Method and kit for detecting protein A

Abstract

The method and the kit for detecting the protein A are used for determining the content of the protein A in a sample, and the method and the kit adopt Fc protein to capture the protein A in the sample and detect the content of the protein A through an anti-protein A antibody coupled with a marker.

Description

[0001] Cross-reference

[0002] This application claims the benefit of Chinese Patent Application No. 202211729373.3, filed on December 30, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention belongs to the field of biotechnology and particularly relates to a method and a kit for detecting Protein A. Background Art

[0004] Protein A is a cell wall protein of Staphylococcus aureus. Due to its high specific affinity for the Fc segment of immunoglobulins, it is used as a packing material for affinity purification columns and widely applied in the purification of antibody drugs. However, during the purification process, Protein A may leach out from the affinity purification column, resulting in the residual Protein A in the antibody drug. When Protein A enters the patient's body along with the drug, it can induce mitosis or immune response in the patient. Therefore, it is crucial to detect and control the residual level of Protein A in the drug.

[0005] Currently, most commercially available kits for detecting residual Protein A are designed for different Protein A ligands. For example, the Repligen 9222-1 kit is for the NGL- A ligand, and the Cygnus F740 kit is for the Amsphere A3 ligand. Additionally, some new Protein A ligands (such as AT Protein A diamond) do not have corresponding detection kits.

[0006] Furthermore, the current technology for detecting residual Protein A is the sandwich immunoenzymatic reaction method using a primary antibody and a secondary antibody. Each ligand produces its own capture antibody, and then the corresponding residual detection kit is developed. Such methods lack generality and the market launch of the kit will theoretically be several months later than the Protein A purification product, thus posing a great challenge to the detection of Protein A residues in newly launched Protein A purification products.

[0007] Therefore, there is a need for a widely applicable kit for detecting Protein A residues to fill the market gap in the detection of residues of different Protein A ligands in newly launched products. Summary of the Invention

[0009] In one aspect, the present invention provides a method for detecting Protein A in a sample, the method comprising:

[0010] Adding the sample and an anti-Protein A antibody conjugated with a label to a plate coated with Fc protein and incubating them, thereby forming a complex of Fc protein, Protein A in the sample, and the anti-Protein A antibody, and

[0011] The content of Protein A in the sample is determined by detecting the signal generated by the marker.

[0012] In some embodiments, the method includes adding the sample to be detected and an anti-Protein A antibody conjugated with a marker to a plate coated with Fc protein, and then incubating them all together.

[0013] In some embodiments, the method includes adding the sample to be detected to a plate coated with Fc protein and performing a first incubation, and then adding an anti-Protein A antibody conjugated with a marker and performing a second incubation. Preferably, one or more washings are also included between the incubation steps.

[0014] In some embodiments, the duration of each incubation ranges from about 30 minutes to about 3 hours, such as about 1 hour to 2 hours, such as about 30 minutes, about 45 minutes, about 1 hour, about 1.25 hours, about 1.5 hours, about 1.75 hours, or about 2 hours. In some embodiments, the first incubation and the second incubation are each carried out for about 40 minutes to 1.5 hours, such as 1 hour.

[0015] In some embodiments, the Fc protein comprises an Fc region derived from an immunoglobulin or a fragment or variant thereof. Preferably, the immunoglobulin is a human immunoglobulin. Any Fc protein capable of binding Protein A can be used in the methods disclosed herein. In some embodiments, the Fc protein comprises or consists of the Fc region of human IgG1, IgG2, IgG3, or IgG4, or a fragment or variant thereof. In some embodiments, the Fc protein is an Fc fusion protein. In some embodiments, the Fc protein is an Fc region fragment as shown in SEQ ID NO: 1. In some embodiments, the Fc protein comprises an amino acid sequence that is at least 80%, 85%, 90%, or 95% identical to SEQ ID NO: 1 and retains the ability to bind Protein A.

[0016] In some embodiments, the anti-Protein A antibody is selected from chicken-derived anti-Protein A antibody, rabbit-derived anti-Protein A antibody, and human-derived anti-Protein A antibody. The anti-Protein A antibody is a monoclonal antibody or a polyclonal antibody. In some specific embodiments, the anti-Protein A antibody is a chicken-derived anti-Protein A polyclonal antibody. In some embodiments, the method includes conjugating the anti-Protein A antibody with the marker before applying it to the plate.

[0017] In some embodiments, the sample to be detected is an elution sample from Protein A affinity chromatography. The elution sample can be the sample eluted for the first time or an elution fraction after multiple rounds of column elution. The sample may contain the target protein (such as an antibody), host cell proteins, and Protein A.

[0018] In some embodiments, the sample to be detected is pretreated before being added to the plate. The pretreatment may be selected from acid treatment, dilution treatment, and heat treatment. In some specific embodiments, the sample is heated at about 95 - 100 °C for about 20 minutes to denature the target protein in the sample, then cooled to room temperature and centrifuged, and the supernatant is harvested as the sample to be added to the plate.

