Analysis method for quantitatively detecting esterase / residual quantity of esterase in antibody and analogue and application of analysis method

Abstract

The invention provides an analysis method for quantitatively detecting the residual quantity of esterase / esterase in an antibody and an analogue and application of the analysis method. The analysis method comprises the following steps: sequentially performing enzymolysis, reduction and antibody removal, vacuum drying and redissolution on a sample to be detected and a standard substance; the standard substance contains an esterase / esterase standard substance and human serum immune globulin G; the amino acid sequence of the esterase / esterase standard substance is shown as SEQ ID NO: 1-8; detecting the redissolved sample by adopting an ultra-high performance liquid chromatograph-mass spectrometer, and collecting mass spectrum data; the method comprises the following steps: performing peptide fragment matching and peak area automatic integration by using analysis software, performing linear fitting by taking logarithms of an extraction chromatogram peak area of a corresponding esterase / lipase characteristic peptide fragment in a standard substance and a doping value, and then substituting the extraction chromatogram peak area of the corresponding esterase / lipase characteristic peptide fragment in a sample to be detected into a linear fitting curve for calculation. And obtaining a corresponding esterase / esterase content measured value.

Description

Technical Field

[0001] The present invention belongs to the field of drug analysis, and specifically relates to an analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs and its application, and in particular to an analytical method for identifying and quantifying antibodies expressed in Chinese hamster (Cricetulus griseus) cell lines, drug conjugates and similar products formed based on the antibodies, or specific residual lipase / esterase derived from host cells during purification. Background Art

[0002] Antibody-based biological drugs are the most cutting-edge products in the current biopharmaceutical industry. Currently, most of the antibodies used in biopharmaceuticals are derived from the Chinese hamster cell expression system (Curr. Opin. Chem. Eng. 2018, 22: 98-106). In the process of antibody production, proteins derived from host cells, namely Chinese hamster cells, are inevitably collected together with the product. Although the production process of antibodies is often accompanied by a purification process for antibodies, a small amount of host cell proteins will still be brought into the final antibody stock product together with the target protein.

[0003] At present, many drug qualification specifications including the Chinese Pharmacopoeia, the United States Pharmacopoeia and the European Pharmacopoeia stipulate that the host cell protein level in the antibody stock solution needs to be monitored. In addition, it is reported that there are many types of host cell proteins that are easy to break through the purification process and enter the downstream. They can be classified into various types such as proteases, lipases / esterases, immunogens and pro-immunogenic factors according to their functions. Depending on their types, their negative effects on drug efficacy, safety, stability and shelf life are also different (Biotechnol Bioeng. 2021; 118: 2870-2885). Therefore, in addition to meeting the basic monitoring requirements for host cell protein content, it is necessary to take additional measures to classify and quantify potential residual host cell proteins.

[0004] Polysorbate (PS) is a class of additives widely used to stabilize high-concentration proteins in biopharmaceuticals. Common types include polysorbate 20 (PS20) and polysorbate 80 (PS80). It is reported that the vast majority (>90%) of antibody biopharmaceuticals on the market currently contain polysorbate compounds in their formulations (J.Pharma.Sci.2021,111,1280-1291). The degradation of polysorbate compounds in antibody biopharmaceuticals can cause the precipitation of drugs as particulate matter, the production of toxic small molecules such as aldehydes, ketones, and acids, and the precipitation of free fatty acids, which are major issues affecting the efficacy, stability and safety of drugs. The investigation of the degradation of polysorbate compounds in preparations is also an important part of the biopharmaceutical production process (J.Pharma.Sci.2022,111,2955-2967). The main cause of polysorbate degradation is the hydrolysis of polysorbate by lipase / esterase proteins in residual host cell proteins (J.Pharma.Sci.2021,111,1280-1291). Such proteins can often cause significant polysorbate degradation problems at a content of several ppm (parts per million). To address this problem, the corresponding esterase / lipase remaining in the stock sample with degradation problems must first be identified and quantified so that the downstream purification process can specifically remove and monitor the corresponding esterase / lipase.

[0005] However, the current mainstream host cell protein analysis methods in the industry often find it difficult to simultaneously identify and quantify esterase / lipase residues. The "gold standard" of host cell protein quantification methods - Enzyme Linked ImmunoSorbent Assay (ELISA) is based on antibodies that can specifically recognize host cell proteins and the accompanying enzymatic reaction, which can achieve high-sensitivity host cell protein quantification. However, since the antibodies used are derived from the immune response to the host cell protein mixture, it is difficult to identify and quantify the types of host cell proteins separately. Targeted ELISA kits for specific host cell proteins on the market can only detect a certain type or certain esterase / lipase selected based on a specific host cell protein in one experiment, and cannot detect certain esterases / lipases that are less immunogenic and cannot produce targeted antibodies. Although the conventional liquid chromatography-data-dependent mode-tandem mass spectrometry method can simultaneously identify and quantify host cell proteins in samples, the data-dependent acquisition mode collects tandem mass spectrometry spectra based on ion intensity, which is not conducive to the analysis of low-abundance components in samples, and therefore often has the problem of insufficient sensitivity. In addition, most current mass spectrometry methods use a unified internal standard method to estimate the host cell protein content, and in terms of quantification, there are also problems such as large batch differences and large deviations between measured values ​​and actual values.

