Method for detecting HCPs in biological protein medicine based on LC-MS / MS method

Abstract

The invention discloses a method for detecting HCPs in a biological protein drug based on an LC-MS / MS method. The method comprises the following steps: covalently binding an HCPs-derived cell line protein sample and a monoclonal antibody protein with the sample by adopting a chemical reagent, mixing, fractionating, and finally performing liquid-phase mass spectrometry to obtain an analysis result of HCPs in the monoclonal antibody drug. Compared with the prior art, the detection signal is enhanced by adding the host cell protein for producing the monoclonal antibody, the detection sensitivity and the identification depth of low-abundance protein are remarkably improved, the important blank of HCPs analysis is filled up, and the monoclonal antibody can be used for detecting and proving HCPs removal, especially extremely-low-level hydrolase in drug process development, and can be applied to detection and proving of HCPs removal. The method has important influence on ensuring the quality of biological protein pharmaceutical products and the safety of patients.

Description

Technical Field

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

[0002] Bioprotein drugs are produced using transgenic prokaryotic or eukaryotic host cells and cell culture technology, which inevitably leads to their co-expression with host cell proteins (HCPs). Host cell proteins (HCPs), as the main type of impurities in the process of bioprotein drugs, may pose risks to patient safety and product efficacy, so continuous monitoring is required to prove that their removal rate is lower than the 100 ng / mg level recognized by regulatory agencies. The risks of host cell proteins are: 1) immune response to the body. Immunogenic HCPs can induce unexpected harmful immune responses in patients. Since HCPs may cause unpredictable immune responses, immune rejection reactions are likely to occur, posing a threat to life safety; 2) Degradation of ingredients in product formulations. HCPs with enzymatic activity can degrade therapeutic antibodies themselves or react with ingredients in the formulation buffer to reduce antibody stability and increase the formation of visible particles. HCPs enzyme activity may directly or indirectly affect the stability of the product. It has been reported that HCPs with inherent hydrolytic activity may have a negative impact on product quality by degrading the surfactant polysorbate, resulting in increased fatty acid release and a tendency to form particles composed of free fatty acids; 3) They are toxic or biologically active. For example, cathepsin D has been shown to cause antibody fragmentation and particle formation through its proteolytic activity; 4) Change antibody binding affinity / potency. The host cell proteins in these recombinant biotherapeutics can significantly affect the efficacy of the drug. In order to reduce the risk of HCPs, strict quality control is required for biopharmaceuticals.

[0003] For decades, enzyme-linked immunosorbent assay (ELISA) has been the gold standard for biotherapeutic process development and quality control to measure the total amount of HCPs in bioprotein drug process development and quality control. ELISA requires polyclonal antibodies produced by immunizing animals with null cell lines, which cannot fully cover all HCPs of production cell lines, resulting in biased detection of more immunogenic HCPs, which reduces its usefulness. Its limitations in HCP identification coverage and inability to identify specific proteins or quantify their individual concentrations have created unacceptable gaps in HCP characterization, encouraging researchers to explore alternative technologies. To meet this challenge, a large number of sample preparation and instrumentation techniques have been developed, and researchers have used a variety of complementary LC-MS / MS methods, including data independent acquisition (DIA) LC-MS / MS methods to obtain all fragment information of all ions in the sample, improve data utilization, fewer missing values, and higher analysis repeatability, two-dimensional high and low pH reverse phase fractionation LC-MS / MS methods to reduce the overlap between HCP and therapeutic protein drug peptide peaks, and molecular weight cutoff enrichment (Molecular Weight Cutoff Enrichment) LC-MS / MS methods to reduce the concentration gap between HCP and therapeutic proteins, etc. So far, more than 800 HCPs have been found 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] In order to solve the above technical problems, the present invention first provides a method for detecting or identifying host cell proteins in a target protein product (biological protein drug), which may include: 1) preparing a mixed covalently bound peptide segment, wherein the mixed covalently bound peptide segment may be composed of a target protein covalently bound peptide segment and a host cell protein covalently bound peptide segment, The target protein covalently bound peptide segment may be a mixture obtained by combining the peptide segment of the target protein product with compound 1, and the host cell protein covalently bound peptide segment may be a mixture obtained by combining the peptide segment of the host cell protein product with compound 2; compound 1 and compound 2 may be isotope-containing compounds with the same molecular weight, and the difference between compound 1 and compound 2 is only that the isotope is an atom located at a different position, and both compound 1 and compound 2 may contain a group of formula 17 to covalently bind to the peptide segment; ; The peptide segments of the target protein product may be a mixture of peptide segments obtained by enzymatically hydrolyzing the target protein product using a digestive enzyme; the peptide segments of the host cell protein product may be a mixture of peptide segments obtained by enzymatically hydrolyzing the host cell protein product; The target protein product can be obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product can be obtained by the following steps: culturing the host cells to obtain a culture of the host cells, and crudely purifying the culture of the host cells to obtain the total protein of the host cells; 2) subjecting the covalently bound peptides to liquid phase secondary mass spectrometry detection to determine a target peptide, wherein the target peptide is a target protein covalently bound peptide and a host cell protein covalently bound peptide having the same mass-to-charge ratio, and using mass spectrometry analysis software to analyze the secondary mass spectrometry detection results of the target peptides, and determining the host cell protein contained in the target protein product based on the signal of the compound 1.

[0006] The cell may be an animal cell or a microbial cell.

[0007] The crude purification is any purification method that can obtain total protein of cells from cell culture.

[0008] The cell culture may be a substance obtained by culturing cells in a culture medium (ie, a culture product, including cells, secretions secreted by the cells into the culture medium, and remaining culture medium).

