Liquid chromatographic analysis method for detecting deamidation and isomerization proportions of antibody and application of liquid chromatographic analysis method

Abstract

The invention relates to a liquid chromatographic analysis method for detecting deamidation and isomerization proportions of an antibody and application of the liquid chromatographic analysis method. The analysis method comprises the following steps: performing denaturation reduction on a sample to be detected, detecting the concentration, performing enzyme digestion, detecting by adopting liquid chromatography, and calculating the deamidation proportion. According to the method disclosed by the invention, the target peptide fragment is obtained by cutting the antibody coupling medicine through trypsin, and separation is carried out through ultra-high performance liquid chromatography within 40 minutes, so that a chromatogram with good reproducibility is obtained. The method can accurately quantify the deamidation and isomerization proportions in the sample, and effectively overcomes the problems of dependence on mass spectrum, long analysis time, spectrum peak drift and large peak shape change in the traditional method. The method is high in accuracy and suitable for high-throughput molecular screening.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical analysis and detection, and in particular to a liquid chromatography analysis method for detecting antibody deamidation and an application thereof. Background Art

[0002] In recent years, the rapid development of antibody-drug conjugates has attracted much attention. Due to their highly complex molecular structure, antibody-drug conjugates are susceptible to various physical, chemical or biochemical factors in multiple links such as production, formulation, storage, and transportation. These factors often lead to post-translational modifications (PTMs) at specific sites, such as glycosylation, oxidation, glycation, deamidation, and isomerization, which in turn produce variants. These variants are important components of the critical quality attributes (CQA) of the product. Even small variations may significantly affect the physical and chemical properties, biological activity, and drug stability of the product, and may even cause immunogenicity problems.

[0003] Among the various post-translational modifications of antibody-drug conjugates, deamidation is a common degradation pathway. PTMs of deamidation and isomerization can significantly lead to changes in protein conformation, decreased stability, and loss of function of antibody-drug conjugates. In particular, these modifications occurring in the complementary determining region (CDR) of antibodies can significantly affect drug activity, change the local hydrophobicity and spatial structure of antibodies, thereby reducing the binding force between antibodies and antigens, leading to off-target effects, and ultimately affecting the safety and effectiveness of drugs in clinical applications. Asparagine (Asn) is a common deamidation modification site. In a low pH environment and high temperature, asparagine is easily deamidated to generate the intermediate succinimide (Sue), which is unstable under alkaline conditions and will be rapidly hydrolyzed to generate aspartic acid (Asp) or isoaspartic acid (isoAsp), the masses of which differ by only 1Da. This modification introduces negative charges and adds a methylene group to the peptide backbone, thereby changing the properties of the protein. The CDR region of an antibody is the main site for binding to the target molecule, and its structure is highly flexible and easily exposed to solvents. Therefore, Asp in the CDR region is more easily degraded to generate isomerized isoAsp, which affects the biological binding activity of the antibody and may induce an immunogenic response.

[0004] Liquid chromatography is one of the commonly used methods for quantitative analysis of antibody-drug conjugates. Ultra-high performance liquid chromatography (UHPLC) has the advantages of accurate quantification, good reproducibility and high economic benefits. At present, there is still a lack of quantitative determination methods for the deamidation and isomerization ratios in the CDR region of antibody-drug conjugates in the biopharmaceutical field. The development is difficult and the durability is poor, which brings challenges to the monitoring of post-translational modifications during the production and transportation of ADC drugs. Therefore, it is urgent to establish a set of mature liquid chromatography analysis techniques for accurately determining the deamidation and isomerization modification ratios in the CDR region of antibody-drug conjugates. This will help support the accuracy of post-translational modification analysis of ADC drugs, effectively identify changes in biological binding activity and quality attributes during drug production, and thus improve the safety and efficacy of antibody-drug conjugates. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a liquid chromatography analysis method for detecting antibody deamidation and its application. The method uses trypsin to cleave the antibody-drug conjugate to obtain the target peptide segment, and separates it by ultra-high performance liquid chromatography within 40 minutes to obtain a chromatogram with good reproducibility.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a liquid chromatography analysis method for detecting antibody deamidation and isomerization ratios, the analysis method comprising denaturing and reducing the sample to be tested, performing enzyme digestion after detecting the concentration, using liquid chromatography for detection, and calculating the deamidation ratio.

[0008] In the present invention, ultra-high performance liquid chromatography separation is performed after trypsin cleavage. This method overcomes the difficulties of traditional mass spectrometry, such as strong dependence, long analysis time, high cost, and large peak drift and peak shape changes, and realizes accurate quantitative determination.

