A method for evaluating the coverage of polyclonal antibodies for HCP detection

Abstract

The present invention provides a method for evaluating the coverage of polyclonal antibodies for HCP detection, wherein the HCP polyclonal antibodies are first coupled to a solid substrate, and then the solid substrate is used to capture proteins in the HCP sample, and then the captured HCP proteins are separated and the total number of proteins in the HCP sample and the number of captured proteins are detected to obtain the coverage of the HCP polyclonal antibodies. The method of the present invention is simple to operate, the conditions are controllable, the results are reliable, and the consistency between each operation is high.

Description

Technical Field

[0001] The present invention relates to the field of detection; in particular, the present invention relates to a method for evaluating the coverage of polyclonal antibodies for HCP detection. Background Art

[0002] Recombinant protein expression technology through host cells (such as bacteria, yeast or mammalian, insect or plant cell lines) has been widely used in many biopharmaceutical products. In the production process of such products, host cell proteins (HCP) will inevitably be introduced into the production process and thus exist in the final drug. Studies have found that the main hazard of residual HCP in biopharmaceutical products is that it may induce the body to produce anti-HCP antibodies, which may cause clinical symptoms in patients. In addition, HCP may act as an immune adjuvant, induce the production of anti-drug antibodies, and affect the safety or efficacy of drugs. Therefore, regulatory agencies in various countries around the world have strict limit requirements for HCP residues.

[0003] At present, HCP ELISA test kits are routinely used in this field. Due to its many advantages such as convenient and fast operation, low cost, and high throughput detection, this kit has been widely used by regulatory agencies and companies in the release inspection of intermediates and finished products. The most important part of the development of HCP ELISA test kits is the characterization of polyclonal antibodies, which includes the coverage verification experiment of polyclonal antibodies, that is, it is necessary to prove that the polyclonal antibodies used in the ELISA kit can react widely with a large number of HCPs. When applying for clinical trials, each company needs to provide relevant data to prove this, and then continue the subsequent research and development work.

[0004] In order to verify the coverage of polyclonal antibodies in HCP ELISA test kits, various technologies have been developed in the field, such as Antibody Affinity Extraction or 2D-WB method. However, each of these methods has obvious shortcomings. For example, the process is complicated, the time cost and economic cost are very high, there are misjudgments in actual application; the detection process is greatly affected by the operation; the points between the blot and the gel are difficult to match and cannot be standardized, etc.

[0005] Therefore, there is an urgent need in the art for a method for evaluating the coverage of polyclonal antibodies for HCP detection that is simple to operate, has controllable conditions, and provides reliable results. Summary of the invention

[0006] The object of the present invention is to provide a method for evaluating the coverage of polyclonal antibodies for HCP detection, which method should have the following advantages:

[0007] 1. Maintain consistency with HCP ELISA testing as much as possible to improve the authenticity of the results;

[0008] 2. Ensure the consistency and repeatability of the results;

[0009] 3. Ensure that small molecular weight proteins are not lost;

[0010] 4. Reduce nonspecific adsorption and ensure that high-affinity HCPs can be eluted;

[0011] 5. Standardization of experimental operations.

[0012] Another object of the present invention is to provide an apparatus for implementing the above-mentioned method of the present invention.

[0013] In a first aspect, the present invention provides a method for evaluating the coverage of polyclonal antibodies for HCP detection, the method comprising the following steps:

[0014] (1) immobilizing the polyclonal antibody for HCP detection on a solid substrate, thereby obtaining a solid substrate on which the polyclonal antibody for HCP detection is immobilized;

[0015] (2) contacting the solid substrate immobilized with the HCP detection polyclonal antibody obtained in step (1) with an HCP sample, so that the polyclonal antibody specifically binds to the protein in the HCP sample;

[0016] (3) dissociating the polyclonal antibody from the bound protein;

[0017] (4) detecting the amount A of the protein specifically bound to the polyclonal antibody and the amount B of the protein in the HCP sample; and

[0018] (5) Calculate the ratio of quantity A to quantity B to obtain the coverage of the polyclonal antibody for HCP detection.

[0019] In a specific embodiment, step (4-1) is performed after step (4): detecting the number C of proteins having the same molecular weight and isoelectric point between the proteins specifically bound by the polyclonal antibody and the proteins in the HCP sample; and

[0020] Accordingly, in step (5), the coverage of the polyclonal antibody for HCP detection is calculated according to the following formula: A / (A+BC).

[0021] In a specific embodiment, the solid substrate includes, but is not limited to, magnetic beads, chips, agarose beads, and resin microspheres.

[0022] In a preferred embodiment, the surface of the solid substrate carries chemical groups such as NH2-, COOH or NHS for coupling with antibodies.

[0023] In a specific embodiment, the solid substrate is magnetic beads.

[0024] In a specific embodiment, step (1) further comprises the step of blocking the solid substrate after the polyclonal antibody for HCP detection is fixed to the solid substrate.

[0025] In a preferred embodiment, the step of blocking the solid substrate includes but is not limited to blocking the solid substrate with bovine serum albumin, Tris-HCl buffer or phosphate buffer.

[0026] In a specific embodiment, in step (2), after the polyclonal antibody specifically binds to the protein in the HCP sample, a step of removing non-specifically bound proteins is optionally included.

[0027] In a preferred embodiment, in step (2), after the polyclonal antibody specifically binds to the protein in the HCP sample, the solid substrate is washed with PBS buffer to remove non-specifically bound proteins.

[0028] In a preferred embodiment, step (3) comprises dissociating the polyclonal antibody from the bound protein and removing the solid substrate to which the polyclonal antibody is immobilized.

[0029] In a preferred embodiment, in step (3), urea buffer and glycine solution (pH 1.0-4.0) are used to dissociate the polyclonal antibody from the bound protein.

[0030] In a preferred embodiment, in step (3), the solid substrate on which the polyclonal antibody is immobilized is removed by magnetic separation.

