Monoclonal antibody relative molecular mass standard substance and preparation method thereof

Abstract

The application discloses a kind of monoclonal antibody relative molecular mass standard substance and preparation method thereof, including raw material selection, qualitative identification, standard substance valuation and uncertainty evaluation etc..The application realizes accurate valuation of complete molecular weight by high performance liquid chromatography-time of flight mass spectrometry; the sample to be measured is separated by size exclusion chromatography, and ammonium acetate system compatible with mass spectrometry is selected as mobile phase, and ionization detection is carried out by cooperating with lower column temperature and mild ionization mode.The method can maintain the natural folded conformation and non-covalent bond combined spatial stereostructure of monoclonal antibody to the maximum extent on the basis of maintaining the complete structure of monoclonal antibody, and then realize high accuracy and high reliability detection of complete molecular weight of monoclonal antibody.The standard substance of the application can be widely applied to performance confirmation of time of flight mass spectrometry, high resolution mass spectrometry and other instruments, detection method validation, and quality control and value traceability of molecular weight detection results of monoclonal antibody, antibody coupling biological product.

Description

Technical Field

[0001] This invention relates to the field of protein and monoclonal antibody standard reference material determination, quality control and traceability technology, and in particular to a monoclonal antibody relative molecular mass standard reference material and its preparation method. Background Technology

[0002] Monoclonal antibodies (mAbs) are highly specific and homogeneous antibodies produced from a single B cell clone. Monoclonal antibody technology has become an important tool in the field of bioscience and is widely used in the research of biopharmaceuticals.

[0003] Relative molecular mass determination is an indispensable core analytical item in the research, development, production, and quality control of monoclonal antibody drugs. It directly determines the structural integrity, post-translational modification level, batch-to-batch consistency, and the safety and efficacy for clinical use. Compared to small molecule compounds, monoclonal antibodies have highly complex structures. Structural variations such as terminal modifications, glycosylation modifications, deamidation, isomerization, oxidation, fragmentation, and polymerization can all lead to significant changes in relative molecular mass. Therefore, accurate determination of relative molecular mass is an important basis for identifying antibody types, confirming primary structural integrity, assessing post-translational modifications, and controlling product quality.

[0004] Currently, ICH Q6, the European Pharmacopoeia, the United States Pharmacopoeia, and the Chinese Pharmacopoeia all explicitly require that mass spectrometry determination of relative molecular mass and post-translational modification analysis be mandatory tests for the physicochemical characterization of monoclonal antibodies. However, domestic and international pharmacopoeias only provide general technical principles and have not yet established standardized methods and supporting standard substances for the determination of molecular weight by mass spectrometry of monoclonal antibodies. This results in a lack of comparability of test results between different laboratories and different instrument platforms, making it difficult to achieve effective traceability of the values.

[0005] Liquid chromatography-time-of-flight mass spectrometry (LC-TOF MS) has become the mainstream technique for the structural characterization and quality control of monoclonal antibodies due to its advantages such as high resolution, strong specificity, fast analysis speed, and high sensitivity.

[0006] Typically, due to differences in glycosylation levels (especially N-glycosylation in the heavy chain constant region) and amino acid sequences, the relative molecular masses of monoclonal antibodies are mostly distributed in the (145–160) kDa range. Existing protein molecular mass standards are only suitable for quality control, method validation, and instrument performance evaluation of protein systems with molecular weights <100 kDa. They are insufficient to meet the needs of high-charge-number, glycosylated monoclonal antibody drugs in terms of instrument calibration, method validation, and quality control of test results. There is a significant gap in relevant standards and corresponding calibration methods. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, the purpose of this invention is to provide a monoclonal antibody relative molecular mass standard and its preparation method. This invention employs high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS) to achieve accurate measurement, ensuring the reliability, consistency, and traceability of the detection results. The standard prepared by this invention can be widely used for performance validation of instruments such as time-of-flight mass spectrometers and high-resolution mass spectrometers, analytical method validation, and quality control and traceability of molecular weight determination for biological products such as monoclonal antibodies and antibody-drug conjugates (ADCs).

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A method for preparing a monoclonal antibody relative molecular mass standard substance, characterized by comprising the following steps:

[0010] (1) Purification of monoclonal antibody raw materials: Using commercially available monoclonal antibodies as raw materials, the monoclonal antibodies with a purity of not less than 99% are obtained through purification treatment, which can be used as candidates for relative molecular mass standard substances;

[0011] (2) Characterization of the purity of monoclonal antibodies: The purity of the purified monoclonal antibody raw material was characterized by size exclusion chromatography and non-reduced sodium dodecyl sulfate capillary electrophoresis.

[0012] (3) Identification of standard material candidates: The amino acid sequence and disulfide bond linkage of monoclonal antibodies were qualitatively analyzed and identified by mass spectrometry to ensure the structural accuracy and specificity of the candidates;

[0013] Amino acid sequence determination: Monoclonal antibodies were specifically digested with trypsin. The digested peptides were separated and detected by high performance liquid chromatography-time-of-flight mass spectrometry. The mass spectrometry data were analyzed using BioPharmaView software. By comparing the molecular weight of the theoretical digested peptides with the precise mass of the measured peptides, the sequence was found to be consistent with the theoretical sequence.

