Host cell protein qualitative and quantitative analysis method based on liquid chromatography-mass spectrometry and application

Abstract

The application belongs to the field of mass spectrometry, and discloses a high-risk host cell protein qualitative and quantitative analysis method based on multi-level liquid chromatography-mass spectrometry technology. Specifically, the application provides a qualitative analysis and quantitative analysis method and application of high-risk host cell proteins in Chinese hamster ovary cell-derived recombinant protein drugs based on multi-level liquid chromatography-mass spectrometry technology such as data-dependent acquisition, parallel reaction monitoring and multiple reaction monitoring. The analysis target includes 28 high-risk host cell proteins, and the analysis method qualitatively and quantitatively analyzes the target host cell proteins through 47 characteristic peptides and 141 ions. The method provides a Chinese hamster ovary host cell protein detection method with high accuracy and sensitivity, and has high industrial application value.

Description

Technical Field

[0001] This invention relates to the field of mass spectrometry detection, specifically to a method for qualitative and quantitative analysis of host cell proteins in recombinant protein drugs derived from Chinese hamster ovary cells based on multi-level liquid chromatography-mass spectrometry (LC-MS) technologies such as data-dependent acquisition, parallel reaction monitoring, and multiple reaction monitoring. Background Technology

[0002] Host cell proteins (HCPs) refer to non-target protein impurities produced by host cells during the production of protein drugs using cell expression systems. Chinese hamster ovary (CHO) cells are the most commonly used production platform. These cells generate HCPs while expressing therapeutic proteins, and some HCPs may be co-purified with the target product during purification, remaining in the final drug formulation. The presence of high-risk HCPs can adversely affect the safety and stability of drug formulations, such as inducing immune responses, triggering drug degradation, or affecting the stability of the drug formulation. Due to these potential risks, HCP control has been considered a critical quality attribute for monoclonal antibody drugs both domestically and internationally, especially during process development and product release stages, requiring rigorous testing methods to ensure that their residual levels are within acceptable ranges. Enzyme-linked immunosorbent assay (ELISA) is commonly used, but it cannot accurately identify and quantify individual weak or non-immunogenic proteins.

[0003] Liquid chromatography-mass spectrometry (LC-MS) is immunogenicity-independent and enables unbiased detection of hepatocellular carcinomas (HCPs). Its high resolution and sensitivity allow for the effective identification, qualitative and quantitative analysis of different HCP species. Mass spectrometry has become a crucial technique for detecting HCPs and guiding downstream purification processes, analyzing changes in HCPs from harvested cell culture medium to the final product to ensure purification accuracy and safety. However, during protein purification, the dynamic range between high-abundance therapeutic proteins and low-abundance HCPs has expanded to more than 5-6 orders of magnitude. Therefore, a highly specific and sensitive mass spectrometry analysis strategy is needed to overcome the bottleneck in the qualitative and quantitative analysis of low-abundance HCPs.

[0004] Most studies focus on qualitative and quantitative analysis of hepatocellular carcinomas (HCPs) in downstream purified samples of monoclonal antibody drugs using single mass spectrometry techniques, with very few involving analysis of whole-cell proteins expressed by the host cell, easily overlooking many low-abundance HCPs. Currently, there is no integrated mass spectrometry technique that combines data-dependent acquisition, parallel reaction monitoring, and multiple reaction monitoring to provide a qualitative and quantitative analysis method for HCPs based on a comprehensive host cell protein spectrum library.

[0005] Bottom-up preprocessing of whole-cell protein samples is an important strategy in proteomics. This involves extraction, denaturation, reduction of disulfide bonds, and alkylation to stabilize proteins. Subsequently, enzymes such as trypsin are used to cleave the proteins into peptides, and finally, solid-phase extraction is used for desalting and purification. This method is efficient and precise, suitable for high-throughput analysis of complex samples, and can comprehensively identify and quantify proteins. Non-denaturing enzymatic digestion is widely used for the detection of hematologic polymerase chains (HCPs) in antibody drugs. It digests HCPs while preserving the native structure of therapeutic proteins, removing interference from undigested or denatured high-abundance proteins, and improving the detection sensitivity of low-abundance HCPs.

[0006] High-performance liquid chromatography (HPLC) is an important sample separation technique, with reversed-phase HPLC being the most commonly used. It separates peptides based on their hydrophobicity. Different peptides are ionized at different times and generate signals, avoiding co-elution inhibition between high-abundance proteins and low-abundance HCPs, thus significantly improving the sensitivity and quantitative accuracy of HCPs mass spectrometry detection.

[0007] High-resolution mass spectrometry (HMS) offers high resolution and mass accuracy, enabling precise detection of the mass-to-charge ratio of theoretical peptides in complex biological samples. Parallel accumulation-serial fragmentation combines ion mobility with HMS, allowing for ion separation based on collision cross-section, significantly improving detection accuracy and sensitivity. Data-dependent acquisition (DDA) scans the sample precursor ion and selects specific precursor ions for fragmentation, suitable for discovering and identifying unknown proteins. Parallel reaction monitoring (PRM) targets specific precursor ions and monitors their daughter ions for highly specific and sensitive quantitative analysis. Combining multistage mass spectrometry and advanced data acquisition techniques, HMS generates comprehensive datasets from which detailed information about target peptides can be extracted, further supporting in-depth data mining and analysis. Triple quadrupole mass spectrometry offers multiple reaction monitoring (MRM) scanning modes, achieving highly specific quantitative analysis by selectively monitoring ion pairs of specific peptides. Combined with stable isotope labeling technology, triple quadrupole mass spectrometry plays an important role in the quantification of multiple targeted proteins, and is particularly suitable for the accurate quantitative analysis of target HCPs in complex biological samples.