[0009] Among the compound 1 and the compound 2, the compound 1 may be a compound C shown in any of the following chemical formulas 1 to 16: 19 H 30 N4O6, the compound 2 can be a compound C represented by formula 1 or formula 16 19 H 30 N4O6, the compound 1 is any one of formula 1 to formula 16 and is different from the compound 2. The atoms with "*" in formula 1 to formula 16 are 13 C or 15 N.

[0010] .

[0011] The culture medium mentioned above may be a liquid culture medium, and the cell culture may be a cell culture fluid.

[0012] The enzymatic hydrolysis method described above may be SP3 enzymatic hydrolysis. The SP3 enzymatic hydrolysis is a Single-Pot Solid-Phase-enhanced Sample Preparation enzymatic hydrolysis technology, which may include the following steps: in the process of enzymatic hydrolysis of the monoclonal antibody host cell protein using a digestive enzyme, the monoclonal antibody host cell protein is non-covalently captured and released by the active groups on the magnetic particles (beads), and the captured monoclonal antibody host protein is concentrated (enriched) by the magnetism of the magnetic particles.

[0013] The above-mentioned digestive enzyme may be trypsin.

[0014] The above-mentioned monoclonal antibody host cell line can be a Chinese hamster ovary cell (CHO-S) line or a mouse myeloma cell (NS-1) line.

[0015] In the above method, Maxquant software can be used to analyze the mass spectrometry detection results.

[0016] The above method further comprises the step of reductively alkylating the target protein product and the host cell protein product.

[0017] In the above method, before the mass spectrometry detection step, the step of performing high pH reverse phase fractionation on the mixed covalently bound peptide segments using a high performance liquid chromatography system may also be included.

[0018] The high pH reverse 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 for reverse phase separation. The mobile phase in the high performance liquid chromatography system can be 98% ammonia water (pH 10).

[0019] The above reductive alkylation includes two steps: reduction and alkylation. The reduction is to reduce the disulfide bonds in the protein to sulfhydryl groups using a reducing agent, and the alkylation is to modify the sulfhydryl groups to prevent them from forming disulfide bonds again.

[0020] In the above method, in the mixed covalently bound peptides, the mass ratio of the target protein covalently bound peptide to the host cell protein covalently bound peptide may be 1:1.

[0021] The mass spectrometry analysis software analysis described above can be performed by Maxquant software; during the analysis, the target-decoy search strategy in the Maxquant software is used to estimate and control the false positive of the detection, and the FDR of the analysis is less than or equal to 1%.

[0022] The above-mentioned target protein product and the host cell protein product may be a protein product that has undergone reductive alkylation.

[0023] In the above method, the target protein product may be a monoclonal antibody.

[0024] The target protein product (biological protein drug) mentioned above may also be a vaccine, insulin or cytokine.

[0025] In order to solve the above 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 segment, and then performing mass spectrometry analysis on the mixed covalently bound peptide segment to improve the detection sensitivity and / or detection depth of host cell proteins in the target protein product; The mixed covalently bound peptide segment may be composed of a target protein covalently bound peptide segment and a host cell protein covalently bound peptide segment. The target protein covalently bound peptide segment may be a mixture obtained by covalently binding the peptide segment of the target protein product with compound 1, and the host cell protein covalently bound peptide segment may be a mixture obtained by covalently binding the peptide segment of the host cell protein product with compound 2; compound 1 and compound 2 are isotope-containing compounds with the same molecular weight, and the difference between compound 1 and compound 2 is only that the isotope is an atom located at a different position, and both compound 1 and compound 2 may contain a group of formula 17 to covalently bind to the peptide segment; .

[0026] The peptide segments of the target protein product may be a mixture of peptide segments obtained by enzymatically hydrolyzing the target protein product using a digestive enzyme; the peptide segments of the host cell protein product may be a mixture of peptide segments obtained by enzymatically hydrolyzing the host cell protein product; The target protein product can be obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product can be obtained by the following steps: culturing the host cells to obtain the culture of the host cells, and crudely purifying the culture of the host cells to obtain the total protein of the host cells.

[0027] In order to solve the above technical problems, the present invention also provides the use of host cell protein peptides in detecting host cell proteins in a target protein product, wherein the host cell protein peptides may be a peptide mixture obtained by enzymatic hydrolysis of the host cell protein product; The target protein product can be obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product can be obtained by the following steps: culturing the host cells to obtain the culture of the host cells, and crudely purifying the culture of the host cells to obtain the total protein of the host cells.

[0028] In order to solve the above technical problems, the present invention also provides the use of host cell protein peptides in the preparation of a product for detecting host cell proteins in a target protein product. The host cell protein peptide segment may be a peptide segment mixture obtained by enzymatic hydrolysis of the host cell protein product; The target protein product can be obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product can be obtained by the following steps: culturing the host cells to obtain the culture of the host cells, and crudely purifying the culture of the host cells to obtain the total protein of the host cells.

[0029] In order to solve the above 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; The host cell protein peptide segment may be a peptide segment mixture obtained by enzymatic hydrolysis of the host cell protein product; The target protein product can be obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product can be obtained by the following steps: culturing the host cells to obtain the culture of the host cells, and crudely purifying the culture of the host cells to obtain the total protein of the host cells.

[0030] The product mentioned above may be a reagent or a kit.

[0031] The target protein product (biological protein drug) mentioned above may be a vaccine, insulin, cytokine or monoclonal antibody).

[0032] The present invention relates to the use of a novel LC-MS / MS method to detect residual host cell proteins (HCPs) in biological protein drugs.

[0033] The purpose of the present invention is to provide a method for detecting HCPs in biological proteins by using High-pH-RPLC-MS / MS technology after covalently binding a labeling reagent to a biological protein sample to be tested, thereby increasing the identification depth of HCPs and improving the identification sensitivity. Using this method, there is no need to enrich host cell proteins in cell / biological protein samples, thus overcoming the dynamic range of more than 6 orders of magnitude between protein drugs and host cell proteins.