[0031] In a preferred embodiment, the detection of the number of proteins specifically binding to the polyclonal antibody, the number of proteins in the HCP sample, and the number of proteins having the same molecular weight and isoelectric point in step (4) can be achieved by various methods known in the art for detecting the number of proteins, including but not limited to 2D electrophoresis, 2D-DIGE, HPLC, mass spectrometry, and UPLC-MS.

[0032] In a preferred embodiment, in step (5), a computer is used to calculate the ratio of quantity A to quantity B plus quantity A minus quantity C, thereby obtaining the coverage of the polyclonal antibody for HCP detection.

[0033] In a preferred embodiment, the method further comprises the step of outputting the obtained polyclonal antibody coverage for HCP detection.

[0034] In a second aspect, the present invention provides a device for evaluating the coverage of polyclonal antibodies for HCP detection, the device comprising:

[0035] (1) a device for fixing the polyclonal antibody for HCP detection on a solid substrate;

[0036] (2) a device for contacting a solid substrate having the HCP detection polyclonal antibody immobilized thereon with an HCP sample;

[0037] (3) a device for dissociating the polyclonal antibody from the bound protein;

[0038] (4) a device for detecting the amount A of proteins specifically bound to the polyclonal antibody and the amount B of proteins in the HCP sample; and

[0039] (5) A device for calculating the ratio of quantity A to quantity B to obtain the coverage of polyclonal antibodies for HCP detection.

[0040] In a specific embodiment, the device (1) comprises a container for holding the solid substrate and the polyclonal antibody and a device for fixing the polyclonal antibody on the solid substrate.

[0041] In a preferred embodiment, the device for fixing the polyclonal antibody on a solid substrate comprises a vortex shaker, a magnetic rack, and a rotary mixer.

[0042] In a preferred embodiment, the solid substrate includes, but is not limited to: magnetic beads, chips, agarose beads, resin microspheres; preferably magnetic beads.

[0043] In a preferred embodiment, the device (1) further optionally comprises a device for sealing the solid substrate after the polyclonal antibody for HCP detection is immobilized on the solid substrate.

[0044] In a preferred embodiment, the device for sealing the solid substrate refers to a device for incubating bovine serum albumin, Tris-HCl buffer or phosphate buffer with the solid substrate on which the polyclonal antibody for HCP detection is immobilized.

[0045] In a specific embodiment, the device (2) includes but is not limited to a container for containing the solid substrate and the HCP sample, a device for transferring the solid substrate and the HCP sample, and a device for contacting the solid substrate with the HCP sample.

[0046] In a preferred embodiment, the device (2) may further include a device for removing non-specifically bound proteins after the polyclonal antibody specifically binds to the proteins in the HCP sample.

[0047] In a preferred embodiment, the device for removing non-specifically bound proteins refers to a device for washing the solid substrate with PBS buffer to remove non-specifically bound proteins.

[0048] In a specific embodiment, the device (3) includes a rotary mixer, a vortex shaker, and a magnetic stand.

[0049] In a specific embodiment, the device (4) includes but is not limited to devices for implementing 2D electrophoresis, 2D-DIGE, HPLC, mass spectrometry, and UPLC-MS.

[0050] In a preferred embodiment, the device (5) is a computer, so that the ratio between the quantity A and the total quantity B can be calculated.

[0051] In a preferred embodiment, the device may optionally further include a device (6) for outputting coverage.

[0052] In a specific embodiment, the device (4) is a device for detecting the number A of proteins specifically bound to the polyclonal antibody, the number B of proteins in the HCP sample, and the number C of proteins having the same molecular weight and isoelectric point between the proteins specifically bound to the polyclonal antibody and the proteins in the HCP sample; and

[0053] Accordingly, the device (5) is a device for calculating the coverage of polyclonal antibodies for HCP detection according to the following formula: A / (A+BC).

[0054] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 Photographs of CHO HCP 2D silver staining are shown;

[0056] Figure 2 2D silver staining photos of HCP captured by immunomagnetic beads are shown;

[0057] Figure 3 2D silver staining photos of HCP captured by immunomagnetic beads are shown;

[0058] Figure 4 2D silver staining photos of HCP captured by immunomagnetic beads are shown;

[0059] Figure 5 The 2D silver staining photos obtained by conventional two-dimensional polyacrylamide gel electrophoresis and protein immunoblotting method (2D-WB method) are shown;

[0060] Figure 6 The Western Blot photos obtained by conventional two-dimensional polyacrylamide gel electrophoresis and protein immunoblotting method (2D-WB method) are shown;

[0061] Figure 7 The schematic diagram of the method of the present invention is shown;

[0062] Figure 8 A schematic diagram of the coverage analysis of the 2D-WB method is shown;

[0063] Fig. 9 A schematic diagram of coverage analysis of the method of the present invention is shown;

[0064] Fig.10 Shown by PDQuest analysis Figure 1 Silver staining results;

[0065] Fig.11 Shown by PDQuest analysis Figure 2 Silver staining results

[0066] Fig.12 The consistency analysis of the experimental results of the method of the present invention by PDQuest software is shown;

[0067] Fig.13 The coverage of polyclonal antibodies used for HCP detection calculated by PDQuest software is shown;

[0068] Fig.14 a schematic diagram showing the apparatus of the present invention; and

[0069] Fig.15 A schematic diagram of a further optimized embodiment of the device according to the invention is shown. DETAILED DESCRIPTION

[0070] After extensive and in-depth research, the inventor unexpectedly found that the coverage of the HCP polyclonal antibody can be obtained by first coupling the HCP polyclonal antibody to a solid substrate, then using the solid substrate to capture the protein in the HCP sample, and then separating the captured HCP protein and detecting the total number of proteins in the HCP sample and the number of captured proteins. The method of the present invention is simple to operate, the conditions are controllable, the results are reliable, and the consistency between each operation is very high. The present invention was completed on this basis.