[0008] This invention establishes a qualitative and quantitative analysis method for HCPs based on multi-level liquid chromatography-mass spectrometry (LC-MS) technologies such as DDA, PRM, and MRM, including absolute quantification of 28 high-risk HCPs and their 47 characteristic peptides. This analytical method improves the specificity and sensitivity of monitoring in multiple steps, providing a novel technical approach and strategy for controlling low-abundance HCPs.

[0009] The analytical samples analyzed in this invention are not limited to whole protein extracts and harvested cell culture media from CHO cells, Protein A affinity purification eluents, deep filtration eluents, cation exchange eluents, and stock solutions, but also include samples from the downstream purification processes of all recombinant protein drugs derived from CHO cells. This means the analytical methods have significant reference value and application prospects in the development, process optimization, and quality control of recombinant protein drugs. Summary of the Invention

[0010] The purpose of this invention is to provide a multi-level analysis method based on a data-dependent acquisition, parallel reaction monitoring, and multiple reaction monitoring workflow, which can be used for qualitative and quantitative analysis of high-risk HCPs in CHO cell-expressed drugs.

[0011] In a first aspect of the present invention, a workflow for qualitative and quantitative analysis of HCPs in different samples derived from CHO cells is provided, characterized by comprising the following steps:

[0012] (1) Bottom-up method for protein sample pretreatment;

[0013] (2) Based on liquid chromatography-mass spectrometry (LC-MS) technology, data-dependent acquisition and parallel reaction monitoring of samples were performed to construct a CHO cell full protein spectrum library for qualitative analysis, screening and verification of characteristic peptides and ion pairs of target high-risk HCPs;

[0014] (3) Based on liquid chromatography-mass spectrometry (LC-MS) technology, multiple reaction monitoring of samples was performed to further verify and optimize the characteristic ion pairs of the target high-risk HCPs, generate quantitative ion pairs, and establish a dynamic multiple reaction monitoring mass spectrometry analysis method to perform quantitative analysis on the screened and verified high-risk HCPs.

[0015] (4) Integrate multi-level liquid chromatography-mass spectrometry (LC-MS) technology to establish qualitative and quantitative analysis methods, and apply them to samples in the purification process of monoclonal antibody drugs.

[0016] In a first aspect of the invention, a bottom-up sample pretreatment method is provided, characterized by comprising pretreatment steps of protein denaturation, reduction, alkylation, and enzymatic digestion:

[0017] After taking an appropriate amount of protein sample, denaturation was performed using urea, reduction under dithiothreitol or tricarboxyethylphosphine conditions, and alkylation under iodoacetamide conditions. The sample was then diluted and digested overnight with trypsin at 37°C. Digestion was terminated by adding formic acid, followed by desalting, vacuum drying, and resolvation in water containing 0.1% formic acid.

[0018] In a second aspect of the present invention, a method for data-dependent acquisition and parallel reaction monitoring analysis based on liquid chromatography-mass spectrometry (LC-MS) technology is provided, the LC-MS analysis method comprising:

[0019] (1) Chromatographic conditions: A reversed-phase nanofluid chromatography column was used as the nanofluid chromatography column; the column length was selected as 5-25 cm, the mobile phase A was an aqueous solution, the mobile phase B was an acetonitrile solution, the mobile phase ratio was A+B=100%, the analysis time was 30-180 min, and the mobile phase B was eluted from 0-20% to 60-100% during the analysis time; the mobile phase additive was 0.1-1% formic acid; the flow rate was 100-500 nL / min; the column temperature was 25-50℃; and the injection volume was 1-20 μL.

[0020] Preferably, the chromatographic analysis conditions for the above-mentioned protein compounds include the following: the reversed-phase nanofluid chromatography column has a column length of 15-25 cm; mobile phase A is an aqueous solution; mobile phase B is an acetonitrile solution; the mobile phase ratio is A+B=100%; the analysis time is 90-180 min; and the elution is performed from 0-10% mobile phase B to 60-90% mobile phase B within the analysis time. The mobile phase additive is 0.1-0.5% formic acid; the flow rate is 300-500 nL / min; the column temperature is 25-50℃; and the injection volume is 1-10 μL.

[0021] More preferably, the chromatographic analysis conditions for the above-mentioned protein compounds include a reversed-phase nanofluid chromatography column, preferably 25 cm in length, packed with octadecylsilane-bonded silica gel, mobile phase A being an aqueous solution, mobile phase B being an acetonitrile solution, mobile phase ratio A+B=100%, analysis time of 120-150 min, eluting from 0-5% mobile phase B to 65-85% mobile phase B within the analysis time; mobile phase additive being 0.1% formic acid, flow rate of 300-500 nL / min; column temperature of 50 °C; and injection volume of 1-5 μL.

