A method for detecting HCP antibody coverage and its application

Abstract

The present invention provides a method for detecting HCP antibody coverage and its application, the method comprising: (1) biotinylating a polyclonal antibody for HCP detection, fixing the biotinylated polyclonal antibody on a solid substrate connected with streptavidin; (2) using the solid substrate obtained in step (1) to capture and enrich HCP protein from a test solution, and then eluting; (3) performing liquid chromatography-mass spectrometry detection and analysis on the eluate obtained in step (2), and calculating the coverage of the polyclonal antibody. The overall experimental operation of the method is easy to achieve standardization, reduce nonspecific adsorption, and ensure that high-affinity HCP can be eluted; thereby improving the authenticity and sensitivity of the HCP coverage result of the detection antibody.

Description

Technical Field

[0001] The present invention belongs to the field of biomacromolecule detection, and specifically relates to a method for detecting HCP antibody coverage and an application thereof. Background Art

[0002] Residual host cell proteins (HCP) in biological products can lead to the risk of adverse clinical reactions such as allergic reactions or potential toxicity. Therefore, it is necessary to establish appropriate HCP detection and verification methods to strictly control the quality of biological products. The residual HCP content in biological products is generally considered to be the critical quality attributes (Critical Quality Attributes, CQA) of the product. It is an important evaluation indicator for process robustness monitoring and an important quality control indicator for the product. HCP is defined as a process-related impurity in many regulations. Relevant guidelines point out that its level should be minimized by using appropriate and controlled manufacturing processes. Biological product manufacturers should evaluate possible impurities and develop quality standards for impurities based on preclinical data and clinical experience. In the residual limit of the final product, the recommended limit value is below 0.01%.

[0003] Currently, the method widely used for HCP detection is the double antibody sandwich enzyme-linked immunosorbent assay (ELISA), but not all HCPs can be used as antigens to produce antibodies. Since HCP components are different, the content of each protein component is also inconsistent, and the ability to produce antibodies as an immunogen is also different, the anti-HCP antibodies prepared in this way cannot fully detect all components in HCP, so there will be "missed detection", so it is necessary to evaluate the coverage of antibodies in the HCP ELISA kit.

[0004] In order to evaluate the coverage of polyclonal antibodies in HCP ELISA detection kits, various technologies have been developed in the art, including: a method combining two-dimensional electrophoresis (2-DE) and protein immunoblotting (Western Blot, WB) technology, fluorescent differential blotting electrophoresis (DIBE, Differential InBlot Electrophoresis), AAE (Antibody Affinity Extraction)-2D, etc.

[0005] Among them, a method combining two-dimensional electrophoresis and protein immunoblotting technology is used to overlay the HCP total protein separation image and the HCP antibody binding image obtained separately, match the gel spots, record the number of matched and unmatched gel spots respectively, and calculate the coverage results. However, this method has poor repeatability and low sensitivity, and cannot accurately match immune spots and gel spots.

[0006] Using fluorescence differential in blot electrophoresis (DIBE) technology, a multiplex fluorescence method based on CyDye pre-labeled protein immunoblotting, Cy3-labeled HCP proteins can be directly compared with protein spots actually detected by Cy5-labeled antibodies on the same membrane, eliminating the error between the gel and the blot membrane in traditional methods.

[0007] AAE-2D is a method based on immunochromatographic columns and two-dimensional electrophoresis. This method couples HCP antibodies to chromatographic fillers, and then loads HCP samples. During the process, HCP antibodies capture and bind recognizable HCPs, while unrecognized HCPs flow through because they are not captured. Finally, recognizable HCPs are collected through elution conditions such as low pH, and the pre-capture HCP samples and captured eluted samples are subjected to two-dimensional electrophoresis and silver staining analysis, respectively, to calculate the antibodies.

[0008] Various techniques have been developed in this field to evaluate the coverage of polyclonal antibodies in HCP ELISA test kits, but these methods each have obvious shortcomings. For example, ① poor experimental repeatability, the coverage of the same material tested in the same laboratory or between different laboratories varies greatly; ② low sensitivity, often resulting in the inability to detect low-concentration HCP; ③ HCP antigen denaturation treatment, the epitope is easily destroyed, which is not conducive to immunoassays; ④ the transfer efficiency is difficult to control, resulting in excessive transfer loss or inability to transfer from the gel; ⑤ data analysis problems, high-concentration points are often blurred and unclear, and cannot accurately match immune points and gel spots; ⑥ anti-HCP resin preparation is difficult, and the process of antibody affinity extraction method is too complicated; ⑦ high-affinity HCP is difficult to elute, resulting in low coverage, and low-concentration proteins may be missed; ⑧ the amount of antibodies used is large, and the time cost and economic cost are very high in actual application. Therefore, this field urgently needs a method for evaluating the coverage of polyclonal antibodies for HCP detection that is simple to operate, controllable conditions, and reliable results. Summary of the invention

[0009] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for detecting HCP antibody coverage and its application. The method combines the capture of HCP protein samples, does not require denaturation, maintains consistency with the HCP ELISA detection conditions, improves the authenticity of the results, has good method repeatability, and improves the detection sensitivity of low-concentration HCPs by enrichment, ensuring that small molecular weight proteins are not lost; the entire experimental operation is easy to standardize, reduces nonspecific adsorption, and ensures that high-affinity HCPs can be eluted; thereby improving the credibility of the detection antibody HCP coverage.

