovine serum albumin (BSA) was used to pre-saturate the centrifuge tubes for subsequent experiments, and the solution in the centrifuge tubes was discarded before use. 500 μg of monoclonal antibody sample was added to the pre-saturated centrifuge tubes, and each internal standard protein working solution was added, that is, 10 ppm (5 ng) of Thioredoxin 1, 15 ppm (7.5 ng) of Myoglobin, 20 ppm (10 ng) of Cytochrome c, 40 ppm (20 ng) of Hemoglobin subunit beta, and 50 ppm (25 ng) of Alpha-lactalbumin and 50 ppm (25 ng) of C-reactive protein were added as internal standard proteins according to the amount of monoclonal antibody protein (that is, for every 500 μg of monoclonal antibody sample added). 25 mM Tris (pH = 8.0) buffer was added to the centrifuge tube to make up the volume to 100 μL, and mixed to obtain a mixed solution. Then, Trypsin was added to the mixture at an enzyme:protein ratio of 1:400 (w / w), and digested at 37°C for 2h; DTT was added to make its final concentration 5mM, denatured at 95°C for 10min, centrifuged at 14000g for 15min, and the supernatant was taken; 10% SDC was added to make its final concentration 1%; DTT was added to make its final concentration 5mM, reacted at 95°C for 10min, and cooled to room temperature; IAA was added to make its final concentration 10mM, reacted in the dark for 30min; Trypsin was added, digested at 37°C overnight to obtain enzymatic peptide fragments. Then, 10% formic acid (FA) was added to the enzymatic peptides to make the final concentration of 1%, mixed, centrifuged at 14000g for 15min, the generated sodium deoxycholate precipitate was removed, and the supernatant was taken; the enzymatic peptides were desalted with a C18 desalting column, the solvent was removed with a evaporator, re-dissolved with 50μL 0.1% formic acid-water, centrifuged at 14000g for 10min, and the supernatant was taken to obtain a mixed peptide solution containing peptides of host residual proteins and peptides of each internal standard protein, and the total peptide content was determined using a quantitative peptide detection kit. Then the mixed peptide solution was transferred to an injection bottle to obtain a sample solution to be tested. The obtained sample solution to be tested was detected and data was collected using nanoliter liquid chromatography-tandem high-resolution mass spectrometry, and the proteomics data processing software Proteome Discoverer 3.0 was used for data analysis.

[0037] 1.6 Proteomics data analysis methods: Proteomics data processing software Proteome Discoverer 3.0 was used for data analysis. Database source UniProt, Taxonomy: Cricetulus griseus (Chinese hamster ovary cells, CHO, Taxonomy ID: 10029, 20231009); Escherichia coli (Escherichia coli, E. coli, Taxonomy ID: 562, 20230704).

[0038] The specific analysis parameters are as follows: Fixed modification (Fix Modification): Carbamidomethyl (C); Enzyme (Enzyme): Trypsin; Maximum Missed Cleavages: 2; Primary mass spectrometry error (Peptide Mass Tolerance): 10ppm; Secondary mass spectrometry error (Fragment Mass Tolerance): 0.02Da.

[0039] Proteomics data processing software Proteome Discoverer 3.0 was used to analyze the data to obtain a data table. The three peptides with the highest response were screened from the data table, and the sum of the response values ​​of the three peptides with the highest response was calculated and used as the response value of the internal standard protein. The response values ​​of the internal standard protein in the samples with and without the monoclonal antibody matrix were screened and calculated respectively, and the ratio of the response values ​​of the internal standard protein in the samples with and without the monoclonal antibody matrix was compared to obtain the internal standard protein recovery correction factor (Recovery Factor), and the calculation formula is as follows:

[0040] By introducing the internal standard protein recovery correction factor, the host residual proteins (HCPs) in monoclonal antibody drugs can be relatively quantified. The proteomics data processing software Proteome Discoverer 3.0 is used for data analysis to obtain a data table. The target protein with a characteristic peptide number of not less than 2 (i.e., the host residual protein to be quantified) is screened from the data table and the three peptides with the highest response (Hi3 Peptides) are selected from the peptides corresponding to the target protein. The sum of the response values ​​of the three peptides with the highest response is calculated and used as the response value of the host residual protein. If there are less than 3 peptides, the sum of the response values ​​of the existing peptides is used for calculation. The calculation formula for the content of host residual protein in the monoclonal antibody sample is as follows: Where: i represents the internal standard protein, and n represents the number of internal standard proteins.