[0022] The most preferred chromatographic analysis conditions for the above-mentioned protein compounds are as follows: the reversed-phase nanofluid chromatography column is preferably an Omicsolution C18 reversed-phase nanofluid chromatography column (25cm×75μm ID, 1.9μm); mobile phase A is an aqueous solution; mobile phase B is an acetonitrile solution; the mobile phase ratio is A+B=100%; the analysis time is 150 min; mobile phase B elutes from 2% to 37% in the first 0-130 min of analysis; mobile phase B elutes from 37% to 80% in the first 130-148 min of analysis; and mobile phase B is maintained at 80% elution in the first 148-150 min of analysis; the mobile phase additive is 0.1% formic acid; the flow rate is 300 nL / min; the column temperature is 50℃; and the injection volume is 1-5 μL.

[0023] (2) Mass spectrometry conditions: High-resolution ion mobility mass spectrometry was used with a Captive Spray ion source in positive ion detection mode; the mass-to-charge ratio (m / z) range was set to 100-1700; the accumulation and cycle time were set to 100 ms; and the ion mobility scan range was 0.6-1.6 Vs / cm. 2 The overall acquisition cycle time was set to 1.16s, including one Tims-MS full scan and 10 parallel cumulative continuous fragmentation scans.

[0024] In a third aspect of the present invention, a method for multiple reaction monitoring analysis based on liquid chromatography-mass spectrometry (LC-MS) technology is provided, the LC-MS analysis method comprising:

[0025] (1) Chromatographic conditions: High performance liquid chromatography was used with a C18 reversed-phase column with a column length of 5 cm; mobile phase A was an aqueous solution and mobile phase B was an acetonitrile solution; the analysis time was 37 min, and the elution was performed from 0.1% mobile phase B to 37% mobile phase B during the analysis time; the mobile phase additive was 0.1% formic acid; the flow rate was 0.3 mL / min; the column temperature was 50 ℃; and the injection volume was 1-20 μL.

[0026] (2) Mass spectrometry conditions: Triple quadrupole mass spectrometry was used with an electrospray ionization source in positive ion detection mode; drying gas temperature: 180℃; drying gas flow rate: 12L / min; nebulizer gas flow rate: 35psi; sheath gas temperature: 400℃; sheath gas flow rate: 12L / min; capillary voltage: 3000V; nozzle voltage: 300V.

[0027] In a fourth aspect of the invention, a multi-level mass spectrometry integration workflow is provided for the qualitative and quantitative analysis of high-risk HCPs, comprising three stages: (1) HCP detection: mass spectrometry analysis is performed using a data-dependent acquisition mode to construct a CHO cell whole protein spectrum library for qualitative analysis; (2) HCP verification: mass spectrometry analysis is performed using a parallel reaction monitoring mode to screen and verify the characteristic peptides and ion pairs of high-risk HCPs; (3) HCP quantification: mass spectrometry analysis is performed using a multiple reaction monitoring mode to further verify and optimize the characteristic ion pairs, generate quantitative ion pairs, and establish a dynamic multiple reaction monitoring mass spectrometry analysis method to perform quantitative analysis on 28 high-risk HCPs screened and optimized. This method is applied to samples in the monoclonal antibody purification process, including harvested cell culture medium, Protein A affinity purification elution buffer, deep filtration elution buffer, cation exchange elution buffer, and stock solution ( Figure 1 ).

[0028] This analytical strategy is characterized by including the following analytical steps:

[0029] (1) Based on a literature review, 38 high-risk HCPs were collected and used as target proteins for subsequent analysis. They can be divided into six categories according to risk type, as shown in Table 1: drug aggregation; polysorbate degradation; drug fragmentation; immune response; drug modification; and N-glycan degradation.

[0030] Table 1 shows the basic information and risk types of 38 high-risk HCPs obtained from the survey.

[0031]

[0032]

[0033]

[0034] (2) By using the data-dependent acquisition mode of ion mobility high-resolution mass spectrometry, a comprehensive CHO cell protein spectrum library was constructed to cover all potential HCPs, thereby improving the accuracy and sensitivity of identification of low-abundance HCPs.

[0035] (3) By using the parallel reaction monitoring mode of a high-resolution ion mobility mass spectrometer, the characteristic peptides and ion pairs of the target high-risk HCPs were verified, and an ion mobility library was established, containing the charge state, collision cross-sectional area, and ion mobility parameters of all precursor ions identified in the experiment. This ensured the accurate identification of each characteristic peptide and excluded peptides with poor signal quality. The results are as follows: Figure 2 As shown;

[0036] (4) The high-confidence characteristic peptides and ion pairs identified in the above steps were further verified using the multiple reaction monitoring mode of a triple quadrupole mass spectrometer. Collision energy optimization was performed, and a dynamic multiple reaction monitoring method with a 2-minute retention time window was developed. By minimizing ion pair overlap and optimizing the residence time, the detection sensitivity and quantitative accuracy of the target peptides were improved. The results are as follows: Figure 3 As shown;

[0037] (5) After final integration and screening and validation of data-dependent acquisition, parallel reaction monitoring, and multiple reaction monitoring methods, 28 high-risk HCPs, 47 characteristic peptides, and 141 ion pairs were retained, as shown in Table 2. The established dynamic multiple reaction monitoring method was applied to samples from the purification process of 5 monoclonal antibody drugs, using exogenous peptides labeled with heavy isotopes, such as LLIYGATNLADGVPSR*.13 C6 15 N4 labeled with R residues) was used as an internal standard to construct a standard curve for quantitative analysis of target HCPs: the peak area ratio of each peptide after internal standard correction was substituted into the standard curve of the corresponding peptide to calculate the concentration of characteristic peptides in the antibody sample.