[0006] Therefore, in order to address the problem of polysorbate degradation in biopharmaceutical preparations, it is necessary to develop a targeted, highly sensitive, high-throughput and highly accurate identification and quantification method to support the root cause of the degradation problem and meet the monitoring needs of samples during process improvement. Summary of the invention

[0007] In view of the shortcomings of the prior art, the purpose of the present invention is to provide an analytical method for quantitatively detecting the amount of esterase / lipase residues in antibodies and analogs and its application, so as to solve the problems of difficulty in identification and inaccurate quantification in the identification, quantification and monitoring of polysorbate degradation-related host cell proteins in early research, process development and clinical stages of antibody biopharmaceutical preparations. The analytical method of the present invention has high sensitivity, high accuracy and good stability, and has important application prospects in the preparation detection of antibodies and analogs.

[0008] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides an analytical method for quantitatively detecting the amount of esterase / lipase residues in antibodies and analogs, the analytical method comprising:

[0010] (S1) enzymatically hydrolyzing, reducing, removing antibodies, vacuum drying and re-dissolving the sample to be tested and the standard substance in sequence; the standard substance contains an esterase / lipase standard substance and human serum immunoglobulin G; the amino acid sequence of the esterase / lipase standard substance includes that shown in SEQ ID NO: 1-8;

[0011] (S2) using ultra-high performance liquid chromatography-mass spectrometry to detect the reconstituted sample and collect mass spectrometry data;

[0012] (S3) Use analysis software to perform peptide matching and automatic peak area integration. Perform linear fitting by taking the logarithm of the peak area of ​​the extracted chromatogram (EIC) of the corresponding esterase / lipase characteristic peptide in the standard and the spiked value. Then substitute the extracted chromatogram peak (EIC peak) area of ​​the corresponding esterase / lipase characteristic peptide in the sample to be tested into the linear fitting curve for calculation to obtain the measured value of the corresponding esterase / lipase content.

[0013] The method of the present invention is conducive to the identification of residual esterases / lipases. Although the traditional ELISA method has a sensitivity similar to that of the present method, it is difficult to specifically identify the type of residual esterases / lipases in the sample to be tested because its detection principle is based on the interaction between the antibody mixture and the host cell protein mixture; and the targeted ELISA method can only detect one esterase / lipase in one experiment, and the targeted ELISA kits on the market cannot cover the 8 esterases / lipases that can be detected by the method of the present invention. The method of the present invention can simultaneously realize the identification and quantification of 8 esterases / lipases in a single experiment, which is helpful to identify the root cause in the investigation of polysorbate degradation problems in biopharmaceutical preparations.

[0014] Preferably, in (S1), the standard is prepared by a method comprising the following steps: mixing human serum immunoglobulin G with an esterase / lipase standard working solution, diluting with a Tris-HCl buffer solution having a pH value of 7.0-8.0 (for example, 7.0, 7.2, 7.4, 7.5, 7.6, 7.8 or 8.0, etc.) and a concentration of 45-55mM (for example, 45mM, 47mM, 49mM, 50mM, 51mM, 53mM or 55mM, etc.), the final concentration of the human serum immunoglobulin G is greater than or equal to 5μg / μL, and the final concentration of the esterase / lipase is 0.5-100ppm (for example, 0.5ppm, 1ppm, 10ppm, 30ppm, 50ppm, 70ppm, 90ppm or 100ppm, etc.).

[0015] In the present invention, the final concentration of the esterase / lipase is 0.5-100 ppm, which means that the ratio of the esterase / lipase standard to human serum immunoglobulin G is (0.5-100):1000000, w / w, for example, it can be 0.5:1000000, 1:1000000, 10:1000000, 30:1000000, 50:1000000, 70:1000000, 90:1000000 or 100:1000000, etc.

[0016] Preferably, the solvent of the esterase / lipase standard working solution is a tris-hydroxymethylaminomethane hydrochloride (Tris-HCl) buffer solution with a pH value of 7.0-8.0 (for example, 7.0, 7.2, 7.4, 7.5, 7.6, 7.8 or 8.0, etc.) and a concentration of 20-200mM (for example, 20mM, 50mM, 100mM, 150mM or 200mM, etc.).

[0017] Preferably, in (S1), the enzymatic hydrolysis is carried out by a method comprising the following steps: vortex mixing the sample and trypsin, centrifuging and incubating at 35-39°C for 2h±5min; 35-39°C can be, for example, 35°C, 36°C, 37°C, 38°C or 39°C, etc., and 2h±5min can be, for example, 2h or 2h 2min or 2h 5min, etc.

[0018] Preferably, the ratio of the sample to trypsin is (3800-4200):3, w / w, for example, it can be 3800:3, 4000:3 or 4200:3, etc.

[0019] In the present invention, the ratio of the above sample to trypsin is selected to ensure that the lipase / esterase is fully hydrolyzed without over-hydrolyzing the antibody. The best performance is achieved under the above conditions. Too high an enzyme content may increase the degree of hydrolysis of the antibody and make it difficult to remove it in the subsequent precipitation step. Too low an enzyme content may result in insufficient hydrolysis of the esterase to be tested and make it difficult to generate a signal that is sensitive enough to be detected.

[0020] Preferably, the reduction and antibody removal are performed by a method comprising the following steps: mixing the enzymatically hydrolyzed sample with dithiothreitol, incubating after mixing, collecting the supernatant by centrifugation, and adding formic acid to terminate the enzyme reaction.

[0021] Preferably, the final concentration of dithiothreitol is 0.003-0.006M, for example, 0.003M, 0.004M, 0.005M or 0.006M.