[0019] In some embodiments, the method of the present invention further includes pre - coating the plate with Fc protein. Specifically, the plate can be incubated with an Fc protein solution for a period of time, washed, and then incubated with a blocking buffer. In some specific embodiments, a plate coated with Fc protein can be prepared by incubating the plate with an Fc protein solution at about 2 - 8 °C overnight, and then blocking and incubating with a BSA solution for a period of time (such as about 30 minutes to 2 hours).

[0020] The signal generated by the label can be detected by enzyme - linked immunosorbent assay, sandwich assay, or immunohistochemical staining. In some embodiments, the label is a label that can generate a detectable signal (such as color development) by adding a chromogenic substrate. In some embodiments, the label is horseradish peroxidase (HRP), biotin, alkaline phosphatase, a dye, or a fluorescent label. In some embodiments, the label is an enzyme label selected from horseradish peroxidase and alkaline phosphatase. The chromogenic substrate can be selected from, for example, TMB, BCIP / NBT, Fast red, and AEC. In some embodiments, the label is a fluorescent label selected from fluorescein isothiocyanate (FITC), tetramethylrhodamine (RB200), tetramethyl rhodamine isothiocyanate (TRITC), and cyanine dyes such as Cy3 and Cy5.

[0021] In some embodiments, the method further includes adding a chromogenic substrate such as TMB, then terminating the color - development reaction and measuring the absorbance value. In the case of using biotin as the label, the method further includes adding streptavidin - HRP to react with biotin, adding a chromogenic solution, then terminating the reaction and measuring the absorbance value.

[0022] In some embodiments, the method further includes preparing a series of concentration solutions of protein A standards as a reference to plot a standard curve.

[0023] In some embodiments, the plate is a microplate such as a multi - well plate, preferably having a high adsorption capacity.

[0024] In one aspect, the present disclosure provides a kit that includes a plate coated with Fc protein and an anti - protein A antibody. Optionally, the anti - protein A antibody is conjugated with a label.

[0025] In one aspect, the present disclosure provides a kit that includes an Fc protein, an anti-protein A antibody conjugated with a label, and optionally a plate. This kit requires the user to coat the plate with the Fc protein before detection.

[0026] In some embodiments, the Fc protein in the kit comprises an Fc region derived from an immunoglobulin or a fragment or variant thereof. Preferably, the immunoglobulin is a human immunoglobulin. In some embodiments, the Fc protein comprises or consists of a human IgG1, IgG2, IgG3, or IgG4 Fc region or a variant thereof. In some embodiments, the Fc protein is an Fc fusion protein. In some embodiments, the Fc protein is a fragment of the Fc region.

[0027] In some embodiments, the kit further comprises one or more of a wash buffer, a blocking buffer, a chromogenic solution, a chromogenic stop solution, and a carbonate-bicarbonate (CBS) buffer. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The following drawings are included to further illustrate certain aspects and features of the present invention. The present invention can be better understood by referring to one or more of these drawings in conjunction with the detailed description of specific embodiments, including examples.

[0030] Figure 1 is a schematic diagram of a conventional sandwich immunoassay using two antibodies employed in current commercial kits, namely a capture antibody that captures protein A in a sample and a detection antibody labeled with biotin.

[0031] Figure 2 is a schematic diagram of a detection method according to some embodiments of the present invention.

[0032] Figure 3 is a flowchart of a detection method according to some embodiments of the present invention. DETAILED DESCRIPTION

[0033] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Additionally, unless the context otherwise requires, singular terms shall include plural forms and plural terms shall include singular forms. More specifically, as used in this specification and the appended claims, unless the context clearly indicates otherwise, the singular forms "a", "an", and "the" include plural referents. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes a mixture of cells, etc. In this application, unless otherwise stated, the use of "or" means "and / or". Additionally, the use of the term "comprising" and other forms (such as "including" and "containing") is not restrictive. Further, the ranges provided in the specification and the appended claims include the endpoints and all values between the endpoints.

[0034] The term "Fc protein" as used herein encompasses various forms of Fc proteins that can bind protein A, including the Fc region or fragments, variants, modified Fc proteins, engineered Fc proteins, fusion proteins of Fc proteins with other peptides, recombinant Fc proteins, etc. of natural immunoglobulins (preferably human immunoglobulins such as human IgG1, IgG2, IgG3, or IgG4). An exemplary Fc protein sequence is as

[0035] shown. Compared to the natural Fc region, Fc variants or modified Fc proteins may contain one or more amino acid additions, substitutions, or deletions while still retaining the ability to bind protein A. The interaction between IgG Fc and protein A consists mainly of hydrophobic interactions as well as some hydrogen bonds and salt bridges.

[0036] The term "anti-protein A antibody" as used herein includes polyclonal and monoclonal anti-protein A antibodies developed in various species such as humans, goats, chickens, mice, and rabbits. Different from Fc proteins, anti-protein A antibodies specifically recognize and bind protein A through the variable regions of their Fab portions. Many anti-protein A antibodies have been developed and are commercially available.