[0038] Table 2. Basic information on internal standard peptides and characteristic peptides.

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

[0045] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions.

[0046] The present invention has at least the following beneficial effects:

[0047] This invention establishes a qualitative and quantitative analysis method for high-risk host cell proteins in recombinant protein drugs based on multi-level liquid chromatography-mass spectrometry (LC-MS) technology. The analytical method of this invention provides a highly accurate and sensitive method for detecting host proteins in Chinese hamster ovary cells, and has high industrial application value.

[0048] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0049] Figure 1A strategy diagram for qualitative and quantitative analysis of high-risk HCPs based on multi-level liquid chromatography-mass spectrometry (LC-MS) technologies, including data-dependent acquisition, parallel reaction monitoring (PRM), and multiple reaction monitoring (MRM): Phase 1: Establish a CHO cell whole-protein map library using the data-dependent acquisition mode for qualitative analysis, including sample preparation, mass spectrometry data acquisition, and library construction using PEAKS Studio software; Phase 2: Screen and validate characteristic peptides and ion pairs of high-risk HCPs using the parallel reaction monitoring mode, and further validate and optimize them using the multiple reaction monitoring mode to generate quantitative ion pairs; Phase 3: Establish a dynamic multiple reaction monitoring method to quantitatively analyze 28 high-risk HCPs selected and optimized, and apply it to the detection of samples in the monoclonal antibody purification process. Figure 2 A schematic diagram of the optimization and validation process for target protein characteristic peptides and ion pairs: taking the characteristic peptide QITINDLPVGR as an example.

[0050] Figure 3Chromatograms of extracts from characteristic peptides of 28 high-risk HCPs using dynamic multiple reaction monitoring: 1, VHNTEPVESAR; 2, SGEGC [+57.021464]VITR; 3, SC [+57.021464]EAGYSPSYK; 4, LQDAEIAR; 5, ELTIGSK; 6, IHFSGTESDK; 7, VLFTALNYGLK; 8, VGDYGSLSGR; 9, HEAGDIMGGHAIR; 10, FNQC [+57.021464]GTC [+57.021464]TEF K;11,GLDSEESYPYEAK;12,SQLEDVFLR;13,LVLVGDGGTGK;14,STC[+57.021464]LYGQLPK;15,FLTESHDR;16,ITLDNAYMEK;17,S EDYVDIVQGR;18,ELDLVSHHVR;19,VSSLPSVTLK;20,ILLADQGQSWK;21,LFVVPADEAQAR;22,QPGITFIAAK;23,SEIDLYNIR;24,SVL LDAASGQLR;25,GTVTAFPGFDSR;26,QITINDLPVGR;27,VTAGISFAIPSDK;28,GAEWHSHLGSMK;29,LTQQYNELLHSLQTK;30,IVIVPS LNPDGR; 31, THILLFLPK; 32, ESLDVFELDPK; 33, QGGLGPMMNIPLVSDPK; 34, IYVDDGLISLQVK; 35, WLSTHVC[+57.021464]NR; 36, HNQ LPLVIEFTEQTAPK;37,FLVEYIAPMTEK;38,ELAVAAAYQSVR;39,MYNEIVNLLR;40,FEELNMDLFR;41,TFAPEEISAMVLTK;42,LVQAQYW HDPIK;43,DYGVLLEGAGIALR;44,GLFIIDPNGVIK;45,DPEC[+57.021464]VNNLLEVSR;46,VIPEFDTPGHTQSWGK;47,NILDIITSLK.

[0051] Figure 4 Extraction chromatogram of high-risk HCPs from harvested cell culture medium samples using multiple reaction monitoring: 1,

[0052] FLTESHDR;2,IHFSGTESDK;3,LQDAEIAR;4,VGDYGSLSGR;5,

[0053] ELDLVSHHVR;6,LVLVGDGGTGK;7,ITLDNAYMEK;8,

[0054] SVLLDAASGQLR;9,QPGITFIAAK;10,QITINDLPVGR;11,

[0055] DPEC[+57.021464]VNNLLEVSR;12,LTQQYNELLHSLQTK;13,

[0056] FEELNMDLFR;14,GLFIIDPNGVIK.

[0057] Figure 5 Chromatograms of high-risk HCPs extracted from Protein A affinity purification eluent samples using dynamic multiple reaction monitoring: 1, FLTESHDR; 2, SVLLDAASGQLR; 3, QPGITFIAAK; 4, DPEC[+57.021464]VNNLLEVSR; 5, ESLDVFELDPK.