[0022] Preferably, the incubation temperature is 90-95°C, for example, 90°C, 91°C, 92°C, 93°C, 94°C or 95°C, and the incubation time is 10-15min, for example, 10min, 11min, 12min, 13min, 14min or 15min.

[0023] Preferably, the centrifugation conditions are: 12000-13000g for 10-15min; 12000-13000g can be, for example, 12000g, 12500g or 13000g, etc., and 10-15min can be, for example, 10min, 11min, 12min, 13min, 14min or 15min, etc.

[0024] Preferably, in (S1), the solvent used for the re-dissolution is a 0.1-0.15% formic acid aqueous solution, for example, 0.1%, 0.12% or 0.15%.

[0025] Preferably, in (S2), the mobile phase used in the ultra-high performance liquid chromatography comprises: mobile phase A: water containing 0.1-0.15% formic acid, mobile phase B: acetonitrile containing 0.1-0.15% formic acid; and the mobile phase is used for gradient elution.

[0026] Among them, 0.1-0.15% formic acid in water can be, for example, 0.1%, 0.12% or 0.15%, etc.; 0.1-0.15% formic acid in acetonitrile can be 0.1%, 0.12% or 0.15%, etc.

[0027] Preferably, the flow rate of the mobile phase is 0.2-0.22 mL / min, for example, 0.2 mL / min, 0.21 mL / min or 0.22 mL / min.

[0028] Preferably, the elution procedure is:

[0029] In the first 0-2 minutes, the volume fraction of mobile phase B was 0.2-0.3%, and the rest was mobile phase A;

[0030] From 2 to 25 minutes, the volume fraction of mobile phase B changes at a constant rate from 0.2-0.3% to 10-10.5%, and the rest is mobile phase A;

[0031] From 25 to 82 minutes, the volume fraction of mobile phase B changed at a constant rate from 10-10.5% to 30-30.5%, and the rest was mobile phase A.

[0032] In the present invention, when selecting characteristic peptides, the peak time in the current elution gradient is optimized, so that in the elution gradient, the number of characteristic peptides selectively collected for mass spectrometry data in each time window is within a reasonable range, thereby balancing the number of sampling points required for quantification and the improvement in signal intensity by extending the acquisition time.

[0033] Preferably, in step (S2), the chromatographic column used in the ultra-high performance liquid chromatography is Agilent / Poroshell120SB-C18, 2.1×150 mm, 2.7 μm, Agilent.

[0034] Preferably, the column temperature of the chromatographic column is set to 60-62°C, for example, 60°C, 61°C or 62°C.

[0035] Preferably, in step (S2), the mass spectrometry conditions include: performing mass spectrometry data acquisition in positive ion mode, with the ion source electrospray ionization source voltage being 4 kV; using high energy collision dissociation (HCD) in secondary fragmentation mode, and performing parallel reaction monitoring acquisition according to the collected ions shown below (Table 1).

[0036] Table 1

[0037]

[0038]

[0039] In the present invention, the instrument does not scan the primary spectrum in mass spectrometry detection, but directly performs parallel reaction monitoring and acquisition.

[0040] The analysis method of the present invention is conducive to the accurate quantification and monitoring of residual esterase / lipase. Although the conventional liquid chromatography-data-dependent mode-tandem mass spectrometry method can realize the identification and quantification of a single host cell protein, the data-dependent mode is not conducive to the data collection of low-abundance components in the sample, so it is not as good as the method of the present invention in terms of monitoring sensitivity. In addition, the conventional method often takes a single internal standard protein as the basis for quantification of host cell proteins, and in actual mass spectrometry detection, factors such as the enzymatic efficiency of different proteins, peptide ionization efficiency, and peak time will affect the ratio of protein content-signal response, and it is often difficult to achieve accurate quantification using a single internal standard protein; while the method of the present invention uses the corresponding esterase / lipase recombinant protein standard to fit the standard curve to quantify the esterase / lipase in the sample to be tested, eliminating the influence of enzymatic efficiency, peptide ionization efficiency, peak time, etc., can achieve more accurate quantification, and is also conducive to the monitoring of the change of the residual esterase / lipase content in the purification process sample.

[0041] Preferably, in step (S3), the analysis software includes Skyline analysis software.

[0042] In a second aspect, the present invention provides the application of the analytical method for quantitatively detecting the amount of esterase / lipase residues in antibodies and analogs described in the first aspect in the detection of biopharmaceutical preparations of antibodies and analogs.

[0043] The numerical range described in the present invention not only includes the point values ​​listed above, but also includes any point values ​​between the above numerical ranges that are not listed. Due to space limitations and for the sake of simplicity, the present invention no longer exhaustively lists the specific point values ​​included in the range.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The analytical method of the present invention is conducive to the identification of residual esterases / lipases. Although the traditional ELISA method has a sensitivity similar to that of the present method, it is difficult to specifically identify the type of residual esterases / lipases in the sample to be tested because its detection principle is based on the interaction between the antibody mixture and the host cell protein mixture; and the targeted ELISA method can only detect one esterase / lipase in one experiment, and the targeted ELISA kits on the market cannot cover the 8 esterases / lipases that can be detected in the method of the present invention. The use of the method of the present invention can simultaneously realize the identification and quantification of 8 esterases / lipases in a single experiment, which is helpful to identify the root cause in the investigation of polysorbate degradation problems in biopharmaceutical preparations.