[0037] As used herein, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, which is determined by aligning and comparing the sequences. "Percentage identity" refers to the percentage of identical residues between amino acids or nucleotides in the molecules being compared, and is calculated based on the size of the smallest molecule being compared. For these calculations, gaps in the alignment (if any) are preferably resolved by a specific mathematical model or computer program (i.e., "algorithm"). Methods for calculating the identity of nucleic acids or polypeptides for alignment include the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), and the Needleman and Wunsch algorithm (J. Mol. Biol. 48: 444-453 (1970)) of the GAP program, which has been incorporated into the GCG software package (available at http: / / www.gcg.com).

[0038] As used herein, the terms "plate / slats" and "sheet" are used interchangeably and refer to a container or material onto which an Fc protein is to be coated on its surface for reaction with a sample to be tested, which is usually in the form of a microplate and a multi-well plate, but is not limited thereto.

[0039] As used herein, the term "ligand" or "packing material" refers to various forms of recombinant protein A, which is used as an affinity ligand conjugated to an affinity chromatography matrix to purify a target antibody by virtue of the binding between the ligand and the Fc region of the antibody. There are currently various protein A ligands for purifying antibodies on the market, such as MabSelect SuRe, NGL- A, Toyopeal, KANEKA KanCapA TM 3G, AT Protein A Diamond, MabSelect Prism A, Amsphere A3, EshmunoA and rProtein A.

[0040] Protein A used in the affinity chromatography column

[0041] Protein A is a single-chain protein extracted from Staphylococcus aureus (also known as SpA), which has 5 IgG-binding regions with highly similar compositions at the N-terminus: E, D, A, B, C. Each of these 5 binding regions can bind to the Fc segment of an IgG antibody, and the adsorption capacities are similar. Protein A can bind to the Fc region of an antibody with high affinity, and thus is used as an affinity ligand conjugated to an agarose matrix.

[0042] In the early days, the protein A columns bound natural protein A. Since other non-binding domains of natural protein A might bind to non-target proteins, the purity of the eluted proteins was insufficient, which would affect subsequent experiments. Therefore, in later affinity chromatography, the ligands used were obtained by cloning the gene of protein A through genetic engineering techniques and modifying it to remove some unimportant non-binding domains. For example, the recombinant protein A column of GE company uses recombinant protein A composed of five binding domains, namely E, D, A, B, and C, coupled with an agarose matrix. Putus developed a recombinant protein A containing three tandem B binding domains coupled with an agarose matrix. Currently, most of the affinity chromatography protein A columns on the market use recombinant protein A.

[0043] Detection of Protein A

[0044] Using protein A affinity chromatography may result in residual protein A in the sample. Currently, most commercial kits for detecting residual protein A are designed for specific recombinant protein A fillers used in affinity chromatography columns. Table 1 below lists various protein A fillers and their corresponding detection kits.

[0045] Table 1. Protein A ligands and corresponding kits

[0046]

[0047] There are various protein A fillers on the market. The mainstream filler is MabSelect SuRe LX, and other companies are also continuously producing new fillers. The commercial kits corresponding to the fillers are mainly Repligen and Cygnus series kits. As shown in Table 2 below, the Cygnus kit uses chicken anti-(IgY) polyclonal antibody as the capture antibody; in terms of detection antibodies, Repligen uses rabbit anti, and Cygnus uses chicken anti. Since it takes time to produce and purify antibodies, it often takes several years to develop corresponding kits for newly launched protein A fillers (the usage instructions of the protein A filler of Prism A were publicly available in July 2020, and the corresponding kit was just developed in 2023).

[0048] In the design of the kits, in order to ensure the specificity of detection, current commercial kits all adopt the sandwich protein A detection method, using a capture antibody and a detection antibody, and further amplifying the detection signal through the binding of a biotin marker conjugated to the detection antibody to streptavidin (SA)-HRP (see Figure 1) Moreover, for the Repligen 9333-1 kit corresponding to MabSelect SuRe LX with the largest market share, since the detection antibody competes with the capture antibody under acidic conditions, it is not feasible to incubate the sample with both the capture antibody and the detection antibody together, that is, they must be incubated step by step.

[0049] The present disclosure provides a detection method using an Fc fusion protein-coated plate and a corresponding kit (referred to as the WuXi Protein A kit herein). Based on the property that Fc and any protein A can bind under neutral pH conditions, a new ELISA method is designed. The detection method of the present invention is not restricted by the protein A ligand of affinity chromatography and can be used for the detection of protein A in post-chromatography samples of various packing materials. This method has strong universality and can quickly ensure a rapid response to the detection of protein A residues in biological drugs.