[0014] Disulfide bond linkage determination: Monoclonal antibodies were non-reductively digested with trypsin, and peptides containing disulfide bonds were collected and analyzed by high performance liquid chromatography-time-of-flight mass spectrometry. The disulfide bond linkage mode was identified using BioPharmaView software, and the measured linkage mode was consistent with the theoretical linkage mode.

[0015] (4) Preparation and dispensing of standard substances: The purified high-purity monoclonal antibody was dissolved in PBS buffer to prepare standard working solutions with a concentration of 0.5 mg / mL to 10 mg / mL. The solutions were dispensed into 200 tubes and stored at -80°C.

[0016] (5) Standard reference determination: The relative molecular mass of monoclonal antibodies was determined by high performance liquid chromatography-time-of-flight mass spectrometry;

[0017] (6) Uncertainty assessment of the determination method: The uncertainty of the relative molecular mass standard of monoclonal antibody determined by high performance liquid chromatography-time-of-flight mass spectrometry is assessed.

[0018] In step (1), a protein purification workstation is used for purification, and the absorbance and elution time are monitored at a wavelength of 280 nm. The eluent within the target elution time period is collected.

[0019] In step (2), the purity of the monoclonal antibody relative molecular mass standard was determined by size exclusion chromatography. The chromatographic column used was an SEC column with specifications of 200 Å or 250 Å, 1.7 μm, and 4.6 mm × 150 mm. The mobile phase A was PBS buffer solution, the mobile phase B was acetonitrile with a volume fraction of 25%, the detection wavelength was 280 nm, the flow rate was 0.2 mL / min, the column temperature was 40℃, and the running time was 20 min.

[0020] In step (2), the purity of the monoclonal antibody raw material is quantitatively determined by non-reduced sodium dodecyl sulfate capillary electrophoresis.

[0021] Sample pretreatment is as follows: Take about 100 μg of the test solution sample, add 85 μL of ultrapure water to mix the sample, then add 5 μL of iodoacetamide aqueous solution and vortex to mix; incubate the test solution at 68℃~72℃ for 10 min, cool to room temperature, centrifuge at 6000 g per minute for 1 min, take 75 μL from the sample tube and put it into the sample bottle for immediate analysis;

[0022] In step (2), the detector used in the non-reduced sodium dodecyl sulfate capillary electrophoresis method is a PDA (220 nm), and the capillary is an uncoated fused silica capillary (50 μm inner diameter). The effective / total length of the capillary is 20 cm / 30.2 cm. Capillary pretreatment: Rinse with 0.1 mol / L sodium hydroxide solution at 70 psi for 3 min, then rinse with 0.1 mol / L hydrochloric acid solution at 70 psi for 2 min, and finally rinse with pure water at 70 psi for 1 min. This should be performed before each run. Capillary pre-filling: Rinse with SDS gel separation buffer at 70 psi for 10 min. This should be performed before each run. Sample injection: 5 kV reverse polarity electrophoresis. Sample injection time: 30 seconds. Separation: 30 min at 15 kV, reverse polarity. Sample chamber temperature: 25℃, capillary temperature: 25℃.

[0023] In the amino acid sequence determination in step (3), the pretreatment method is as follows:

[0024] (1) Desalting: Add 100 μL of ultrapure water to a 10 kD ultrafiltration tube, centrifuge at 11,000 g for 1 min to remove glycerol; add 100 μg of monoclonal antibody sample, centrifuge at 11,000 g for (3-5) min; add 100 μL of ultrapure water and centrifuge to desalt;

[0025] (2) Denaturation: Add 98 μL of 6 M guanidine hydrochloride to fully denature the protein;

[0026] (3) Reduction: Add 2 μL of 500 mM DTT to a final concentration of 10 mM, and react at 42℃ for (0.5-2) h;

[0027] (4) Alkylation: Add 2 μL of 1 M iodoacetamide (IAA) to a final concentration of 20 mM, and react at room temperature in the dark for (30-60) min;

[0028] (5) Solution replacement: Add 100 μL of 50 mM ammonium bicarbonate and centrifuge at 16000 g for (10-20) 15 min. Repeat three times.

[0029] (6) Enzymatic digestion: Redissolve in 100 μL 50 mM ammonium bicarbonate, add trypsin (enzyme:protein = 1:30~1:50), and digest overnight at 37℃;

[0030] (7) Acidification: After centrifugation by inversion, collect the filtrate, add 10 μL of 10% formic acid to acidify it to a final concentration of about 1%, mix well and then test.

[0031] In step (3), the pretreatment method for determining the disulfide bond linkage is as follows:

[0032] (1) Desalting: Add 100 μL of ultrapure water to a 10 kD ultrafiltration tube, centrifuge at 11,000 g for 1 min to remove glycerol; add 100 μg of monoclonal antibody sample, centrifuge at 11,000 g for (3-5) min; add 100 μL of ultrapure water to replace and desalt.

[0033] (2) Free thiol blocking: Add 200 μL 8 M urea and 100 mM Tris (pH 6.5), centrifuge at 11,000 g for (5-15) min; add 200 μL 8 M urea, 100 mM Tris (pH 6.5) and N-ethylmaleimide to a final concentration of 2 mM, and incubate at 37 °C for (1-2) h;

[0034] (3) Solution replacement: Add 100 μL of 50mM ammonium bicarbonate and centrifuge at 16000g for (10-20) min. Repeat three times.

