A method for detecting HCPs in biological protein drugs based on LC-MS / MS

Abstract

This invention discloses a method for detecting hepatocellular carcinoma (HCPs) in biological protein drugs based on LC-MS / MS. The method involves covalently binding HCP-derived cell line protein samples and monoclonal antibody proteins to the samples using chemical reagents, followed by mixing and fractionation. Finally, liquid chromatography-mass spectrometry (LC-MS / MS) analysis is performed to obtain the analytical results for HCPs in the monoclonal antibody drug. Compared with existing technologies, this invention enhances the detection signal by adding host cell proteins from monoclonal antibody production, significantly improving detection sensitivity and the depth of identification of low-abundance proteins. This fills a significant gap in HCP analysis and can be used to detect and demonstrate HCP clearance, particularly extremely low-level hydrolases in drug manufacturing processes, which has a crucial impact on ensuring the quality of biological protein pharmaceutical products and patient safety.

Description

Technical Field

[0001] This invention belongs to the field of analytical chemistry, specifically relating to a method for detecting HCPs in biological protein drugs based on LC-MS / MS. Background Technology

[0002] Biological protein drugs are produced using transgenic prokaryotic or eukaryotic host cells and cell culture technology, inevitably leading to co-expression of host cell proteins (HCPs). HCPs are a major type of impurity in the production of biological protein drugs and may pose risks to patient safety and product efficacy. Therefore, continuous monitoring is required to demonstrate that their removal rate is below the regulatory-approved level of 100 ng / mg. The risks associated with HCPs include: 1) Impact on the body's immune response. Immunogenic HCPs can trigger unexpected and harmful immune responses in patients. Because HCPs may cause unpredictable immune reactions, immune rejection is highly likely, potentially threatening life; 2) Degradation of product formulation components. Enzymatically active HCPs can degrade therapeutic antibodies themselves or react with components in formulation buffers, reducing antibody stability and increasing the formation of visible particles. HCP enzymatic activity may directly or indirectly affect product stability. It has been reported that HCPs with inherent hydrolytic activity may negatively impact product quality by degrading the surfactant polysorbate, leading to increased fatty acid release and a tendency to form particles composed of free fatty acids; 3) they may possess toxicity or biological activity themselves. For example, cathepsin D has been shown to cause antibody fragmentation and particle formation through its proteolytic activity; 4) they may alter antibody binding affinity / potency. These host cell proteins in recombinant biological therapeutics can significantly affect drug efficacy. Therefore, strict quality control is essential for biopharmaceuticals to reduce the risks associated with HCPs.

[0003] For decades, enzyme-linked immunosorbent assay (ELISA) has been the gold standard for biotherapeutic process development and quality control, used to measure the total number of hematologic proteins (HCPs) in biopharmaceutical process development and quality control. ELISA requires polyclonal antibodies produced by immunizing animals with zero cell lines, which does not fully cover all HCPs in the production cell lines, leading to biased detection of more immunogenic HCPs. These limitations reduce its practicality. Its limitations in HCP identification coverage, such as the inability to identify specific proteins or quantify their individual concentrations, create unacceptable gaps in HCP characterization, encouraging researchers to explore alternative technologies. To address this challenge, numerous sample preparation and instrumentation techniques have been developed. Researchers employ a variety of complementary LC-MS / MS methods, including data-independent acquisition (DIA) LC-MS / MS, which obtains complete fragmentation information for all ions in the sample, improving data utilization, reducing missing values, and increasing analytical reproducibility; two-dimensional high- and low-pH reverse-phase fractionation LC-MS / MS, which reduces overlap between HCPs and therapeutic protein drug peptide peaks; and molecular weight cutoff enrichment LC-MS / MS, which reduces the concentration gap between HCPs and therapeutic proteins. To date, more than 800 HCPs have been identified in human NIST mAb (RM 8671). Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to detect residual host cell proteins (HCPs) in biological protein drugs and / or how to improve the identification depth of residual host cell proteins (HCPs) in biological protein drugs.

[0005] To address the aforementioned technical problems, the present invention first provides a method for detecting or identifying host cell proteins in a target protein product (biological protein drug), the method comprising:

[0006] 1) Preparation of mixed covalently bound peptides, wherein the mixed covalently bound peptides may be composed of target protein covalently bound peptides and host cell protein covalently bound peptides.

[0007] The target protein covalently bound peptide can be a mixture obtained by binding the peptide of the target protein product with compound 1, and the host cell protein covalently bound peptide can be a mixture obtained by binding the peptide of the host cell protein product with compound 2; compound 1 and compound 2 can be isotope-containing compounds with the same molecular weight, the only difference between compound 1 and compound 2 is that the isotopes are atoms located at different positions, and both compound 1 and compound 2 can contain groups of formula 17 to covalently bind with the peptide;

[0008] ;

[0009] The peptides of the target protein product may be a mixture of peptides obtained by enzymatic hydrolysis of the target protein product using digestive enzymes; the peptides of the host cell protein product are a mixture of peptides obtained by enzymatic hydrolysis of the host cell protein product.

[0010] The target protein product can be obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells.

[0011] The host cell protein product can be obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells;

[0012] 2) The covalently bound peptide is detected by liquid chromatography-mass spectrometry (LC-MS) to identify the target peptide, which is the covalently bound peptide of the target protein and the covalently bound peptide of the host cell protein with the same mass-to-charge ratio. The results of the LC-MS detection of the target peptide are analyzed using mass spectrometry analysis software. Based on the signal of compound 1, the host cell protein contained in the target protein product is identified.

[0013] The cells may be animal cells or microbial cells.

[0014] The crude purification can be any purification method that yields total protein from cells obtained from cell cultures.

[0015] The cell culture may be a substance obtained by culturing cells in a culture medium (i.e., a culture product, consisting of cells, secretions from the cells into the culture medium, and the remaining culture medium).

[0016] Of the compounds 1 and 2, compound 1 may be compound C represented by any of the chemical formulas 1-16 below. 19 H 30 N4O6, wherein compound 2 may be compound C shown in formula 1 or formula 16. 19 H 30 N4O6, wherein compound 1 is any one of formulas 1-16 and is different from compound 2. The atoms marked with "*" in formulas 1-16 are... 13 C or 15 N.