[0050] In addition, different from the form of biotin-conjugated antibody and streptavidin (SA)-HRP used in current commercial kits, the detection method of the present invention greatly simplifies the experimental operation process. It uses an Fc protein to coat the plate and uses a chicken antibody as the detection antibody for incubation under neutral conditions, which can avoid possible cross-reactions. At the same time, since the Fc fusion protein, the sample, and the detection antibody conjugated with HRP can be incubated together (i.e., the "one-pot" method), the number of incubations is reduced, and the complexity of the experimental operation is greatly reduced.

[0051] Method for detecting residual Protein A in the test sample

[0052] In some aspects, the present invention provides a method for detecting protein A in a sample, the method comprising: adding a sample to be detected and an anti-protein A antibody conjugated with a label to a plate coated with an Fc protein and incubating, and then detecting the content of protein A in the sample to be detected by detecting the signal or chromogenic substance generated by the label.

[0053] In some embodiments, the method further comprises adding a series of concentrations of protein A standard solution to the plate coated with an Fc protein in parallel to plot a standard curve.

[0054] In some embodiments, the anti-protein A antibody is conjugated with horseradish peroxidase (HRP). Accordingly, the method further comprises adding a TMB substrate for color development. TMB is converted into blue under the catalysis of HRP enzyme and finally converted into yellow under the action of acid treatment. Finally, the absorbance is measured at 450 / 650 nm, and the color intensity is positively correlated with the amount of protein A in the sample.

[0055] In some embodiments, the anti-Protein A antibody is conjugated with biotin. Accordingly, the method further includes adding streptavidin-HRP for incubation, and then adding TMB substrate for color development.

[0056] In some embodiments, the plate coated with Fc protein is a microplate, such as a multi-well plate, such as a 48-well plate, a 96-well plate, etc. It can be a V-bottom, U-bottom, flat-bottom or other bottom shape, and can be transparent, semi-transparent or opaque. The plate is preferably highly adsorptive.

[0057] In some embodiments, the method further includes pre-coating the plate with Fc protein. The method of coating the plate with protein is commonly used and publicly available in the art. For example, an Fc protein solution can be added to the plate and incubated overnight at 2-8°C. After the coating is completed, the 96-well plate is washed with 1x wash buffer, and a sample blocking solution is added for blocking. Optionally, after blocking, the plate strips are washed, dried, covered with a sealing film and reserved for use.

[0058] In some embodiments, the method further includes pre-treating the sample to be detected before adding it to the plate. The pre-treatment can be selected from dilution, acid treatment and heat treatment. One reason for the pre-treatment is that the sample to be detected often contains the target antibody to be purified that itself has an Fc region, and the shed Protein A can bind to the Fc region of the target antibody, which may affect the accuracy of quantification. Another reason is that if the concentration of the target antibody in the sample is high, it may interfere with the detection and needs to be diluted.

[0059] In some embodiments, the method includes denaturing the protein in the sample by heating and then separating the target protein and Protein A in the sample by centrifugation. Specifically, the Protein A standard solution or the sample can be placed at 100°C and heated for 20 minutes to denature the target protein in the sample (Protein A is not easily denatured due to its high temperature resistance), so as to separate it from the residual Protein A; then it is cooled to room temperature, centrifuged for 4 minutes, and the supernatant is taken as the sample solution to be detected.

[0060] In some embodiments, the method further includes preparing a solution of Protein A standard. For example, the Protein A standard is serially diluted with a sample diluent to prepare Protein A standards with different concentrations (Protein A standard solutions).

[0061] Kit and its preparation method

[0062] In some aspects, the present invention provides a kit for detecting Protein A in a sample. The kit may contain a plate coated with Fc protein, or a plate to be coated and Fc protein. The plate can be a microplate, such as a 96-well plate, and is preferably of high adsorption capacity.

[0063] In some embodiments, the kit comprises a plate coated with an Fc protein and an anti-Protein A antibody conjugated with a label. In some alternative embodiments, the kit comprises an Fc protein, an anti-Protein A antibody conjugated with a label, and a plate not coated with the Fc protein. In some alternative embodiments, the kit comprises an Fc protein and an anti-Protein A antibody conjugated with a label. In the latter two cases, the user can coat the plate with the Fc protein immediately before applying the methods disclosed herein.

[0064] In some embodiments, the kit further comprises one or more of the following solutions: a washing buffer, a blocking buffer, a chromogenic solution, and a chromogenic stop solution. Additionally, the kit may further comprise a carbonate-bicarbonate (CBS) buffer.

[0065] In some specific embodiments, the washing buffer is a PBST solution. In some embodiments, the blocking buffer is a bovine serum albumin solution, such as a solution of bovine serum albumin in PBST. In some specific embodiments, the chromogenic solution is a TMB solution, and the chromogenic stop solution is an HCL or H 2 SO 4 solution.

[0066] Multi - aspect evaluation of the detection method

[0067] One concern of commercial kits is specificity, and it is necessary to ensure that the correct concentration of residual Protein A can be accurately measured in various samples. Therefore, current commercial kits mainly use Fab fragment antibodies with stronger immune specificity as the coating antibody to capture specific Protein A molecules.