[0010] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0011] In a first aspect, the present invention provides a method for detecting HCP antibody coverage, the method comprising:

[0012] (1) biotinylating a polyclonal antibody for HCP detection, wherein the molar ratio between the polyclonal antibody and biotin is 1:(1-10); the reaction conditions of the biotinylation are: the reaction temperature is 4-37° C., and the reaction time is 1-3 h; the biotinylated polyclonal antibody is fixed on a solid substrate connected with streptavidin, and the ratio of the biotinylated polyclonal antibody to the solid substrate connected with streptavidin is 1:(5-20);

[0013] (2) using the solid substrate obtained in step (1) to capture and enrich HCP proteins from the test solution, and then eluting;

[0014] (3) Performing liquid chromatography-mass spectrometry (LC-MS) analysis on the eluate obtained in step (2) to calculate the coverage of the polyclonal antibody.

[0015] The present invention creatively biotinylates a polyclonal antibody for HCP detection at a certain molar ratio, and utilizes the extremely high affinity of streptavidin for biotin, so that the biotinylated polyclonal antibody for HCP detection can be irreversibly fixed to a streptavidin solid substrate; the solid substrate fixed with the polyclonal antibody is brought into contact with an HCP sample, thereby capturing HCP proteins in the HCP sample that can specifically bind to the polyclonal antibody, washing away unrecognized HCP proteins, collecting recognizable HCP proteins by elution, and comparing the eluted HCP protein sample with the total HCP sample by high-resolution mass spectrometry detection and analysis (such as Figure 1 ).

[0016] The method of the present invention combines the capture of HCP protein samples without denaturation, maintains consistency with HCP ELISA detection conditions, and improves the authenticity of the results; and improves the detection sensitivity of low-concentration HCPs through enrichment, ensuring that small molecular weight proteins are not lost; the entire experimental operation is standardized, nonspecific adsorption is reduced, and high-affinity HCPs can be eluted, thereby improving the authenticity of the HCP coverage results of the detection antibody.

[0017] In the present invention, in step (1), the molar ratio between the polyclonal antibody and biotin is 1:(1-10), for example, it can be 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, etc.

[0018] Biotinylation of polyclonal antibodies for HCP detection utilizes the extremely high affinity of streptavidin for biotin, which can be irreversibly fixed to the streptavidin solid substrate; despite this, the method of directly labeling polyclonal antibodies for HCP detection has not been widely popularized. This is because this method still has problems such as cumbersome labeling operation and decreased activity of the obtained labeled antibodies. Most experimenters often give up the technology of directly labeling antibodies due to its difficult operation.

[0019] In the present invention, in step (1), the biotinylation reaction conditions are as follows: the reaction temperature is 4-37°C, for example, it can be 4°C, 10°C, 15°C, 20°C, 25°C, 30°C or 37°C, and the reaction time is 1-3h, for example, it can be 1h, 1.5h, 2h, 2.5h or 3h, etc.

[0020] In the present invention, the molar ratio of biotin to antibody and the reaction temperature will affect the coupling effect. The binding condition is better when the molar ratio of antibody to biotin is 1:1-1:5, the binding condition is better when the reaction temperature is 20-37°C, and the binding condition is better when the reaction time is 1-3h.

[0021] In the present invention, the antibody is biotinylated and then combined with a solid substrate connected with streptavidin to capture the antigen, and the separate antigen can be obtained after elution.

[0022] In the present invention, in step (1), the ratio of the biotinylated polyclonal antibody to the solid substrate connected with streptavidin is 1:(5-20), for example, it can be 1:5, 1:10, 1:15 or 1:20.

[0023] Preferably, in step (1), the biotin is selected from N-hydroxysulfosuccinimide (NHS) ester-biotin.

[0024] In the present invention, biotin N-hydroxysulfosuccinimide (NHS) ester is used to efficiently react with primary amine groups (-NH2) in antibodies, such as the side chain of lysine (K), to form a stable amide bond. In the present invention, the molar ratio and reaction conditions between the polyclonal antibody for HCP detection and biotin are optimized. The present invention directly labels antibodies simply and quickly without compromising specificity and activity, and takes advantage of the specific binding of biotin and streptavidin.

[0025] Preferably, in step (2), the capture and enrichment reaction temperature is 4-37°C, for example, it can be 4°C, 10°C, 15°C, 20°C, 25°C, 30°C or 37°C.

[0026] Preferably, in step (2), the elution solution used for elution comprises, in terms of concentration, 0.05-0.15 M glycine (for example, 0.05 M, 0.1 M or 0.15 M, etc.) and 1.5-2.5 M Tris (for example, 1.5 M, 2 M or 2.5 M, etc.), the solvent is water, and the pH value is 1-3 (for example, 1, 2 or 3, etc.).

[0027] Preferably, in step (2), the elution time is 10-20 minutes, for example, 10, 15 or 20 minutes.

[0028] Preferably, in step (2), the elution temperature is 4-37°C, for example, 4°C, 10°C, 15°C, 20°C, 25°C, 30°C or 37°C.

[0029] In the present invention, an elution solution with a low pH is used to elute the antigen bound to the antibody.

[0030] Preferably, in step (3), the LC-MS detection and analysis further includes a step of pre-treating the eluate.