[0071] Definition of terms

[0072] HCP

[0073] As used herein, “HCP” and “host cell protein” have the same meaning and refer to proteins derived from host cells.

[0074] Currently, different cell expression systems are used to produce pharmaceutical products, such as bacteria (Escherichia coli, Pseudomonas fluorescens), yeast (Saccharomyces cerevisiae, Pichia pastoris), mammalian cells (e.g., Chinese hamster ovary cells (CHO), mouse myeloma cell line NS0, etc.), insect cells (baculovirus-infected Spodoptera frugiperda cells) and cells of plants (tobacco, Arabidopsis, rice). Under specific culture conditions and manufacturing processes, the specific HCP spectrum of a specific host cell is unique and specific. Depending on the host cell and production process used, HCPs can vary in isoelectric point (3-11) and hydrophobicity, and HCPs show a wide range of molecular weights (from ~5 kDa to at least ~250 kDa). Depending on the host cell and culture conditions, the number of HCPs in an upstream sample can range from a few hundred to more than a thousand proteins.

[0075] In mammalian cells, recombinant proteins are usually secreted from cells into the cell culture medium together with many HCPs. Residual HCPs may affect the quality, safety and efficacy of the product. The main problem with HCPs in biological products is that they may induce the body to produce anti-HCP antibodies, which may cause clinical symptoms in patients. In addition, HCPs may act as immune adjuvants to induce anti-drug antibodies, affecting drug safety or efficacy. HCPs also have a direct impact on the quality of the product itself. For example, HCPs with proteolytic functions can gradually hydrolyze product proteins, weaken or eliminate the biological efficacy of the product, or change the stability of the product even if there are very small amounts of HCPs.

[0076] HCP detection methods

[0077] Given that residual host cell proteins in biological products can lead to adverse clinical reaction risks such as allergic reactions or potential toxicity, it is necessary to establish appropriate HCP detection and verification methods to strictly control the quality of biological products. Sandwich immunoassay is the most commonly used HCP detection method, including colorimetry, electrochemiluminescence (ECL), chemiluminescence, radioactivity, etc. At present, the biological products industry mostly uses commercial universal HCP ELISA kits (such as Cygnus Technologies) to detect host protein residues in products.

[0078] Immunoassays rely on antibodies that recognize as broadly as possible the HCP population that enters the downstream purification process; therefore, sandwich immunoassays designed with polyclonal antibodies are the preferred method for HCP monitoring and quantification. However, the secretory proteins of host cells and the structural proteins released by some dead cells are complex, and the different processes, coupled with the need for unique post-translational modifications of related gene products, have greatly increased the number of HCPs and their biochemical complexity. Therefore, it is difficult to evaluate the specificity of polyclonal antibodies in general commercial HCP ELISA kits for residual host proteins. If the specificity and applicability of polyclonal antibodies in commercial ELISA kits are not high enough, there will be a risk of missing HCPs, thereby posing a risk to the quality and safety of biological products.

[0079] Therefore, regulatory authorities around the world, such as the FDA and CFDA, require relevant certification for commercial HCP ELISA kits used by biopharmaceutical companies. They need to prove that the antibodies used in the ELISA kits can react extensively with a large number of HCPs found in known process flows, i.e., kit applicability or antigen coverage verification. When applying for clinical trials, each company needs to provide relevant data to prove this, and then continue with the next research and development work.

[0080] At present, various technologies have been developed in this field for this verification, such as antibody affinity extraction method or 2D-WB method. However, the preparation of anti-HCP resin is difficult, the process of antibody affinity extraction method is too complicated, the result depends on the resin load and elution conditions, and it is difficult to repeat; the amount of antibody used is large, and the time cost and economic cost are very high; high-affinity HCP is difficult to elute, resulting in low coverage, and low-concentration proteins may be missed. The way the antibody is fixed to HCP leads to a significant reduction in the efficiency of binding. Therefore, the method for verifying the coverage of polyclonal antibodies for HCP detection widely used in this field is two-dimensional polyacrylamide gel electrophoresis and protein immunoblotting (2D SDS-PAGE / Western blot, 2D-WB). In this method, proteins are separated on the gel by SDS-PAGE method based on the difference in isoelectric point and molecular weight. One gel is silver stained, and the protein of the other gel is transferred to PVDF membrane or nitrocellulose membrane, and then Western Blotting is performed in combination with the antibody in the ELISA kit. The two pictures are superimposed by electronic means, and then the coverage is calculated and analyzed.

[0081] However, the experimental method itself has certain limitations and unavoidable defects. For example, in actual application, this method has many shortcomings, such as HCP antigens are denatured and may not represent the antigens seen in ELISA tests, which may misjudge qualified products as unqualified; the transfer process will cause protein loss, which may misjudge unqualified products as qualified; the detection process is greatly affected by the operation, resulting in poor repeatability of the test results, and the results obtained by different laboratories and personnel vary greatly; the wide HCP spectrum makes it difficult to optimize the transfer efficiency, resulting in underestimation due to excessive transfer through the membrane or failure to transfer from the gel; after Western Blotting on the blot membrane, the high-concentration points are often blurred and difficult to distinguish, resulting in difficulty in matching the points between the blot and the gel and no standardization, etc.

[0082] In order to overcome the defects of various HCP detection polyclonal antibody coverage verification methods in the prior art, the inventors creatively fixed the HCP detection polyclonal antibody on a solid substrate; contacted the solid substrate fixed with the polyclonal antibody with the HCP sample, thereby capturing the protein in the HCP sample that can specifically bind to the polyclonal antibody; then separated the captured protein from the polyclonal antibody; then detected the number A of proteins specifically bound to the polyclonal antibody and the total number B of proteins in the HCP sample; finally, the coverage of the polyclonal antibody was obtained using the ratio of the number A to the total number B.