[0046] (2) The analytical method of the present invention is conducive to the accurate quantification and monitoring of residual esterase / lipase. Although the conventional liquid chromatography-data-dependent mode-tandem mass spectrometry method can realize the identification and quantification of a single host cell protein, the data-dependent mode is not conducive to the data collection of low-abundance components in the sample, so it is not as sensitive as the method of the present invention in monitoring. In addition, the conventional method often uses a single internal standard protein as the basis for the quantification of host cell proteins. In actual mass spectrometry detection, factors such as the enzymatic efficiency, peptide ionization efficiency, and peak time of different proteins will affect the ratio of protein content-signal response. It is often difficult to achieve accurate quantification using a single internal standard protein; while the method of the present invention uses the recombinant protein standard corresponding to the esterase / lipase to fit the standard curve to quantify the esterase / lipase in the sample to be tested, eliminating the influence of enzymatic efficiency, peptide ionization efficiency, peak time, etc., and can achieve more accurate quantification, and is also conducive to the monitoring of changes in the residual esterase / lipase content in the purification process sample.

[0047] (3) The analytical method of the present invention is highly versatile and can be fully applied to the detection of residual esterase / lipase content in all monoclonal antibody-based biopharmaceutical products. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 Flow chart of the esterase / lipase analysis method based on parallel reaction monitoring mass spectrometry. DETAILED DESCRIPTION

[0049] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0050] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0051] The detailed information of the instruments and reagents used in the following examples are shown in Tables 2 and 3.

[0052] Table 2

[0053]

[0054] Table 3

[0055]

[0056]

[0057] Example 1

[0058] Screening of characteristic peptides

[0059] 1. Sample

[0060] (1) The list of eight esterases / lipases and their sequence numbers in the database are shown in Table 4.

[0061] Table 4

[0062]

[0063]

[0064] 2. Sample preparation

[0065] Low-absorption centrifuge tubes and low-absorption pipette tips must be used throughout the following preparation process.

[0066] (1) Preparation of esterase / lipase standards

[0067] The eight recombinantly expressed esterase / lipase standards were diluted to 0.2 mg / mL using a 50 mM Tris-HCl buffer solution at pH 8.0 and then mixed in equal volumes to obtain a 25 ng / μL lipase and esterase standard working solution.

[0068] (2) Preparation of standard products

[0069] Take some human serum immunoglobulin G, package it into 1 mg per tube, add lipase and esterase standard working solution with a final concentration of 100 ppm and 250 ppm, dilute it to 200 μL with 50 mM Tris-HCl buffer solution with a pH value of 8.0 and a concentration of 50 mM, and record it as "data-dependent mode sample" for later use.

[0070] The same method was used to prepare samples of lipase and esterase standard working solutions with final concentrations of 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0 and 100.0 ppm. Each sample was prepared in triplicate and recorded as "parallel reaction monitoring mode samples" for later use. The same method was used to prepare samples of lipase and esterase standard working solutions with final concentrations of 0.8 and 4.0 ppm. Each sample was prepared in triplicate and recorded as "method validation samples" for later use.

[0071] (3) Sample dilution: Take 1 mg of the above sample and dilute it to 200 μL with 50 mM Tris-HCl buffer solution with a pH value of 8.0 for later use.

[0072] (4) Enzymatic hydrolysis: 1.5 μL of 0.5 mg / mL trypsin (product protein: enzyme = 4000:3, w / w) was added to all samples, then vortexed to mix, centrifuged, and incubated at 37° C. for 2 h±5 min in a thermostat.

[0073] (5) Reduction and antibody removal: Add 1 μL of 1M dithiothreitol to the enzymatically hydrolyzed sample, mix well and incubate in a heating apparatus at 90°C for 10 min. Centrifuge all samples at 13,000 g for 10 min. Carefully transfer 180 μL of supernatant from the sample to a new 0.6 mL centrifuge tube and discard the precipitate. If precipitated particles are obtained in any sample, the supernatant taken from all samples needs to be centrifuged again under the same conditions, and an equal volume of supernatant is taken (recorded) until no precipitated particles are obtained in any sample. Then add 2.5 μL of 20% formic acid to terminate the enzyme reaction.

[0074] (6) Vacuum drying: The supernatant obtained in the previous step is centrifuged using a centrifugal concentrator at room temperature, a rotation speed of 1500 rpm or more, and an air pressure of less than 75 mbar until the sample is dried or the volume no longer decreases significantly (at least 1 hour).

[0075] (7) Peptide reconstitution: Add 50 μL of 0.1% formic acid aqueous solution to all samples dried in the previous step, vortex to fully dissolve and mix, and centrifuge at 13000g for 10 min to remove bubbles and insoluble matter that appear during the mixing process. Then, transfer 45 μL of the supernatant to a glass insert vial for injection and detection. Depending on the sample to be detected, data-dependent mode acquisition and parallel reaction monitoring mode acquisition were used.

[0076] 3. Detection steps

[0077] The analysis of the samples described in the following (1) data-dependent mode and (2) parallel reaction monitoring mode are based on the chromatographic instrument, mass spectrometer, chromatographic column and liquid phase parameters described below. The difference lies in the different modes in which the mass spectrometer collects data.