[0041] 2.1 Methodological Validation: 2.1.1 Method reproducibility: 10ppm Thioredoxin 1, 15ppm Myoglobin, 20ppm Cytochrome c, 40ppm Hemoglobin subunit beta, and 50ppm Alpha-lactalbumin and C-reactive protein were added to 500μg monoclonal antibody sample according to the amount of total protein as internal standard proteins, and enzymatic hydrolysis was performed under non-denaturing conditions. The supernatant was taken and then subjected to SDC denaturing enzymatic hydrolysis. Finally, high-resolution mass spectrometry was used for detection. The TIC diagram (i.e., its characteristic total ion current diagram) of the monoclonal antibody injection non-denaturing enzymatic hydrolysis combined with the SDC denaturing enzymatic hydrolysis is shown in Figure 1 As shown in Figure 2, the peptide chromatographic peak shape and response are good. The retention time (RT) distribution of the identified peptides is shown in Figure 2. Figure 2 As shown, the overall distribution is normal. The peptide mass deviation (ΔM) is shown in Figure 3 As shown, the deviation between the molecular weight of the peptide and the theoretical molecular weight is basically within 10 ppm, indicating that the mass axis of the high-resolution mass spectrometry is stable and the identification results are good and reliable.

[0042] The collected data were analyzed using proteomics data processing software, and the Cricetulus griseus protein database was selected, which contained 83,455 protein sequence information. The proteins identified in the three parallel samples were compared, and the screening condition was that the characteristic peptide segment was not less than 2. The number of proteins identified in the three samples was 9, 10, and 9, respectively. Among them, a total of 8 proteins were identified in the three samples at the same time, with an average reproducibility of 85.9% (see Figure 4 Similarly, the peptides corresponding to the proteins that meet the screening condition of no less than 2 characteristic peptides were counted, and the number of peptides identified in the three samples was 40, 40 and 43 respectively, of which 34 peptides were identified in the three samples at the same time, with an average recurrence rate of 91.1% (see Figure 4 Figure B in Figure 2). The identification quantity Venn diagram is shown in Figure 2. Figure 4 As shown. Among them, Figure 4 Figure A is a Venn diagram of the number of HCPs proteins identified. Figure 4 Figure B is a Venn diagram of the number of HCPs peptides identified. The results show that the proteins and peptides identified by this method have good reproducibility.

[0043] 2.1.2 Detection limit: 100 ppm of exogenous E. coli reference protein mixture was added to the monoclonal antibody sample, and after non-denaturing enzymatic hydrolysis, the supernatant was taken, and then SDC denaturing enzymatic hydrolysis was performed. After desalting, mass spectrometry analysis was performed. The data was analyzed using proteomics data processing software, and the Escherichia coli database was selected. The characteristic peptide segment was not less than 2 as the screening condition, and there were 10 exogenous proteins that met the conditions. At the same time, the present invention prepared E. coli reference samples for mass spectrometry detection, and analyzed them using proteomics data processing software to obtain the sum of the response values ​​of the three peptide segments (Hi3 Peptides) with the highest response for each protein. By comparing the sum of the Hi3 peptide segment responses of a single protein with the sum of the Hi3 peptide segment responses of all proteins, the relative content of the single protein in the E. coli reference can be obtained. In this way, the amount of the 10 exogenous proteins screened out in the monoclonal antibody sample was calculated, and their total content accounted for 11.21% of the total protein of the E. coli reference sample. The lower limit of protein detection is shown in FIG. Figure 5 The lowest exogenous protein concentration that can be identified is about 0.3 ppm, which can be used as the detection limit of this method.