[0058] Figure 6 Chromatograms of high-risk HCPs extracted by dynamic multiple reaction monitoring in deep filtration eluent samples: 1, SVLLDAASGQLR; 2, QPGITFIAAK; 3, DPEC[+57.021464]VNNLLEVSR; 4, ESLDVFELDPK.

[0059] Figure 7 Chromatograms of high-risk HCPs extracted by dynamic multiple reaction monitoring in cation exchange eluent samples: 1, SVLLDAASGQLR; 2, DPEC[+57.021464]VNNLLEVSR.

[0060] Figure 8 Chromatograms of high-risk HCPs extracted from the stock solution sample under dynamic multiple reaction monitoring: 1, ELAVAAAYQSVR; 2, SVLLDAASGQLR; 3, DPEC[+57.021464]VNNLLEVSR. Detailed Implementation

[0061] Through extensive and in-depth research and screening, the inventors have developed for the first time a qualitative and quantitative analysis method for high-risk HCPs in CHO cell-derived recombinant protein drugs based on multi-level liquid chromatography-mass spectrometry (LC-MS) technologies, including data-dependent acquisition, parallel reaction monitoring, and multiple reaction monitoring. The method includes an analytical procedure, pretreatment conditions, chromatographic conditions, mass spectrometry conditions, and data processing and analysis methods. This method not only simplifies the operation but also enables simultaneous qualitative and quantitative analysis of 28 high-risk HCPs in a single test.

[0062] The present invention will now be described in further detail with reference to the accompanying drawings and examples, so that those skilled in the art can implement it based on the description.

[0063] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0064] Example 1: Qualitative and quantitative analysis of high-risk HCPs in samples from the CD20 monoclonal antibody drug purification process

[0065] I. Experimental Apparatus, Controls and Reagents

[0066] The experimental instruments included a Bruker nanoElute nanofluid chromatography system tandem with a Tims TOF Pro ion mobility high-resolution mass spectrometer (Bruker Tims TOF Pro nanoLC-MS / MS system, Bruker Daltonics, Germany), equipped with a high-pressure pump and autosampler, and a Captive Spray source for the MS system, along with Compass Data Analysis 5.3 data processing system; an Agilent 1290 liquid chromatography system tandem with a triple quadrupole mass spectrometer (Agilent 6490QQQ LC-MS / MS system, Agilent Technologies, Santa Clara, CA, USA), equipped with a high-pressure pump and autosampler, and an AJS-ESI source for the MS system, along with MassHunter 8.0 data processing system; Skyline 23.1 software from MacCoss Labs, USA; and PEAKS studio. Software 10.6, BSI (Canada); Thermostatic water bath, Shanghai Lichen Bangxi Instrument Technology Co., Ltd.; High-speed centrifuge, Eppendorf (USA); Vortex mixer, Scientific Industry; METLER TOLEDO AG135 electronic balance, Mettler AG (Switzerland); OasisHLB solid-phase extraction column, Waters Corporation (USA); NanoDrop One micro spectrophotometer, Thermo Fisher Scientific (USA).

[0067] Furthermore, the samples and reagents included the CHO-K1 cell line (Peking Union Medical College); CD20 monoclonal antibody harvested cell culture medium, Protein A affinity purification elution buffer, deep filtration elution buffer, cation exchange elution buffer, and stock solution (China National Institutes for Food and Drug Control); deionized water (Hangzhou Wahaha Group Co., Ltd.); methanol, formic acid, acetonitrile, Trypsin / Lys-C enzyme (mass spectrometry grade, Thermo Fisher Scientific, USA); tricarboxyethylphosphine, iodoacetamide, sodium deoxycholate, and Tris-HCl buffer (Sigma-Aldrich, USA); RIPA lysis buffer (Solepro); protease inhibitors and phosphatase inhibitors (Shanghai Taoshu Biotechnology Co., Ltd.); and a BCA protein concentration assay kit (Thermo Fisher Scientific, USA).

[0068] II. Sample Pretreatment

[0069] A 100 μg protein sample was taken and diluted to a final concentration of 50 mM Tris-HCl buffer with water and Tris / HCl buffer (pH 8.0). The sample was then reduced with 10 mM tricarboxyethylphosphine at 95 °C for 15 min. Subsequently, alkylation was performed with 20 mM iodoacetamide at room temperature in the dark for 30 min. The sample was diluted 4-fold with LC-MS grade water and digested overnight at 37 °C using trypsin / Lys-C (enzyme to protein ratio 1:50, w / w). Digestion was terminated by adding 0.5% formic acid. The sample was purified and desalted using Oasis HLB solid-phase extraction, eluted with 1 mL of 50% acetonitrile, vacuum dried, and redissolved in 100 μL of 0.1% formic acid water. The injection concentration was measured using a NanoDrop One micro-spectrophotometer.