[0017] .

[0018] The culture medium mentioned above can be a liquid culture medium, and the cell culture can be a cell culture medium.

[0019] The enzymatic digestion method described above can be SP3 digestion. SP3 digestion is a Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) digestion technique, which may include the following steps: during the digestion of the monoclonal antibody host cell protein using digestive enzymes, the monoclonal antibody host cell protein is non-covalently captured and released by active groups on magnetic beads, and the captured monoclonal antibody host protein is concentrated (enriched) by the magnetism of the magnetic beads.

[0020] The digestive enzyme mentioned above may be trypsin.

[0021] The host cell lines for the above-mentioned monoclonal antibodies can be Chinese hamster ovary cells (CHO-S) or mouse myeloma cells (NS-1).

[0022] In the above method, Maxquant software can be used to analyze the mass spectrometry detection results.

[0023] The method described above also includes the step of reducing and alkylating the target protein product and the host cell protein product.

[0024] In the above method, the mass spectrometry detection step may be preceded by a step of high-pH reverse-phase fractionation of the mixed covalently bound peptides using a high-performance liquid chromatography system.

[0025] The high-pH reversed-phase fractionation can be performed on an Eksigent XBridge BEH C18 column (3 μm, 130 Å, 0.3 × 150 mm) using an Ekspert nanoLC 4000 high-performance liquid chromatography system. The mobile phase in the high-performance liquid chromatography system can be 98% ammonia (pH 10).

[0026] The reductive alkylation described above includes two steps: reduction and alkylation. The reduction involves using a reducing agent to reduce the disulfide bonds in the protein to thiol groups, and the alkylation involves modifying the thiol groups to prevent them from reforming disulfide bonds.

[0027] In the above method, the mass ratio of the target protein covalently bound peptide to the host cell protein covalently bound peptide in the mixed covalently bound peptides can be 1:1.

[0028] The mass spectrometry analysis software described above can be Maxquant software; during the analysis, the target-decoy search strategy in Maxquant software is used to estimate and control false positives, and the FDR of the analysis is less than or equal to 1%.

[0029] The target protein product and the host cell protein product mentioned above may be protein products that have undergone reductive alkylation.

[0030] In the above method, the target protein product can be a monoclonal antibody.

[0031] The products containing the target protein mentioned above (biological protein drugs) can also be vaccines, insulin, or cytokines.

[0032] To address the aforementioned technical problems, the present invention also provides a method for improving the detection sensitivity and / or detection depth of host cell proteins in a target protein product. The method may include the steps of preparing a mixed covalently bound peptide and then performing mass spectrometry analysis on the mixed covalently bound peptide to improve the detection sensitivity and / or detection depth of host cell proteins in the target protein product.

[0033] The mixed covalently bound peptides may consist of target protein covalently bound peptides and host cell protein covalently bound peptides.

[0034] The target protein covalently bound peptide can be a mixture obtained by covalently binding the peptide of the target protein product with compound 1, and the host cell protein covalently bound peptide can be a mixture obtained by covalently binding the peptide of the host cell protein product with compound 2; compound 1 and compound 2 are isotopic compounds with the same molecular weight, the only difference between compound 1 and compound 2 is that the isotopes are atoms located at different positions, and both compound 1 and compound 2 can contain groups of formula 17 to covalently bind with the peptide;

[0035] .

[0036] The peptides of the target protein product may be a mixture of peptides obtained by enzymatic hydrolysis of the target protein product using digestive enzymes; the peptides of the host cell protein product are a mixture of peptides obtained by enzymatic hydrolysis of the host cell protein product.

[0037] The target protein product can be obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells.

[0038] The host cell protein product can be obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells.

[0039] To address the aforementioned technical problems, the present invention also provides the application of host cell protein peptides in detecting host cell proteins in target protein products, wherein the host cell protein peptides may be a mixture of peptides obtained by enzymatic hydrolysis of host cell protein products.

[0040] The target protein product can be obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells.

[0041] The host cell protein product can be obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells.

[0042] To address the aforementioned technical problems, this invention also provides the application of host cell protein peptides in the preparation of products for detecting host cell proteins in target protein products.

[0043] The host cell protein peptides may be a mixture of peptides obtained by enzymatic hydrolysis of host cell protein products;

[0044] The target protein product can be obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells.

[0045] The host cell protein product can be obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells.

[0046] To address the aforementioned technical problems, the present invention also provides a product for detecting host cell proteins in a target protein product, wherein the product may contain host cell protein peptides;

[0047] The host cell protein peptides may be a mixture of peptides obtained by enzymatic hydrolysis of host cell protein products;

[0048] The target protein product can be obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells.

[0049] The host cell protein product can be obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells.

[0050] The products mentioned above may be reagents or kits.

[0051] The target protein products (biological protein drugs) mentioned above can be vaccines, insulin, cytokines, or monoclonal antibodies.

[0052] This invention relates to the use of a novel LC-MS / MS method to detect residual host cell proteins (HCPs) in biological protein drugs.

[0053] The purpose of this invention is to provide a method for detecting hematologic cytokines (HCPs) in biological proteins using High-pH-RPLC-MS / MS technology after covalently binding a tag reagent to the sample. This method increases the depth of HCP identification and improves the sensitivity of identification. Using this method, there is no need to enrich host cell proteins in the cell / biological protein sample, overcoming the dynamic range of more than six orders of magnitude between protein drugs and host cell proteins.

[0054] The present invention further provides a method for detecting HCPs in the sample, comprising the following steps:

[0055] (1) Select the cell line for producing protein drugs, and lyse the harvested cells into peptides as the source sample for host cell proteins;

[0056] (2) Covalently bind the source samples of host cell proteins and biological protein samples with chemical tagging reagents respectively, and mix the bound samples for later use;

[0057] (3) The mixed sample described in (2) was subjected to high pH reverse phase fractionation, and after being combined into 8 fractions, mass spectrometry was performed to obtain the quantitative results of host cell protein.