[0034] The present invention further provides a method for detecting HCPs in the sample, comprising the following steps: (1) Select the production cell line of protein drugs, lyse the harvested cells into peptide fragments and use them as the source sample of host cell protein; (2) Covalently binding the source sample of the host cell protein and the biological protein sample with the chemical labeling reagent respectively, and mixing the bound samples for later use; (3) subjecting the mixed sample described in (2) to high pH reverse phase fractionation, combining the fractions into 8 fractions, and then subjecting the fractions to mass spectrometry to obtain quantitative results of host cell proteins; (4) Peptide quantification of HCPs was achieved using MaxQuant, the most commonly used platform for mass spectrometry (MS)-based proteomics data analysis. It has its own peptide search engine, Andromeda, which uses a target-decoy search strategy to estimate and control false positives, keeping the FDR within 1%; The source sample of the host cell protein in step 1) is derived from biological samples such as cells, tissues, and blood, specifically cell samples, and the sample volume is 10 μg of peptide samples; Compared with the existing HCPs analysis method, the present invention has the following beneficial effects: By introducing HCPs source samples to improve the mass spectrometry signal response of trace samples, the sensitivity is significantly improved, overcoming the ultra-wide dynamic range between drugs and low-abundance protein HCPs; at the same time, the fractionation technology is combined to increase the load of mass spectrometry detection, greatly improving the identification depth of HCPs; MaxQuant software is used to analyze HCPs, and the target-decoy search strategy is used to estimate and control false positives, ensuring the reliability of quantitative results. This method realizes the HCPs analysis of biological protein drugs, which has very important application value for the quality control and detection of HCPs in biological protein pharmaceutical products.

[0035] In the above method, the mass ratio of the cell lysate peptide fragment to the biological protein drug is 1:1, and the ratio value calculated by SCPComponsion is about 114.

[0036] After the above covalent binding and fractionation, the biopharmaceutical protein samples (enzymatically cleaved into monoclonal antibody peptides and HCPs peptides) can be directly injected into the mass spectrometer and analyzed by mass spectrometry following the mobile phase without the need for desalting again, thus reducing the sample loss caused by pre-treatment. Single-column or one-dimensional (1D) mass spectrometry / mass spectrometry with data-dependent acquisition (DDA) has been described for HCP detection in biopharmaceutical products, but to achieve higher sensitivity, two-dimensional reversed-phase liquid chromatography (RPLC) can improve peak capacity and resolution to separate such highly complex peptide samples with a wide dynamic range.

[0037] The application of the above analytical method in the detection of HCPs or standard proteins in biological protein drugs is specifically as follows: 1) Applied to the characterization and identification of residual host cell proteins (HCPs) in biopharmaceutical products.

[0038] More specific applications are: 4.6µL of mAb-1 injection solution was dissolved in 100mM TEAB, reduced and alkylated, and then cleaved into peptides with trypsin, and the peptide samples were covalently bound with reagent labels; the mammalian expression system Chinese hamster ovary cells (CHO-S) were lysed, reduced and alkylated, and then enzymatically digested, and the peptides after enzymatic digestion were covalently bound, and the combined mAb and cell peptides were mixed. More than 1,000 HCPs can be identified in the unfractionated mAb-1 sample; 2) This method was applied to the study of HCPs in the NIST monoclonal antibody (NISTmAb) standard RM8671, and 3475 HCPs were identified with high confidence, which is approximately 4 times the number of HCPs identified previously reported.

[0039] More specifically, NIST monoclonal antibody RM8671 was dissolved in 100 mM TEAB, reduced and alkylated, and then cleaved into peptides using Trypsin enzyme. 19 H 30 The peptide was treated with N4O6; the mouse myeloma cell line NS-1 cells were lysed, reduced and alkylated, and then treated with enzymes to separate the peptide from the C 19 H 30 N4O6 is covalently bound, and the bound monoclonal antibody and cell peptides are mixed for fractionation; 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-level HCPs, and all three proteins were identified without fractionation.

[0040] More specifically, the (yeast alcohol dehydrogenase) ADH1, (yeast enolase) ENO1, (bovine serum albumin) ALB proteins were dissolved in 100 mM TEAB, reduced and alkylated, and then cleaved into peptides with Trypsin. 19 H 30 N4O6 was covalently bound to the peptide; the three proteins were added to mAb-1 at a level of 1 ppm, and after reduction and alkylation, the peptide was treated with Trypsin to bind to C 19 H 30 N4O6 is covalently bonded and the bound peptides are mixed 4) Using the more complex Universal Proteomics Standard 1 (UPS-1) as the HCP model, 17 out of 23 proteins in the range of 0.32-4.15ppm (<1ppm) were confidently identified (≥2 unique peptides), and 24 out of 25 proteins in the range of (>1ppm) were confidently identified (≥2 unique peptides).

[0041] More specifically, UPS-1 was dissolved in 100 mM TEAB, cut into peptides using Trypsin enzyme + Lys-C, and covalently bound to the peptides using reagent labels; UPS-1 in the range of 0.32-4.15 ppm was added to mAb-1, and the peptides were bound to C 19 H 30 N4O6 is covalently bound, and the bound peptides are mixed for fractionation; The present invention is a method for first enzymatically cleaving protein samples into peptide segments and then covalently binding and separating them before using mass spectrometry to analyze and identify biological protein pharmaceutical samples. That is, a method for increasing the MS signal intensity of low-concentration HCPs peptides, and then triggering MS / MS fragmentation by the "total" peptide signal from the mixed sample (or samples) for peptide identification, while the reporter ion provides quantitative information.