[0078] An ultra-high performance liquid chromatography-mass spectrometer was used for data acquisition, and 20 μL of the enzymatically hydrolyzed sample was detected on an Agilent / UHPLC (Ultra-High Performance Liquid Chromatography, UPLC) 1290 InfinityII or an equivalent ultra-high performance liquid chromatography instrument. An Agilent / Poroshell 120SB-C18 (2.1×150mm, 2.7μm, Agilent) reverse phase column was used for sample separation. During the liquid phase separation process, the column temperature was 60°C, the liquid phase flow rate was 0.2mL / min, and 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B) system was used for elution, and the elution gradient program was:

[0079] From 0 to 2 minutes, the volume fraction of mobile phase B was 0.2%, and the rest was mobile phase A;

[0080] From 2 to 25 minutes, the volume fraction of mobile phase B was changed from 0.2% to 10% at a constant rate, and the rest was mobile phase A;

[0081] From 25 to 82 minutes, the volume fraction of mobile phase B was changed from 10% to 30% at a constant rate, and the rest was mobile phase A.

[0082] After elution is completed, the column is flushed with 90% phase B for 3 minutes. After the flushing is completed, the proportion of phase B is restored to the initial state, and the column is balanced.

[0083] (1) Obtaining a total peptide list based on data-dependent model acquisition and performing preliminary screening

[0084] For the prepared "data-dependent mode samples", data-dependent mode mass spectrometry data acquisition was performed in positive ion mode based on mass spectrometers such as Thermo / Orbitrap Exploris 480. The instrument scanned the primary spectrum according to the preset parameters, the ion source electrospray ionization source voltage was 4kV, and the parent ions were fragmented in the secondary mode according to the signal intensity sorting and dynamic exclusion principles. The secondary fragmentation mode used high energy collision dissociation (HCD).

[0085] The data obtained were analyzed using Thermo / Proteome Discoverer 2.3 software. The list of characteristic peptides of esterase and lipase detectable in the sample was obtained through database retrieval, and its mass-to-charge ratio, charge number and peak time were derived as a reference for subsequent peptide screening. At the same time, the first round of screening will be carried out at this stage based on the peak type and width of the peptide, whether there is a high proportion of missed cleavage peptides and signal intensity, and the screened peptides will enter the next round of screening based on parallel reaction monitoring mode collection.

[0086] (2) Further screening of peptides based on parallel reaction monitoring mode acquisition

[0087] For the prepared "parallel reaction monitoring samples" and "method validation samples", data-dependent mass spectrometry data acquisition was performed in positive ion mode based on a mass spectrometer such as Thermo / OrbitrapExploris 480. The instrument did not scan the primary spectrum, the ion source electrospray ionization source voltage was 4 kV, and based on the mass-to-charge ratio, charge number and peak time of each peptide segment obtained in (1), the specific parent ion was selectively fragmented in the specific retention time period in the elution gradient. The secondary fragmentation mode used high energy collision dissociation (HCD).

[0088] The obtained data were analyzed using Skyline software to confirm the ion flow peak shape and fragmentation of each peptide in the total peptide list in the parallel reaction monitoring mode, as well as the peak area of ​​its daughter ion in samples with different concentrations; for the "parallel reaction monitoring samples", the concentration and peak area were linearly fitted to obtain the standard curve by statistical methods, and the peak conditions and peak areas of the corresponding peptides of the parallel prepared samples were statistically evaluated. Based on the above results and the peak time of each peptide in the elution gradient, comprehensive evaluation was performed for screening.

[0089] 4. Characteristic peptide screening results

[0090] According to the above screening method, the peptides screened from the total peptide list generated by 8 esterases and lipases under trypsin hydrolysis are shown in Table 1, and the sequence numbers of the peptides are shown in Table 5. The detection limit of each peptide is 0.25ppm, and the peak area and concentration of the selected daughter ions have good linearity in the range of 0.5ppm to 100ppm. The relative average error of the concentration value obtained by substituting the measured values ​​of the "method verification sample" into the standard curve is less than 25%, and the relative average deviation of the results of three repeated experiments at each concentration is less than 20%. It shows that the screened peptides have good performance in the quantification of the corresponding esterase / lipase.

[0091] Table 5

[0092]

[0093]

[0094] Example 2

[0095] This embodiment provides an analytical method for quantitatively detecting the amount of esterase / lipase residues in antibodies and analogs. Figure 1 The flowchart of the esterase / lipase analysis method based on parallel reaction monitoring mass spectrometry in this embodiment. After non-denaturing enzyme digestion and heat denaturation precipitation of product protein, the dynamic range of protein in the sample is reduced, and the introduction of parallel reaction monitoring acquisition mode is conducive to the monitoring of low-abundance components. The introduction of recombinant protein standards is conducive to more accurate quantification.

[0096] like Figure 1 As shown, first prepare a human serum immunoglobulin G standard mixed with a recombinant esterase / lipase standard in a certain proportion, and hydrolyze it with the sample to be tested under non-denaturing conditions using trypsin. In this process, only a low proportion of antibody product proteins are cleaved, while host cell proteins including recombinant esterase / lipase are cleaved into peptides. After adding dithiothreitol to open the disulfide bonds of the protein, a thermal denaturation method is used to precipitate the antibody product proteins that are not cleaved in the sample to reduce the dynamic range of protein concentration in the sample. Subsequently, according to the ion pair list recorded in the method of the present invention, liquid chromatography-parallel reaction monitoring-tandem mass spectrometry is used to selectively collect the characteristic peptides of specific esterases / lipases in the sample. According to the extraction chromatogram (EIC) peak area of ​​the corresponding esterase / lipase characteristic peptide in the standard and the logarithm of the incorporation value, a linear fit is performed, and then the EIC peak area of ​​the corresponding esterase / lipase characteristic peptide in the sample to be tested is substituted into the linear fitting curve for calculation to obtain the measured value of the corresponding esterase / lipase content.