[0083] The inventors further noted that when the two-dimensional electrophoresis method is used to detect the total number of proteins in the HCP sample, the bands or spots on the electrophoresis gel may be indistinguishable due to the excessively high concentration of the HCP sample or the insufficient sensitivity of the electrophoresis detection, that is, a band or spot may contain multiple proteins, resulting in the total number of proteins in the HCP sample detected being lower than the actual total number of proteins; and the sensitivity of antibody detection is higher than that of electrophoresis, so some proteins may be missed in the total number of proteins in the HCP sample detected, and these missed proteins can bind to the antibody, and are thus included in the number of proteins that can specifically bind to the antibody, thereby over-evaluating the coverage of the polyclonal antibody for HCP detection, that is, it is possible to evaluate products with unqualified coverage as qualified products.

[0084] To this end, the present invention can adopt a further preferred embodiment, that is, in addition to detecting the number A of proteins specifically binding to the polyclonal antibody and the total number B of proteins in the HCP sample, further detecting the number C of proteins having the same molecular weight and isoelectric point between the two; finally, the coverage of the polyclonal antibody is obtained by using the ratio of the number A to the total number B plus the number A minus the number C, that is, according to the formula: A / (A+BC).

[0085] Those skilled in the art will appreciate that the above preferred embodiments can also be used as a verification of the basic method of the present invention, i.e., detecting the ratio of A to B, so as to more objectively and accurately verify the coverage of the polyclonal antibody for HCP detection. Fig.13 shown.

[0086] Based on the teachings of the present invention, it will be appreciated by those skilled in the art that the solid substrate used in the method of the present invention may be various solid substrates conventionally used in the art, as long as the solid substrate is suitable for fixing antibodies. For example, the solid substrate includes, but is not limited to, magnetic beads, chips, agarose beads, resin microspheres in the form of NH2-, COOH or NHS chemical groups thereon. In a preferred embodiment, the solid substrate is a magnetic bead, so that after the polyclonal antibody on the solid substrate specifically captures the protein from the HCP sample, the solid substrate and the HCP sample can be separated magnetically without centrifugation. Such a liquid phase reaction has the advantages of sufficient reaction, fast speed, high efficiency, short time (HCP capture can be completed within 3 hours), etc., which reduces the amount of antibody used and the experimental time.

[0087] Based on the teachings of the present invention and the common knowledge in the art, those skilled in the art can also understand that after the polyclonal antibody is fixed to the solid substrate, other active sites on the solid substrate can be blocked to prevent the capture of proteins in the HCP sample that cannot specifically bind to the polyclonal antibody. For example, the solid substrate is blocked using a bovine serum albumin or casein solution prepared with a Tris-HCl buffer or a phosphate buffer.

[0088] After the protein that can specifically bind to the polyclonal antibody is captured from the HCP sample using the solid substrate on which the polyclonal antibody is fixed, the solid substrate can also be washed to remove non-specifically bound proteins. Therefore, in a preferred embodiment, the method of the present invention optionally includes a step of washing to remove non-specifically bound proteins.

[0089] After the polyclonal antibody specifically binds to the protein in the HCP sample, the polyclonal antibody and the bound protein can be dissociated in a conventional manner. For example, a urea solution or a glycine solution (pH 1.0-4.0) is used to dissociate the polyclonal antibody and the bound protein. After the polyclonal antibody and the bound protein are dissociated, a conventional method, such as magnetic separation, can be used to remove the solid substrate to which the polyclonal antibody is fixed, thereby separating the protein bound to the polyclonal antibody from the solid substrate to which the polyclonal antibody is fixed, so as to detect the number A of proteins specifically bound to the polyclonal antibody.

[0090] Those skilled in the art are aware of various methods for detecting the amount of protein specifically captured by the polyclonal antibody and the total amount of protein in the HCP sample, including but not limited to: 2D electrophoresis, 2D-DIGE, HPLC, mass spectrometry, UPLC-MS, etc.

[0091] Based on the teachings of the present invention, those skilled in the art can think of various ways to implement the present invention. For example, in a specific embodiment, HCP polyclonal antibodies can be prepared by coupling magnetic beads to bind to proteins in HCP, and then the bound HCP proteins are eluted, and finally the eluate is compared and analyzed by two-dimensional electrophoresis and silver staining to calculate the coverage of HCP antibodies.

[0092] On the basis of the method for verifying the coverage of polyclonal antibodies for HCP detection of the present invention, the inventors also provide an apparatus for implementing the method; that is, an apparatus for evaluating the coverage of polyclonal antibodies for HCP detection, the apparatus comprising various specific devices for implementing the above-mentioned method of the present invention, for example, a device for fixing the polyclonal antibodies for HCP detection on a solid substrate; a device for contacting the solid substrate fixed with the polyclonal antibodies with an HCP sample; a device for dissociating the polyclonal antibodies from the bound proteins; a device for detecting the number A of proteins specifically bound to the polyclonal antibodies and the number B of proteins in the HCP sample; and a device for calculating the ratio of the number A to the number B to obtain the coverage of the polyclonal antibodies for HCP detection.

[0093] Based on the teachings of the present invention, those skilled in the art are aware of the various devices in the above-mentioned apparatus. For example, the device for fixing the polyclonal antibody for HCP detection on a solid substrate may be equipped with a suitable solid substrate including but not limited to: magnetic beads, chips, agarose beads, resin microspheres with NH2-, COOH or NHS chemical groups thereon, and the necessary devices for fixing the polyclonal antibody on the solid substrate, such as a vortex oscillator, a magnetic rack, and a rotary mixer; the device for fixing the polyclonal antibody for HCP detection on a solid substrate may also optionally include a device for sealing the solid substrate after the polyclonal antibody is fixed on the solid substrate.

[0094] For another example, the device for contacting the solid substrate immobilized with the polyclonal antibody with the HCP sample may be equipped with, for example (but not limited to), a container for accommodating the solid substrate and the HCP sample, a device for transferring the solid substrate and the HCP sample, and a device for contacting the solid substrate with the HCP sample. The device for contacting the solid substrate with the HCP sample may also optionally include a device for removing non-specific binding proteins.