[0035] (4) Enzyme digestion: Redissolve in 100 μL 50 mM ammonium bicarbonate, add trypsin (enzyme: protein = 1:30~1:50), and incubate overnight at 37°C;

[0036] (5) Sample preparation: The enzyme digest was divided into two portions. One portion was acidified with 1% formic acid and used directly for disulfide bond analysis. The other portion was reduced with 10 mM DTT (42℃, 1h), then alkylated with 20 mM iodoacetamide (room temperature, protected from light for 30 min), and finally acidified with 1% formic acid as a control sample.

[0037] In step (3), during the qualitative analysis and identification of the amino acid sequence and disulfide bond linkage of the monoclonal antibody, the injection conditions for high-performance liquid chromatography-time-of-flight mass spectrometry are as follows:

[0038] (1) The high performance liquid chromatography conditions are as follows:

[0039] Chromatographic column: Peptide BEH C18 column, 130 Å, 1.7 μm, 2.1 mm × 100 mm, 1 / pkg, column temperature 30℃, mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile, injection volume 2 μL; the time-of-flight mass spectrometry mobile phase gradient is shown in the table below:

[0040] 0 0.3 98.0 2.0 1.00 0.3 98.0 2.0 20.00 0.3 65.0 35.0 50.0 0.3 55.0 45.0 52.0 0.3 20.0 80.0 55.10 0.3 98.0 2.0 60.0 0.3 98.0 2.0

[0041] (2) Mass spectrometry conditions

[0042] Mass spectrometry conditions: Ion source: ESI source, positive ion scan, IDA mode, TOF MS scan range: (200~2000) Da, TOF MS / MS scan range: (100~2000) Da, DP: 100V, CE: 15V.

[0043] In step (5), the relative molecular mass standard of the monoclonal antibody is determined by high performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS). The determination method is a non-denaturing mass spectrometry method, and the determination result is the relative molecular mass of a single isotope, which is ultimately traced back to the international relative atomic mass. The injection and detection conditions are as follows:

[0044] (1) The high performance liquid chromatography conditions are as follows:

[0045] (a) Chromatographic column: SEC column, 200 Å or 250 Å, 1.7 μm, 4.6 mm × 150 mm;

[0046] (b) Mobile phase: (20~30) mM ammonium acetate solution, isocratic elution, run time 15 min;

[0047] (c) Flow rate: (0.15~0.3) mL / min;

[0048] (d) Column temperature: (20~30)℃;

[0049] (e) Injection volume: Adjusted according to the sample response value, to (2–30) μL.

[0050] (2) The time-of-flight mass spectrometry injection conditions are as follows:

[0051] (a) Ionization mode: Electrospray ionization mode (ESI);

[0052] (b) Scanning mode: Positive ion mode (+);

[0053] (c) Mass spectrometry calibration method: ESI ion source positive ion calibration reagent;

[0054] (d) Acquisition mode: Time-of-flight mass spectrometry full scan (TOF MS);

[0055] (e) Scan quality range: (1000~7000) Da;

[0056] (f) Electrospray voltage (IS): 5500V;

[0057] (g) Ion source temperature (TEM): 650℃;

[0058] (h) Nebulizer gas pressure (GS1): 60 psi;

[0059] (i) Auxiliary gas pressure (GS2): 60 psi;

[0060] (j) Air curtain pressure (GUR): 35 psi

[0061] (k) Declustering voltage (DP): 100V;

[0062] (l) Collision voltage (CE): 7V;

[0063] (m) Ion accumulation time: 1s;

[0064] (n) Number of points for unimodal summation: 80.

[0065] The present invention also provides a monoclonal antibody relative molecular mass standard material prepared by the above preparation method.

[0066] Compared with the prior art, the outstanding effect of the present invention is as follows:

[0067] (1) This invention employs high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS) to achieve accurate determination of the complete molecular weight. The sample to be tested is separated by size exclusion chromatography (SEC), and an ammonium acetate system compatible with mass spectrometry under neutral pH conditions is selected as the mobile phase. Ionization detection is performed using a lower column temperature and a mild ionization mode. This method is based on non-denaturing mass spectrometry, which can maintain the monoclonal antibody's complete structure while maximizing the preservation of its native folded conformation and non-covalently bonded spatial stereostructure, thereby achieving high accuracy and high reliability in detecting the complete molecular weight of the monoclonal antibody.

[0068] (2) The monoclonal antibody relative molecular mass standard material prepared by the present invention can be widely used for performance confirmation of instruments such as time-of-flight mass spectrometry and high-resolution mass spectrometry, verification of detection methodologies, and quality control and traceability of molecular weight detection results of monoclonal antibodies and antibody-drug conjugates.

[0069] The following description, in conjunction with the accompanying drawings and specific embodiments, further illustrates the monoclonal antibody relative molecular mass standard material and its preparation method according to the present invention. Attached Figure Description

[0070] Figure 1 This is the ultra-high performance liquid chromatogram of purified trastuzumab.

[0071] Figure 2 This is an enlarged view of the ultra-high performance liquid chromatogram of purified trastuzumab.

[0072] Figure 3 This is a capillary electrophoresis image of purified trastuzumab sodium dodecyl sulfate.

[0073] Figure 4 This is a graph showing the amino acid coverage of trastuzumab.

[0074] Figure 5 Deconvolution mass spectra for determining the relative molecular weight of bovine serum albumin using high performance liquid chromatography-time-of-flight mass spectrometry.