[0070] III. Chromatographic Conditions

[0071] Nano-fluid chromatography: nanoElute liquid chromatograph; Column: C18 reversed-phase column (25cm×75μm ID, 1.9μm, C18, Omicsolution); Mobile phase: Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid; Analysis time: 150 min; During the analysis time 0-130 min, mobile phase B was gradually eluted from 2% to 37%; During the analysis time 130-148 min, mobile phase B was gradually eluted from 37% to 80%; During the analysis time 148-150 min, mobile phase B was maintained at 80% elution; Flow rate: 300 nL / min; Column temperature: 50℃; Injection volume: 1 μL.

[0072] High-performance liquid chromatography: Agilent 1290 high-performance liquid chromatograph; Column: Agilent Zorbax RRHDEclipse Plus C18 column (2.1×50mm, 1.8μm, Mobile phase: Mobile phase A is an aqueous solution containing 0.1% formic acid, and mobile phase B is an acetonitrile solution containing 0.1% formic acid; Analysis time is 37 min, during which the solution is eluted from 0.1% mobile phase B to 37% mobile phase B at a flow rate of 0.3 mL / min; Column temperature: 50℃; Injection tray temperature: 4℃; Injection volume is 10 μL.

[0073] IV. Mass Spectrometry Conditions

[0074] High-resolution mass spectrometry of ion mobility: Bruker Tims TOF Pro high-resolution mass spectrometer of ion mobility;

[0075] CaptiveSpray source parameters: positive ion detection mode; mass-to-charge ratio (m / z) range: 100-1700; accumulation and cycle time: 100 ms; ion mobility scan range: 0.6 -1.6 Vs / cm 2 Overall acquisition cycle time: 1.16s, including one Tims-MS full scan and 10 parallel cumulative continuous fragmentation scans.

[0076] Triple quadrupole mass spectrometer: Agilent 6490 triple tandem quadrupole mass spectrometer; AJS ESI source parameters: positive ion detection mode; drying gas temperature: 180℃; drying gas flow rate: 12L / min; nebulizer gas flow rate: 35psi; sheath gas temperature: 400℃; sheath gas flow rate: 12L / min; capillary voltage: 3000V; auxiliary nozzle voltage: 300V.

[0077] V. Data Processing and Analysis

[0078] Whole protein samples from CHO cells were analyzed using data-dependent acquisition mode on high-resolution ion mobility mass spectrometry (HMAC). The acquired mass spectrometry data were imported into PEAKS Studio software for library search and identification. Database: UniProt Cricetulusgriseus CHO K1 (UP000001075); Enzyme: Trypsin; Maximum missed cleavage sites: 2; Precursor ion mass error: 10 ppm; Fragment ion mass error: 0.05 Da; Fixed modification: carbamide methylation (cysteine); Variable modification: methionine oxidation, acetylation (protein N-terminus); False detection rate: 1%; Minimum number of characteristic peptides: 2. The constructed spectral library identified 6,845 proteins and 142,652 peptides. The results were exported in pepXML format and then imported into Skyline software to generate a library. In Skyline software, 38 high-risk HCPs (as shown in Table 1) were imported as target proteins, and their characteristic peptides and ion pairs were predicted.

[0079] Parallel reaction monitoring mode was used on high-resolution ion mobility mass spectrometry to verify the characteristic peptides and ion pairs of the target high-risk HCPs. The chromatograms of each peptide were manually checked, and an ion mobility library was established, which included the charge state, collision cross-sectional area value and ion mobility parameters of all precursor ions identified in the experiment. This ensured the accurate identification of each characteristic peptide, excluded peptides with poor signal quality, corrected errors in peak selection or integration boundaries, and focused on peptides with dotp and idotp values ​​higher than 0.6.

[0080] Multiple reaction monitoring (MRM) methods were established using triple quadrupole mass spectrometry to further validate target characteristic peptides and ion pairs, and collision energies were optimized. After screening and validation using DDA, PRM, and MRM methods, 28 high-risk HCPs, 47 characteristic peptides, and 141 ion pairs were retained, as shown in Table 2. Dynamic multiple reaction monitoring methods were then developed for each of the 28 high-risk HCPs, and the methodology was validated and applied.

[0081] VI. Methodological Validation

[0082] Twenty-eight synthetic peptides were purchased as standards and dissolved in a 50:50 acetonitrile / water solution to prepare a 10 μmol / L standard stock solution. Heavy isotope-labeled exogenous peptides were also used.

[0083] LLIYGATNLADGVPSR*( 13 C6 15An internal standard stock solution of 10 μmol / L was prepared using the same method (N4 labeled on R residues). IgG without target HCPs was used as the matrix and enzymatically digested according to the sample pretreatment method to obtain a matrix hydrolysate with a concentration of 10 mg / mL. The internal standard stock solution was added to the matrix hydrolysate to achieve a final concentration of 20 nmol / L. Standard curves were prepared by serial dilution of the standard stock solution to the matrix hydrolysate containing the internal standard to the following concentrations: 0.1, 0.2, 0.4, 0.5, 1, 2, 2.5, 5, 10, 20, 25, 50, 75, and 100 nmol / L. Quality control (QC) peptides were prepared by serial dilution of individual stock solutions to concentrations of LLOQ, twice the LLOQ, the intermediate concentration of the standard curve, and 80% of the upper limit of the standard curve range.