[0044] 2.1.3 Precision: 10ppm Thioredoxin 1, 15ppm Myoglobin, 20ppm Cytochrome c, 40ppm Hemoglobin subunit beta, and 50ppm Alpha-lactalbumin and C-reactive protein were added to the monoclonal antibody sample as internal standard proteins, and 6 samples were prepared in parallel. The responses of the internal standard proteins between the parallel samples were calculated, and the coefficients of variation were all lower than 15%. The precision of the internal standard proteins is shown in Table 2 below. The results show that the mass spectrometry analysis method established by the present invention has good precision.

[0045] Table 2 Note: Standardized abundance is the data standardized abundance value exported by Proteome Discoverer software, which is dimensionless so that all data are in the same order of magnitude.

[0046] 2.1.4 Accuracy: 10ppm Thioredoxin 1, 15ppm Myoglobin, 20ppm Cytochrome c, 40ppm Hemoglobin subunit beta, and 50ppm Alpha-lactalbumin and C-reactive protein were added to the monoclonal antibody sample as internal standard proteins. After non-denaturing enzymatic hydrolysis, the supernatant was taken and then subjected to SDC denaturing enzymatic hydrolysis. After desalting, the sample was loaded for analysis. Similarly, a sample without monoclonal antibody matrix was prepared for mass spectrometry analysis. The sum of the response values ​​of the three peptides (Hi3 Peptides) with the highest response of the internal standard protein was calculated, and the ratio of the response values ​​of the internal standard protein in the samples with and without the monoclonal antibody matrix was compared to obtain the recovery correction factor of the internal standard protein. The recovery correction factor (Recovery Factor) of the internal standard protein is calculated as shown in Formula 1 above. The recovery correction factors of each internal standard protein under the influence of the matrix are shown in Figure 6 The quantitative heat map of internal standard protein is shown in Figure 7 As shown. Among them, Figure 7 Figure A is the internal standard protein quantification heat map without recovery correction factor correction. Figure 7 Figure B is the quantitative heat map of the internal standard protein when corrected with the recovery correction factor.

[0047] Without introducing the internal standard protein recovery correction factor, each internal standard protein is used in turn to calculate other internal standard proteins to obtain a heatmap, see Figure 7 After the internal standard protein recovery correction factor was introduced, each internal standard protein was used in turn to perform quantitative calculations on other internal standard proteins to obtain a heat map, see Figure 7 As shown in Figure B. Figure 7 Figure A and Figure 7 In Figure B, after the internal standard protein recovery correction factor was introduced, the quantitative accuracy between the internal standard proteins was significantly better than that without the internal standard protein recovery correction factor. The results show that when the internal standard protein recovery correction factor was introduced, the overall accuracy of this group of internal standard proteins for quantitative calculation is good.

[0048] 3.1 Sample testing: This method was used to detect the host cell residual protein in 26 batches of monoclonal antibody injection samples. The sum of the response values ​​of the three peptides (Hi3 Peptides) with the highest response of the internal standard protein was calculated as the response value of the internal standard protein. The ratio of the response values ​​of the internal standard protein in the samples with and without the monoclonal antibody matrix was compared to obtain the internal standard protein recovery correction factor (Recovery Factor), and its calculation formula is shown in the above formula 1. By introducing the internal standard protein recovery correction factor, the host residual protein (HCPs) in monoclonal antibody drugs can be relatively quantified. The target protein with a characteristic peptide number of not less than 2 was screened out and the three peptides (Hi3 Peptides) with the highest response were selected from the peptides corresponding to the target protein. The sum of the response values ​​of the three peptides with the highest response was calculated and used as the response value of the host residual protein. If there are less than 3 peptides, the sum of the response values ​​of the existing peptides is used for calculation. The calculation formula for the content of host residual protein in the monoclonal antibody sample is shown in the above formula 2. The quantitative list of residual protein in 26 monoclonal antibody samples is shown in Tables 3 to 28 below. After excluding possible abnormal contaminating proteins, the total residual HCPs in each sample ranged from 47.0 to 103.5 ppm.