[0075] Figure 6 Deconvolution mass spectra of the relative molecular weight of NIST 8671 determined by high performance liquid chromatography-time-of-flight mass spectrometry.

[0076] Figure 7 This study describes the unconvolution mass spectrum of a trastuzumab relative molecular mass standard determined by high performance liquid chromatography-time-of-flight mass spectrometry. Detailed Implementation

[0077] The reagent used in this embodiment is:

[0078] GBW(E)100151 Bovine serum albumin relative molecular mass standard material, (66455±32) Da (k=2), National Institute of Metrology, China.

[0079] NIST8671 is a standard developed by the National Institute of Standards and Technology (NIST) in the United States.

[0080] Acetonitrile, Merck GmbH, Germany, chromatographic grade;

[0081] Formic acid, Merck GmbH, Germany, chromatographic grade;

[0082] Trypsin, Promega (USA);

[0083] Dithiothreitol (DTT) is produced by Inalco in the United States.

[0084] Iodoacetamide (IAM), manufactured by Inalco, USA;

[0085] Ammonium acetate, Red Supelco

[0086] Ammonium bicarbonate, Sigma-Aldrich;

[0087] N-Ethylmaleimide, Sigma-Aldrich;

[0088] SDS sample buffer, AB SCIEX

[0089] SDS gel separation buffer, AB SCIEX

[0090] 0.1 mol / L hydrochloric acid solution, AB SCIEX

[0091] 0.1 mol / L sodium hydroxide solution, AB SCIEX

[0092] instrument:

[0093] High-performance liquid chromatography-time-of-flight mass spectrometry, AB SCIEX Q-TOF X500B

[0094] Electronic balance: Sartorius

[0095] High-resolution mass spectrometry and liquid chromatography (nanoliter liquid chromatography), Thermo Fisher Orbitrap Exploris 240

[0096] Ultra-high performance liquid chromatograph, Waters BIO H-CLASS

[0097] High Performance Liquid Chromatography (HPLC) System, Waters Alliance e2695

[0098] Capillary electrophoresis apparatus, AB SCIEX CESI 8000 plus

[0099] Rapid purification liquid chromatography system, GE AKTA avant 25

[0100] Example

[0101] A method for preparing a monoclonal antibody relative molecular mass standard substance (Note: This embodiment only uses trastuzumab as an example for preparation; this method is applicable to other commercially available monoclonal antibodies). The specific steps are as follows:

[0102] I. Purification of Trastuzumab Raw Material

[0103] The GE Avant 25 protein purification workstation was used for the purification and preparation of trastuzumab. A Superdex 200 gel filtration column was used for molecular sieve chromatography separation, with PBS buffer as the elution mobile phase. The protein absorption signal of the chromatographic eluent was monitored in real time at a UV wavelength of 280 nm throughout the process. The main peak of the target protein was accurately identified based on the changes in the UV absorption peak shape. The elution fractions corresponding to the main peaks were collected in segments to complete the separation, purification and enrichment of monoclonal antibodies.

[0104] II. Characterization of the purity of trastuzumab raw material

[0105] 1. Purity analysis of the purified trastuzumab sample was performed using size exclusion chromatography.

[0106] Purity analysis of purified monoclonal antibody samples was performed using size exclusion chromatography. The chromatographic conditions were as follows: a 200 Å, 1.7 μm, 4.6 mm × 150 mm SEC column; mobile phase A was PBS buffer solution (8 g NaCl, 0.2 g KCl, 0.24 g KH2PO4, 1.44 g Na2HPO4, adjusted to pH 7.2, and diluted to 1 L); mobile phase B was acetonitrile. The volume fraction of mobile phase B was optimized by gradient (set to 0%, 5%, 10%, 15%, 20%, and 25%), and the optimal conditions for purity determination were finally determined to be: mobile phase B volume fraction of 25%, detection wavelength of 280 nm, flow rate of 0.2 mL / min, column temperature of 40℃, and run time of 20 min.

[0107] The results are as follows Figure 1 and Figure 2 As shown in the figure. The results indicate that the purity of the monoclonal antibody relative molecular mass standard material used in this embodiment is 99.8%, which is a high purity value.

[0108] 2. The purity of monoclonal antibody raw materials was quantitatively determined using non-reducing sodium dodecyl sulfate capillary electrophoresis.

[0109] (1) Sample pretreatment: Take about 100 μg of the test sample, add 85 μL of ultrapure water to mix the sample, then add 5 μL of iodoacetamide aqueous solution and vortex to mix. Incubate the test sample solution at 72 ℃ for 10 min. After cooling to room temperature, centrifuge at 6000 g per minute for 1 min. Remove from the sample tube and transfer to the injection bottle for immediate analysis.

[0110] (2) Sample introduction method: Non-reduced sodium dodecyl sulfate capillary electrophoresis. Detector: PDA (220 nm), capillary: uncoated fused silica capillary (inner diameter 50 μm), effective capillary length / total length: 20 cm / 30.2 cm. Capillary pretreatment: Rinse with 0.1 mol / L sodium hydroxide solution at 70 psi for 3 min, then rinse with 0.1 mol / L hydrochloric acid solution at 70 psi for 2 min, and finally rinse with pure water at 70 psi for 1 min. This should be done before each run. Capillary pre-filling: Rinse with SDS gel separation buffer at 70 psi for 10 min. This should be done before each run. Sample introduction: 5 kV reverse polarity electrophoresis. Sample introduction time: 30 seconds. Separation: 30 min at 15 kV, reverse polarity. Sample chamber temperature: 25℃, capillary temperature: 25℃.