[0084] Method validation was conducted in accordance with the guidelines for validation of quantitative analysis methods for biological samples in the Pharmacopoeia of the People's Republic of China (2020 edition). The parameters evaluated included standard curve, accuracy, precision, residues, matrix effects, and stability.

[0085] The calibration curve is obtained by fitting the relationship between the peak area ratio of the analyte to the internal standard and the corresponding concentration using a least-squares linear regression, with 1 / X. 2 R is derived from the weighting factors. 2 >0.98. The concentration calculated from the calibration standard should be within ±15% of the labeled value, and the lower limit of quantitation should be within ±20%, containing at least 6 effective concentrations.

[0086] The standard curves for the 28 quantitative characteristic peptides are shown in Table 3 below, and the corresponding proteins for the characteristic peptides in the table are shown in Table 2.

[0087] Table 3 Standard curves for 28 characteristic peptides used for quantification

[0088]

[0089]

[0090] The accuracy (relative error, RE) of an analytical method describes how close the measured value is to the labeled concentration of the analyte, expressed as: measured value / labeled value × 100%. The precision (relative standard deviation, RSD) of an analytical method describes how close the repeatability of analyte determinations is. Precision is obtained within the same batch and between different batches using the same batch of samples used to demonstrate accuracy. Quality control samples were analyzed according to standard curves, and accuracy and precision were evaluated using single and different batches (at least 2 days), with 6 samples measured for each concentration. The intra-batch (n=6) accuracy for the 28 characteristic peptides was 89.72%–113.37%, and the precision was 1.12–10.74; the inter-batch (n=18) accuracy was 90.58%–112.86%, and the precision was 2.67%–12.66%. The standard curves for the 28 peptides are shown in Table 4 below, all of which meet the requirements for biological sample determination in the Chinese Pharmacopoeia.

[0091] Table 4. Accuracy and Precision of 28 Characteristic Peptides Used for Quantification

[0092]

[0093]

[0094]

[0095]

[0096]

[0097] Residues were evaluated by injecting a blank sample after injecting a high-concentration sample or calibration standard. Residues in the blank sample following the high-concentration sample did not exceed 20% of the lower limit of quantitation and 5% of the internal standard. Residues met the requirements for biological sample determination in the Chinese Pharmacopoeia.

[0098] For quality control samples, the ratio of the peak area of ​​the analyte in the presence of the matrix to the corresponding peak area without the matrix was calculated. The peak areas of the analyte and internal standard were then compared with the corresponding matrix-free samples prepared with blank solvent, and the matrix factor corrected by the internal standard was calculated. The matrix factors corrected by the internal standard for the 28 characteristic peptides ranged from 85.06% to 113.53%, with RSDs ranging from 3.26% to 13.67%. The matrix effects of the 28 peptides are shown in Table 5 below, all of which meet the requirements for biological sample determination in the Chinese Pharmacopoeia.

[0099] Table 5. Matrix effects of 28 characteristic peptides used for quantification

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] For quality control samples, stability was assessed after short-term storage at room temperature for 24 hours, repeated freeze-thaw cycles at -80℃ three times, and long-term storage at 4℃ for 7 days. Each sample was injected six times (n=6), and RSD and RE were calculated. The RE for short-term storage at room temperature for 24 hours was 90.76%–113.60%, and the RSD was 0.34%–12.45%; the RE for repeated freeze-thaw cycles at -80℃ three times was 87.76%–113.45%, and the RSD was 1.13%–12.41%; the RE for long-term storage at 4℃ for 7 days was 87.35%–112.36%, and the RSD was 1.39%–14.70%. The stability of the 28 peptides is shown in Table 6 below, and all meet the requirements for biological sample determination in the Chinese Pharmacopoeia.

[0106] Table 6. Stability of 28 characteristic peptides used for quantification

[0107]

[0108]

[0109]

[0110]

[0111]

[0112] VII. Application of the Method

[0113] Dilute 2 mg of antibody sample to a protein concentration of 5 g / L with water and 1 M Tris / HCl buffer (pH 8.0), and then digest the sample with Trypsin / Lys-C at an enzyme-to-protein ratio of 1:2000 (w / w) at 37 °C for 2 hours. Then, reduce the sample by adding 0.9% sodium deoxycholate (SDC) and 11 mM TCEP, and heat at 90 °C for 10 minutes. After cooling to room temperature, acidify the sample to pH <2 with 7% formic acid (FA) to terminate digestion and precipitate SDC. The SDC precipitate, along with undigested and denatured proteins, is precipitated by centrifugation at 20,000 g for 30 minutes at 4 °C. Transfer the supernatant to a new 1.5 mL low-binding microcentrifuge tube and centrifuge at 20,000 g for 10 minutes at 4 °C. Finally, the supernatant was purified using an Oasis HLB column, eluted with 1 mL of 50% ACN solution, dried under vacuum, and redissolved in 100 μL of 0.1% formic acid water containing 40 nmol / L internal standard.