[0068] The Fab fragment anti-Protein A antibodies used in the Repligen and Enzo kits are chicken antibodies, which have a single immune site for Protein A. If the capture antibody, sample, and detection antibody are incubated together, there is a possibility that the capture antibody and the detection antibody will competitively bind to Protein A in the sample, resulting in a decrease in the signal value. Therefore, it is necessary to incubate and wash the unbound part separately multiple times, and then add the next reagent for incubation. A total of three incubations (sample, detection antibody, HRP conjugate) may be required before color development. Since the Cygnus kit uses a plate coated with a polyclonal antibody with multiple recognition sites as the capture antibody, there is a possibility of being recognized regardless of whether the detection antibody is monoclonal or polyclonal.

[0069] The inventors unexpectedly found that coating the plate with a human-derived Fc fusion protein can capture various types of affinity chromatography Protein A ligands, and selecting a chicken-derived polyclonal anti-Protein A antibody as the detection antibody can avoid cross-reaction and ensure that the binding sites corresponding to Protein A are not obscured. The methods and kits disclosed in the present invention can achieve good accuracy, precision, sensitivity, and linearity within a wide range.

[0070] A. Anti-interference performance verification

[0071] Since the double antibody sandwich detection method theoretically has higher specificity than the Fc-coated plate, it has stronger anti-interference ability. Therefore, it was verified whether the detection method of the present invention would be affected by and to what extent it would be affected by impurity proteins (mainly host cell proteins) and mAb concentration. As verified in Example 5.1, for the affinity chromatography harvest solution (AC harvest solution) containing a large amount of impurity proteins, the detection method of the present invention can still obtain linear measurement results. The fluctuation of the final protein A content level measured after diluting the same sample at different concentrations is very small, indicating that the interference by impurity proteins is extremely small. For samples containing a high antibody content, accurate measurement values can still be maintained when the antibody concentration reaches 1 mg / mL.

[0072] As the linearity evaluated herein refers to the degree to which the dilution factor is directly proportional to the concentration of the analyte within the designed range. The correlation coefficient R 2 The larger it is, the stronger the correlation between the two variables. The range refers to the relationship between the upper and lower limit concentrations or amounts applicable to the method, and the verification range can be determined prior to the start of experimental research.

[0073] B. Verification of universality and sensitivity

[0074] To test whether the detection method of the present invention is applicable to detecting different protein A ligands, experiments were respectively carried out on Mabselect SuRe, NGL- A, ToYoPEARL, KANEKA KanCapA TM 3G, AT ProteinA Diamond, MabSelectPrism A, Amsphere A3, Eshmuno A and rProtein A packing materials. The results showed (Table 7) that the method of the present invention can be applicable to various packing materials, and the limit of detection (LOD) is at the level of 4 - 14 pg / mL.

[0075] C. Accuracy of detection

[0076] Accuracy refers to the degree to which the result measured by the method is close to the true value or reference value. Accuracy can be expressed by the recovery rate (%). To test whether the detection method of the present invention can obtain accurate results on the eluted samples of different protein A packing materials, on Mabselect SuRe, NGL- A, ToYoPEARL, KANEKA KanCapA TMRecovery rates of Protein A samples at high (600 pg / mL), medium (200 pg / mL), and low (60 pg / mL) concentrations were tested on 3G, ATProteinA Diamond, MabSelect Prism A, Amsphere A3, Eshmuno A, and rProtein A ligands. The results showed (Table 9-26) that at least 90% recovery was maintained at medium and high concentrations on all ligands, and at least 85% recovery was maintained at low concentration.

[0077] D. Repeatability of Detection

[0078] Repeatability can be evaluated by multiple sampling measurements in different laboratory environments (such as different instruments, operating personnel, analysis batches, and times), and the results can be expressed by the coefficient of variation (%, CV). The coefficient of variation is the value obtained by dividing the standard deviation by the corresponding mean and multiplying by 100%, and can also be called the relative standard deviation. As verified in the examples, the detection results of the method and kit of the present invention show high consistency under different batches.

[0079] Advantages of the present invention

[0080] 1. Compared with traditional Protein A ELISA kits, the present invention uses Fc protein to replace the ligand-specific anti-Protein A antibody as the coating antibody, avoiding the problem that the kit cannot be generally applicable to different fillers.

[0081] 2. The present invention has a wide adaptability and is applicable to Protein A ligands of affinity chromatography columns for antibody purification (including MabSelectSuRe, MabSelect Prism A, Amsphere A3, NGL- A, AT Protein A diamond, Toyopearl, KANEKA KanCapA TM 3G, and Eshmuno A) and shows good performance. If there are new Protein A ligands in the future, the present invention can also be used for detection.

[0082] 3. The detection method of the present invention has higher sensitivity than Cygnus and Repligen, is more convenient to operate than Repligen and ENZO, and has a shorter operation time than ENZO.