[0111] The results are as follows Figure 3 As shown in the figure. The results indicate that the purity of the glycosylated monoclonal antibody relative molecular mass standard material used in this embodiment is 95.73%, and the purity of the non-glycosylated monoclonal antibody is 3.32%, with a purity ratio of 99.05% between glycosylated and non-glycosylated monoclonal antibodies.

[0112] III. Identification of Standard Material Candidates: The amino acid sequence and disulfide bond linkage of trastuzumab were qualitatively identified by mass spectrometry.

[0113] 1. Amino acid sequence determination: Trastuzumab was specifically digested with trypsin. The digested peptides were separated and detected by high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS). The mass spectrometry data were analyzed using BioPharmaView software. By comparing the theoretical molecular weight of the digested peptides with the precise mass of the measured peptides, consistency with the theoretical sequence was confirmed. The peptide coverage pretreatment method is as follows:

[0114] (1) Desalting: Add 100 μL of ultrapure water to a 10 kD ultrafiltration tube, centrifuge at 11,000 g for 1 min to remove glycerol; add 100 μg of monoclonal antibody sample, centrifuge at 11,000 g for 3 min; add 100 μL of ultrapure water and centrifuge to desalt;

[0115] (2) Denaturation: Add 98 μL of 6 M guanidine hydrochloride to fully denature the protein;

[0116] (3) Reduction: Add 2 μL of 500 mM DTT to a final concentration of 10 mM, and react at 42℃ for 1 h;

[0117] (4) Alkylation: Add 2 μL of 1 M iodoacetamide (IAA) to a final concentration of 20 mM, and react at room temperature in the dark for 30 min;

[0118] (5) Solution replacement: Add 100 μL of 50 mM ammonium bicarbonate and centrifuge at 16000 g for 15 min. Repeat three times.

[0119] (6) Enzymatic digestion: Redissolve in 100 μL of 50mM ammonium bicarbonate, add trypsin (enzyme:protein = 1:30), and digest overnight at 37°C;

[0120] (7) Acidification: After centrifugation by inversion, collect the filtrate, add 10 μL of 10% formic acid to acidify it to a final concentration of about 1%, mix well and then test.

[0121] 2. Determination of disulfide bond linkage: Trastuzumab was non-reductively digested with trypsin, and peptides containing disulfide bonds were collected and analyzed by high-performance liquid chromatography-time-of-flight mass spectrometry. The disulfide bond linkage mode was identified using BioPharmaView software, and the measured linkage mode was consistent with the theoretical linkage mode. The pretreatment method for disulfide bond linkage is as follows:

[0122] (1) Desalting: Add 100 μL of ultrapure water to a 10 kD ultrafiltration tube, centrifuge at 11,000 g for 1 min to remove glycerol; add 100 μg of monoclonal antibody sample, centrifuge at 11,000 g for 3 min; add 100 μL of ultrapure water to replace and desalt.

[0123] (2) Free thiol blocking: Add 200 μL of 8 M urea and 100 mM Tris (pH 6.5), centrifuge at 11,000 g for 10 min; add 200 μL of 8 M urea, 100 mM Tris (pH 6.5) and N-ethylmaleimide to a final concentration of 2 mM, and incubate at 37 °C for 2 h;

[0124] (3) Solution replacement: Add 100 μL of 50 mM ammonium bicarbonate and centrifuge at 16000 g for 15 min. Repeat three times.

[0125] (4) Enzyme digestion: Redissolve in 100 μL of 50mM ammonium bicarbonate, add trypsin (enzyme:protein = 1:30), and incubate overnight at 37°C;

[0126] (5) Sample preparation: The enzyme digest was divided into two portions. One portion was acidified with 1% formic acid and used directly for disulfide bond analysis. The other portion was reduced with 10 mM DTT (42℃, 1h), then alkylated with 20 mM iodoacetamide (room temperature, protected from light for 30 min), and finally acidified with 1% formic acid as a control sample.

[0127] 3. The conditions for high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS) of amino acid sequences and disulfide bond linkages are as follows:

[0128] (1) High performance liquid chromatography is as follows:

[0129] Chromatographic column: Peptide BEH C18 Column, 130 Å, 1.7 μm, 2.1 mm × 100 mm, 1 / pkg, column temperature 30 °C, mobile phase A: 0.1% formic acid water, mobile phase B: 0.1% formic acid acetonitrile, injection volume 2 μL.

[0130] Table 1. Time-of-flight mass spectrometry mobile phase gradient table

[0131]

[0132] (2) Mass spectrometry conditions:

[0133] Mass spectrometry conditions: Ion source: ESI source, positive ion scan, IDA mode, TOF MS scan range: (200~2000) Da, TOF MS / MS scan range: (100~2000) Da, DP: 100V, CE: 15V.