[0095] In order to verify the coverage of the polyclonal antibody for HCP detection more objectively and accurately, as described above, the method of the present invention also includes detecting the number of proteins that specifically bind to the polyclonal antibody and the total proteins in the HCP sample that have the same molecular weight and isoelectric point; finally, the coverage of the polyclonal antibody is obtained by using the ratio of the number A to the total number B plus the number A minus the number C, that is, according to the formula: A / (A+BC). Accordingly, the equipment for implementing the method of the present invention also includes a device for detecting the number C of proteins that specifically bind to the polyclonal antibody and the proteins in the HCP sample that have the same molecular weight and isoelectric point; and a device for calculating the coverage of the polyclonal antibody for HCP detection according to the formula: A / (A+BC).

[0096] In a specific embodiment, the device for fixing the polyclonal antibody for HCP detection on a solid substrate comprises a solid substrate and a device necessary for fixing the polyclonal antibody on the solid substrate; the device necessary for fixing the polyclonal antibody on the solid substrate includes but is not limited to a vortex shaker, a magnetic stand, and a rotary mixer;

[0097] The device for contacting the solid substrate fixed with the polyclonal antibody for HCP detection with the HCP sample may be equipped with, for example (but not limited to), a container for accommodating the solid substrate and the HCP sample, a device for transferring the solid substrate and the HCP sample, and a device for contacting the solid substrate with the HCP sample, and may also optionally include a device for removing non-specifically bound proteins after the polyclonal antibody specifically binds to the protein in the HCP sample; for example, a device for washing the solid substrate with PBS buffer to remove non-specifically bound proteins after the polyclonal antibody specifically binds to the protein in the HCP sample;

[0098] The device for dissociating the polyclonal antibody from the bound protein includes but is not limited to a rotary mixer, a vortex shaker, a magnetic stand, etc.;

[0099] The device for detecting the amount of protein specifically bound to the polyclonal antibody and the amount of protein in the HCP sample includes, but is not limited to, a device for implementing 2D electrophoresis, 2D-DIGE, HPLC, mass spectrometry, and UPLC-MS.

[0100] In a specific embodiment, various conventional devices in the art, such as a computer, can be used to calculate the ratio of each quantity to obtain the coverage of the polyclonal antibody. After obtaining the coverage, the coverage can also be output by an output device. Therefore, the device of the present invention can also optionally include a device for outputting the obtained coverage and whether the coverage of the evaluated polyclonal antibody for HCP detection is qualified.

[0101] The schematic diagram of the equipment for implementing the method of the present invention and further optimizing the equipment is as follows Fig.14 and 15 shown.

[0102] Advantages of the present invention:

[0103] 1. The method of the present invention is similar to the actual test method ELISA sandwich immunoassay, so HCP can bind to the antibody coupled to the solid substrate under native conditions and can therefore be used for actual detection;

[0104] 2. The method of the present invention only requires two-dimensional electrophoresis and silver staining, and does not require membrane transfer, so it is easy to operate and can be automated;

[0105] 3. The coupling and elution conditions of the method of the present invention are repeatable, so the results have good repeatability and high reproducibility; and

[0106] 4. The method of the present invention preferably uses immunomagnetic separation technology, which does not require centrifugation and liquid phase reaction, has sufficient reaction, fast speed and high efficiency, and can complete HCP capture within 3 hours, so the capture efficiency is high; and

[0107] 5. The method of the present invention can more objectively and accurately verify the coverage of polyclonal antibodies for HCP detection, and will not misjudge products with unqualified coverage as qualified, nor will it misjudge products with qualified coverage as unqualified.

[0108] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are generally carried out according to conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0109] Materials and Methods

[0110] Materials for the experiment:

[0111] NHS group-modified magnetic beads: purchased from Thermo Fisher Scientific, USA;

[0112] CHO HCP protein: CHO K1 cell culture supernatant was collected;

[0113] HCP polyclonal antibody: purchased from Cygnus, anti-CHO HCP Ab (3G-0016-AF);

[0114] Magnetic bead washing solution: 1mM HCl solution;

[0115] Coupling buffer: 200 mM NaHCO3, pH 8.3;

[0116] Blocking buffer: 100 mM Tris-HCl, 150 mM NaCl, pH 8.0;

[0117] Eluent: 8M urea.

[0118] method

[0119] Analysis of HCP antibody coverage based on magnetic bead-coupled HCP polyclonal antibody method

[0120] 1. Preparation of magnetic bead-coupled HCP polyclonal antibodies

[0121] 1.1 Preparation of CHO HCP solution:

[0122] Take an appropriate amount of CHO HCP to be coupled and dissolve it in coupling buffer to prepare a concentration greater than 3 mg / ml. Note: It is necessary to remove the substances containing primary amine groups in the protein stock solution in advance by dialysis or desalting.

[0123] 1.2 Take 500 μl of the magnetic bead suspension into a 1.5 ml centrifuge tube, place the centrifuge tube on a magnetic separator, enrich the magnetic beads, and remove the supernatant.

[0124] 1.3 Add 1 ml of 4°C pre-cooled magnetic bead washing buffer and vortex for 15 seconds to fully mix the magnetic beads.

[0125] 1.4 Place the centrifuge tube on a magnetic separator to enrich the magnetic beads and remove the supernatant.

[0126] 1.5 Add a total of 0.2 mg-0.6 mg of CHO HCP antibody in a volume of 200 μl-400 μl, vortex for 30 seconds to mix thoroughly, incubate on a rotary mixer at room temperature for 2-4 hours, and then incubate at 4°C overnight.

[0127] 1.6 Place the centrifuge tube on a magnetic separator to enrich the magnetic beads and remove the supernatant.

[0128] 1.7 Add 1 ml of blocking buffer to the centrifuge tube, vortex for 30 min, place the centrifuge tube on a magnetic separator to enrich the magnetic beads, and discard the supernatant.