[0083] Examples

[0084] The following examples are provided only for the purpose of illustrating various embodiments of the present invention, and they do not mean to limit the present invention.

[0085] Example 1. Preparation of Fc Protein-Coated Plate

[0086] Preparation of reagents:

[0087]

[0088] Dilute human IgG1 Fc protein (WuXi Biologics, SEQ ID NO: 1, approximately 32 kDa) to 4 μg / mL with coating buffer, and add 100 μL per well to a high-binding 96-well plate (Thermo / 442404). Incubate overnight at 2 - 8°C. The next day, take out the 96-well plate, discard the coating solution in the 96-well plate, then add 300 μL of 1x wash solution per well and wash once. Pat dry the residual wash solution on the blotting paper. Then add 200 μL of 1% BSA to each well of the 96-well plate, stick on the sealing film, and incubate in a thermostatic mixer at 25°C and 300 rpm for 60 minutes. Take out the 96-well plate, discard the blocking solution in the 96-well plate, then add 300 μL of 1x wash solution per well and wash three times. Pat dry the residual wash solution on the blotting paper. It can be used immediately or placed in the fume hood for about 5 minutes to dry the residual moisture. Then seal with the sealing film and store in the dark at 2 - 8°C for use within 7 days.

[0089] Example 2. Detection of residual Protein A in samples

[0090] Preparation of Protein A standard solution: Gradient dilute the Protein A standard with sample diluent to prepare Protein A standards with different concentrations.

[0091] Place the standard and the sample to be detected at 100°C for 20 minutes to denature the target protein in the sample, so as to separate it from the residual Protein A. Then cool to room temperature, centrifuge at 13800 rfc for 4 minutes, and take the supernatant for use.

[0092] 2.1 Example 1 (Add sample first for incubation, then add anti-Protein A antibody for incubation)

[0093] a) Add the standard solution or sample to the Fc protein-coated well plate, 100 μL per well. Cover with the sealing film and incubate at 25°C and 500 rpm for 60 minutes.

[0094] b) Wash the 96-well plate with 1x wash solution, add 300 μL of 1x wash solution per well and wash once. Pat the plate on the blotting paper to completely remove the residual liquid in the wells. Then add 100 μL of anti-Protein A antibody conjugated with HRP to each well, cover with the sealing film, and incubate at 25°C and 500 rpm for 60 minutes.

[0095] c) Wash the 96-well plate with 1x wash buffer. Add 300 μL of 1x wash buffer to each well, wash once, tap the plate on absorbent paper to completely remove the residual liquid in the wells, then add 100 μL of chromogenic solution to each well, seal the plate with a cover film, and incubate at 25 °C and 500 rpm for 15 - 20 minutes.

[0096] d) Add 100 μL of stop solution to each well in the 96-well plate to terminate the reaction. Measure the absorbance at 450 / 650 nm with a microplate reader within 30 minutes.

[0097] 2.2 Example 2 (Incubate after adding both anti-Protein A antibody and sample together, i.e., the "one-pot" method)

[0098] a) Add anti-Protein A antibody conjugated with HRP to the Fc protein-coated well plate, 100 μL per well. Then add the sample or standard solution, 100 μL per well. Cover the plate with a sealing film and incubate at 25 °C and 500 rpm for 120 minutes.

[0099] b) Wash the 96-well plate with 1x wash buffer. Add 300 μL of 1x wash buffer to each well, wash once, tap the plate on absorbent paper to completely remove the residual liquid in the wells, then add 100 μL of chromogenic solution to each well, cover the plate with a sealing film, and incubate at 25 °C and 500 rpm for 15 - 20 minutes.

[0100] c) Add 100 μL of stop solution to each well in the 96-well plate to terminate the reaction. Measure the absorbance at 450 / 650 nm with a microplate reader within 30 minutes.

[0101] Draw a standard curve based on the results of the standard solutions at each concentration, and then calculate the protein A content level in the sample based on the standard curve and the absorbance value of the sample to be detected. The standard curve range obtained in Example 2 is smaller than that in Example 1, indicating higher sensitivity. The highest OD values in both Example 1 and Example 2 can reach above 2.0. Generally speaking, both methods can be used for subsequent detection.

[0102] Example 3: Prepare a kit (WuXi Protein A Kit) according to the method of the present invention

[0103] Prepare each component as follows:

[0104] CBS solution: Take one carbonate buffer (CBS) capsule, dissolve the powder in 100 mL of ultrapure water, and store at 2 - 8 °C. The solution is valid for 1 month.

[0105] 1x Wash Solution: Prepare 1x PBST as the wash solution and store it at room temperature. The solution is valid for 1 month. For example: Take 50 ml of 20×PBST and add it to 950 ml of deionized water to make 1000 ml of wash solution, and store it at room temperature. Or, dilute 10x PBS and add a certain amount of Tween 20 to prepare 1x PBST.