[0058] (4) Peptide quantification of HCPs was achieved using MaxQuant, the most commonly used platform for proteomics data analysis based on mass spectrometry (MS), which has its own peptide search engine, Andromeda. The target-decoy search strategy was used to estimate and control false positives, keeping the FDR below 1%.

[0059] The source sample of the host cell protein mentioned in step 1) is derived from biological samples such as cells, tissues, and blood, specifically cell samples, with a sample size of 10 μg peptide samples.

[0060] Compared with existing HCP analysis methods, the present invention has the following advantages:

[0061] By introducing samples from HCP sources to enhance the mass spectrometry signal response of trace samples, the sensitivity was significantly improved, overcoming the extremely wide dynamic range between drugs and low-abundance protein HCPs. Simultaneously, fractionation techniques were combined to increase the detection loading of mass spectrometry, greatly enhancing the identification depth of HCPs. MaxQuant software was used for HCP analysis, employing a target-decoy search strategy to estimate and control false positives, ensuring the reliability of the quantitative results. This method enables HCP analysis of biological protein drugs and has significant application value for the quality control and detection of HCPs in biological protein pharmaceutical products.

[0062] In the above method, the mass ratio of the cell lysate peptides to the biological protein drug is 1:1, and the ratio value calculated using SCPComponsion is approximately 114.

[0063] Biopharmaceutical samples, after covalent binding and fractionation (enzyme-digested into monoclonal antibody peptides and HCP peptides), can be directly injected into a mass spectrometer and analyzed along with the mobile phase without the need for re-desalting, thus reducing sample loss caused by pretreatment. Single-column or data-dependent acquisition (DDA) one-dimensional (1D) mass spectrometry / mass spectrometry has been described for HCP detection in biopharmaceutical products, but for higher sensitivity, two-dimensional reversed-phase liquid chromatography (RPLC) can improve peak capacity and resolution to separate these highly complex peptide samples with a wide dynamic range.

[0064] The specific applications of the above analytical methods in the detection of HCPs or standard proteins in biological protein drugs are as follows:

[0065] 1) Applied to the characterization and identification of residual host cell proteins (HCPs) in biopharmaceutical products.

[0066] A more specific application is as follows: 4.6 µL of monoclonal antibody 1 (mAb-1) injection solution was dissolved in 100 mM TEAB, reduced and alkylated, then digested into peptides with trypsin, and the peptides were covalently bound to the sample with a reagent tag; Chinese hamster ovary cells (CHO-S) from the mammalian expression system were lysed, reduced and alkylated, and then digested with enzymes. The digested peptides were covalently bound, and the combined monoclonal antibody and cellular peptides were mixed. More than 1000 HCPs could be identified in the unfractionated mAb-1 sample.

[0067] 2) The method was applied to the study of HCPs in NIST monoclonal antibody (NISTmAb) standard RM8671, and 3475 HCPs were identified with high confidence, which is about 4 times the number of HCPs previously reported.

[0068] A more specific application is as follows: NIST monoclonal antibody RM8671 is dissolved in 100mM TEAB, reduced and alkylated, then digested into peptides with Trypsin, and then... 19 H 30 N4O6 treatment of peptides; lysis of mouse myeloma cell line NS-1, reductive alkylation, followed by enzymatic digestion, and recombining of peptides with C 19 H 30 N4O6 covalently binds the monoclonal antibody and cellular peptides, which are then mixed and fractionated.

[0069] 3) Three standard proteins with a size range of 36.8-66.3 kDa were added to the purified antibody 1 product at a level of 1 ppm to simulate the presence of low levels of HCPs. All three proteins were identified without fractionation.

[0070] A more specific application involves dissolving yeast alcohol dehydrogenase (ADH1), yeast enolase (ENO1), and bovine serum albumin (ALB) proteins in 100 mM TEAB, reductively alkylating them, then cleaving them into peptides with Trypsin, and then... 19 H 30 N4O6 covalently binds to the peptide; three proteins are added to mAb-1 at a level of 1 ppm, followed by reductive alkylation and Trypsin digestion to bind the peptide with C. 19 H 30 N4O6 is covalently bonded, and the resulting peptides are mixed.

[0071] 4) Using the more complex Universal Proteomics Standard 1 (UPS-1) as the HCP model, 17 out of 23 proteins in the 0.32-4.15 ppm (<1 ppm) range (≥2 unique peptides) were confidently identified, and 24 out of 25 proteins in the (>1 ppm) range (≥2 unique peptides) were reliably identified.

[0072] A more specific application involves: dissolving UPS-1 in 100mM TEAB, digesting it into peptides using Trypsin + Lys-C, and covalently binding the peptides with reagent tags; adding 0.32-4.15ppm of UPS-1 to mAb-1 for enzymatic digestion, and then binding the peptides with C... 19 H 30 N4O6 covalently binds the peptides, and the resulting peptides are mixed and fractionated.

[0073] This invention is a method for analyzing and identifying biological protein pharmaceutical samples using mass spectrometry. The method involves first enzymatically cleaving the protein sample into peptides, then covalently binding them with the actual peptides, and finally separating them. Specifically, it involves increasing the MS signal intensity of low-concentration HCP peptides, and then triggering MS / MS fragments with the "total" peptide signal from the mixed sample (or sample) for peptide identification, while the reporter ion provides quantitative information.

[0074] This invention has the following advantages:

[0075] 1) This detection method overcomes the problem of a wide dynamic range (more than 6 orders of magnitude) between low ppm levels of HCPs and high levels of therapeutic antibodies, thus exhibiting high sensitivity for protein identification.

[0076] 2) Compared with enzyme-linked immunosorbent assay (ELISA), it does not require the use of polyclonal antibodies produced by immunizing animals with zero cell lines, can completely cover all HCPs of the production cell lines, avoids biased detection of more immunogenic HCPs, and can quickly quantify the relative content of individual HCP components in monoclonal antibody drugs.

[0077] 3) For the first time, an effective LC-MS / MS strategy was demonstrated. This strategy not only has high sensitivity but also high reliability for HCP detection, which greatly increases the number of peptides identified in HCP. Moreover, the sample processing steps in this method are simple and do not require additional desalting, thus reducing sample loss.