[0009] By protease hydrolysis, specific peptides of post-translational modification of deamidation and isomerization in the CDR region of antibody-drug conjugates are obtained, separated and analyzed by ultra-high performance liquid chromatography, and peak attribution is performed by mass spectrometry identification, which can realize the analysis and quantification of deamidation and isomerization modification of specific peptides in the CDR region. This method has the characteristics of high throughput, high sensitivity, good reproducibility, low technical cost, simple operation, and easy promotion.

[0010] Preferably, the sample to be tested includes any one of an antibody-drug conjugate, a monospecific antibody or a bispecific antibody, or a combination of at least two of them.

[0011] Preferably, the added amount of the sample to be tested is 100-300 μg, for example, it can be 100 μg, 120 μg, 140 μg, 160 μg, 170 μg, 200 μg, 220 μg, 240 μg, 260 μg, 280 μg or 300 μg, etc.

[0012] Preferably, the denaturation reduction step comprises adding the sample to be tested into a denaturing solution and then adding a reducing agent for reduction incubation.

[0013] Preferably, the denaturing fluid comprises 6-8 mol / L guanidine hydrochloride, for example, it may be 6 mol / L, 6.2 mol / L, 6.4 mol / L, 6.6 mol / L, 6.8 mol / L, 7 mol / L, 7.2 mol / L, 7.4 mol / L, 7.6 mol / L, 7.8 mol / L or 8 mol / L.

[0014] Preferably, the preparation method of the denaturing solution comprises dissolving guanidine hydrochloride in a sodium acetate solution with a concentration of 0.2-0.4 mol / L. The 0.2-0.4 mol / L may be, for example, 0.2 mol / L, 0.25 mol / L, 0.3 mol / L, 0.35 mol / L or 0.4 mol / L.

[0015] Preferably, the pH value of the denaturing liquid is 4.5-5.5, for example, it can be 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4 or 5.5.

[0016] Preferably, the reducing agent comprises any one of trichloroethyl phosphate, dithiothreitol or mercaptoethanol, or a combination of at least two thereof.

[0017] Preferably, the final concentration of the reducing agent is 24-36 mM, for example, it can be 24 mM, 25 mM, 26 mM, 27 mM, 28 mM, 29 mM, 30 mM, 31 mM, 32 mM, 33 mM, 34 mM, 35 mM or 36 mM, etc.

[0018] Preferably, the reduction incubation time is 10-20 min, and the temperature is 35-40° C. The 10-20 min may be, for example, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, etc. The 35-40° C. may be, for example, 35° C., 36° C., 37° C., 38° C., 39° C. or 40° C., etc.

[0019] Preferably, the step of detecting the concentration comprises adding a liquid replacement buffer to the sample after denaturation and reduction, centrifuging the sample, and performing ultraviolet detection after ultrafiltration.

[0020] Preferably, the fluid replacement buffer comprises any one of urea, Tris hydrochloride or phosphate buffer or a combination of at least two thereof.

[0021] Preferably, the concentration of the liquid exchange buffer is 1-2M, for example, it can be 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, 1.6M, 1.7M, 1.8M, 1.9M or 2M, etc.

[0022] Preferably, the centrifugal speed is 10000-15000 rpm, and the time is 10-20 min. The 10000-15000 rpm may be, for example, 10000 rpm, 11000 rpm, 12000 rpm, 13000 rpm, 14000 rpm or 15000 rpm, etc. The 10-20 min may be, for example, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min, etc.

[0023] Preferably, the ultrafiltration speed is 3000-5000 rpm, and the time is 1-5 min. The 3000-5000 rpm may be, for example, 3000 rpm, 3200 rpm, 3400 rpm, 3600 rpm, 3700 rpm, 4000 rpm, 4200 rpm, 4400 rpm, 4600 rpm, 4800 rpm or 5000 rpm, etc. The 1-5 min may be, for example, 1 min, 2 min, 3 min, 4 min or 5 min, etc.

[0024] Preferably, the ultraviolet detection is to detect the absorbance value of the sample after ultrafiltration at 280nm.

[0025] Preferably, the formula for calculating protein concentration after ultraviolet detection is:

[0026] c = A280 / ε.

[0027] Wherein, c: protein concentration; A280: protein absorbance at UV 280nm; ε: protein extinction coefficient.

[0028] Preferably, the enzyme cleavage step comprises diluting the sample to be tested, adding a first portion of trypsin for a first incubation, and then adding an enzyme cleavage buffer and a second portion of trypsin for a second incubation.

[0029] Preferably, the concentration of the diluted sample is 0.5-1.5 mg / mL, for example, it can be 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL or 1.5 mg / mL, etc.

[0030] Preferably, the mass ratio of the diluted sample to the first portion of trypsin is (10-40):1, and the (10-40) can be, for example, 10, 15, 20, 25, 30, 35 or 40, etc.