[0097] The analytical method comprises the following steps:

[0098] 1. Sample preparation

[0099] Low-absorption centrifuge tubes and low-absorption pipette tips must be used throughout the following preparation process.

[0100] (1) Preparation of esterase / lipase standards

[0101] The eight recombinantly expressed esterase / lipase standards were diluted to 0.2 mg / mL using a 50 mM Tris-HCl buffer solution at pH 8.0 and then mixed in equal volumes to obtain a 25 ng / μL lipase and esterase standard working solution.

[0102] (2) Preparation of standard: Take a certain amount of human serum immunoglobulin G and package it into 1 mg per tube. After adding lipase and esterase standard working solution in a certain ratio (such as 0.5 ppm, 1 ppm or 2.5 ppm, etc.), dilute it to 200 μL with 50 mM Tris-HCl buffer solution with a pH value of 8.0 and a concentration of 50 mM for later use.

[0103] (3) Sample dilution: Take 1 mg of sample and dilute it to 200 μL with 50 mM Tris-HCl buffer solution with a pH value of 8.0 for later use.

[0104] (4) Enzymatic hydrolysis: Add 1.5 μL of 0.5 mg / mL trypsin (product protein: enzyme = 4000:3, w / w) to all samples (samples to be tested and standards), then vortex to mix, centrifuge, and incubate at 37°C for 2 h±5 min.

[0105] (5) Reduction and antibody removal: Add 1 μL of 1M dithiothreitol to the enzymatically hydrolyzed sample, mix well and incubate in a heating apparatus at 90°C for 10 min. Centrifuge all samples at 13,000 g for 10 min. Carefully transfer 180 μL of supernatant from the sample to a new 0.6 mL centrifuge tube and discard the precipitate. If precipitated particles are obtained in any sample, the supernatant taken from all samples needs to be centrifuged again under the same conditions, and an equal volume of supernatant is taken (recorded) until no precipitated particles are obtained in any sample. Then add 2.5 μL of 20% formic acid to terminate the enzyme reaction.

[0106] (6) Vacuum drying: The supernatant obtained in the previous step is centrifuged using a centrifugal concentrator at room temperature, a rotation speed of 1500 rpm or more, and an air pressure of less than 75 mbar until the sample is dried or the volume no longer decreases significantly (at least 1 hour).

[0107] (7) Peptide reconstitution: Add 50 μL of 0.1% formic acid aqueous solution to all samples dried in the previous step, vortex to fully dissolve and mix, and centrifuge at 13000g for 10 min to remove bubbles and insoluble matter that appear during the mixing process. Then transfer 45 μL of the supernatant to a glass insert vial for injection and testing.

[0108] 2. Instrument detection and collection method

[0109] An ultra-high performance liquid chromatography-mass spectrometer was used for data acquisition, and 20 μL of the enzymatically hydrolyzed sample was detected on an Agilent / UHPLC (Ultra-High Performance Liquid Chromatography, UPLC) 1290 InfinityII or an equivalent ultra-high performance liquid chromatography instrument. An Agilent / Poroshell 120SB-C18 (2.1×150mm, 2.7μm, Agilent) reverse phase column was used for sample separation. During the liquid phase separation process, the column temperature was 60°C, the liquid phase flow rate was 0.2mL / min, and 0.1% formic acid in water (mobile phase A) and acetonitrile (mobile phase B) system was used for elution, and the elution gradient program was:

[0110] From 0 to 2 minutes, the volume fraction of mobile phase B was 0.2%, and the rest was mobile phase A;

[0111] From 2 to 25 minutes, the volume fraction of mobile phase B was changed from 0.2% to 10% at a constant rate, and the rest was mobile phase A;

[0112] From 25 to 82 minutes, the volume fraction of mobile phase B was changed from 10% to 30% at a constant rate, and the rest was mobile phase A.

[0113] After elution is completed, the column is flushed with 90% phase B for 3 minutes. After the flushing is completed, the proportion of phase B is restored to the initial state, and the column is balanced.

[0114] Mass spectrometry data were collected in positive ion mode using a mass spectrometer such as Thermo / Orbitrap Exploris 480. The instrument did not scan the primary spectrum, and the ion source electrospray ionization source voltage was 4 kV. High energy collision dissociation (HCD) was used in the secondary fragmentation mode, and parallel reaction monitoring was performed according to the list of collected ion pairs shown in Table 1.

[0115] 3. Data analysis methods

[0116] When analyzing the collected data, the Skyline analysis software (MacCoss Lab of University of Washington, Washington, USA) was used for peptide matching and automatic integration of peak areas. When reporting the level of each esterase / lipase, the peak area of ​​the extraction chromatogram (EIC) of the corresponding esterase / lipase characteristic peptide in the standard was taken to be linearly fitted with the logarithm of the spiked value, and then the EIC peak area of ​​the corresponding esterase / lipase characteristic peptide in the sample to be tested was substituted into the linear fitting curve for calculation to obtain the measured value of the corresponding esterase / lipase content.

[0117] Example 3

[0118] This example compares and examines mass spectrometry methods using a parallel reaction monitoring acquisition mode and a data-dependent acquisition mode to verify the effect of the parallel reaction monitoring acquisition mode on the sensitivity of the method.