[0106] Blocking Solution / Sample Diluent: Dissolve a certain concentration of BSA in 100 mL of 1x PBST and mix well. Store at 2 - 8°C. The solution is valid for 7 days.

[0107] Fc Protein-Coated Strip: Dilute Fc protein to a certain concentration using CBS solution. Add Fc protein to a high-binding 96-well plate and incubate overnight at 2 - 8°C. After coating, wash the 96-well plate with 1x wash solution and add blocking solution for blocking. After blocking, wash the strip, dry it, cover it with a sealing film and set aside for use.

[0108] Antibody-HRP Conjugate: Dilute HRP anti-protein A antibody to a certain concentration using sample diluent.

[0109] Chromogenic Solution: TMB, use directly.

[0110] Stop Solution (2M Sulfuric Acid): Take 820 mL of ultrapure water and add it to a glass beaker of appropriate size. Slowly add 100 mL of 98% concentrated sulfuric acid along the wall of the bottle, stirring while adding to dissipate heat. Transfer the prepared stop solution to a clean solution bottle and store at room temperature. The solution is valid for one year.

[0111] According to needs, the kit can contain one or more of the above components. The kit can contain pre-prepared solutions and buffers, or provide stock solutions or preparations, which are to be formulated into solutions and buffers by the user before use.

[0112] Example 4: Comparison of Detection Methods for Various Kits

[0113] Use different kits and corresponding detection methods in the following table to compare the results. Cygnus, Repligen, and Enzo are commonly used kits for measuring residual protein A in samples. The information of these kits is summarized as follows:

[0114] Table 2. Antibodies Used in Commercial Kits

[0115] Kit Cygnus Repligen Enzo Capture antibody Polyclonal antibody against Protein A Chicken anti - Protein A Chicken anti - Protein A Detection antibody Chicken anti - Protein A Rabbit anti - Protein A Chicken anti - Protein A

[0116] Table 3. Processing Procedures for Various Kits

[0117]

[0118] The results show that the standard curve range of the method of the present invention is at the pg / mL level, indicating higher sensitivity than the methods of the Cygnus kit and the Repligen kit. Moreover, the incubation step can be no more than 3 times, which is more convenient to operate than Repligen and ENZO, and the experimental time is also shorter than that of the ENZO operation. The recommended anti-mAb interference concentration of the kit is the concentration of the antibody in the sample when applying the method of the kit provided in the corresponding instruction manual of the kit. The larger the value, the stronger the anti-mAb interference ability of the kit.

[0119] Example 5. Comparison and Effect Evaluation of Detection Methods

[0120] 5.1 Interference Verification

[0121] Interferences that may be involved in the detection method include: interferences from impurities such as host cell proteins and interferences from the monoclonal antibody (mAb) to be purified (many kits evaluate their performance based on the interference of human-derived mAbs). In this regard, corresponding interference experiments were carried out, including the elution sample of the AC affinity column for impurity enrichment and the spike recovery rate of increasing the mAb concentration.

[0122] Since most projects have used the MabSelece SuRe (Lx) packing column, in the accuracy verification of the kit of the present invention, the comparative experiments all used the Repligen 9333-1 kit.

[0123] The double antibody sandwich detection method theoretically has higher specificity than the Fc-coated plate, and thus stronger anti-interference ability. Therefore, the kit verification will be carried out from two aspects: impurity protein interference and mAb concentration interference.

[0124] 5.1.1 Impurity Interference Verification

[0125] After the affinity chromatography (AC) of two monoclonal antibodies, the sample of the first-step harvest solution (this sample contains a large amount of impurity proteins, such as host cell proteins) and the sample of the final purified product after multiple rounds of purification are used to test the protein A content at different mAb protein concentrations. The experimental results are shown in Tables 4 and 5. The sample concentration in the second column represents the concentration of the interfering protein in the sample to be tested, which is obtained by diluting the sample whose protein concentration has been determined by a spectrophotometer. The protein A concentration in the third column represents the measured protein A concentration in the corresponding sample, the result in the fourth column represents the ratio of the third column to the second column, and the linearity in the fifth column represents the fitting degree of the obtained result with the standard curve. The final result represents the ratio of protein A to the monoclonal antibody in the sample after averaging 4 tests. Among them, if the detected protein A concentration exceeds the highest point of the standard curve, 1000 pg / mL, the result calculation is not carried out; "Range?" appearing in all the following tables means below the detection limit and is calculated as 0.

[0126] The experimental results show that the WuXi Protein A Kit and the Repligen 9333-1 Kit can achieve equivalent detection results, and the sample mAb concentration can reach at least 0.5 mg / mL (500 μg / mL). Whether impurities are present or not does not affect the detection results.

[0127] Table 4. Test Results of the Method of the Present Invention

[0128]

[0129]

[0130] Table 5. Detection Results of the Method of the Repligen 9333-1 Kit

[0131]

[0132] * 0.05 ng / mL is the lowest point of the standard curve of the Repligen 9333-1 Kit, so the results were not calculated.