[0031] Preferably, the pretreatment comprises: sequentially denaturing, reducing, alkylating, enzymatically cleaving, acid hydrolyzing and concentrating and re-dissolving the HCP protein in the eluate.

[0032] Preferably, the denaturing agent used in the denaturation step is RapiGest SF proteolytic surfactant.

[0033] Preferably, the final concentration of the RapiGest SF proteolytic surfactant in the reaction solution is 0.025-0.075%, for example, 0.025%, 0.05% or 0.075%.

[0034] Preferably, the reaction time of the denaturation step is 10-30 minutes, for example, 10, 15, 20, 25 or 30, etc., and the reaction temperature is 60±5°C, for example, 55°C, 60°C or 65°C, etc.

[0035] Preferably, the reducing agent used in the reduction step is dithiothreitol.

[0036] Preferably, the final concentration of dithiothreitol in the reaction solution is 15-25 mM, for example, 15 mM, 20 mM or 25 mM.

[0037] Preferably, the reaction time of the reduction step is 50-70 minutes, for example, 50, 55, 60, 65 or 70, etc., and the reaction temperature is 60±5°C, for example, 55°C, 60°C or 65°C, etc.

[0038] Preferably, the alkylating agent used in the alkylation step is iodoacetamide.

[0039] Preferably, the final concentration of iodoacetamide in the reaction solution is 8-12 mM, for example, 8 mM, 9 mM, 10 mM, 11 mM or 12 mM.

[0040] Preferably, the reaction time of the alkylation step is 25-35 minutes, for example, 25, 30 or 35, etc., and the reaction temperature is 20-25°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C or 25°C, etc., and the reaction is carried out in the dark.

[0041] Preferably, the enzyme used in the enzymatic cleavage step is trypsin.

[0042] Preferably, the ratio of trypsin to HCP protein is 1:(30-50), for example, it can be 1:30, 1:35, 1:40, 1:45 or 1:50, etc.

[0043] Preferably, the reaction time of the enzyme cleavage step is 4-20 h, for example, 4 h, 10 h, 15 h or 20 h, and the reaction temperature is 37±2°C, for example, 35°C, 36°C, 37°C, 38°C or 39°C.

[0044] Preferably, the acid used in the acid hydrolysis step is formic acid.

[0045] Preferably, the final concentration of formic acid in the reaction solution is 0.25-0.75%, for example, it can be 0.25%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7% or 0.75%.

[0046] Preferably, the concentration and reconstitution step comprises: vacuum concentrating the acidified sample at 45±2°C (for example, 43°C, 45°C or 47°C, etc.), reconstituted with mobile phase to 50-200 μL (for example, 50 μL, 80 μL, 100 μL, 120 μL, 150 μL, 180 μL or 200 μL, etc.), centrifuging and taking the supernatant for LC-MS detection.

[0047] In the present invention, in the pretreatment step, the HCP treatment amount is 1-3 mg, and the injection amount for detection on the machine after pretreatment is 15-50 μg. The present invention also examines the effects of treatment amount and injection amount on the detection results. Based on the existing data and comprehensive consideration of sample usage, material cost, instrument limit and other aspects, the recommended treatment amount can be in the range of 1-3 mg, and the recommended injection amount is not more than 50 μg.

[0048] Preferably, in step (3), the formula for calculating the coverage of the polyclonal antibody is:

[0049] Coverage (%) = A / (A+BC)

[0050] Wherein, A is the number of proteins specifically bound by the polyclonal antibody, B is the total number of proteins in the HCP sample, and C is the number of proteins that are identical between the two.

[0051] In a second aspect, the present invention provides application of the method for detecting HCP antibody coverage described in the first aspect in the detection of biological products.

[0052] 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.

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

[0054] The method of the present invention combines the capture of HCP protein samples without denaturation, maintains consistency with HCP ELISA detection conditions, and improves the authenticity of the results; and improves the detection sensitivity of low-concentration HCPs through enrichment, ensuring that small molecular weight proteins are not lost; the entire experimental operation can be standardized, reducing nonspecific adsorption, and ensuring that high-affinity HCPs can be eluted; thereby improving the authenticity of the HCP coverage results of the detection antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 It is a flow chart of the method principle.

[0056] Figure 2 It is the result of SDS-PAGE and UV detection of N-hydroxysuccinimide magnetic beads (NHS magnetic beads) solid substrate.

[0057] Figure 3 It is the result of SDS-PAGE and UV detection of Protein A magnetic beads solid substrate.

[0058] Figure 4 It is the result of SDS-PAGE and UV detection of mycoavidin magnetic beads (SA magnetic beads) solid substrate.

[0059] Figure 5 It is the detection spectrum of different molar ratios of antibody and biotin.

[0060] Figure 6 It is a detection graph of antibodies and biotin at different reaction temperatures.

[0061] Figure 7 It is a detection graph of different reaction times of antibodies and biotin.

[0062] Figure 8 This is a Veen comparison chart of the results of detecting HCP coverage peptides ≥1 with the same pretreatment amount (3 mg) and different loading amounts (15 μg / 50 μg / 150 μg).

[0063] Fig. 9 This is a Veen comparison chart of the results of detecting HCP coverage peptides ≥ 2 using the same pretreatment amount (3 mg) but different loading amounts (15 μg / 50 μg / 150 μg).

[0064] Fig.10 This is a Veen comparison chart of the results of detecting HCP coverage peptides ≥1 with different pre-treatment amounts (1mg / 3mg / 5mg) and the same amount on the machine (50μg).