[0129] 1.8 Repeat step 1.7 four times

[0130] 1.9 Add 1 ml of blocking buffer to the centrifuge tube, vortex for 30 min, and place the centrifuge tube on a rotary mixer for 2 h.

[0131] 1.10 Place the centrifuge tube on a magnetic separator to collect the magnetic beads and remove the supernatant.

[0132] 1.11 Add 1 ml of ultrapure water to the centrifuge tube, mix thoroughly, use a magnetic separator to enrich the magnetic beads, and discard the supernatant.

[0133] 1.12 Add 1 ml of PBS buffer to a centrifuge tube, wash twice, and for the last time add 500 μl of PBS buffer containing 0.01%-0.05% thimerosal, and store at 4°C for a long term.

[0134] 2. Evaluate the coverage of HCP antibody by coupling CHO HCP antibody to magnetic beads.

[0135] 2.1 Place the magnetic beads prepared above on a magnetic separator to enrich the magnetic beads and remove the supernatant.

[0136] 2.2 Add 5% BSA solution and incubate on a rotary mixer at room temperature for 2 hours.

[0137] 2.3 Place the centrifuge tube on a magnetic separator to enrich the magnetic beads and discard the supernatant.

[0138] 2.4 Add 1 ml PBS buffer and wash for 15 seconds. Place the centrifuge tube on a magnetic separator to enrich the magnetic beads and remove the supernatant.

[0139] 2.5 Repeat the above steps 4 times.

[0140] 2.6 Add CHO HCP antigen (volume 200 μl-400 μl, concentration greater than 1 mg / ml) and incubate on a rotary mixer at room temperature for 2 h.

[0141] 2.7 Place the centrifuge tube on a magnetic separator to enrich the magnetic beads and remove the supernatant.

[0142] 2.8 Add 1 ml PBS buffer and wash for 15 seconds. Place the centrifuge tube on a magnetic separator to enrich the magnetic beads and remove the supernatant.

[0143] 2.9 Repeat the above steps 3 times.

[0144] 2.10 Add 200 μl of elution buffer and incubate on a rotary mixer for 15 min at room temperature.

[0145] 2.11 Place the centrifuge tube on a magnetic separator, enrich the magnetic beads, and collect the supernatant.

[0146] 2.12 Repeat the above steps twice, and mix the supernatants from the three times and concentrate them by ultrafiltration.

[0147] 2.13 Take an appropriate amount of supernatant and determine the protein concentration using the Bradford method.

[0148] 3. The supernatant obtained above was analyzed according to the following experimental method:

[0149] 3.1 Two-dimensional electrophoresis and silver staining

[0150] 3.1.1 First-axis isoelectric focusing

[0151] 1) Take out two 18 cm strips from the -20℃ freezer, place them on an IPG expansion plate, and equilibrate at room temperature for 20 minutes.

[0152] 2) Centrifuge the sample at 12000rpm for 5min, then add 340ul of hydration solution according to the optimized loading volume of 18cm strip, and carefully add the mixed solution into the strip tank with a gun.

[0153] 3) Use tweezers to hold the alkaline end of the IPG strip and tear off the protective film of the strip, with the strip facing down. First, place the acidic end of the IPG strip into the strip slot toward the tip (positive electrode) of the strip slot and slowly press down the strip to avoid bubbles. Finally, put down the alkaline end of the IPG strip to allow the hydration solution to soak the entire strip and leave it overnight (the strip marked + is the acidic end, and marked - is the alkaline end).

[0154] 4) Adjust the level of the isoelectric focusing electrophoresis instrument (IPGphor3) first, place the ceramic focusing tank on the isoelectric focusing electrophoresis instrument, transfer the gel strip into the focusing tank with the gel surface facing up, and make sure that the gel strip is located in the center of the gel strip tank, place the acidic end of the gel strip on the positive electrode of the isoelectric focusing electrophoresis instrument, and the alkaline end of the gel strip on the negative electrode of the isoelectric focusing electrophoresis instrument, and place the wet electrode filter paper (moistened with 150ulddH2O) on both ends of the gel strip (approximately one-third of the filter paper is placed on the gel surface).

[0155] 5) Place the electrode clamps at both ends of the rubber strips so that the electrode wires of the electrode clamps are pressed at about 1 / 3 of the outer edge of the two electrode filter papers. Rotate the cam to the position under the outer edge of the Manifold rubber strips, install the electrodes in place, and cover each rubber strip with enough mineral oil.

[0156] 6) Set the program to focus the instrument on an 18 cm strip as follows:

[0157] Focusing program (2gels)

[0158]

[0159] 7) After focusing begins, prepare the SDS-PAGE gel and run the second dimension SDS-PAGE electrophoresis.

[0160] 3.1.2 Second dimension SDS-PAGE electrophoresis

[0161] 1) Install the glass plate according to the Bio-Rad vertical electrophoresis system glass plate installation instructions.

[0162] 2) Prepare separation gel (15%), mix the separation gel and immediately pour it into the gap between the glass plates, carefully cover the upper layer with a layer of isopropanol, place the gel plate vertically at room temperature, wait until the separation gel is completely polymerized, pour off the isopropanol on top, and absorb it dry with filter paper.

[0163] 3) After focusing, wash the strips with double distilled water three times, hold the strips upright with tweezers to control the water, place the strips in the strip tank, add 5 ml / 1 strip balancing solution with DTT, shake on a shaker for 15 minutes, discard the balancing solution with DTT, then add 5 ml / 1 strip balancing solution with iodoacetamide, shake on a shaker for 16 minutes, and discard the balancing solution. (Add 0.05 g of DTT to 5 ml of balancing solution stock solution, and add 0.125 g of iodoacetamide to 5 ml of balancing solution stock solution)

[0164] 4) Take out the equilibrated gel strip from the equilibration solution containing iodoacetamide, drop the equilibration solution on the filter paper, and rinse it with 1X electrophoresis buffer.