[0134] The results are as follows Figure 4 As shown in the figure. The sequences of HC1, HC2, LC1, and LC2 are shown in SEQ ID NO:1-4. The results indicate that the monoclonal antibody relative molecular mass standard material used in this embodiment has 100% amino acid sequence coverage, with a total of 16 pairs of disulfide bonds. The disulfide bond linkage is as follows: light chain: Cys23-Cys88, Cys134-Cys194 (2 pairs for each of the two light chains); heavy chain: Cys22-Cys96, Cys147-Cys203, Cys264-Cys324, Cys370-Cys428 (4 pairs for each of the two heavy chains); interchain disulfide bonds: heavy chain-light chain: Cys214-Cys223; heavy chain-heavy chain: Cys229-Cys229, Cys232-Cys232. The disulfide bond linkage is correct.

[0135] IV. Preparation and Dispensing of Standard Substances: The purified high-purity trastuzumab was dissolved in PBS buffer to prepare a standard working solution with a concentration of (0.5–10) mg / mL. The solution was dispensed into 200 tubes and stored at -80℃.

[0136] V. Standard Reference Material Determination: The relative molecular mass of trastuzumab was determined using high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS / MS). This determination method is a non-denaturing mass spectrometry method, and the injection and detection conditions are as follows:

[0137] 1. Establishment of a high-performance liquid chromatography-time-of-flight mass spectrometry method for value determination

[0138] (1) The high performance liquid chromatography conditions are as follows:

[0139] (a) Chromatographic column: SEC Column, 200 Å, 1.7 μm, 4.6 mm × 150 mm;

[0140] (b) Mobile phase: 25 mM ammonium acetate solution, isocratic elution, run time 15 min;

[0141] (c) Flow rate: 0.2 mL / min;

[0142] (d) Column temperature: 30 ℃;

[0143] (e) Injection volume: Adjusted according to the sample response value, to 15 μL.

[0144] (2) The time-of-flight mass spectrometry injection conditions are as follows:

[0145] (a) Ionization mode: Electrospray ionization mode (ESI);

[0146] (b) Scanning mode: Positive ion mode (+);

[0147] (c) Mass spectrometry calibration method: ESI ion source positive ion calibration reagent;

[0148] (d) Acquisition mode: Time-of-flight mass spectrometry full scan (TOF MS);

[0149] (e) Scan quality range: (1000~7000) Da;

[0150] (f) Electrospray voltage (IS): 5500V;

[0151] (g) Ion source temperature (TEM): 650℃;

[0152] (h) Nebulizer gas pressure (GS1): 60 psi;

[0153] (i) Auxiliary gas pressure (GS2): 60 psi;

[0154] (j) Air curtain pressure (GUR): 35 psi

[0155] (k) Declustering voltage (DP): 100V;

[0156] (l) Collision voltage (CE): 7V;

[0157] (m) Ion accumulation time: 1s;

[0158] (n) Number of points for unimodal summation: 80.

[0159] 2. Verification of the correctness of the method

[0160] The accuracy of the method was verified using GBW(E)100151 bovine serum albumin relative molecular mass standard and NIST8671. The average value of the measurement results of GBW(E)100151 bovine serum albumin relative molecular mass standard was within the range of the standard's set values, and the results measured by NIST8671 were consistent with the theoretical values. Therefore, the high-performance liquid chromatography-time-of-flight mass spectrometry method for determining the molecular weight of bovine serum albumin is reliable. Figure 5-6 (As shown). The results are shown in Tables 2 and 3:

[0161] Table 2 Validation results of bovine serum albumin relative molecular weight standard material

[0162]

[0163] Table 3. Validation results of NIST8671 high-performance liquid chromatography-time-of-flight mass spectrometry (Da)

[0164]

[0165] 3. Method repeatability

[0166] The optimized high-performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS) method was used to perform six repeated determinations of the monoclonal antibody sample. The average values ​​obtained were 148061.87 Da, 148221.48 Da, 148381.43 Da, 148535.27 Da, 148674.08 Da, 148825.69 Da, and 148971.52 Da, with RSDs of 0.00070%, 0.00065%, 0.00043%, 0.00048%, 0.00069%, 0.00065%, and 0.00057%, respectively. This indicates that the established method has good repeatability. The relative molecular weight of trastuzumab determined by HPLC-TOF-MS is shown below. Figure 7 As shown.

[0167] Table 4. Results of repeatability determination of relative molecular mass of monoclonal antibodies (Da)

[0168]

[0169] VI. Uncertainty Assessment of the Determination Method

[0170] The uncertainty introduced by high performance liquid chromatography-time-of-flight mass spectrometry in determining the relative molecular mass of monoclonal antibodies comes from Type A uncertainty introduced by repeated measurements and Type B uncertainty introduced by instrument drift.

[0171] 1. Type A standard uncertainty component introduced by repeatability of time-of-flight mass spectrometry measurements

[0172] The average of the six measurements was used as the estimated value. The experimental standard deviation s of the measurement results is shown in Table 5.

[0173] Table 5. Results of repeatability determination of relative molecular mass of trastuzumab (Da)

[0174]

[0175] 2. Type B standard uncertainty component introduced by time-of-flight mass spectrometer drift

[0176] Table 6 shows the drift of the relative molecular mass of the standard substance within 2 hours. Generally, the instrument needs to be recalibrated every 2 hours; therefore, the actual drift during testing is less than the instrument drift over 2 hours. The instrument drift is denoted as... Its half-width is / 2, Assuming uniform distribution, the standard uncertainty components introduced by instrument drift are also listed in Table 8.