[0065] Fig.11 This is a Veen comparison chart of the results of detecting HCP coverage peptides ≥ 2 with different pre-treatment amounts (1mg / 3mg / 5mg) and the same amount on the machine (50μg).

[0066] Fig.12 This is a Veen comparison chart of the results of detecting HCP coverage peptides ≥1 at different enzyme digestion incubation times.

[0067] Fig.13 This is a Veen comparison chart of the results of detecting HCP coverage peptides ≥ 2 at different enzyme digestion incubation times.

[0068] Fig.14 This is a Veen comparison chart of the results of detecting HCP coverage peptides ≥1 at different enzyme cleavage ratios.

[0069] Fig.15 This is a Veen comparison chart of the results of detecting HCP coverage peptides ≥ 2 at different enzyme cleavage ratios.

[0070] Fig.16 It is the detection result spectrum of HCP before and after affinity capture.

[0071] Fig.17 It is a 2D-WB Ruby fluorescence staining map.

[0072] Fig.18 It is a 2D-WB immunoblot profile.

[0073] Fig.19 This is a graph of the precision results of three tests of HCP antibody coverage. DETAILED DESCRIPTION

[0074] 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.

[0075] 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.

[0076] The relevant instruments and equipment information used in the following specific implementations are shown in Table 1, the relevant consumables information is shown in Table 2, and the sample information is shown in Table 3.

[0077] Table 1

[0078] Serial number Instrument Description Manufacturer / Model 1 High Resolution Mass Spectrometry Thermo / Q Exactive Plus 2 High performance liquid chromatography Thermo / Vanquish F 3 Label-free biomolecule analyzer Octet / Octet R4 4 Vortex mixer Thermo / Digital Vortex Mixer 5 Vacuum Concentrator Eppendorf / Concentrator plus 6 Micro centrifuge Thermo / MySPIN12 7 Constant temperature metal bath Shanghai Yiheng Scientific Instrument Co., Ltd. / TU-100C 8 Electric constant temperature incubator Shanghai Yiheng Scientific Instrument Co., Ltd. DHP-9032B

[0079] Table 2

[0080]

[0081] Table 3

[0082] Sample name Sample batch number Sample concentration Anti-CHO HCP 3G-0016-1-AF 2mg / mL HCP proteins 2084-R2301-1-B6-UF / DF 1.61mg / mL

[0083] Example 1

[0084] This example optimizes the selection conditions for three different solid substrates.

[0085] Analytical methods:

[0086] (1) Buffer preparation:

[0087] PBS solution (pH 7.4): Take a packet of phosphate granules, pour it into 1000mL of purified water, dissolve and mix well, store at 2-8℃, and the validity period is 1 month.

[0088] 0.1M Glycine (pH 2.0): Weigh 0.7507g of glycine and put it into a beaker. Use a measuring cylinder to measure 100mL of water and add it to the beaker. Mix well and adjust the pH to 2.0. Store at 2-8℃. The shelf life is 3 months.

[0089] (2) Experimental procedures

[0090] Wash different solid substrates: Place three different solid substrates, N-hydroxysuccinimide magnetic beads (NHS magnetic beads), protein A magnetic beads (ProteinA magnetic beads), and streptavidin magnetic beads (SA magnetic beads) into a magnetic stand, collect the beads and discard the supernatant. Add 300-500 μL PBS solution (pH 7.4) and wash repeatedly for 3 times. After the last wash, discard the supernatant.

[0091] Conjugated antibodies: Add 0.1-0.5 mg of total antibody in a volume of 200-600 μL to three different solid substrates, NHS magnetic beads, Protein A magnetic beads, and SA magnetic beads, and mix the magnetic beads and antibodies through a mixer and incubate at room temperature for 100-200 minutes. After the reaction, add 300-500 μL PBS solution (pH 7.4) and wash repeatedly for 3 times. After the last wash, discard the supernatant. (NHS magnetic beads are blocked at room temperature for 100-200 minutes after the reaction is completed, and the antibodies need to be biotinylated before SA magnetic beads are conjugated to antibodies).

[0092] Enrichment of antigen: After coupling the above three different solid substrates with antibodies, add a total of 0.2-1 mg and a volume of 200-600 μL of antigen, and mix the magnetic beads and antigen through a mixer, and incubate at room temperature for 100-200 minutes. After the reaction is completed, add 300-500 μL PBS solution (pH 7.4) and wash repeatedly for 3 times. After the last wash, discard the supernatant.

[0093] Elution of antigen: Add 50-200 μL of elution buffer 0.1 M glycine (pH 2.0) to the three different solid substrate test tubes above, cap the tubes on a rotator, and incubate on a mixer at room temperature for 10-20 minutes.

[0094] Collect samples for testing: Perform SDS-PAGE and UV detection on samples from three different solid substrates, NHS magnetic beads, Protein A magnetic beads, and SA magnetic beads, before and after antibody coupling, before and after antigen coupling, and in combination with the elution solution.