[0165] 5) Use tweezers to clamp both ends of the rubber strip, with the support surface facing down and the rubber surface facing up, and place it flat on the raised part of the sandwich long board. At this time, it should be tightly combined with the glass long board.

[0166] 6) Place the sandwich glass plate vertically on the rack, use the syringe needle to push the adhesive strip down along the glass plate until it is in close contact with the adhesive surface (the needle should be applied on the supporting surface of the adhesive strip and should not touch the adhesive surface to avoid scratching it), cut a small piece of filter paper and place it next to the + electrode of the adhesive strip and add 10μl of Marker.

[0167] 7) Use a 1 ml pipette to add the prepared low melting point agarose into the sandwich gel along one end. The low melting point agarose solution will move along the gel surface to the other end, eliminating the bubbles between the gel strip and the SDS-PAGE gel surface.

[0168] 8) When the low melting point agarose has not solidified, use a needle to push the strip down until it is in close contact with the SDS-PAGE gel surface and remove bubbles.

[0169] 9) After the low melting point agarose solidifies, fix the gel plate on the electrophoresis device and add electrophoresis buffer to the upper and lower tanks. Set the program: 2w-1h (1w per gel strip), 28w-5h (14w per gel strip), start electrophoresis until bromophenol blue reaches the bottom of the gel, and turn off the power.

[0170] 10) After the electrophoresis is completed, the gel is pulled out and one of the gels is stained using a low background silver staining kit according to the kit instructions.

[0171] 11) Results Antibody coverage was assessed by PDQuest analysis.

[0172] 4. Two-dimensional electrophoresis and Western Blot experiments to analyze the coverage of CHO HCP antibodies

[0173] 4.1 Run two large gels in two-dimensional electrophoresis. The specific steps are shown in the two-dimensional electrophoresis section in 3 above. One gel is silver stained and the other gel is used for Western blot analysis.

[0174] 4.2 Western blot experimental steps are as follows:

[0175] 1) After electrophoresis is completed, carefully pull out the gel and place it in the prepared transfer solution. Cut the filter paper and nitrocellulose membrane to the same size as the gel and place them in the transfer solution.

[0176] 2) Open the cover of the semi-dry transfer instrument and place the filter paper-membrane-gel-filter paper in the order from bottom to top, remove bubbles and put the cover on, and transfer at a constant voltage of 20V for 40 minutes.

[0177] 3) Take out the membrane and wash it once in PBST. After blocking it in 5% BSA (prepared with PBST) for 1 hour, apply HCP antibody of appropriate concentration using 1% BSA (prepared with PBST) and incubate it at 4 degrees overnight.

[0178] 4) The next day, take out the membrane from the 4°C refrigerator, incubate on a shaker for 30 min, then discard the primary antibody and wash three times in PBST, 5 min each time.

[0179] 5) Dilute the fluorescent secondary antibody at 1:15000 in PBST and incubate at room temperature in the dark for 1 h.

[0180] 6) Remove the membrane and wash it in PBST in the dark for 3 times, 5 min each time.

[0181] 7) Use a dual-mode multispectral laser imaging system to scan the nitrocellulose membrane on the instrument and take photos for preservation.

[0182] 8) Results Antibody coverage was assessed by PDQuest analysis.

[0183] Example

[0184] Example 1

[0185] As described in the "Materials and Methods" section, the present inventors analyzed the coverage of HCP polyclonal antibodies.

[0186] The results are as follows Figure 1 and Figure 2 As shown (where Figure 1 The 2D silver staining image of CHO HCP is shown. Figure 22D silver staining images of protein capture by immunomagnetic beads coupled to HCP polyclonal antibodies were shown) and analyzed by PDQuest software (results in Fig.10 and Fig.11 ) The coverage of the detected HCP polyclonal antibodies can reach 78.5%, and the software analysis results are shown in the figure.

[0187] Example 2.

[0188] The inventors repeated the method of the present invention, using HCP polyclonal antibodies coupled to magnetic beads to capture proteins in CHO HCP, and then performing 2D silver staining on the captured proteins. The results of the two experiments are shown in Figure 2. Figure 3 and Figure 4 .

[0189] The experimental results show that the comparison rate between the two different experimental results is 82.3% (analysis results see Fig.12 ), thus proving that the consistency of the results obtained by the method of the present invention is very high.

[0190] Example 3.

[0191] The present inventors used the mainstream method in the field, i.e., 2D-WB method, to analyze the coverage of HCP polyclonal antibodies using the same samples. The results are shown in Figure 2. Figure 5 and 6 As shown, Figure 5 2D silver staining photos showing CHO HCP, Figure 6 Western Blot photos are shown.

[0192] From the results of this example, it can be seen that the coverage of CHO HCP antibody obtained by 2D-WB method is only 35.5%. Figure 6 The photos shown also clearly show that small molecular weight protein spots are not visible in the 2D / WB experiment.

[0193] Therefore, conventional 2D-WB methods will misjudge qualified products as unqualified products.

[0194] Example 4.

[0195] Considering that the sensitivity of antibody detection is higher than that of electrophoresis, the total amount of protein in the HCP sample detected by electrophoresis may be lower than the actual total amount of protein.

[0196] To this end, the inventors took another HCP sample to detect the number A of proteins specifically bound to the polyclonal antibody, the number B of proteins in the HCP sample, and the number C of proteins having the same molecular weight and isoelectric point between the proteins specifically bound to the polyclonal antibody and the total proteins in the HCP sample; and the coverage rate of the polyclonal antibody was obtained according to the formula: A / (A+BC).

[0197] The experimental results are as follows Fig.13 As shown, the number A of proteins specifically binding to the polyclonal antibody is "334+142", the number B of proteins in the HCP sample is "380+334", the number C of proteins having the same molecular weight and isoelectric point as the total proteins in the HCP sample that specifically bind to the polyclonal antibody is "334", and the coverage rate of the polyclonal antibody is calculated to be (334+142) / ("334+142"+"380+334"-"334")=55.6%.