[0042] The invention has the following advantages: 1) Overcoming the wide dynamic range (over 6 orders of magnitude) between low ppm levels of HCPs and high levels of therapeutic antibodies, so the detection method has high protein identification sensitivity; 2) Compared with enzyme-linked immunosorbent assay (ELISA), it does not require the use of polyclonal antibodies produced by immunizing animals with null cell lines, can fully cover all HCPs of the production cell line, avoid biased detection of more immunogenic HCPs, and can quickly quantify the relative content of a single HCP component in a monoclonal antibody drug; 3) For the first time, an effective LC-MS / MS strategy was demonstrated, which not only has high sensitivity but also high reliability for HCPs detection, greatly increasing the number of peptide identifications of HCPs. In addition, the sample processing steps in this method are simple and do not require additional desalting treatment, thus reducing sample loss. 4) Use a micro-fractionation device to grade and separate the covalently bound mixed peptides, with almost zero sample loss and effectively increase the sample load. The complex mixed sample is converted into multiple relatively simple samples through high-performance liquid chromatography (HPLC) pre-separation and then LC-MS detection is performed separately, reducing the sample complexity of a single test and increasing the depth of sample analysis.

[0043] The present invention relates to a method for detecting host cell proteins in monoclonal antibody drugs / cell samples based on mass spectrometry, wherein the lysate of the monoclonal antibody drug production cell line is processed into peptides as the source sample of the host cell protein, the signal response of the host cell protein peptides in the monoclonal antibody drug is improved, and the detection of low-abundance host cell proteins in the constant sample is realized. The innovation lies in finding the source of the host cell protein, without the need for additional enrichment of the host cell protein, the source sample of the host cell is processed into peptides and covalently bound to the monoclonal antibody drug, and then mixed and subjected to high pH reverse phase fractionation, and finally mass spectrometry detection. The host cell protein is analyzed by MaxQuant software, and the false positive is controlled based on target-decoy, and the false positive is monitored at the PSM level, and the PSM FDR is within 1%, thereby ensuring the reliability of the identification result. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a flow chart of the technical solution of the present invention.

[0045] Figure 2 The experimental design and effect diagram of the feasibility of this technology. A) is the quantitative quantity of each host cell protein in the three repeated monoclonal antibody samples and source samples provided by the present invention. B) is the sum of the abundance of peptides in each sample.

[0046] Figure 3 Comparison results of the identification depth of the method of the present invention in NIST monoclonal antibodies compared with the prior art. 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.

[0047] Figure 4 The names of the protein standards, protein abbreviations, molecular weights and unique peptide quantities were detected for the three standard proteins at a 1 ppm spike level.

[0048] Figure 5 The sensitivity and reliability of the method of the present invention were investigated. 48 UPS-1 proteins of different concentrations (4.15 ~ 0.32 ppm, sorted from high to low ppm) were detected in mAb1. The pink bar indicates the number of unique peptides identified from the UPS-1 protein, and the green dot indicates the concentration level of the UPS-1 protein. The left ordinate is the unique peptides identified from the UPS-1 protein; the right ordinate is the concentration level of the UPS-1 protein spiked in the monoclonal antibody; the abscissa is the 48 UPS-1 proteins of different concentrations (4.15 ~ 0.32 ppm, sorted from high to low ppm). DETAILED DESCRIPTION

[0049] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0050] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0051] Example 1. Process and feasibility verification of the LC-MS / MS method of the present invention for detecting HCPs in biological protein drugs 1. Preparation of peptides from host cell samples 1.1 Cell culture and treatment: CHO-S cell line (Shanghai Yuchi Biotechnology Co., Ltd.) producing 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, 5% CO2. CHO-S cell line was adapted to suspension culture in chemically defined serum-free medium. NS-1 cells (Shanghai Yuchi Biotechnology Co., Ltd.), the culture cell line of NIST RM8671, were maintained in a humidified atmosphere of 5% CO2 at 37°C. Dulbecco's modified Eagle's medium (DMEM, Gibco, UK) was used for culture, 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 by trypan blue staining. When 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 into a 2 mL centrifuge tube, centrifuged at 1000×g, 4°C for 10 min, discarded the supernatant to obtain the cell pellet, and stored at -80°C or at 4°C for subsequent operations.

[0052] 1.2 Cell lysis: Add the cell pellet obtained in step 1.1 to the lysis buffer (0.1% SDS, 8 M urea, 100 mMTris-HCl, pH 8.0) and resuspend it. Lyse on ice for 15 minutes. Repeatedly blow and aspirate the sample to assist lysis and make the sample suspended. Use an ultrasonic cell disruptor to dissolve, place the lysate in an ice-water mixture, set the power to 100w, perform three times, work for 2s, interval for 2s, and work 99 times. Centrifuge the broken sample at 16000×g, 4℃ for 30 min, then remove it. Take the supernatant as the cell protein extract, use BCA to measure the protein concentration, and store it at -80℃ for later use.

[0053] 1.3 Protein reductive alkylation: 20 mM DTT (dithiothreitol) was added to the cell protein extract and incubated at 56°C for 1 h to reduce the disulfide bonds in the protein. CAA was then added to a final concentration of 40 mM and reacted at room temperature for 45 min to obtain a protein reduction alkylation solution.