[0095] (3) Data analysis

[0096] The results of SDS-PAGE and UV detection of N-hydroxysuccinimide magnetic beads (NHS magnetic beads) solid substrate are as follows Figure 2 As shown, the results of SDS-PAGE and UV detection of protein A magnetic beads solid substrate are as follows Figure 3 As shown, the results of SDS-PAGE and UV detection of streptavidin magnetic beads (SA magnetic beads) solid substrate are as follows Figure 4 As shown in the figure, it can be seen that the NHS magnetic bead solid substrate can couple with 50-80μg of antibody, and the antibody coupled with the magnetic beads captures 5-10μg of antigen, and less antigen is obtained after elution; the Protein A magnetic bead solid substrate can couple with 100-500μg of antibody, and the antibody coupled with the magnetic beads captures 50-200μg of antigen, but after elution, not only the antigen is obtained, but also the antibody is eluted; the SA magnetic bead solid substrate can couple with 50-200μg of antibody, and the antibody coupled with the magnetic beads captures 20-100μg of antigen, and a single antigen can be obtained after elution.

[0097] Example 2

[0098] This example optimizes the biotinylation conditions of polyclonal antibodies for HCP detection.

[0099] Analytical methods:

[0100] (1) Buffer preparation:

[0101] PBS solution (pH 7.4) was prepared according to Example 1.

[0102] Q buffer: PBS + 0.02% Tween-20, stored at 2-8°C, valid for 14 days.

[0103] (2) Experimental steps:

[0104] Anti-CHO HCP antibody biotin labeling: Add biotin EZ-Link to the Anti-CHO HCP antibody solution at a molar ratio of antibody to biotin of 1:1-1:40 TM NHS-LC-LC-biotin, after the two are mixed, reacted under different reaction temperatures and reaction times, the excess biotin is removed through a vertical membrane ultrafiltration tube, and the UV value is detected.

[0105] Octet R4 non-labeled biomolecule analyzer detection: biotinylated Anti-CHO HCP antibody was diluted to 10-100 μg / mL with Q buffer. After coupling with streptavidin sensor (SA sensor), the HCP protein was diluted to 50-200 nM with Q buffer.

[0106] (3) Data analysis

[0107] The molar ratio of biotin to antibody is 1:1-1:40. The detection spectrum is as follows Figure 5 As shown, the detection spectrum of different reaction temperatures is as follows Figure 6 As shown, the detection spectrum of different reaction times is as follows Figure 7 As shown in the figure, according to the comparison of signal response values, the binding conditions are better when the molar ratio of antibody to biotin is 1:1 to 1:5; the binding conditions are better when the reaction temperature is 20-37°C; and the binding conditions are better when the reaction time is 1-3h.

[0108] Example 3

[0109] This example optimizes the sample pretreatment conditions and related parameters.

[0110] 1. Analysis methods

[0111] (1) Reagent preparation:

[0112] 5% RapiGest: Dissolve 3 mg of RapiGest from one bottle in 60 μL of 5x GlycoWorks Rapid buffer to prepare 5% (w / v) RapiGest buffer. Vortex and mix thoroughly. Store at -80°C. The shelf life is 1 month.

[0113] 1M DTT: Weigh 0.0157 g DTT and add 100 μL mass spectrometry grade water, mix well, store at 2-8°C away from light, valid for 1 day.

[0114] 1M IAM: Weigh 0.0185g IAM and add 100μL mass spectrometry grade water, mix well, store at 2-8℃ away from light, valid for 1 day.

[0115] 1μg / μL Trypsin solution: Weigh 20μg trypsin powder, dissolve in 20μL water, vortex to mix, store at -20℃, valid for 1 year.

[0116] Mass spectrometry mobile phase A 0.1% FA·H2O: Measure 999 mL of mass spectrometry grade water (Fisher Chemical, W6-4), add 1 mL of FA, mix well, and then degas by ultrasonication for 15 min. Store at room temperature. The shelf life is 1 month.

[0117] Mass spectrometry mobile phase B 0.1% FA·ACN: Measure 999 mL of mass spectrometry grade acetonitrile (Fisher Chemical, A955-4), add 1 mL of FA, mix well, and then degas by ultrasonic for 15 minutes. Store at room temperature. The shelf life is 1 month.

[0118] (2) Investigation of pre-treatment conditions and parameters

[0119] ①Based on sample quantity investigation:

[0120] 1 mg, 3 mg, and 5 mg of empty samples were subjected to the same denaturation, reduction, and alkylation treatments, and incubated with the same Trypsin protease ratio for enzyme digestion. After the end of the enzyme digestion and incubation, acidification and enrichment were performed simultaneously.

[0121] Two methods were used for loading the machine: 50 μg of 1 mg, 3 mg, and 5 mg processed samples were injected respectively to investigate the difference in pre-treatment amounts; 15 μg, 50 μg, and 150 μg of 3 mg samples were injected respectively.

[0122] ②Investigation of enzyme digestion incubation time:

[0123] Take two groups of empty samples of the same amount, perform the same denaturation, reduction, and alkylation treatments, and add the same proportion of Trypsin protease for enzymatic incubation; one group is acidified and enriched after 4 hours of enzymatic incubation, and the other group is acidified and enriched after 16 hours of enzymatic incubation. Finally, all samples are loaded with the same amount.

[0124] ③Investigation of enzyme cutting ratio:

[0125] Three groups of empty samples of the same amount were taken and subjected to the same denaturation, reduction and alkylation treatments, and then incubated for 16 h with protein (m): enzyme (m) ratios of 40:1, 100:1 and 300:1, respectively. After the incubation, acidification and enrichment were performed simultaneously, and finally all samples were loaded onto the machine in the same amount.