[0031] Preferably, the temperature of the primary incubation is 35-40°C, and the time is 0.5-1.5h. The 35-40°C may be, for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C. The 0.5-1.5h may be, for example, 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1.0h, 1.1h, 1.2h, 1.3h, 1.4h or 1.5h.

[0032] Preferably, the enzyme digestion buffer comprises any one of Tris hydrochloride buffer, phosphate buffer or sodium acetate-acetate buffer or a combination of at least two thereof.

[0033] Preferably, the mass ratio of the diluted sample to the comprehensive trypsin is (2.5-10): 1. The (2.5-10) may be, for example, 2.5, 3, 4, 5, 6, 7, 8, 9 or 10, etc.

[0034] Preferably, the comprehensive trypsin comprises a first part of trypsin and a second part of trypsin.

[0035] Preferably, the pH value of the enzyme cleavage buffer is 6-7, for example, it can be 6, 6.2, 6.4, 6.8 or 7.

[0036] Preferably, the secondary incubation temperature is 35-40°C and the time is 2.5-3.5h. The 35-40°C may be, for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C. The 2.5-3.5h may be, for example, 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3.0h, 3.1h, 3.2h, 3.3h, 3.4h or 3.5h.

[0037] Preferably, the mobile phase A for the liquid chromatography detection is 0.05%-0.2% formic acid solution, and the 0.05%-0.2% may be, for example, 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%, etc.

[0038] Preferably, the mobile phase A for the liquid chromatography detection is 0.05%-0.2% formic acid-acetonitrile solution, and the 0.05%-0.2% may be, for example, 0.05%, 0.06%, 0.08%, 0.1%, 0.12%, 0.14%, 0.16%, 0.18% or 0.2%, etc.

[0039] Preferably, the sample loading volume for liquid chromatography detection is 5-15 μL, for example, it can be 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL or 15 μL, etc.

[0040] Preferably, the column temperature of the liquid chromatography detection is 50-70° C. The 50-70° C. may be, for example, 50° C., 52° C., 54° C., 56° C., 58° C., 60° C., 62° C., 64° C., 66° C., 68° C. or 70° C.

[0041] Preferably, the elution time of the liquid chromatography detection is 30-90 min, for example, it can be 30 min, 40 min, 50 min, 60 min, 70 min, 80 min or 90 min.

[0042] Preferably, the elution procedure of the gradient elution of the liquid chromatography is as follows:

[0043] At 0min-2min, mobile phase A was 95%;

[0044] From 2min to 42min, mobile phase A was changed from 95% to 77% at a constant rate;

[0045] At 42-44 min, mobile phase A was changed from 77% to 5% at a constant rate;

[0046] At 44min-50min, mobile phase A was 5%;

[0047] At 50min-50.1min, mobile phase A was changed from 5% to 95% at a constant rate;

[0048] At 50.1min-60min, mobile phase A was 95%.

[0049] Preferably, the method for calculating the deamidation ratio is: liquid chromatography detection obtains peptide peaks of asparagine, aspartic acid and isoaspartic acid, the target peak is integrated to obtain the peak areas of different amino acids, and the deamidation ratio is calculated by the peak area.

[0050] Preferably, the calculation formula for the deamidation ratio is:

[0051]

[0052] In a second aspect, the present invention provides an application of the liquid chromatography analysis method for detecting antibody deamidation and isomerization ratios according to the first aspect in detecting the deamidation ratio of the CDR region of an antibody-drug conjugate.

[0053] Compared with the prior art, the present invention has at least the following beneficial effects:

[0054] 1. The present invention provides a method for analyzing the deamidation and isomerization ratio of the CDR region of an antibody-drug conjugate, which is separated by ultra-high performance liquid chromatography after trypsin cleavage. This method overcomes the difficulties of traditional mass spectrometry, such as strong dependence, long analysis time, high cost, and large peak drift and peak shape changes, and achieves accurate quantitative determination.

[0055] 2. The method of the present invention has good specificity, repeatability, accuracy, linearity and method durability. Its high accuracy, excellent solution stability, simple operation process and economical cost make it extremely advantageous in practical applications.

[0056] 3. The method of the present invention solves the problem that traditional mass spectrometry and peptide map analysis are expensive and time-consuming, and is particularly unsuitable for early molecular post-translational modification screening. By using samples with a high Asp ratio as a control, the deamidation target peak is quickly located, which greatly saves detection and analysis time and is suitable for high-throughput target antibody-drug conjugate molecule screening detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Schematic diagram of the structure of camptothecin ADC drugs.

[0058] Figure 2 Schematic diagram for detecting the deamidation ratio of antibodies.

[0059] Figure 3 The results of the control sample are shown in Figure 2.

[0060] Figure 4 This is the mass spectrum of Example 1.