[0198] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A method for evaluating the coverage of polyclonal antibodies for HCP detection, the method comprising the following steps: (1) immobilizing the polyclonal antibody for HCP detection on a solid substrate, thereby obtaining a solid substrate on which the polyclonal antibody for HCP detection is immobilized; (2) contacting the solid substrate immobilized with the HCP detection polyclonal antibody obtained in step (1) with an HCP sample, so that the polyclonal antibody specifically binds to the protein in the HCP sample; (3) dissociating the polyclonal antibody from the bound protein; (4) detecting the amount A of the protein specifically bound to the polyclonal antibody and the amount B of the protein in the HCP sample; (4-1): Detecting the protein specifically bound by the polyclonal antibody and the number C of proteins having the same molecular weight and isoelectric point among the proteins in the HCP sample; and (5) The coverage of the polyclonal antibody for HCP detection was calculated according to the following formula: A / (A+BC).

2. The method according to claim 1, characterized in that The solid substrate includes magnetic beads, chips, agarose beads or resin microspheres.

3. The method according to claim 2, characterized in that The surface of the solid substrate carries NH2-, COOH or NHS chemical groups for coupling with antibodies.

4. The method according to claim 2, characterized in that The solid substrate is magnetic beads.

5. The method according to claim 1, characterized in that Step (1) also includes the step of blocking the solid substrate after the HCP detection polyclonal antibody is fixed to the solid substrate.

6. The method according to claim 5, characterized in that The step of blocking the solid substrate comprises blocking the solid substrate with bovine serum albumin, Tris-HCl buffer or phosphate buffer.

7. The method according to claim 1, characterized in that In step (2), after the polyclonal antibody specifically binds to the protein in the HCP sample, the method optionally further includes a step of removing non-specifically bound proteins.

8. The method according to claim 7, characterized in that In step (2), after the polyclonal antibody specifically binds to the protein in the HCP sample, the solid substrate is washed with PBS buffer to remove non-specifically bound proteins.

9. The method according to claim 1, characterized in that Step (3) includes dissociating the polyclonal antibody from the bound protein and removing the solid substrate on which the polyclonal antibody is fixed.

10. The method according to claim 9, characterized in that In step (3), the polyclonal antibody is dissociated from the bound protein using a urea buffer or a glycine solution with a pH of 1.0 to 4.

0.

11. The method according to claim 1, characterized in that In step (3), the solid substrate on which the polyclonal antibody is fixed is removed by magnetic separation.

12. The method according to claim 1, characterized in that In step (4), the number of proteins specifically bound to the polyclonal antibody, the number of proteins in the HCP sample, and the number of proteins having the same molecular weight and isoelectric point can be detected by the following methods, including 2D electrophoresis, 2D-DIGE, HPLC, mass spectrometry or UPLC-MS.

13. The method according to claim 12, characterized in that In step (5), a computer is used to calculate the ratio of quantity A to quantity B plus quantity A minus quantity C, thereby obtaining the coverage of the polyclonal antibody for HCP detection.

14. The method according to claim 1, wherein: The method further comprises the step of outputting the obtained polyclonal antibody coverage for HCP detection.

15. A device for evaluating the coverage of polyclonal antibodies for HCP detection, the device comprising: (1) a device for fixing the polyclonal antibody for HCP detection on a solid substrate; (2) a device for contacting a solid substrate having the HCP detection polyclonal antibody immobilized thereon with an HCP sample; (3) a device for dissociating the polyclonal antibody from the bound protein; (4) a device for detecting the number A of proteins specifically bound to the polyclonal antibody, the number B of proteins in the HCP sample, and the number C of proteins having the same molecular weight and isoelectric point as the proteins specifically bound to the polyclonal antibody and the proteins in the HCP sample; and (5) A device for calculating the coverage of polyclonal antibodies for HCP detection according to the following formula: A / (A+BC).

16. The device according to claim 15, characterized in that The device (1) comprises a container for holding the solid substrate and the polyclonal antibody and a device for fixing the polyclonal antibody on the solid substrate.

17. The device according to claim 16, characterized in that The device for fixing the polyclonal antibody on the solid substrate includes a vortex shaker, a magnetic rack, and a rotary mixer.

18. The device according to claim 17, characterized in that The solid substrate includes magnetic beads, chips, agarose beads or resin microspheres.

19. The device according to claim 18, characterized in that The solid substrate is magnetic beads.

20. The device according to claim 15, characterized in that The device (1) may further optionally include a device for sealing the solid substrate after the polyclonal antibody for HCP detection is immobilized on the solid substrate.

21. The device according to claim 20, characterized in that The device for sealing the solid substrate refers to a device in which bovine serum albumin, Tris-HCl buffer or phosphate buffer are incubated with the solid substrate on which the polyclonal antibody for HCP detection is immobilized.

22. The device according to claim 15, characterized in that The device (2) comprises a container for containing the solid substrate and the HCP sample, a device for transferring the solid substrate and the HCP sample, and a device for contacting the solid substrate with the HCP sample.

23. The device according to claim 22, characterized in that The device (2) may also optionally include a device for removing non-specifically bound proteins after the polyclonal antibody specifically binds to the proteins in the HCP sample.

24. The device according to claim 23, characterized in that The device for removing non-specifically bound proteins refers to a device in which the solid substrate is washed with a PBS buffer to remove non-specifically bound proteins.

25. The device according to claim 15, characterized in that The device (3) comprises a rotary mixer, a vortex oscillator and a magnetic stand.

26. The device of claim 15, wherein: The apparatus (4) comprises a device for performing 2D electrophoresis, 2D-DIGE, HPLC, mass spectrometry or UPLC-MS.

27. The apparatus of claim 15, wherein: The device may also optionally include a device (6) for outputting the coverage ratio.

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