[0049] Table 3: Quantitative list of residual protein in sample No. 483 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0050] Table 4: Quantitative list of residual protein in sample No. 484 .

[0051] Table 5: Quantitative list of residual protein in sample No. 672 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0052] Table 6: Quantitative list of residual protein in sample No. 737 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0053] Table 7: Quantitative list of residual protein in sample No. 839 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0054] Table 8: Quantitative list of residual protein in sample No. 840 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0055] Table 9: Quantitative list of residual protein in sample No. 881 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0056] Table 10: Quantitative list of residual protein in sample No. 882 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0057] Table 11: Quantitative list of residual protein in sample No. 883 .

[0058] Table 12: Quantitative list of residual protein in sample No. 947 .

[0059] Table 13: Quantitative list of residual protein in sample 1007 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0060] Table 14: Quantitative list of residual protein in sample No. 1008 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0061] Table 15: Quantitative list of residual protein in sample No. 1031 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0062] Table 16: Quantitative list of residual protein in sample No. 1065 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0063] Table 17: Quantitative list of residual protein in sample No. 1087 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0064] Table 18: Quantitative list of residual protein in sample No. 1098 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0065] Table 19: Quantitative list of residual protein in sample No. 1099 .

[0066] Table 20: Quantitative list of residual protein in sample No. 1121 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0067] Table 21: Quantitative list of residual protein in sample No. 1122 .

[0068] Table 22: Quantitative list of residual protein in sample No. 1123 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0069] Table 23: Quantitative list of residual protein in sample No. 1025 .

[0070] Table 24: Quantitative list of residual protein in sample No. 1026 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0071] Table 25: Quantitative list of residual protein in sample No. 1286 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0072] Table 26: Quantitative list of residual protein in sample No. 1592 .

[0073] Table 27: Quantitative list of residual protein in sample No. 2469 .

[0074] Table 28: Quantitative list of residual protein in sample No. 2750 Note: Proteins marked with * are not included in the calculation of total HCPs residues.

[0075] The above description is only a detailed description of the preferred embodiments and principles of the present invention. For ordinary technicians in this field, according to the ideas provided by the present invention, there will be changes in the specific implementation methods, and these changes should also be regarded as the protection scope of the present invention.

Claims

1. A method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs, characterized in that: The following steps are involved: (S.1) Weigh each internal standard protein respectively and prepare and dilute with a buffer to obtain a working solution of each internal standard protein; (S.2) adding ultrapure water to the ultrafiltration tube and centrifuging to remove glycerol, then adding the monoclonal antibody sample and centrifuging, adding a buffer solution for substitution, obtaining the monoclonal antibody sample solution after substitution and determining the total protein content of the monoclonal antibody sample solution; (S.3) adding the replaced monoclonal antibody sample solution obtained in step (S.2) to the pre-saturated centrifuge tube, adding each internal standard protein working solution obtained in step (S.1), and then adding a buffer solution to mix well to obtain a mixed solution; the mixed solution is subjected to non-denaturing enzymatic hydrolysis and precipitation to remove the main monoclonal antibody protein, and sodium deoxycholate is used for denaturing enzymatic hydrolysis to obtain enzymatic peptides; then formic acid is added to the enzymatic peptides to remove the generated sodium deoxycholate precipitate, and then the enzymatic peptides are desalted and redissolved, centrifuged, and the supernatant is taken to obtain a mixed peptide solution containing peptides of the host residual protein and peptides of each internal standard protein, and the total peptide content is determined, and the mixed peptide solution is transferred to an injection bottle to obtain an injection solution to be tested; (S.4) The test injection solution obtained in step (S.3) is detected and data is collected using nano-liquid chromatography-tandem high-resolution mass spectrometry, and the proteomics data processing software Proteome Discoverer 3.0 is used for data analysis. The response values ​​of the internal standard protein in the samples with and without the monoclonal antibody matrix are screened and calculated, respectively, so as to calculate the internal standard protein recovery correction factor, and the response values ​​of the host residual protein are screened and calculated, and the content of the host residual protein in the monoclonal antibody sample is further calculated.