[0061] Figure 5 The figures are superimposed on the results of Example 4 and Example 1 under different chromatographic elution gradients.

[0062] Figure 6 The figures are superimposed on the results of Example 5 and Example 1 under different chromatographic elution gradients.

[0063] Figure 7 The chromatograms of peptide segments obtained in Example 7 and Example 1 under different enzyme cleavage modes are compared.

[0064] Figure 8 This is the result diagram of Comparative Example 1.

[0065] Fig. 9 The peptide chromatograms of the samples of Example 1 under strong conditions, normal conditions and standard control (containing high proportions of Asp and Iso-Asp peptides) are shown in Figure 1.

[0066] Fig.10 The peptide chromatograms of Example 2 under strong conditions, normal conditions and blank control are shown.

[0067] Fig.11 This is an overlay of the six injections of Example 1.

[0068] Fig.12 This is the linear result diagram of Example 1.

[0069] Fig.13 The chromatograms of the solution stable peptides at different injection times of Example 1 are shown. DETAILED DESCRIPTION

[0070] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0071] Example 1

[0072] This example provides a liquid chromatography analysis method for detecting antibody deamidation

[0073] (1) Sample denaturation and reduction

[0074] In this embodiment, camptothecin ADC drugs are used. The structure of the camptothecin ADC drugs is as follows: Figure 1 As shown, the antibody used is an IgG1 antibody containing a HSNGNTYLQWYQQKPGQAPR fragment, and the conjugate-antibody ratio is 4:1.

[0075] Take 200 μg of ADC and add it to a 1.5 mL centrifuge tube. Add 7 mol / L guanidine hydrochloride solution dissolved in 0.3 mol / L sodium acetate solution to 94 μL, pH 5.0, and add 6 μL trichloroethyl phosphate (TCEP). The final concentration of guanidine hydrochloride in the solution is 20 mmol / L, and the final concentration of TCEP is 30 mM. After vortex mixing, place it in a 37°C constant temperature metal bath for incubation for 15 minutes and then cool it to room temperature.

[0076] (2) Concentration detection

[0077] Add 400 μL of 1.6 mol / L urea buffer solution to the ultrafiltration tube, centrifuge at 13000 rpm for 10 min to rinse, and repeat this operation once. Then add 1.6 mol / L urea buffer solution to the sample obtained by denaturation and reduction to a final volume of 400 μL, transfer to the rinsed ultrafiltration tube, centrifuge at 13000 rpm for 15 min, and repeat this operation three times. Invert the ultrafiltration tube into a clean centrifuge tube and ultrafilter at 4000 rpm for 2 min.

[0078] The protein absorption value at 280 nm was measured using a micro-UV-visible spectrophotometer or a microfluidic UV-visible spectrometer, and the protein concentration was calculated according to the formula c=A280 / ε (c: protein concentration; A280: protein absorbance at UV 280 nm; ε: protein extinction coefficient). The protein solution was then diluted to 1.0 mg / mL with 1.6 mol / L urea buffer solution for the following enzymatic hydrolysis step.

[0079] (3) Enzyme digestion

[0080] Take 25 μg of the sample obtained by the replacement solution and add it to a 1.5 mL centrifuge tube, add 2.5 μL of 0.5 mg / mL trypsin (protein: first trypsin w / w = 20:1), vortex mix for a few seconds, and place it in a constant temperature mixer at 37°C for incubation for 1 hour. Then add 7.5 μL of 0.5 mg / mL second trypsin (protein: total trypsin w / w = 5:1), the second trypsin is dissolved in 25 μL of 50 mM Tris-HCl buffer solution at pH 6.8, vortex mix, and place it in a constant temperature mixer at 37°C for further incubation for 3 hours.

[0081] (4) Liquid chromatography detection

[0082] Take 10 μL of the digested sample and use a liquid chromatography analyzer and a UV detector to detect the sample. The ultra-high performance liquid chromatography model used is Agilent; the chromatographic column is ACQUITY Peptide CSHC18 Column, 1.7μm, 2.1mm x 150mm; column temperature 60℃; mobile phase A 0.1% formic acid-water; mobile phase B 0.1% formic acid-acetonitrile, gradient elution, injection volume 10μL. UV detector is DAD detector, and its detection wavelength is 280nm.

[0083] The elution procedure of the gradient elution is:

[0084] At 0min-2min, mobile phase A was 95%;

[0085] From 2min to 42min, mobile phase A was changed from 95% to 77% at a constant rate;

[0086] At 42-44 min, mobile phase A was changed from 77% to 5% at a constant rate;

[0087] At 44min-50min, mobile phase A was 5%;

[0088] At 50min-50.1min, mobile phase A was changed from 5% to 95% at a constant rate;

[0089] At 50.1min-60min, mobile phase A was 95%.