[0054] 1.4 SP3 digestion: SP3 (Single-Pot Solid-Phase-enhanced Sample Preparation) enzymatic hydrolysis technology is used to enzymatically hydrolyze proteins. The principle is to non-covalently capture and release proteins through the active groups on magnetic particles (beads), and to concentrate the captured proteins through their magnetism. This can more thoroughly and flexibly remove or replace the different chemical solvents required in each step of the enzymatic hydrolysis operation, and has a very good removal effect on detergents, etc. The specific steps are: Two beads (Cytiva, USA) were mixed at a ratio of 1:1, vortexed lightly for 30 seconds, placed on a magnetic rack, and the supernatant was discarded; 5 times the volume of ultrapure water was added, vortexed lightly for 30 seconds, placed on a magnetic rack, and the supernatant was discarded. Wash three times; finally, a 100µg / µL beads stock solution was prepared with water and stored at 4°C; the beads stock solution was added to the protein reduction alkylation solution at a mass ratio of beads: protein = 10:1, and ACN was added to make the final concentration of the organic solvent reach 80%, inducing the binding of beads and proteins, and the binding was carried out at 37°C, 1500rpm for 18 minutes; The sample was placed on a magnetic rack and allowed to stand for 2 minutes. After the solution was clarified, the supernatant was discarded with a pipette, ACN was added, adsorbed in staggered wells for 30 seconds, the supernatant was discarded, rinsed 3 times, dried at room temperature for 30 seconds, 100 mM TEAB was added, and trypsin was added at a mass ratio of protein: trypsin = 100:1. The reaction was carried out at 37°C for 18 hours for enzyme digestion to obtain host cell protein (HCPs) peptides.

[0055] After the digestion was completed, the supernatant was collected into a new tube by centrifugation at 16000×g for 10 min. The concentration of the collected peptide solution was measured at 280 nm using a NanoDrop 2000C UV spectrophotometer, and 10 μg of peptide was divided into an EP tube and stored at -80°C for later use.

[0056] 2. Monoclonal antibody sample preparation Take 100ug of monoclonal antibody preparation (NIMCmAb, from China Institute of Metrology; NIST RM 8671, Sigma-Aldrich) and dilute it with 100mM TEAB, add 20mM reducing agent DTT, so that the final concentration of monoclonal antibody is 5µg / µL, incubate at 56℃ for 1h to reduce the disulfide bonds in the protein and make the protein denatured completely. Then add CAA to a final concentration of 40mM and react at room temperature for 45min. Then perform SP3 enzyme cleavage according to the steps described in step 1.4 to obtain a solution containing monoclonal antibody peptides.

[0057] 3. Covalent binding of samples to isotope-containing compounds The above two peptides are respectively combined with an isotope-containing compound C containing a group of formula 17 (which can be covalently bonded to the peptide) 19 H 30 N4O6 (Thermo Fisher Scientific - CN) (shown in formula 1 to formula 16, the atoms with "*" in formula 1 to formula 16 are 13 C or 15 N) After covalent bonding to form a covalent bond, they were mixed at a mass ratio of 1:1, and the ratio value calculated using SCPComponsion was approximately 114.

[0058] Specifically, the HCPs peptide obtained in step 1 was placed in a 0.2 mL PCR tube, and the C 19 H 30 N4O6 (dissolved in anhydrous acetonitrile) was added to the PCR tube and mixed and incubated at room temperature for 1.5 h for covalent binding, and then 5% hydroxylamine solution was added and incubated at room temperature for 18 min to quench the covalent binding reaction to obtain HCPs covalently bound peptides; the monoclonal antibody peptide obtained in step 2 was placed in a 0.2 mL PCR tube, and the C 19 H 30 N4O6 (dissolved in anhydrous acetonitrile) was added to the PCR tube, mixed and incubated at room temperature for 1.5 h for covalent binding, and then a 5% hydroxylamine solution was added and incubated at room temperature for 18 min to quench the covalent binding reaction to obtain the monoclonal antibody covalently bound peptide segment; the HCPs covalently bound peptide segment and the monoclonal antibody covalently bound peptide segment were mixed and heat-dried to obtain the (mixed) covalently bound peptide segment for use.

[0059] ; .

[0060] 4. Use a chromatographic column to perform high pH reverse phase fractionation on the mixed covalently bound peptides Using a micro-fractionation device to grade and separate the covalently bound mixed peptides can reduce sample loss and increase sample loading. The complex mixed sample is converted into multiple relatively simple samples through pre-separation (fractionation) of high performance liquid chromatography (HPLC) and then tested by LC-MS separately, which reduces the sample complexity of a single test and increases the depth of sample analysis. Specifically: Dissolve the mixed covalently bound peptides obtained in step 3 in water, add them to the injection vial (injection volume ≤ 10 µL), and collect them according to the following gradient: reverse 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.

[0061] The mobile phases were 98% ammonia (pH 10) plus 2% acetonitrile (mobile phase A) and 98% acetonitrile plus 2% ammonia (pH 10) (mobile phase B) at a flow rate of 2 uL / min. A total of 120 min LC method with a 5 min wash at 3.3% B, 3.3 - 43% B in 68 min, 43 - 56% B in 22 min, 56 - 82% B in 5 min, column wash (5 min, 100% B), 82 - 3.3% B in 5 min, column re-equilibration (10 min, 0% B) step, and the number of cycles was set to 5. Each well contained 300 μL of acetonitrile. Between 5 and 105 min of LC separation time, 40 high pH RP fractions (5 μL each) were combined into 8 fractions (pool 1-pool 8). Fractions 1, 9, 17, 25, and 33 were combined into pool 1; 2, 10, 18, 26, and 34 were pool 2; and so on until fractions 8, 16, 24, 32, and 40 were pool 8. The fractions were dried in a high-speed vacuum concentrator for about 0.5 h to obtain the fractionated heat-dried samples, which were stored at - 80°C for LC-MS / MS analysis.

[0062] The bioprotein pharmaceutical sample (fractionated hot-dried sample) after the above-mentioned covalent binding and fractionation can be directly injected into the mass spectrometer and subjected to mass spectrometry analysis following the mobile phase without the need for re-desalting treatment, thereby reducing sample loss caused by pre-treatment.