[0078] 4) Micro-fractionation is used to fractionate covalently bound mixed peptides, resulting in almost zero sample loss and effectively increasing sample loading. Complex mixed samples are pre-separated by high-performance liquid chromatography (HPLC) into multiple relatively simple samples, which are then analyzed separately by LC-MS, reducing the sample complexity of a single detection and increasing the depth of sample analysis.

[0079] This invention relates to a method for detecting host cell proteins in monoclonal antibody drugs / cell samples based on mass spectrometry. The method involves processing the lysate of the monoclonal antibody drug production cell line into peptides as the source sample for host cell proteins, thereby enhancing the signal response of host cell protein peptides in the monoclonal antibody drug and enabling the detection of low-abundance host cell proteins in constant-volume samples. The innovation lies in identifying the source of the host cell protein, eliminating the need for additional enrichment. The source sample of the host cell is covalently bound to the monoclonal antibody drug peptides, then mixed and subjected to high-pH reverse-phase fractionation, followed by mass spectrometry detection. MaxQuant software is used for host cell protein analysis, utilizing target-decoy control to monitor false positives at the PSM level, with a PSM FDR within 1%, ensuring the reliability of the identification results. Attached Figure Description

[0080] Figure 1 This is a flowchart of the technical solution of the present invention.

[0081] Figure 2 Experimental design and results for the feasibility of this technology. A) Quantitative quantity of host cell protein in each of the three replicate monoclonal antibody samples and the source sample provided by this invention. B) Sum of peptide abundance in each sample.

[0082] Figure 3 This is a comparison of the identification depth of the present invention in NIST monoclonal antibodies with existing technologies. A) The number of HCPs and peptides identified from NIST monoclonal antibodies by different methods. B) Venn diagram of HCPs identified from NIST monoclonal antibodies by different methods.

[0083] Figure 4 The names of the protein standards, abbreviations of the protein names, molecular weights, and number of unique peptides were detected for three standard proteins at a 1 ppm spiking level.

[0084] Figure 5To assess the sensitivity and reliability of the method of this invention, 48 UPS-1 proteins at different concentrations (4.15 ~ 0.32 ppm, sorted from highest to lowest ppm) were detected in mAb1. The pink bars represent the number of unique peptides identified from UPS-1 proteins, and the green dots represent the concentration levels of UPS-1 proteins. The left ordinate represents the unique peptides identified from UPS-1 proteins; the right ordinate represents the concentration levels of UPS-1 proteins spiked in the monoclonal antibody; and the x-axis represents the 48 UPS-1 proteins at different concentrations (4.15 ~ 0.32 ppm, sorted from highest to lowest ppm). Detailed Implementation

[0085] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0086] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0087] Example 1. Procedure and feasibility verification of the LC-MS / MS method for detecting HCPs in biological protein drugs according to the present invention.

[0088] 1. Preparation of peptides from host cell-derived samples

[0089] 1.1 Cell Culture and Treatment:

[0090] The CHO-S cell line (Shanghai Yuchi Biotechnology Co., Ltd.) that produced IgG1 antibody (mAb-1, NIMCmAb human IgG1κ monoclonal antibody, National Institute of Metrology, China, reference material number: GBW(E)091164) was cultured at 37°C, 30% oxygen, and 5% CO2. The CHO-S cell line is adapted for chemically defined serum-free suspension culture. The NIST RM8671 cell line NS-1 (Shanghai Yuchi Biotechnology Co., Ltd.) was maintained in a humidified atmosphere of 5% CO2 at 37°C. Cells were cultured using Dulbecco's modified Eagle's medium (DMEM, Gibco, UK) supplemented with 10% (v / v) fetal bovine serum and penicillin-streptomycin (100 U / mL, Thermo Fisher Scientific). Cell concentration and viability were monitored daily using a Vi-Cell cell viability analyzer (Beckman Coulter, Brea, CA, USA). Live and dead cells were separated using trypan blue staining. Once the cell growth density reaches 80%-90%, the collected cells are washed three times with pre-cooled 4°C phosphate-buffered saline (PBS), centrifuged at 1000 rpm for 3 minutes to discard the culture medium, collected in 2 mL centrifuge tubes, centrifuged at 1000×g at 4°C for 10 min, the supernatant is discarded to obtain the cell pellet, which is then stored at -80°C or used for subsequent operations at 4°C.

[0091] 1.2 Cell lysis:

[0092] The cell pellet obtained in step 1.1 was resuspended in lysis buffer (0.1% SDS, 8 M urea, 100 mM Tris-HCl, pH 8.0) and lysed on ice for 15 min. The sample was repeatedly pipetted to aid lysis and maintain suspension. An ultrasonic cell disruptor was used to aid dissolution. The lysate was placed in an ice-water mixture and subjected to 100 W for 99 cycles (2 seconds each, 2 seconds interval). The disrupted sample was centrifuged at 16000 × g at 4 °C for 30 min, then discarded. The supernatant was collected as the cell protein extract. Protein concentration was measured using BCA, and the extract was stored at -80 °C for later use.

[0093] 1.3 Protein reductive alkylation:

[0094] Add 20 mM DTT (dithiothreitol) to the cell protein extract and incubate at 56 °C for 1 h to reduce the disulfide bonds in the protein. Then add CAA to a final concentration of 40 mM and react at room temperature for 45 min to obtain a protein reductive alkylation solution.

[0095] 1.4 SP3 digestion:

[0096] The SP3 (Single-Pot Solid-Phase-enhanced Sample Preparation) enzymatic hydrolysis technology is used to hydrolyze proteins. The principle involves the non-covalent capture and release of proteins through active groups on magnetic beads, and the magnetic properties of these beads concentrate the captured proteins. This allows for a more thorough and flexible removal or replacement of different chemical solvents required in each step of the enzymatic hydrolysis process, and it exhibits excellent removal effects on detergents and other solvents. The specific steps are as follows:

[0097] Two types of beads (Cytiva, USA) were mixed in a 1:1 ratio, gently vortexed for 30 seconds to mix, placed on a magnetic rack, and the supernatant was discarded. Five volumes of ultrapure water were added, gently vortexed for 30 seconds to mix, placed on a magnetic rack, and the supernatant was discarded. The mixture was washed three times. Finally, a bead stock solution of 100 µg / µL was prepared with water and stored at 4 °C. In the protein reductive alkylation solution, the bead stock solution was added at a mass ratio of beads:protein = 10:1. ACN was added to bring the final concentration of organic solvent to 80%, and the binding of beads to protein was induced at 37 °C and 1500 rpm for 18 min.