[0090] Example 2

[0091] This example provides a liquid chromatography analysis method for detecting antibody deamidation

[0092] (1) Sample denaturation and reduction

[0093] In this embodiment, camptothecin ADC drugs are used. The structure of the camptothecin ADC drugs is as follows: Figure 1 As shown, the antibody used is an IgG1 antibody containing a HSNGNTYLQWYQQKPGQAPR fragment, and the conjugate-antibody ratio is 4:1.

[0094] Take 200 μg of ADC and add it to a 1.5 mL centrifuge tube. Add guanidine hydrochloride solution dissolved in 0.3 mol / L sodium acetate solution to 94 μL, pH 5.0, and add 6 μL trichloroethyl phosphate (TCEP). The final concentration of guanidine hydrochloride in the solution is 30 mmol / L, and the final concentration of TCEP is 36 mM. After vortex mixing, place it in a 40°C constant temperature metal bath for incubation for 10 minutes and then cool it to room temperature.

[0095] (2) Concentration detection

[0096] Add 400 μL of 2 mol / L urea buffer solution to the ultrafiltration tube, centrifuge at 10000 rpm for 15 min to rinse, and repeat this operation once. Then add 2 mol / L urea buffer solution to the sample obtained by denaturation and reduction to a final volume of 400 μL, transfer to the rinsed ultrafiltration tube, centrifuge at 15000 rpm for 20 min, and repeat this operation three times. Invert the ultrafiltration tube into a clean centrifuge tube and ultrafilter at 5000 rpm for 1 min.

[0097] The protein absorption value at 280 nm was measured using a micro-UV-visible spectrophotometer or a microfluidic UV-visible spectrometer, and the protein concentration was calculated according to the formula c=A280 / ε (c: protein concentration; A280: protein absorbance at UV 280 nm; ε: protein extinction coefficient). The protein solution was then diluted to 1.0 mg / mL with a 2 mol / L urea buffer solution for the following enzymatic hydrolysis step.

[0098] (3) Enzyme digestion

[0099] Take 25 μg of the sample obtained by liquid exchange and add it to a 1.5 mL centrifuge tube, add 1.25 μL of 0.5 mg / mL trypsin (protein: first trypsin w / w = 40:1), vortex mix for a few seconds, place it in a constant temperature mixer at 35°C and incubate it for 1.5 h, then add 5 μL of 0.5 mg / mL second trypsin (protein: total trypsin w / w = 8:1), the second trypsin is dissolved in 25 μL 50 mM Tris-HCl buffer solution at pH 6.8, vortex mix for a few seconds, and place it in a constant temperature mixer at 37°C and continue incubating for 3.5 h.

[0100] (4) Liquid chromatography detection

[0101] Take 10 μL of the digested sample and use a liquid chromatography analyzer and a UV detector to detect the sample. The ultra-high performance liquid chromatography model used is Waters / ACQUITY Premier; the chromatographic column is ACQUITY Peptide CSHTM C18 Column, 1.7μm, 2.1mm X150mm; column temperature is 60℃; mobile phase A is 0.1% formic acid-water; mobile phase B is 0.1% formic acid-acetonitrile, gradient elution is performed, and the injection volume is 10μL. The UV detector is a DAD detector, and its detection wavelength is 280nm.

[0102] The elution procedure of the gradient elution is:

[0103] At 0min-2min, mobile phase A was 99%;

[0104] From 2min to 62min, mobile phase A was changed from 99% to 65% at a constant rate;

[0105] At 62-64 min, mobile phase A was changed from 65% to 55% at a constant rate;

[0106] At 64-66 min, mobile phase A was changed from 55% to 5% at a constant rate;

[0107] At 66-68 min, mobile phase A was 5%;

[0108] At 68-72 min, mobile phase A was changed from 5% to 99% at a constant rate;

[0109] At 72min-85min, mobile phase A was 99%.

[0110] Example 3

[0111] This example provides a liquid chromatography analysis method for detecting antibody deamidation, which differs from Example 1 only in that the concentration of the urea buffer is 1.6 mol / L, and the rest is consistent with Example 1.

[0112] Example 4

[0113] This example provides a liquid chromatography analysis method for detecting antibody deamidation, which differs from Example 1 only in that the elution procedure of the gradient elution of the liquid chromatography is:

[0114] At 0min-2min, mobile phase A was 95%;

[0115] From 2min to 62min, mobile phase A was changed from 95% to 65% at a constant rate;

[0116] At 62-64 min, mobile phase A was changed from 65% to 5% at a constant rate;

[0117] At 64-68 min, mobile phase A was 5%;

[0118] At 68min-68.1min, mobile phase A was changed from 5% to 95% at a constant rate;

[0119] At 68.1min-80min, mobile phase A was 95%.