[0177] Table 6 Drift values ​​and calculation results

[0178]

[0179] 3. The standard combined uncertainty is

[0180]

[0181] Table 7 Standard uncertainty components and combined standard uncertainty of measurement results

[0182]

[0183] 4. Expanded uncertainty

[0184] Expanded uncertainty according to formula Calculate, taking k=2, then we have:

[0185]

[0186] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a monoclonal antibody relative molecular mass standard substance, characterized in that... Includes the following steps: (1) Purification of monoclonal antibody raw materials: Using commercially available monoclonal antibodies as raw materials, the monoclonal antibodies with a purity of not less than 99% are obtained through purification treatment, which can be used as candidates for relative molecular mass standard substances; (2) Characterization of the purity of monoclonal antibodies: The purity of the purified monoclonal antibody raw material was characterized by size exclusion chromatography and non-reduced sodium dodecyl sulfate capillary electrophoresis. (3) Identification of standard substance candidates: The amino acid sequence and disulfide bond linkage of monoclonal antibodies were qualitatively analyzed and identified by mass spectrometry to ensure the structural accuracy and specificity of the candidates; Amino acid sequence determination: Monoclonal antibodies were specifically digested with trypsin. The digested peptides were separated and detected by high performance liquid chromatography-time-of-flight mass spectrometry. The mass spectrometry data were analyzed using BioPharmaView software. By comparing the molecular weight of the theoretical digested peptides with the precise mass of the measured peptides, the sequence was found to be consistent with the theoretical sequence. Disulfide bond linkage determination: Monoclonal antibodies were non-reductively digested with trypsin, and peptides containing disulfide bonds were collected and analyzed by high performance liquid chromatography-time-of-flight mass spectrometry. The disulfide bond linkage mode was identified using BioPharmaView software, and the measured linkage mode was consistent with the theoretical linkage mode. (4) Preparation and dispensing of standard substances: The purified high-purity monoclonal antibody was dissolved in PBS buffer to prepare standard working solutions with a concentration of 0.5 mg / mL to 10 mg / mL. The solutions were dispensed into 200 tubes and stored at -80℃. (5) Standard reference determination: The relative molecular mass of monoclonal antibodies was determined by high performance liquid chromatography-time-of-flight mass spectrometry; (6) Uncertainty assessment of the determination method: The uncertainty of the relative molecular mass standard of monoclonal antibody determined by high performance liquid chromatography-time-of-flight mass spectrometry is assessed.

2. The method for preparing the monoclonal antibody relative molecular mass standard material according to claim 1, characterized in that: In step (1), a protein purification workstation is used for purification, and the absorbance and elution time are monitored at a wavelength of 280 nm. The eluent within the target elution time period is collected.

3. The method for preparing the monoclonal antibody relative molecular mass standard material according to claim 1, characterized in that: In step (2), the purity of the monoclonal antibody relative molecular mass standard was determined by size exclusion chromatography. The chromatographic column used was an SEC column with specifications of 200 Å or 250 Å, 1.7 μm, and 4.6 mm × 150 mm. The mobile phase A was PBS buffer solution, the mobile phase B was acetonitrile, the volume fraction of mobile phase B was 25%, the detection wavelength was 280 nm, the flow rate was 0.2 mL / min, the column temperature was 40℃, and the running time was 20 min.

4. The method for preparing the monoclonal antibody relative molecular mass standard material according to claim 1, characterized in that: In step (2), the purity of the monoclonal antibody raw material is quantitatively determined by non-reduced sodium dodecyl sulfate capillary electrophoresis. Sample pretreatment was performed as follows: Take about 100 μg of the test solution sample, add 85 μL of ultrapure water to mix the sample, then add 5 μL of iodoacetamide aqueous solution and vortex to mix. Incubate the test solution at 68℃~72℃ for 10 min, cool to room temperature, centrifuge at 6000 g per minute for 1 min, and take 75 μL from the sample tube into the sample vial for immediate analysis.

5. The method for preparing the monoclonal antibody relative molecular mass standard material according to claim 1, characterized in that: In step (2), the detector used in the non-reduced sodium dodecyl sulfate capillary electrophoresis method is a PDA 220nm; the capillary is an uncoated fused silica capillary with an inner diameter of 50μm and an effective / total capillary length of 20 cm / 30.2 cm. The capillary pretreatment involves rinsing with 0.1 mol / L sodium hydroxide solution at 70 psi for 3 min, then rinsing with 0.1 mol / L hydrochloric acid solution at 70 psi for 2 min, and finally rinsing with pure water at 70 psi for 1 min. This process should be performed before each run. The capillary pre-filling involves rinsing with SDS gel separation buffer at 70 psi for 10 min. This process should be performed before each run. Sample injection is performed using a 5kV reverse-phase polarity electric injection system. Sample injection lasts 30 seconds. Separation is performed at 15kV for 30 min using reverse-phase polarity. The sample chamber temperature is 25℃, and the capillary temperature is 25℃.