[0098] Place the sample on a magnetic rack and let it stand for 2 minutes. After the solution becomes clear, discard the supernatant with a pipette, add ACN, and adsorb through the pores for 30 seconds. Discard the supernatant, rinse 3 times, and dry at room temperature for 30 seconds. Add 100 mM TEAB and trypsin at a mass ratio of protein:trypsin = 100:1. Incubate at 37°C for 18 hours to digest the enzyme and obtain host cell protein (HCP) peptides.

[0099] After enzyme digestion, centrifuge at 16000×g for 10 min and collect the supernatant into a new tube. Measure the concentration of the collected peptide solution at 280 nm using a NanoDrop 2000C UV spectrophotometer. Aliquot 10 μg of peptide into an EP tube and store at -80℃ for later use.

[0100] 2. Monoclonal antibody sample preparation

[0101] 100 μg of monoclonal antibody formulation (NIMCmAb, sourced from the National Institute of Metrology, China; NIST RM 8671, Sigma-Aldrich) was diluted with 100 mM TEAB, and 20 mM DTT was added to bring the final concentration of the monoclonal antibody to 5 µg / µL. The mixture was incubated at 56 °C for 1 h to reduce disulfide bonds in the protein, ensuring complete protein denaturation. Then, CAA was added to a final concentration of 40 mM, and the reaction was carried out at room temperature for 45 min. Finally, SP3 digestion was performed as described in step 1.4 to obtain a solution containing the monoclonal antibody peptide.

[0102] 3. The sample covalently binds to the isotopic compound.

[0103] The two peptides mentioned above were respectively coupled with an isotopic compound C containing a group of formula 17 (which can covalently bind to the peptide). 19 H 30 N4O6 (Thermo Fisher Scientific - CN) (as shown in Equations 1-16, where the atoms marked with "*" in Equations 1-16 are...) 13 C or 15 N) After covalent bonding to form covalent bonds, they are mixed in a 1:1 mass ratio, and the ratio calculated using SCPComponsion is approximately 114.

[0104] Specifically: the HCP peptide obtained in step 1 is placed in a 0.2 mL PCR tube, and the C shown in Formula 16 is added. 19 H 30 N4O6 (dissolved in anhydrous acetonitrile) was added to a PCR tube and incubated at room temperature for 1.5 h for covalent binding. Then, 5% hydroxylamine solution was added and incubated at room temperature for 18 min to quench the covalent binding reaction, yielding the HCPs covalently bound peptide. The monoclonal antibody peptide obtained in step 2 was placed in a 0.2 mL PCR tube, and the C (as shown in Formula 6) was added... 19 H 30 N4O6 (dissolved in anhydrous acetonitrile) was added to a PCR tube and incubated at room temperature for 1.5 h for covalent binding. Then, 5% hydroxylamine solution was added and incubated at room temperature for 18 min to quench the covalent binding reaction, yielding the monoclonal antibody covalently bound peptide. The HCPs covalently bound peptide and the monoclonal antibody covalently bound peptide were mixed and heat-dried to obtain the (mixed) covalently bound peptide for later use.

[0105] ;

[0106] .

[0107] 4. High-pH reversed-phase fractionation of the mixed covalently bound peptides using a chromatographic column.

[0108] Using a micro-fractionation device to fractionate covalently bound mixed peptides can reduce sample loss and increase sample loading. Complex mixed samples are pre-separated (fractionated) by high-performance liquid chromatography (HPLC) into multiple relatively simple samples, which are then analyzed separately by LC-MS. This reduces the sample complexity of a single analysis and increases the depth of sample analysis. Specifically:

[0109] Dissolve the mixed covalently bound peptides obtained in step 3 in water and add them to a sample vial (injection volume ≤ 10 µL). Elute and collect the peptides according to the following gradient: perform reversed-phase separation on an Eksigent XBridge BEH C18 column (3 μm, 130 Å, 0.3 × 150 mm) using an Ekspert nanoLC 4000 high-performance liquid chromatography system.

[0110] The mobile phase consisted of 98% ammonia (pH 10) with 2% acetonitrile (mobile phase A) and 98% acetonitrile with 2% ammonia (pH 10) (mobile phase B), with a flow rate of 2 μL / min. The total LC process lasted 120 minutes. The steps included: 5 minutes for washing 3.3% B, 68 minutes for washing 3.3-43% B, 22 minutes for washing 43-56% B, 5 minutes for washing 5% B, 5 minutes for washing 100% B, and 5 minutes for washing 82-3.3% B, followed by column rebalancing (10 minutes, 0% B). The number of cycles was set to 5. Each well contained 300 μL of acetonitrile. Forty high-pH RP fractions (5 μL each) were combined into eight fractions (pool 1-pool 8) during LC separation times ranging from 5 to 105 min. Fractions 1, 9, 17, 25, and 33 were grouped into pool 1; 2, 10, 18, 26, and 34 into pool 2; and so on until fractions 8, 16, 24, 32, and 40 were grouped into pool 8. The fractions were dried in a high-speed vacuum concentrator for approximately 0.5 h to obtain fractionated heat-dried samples, which were then stored at -80 °C for LC-MS / MS analysis.

[0111] The biological protein pharmaceutical samples (fractionated hot-dry samples) after the above covalent bonding and fractionation can be directly injected into the mass spectrometer and analyzed by mass spectrometry along with the mobile phase. No further desalting is required, which reduces sample loss caused by pretreatment.