[0114] The established dynamic multiple reaction monitoring (MRM) method was applied to monitor 28 high-risk hepatocellular carcinomas (HCPs) in CD20 monoclonal antibody samples. A standard curve was constructed using synthetic peptide standards to perform absolute quantitative analysis of the high-risk HCPs, and the concentration of each peptide in the sample was calculated (n=3). The results are shown in Table 7. Figure 4 As shown, the peptides identified in the harvested cell culture medium samples are: FLTESHDR, IHFSGTESDK, LQDAEIAR, VGDYGSLSGR, ELDLVSHHVR, LVLVGDGGTGK, ITLDNAYMEK, SVLLDAASGQLR, QPGITFIAAK, QITINDLPVGR, DPEC[+57.021464]VNNLLEVSR, LTQQYNELLHSLQTK, FEELNMDLFR, GLFIIDPNGVIK; Figure 5 As shown, the peptides identified in the Protein A affinity purification eluent sample were: FLTESHDR, SVLLDAASGQLR, QPGITFIAAK, DPEC[+57.021464]VNNLLEVSR, ESLDVFELDPK; Figure 6 As shown, the peptides identified in the deep filtration eluent sample are: SVLLDAASGQLR, QPGITFIAAK, DPEC[+57.021464]VNNLLEVSR, ESLDVFELDPK; (The text abruptly ends here, so the translation stops as well.) Figure 7 As shown, the peptides identified in the cation exchange eluent sample were: SVLLDAASGQLR, DPEC[+57.021464]VNNLLEVSR; Figure 8 As shown, the peptides identified in the original sample are: ELAVAAAYQSVR, SVLLDAASGQLR, and DPEC[+57.021464]VNNLLEVSR.

[0115] Table 7 High-risk HCPs detected in antibody samples (n=3)

[0116]

[0117]

[0118] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. After reading the above teachings of the present invention, those skilled in the art can make various modifications or alterations to the present invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for qualitative and quantitative analysis of high-risk host cell proteins in Chinese hamster ovary host cells based on multi-level liquid chromatography-mass spectrometry (LC-MS / MS), characterized in that, The analytical method includes the following steps: (1) sample preparation and pretreatment; (2) chromatographic detection; (3) multi-level mass spectrometry detection; (4) constructing a complete protein spectrum library of Chinese hamster ovary cells and qualitatively identifying and quantitatively analyzing HCP protein.

2. The method as described in claim 1, characterized in that, By employing multi-level liquid chromatography-mass spectrometry (LC-MS) technology, integrating mass spectrometry techniques such as data-dependent acquisition, parallel reaction monitoring, and multiple reaction monitoring, a complete protein spectrum library of Chinese hamster ovary cells was obtained. Host cell proteins were qualitatively and quantitatively analyzed, and 28 high-risk host cell proteins were quantitatively analyzed using the standard curve method.

3. The method as described in claim 1, characterized in that, The samples analyzed include, but are not limited to, samples from the downstream purification process of recombinant protein drugs, such as CHO cell whole protein extract, harvested cell culture medium, Protein A affinity purification elution buffer, deep filtration elution buffer, cation exchange elution buffer, and stock solution.

4. The method as described in claim 3, characterized in that, The analyzed samples were derived from CHO cells, including all biotherapeutic drugs derived from Chinese hamster ovary cells and samples of their purification processes.

5. The method as described in claim 1, characterized in that, The sample pretreatment method was the classic bottom-up protein digestion method.

6. The method as described in claim 1, characterized in that, This includes nanofluid chromatography and high-performance liquid chromatography (HPLC). The nanofluid chromatography conditions were set as follows: a 25 cm C18 reversed-phase column was used; mobile phase A was an aqueous solution, mobile phase B was an acetonitrile solution, the analysis time was 150 min, with mobile phase B eluting from 2% to 37% in a gradient elution from 130 to 148 min, and then maintaining 80% elution from 148 to 150 min; the mobile phase additive was 0.1% formic acid; the flow rate was 300 nL / min; the column temperature was 50 °C; and the injection volume was 1-20 μL. The high-performance liquid chromatography (HPLC) conditions were set as follows: a C18 reversed-phase column with a column length of 5 cm was used; mobile phase A was an aqueous solution, mobile phase B was an acetonitrile solution, the analysis time was 37 min, and during the analysis time, the solution was eluted from 0.1% mobile phase B to 37% mobile phase B using a gradient elution; the mobile phase additive was 0.1% formic acid; the flow rate was 0.3 mL / min; the column temperature was 50 ℃; and the injection volume was 1-20 μL.

7. The method as described in claim 1, characterized in that, This includes high-resolution mass spectrometry (HMS) of ion mobility and triple quadrupole mass spectrometry (TQMS). The parameters for HMS of ion mobility were set as follows: Captive Spray ion source, positive ion detection mode; mass-to-charge ratio (m / z) range of 100-1700; accumulation and cycle time of 100 ms; and ion mobility scan range of 0.6-1.6 Vs / cm. 2 The overall acquisition cycle time was set to 1.16 s, including one Tims-MS full scan and 10 parallel cumulative fragmentation scans. Triple quadrupole mass spectrometry parameters were set as follows: electrospray ionization source, positive ion detection mode; drying gas temperature: 180℃; drying gas flow rate: 12 L / min; nebulizer gas flow rate: 35 psi; sheath gas temperature: 400℃; sheath gas flow rate: 12 L / min; capillary voltage: 3000 V; nozzle voltage: 300 V.

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