[0126] (3) Data processing: After the original data of the samples are collected, the data are imported into the data processing workstation and processed using relevant software such as BioPharmaFinder.

[0127] 2. Experimental results and data analysis

[0128] (1) The sample volume investigation results are as follows: The same treatment of the empty load 3mg investigation sample volume results are shown in Table 4 and Figure 8 , Fig. 9 The same injection of 50 μg was used to investigate the pre-treatment empty load volume, as shown in Table 5 and Fig.10 , Fig.11 shown.

[0129] Table 4

[0130]

[0131] Table 5

[0132]

[0133] From the above results, it can be seen that under the conditions of the same processing volume (3 mg) and different injection volumes (15μg / 50μg / 150μg), the number of identified HCPs showed an upward trend with the increase of injection volume. Under the specific peptide identification conditions (from ≥1 to ≥2), the corresponding HCPs all showed an upward trend. Among the three conditions investigated, the point with the largest identification amplitude was the injection of 50μg; under the conditions of different processing volumes (1mg / 3mg / 5mg) and the same injection volume (50μg), with the increase of processing volume, the detection results did not show a relevant growth trend. The results under the conditions of 5mg, 1mg and 3mg were slightly different, and the number of identified HCPs showed a downward trend.

[0134] Theoretically, increasing the sample volume can increase the detectability of more HCPs and improve the credibility of peptide identification results. Based on existing data and comprehensive considerations of sample usage, material costs, instrument limitations, etc., the enzyme cleavage efficiency under 5mg conditions is not as good as that of 1mg or 3mg, and the recommended processing volume can be in the range of 1-3mg. A significant increase can be seen in the results when the injection volume increases from 15μg to 50μg. When the injection volume is greater than 50μg, the identification results show a certain marginal effect, and the increase is not obvious. The recommended injection volume should not exceed 50μg.

[0135] (2) The results of the enzyme digestion incubation time are shown in Table 6 and Fig.12 , Fig.13 shown.

[0136] Table 6

[0137]

[0138] From the above results, it can be seen that under the conditions of different enzyme cutting time, there is no significant difference in the identification results between the 4h group and the 16h group. The overall results of this round show that enzyme cutting time is not the main influencing factor.

[0139] (3) The results of the enzyme cutting ratio investigation are shown in Table 7 and Fig.14 , Fig.15 shown.

[0140] Table 7

[0141]

[0142] From the above results, it can be seen that with the increase of enzyme addition, the number of identified HCPs increased (see the non-overlapping part); there was no obvious increase after the enzyme addition exceeded 100:1, and it had little effect on the overall identification results. The overall results of this round showed that the enzyme cutting ratio was not the main influencing factor.

[0143] Example 4

[0144] This embodiment provides a method for detecting HCP antibody coverage, the method comprising:

[0145] Analytical methods:

[0146] (1) Buffer preparation:

[0147] PBS solution (pH 7.4) and 0.1 M glycine (pH 2.0) were prepared according to Example 1.

[0148] 5% RapiGest, 1M DTT, 1M IAM, 1 μg / μL Trypsin, mass spectrometry mobile phase A 0.1% FA·H2O, and mass spectrometry mobile phase B 0.1% FA·ACN solutions were prepared according to Example 3.

[0149] (2) Experimental procedures

[0150] Anti-CHO HCP antibody biotin labeling: Add biotin EZ-Link to the Anti-CHO HCP antibody solution at a molar ratio of antibody to biotin of 1:1-1:40 TM NHS-LC-LC-biotin, after the two are mixed, react at a temperature of 20-37°C for 1-3 hours, remove excess biotin through a vertical membrane ultrafiltration tube, and then detect the UV value.

[0151] Wash the solid substrate: Place the streptavidin magnetic beads (SA magnetic beads) solid substrate into a magnetic stand, collect the beads and discard the supernatant. Add 300-500 μL PBS solution (pH 7.4) and wash repeatedly for 3 times. After the last wash, discard the supernatant.

[0152] Conjugated antibody: Add 0.1mg-0.5mg of SA magnetic bead solid substrate and 200-600μL of antibody, mix the magnetic beads and antibody by mixer, and incubate at room temperature for 100-200 minutes. After the reaction, add 300-500μL PBS solution (pH 7.4) and wash repeatedly for 3 times. After the last wash, discard the supernatant.

[0153] Enrichment of HCP protein: After coupling the SA magnetic bead solid substrate with antibodies, add a total of 0.2-1 mg of HCP protein in a volume of 200-600 μL, mix the magnetic beads and HCP protein through a mixer, and incubate at room temperature for 100-200 minutes. After the reaction is completed, add 300-500 μL PBS solution (pH 7.4) and wash repeatedly for 3 times. After the last wash, discard the supernatant.

[0154] Elution of HCP proteins: Add 50-200 μL of elution buffer 0.1 M glycine (pH 2.0) to the above SA magnetic bead solid substrate test tube, cover the tube on a rotator, and incubate on a mixer at room temperature for 10-20 minutes.

[0155] Denaturation and reduction: Take a total of 1-5 mg of HCP protein before affinity capture and a total of 0.1-0.5 mg of HCP protein after affinity capture, add 5% RapiGest SF to a final concentration of 0.05%, react at 60°C for 15 minutes, then add 1M DTT to a final concentration of 20mM, react at 60°C for 60 minutes, and cool to room temperature.