[0112] 5. Mass spectrometry detection and data analysis

[0113] The fractionated and heat-dried sample obtained in step 4 was reconstituted with 0.1% formic acid solution. Liquid chromatography-mass spectrometry (LC-MS / MS) analysis was performed on a Q Exactive HF Hybrid quadrupole orbital mass spectrometer (Thermo Fisher Scientific), coupled online with a nanoflow LC system (EASY-nLC 1200, Thermo Fisher Scientific), with a full MS scan range of 375 to 1400 m / z. All separations were performed on a 1 cm self-packed trap column (150 μm inner diameter, 1.9 μm resin, ReproSil-Pur C18-AQ, Dr Maisch GmbH) with solvent A (0.1% formic acid FA in HPLC-grade water). After loading and washing, the peptides were transferred to a 15 cm column (150 μm inner diameter, 1.9 μm resin, ReproSil-Pur C18-AQ, Dr Maisch GmbH). The mobile phase was either a 0.1% formic acid aqueous solution (mobile phase A, pH ~ 2.7) or an 80% acetonitrile solution containing 0.1% formic acid (mobile phase B). The flow rate was 600 nL / min, and the column temperature was 60 °C. The LC gradients were as follows: B at 4–7% for 1 min, B at 7–13% for 5 min, B at 13%–25% for 30 min, B at 25–45% for 18 min, B at 45–95% for 1 min, and B at 95% for 5 min. Under the following mass spectrometry settings: 120,000 m / s resolution, 300% normalized automatic gain control (AGC) target, and 80 ms maximum injection time; and 60,000 m / s resolution, 100% normalized automatic gain control (AGC) target, 100 ms maximum injection time, and 0.7 m / z isolation window, the peptides were subjected to high-energy collisional dissociation fragmentation, with a normalized collision energy of 30% for each full MS scan. The peptide was fragmented by high-energy collisions, and the MS / MS normalized collision energy was 32%.

[0114] LC-MS / MS DDA data files were used for SEQUEST search using Maxquant software (version 2.4.2.0) to retrieve sequences containing all internal biotherapeutic products and all spiked protein standards, and were linked to the Cricetulus griseus database (downloaded from UniProt on April 20, 2023, with 58,104 entries). The original NIST files were directly linked to the Uniprot.org mouse (mus musculus) Swiss-Prot database (October 2023 version; 17,734 entries) for HCP analysis in NIST mAb.

[0115] The initial maximum precursor mass tolerance was set to 20 ppm in the first search and 4.5 ppm in the main search, with a fragment mass tolerance of 10 ppm. Search criteria included static aminomethylation of cysteine ​​(+57.0214 Da), N-terminal reagent tags of lysine (K) and peptides (+304.207 Da), oxidative modification of methionine residues (+15.9949 Da), and N-terminal acetylation of proteins (+42.011 Da). Searches were performed using trypsin / P digestion, allowing a maximum of two missing cleavages on peptides analyzed in the sequence database. The false discovery rate for proteins and peptides was set to 0.01.

[0116] The intensity of the obtained HCP peptides was relatively quantified by the intensity of the peptide signal from the HCP source samples, then Log10 transformation and normalization were performed, and subsequent data analysis was conducted using Origin 2021 and GraphPad Prism (Version 9.5).

[0117] 6. Feasibility Analysis of the Method of the Invention

[0118] As a proof of concept, three samples were prepared to test the feasibility of the method of the present invention for deep HCPs identification in the research samples. Figure 2 (A)

[0119] For sample I (mAb-1, NIMCmAb human IgG1κ monoclonal antibody, National Institute of Metrology, China), such as Figure 2 As shown in A, C is represented by equations 6, 7, and 8 in step 3. 19 H 30 The N4O6 was covalently bound to the peptide of 200 ng monoclonal antibody (mAb-1) to tag the sample, resulting in the covalently bound monoclonal antibody peptide. Three replicate samples (mAb-1-1, mAb-1-2, and mAb-1-3) of the covalently bound monoclonal antibody peptide were combined for LC-MS analysis.

[0120] For sample II, the cell protein peptides (HCPs) harvested from the CHO-S cell line used to produce mAb-1 were compared with the C protein peptides shown in Formula 16 of step 3. 19 H 30 N4O6 undergoes covalent bonding.

[0121] Sample III is a combination of Sample I and Sample II to assess the feasibility of using host cell protein-derived samples to facilitate the identification of HCPs in trace samples in mAb-1.

[0122] For sample I, 10 HCPs were identified due to their low abundance in mAb-1. However, after carriers were added to the host cell protein-derived sample, sample III showed HCPs ( Figure 2 The number of HCPs source samples (represented by A) increased by at least 100-fold. Figure 2 (A). The results of this invention show that the intensity of HCPs found in mAb-1 of sample III was reduced compared with sample II, which is due to the inhibitory effect of the high abundance of mAb-1 in the study channel of sample III. Figure 2 The total intensity in each sample of the B group increased by at least 32-fold, indicating that using enhanced samples (HCP peptides) significantly improves the possibility of detecting and quantifying HCPs in biopharmaceutical (monoclonal antibody) samples. Figure 2 (B) This indicates that using source samples of host cell proteins greatly increases the chances of identifying and quantifying HCPs in trace biological protein samples.

[0123] Example 2. The identification depth of this technique was examined using the NIST standard antibody RM 8671.

[0124] When the method of this invention was applied to the study of HCPs in NIST monoclonal antibody (NISTmAb) standard RM8671, 3475 HCPs were identified with high confidence, which is about four times the maximum number of HCPs identified in the prior art.