[0063] 5. Mass spectrometry detection and data analysis The fractionated heat-dried samples obtained in step 4 were reconstituted with 0.1% formic acid solution. LC-MS / MS analysis was performed on a Q Exactive HF Hybrid quadrupole orbital mass spectrometer (Thermo Fisher Scientific) coupled online to 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 0.1% formic acid in water (mobile phase A, pH ~ 2.7) or 80% acetonitrile containing 0.1% formic acid (mobile phase B). The flow rate was 600 nL / min and the column temperature was 60°C. The LC gradient included: 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. The peptides were fragmented by high-energy collisional dissociation under the settings of 120,000 resolution, 300% normalized automatic gain control (AGC) target, 80 ms maximum injection time for the primary MS scan, 60,000 resolution, 100% normalized automatic gain control (AGC) target, 100 ms maximum injection time, and 0.7 m / z isolation window for the secondary MS scan. The normalized collision energy for each full MS scan was 30%. The peptides were subjected to high-energy collision dissociation fragmentation, and the MS / MS normalized collision energy was 32%.

[0064] LC-MS / MS DDA data files were searched using Maxquant software (version 2.4.2.0) with a SEQUEST search of the database containing sequences of all in-house biotherapeutic products, as well as sequences of all spiked protein standards, and linked to the Cricetulus griseus database (downloaded from UniProt on April 20, 2023, 58,104 entries). NIST raw files were linked directly to the mouse (mus musculus) Swiss-Prot database (October 2023 version; 17,734 entries) from Uniprot.org for HCP analysis in NIST mAbs: The initial maximum precursor mass tolerance was set to 20 ppm in the first search and 4.5 ppm in the main search, and the fragment mass tolerance was set to 10 ppm. Search criteria. The search criteria included static carbamidomethylation of cysteine ​​(+57.0214 Da), reagent tags of lysine (K) and peptide N-termini (+304.207 Da), oxidative modifications of methionine residues (+15.9949 Da) and protein N-terminal acetylation modification (+42.011 Da). Searches were performed with trypsin / P digestion, and a maximum of two missed cleavages were allowed on the peptides analyzed in the sequence database. The false discovery rate for proteins and peptides was set to 0.01.

[0065] The intensity of the obtained HCPs peptides was relatively quantified by the intensity of the peptide signals of the HCPs-derived samples, and then Log10 transformed and standardized. Origin 2021 and GraphPad Prism (Version 9.5) were used for subsequent data analysis.

[0066] 6. Feasibility Analysis of the Method of the Invention As a proof of concept, three samples were prepared to test the feasibility of the method of the present invention for in-depth HCPs identification of research samples ( Figure 2 (A).

[0067] For sample I (mAb-1, NIMCmAb human IgG1κ monoclonal antibody, China National Institute of Metrology), Figure 2 As shown in A, use C shown in formulas 6, 7 and 8 in step 3 19 H 30 N4O6 was covalently bound to 200 ng of monoclonal antibody (mAb-1) peptides to label the samples, and three replicate samples (mAb-1-1, mAb-1-2, and mAb-1-3) with covalently bound monoclonal antibody peptides were combined for LC-MS analysis.

[0068] For sample II, the cell protein peptides (HCPs peptides) harvested from the CHO-S cell culture of the mAb-1 production cell line were mixed with the C 19 H 30 N4O6 is covalently bonded.

[0069] Sample III was a combination of Samples I and II to evaluate the feasibility of using host cell protein source samples to facilitate the identification of HCPs in trace samples of mAb-1.

[0070] For sample I, 10 HCPs were identified due to their low abundance in mAb-1. However, after the carrier was added to the host cell protein source sample, sample III showed HCPs ( Figure 2 The number of HCPs in A (representative of the source sample) increased by at least 100 times ( Figure 2 The results of the present invention show that the intensity of HCPs found in mAb-1 in sample III is reduced compared to sample II, which is the result of the inhibitory effect of mAb-1 with high abundance in the study channel of sample III. Figure 2 The total intensity in each sample in Figure B increased by at least 32-fold, indicating that the use of enhanced samples (HCPs peptides) greatly improved the detection and quantification of HCPs in biopharmaceutical (monoclonal antibody) samples. Figure 2 (B), indicating that using source samples of host cell proteins greatly increases the chances of identifying and quantifying HCPs in trace amounts of biological protein samples.

[0071] Example 2. Using NIST standard antibody RM 8671 to investigate the identification depth of this technology The method of the present invention was applied to the study of HCPs in the NIST monoclonal antibody (NISTmAb) standard RM8671, and 3475 HCPs were identified with high confidence, which is about 4 times the maximum number of HCPs identified reported in the prior art.