[0156] Alkylation: Add 1M IAM to the denatured and reduced sample to a final concentration of 10mM and react at room temperature in the dark for 30 minutes.

[0157] Sample enzymatic digestion: Take Trypsin at a ratio of protein: enzyme = 40:1, vortex mix, add to the treated sample, and place in a constant temperature incubator at 37°C for 16 hours.

[0158] Acid hydrolysis of the enzymatic solution: Add formic acid to the enzymatic solution to a final concentration of 0.5%.

[0159] Concentration, reconstitution and loading: Place the acidified sample in a vacuum concentrator, concentrate to powder at 45°C, reconstitute to 50-200 μL with phase A (0.1% FA·H2O), centrifuge at 13000 rpm for 5 minutes, take the supernatant into an injection vial, and load for LC-MS detection.

[0160] Data processing: After the original data of the samples are collected, the data are imported into the data processing workstation and processed using relevant software such as BioPharmaFinder.

[0161] (3) Data analysis

[0162] Fig.16 The results of HCP detection before and after affinity capture are shown in Figure 1. In the figure, A represents the number of HCP proteins before affinity capture, B represents the number of HCP proteins eluted after affinity capture (magnetic bead elution sample); C represents the number of HCP proteins that are the same in A and B.

[0163] The formula for calculating the coverage of the polyclonal antibody is:

[0164] Coverage (%) = A / (A+BC)

[0165] Through high-resolution mass spectrometry analysis, the number A of proteins specifically bound to the polyclonal antibody was 1486 and the total number B of proteins in the HCP sample was 1698; further analysis showed that the number C of proteins identical between the two was 1462; substituting the data in the figure into the formula, according to the formula: A / (A+BC), the coverage rate of the polyclonal antibody was obtained to be 86.3%.

[0166] Comparative Example 1

[0167] In this embodiment, two-dimensional electrophoresis (2-DE) and protein immunoblotting (WB) methods are used to detect HCP antibody coverage, and the method includes:

[0168] Analytical methods:

[0169] (1) Buffer preparation:

[0170] Tris-Glycine Electrophoresis Buffer: Take 100 mL of 10× Tris-Glycine Electrophoresis Buffer, pour it into 900 mL of purified water, dissolve and mix well, store at 2-8°C, and the validity period is 1 month.

[0171] Wet Transfer Buffer for Western Blot: Take 100 mL of 10×Western Blot Wet Transfer Buffer, pour it into 900 mL of purified water, dissolve and mix well, store at 2-8℃, and the validity period is 1 month.

[0172] (2) Experimental procedures

[0173] Isoelectric focusing electrophoresis: Add an appropriate amount of sample linearly from left to right along the edge of the groove in the focusing disk. After removing the protective layer on the prefabricated IPG strip, place the IPG strip with the adhesive surface facing down on the sample solution in the focusing disk so that the positive electrode of the strip (marked with +) corresponds to the positive electrode of the focusing disk for isoelectric focusing electrophoresis.

[0174] Vertical electrophoresis: Place the processed gel strip with the gel surface facing up on the long glass plate of the gel, add low melting point agarose sealing solution on top of the gel. Transfer the gel to the electrophoresis tank, add electrophoresis buffer to the electrophoresis tank and perform vertical electrophoresis.

[0175] Western Blot: The gel was loaded into the transfer cassette, and the protein was transferred to the PVDF membrane. After the transfer, the PVDF membrane was removed and stained with SYPRO Ruby protein blotting staining reagent, and the chemiluminescence imaging system was used to take pictures and save them. After staining and taking pictures, the PVDF membrane was treated with primary and secondary antibodies, and the chemiluminescence imaging system was used to take pictures and save them.

[0176] (3) Data analysis

[0177] The HCP total protein fluorescence staining map and the immunoblot map were matched, the proportion of protein spots detected by the immunoblot map to the total protein spots was calculated, and the matching results were exported. Fig.17 The immunoblot profile is shown in Fig.18 As shown. The green “+” sign represents all identifiable protein spots, and the red The protein spots that match the two pictures.

[0178] The comparison results of the methods in Example 4 and Comparative Example 1 are shown in Table 8.

[0179] Table 8

[0180] / 2D-WB-Method MS method Number of protein spots detected by blot staining 739 1698 Number of protein spots detected by the antibody 604 1486 Number of protein spots detected by both assays 376 1462 The total number of protein spots detected 967 1722 Coverage 62.46％ 86.30％

[0181] Through the optimization conditions of biotinylation of polyclonal antibodies for HCP detection in the present invention, the HCP protein sample and the total HCP sample were respectively digested after the specific binding HCP protein was eluted, and the number of proteins A specifically bound to the polyclonal antibody and the total number of proteins B in the HCP sample were detected by high-resolution mass spectrometry analysis, and the number of proteins A specifically bound to the polyclonal antibody was 1486 and the total number of proteins B in the HCP sample was 1698; further analysis showed that the number of proteins C with the same content between the two was 1462; according to the formula: A / (A+BC), the coverage of the polyclonal antibody was 86.3%. The inventors used the traditional 2D-WB method in the art to analyze the coverage of HCP polyclonal antibodies using the same sample. The number of protein spots detected by the cloned antibody specific binding immunoblot map is 604, the total number of proteins in the HCP sample B is 739, and the number of proteins that are identical between the two C is 376; according to the formula: A / (A+BC), the coverage of the polyclonal antibody is 62.46%. In this method, the HCP protein sample is captured without denaturation, and the detection conditions of HCP ELISA are kept consistent to improve the authenticity of the results; the detection sensitivity of low-concentration HCP is improved by enrichment to ensure that small molecular weight proteins are not lost; the standardization of the entire experimental operation reduces nonspecific adsorption and ensures that high-affinity HCP can be eluted; thereby improving the authenticity of the HCP coverage results of the detection antibody.