[0125] Specifically, the method of this invention is applied to the peptide profiling analysis of HCPs in the NIST standard antibody RM 8671. The specific method is as follows: Steps 1-5 are the same as in Example 1: NIST monoclonal antibody 8671 is dissolved in 100mM TEAB, reductively alkylated, and then digested into peptides with Trpsin. The peptides are then analyzed using the C6400 peptides shown in Formulas 6, 7, and 8 of Example 1. 19 H 30 N4O6 was used to treat the monoclonal antibody peptide; the mouse myeloma cell line NS-1, used to produce monoclonal antibody 8671, was lysed, reduced and alkylated, and then enzymatically digested. The peptide was then processed using the C4O6 method shown in Example 1. 19 H 30N4O6 treatment yielded covalently bound peptides of HCPs. The covalently bound monoclonal antibody peptides and HCP peptides were mixed at a 1:1 mass ratio and fractionated; then mass spectrometry detection and data analysis were performed. Since other researchers typically cannot obtain specific biopharmaceutical products from most MS-based HCP samples, direct comparisons of results from different methods are nearly impossible. The key to achieving these goals is to achieve higher identification through detection standards. NISTmAb, a humanized IgG1κ antibody produced in mouse cell culture and purified using standard biopharmaceutical manufacturing methods, developed by the National Institute of Standards and Technology (NIST), can be used as a reference. This invention can be compared with recent reports and analyzed using RM8671 NIST antibodies. Recently, Song Nie et al. and Feng Yang et al. developed HCP identification strategies based on NISTmAb standards, identifying 453 and 746 HCPs, respectively. In the results of this invention, a total of 3475 HCPs were identified (2851, unique peptides ≥ 2), with a false positive rate ≤ 0.01. Figure 3 Institute A, and existing technology 1 (Song Nie et al., Figure 3 Nie et al. (representative of A) and prior art 2 (Feng Yang et al.) Figure 3 Compared to Yang et al. (represented by A), the method of this invention identifies HCPs without fractionation. Figure 3 The efficiency (number of proteins) of non-fractionation in A. Figure 3 The Proteins in A are 1.7 times (compared to prior art 1) or 0.9 times (compared to prior art 2). In the fractionation method of the present invention ( Figure 3 The results of our Fractionation (represented by A) indicate the HCP identification rate (number of proteins). Figure 3 The Proteins in A (representing the HCP) improved by 7.7 times and 4.1 times respectively compared to the two existing technologies mentioned above, demonstrating the strategy's ability to achieve deep coverage of HCP maps. Figure 3 As shown in B, prior art 1 ( Figure 3 Of the 453 HCPs detected by Nie, S. (represented by B), 328 were found in existing technology 2 ( Figure 3 Of the 848 HCPs detected by Yang, F. (represented by B), 600 were also detected by the method of this invention. Figure 3The results of our results (fractionation) and our results (no-fractionation) are covered by the method described in this invention. In summary, the 2869 mouse HCPs identified by the method of this invention in the NIST mAb criteria were not reported in the previous two studies.

[0126] Therefore, the method of the present invention increases the depth of HCP identification, and can identify more than 2,000 HCPs independently, in addition to repeating the identification of more than 70% of the HCPs reported in the prior art on NIST mAb (RM 8671). This promotes LC-MS / MS as a more powerful tool for HCP analysis and can be applied to the preparation of products that detect HCPs in monoclonal antibody drugs.

[0127] Example 3. Detection limit of fractionation-free LC-MS / MS

[0128] To evaluate the detection limit of the method of the present invention, three purified proteins—bovine serum albumin ALB (LABLEAD BIOTECH CO.LTD, #0332), yeast enolase ENO1 (Sigma Aldrich #E6126), and yeast alcohol dehydrogenase ADH1 (Sigma Aldrich, #A7011)—with molecular weights between 36.8 and 66.3 kDa, were added to a purified monoclonal antibody (mAb-1) containing very low levels of endogenous HCPs. After sample preparation using steps 2 and 3 of Example 1, the samples were analyzed using liquid chromatography-mass spectrometry (LC-MS / MS) in step 5, without using the fractionation step in step 4 of Example 1.

[0129] Specifically, three standard proteins with a size range of 36.8-66.3 kDa—yeast alcohol dehydrogenase ADH1, yeast enolase ENO1, and bovine serum albumin ALB—were dissolved in 100 mM TEAB, reduced and alkylated, and then cleaved into peptides using Trypsin. The peptides were then processed using the C16 formula shown in Example 1. 19 H 30 N4O6 was used to treat the peptides; it was added at a level of 1 ppm to the purified antibody mAb-1 product to simulate the presence of low levels of HCPs. After reductive alkylation, the peptides were cleaved with Trypsin and processed using C as shown in Formulas 2, 3, and 5 in Example 1. 19 H 30 The peptides treated with N4O6 were subjected to mass spectrometry detection and data analysis without fractionation, and all three proteins were identified.

[0130] Figure 4The results show the number of three standard protein peptides identified by mAb-1 at a peak level of 1 ppm (1 ng / mg) relative to monoclonal antibody-1. In three replicate mAb-1 peptide samples, all other proteins (ADH1, ENO1, and ALB) were identified as having ≥2 unique peptides. This demonstrates the good reproducibility and accuracy of the method of this invention. The method of this invention describes sensitivity based on minimum levels of spiked protein detection and demonstrates comprehensiveness or consistency in the detection of different proteins at a given level.

[0131] This invention defines the detection limit as the ability to consistently detect all spiked proteins in each sample (n=3) at or above a specific peak level. Therefore, 1 ppm is considered the detection limit for reliable HCP detection in monoclonal antibody products.

[0132] Example 4: Identification of low-level UPS-1 protein spiked in monoclonal antibody-1

[0133] To further verify the reliability of the method of this invention in detecting low levels of HCPs in biopharmaceutical products, mAb-1 was detected under optimal conditions (8 tandem components, 15 cm long column, 60 min gradient). Forty-eight known UPS-1 proteins (Universal Proteomics Standard, Sigma-Aldrich) (molecular weight range 6.3–82.9 kDa) were added to mAb-1 to simulate the presence of trace HCPs in the product. Using the more complex Universal Proteomics Standard 1 (UPS-1) as the HCP model, this invention added 48 UPS-1 proteins (25 proteins ≥ 1 ppm, 23 proteins < 1 ppm) at concentrations ranging from 0.32 to 4.15 ppm to the monoclonal antibody-1 to simulate trace HCP contamination. Figure 5 The ppm level of UPS-1 protein and the number of unique peptides in monoclonal antibody-1 were shown. 44 (91.7%) UPS-1 proteins were identified, demonstrating the stability of the method of this invention in identifying sub-ppm level HCPs over a wide molecular weight range (6.3 to 82.9 kDa).

[0134] Specifically, using the method in Example 1 of this invention, UPS-1 was dissolved in 100mM TEAB, cleaved into peptides using Trypsin enzyme + Lys-C, and then processed using the C shown in Formula 16 of Example 1. 19 H 30 N4O6 was used to treat UPS-1 peptides; UPS-1 in the range of 0.32-4.15 ppm was added to mAb-1 for enzymatic digestion, and the peptides were processed using C as shown in Formulas 2, 3, and 5 in Example 1. 19 H 30N4O6 treatment was used to mix and fractionate the covalently bound peptides; then mass spectrometry detection and data analysis were performed.