[0120] The rest are consistent with Example 1.

[0121] Example 5

[0122] This example provides a liquid chromatography analysis method for detecting antibody deamidation, which differs from Example 1 only in that the elution procedure of the gradient elution of the liquid chromatography is:

[0123] At 0min-2min, mobile phase A was 100%;

[0124] From 2min to 10min, mobile phase A was changed from 100% to 93% at a constant speed;

[0125] From 10min to 100min, mobile phase A was changed at a constant rate from 93% to 63%;

[0126] At 100-110 min, mobile phase A was changed from 63% to 50% at a constant speed;

[0127] At 110-111 min, mobile phase A was changed from 50% to 10% at a constant speed;

[0128] From 111min to 120.5min, the mobile phase A was 10%.

[0129] From 111min to 120.5min, the mobile phase A was changed at a constant speed from 10% to 100%.

[0130] At 121min-130min, mobile phase A was 100%.

[0131] The rest are consistent with Example 1.

[0132] Example 6

[0133] This example provides a liquid chromatography analysis method for detecting antibody deamidation, which differs from Example 1 only in that the drug used is a monoclonal antibody, which is the same antibody as the ADC drug in Example 1, and the antibody used is an IgG1 antibody containing a HSNGNTYLQWYQQKPGQAPR fragment, and the rest is consistent with Example 1.

[0134] Example 7

[0135] This comparative example provides a liquid chromatography analysis method for detecting antibody deamidation, which differs from Example 1 only in that the second step of enzymatic digestion uses a mixed enzymatic digestion of 5 μL 0.5 mg / mL lysine protease (protein: lysine protease w / w = 20:1) and 5 μL 0.5 mg / mL trypsin (protein: total trypsin w / w = 10:1), and the rest is consistent with Example 1.

[0136] Comparative Example 1

[0137] This comparative example provides a liquid chromatography analysis method for detecting antibody deamidation, which differs from Example 1 in that different enzyme cleavage steps are used. The specific enzyme cleavage steps are as follows:

[0138] (3) Enzyme digestion

[0139] 50 μg of the sample obtained by replacing the liquid was added to a 1.5 mL centrifuge tube, and 2.5 μg of 1.0 mg / mL Lys-C lysine protease (protein: lysine protease w / w = 20:1) and 50 μL of buffer (8 mol / L urea dissolved in 50 mM Tris-HCl buffer solution at pH 6.8, 0.5 mM TCEP) were added, vortexed and placed in a constant temperature mixer at 37°C for 1 hour. 6 μL of 20% trifluoroacetic acid (TFA) was added to terminate the reaction.

[0140] Test Example 1

[0141] This test example calculates the deamidation percentage

[0142] The detection principle of this test case is as follows Figure 2 As shown, asparagine is easily deamidated to generate an intermediate succinimide (Sue), which is unstable under alkaline conditions and will be rapidly hydrolyzed to generate aspartic acid (Asp) or isoaspartic acid (isoAsp). The liquid phase detection in the above embodiments and comparative examples obtains the peptide peaks of asparagine, aspartic acid and isoaspartic acid, and the peak areas of different amino acids are obtained by integrating the target peaks. The deamidation ratio is calculated by the peak area. The calculation formula of the deamidation ratio is:

[0143]

[0144] In this test example, the control sample, i.e. the sample with a high Asp+isoAsp ratio, is first tested to locate the Asp and isoAsp peaks. Samples with a high Asp ratio: The structure of camptothecin ADC drugs is as follows Figure 1 As shown, the only difference is that the antibody used is an IgG1 antibody containing the HSDGNTYLQWYQQKPGQAPR fragment, and the conjugate antibody ratio is 4:1. The above examples and comparative examples were then tested, and the specific results are shown in Table 1 below:

[0145] Table 1

[0146]

[0147] The results show that:

[0148] (1) The test results of the control sample are shown in the figure Figure 3 As shown, it can be seen that the control sample contains a high proportion of Asp peptides, which can be used to locate the Asp peak of the normal sample.

[0149] (2) The mass spectrum peak identification results of Example 1 are as follows Figure 4As shown, it can be seen that through mass spectrometry identification of the peptide segment with the UV signal peak at 19.5 minutes, b3 confirmed that Asn was deamidated to Asp, and b3 and b4 jointly verified that the site of deamidation occurred at the N3 position.

[0150] (3) The robustness of the method can be seen from the comparison between Example 1 and Example 2. Similar results can be obtained on equivalent instrument brands and chromatographic column instruments after elution gradient optimization.

[0151] (4) By comparing Example 1 with Example 4, it can be seen that the specific results are as follows Figure 5 As shown in the figure, changing the liquid phase elution gradient will result in different peak separations, especially the Asp peptide segment will be interfered by the co-elution of other peptide segments, thus affecting the accuracy of the results.