[0072] Specifically, the method of the present invention is applied to the peptide spectrum analysis of HCPs in the NIST standard antibody RM 8671. The specific method is as follows: Steps 1 to 5 are the same as in Example 1: NIST monoclonal antibody 8671 is dissolved in 100 mM TEAB, reduced and alkylated, and then cleaved into peptide segments using Trpsin enzyme, and C shown in Formulas 6, 7 and 8 in Example 1 is used to obtain peptide segments. 19 H 30 The monoclonal antibody peptide was treated with N4O6; the mouse myeloma cell line NS-1 cells, which was the production cell line of monoclonal antibody 8671, were lysed, reduced and alkylated, and then treated with enzymes to obtain the peptide using C shown in Formula 16 in Example 1. 19 H 30The peptides after HCPs covalent binding were obtained by N4O6 treatment, and the covalently bound monoclonal antibody peptides and HCPs peptides were mixed in a mass ratio of 1:1 and fractionated; then mass spectrometry detection and data analysis were performed. Since other researchers usually cannot obtain specific biopharmaceutical products for most MS-based HCPs samples, it is almost impossible to directly compare the results of different methods. The key to achieving these goals is to achieve higher identification through the detection of standard substances. NISTmAb is a humanized IgG1κ antibody produced in mouse cell culture and purified by standard biopharmaceutical production methods. It was developed by the National Institute of Standards and Technology (NIST) and can be used as a reference material. The present invention can be compared with recent reports and RM8671 NIST antibody analysis was performed. Recently, Song Nie et al. and Feng Yang et al. developed HCP identification strategies based on the NISTmAb standard and identified 453 HCPs and 746 HCPs, respectively. In the results of the present invention, a total of 3475 HCPs (2851, unique peptides ≥ 2) were identified, and the false positive rate was ≤ 0.01. As Figure 3 China A Institute, and the prior art 1 (Song Nie et al., Figure 3 Nie et al. representative of A) and prior art 2 (Feng Yang et al., Figure 3 Compared with Yang et al. (represented in Figure A), the method of the present invention can identify HCPs without fractionation ( Figure 3 The non-fractionation of A represents the efficiency (protein quantity, Figure 3 The Proteins represented by A in the fractionation method of the present invention is 1.7 times (compared with the prior art 1) or 0.9 times (compared with the prior art 2). Figure 3 Our Results Fractionation represents in A), HCP identification rate (protein number, Figure 3 The protein represented by A in the figure is 7.7 times and 4.1 times higher than the above two existing technologies, respectively, showing the ability of this strategy to deeply cover the HCP map. Figure 3 As shown in B, prior art 1 ( Figure 3 Among the 453 HCPs detected by Nie, S. in B, 328 of them were detected by the prior art 2 ( Figure 3 Among the 848 HCPs detected by Yang, F. (B), 600 were also detected by the method of the present invention ( Figure 3In summary, 2869 mouse HCPs in the NISTmAb standard identified by the method of the present invention were not reported in the previous two studies.

[0073] Therefore, the method of the present invention increases the depth of HCPs identification, and can identify more than 2,000 HCPs separately in addition to repeating the identification of more than 70% of HCPs reported in the prior art in NIST mAb (RM 8671), which promotes LC-MS / MS method to become a more powerful tool for HCP analysis and can be applied to the preparation of products for detecting HCPs in monoclonal antibody drugs.

[0074] Example 3. Detection Limits of Non-Fractionated LC-MS / MS In order to evaluate the detection limit of the method of the present invention, three purified proteins of standard proteins with molecular weights between 36.8 and 66.3 kDa, bovine serum albumin ALB (LABLEAD BIOTECH CO.LTD, #0332), yeast enolase ENO1 (Sigma Aldrich #E6126) and yeast alcohol dehydrogenase ADH1 (SigmaAldrich, #A7011) were added to the purified monoclonal antibody (mAb-1) containing extremely low levels of endogenous HCPs, and the samples were directly prepared in steps 2 and 3 of Example 1, and then analyzed by liquid chromatography-mass spectrometry (LC-MS / MS) in step 5, without using the fractionation step in step 4 of Example 1.

[0075] Specifically, three standard proteins (yeast alcohol dehydrogenase) ADH1, (yeast enolase) ENO1, and (bovine serum albumin) ALB with a size range of 36.8-66.3 kDa were dissolved in 100 mM TEAB, reduced and alkylated, and then cleaved into peptides using Trypsin. 19 H 30 N4O6 treated peptides; added to the purified mAb-1 product at a level of 1 ppm to simulate the presence of low-level HCPs, and after reductive alkylation, cleaved into peptides with Trypsin, and then cleaved with C shown in formulas 2, 3 and 5 in Example 1. 19 H 30 The N4O6-treated peptides were subjected to mass spectrometry detection and data analysis without fractionation, and all three proteins were identified.

[0076] Figure 4The number of peptides of the three standard proteins identified relative to mAb-1 at a peak level of 1 ppm (1 ng / mg) is shown. All other proteins (ADH1, ENO1 and ALB proteins) were identified as having ≥2 unique peptides in the three replicate mAb-1 peptide samples. This shows that the method of the present invention has good repeatability and accuracy. The method of the present invention describes the sensitivity based on the lowest level of spiked protein detection and demonstrates the comprehensiveness or consistency of detection of different proteins at a given level.

[0077] The present invention defines the detection limit as the ability to consistently detect all spiked proteins in each sample (n=3) at and above a specific peak level. Therefore, 1 ppm is considered to be the detection limit for reliable HCPs detection in monoclonal antibody products.

[0078] Example 4: Identification of low-level UPS-1 protein spiked in mAb-1 To further verify the reliability of the method of the present invention for detecting low-level HCPs in biopharmaceutical products, mAb-1 was detected under optimal conditions (8 series components, 15 cm long column, 60 min gradient). 48 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 an HCP model, the present invention added 48 UPS-1 proteins (25 proteins ≥ 1 ppm, 23 proteins < 1 ppm) with a concentration range of 0.32 to 4.15 ppm to mAb-1 to simulate trace HCP contamination. Figure 5 The ppm levels of UPS-1 proteins and the number of unique peptides in mAb-1 are shown. 44 (91.7%) UPS-1 proteins were identified, demonstrating the robustness of the method of the present invention to identify HCPs at sub-ppm levels over a wide molecular weight range (6.3 to 82.9 kDa).

[0079] Specifically, the method in Example 1 of the present invention was used to dissolve UPS-1 in 100 mM TEAB, cut into peptide segments using Trypsin enzyme + Lys-C, and then the C 19 H 30 N4O6 was used to treat the UPS-1 peptide; UPS-1 in the range of 0.32-4.15 ppm was added to mAb-1, and the peptide was treated with C shown in formulas 2, 3 and 5 in Example 1. 19 H 30After N4O6 treatment, the covalently bound peptides were mixed and fractionated; followed by mass spectrometry detection and data analysis.