6. The method for preparing the monoclonal antibody relative molecular mass standard material according to claim 1, characterized in that: In the amino acid sequence determination in step (3), the pretreatment method is as follows: (1) Desalting: Add 100 μL of ultrapure water to a 10 kD ultrafiltration tube, centrifuge at 11,000 g for 1 min to remove glycerol; add 100 μg of monoclonal antibody sample, centrifuge at 11,000 g for 3 min-5 min; add 100 μL of ultrapure water and centrifuge to desalt; (2) Denaturation: Add 98 μL of 6 M guanidine hydrochloride to fully denature the protein; (3) Reduction: Add 2 μL of 500 mM DTT to a final concentration of 10 mM, and react at 42℃ for 0.5 h. -2h； (4) Alkylation: Add 2 μL of 1 M iodoacetamide to a final concentration of 20 mM, and react at room temperature in the dark for 30 min-60 min; (5) Solution replacement: Add 100 μL of 50 mM ammonium bicarbonate and centrifuge at 16000 g for 10-20 min, repeat three times; (6) Enzymatic digestion: Redissolve in 100 μL of 50 mM ammonium bicarbonate, add trypsin, enzyme:protein = 1:30~1:50, and digest overnight at 37℃; (7) Acidification: After centrifugation by inversion, collect the filtrate, add 10 μL of 10% formic acid to acidify it to a final concentration of about 1%, mix well and then test.

7. The method for preparing the monoclonal antibody relative molecular mass standard material according to claim 1, characterized in that: In the determination of disulfide bond linkage in step (3), the pretreatment method is as follows: (1) Desalting: Add 100 μL of ultrapure water to a 10 kD ultrafiltration tube, centrifuge at 11,000 g for 1 min to remove glycerol; add 100 μg of monoclonal antibody sample, centrifuge at 11,000 g for 3 min-5 min; add 100 μL of ultrapure water and centrifuge to desalt; (2) Free thiol blocking: Add 200 μL 8 M urea and 100 mM Tris, centrifuge at 11,000 g for 5-15 min; add 200 μL 8 M urea, 100 mM Tris and N-ethylmaleimide with a final concentration of 2 mM, and incubate at 37℃ for 1-2 h. (3) Solution replacement: Add 100 μL of 50 mM ammonium bicarbonate and centrifuge at 16000 g for 10-20 min. Repeat three times. (4) Enzyme digestion: Redissolve in 100 μL of 50mM ammonium bicarbonate, add trypsin, enzyme:protein = 1:30~1:50, incubate overnight at 37 ℃; (5) Sample preparation: The enzyme digest was divided into two portions. One portion was acidified with 1% formic acid and used directly for disulfide bond analysis. The other portion was reduced with 10 mM DTT at 42℃ for 0.5 h-2 h. Then, alkylation was performed with 20 mM iodoacetamide at room temperature in the dark for 30 min-60 min. Finally, 1% formic acid was added for acidification as a control sample.

8. The method for preparing the monoclonal antibody relative molecular mass standard material according to claim 1, characterized in that: In step (3), during the qualitative analysis and identification of the amino acid sequence and disulfide bond linkage of the monoclonal antibody, the injection conditions for high-performance liquid chromatography-time-of-flight mass spectrometry are as follows: (1) The high performance liquid chromatography conditions are as follows: Chromatographic column: Peptide BEH C18 column, 130 Å, 1.7 μm, 2.1 mm × 100 mm, 1 / pkg, column temperature 30℃, mobile phase A: 0.1% formic acid in water, mobile phase B: 0.1% formic acid in acetonitrile, injection volume 2 μL; the time-of-flight mass spectrometry mobile phase gradient is shown in the table below: (2) Mass spectrometry conditions Mass spectrometry conditions: Ion source: ESI source, positive ion scan, IDA mode, TOF MS scan range: 200 Da~2000 Da, TOFMS / MS scan range: 100 Da~2000 Da, DP: 100 V, CE: 15 V.

9. The method for preparing the monoclonal antibody relative molecular mass standard material according to claim 1, characterized in that: In step (5), the relative molecular mass standard of the monoclonal antibody is determined by high performance liquid chromatography-time-of-flight mass spectrometry (HPLC-TOF-MS). The determination method is a non-denaturing mass spectrometry method, and the determination result is the relative molecular mass of a single isotope, which is ultimately traced back to the international relative atomic mass. The injection and detection conditions are as follows: (1) The high performance liquid chromatography conditions are as follows: (a) Chromatographic column: SEC column, 200 Å or 250 Å, 1.7 μm, 4.6 mm × 150 mm; (b) Mobile phase: 20mM~30mM ammonium acetate solution, isocratic elution, running time 15 min; (c) Flow rate: 0.15 mL / min ~ 0.3 mL / min; (d) Column temperature: 20℃~30℃; (e) Injection volume: Adjusted according to the sample response value, ranging from 2 μL to 30 μL; (2) The time-of-flight mass spectrometry injection conditions are as follows: (a) Ionization mode: Electrospray ionization mode; (b) Scanning mode: positive ion mode; (c) Mass spectrometry calibration method: ESI ion source positive ion calibration reagent; (d) Acquisition mode: Time-of-flight mass spectrometry, full scan, first stage; (e) Scan quality range: 1000 Da ~ 7000 Da; (f) Electrospray voltage: 5500V; (g) Ion source temperature: 650℃; (h) Nebulizer gas pressure: 60 psi; (i) Auxiliary gas pressure: 60 psi; (j) Air curtain pressure: 35psi (k) Declustering voltage: 100V; (l) Collision voltage: 7V; (m) Ion accumulation time: 1s; (n) Number of points for unimodal summation:

80.

10. The monoclonal antibody relative molecular mass standard material prepared by the preparation method according to any one of claims 1-9.

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