[0152] (5) By comparing Example 1 with Example 5, the results are as follows Figure 6 As shown, the Asp peptide segment co-eluted with the previous peptide segment under the elution gradient of Example 5, and the gradient of Example 5 took as long as 130 minutes, so Example 1 was selected as the optimal condition.

[0153] (6) By comparing Example 1 with Example 6, it can be seen that this method is applicable to both antibody-drug conjugates and monoclonal antibody protein samples.

[0154] (7) By comparing Example 1 with Example 7, the results are as follows Figure 7 As shown in the figure, the Asp peptide co-eluted with other peptides at 22.6 min in the mixed enzyme digestion, resulting in a larger UV quantification and reduced accuracy of the results.

[0155] (8) By comparing Example 1 with Comparative Example 1, the results are as follows Figure 8 As shown, by using lysine protease cleavage and changing the enzymatic cleavage treatment method, the deamidated Asp and deamidated isomerized IsoAsp cannot be quantified separately in the spectrum, and the Asp and Iso-Asp peaks cannot be separated.

[0156] Test Example 2

[0157] This test case is tested under strong conditions

[0158] The samples of the above embodiments and comparative examples were incubated at 45°C for 3 days and then tested. The specific test results are shown in Table 2:

[0159] Table 2

[0160]

[0161] The results show that:

[0162] (1) By comparing the strong conditions and normal conditions of Example 1, Fig. 9 As shown in the figure, the total deamidation ratio increased under strong conditions, indicating that this method can monitor and quantitatively analyze the changes in the deamidation and isomerization ratios in the sample.

[0163] (2) By comparing Example 1 with Example 2, it can be seen that Fig.10 As shown, the instrument model is changed to Waters / ACQUITY Premier; the chromatographic column is ACQUITY Peptide CSHTM C18 Column, 1.7μm, 2.1mm X 150mm, deamidation and isomerization results are similar.

[0164] (3) By comparing Example 1 with Example 3, it can be seen that changing the urea buffer concentration to 1.6 mol / L does not significantly change the deamidation and isomerization results.

[0165] (4) By comparing Example 1 with Example 6, it can be seen that this method is applicable to both antibody-drug conjugates and monoclonal antibody protein samples.

[0166] (5) By comparing Example 1 with Example 7, it can be seen that when the mixed enzyme cleavage method is used, the Asp peptide segment and other peptide segments co-elute, resulting in a larger UV quantification and inaccurate quantification.

[0167] Test Example 3

[0168] This test case is used to test stability

[0169] (1) Repeatability

[0170] The sample of Example 1 was prepared in six parallel batches, and one injection was made for each sample under the same conditions to obtain stable peptide spectrum data. The specific results are as follows: Fig.11 shown.

[0171] (2) Linear

[0172] The injection volume was changed to adjust the injection volume of the sample of Example 1 to 50% (12.5 μg), 75% (18.75 μg), 100% (25 μg), 125% (31.25 μg), and 150% (37.5 μg). Under these conditions, one injection was performed respectively to obtain good linear data. Fig.12 This is the linear result of Example 1.

[0173] (3) Solution stability

[0174] The sample prepared in Example 1 was injected once at 0h, 24h, 48h, and 72h to obtain stable peptide spectrum data. The specific results are as follows Fig.13 shown.

[0175] The above test results are shown in Table 3:

[0176] Table 3

[0177] Peak group name Deamidation ratio% Repeatability RSD% (n=6) 0.5 Intermediate precision RSD% (n=12) 0.8 Solution stability D% (T0,24h) 0.2 Solution stability D% (T0,48h) 1.7 Solution stability D% (T0,72h) 0.8

[0178] The above results show that the technical indicators of the method of the present invention, such as repeatability, solution stability, and linearity, all meet the requirements. The method is used to detect the deamidation and isomerization ratio content of antibody-drug conjugates, and has good solution stability and good reproducibility.

[0179] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A liquid chromatography analysis method for detecting antibody deamidation and isomerization ratio, characterized in that: The analysis method comprises denaturing and reducing the sample to be tested, performing enzyme digestion after detecting the concentration, using liquid chromatography for detection, and calculating the deamidation ratio.