[0080] These UPS-1 proteins have the same molar concentration; therefore, their peak levels range from 0.32 to 4.15 ppm (25 proteins are present at ≥1 ppm levels and 23 proteins are present at <1 ppm levels). The spike ppm levels and number of peptides identified for each UPS-1 protein at 10 μg target sample load are shown in Figure 2. Figure 5 The results show that Figure 5 As shown, 41 (85.4%) UPS-1 (0.32 to 4.15 ppm) proteins were reliably and consistently identified (≥2 unique peptides), demonstrating robustness in identifying HCPs down to 1 ppm (replicate = 3) across the molecular weight range (6.3 to 82.9 kDa). Consistent and 100% success rate (25 / 25) was achieved in detecting UPS-1 proteins at ≥1 ppm peak levels, and ≥82.6% success rate (19 / 23) was achieved in detecting UPS-1 proteins at <1 ppm levels. This method was reaffirmed to have a high success rate in detecting HCPs <1 ppm in monoclonal antibody drugs. Four unidentified UPS-1 spike proteins ( Figure 5 40, 46, 47, and 48) are all small in size (6–13 kDa) and low in abundance relative to most proteins in the sample (1 < 0.61 ppm; 3 < 0.42 ppm).

[0081] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for detecting or identifying host cell proteins in a target protein product, characterized in that: The method comprises: 1) preparing a mixed covalently bound peptide segment, wherein the mixed covalently bound peptide segment is composed of a target protein covalently bound peptide segment and a host cell protein covalently bound peptide segment, The target protein covalently bound peptide segment is a mixture obtained by covalently binding the peptide segment of the target protein product with compound 1, and the host cell protein covalently bound peptide segment is a mixture obtained by covalently binding the peptide segment of the host cell protein product with compound 2; compound 1 and compound 2 are isotope-containing compounds with the same molecular weight, and the difference between compound 1 and compound 2 is only that the isotope is an atom located at a different position, and both compound 1 and compound 2 contain a group of formula 17 to covalently bind to the peptide segment; ； The peptide segments of the target protein product are a mixture of peptide segments obtained by enzymatic hydrolysis of the target protein product using digestive enzymes; the peptide segments of the host cell protein product are a mixture of peptide segments obtained by enzymatic hydrolysis of the host cell protein product; The target protein product is obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product is obtained by the following steps: culturing the host cells to obtain the culture of the host cells, and crudely purifying the culture of the host cells to obtain the total protein of the host cells; 2) subjecting the mixed covalently bound peptides to liquid phase secondary mass spectrometry detection to determine a target peptide, wherein the target peptide is the target protein covalently bound peptide and the host cell protein covalently bound peptide having the same mass-to-charge ratio, and using mass spectrometry analysis software to analyze the secondary mass spectrometry detection results of the target peptide, and determining the host cell protein contained in the target protein product based on the signal of the compound 1.

2. The method according to claim 1, characterized in that: The mass spectrometry detection step also includes a step of using a high performance liquid chromatography system to perform high pH reverse phase fractionation on the mixed covalently bound peptide segments.

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

1.

4. The method according to any one of claims 1 to 3, 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 any one of claims 1 to 4, 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 comprises the steps of preparing a mixed covalently bound peptide segment, and then subjecting the mixed covalently bound peptide segment to mass spectrometry analysis to improve the detection sensitivity and / or detection depth of host cell proteins in the target protein product; The mixed covalently bound peptide segment is composed of a target protein covalently bound peptide segment and a host cell protein covalently bound peptide segment. The target protein covalently bound peptide segment is a mixture obtained by covalently binding the peptide segment of the target protein product with compound 1, and the host cell protein covalently bound peptide segment is a mixture obtained by covalently binding the peptide segment of the host cell protein product with compound 2; compound 1 and compound 2 are isotope-containing compounds with the same molecular weight, and the difference between compound 1 and compound 2 is only that the isotope is an atom located at a different position, and both compound 1 and compound 2 contain a group of formula 17 to covalently bind to the peptide segment; ； The peptide segments of the target protein product are a mixture of peptide segments obtained by enzymatic hydrolysis of the target protein product using digestive enzymes; the peptide segments of the host cell protein product are a mixture of peptide segments obtained by enzymatic hydrolysis of the host cell protein product; The target protein product is obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product is obtained by the following steps: culturing the host cells to obtain the host cell culture, and crudely purifying the host cell culture to obtain the total protein of the host cells.

7. Use of host cell protein peptides in detecting host cell proteins in target protein products, wherein the host cell protein peptides are a peptide mixture obtained by enzymatic hydrolysis of the host cell protein product; The target protein product is obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product is obtained by the following steps: culturing the host cells to obtain the host cell culture, and crudely purifying the host cell culture to obtain the total protein of the host cells.

8. Application of host cell protein peptides in the preparation of products for detecting host cell proteins in target protein products, The host cell protein peptide segment is a peptide segment mixture obtained by enzymatic hydrolysis of the host cell protein product; The target protein product is obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product is obtained by the following steps: culturing the host cells to obtain the host cell culture, and crudely purifying the host cell culture to obtain the total protein of the host cells.

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 peptide segment is a peptide segment mixture obtained by enzymatic hydrolysis of the host cell protein product; The target protein product is obtained by the following steps: culturing a recombinant cell containing a gene encoding the target protein to obtain a culture of the recombinant cell, crudely purifying the culture of the recombinant cell to obtain a total protein of the recombinant cell, and further purifying the total protein of the recombinant cell to obtain the target protein product; the recombinant cell is a recombinant cell expressing the coding gene obtained by introducing the coding gene into a host cell; The host cell protein product is obtained by the following steps: culturing the host cells to obtain the host cell culture, and crudely purifying the host cell culture to obtain the total protein of the host cells.

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

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