[0135] These UPS-1 proteins had the same molar concentration; therefore, their peak levels ranged from 0.32 to 4.15 ppm (25 proteins were present at ≥1 ppm levels, and 23 proteins were present at <1 ppm levels). The spiking ppm levels and the number of identified peptides for each UPS-1 protein at a 10 μg target sample load are as follows: Figure 5 As shown. The results indicate that, as Figure 5 As shown, 41 (85.4%) UPS-1 proteins (0.32 to 4.15 ppm) were reliably and consistently identified (≥2 unique peptides), demonstrating stability in identifying HCPs down to 1 ppm (replicas = 3) within the molecular weight range (6.3 to 82.9 kDa). Consistency and 100% success rate (25 / 25) were achieved when detecting UPS-1 proteins at peak levels ≥1 ppm, and a success rate ≥82.6% (19 / 23) was achieved when detecting UPS-1 proteins at levels <1 ppm. This invention further validates the reliability of this method for detecting <1 ppm HCPs in monoclonal antibody drugs with high success rates. Four unidentified UPS-1 spiked proteins ( Figure 5 Among them, 40, 46, 47 and 48 are small in size (6-13 kDa) and have low abundance relative to most proteins in the sample (1 <0.61 ppm; 3 <0.42 ppm).

[0136] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for detecting or identifying host cell proteins in a target protein product, characterized in that: The method includes: 1) Preparation of mixed covalently bound peptides, wherein the mixed covalently bound peptides consist of target protein covalently bound peptides and host cell protein covalently bound peptides. The target protein covalently bound peptide is a mixture obtained by covalently binding the peptide of the target protein product with compound 1, and the host cell protein covalently bound peptide is a mixture obtained by covalently binding the peptide of the host cell protein product with compound 2; compound 1 and compound 2 are isotopic compounds with the same molecular weight, the only difference between compound 1 and compound 2 is that the isotopes are atoms located at different positions, and both compound 1 and compound 2 contain groups of formula 17 to covalently bind with the peptide; ； The peptides of the target protein product are a mixture of peptides obtained by enzymatic hydrolysis of the target protein product using digestive enzymes; the peptides of the host cell protein product are a mixture of peptides obtained by enzymatic hydrolysis of the host cell protein product. The target protein product is obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells. The host cell protein product is obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells; 2) The mixed covalently bound peptides are subjected to liquid chromatography-mass spectrometry (LC-MS) to identify the target peptide, which is the covalently bound peptide of the target protein and the covalently bound peptide of the host cell protein with the same mass-to-charge ratio. The LC-MS detection results of the target peptide are analyzed using mass spectrometry analysis software. Based on the signal of compound 1, the host cell protein contained in the target protein product is identified.

2. The method according to claim 1, characterized in that: The mass spectrometry detection step is preceded by a step of high-pH reverse-phase fractionation of the mixed covalently bound peptides using a high-performance liquid chromatography system.

3. The method according to claim 1 or 2, characterized in that: In the aforementioned mixed covalently bound peptides, the mass ratio of the target protein covalently bound peptide to the host cell protein covalently bound peptide is 1:

1.

4. The method according to claim 1 or 2, characterized in that: The target protein product and the host cell protein product are protein products that have undergone reductive alkylation.

5. The method according to claim 1 or 2, characterized in that: The target protein product is a monoclonal antibody.

6. A method for improving the detection sensitivity and / or detection depth of host cell proteins in a target protein product, characterized in that: The method includes the steps of preparing a mixed covalently bound peptide and then performing mass spectrometry analysis on the mixed covalently bound peptide to improve the detection sensitivity and / or detection depth of host cell proteins in the target protein product. The mixed covalently bound peptides consist of target protein covalently bound peptides and host cell protein covalently bound peptides. The target protein covalently bound peptide is a mixture obtained by covalently binding the peptide of the target protein product with compound 1, and the host cell protein covalently bound peptide is a mixture obtained by covalently binding the peptide of the host cell protein product with compound 2; compound 1 and compound 2 are isotopic compounds with the same molecular weight, the only difference between compound 1 and compound 2 is that the isotopes are atoms located at different positions, and both compound 1 and compound 2 contain groups of formula 17 to covalently bind with the peptide; ； The peptides of the target protein product are a mixture of peptides obtained by enzymatic hydrolysis of the target protein product using digestive enzymes; the peptides of the host cell protein product are a mixture of peptides obtained by enzymatic hydrolysis of the host cell protein product. The target protein product is obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells. The host cell protein product is obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells.

7. Application of host cell protein peptides in the detection of host cell proteins in target protein products, wherein the host cell protein peptides are a mixture of peptides obtained by enzymatic hydrolysis of host cell protein products; The target protein product is obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells. The host cell protein product is obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells; The application is implemented through the steps of the method described in any one of claims 1-5.

8. Application of host cell protein peptides in the preparation of products containing host cell proteins for the detection of target proteins. The host cell protein peptides are a mixture of peptides obtained by enzymatic hydrolysis of host cell protein products; The target protein product is obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells. The host cell protein product is obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells; The application is implemented through the steps of the method described in any one of claims 1-5.

9. A product for detecting host cell proteins in a target protein product, characterized in that: The product contains host cell protein peptides; The host cell protein peptides are a mixture of peptides obtained by enzymatic hydrolysis of host cell protein products; The target protein product is obtained through the following steps: culturing recombinant cells containing the coding gene of the target protein to obtain a culture of the recombinant cells; crudely purifying the culture of the recombinant cells to obtain the total protein of the recombinant cells; and further purifying the total protein of the recombinant cells to obtain the target protein product; wherein the recombinant cells are recombinant cells expressing the coding gene obtained by introducing the coding gene into host cells. The host cell protein is obtained through the following steps: culturing the host cells to obtain a culture of the host cells, and then performing crude purification on the culture of the host cells to obtain the total protein of the host cells; The product achieves the detection of the host cell protein in the target protein product through the steps of the method described in any one of claims 1-5.

10. The product according to claim 9, characterized in that: The product is a reagent or kit.

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