[0182] Comparative Example 2

[0183] This example repeated the method of detecting HCP antibody coverage three times to investigate the precision of the detection. All steps refer to Example 4. The precision results are shown in Fig.19 As shown, the HCPs identified in the empty samples and elution samples of the three experiments were reproducible, and the HCP coverage results of the three experiments were around 80% to 90%.

[0184] In summary, the present invention provides a method for detecting HCP antibody coverage, which combines the capture of HCP protein samples without denaturation, maintains consistency with HCP ELISA detection conditions, and improves the authenticity of the results; and improves the detection sensitivity of low-concentration HCPs by enrichment, ensuring that small molecular weight proteins are not lost; the entire experimental operation is easy to standardize, reduces nonspecific adsorption, and ensures that high-affinity HCPs can be eluted; thereby improving the authenticity of the HCP coverage results of the detection antibody.

[0185] 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 detecting HCP antibody coverage, characterized in that: The method comprises: (1) Biotinylating a polyclonal antibody for HCP detection, wherein the molar ratio between the polyclonal antibody and biotin is 1:(1-10); the reaction conditions of the biotinylation are: the reaction temperature is 4-37°C, and the reaction time is 1-3 h; the biotinylated polyclonal antibody is fixed on a solid substrate connected with streptavidin, and the ratio of the biotinylated polyclonal antibody to the solid substrate connected with streptavidin is 1:(5-20); the biotin is selected from N-hydroxysulfosuccinimide ester-biotin; (2) using the solid substrate obtained in step (1) to capture and enrich HCP proteins from the test solution, and then eluting; the reaction temperature of the capture and enrichment is 4-37°C; the elution solution used for the elution includes, by concentration, 0.05-0.15 M glycine and 1.5-2.5 M Tris, the solvent is water, and the pH value is 1-3; the temperature of the elution is 4-37°C; (3) Performing liquid chromatography-mass spectrometry analysis on the eluate obtained in step (2) to calculate the coverage of the polyclonal antibody.

2. The method for detecting HCP antibody coverage according to claim 1, characterized in that: In step (2), the elution time is 10-20 minutes.

3. The method for detecting HCP antibody coverage according to claim 1, characterized in that: In step (3), the LC-MS detection and analysis also includes a step of pre-treating the eluate.

4. The method for detecting HCP antibody coverage according to claim 3, characterized in that: The pretreatment comprises: sequentially denaturing, reducing, alkylating, enzymatically cleaving, acid hydrolyzing and concentrating and re-dissolving the HCP protein in the eluate.

5. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The denaturing agent used in the denaturation step is RapiGest SF proteolytic surfactant.

6. The method for detecting HCP antibody coverage according to claim 5, characterized in that: The final concentration of the RapiGestSF proteolysis surfactant in the reaction solution is 0.025-0.075%.

7. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The reaction time of the denaturation step is 10-30 minutes, and the reaction temperature is 60±5°C.

8. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The reducing agent used in the reduction step is dithiothreitol.

9. The method for detecting HCP antibody coverage according to claim 8, characterized in that: The final concentration of the dithiothreitol in the reaction solution is 15-25 mM.

10. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The reaction time of the reduction step is 50-70 minutes, and the reaction temperature is 60±5°C.

11. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The alkylating agent used in the alkylation step is iodoacetamide.

12. The method for detecting HCP antibody coverage according to claim 11, characterized in that: The final concentration of iodoacetamide in the reaction solution is 8-12 mM.

13. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The reaction time of the alkylation step is 25-35 minutes, the reaction temperature is 20-25° C., and the reaction is carried out in the dark.

14. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The enzyme used in the enzymatic cleavage step is trypsin.

15. The method for detecting HCP antibody coverage according to claim 14, characterized in that: The ratio of trypsin to HCP protein is 1:(30-50).

16. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The reaction time of the enzyme cleavage step is 4-20 h, and the reaction temperature is 37±2°C.

17. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The acid used in the acid hydrolysis step is formic acid.

18. The method for detecting HCP antibody coverage according to claim 17, characterized in that: The final concentration of the formic acid in the reaction solution is 0.25-0.75%.

19. The method for detecting HCP antibody coverage according to claim 4, characterized in that: The step of concentration and reconstitution comprises: vacuum concentrating the acidified sample at 45±2° C., reconstituted to 50-200 μL using a mobile phase, centrifuged and collected the supernatant for LC-MS detection.

20. The method for detecting HCP antibody coverage according to claim 1, characterized in that: In step (3), the calculation formula for the coverage of the polyclonal antibody is: Coverage rate (%) = A / (A+BC) Wherein, A is the number of proteins specifically bound by the polyclonal antibody, B is the total number of proteins in the HCP sample, and C is the number of proteins that are identical between the two.

21. Use of the method for detecting HCP antibody coverage as described in any one of claims 1 to 20 in the detection of biological products.

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