2. The method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs according to claim 1, characterized in that: The internal standard protein in step (S.1) is any one or more combinations of cytochrome c, α-lactoprotein, C-reactive protein, thioredoxin 1, myoglobin, and hemoglobin S.

3. The method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs according to claim 1, characterized in that: The specific method for pre-saturating the centrifuge tube in step (S.3) is as follows: Pre-saturate the centrifuge tube with bovine serum albumin and discard the solution in the centrifuge tube before use.

4. The method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs according to claim 1, characterized in that: In step (S.3), 10 ppm of thioredoxin 1, 15 ppm of myoglobin, 20 ppm of cytochrome c, 40 ppm of hemoglobin S, 50 ppm of α-lactoprotein and 50 ppm of C-reactive protein are added to the monoclonal antibody sample solution after replacement for every 500 μg of monoclonal antibody protein.

5. The method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs according to claim 1, characterized in that: The specific process of obtaining the enzymatic peptide fragments in step (S.3) is as follows: Trypsin was added to the mixture at an enzyme:protein ratio of 1:400 (w / w), and digested at 37°C for 2h; dithiothreitol was added to make the final concentration 5mM, denatured at 95°C for 10min, centrifuged at 14000g for 15min, and the supernatant was taken; 10% sodium deoxycholate was added to make the final concentration 1%; dithiothreitol was added to make the final concentration 5mM, reacted at 95°C for 10min, and cooled to room temperature; iodoacetamide was added to make the final concentration 10mM, and reacted in the dark for 30min; trypsin was added, and digested at 37°C overnight; 10% formic acid was added to make the final concentration 1%, mixed, centrifuged at 14000g for 15min, and the supernatant was taken.

6. The method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs according to claim 1, characterized in that: The specific process of desalting and re-dissolving in step (S.3) is as follows: After desalting the enzymatic peptides using a C18 desalting column, the solvent was removed and the peptides were reconstituted with formic acid-water solution.

7. The method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs according to claim 1, characterized in that: The calculation formula for the internal standard protein recovery correction factor calculated in step (S.4) is as follows:

8. The method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs according to claim 1 or 7, characterized in that: The method for respectively screening and calculating the response values ​​of the internal standard protein in the samples with and without the monoclonal antibody matrix in step (S.4) is as follows: Proteomics data processing software Proteome Discoverer 3.0 was used to analyze the data to obtain a data table. The three peptides with the highest response were screened out from the data table, and the sum of the response values ​​of the three peptides with the highest response was calculated and used as the response value of the internal standard protein.

9. The method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs according to claim 1, characterized in that: The calculation formula for the content of host residual protein in the monoclonal antibody sample calculated in step (S.4) is as follows: Where: i represents the internal standard protein, and n represents the number of internal standard proteins.

10. The method for quantitatively detecting low-abundance host cell residual proteins of monoclonal antibody drugs according to claim 1, characterized in that: The method for screening and calculating the response value of the host residual protein in step (S.4) is as follows: Proteomics data processing software Proteome Discoverer 3.0 was used to analyze the data to obtain a data table. Target proteins with no less than 2 characteristic peptides were screened from the data table, and peptides were selected from the peptides corresponding to the target proteins. The sum of the response values ​​of the selected peptides was calculated and used as the response value of the host residual protein.

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