2. The liquid chromatography analysis method for detecting antibody deamidation and isomerization ratio according to claim 1, characterized in that: The sample to be tested includes any one or a combination of at least two of an antibody-drug conjugate, a monospecific antibody or a bispecific antibody; Preferably, the amount of the sample to be tested is 100-300 μg; Preferably, the denaturation reduction step comprises adding the sample to be tested into a denaturing solution and then adding a reducing agent for reduction incubation; Preferably, the denaturing solution comprises 6-8 mol / L guanidine hydrochloride; Preferably, the method for preparing the denaturing solution comprises dissolving guanidine hydrochloride in a sodium acetate solution having a concentration of 0.2-0.4 mol / L; Preferably, the pH value of the denaturing solution is 4.5-5.5; Preferably, the reducing agent comprises any one or a combination of at least two of trichloroethyl phosphate, dithiothreitol or mercaptoethanol; Preferably, the final concentration of the reducing agent is 24-36 mM; Preferably, the reduction incubation time is 10-20 min and the temperature is 35-40°C.

3. The liquid chromatography analysis method for detecting antibody deamidation and isomerization ratio according to claim 1 or 2, characterized in that: The step of detecting the concentration comprises adding a liquid replacement buffer to the sample after denaturation and reduction, centrifuging, and performing ultraviolet detection after ultrafiltration; Preferably, the fluid replacement buffer comprises any one of urea, Tris hydrochloride or phosphate buffer or a combination of at least two thereof; Preferably, the concentration of the liquid replacement buffer is 1-2M; Preferably, the centrifugal speed is 10000-15000 rpm, and the time is 10-20 min; Preferably, the ultrafiltration speed is 3000-5000 rpm, and the time is 1-5 min.

4. The liquid chromatography analysis method for detecting antibody deamidation and isomerization ratio according to claim 3, characterized in that: The ultraviolet detection is to detect the absorbance value of the sample after ultrafiltration at 280nm; Preferably, the formula for calculating protein concentration after ultraviolet detection is: c = A280 / ε; Wherein, c: protein concentration; A280: protein absorbance at UV 280nm; ε: protein extinction coefficient.

5. The liquid chromatography analysis method for detecting antibody deamidation and isomerization ratio according to any one of claims 1 to 4, characterized in that: The enzyme cleavage step comprises diluting the sample to be tested, adding a first portion of trypsin for a first incubation, and then adding an enzyme cleavage buffer and a second portion of trypsin for a second incubation; Preferably, the concentration of the diluted sample is 0.5-1.5 mg / mL; Preferably, the mass ratio of the diluted sample to the first portion of trypsin is (10-40):1; Preferably, the temperature of the primary incubation is 35-40°C and the time is 0.5-1.5h.

6. The liquid chromatography analysis method for detecting antibody deamidation and isomerization ratio according to any one of claims 1 to 5, characterized in that: The enzyme digestion buffer comprises any one of tris(hydroxymethylaminomethane) hydrochloride buffer, phosphate buffer or sodium acetate-acetate buffer or a combination of at least two thereof; Preferably, the mass ratio of the diluted protein sample to comprehensive trypsin is (2.5-10):1; Preferably, the comprehensive trypsin comprises a first portion of trypsin and a second portion of trypsin; Preferably, the pH value of the enzyme digestion buffer is 6-7; Preferably, the secondary incubation temperature is 35-40°C and the time is 2.5-3.5 hours.

7. The liquid chromatography analysis method for detecting antibody deamidation and isomerization ratio according to any one of claims 1 to 6, characterized in that: The mobile phase A of the liquid chromatography detection is 0.05%-0.2% formic acid solution; Preferably, the mobile phase A of the liquid chromatography detection is 0.05%-0.2% formic acid-acetonitrile solution; Preferably, the sample loading volume for liquid chromatography detection is 5-15 μL; Preferably, the column temperature of the liquid chromatography detection is 50-70°C.

8. The liquid chromatography analysis method for detecting antibody deamidation and isomerization ratio according to any one of claims 1 to 7, characterized in that: The elution time of the liquid chromatography detection is 30-90min; Preferably, the elution procedure of the gradient elution of the liquid chromatography is as follows: At 0min-2min, mobile phase A was 95%; From 2min to 42min, mobile phase A was changed from 95% to 77% at a constant rate; At 42-44 min, mobile phase A was changed from 77% to 5% at a constant rate; At 44min-50min, mobile phase A was 5%; At 50min-50.1min, mobile phase A was changed from 5% to 95% at a constant rate; At 50.1min-60min, mobile phase A was 95%.

9. The liquid chromatography analysis method for detecting antibody deamidation and isomerization ratio according to any one of claims 1 to 8, characterized in that: The deamidation ratio is calculated by: liquid phase detection to obtain the peptide peaks of asparagine, aspartic acid and isoaspartic acid, integrating the target peak to obtain the peak areas of different amino acids, and calculating the deamidation ratio by the peak areas; Preferably, the calculation formula for the deamidation ratio is:

10. Use of the liquid chromatography analysis method for detecting antibody deamidation and isomerization ratios according to any one of claims 1 to 9 in detecting the deamidation ratio of the CDR region of an antibody-drug conjugate.

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