[0119] 1. Sample preparation

[0120] Low-absorption centrifuge tubes and low-absorption pipette tips must be used throughout the following preparation process.

[0121] (1) Preparation of esterase / lipase standard refers to Example 2.

[0122] (2) Preparation of standard: Take a certain amount of human serum immunoglobulin G and dispense it into 1 mg per tube. Add lipase and esterase standard working solution with final concentrations of 0.25 ppm, 0.5 ppm, 1.0 ppm, 2.5 ppm, 5.0 ppm, 10.0 ppm, 25.0 ppm, 50.0 ppm and 100.0 ppm, and dilute to 200 μL with 50 mM Tris-HCl buffer solution at pH 8.0.

[0123] (3) Sample dilution: Take 1 mg of the above sample and dilute it to 200 μL with 50 mM Tris-HCl buffer solution with a pH value of 8.0 for later use.

[0124] (4) Enzymatic hydrolysis: 1.5 μL of 0.5 mg / mL trypsin (product protein: enzyme = 4000:3, w / w) was added to all samples, then vortexed to mix, centrifuged, and incubated at 37° C. for 2 h±5 min in a thermostat.

[0125] (5) Reduction and antibody removal: Add 1 μL of 1M dithiothreitol to the enzymatically hydrolyzed sample, mix well and incubate in a heating apparatus at 90°C for 10 min. Centrifuge all samples at 13,000 g for 10 min. Carefully transfer 180 μL of supernatant from the sample to a new 0.6 mL centrifuge tube and discard the precipitate. If precipitated particles are obtained in any sample, the supernatant taken from all samples needs to be centrifuged again under the same conditions, and an equal volume of supernatant is taken (recorded) until no precipitated particles are obtained in any sample. Then add 2.5 μL of 20% formic acid to terminate the enzyme reaction.

[0126] (6) Vacuum drying: The supernatant obtained in the previous step is centrifuged using a centrifugal concentrator at room temperature, a rotation speed of 1500 rpm or more, and an air pressure of less than 75 mbar until the sample is dried or the volume no longer decreases significantly (at least 1 hour).

[0127] (7) Peptide reconstitution: Add 50 μL of 0.1% formic acid aqueous solution to all samples dried in the previous step, vortex to fully dissolve and mix, and centrifuge at 13000g for 10 min to remove bubbles and insoluble matter that appear during the mixing process. Then transfer 45 μL of the supernatant to a glass insert injection vial for injection and detection. The same sample was collected in data-dependent mode and parallel reaction monitoring mode.

[0128] 2. Detection steps

[0129] An ultra-high performance liquid chromatography-mass spectrometer was used for data acquisition, and the detection conditions were as described in Example 2.

[0130] When data-dependent mode is used for acquisition, data-dependent mode mass spectrometry data acquisition is performed in positive ion mode based on mass spectrometers such as Thermo / Orbitrap Exploris 480. The instrument scans the primary spectrum according to preset parameters, the ion source electrospray ionization source voltage is 4kV, and the parent ion is fragmented for the secondary stage according to the signal intensity sorting and dynamic exclusion principles. The secondary fragmentation mode uses high energy collision dissociation (HCD).

[0131] When parallel reaction monitoring mode is used for acquisition, data-dependent mode mass spectrometry data acquisition is performed based on the contents of Table 1 in positive ion mode on a mass spectrometer such as Thermo / Orbitrap Exploris 480, the instrument does not scan the primary spectrum, and the ion source electrospray ionization source voltage is 4 kV.

[0132] 3. Comparison results

[0133] Skyline software and Thermo / Proteome Discoverer software were used to analyze the data collected in parallel reaction monitoring mode and data collected in data-dependent mode, respectively. For data collected in parallel reaction monitoring mode, if three sub-ions with overlapping peak areas and similar peak shapes appeared in the corresponding retention time of the candidate peptide of a certain protein, it was considered to be detected. For data collected in data-dependent mode, if a protein had confidence = high in the search results, it was considered to be detected. The detection limit of each esterase / lipase was determined according to the lowest sample concentration that could be detected in different modes.

[0134] The detection limits of the two acquisition modes for eight esterases / lipases are shown in Table 6.

[0135] Table 6

[0136]

[0137]

[0138] The data show that this embodiment uses the characteristic of the parallel reaction monitoring acquisition mode that can target and fragment specific ions to improve the detection sensitivity. The parallel reaction monitoring acquisition mode significantly improves the detection sensitivity of 8 esterases / lipases.

[0139] Example 4

[0140] A total of 24 samples prepared in parallel at 8 concentration points were subjected to quantitative analysis of 8 esterases / lipases to verify the precision of the analytical method.

[0141] 1. Sample preparation was carried out according to Example 1.

[0142] 2. The instrument detection collection method and data analysis method are carried out with reference to Example 1.

[0143] 3. Experimental results

[0144] The collected data were analyzed by using Skyline data analysis software, and the results showed that the peak areas of representative peptides of 8 esterases / lipases were shown in Table 7. The relative average deviation of the peak areas of all peptides was within 20% after three replicates, which proved the high accuracy of this method.

[0145] Table 7 is a summary of the results of three repeated experiments on the peak areas of representative peptides of different esterases / lipases.

[0146] Table 7

[0147]

[0148]

[0149] Note: The 8 esterases / lipases are named protein 1-8 in the order listed in the reagent / consumables table.

[0150] Example 5

[0151] This example conducts a stability experiment.

[0152] Experimental steps:

[0153] 1. Sample preparation was carried out according to Example 1, and the final concentration of each lipase / esterase standard was set to 10 ppm.

[0154] 2. The instrument detection collection method and data analysis method are carried out in accordance with Example 1. For the same sample, the sample is injected again 24 hours and 48 hours after injection. The peak areas of the candidate peptides measured are compared.

[0155] Experimental results: For the injection results of the sample at T0, 24 hours, and 48 hours, the relative average deviation of the peak area of ​​each candidate peptide segment was less than 20%, indicating that the sample prepared by the patent method has good stability within 48 hours.

[0156] In summary, the present invention provides an analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs and its application. The method can simultaneously detect the contents of eight esterases / lipases. The analytical method of the present invention has high sensitivity, high detection throughput and high accuracy, and has important application value in the identification, quantification and monitoring of host cell proteins related to polysorbate degradation.

[0157] 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 method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs, characterized in that: The analysis method comprises: (S1) enzymatically hydrolyzing, reducing, removing antibodies, vacuum drying and re-dissolving the sample to be tested and the standard substance in sequence; the standard substance contains an esterase / lipase standard substance and human serum immunoglobulin G; the amino acid sequence of the esterase / lipase standard substance includes that shown in SEQ ID NO: 1-8; (S2) using ultra-high performance liquid chromatography-mass spectrometry to detect the reconstituted sample and collect mass spectrometry data; (S3) Use analysis software to perform peptide matching and automatic peak area integration, and perform linear fitting by taking the logarithm of the peak area of ​​the extracted chromatogram flow chart of the corresponding esterase / lipase characteristic peptide in the standard and the spiked value. Then, substitute the peak area of ​​the extracted chromatogram flow chart of the corresponding esterase / lipase characteristic peptide in the sample to be tested into the linear fitting curve for calculation to obtain the measured value of the corresponding esterase / lipase content.

2. The analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs according to claim 1, characterized in that: In (S1), the standard is prepared by a method comprising the following steps: mixing human serum immunoglobulin G with an esterase / lipase standard working solution, diluting with a Tris-HCl buffer solution having a pH value of 7.0-8.0 and a concentration of 45-55 mM, wherein the final concentration of the human serum immunoglobulin G is greater than or equal to 5 μg / μL, and the final concentration of the esterase / lipase is 0.5-100 ppm.

3. The analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs according to claim 2, characterized in that: The solvent of the esterase / lipase standard working solution is a tris(hydroxymethyl)aminomethane hydrochloride buffer solution with a pH value of 7.0-8.0 and a concentration of 20-200 mM.

4. The analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs according to any one of claims 1 to 3, characterized in that: In (S1), the enzymatic hydrolysis is performed by a method comprising the following steps: vortexing the sample and trypsin, centrifuging, and incubating at 35-39°C for 2h±5min; Preferably, the ratio of the sample to trypsin is (3800-4200):3, w / w; Preferably, the reduction and antibody removal are performed by a method comprising the following steps: mixing the enzymatically hydrolyzed sample with dithiothreitol, incubating after mixing, collecting the supernatant by centrifugation, and adding formic acid to terminate the enzyme reaction; Preferably, the final concentration of dithiothreitol is 0.003-0.006M; Preferably, the incubation temperature is 90-95°C and the time is 10-15 min; Preferably, the centrifugation condition is: 12000-13000g for 10-15min.

5. The analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs according to any one of claims 1 to 4, characterized in that: In (S1), the solvent used for the re-dissolution is a 0.1-0.15% formic acid aqueous solution.

6. The analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs according to any one of claims 1 to 5, characterized in that: In (S2), the mobile phase used in the ultra-high performance liquid chromatography includes: mobile phase A: water with 0.1-0.15% formic acid, mobile phase B: acetonitrile with 0.1-0.15% formic acid; the mobile phase is used for gradient elution; Preferably, the flow rate of the mobile phase is 0.2-0.22 mL / min.

7. The analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs according to claim 6, characterized in that: The elution procedure is: In the first 0-2 minutes, the volume fraction of mobile phase B was 0.2-0.3%, and the rest was mobile phase A; From 2 to 25 minutes, the volume fraction of mobile phase B changes from 0.2-0.3% to 10-10.5% at a constant rate, and the rest is mobile phase A; From 25 to 82 minutes, the volume fraction of mobile phase B changed at a constant rate from 10-10.5% to 30-30.5%, and the rest was mobile phase A.

8. The analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs according to any one of claims 1 to 7, characterized in that: In step (S2), the chromatographic column used in the ultra-high performance liquid chromatography is Agilent / Poroshell 120SB-C18, 2.1×150 mm, 2.7 μm, Agilent; Preferably, the column temperature of the chromatographic column is set at 60-62°C.

9. The analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs according to any one of claims 1 to 8, characterized in that: In step (S2), the mass spectrometry conditions include: performing mass spectrometry data acquisition in positive ion mode, with an ion source electrospray ionization source voltage of 4 kV; using high-energy collision dissociation in secondary fragmentation mode, and performing parallel reaction monitoring acquisition according to the collected ions as shown below; Preferably, in step (S3), the analysis software includes Skyline analysis software.

10. Use of the analytical method for quantitatively detecting the residual amount of esterase / lipase in antibodies and analogs according to any one of claims 1 to 9 in the detection of biological pharmaceutical preparations of antibodies and analogs.

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