[0133] 5.1.2 Verification of mAb Concentration Interference

[0134] In this experiment, the content of Protein A at different mAb concentrations was detected. The content of 5 final purified solutions at different mAb concentrations was detected, and the results are shown in Table 6A. The results show that when the mAb concentration of the sample reaches 1 mg / mL (1000 μg / mL), accurate measured values can still be maintained.

[0135] For the AC harvest solution, Protein A detection at an mAb concentration of 1 mg / mL was also performed. Since it exceeded the upper limit value of the standard curve, the data was not adopted.

[0136] The data in Tables 6A-6B below show that the kit of the present invention has a lower detection limit and a wider linear detection range than the Repligen 9333-1 Kit.

[0137] Table 6A. Detection Results of the Kit of the Present Invention

[0138]

[0139] Table 6B. Detection Results of the Repligen 9333-1 Kit

[0140]

[0141]

[0142] * 0.05 ng / mL is the lowest point of the standard curve of the Repligen 9333-1 Kit, so the results were not calculated.

[0143] 5.2 Verification of Universality and Sensitivity

[0144] Experiments were conducted on commonly used ligands and ligands for which there are no corresponding kits on the newly launched market. Standard curves were made using different Protein A ligands respectively, and the recovery rate of each point was calculated. In terms of sensitivity, since the calibration curve range is 0 - 1000 pg / mL, data could not be obtained for two ligands according to the detection limit (LOD) calculation method of Cygnus (0 - 10 ng / mL). Therefore, the calculation method of the Enzo (0 - 1000 pg / mL) kit was adopted to calculate the detection limit:

[0145]

[0146] The experimental results are shown in Table 7 below.

[0147] Table 7. Detection Results of the Kit of the Present Invention for Different Protein A Ligands

[0148]

[0149] 5.3 Verification of Repeatability

[0150] In this experiment, a sample with a relatively high impurity content was used, and different experimenters detected it on different days to verify the data repeatability. Different experimenters detected the AC harvest solution with different mAb concentrations of monoclonal antibodies on 3 different natural days, and the results are shown in Tables 8A - B below. CV% represents the coefficient of variation. The detection results show high consistency.

[0151] Table 8A. Detection Results of the Kit of the Present Invention

[0152]

[0153] Table 8B. Detection Results of the Kit of the Present Invention

[0154]

[0155]

[0156] 5.4 Verification of Detection Accuracy of Each Protein A Ligand

[0157] In this experiment, the recovery rates of 4 detections each at high, medium, and low concentrations of different Protein A ligands were verified.

[0158] Prepare the standard curve of MabSelect SuRe ligand (see Table 9 below) and perform repeated detections on MabSelect SuRe at high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) respectively. The results are shown in Table 10 below. The experimental results show that high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) can all ensure high recovery rates.

[0159] Table 9. Standard Curve of MabSelect SuRe

[0160]

[0161] Table 10. Detection Results of MabSelect SuRe at High, Medium, and Low Concentrations

[0162]

[0163] Prepare the standard curve of NGL- A ligand (see Table 11 below) and perform repeated detections on NGL-Impact A at high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) respectively. The results are shown in Table 12 below. The experimental results show that high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) can all ensure high recovery rates.

[0164] Table 11. Standard Curve of NGL- A

[0165]

[0166]

[0167] Table 12. Detection Results of NGL- A at High, Medium, and Low Concentrations

[0168]

[0169] Prepare the standard curve of Toyopearl ligand (see Table 13 below) and perform repeated detections on Toyopearl at high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) respectively. The results are shown in Table 14 below. The experimental results show that high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) can all ensure high recovery rates.

[0170] Table 13. Standard Curve of Toyopearl

[0171]

[0172]

[0173] Table 14. Detection results of Toyopearl at high, medium, and low concentrations

[0174]

[0175] Production of KANEKA KanCapA TM Prepare the standard curve of 3G ligand (see Table 15 below) and perform repeated detections on KANEKA KanCapA at high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) respectively. The results are shown in Table 16 below. The experimental results show that high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) can all ensure a high recovery rate. TM

[0176] Table 15. Standard curve of KANEKA KanCapA TM 3G

[0177]

[0178] Table 16. Detection results of KANEKA KanCapA TM 3G at high, medium, and low concentrations

[0179]

[0180] Prepare the standard curve of AT Protein A Diamond ligand (see Table 17 below) and perform repeated detections on AT Protein A Diamond at high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) respectively. The results are shown in Table 18 below. The experimental results show that high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) can all ensure a high recovery rate.

[0181] Table 17. Standard curve of AT Protein A Diamond

[0182]

[0183] Table 18. Detection results of AT Protein A Diamond at high, medium, and low concentrations

[0184] ​

[0185]

[0186] Prepare the standard curve of MabSelect Prism A (see Table 19 below) and perform repeated detections on MabSelect Prism A at high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) respectively. The results are shown in Table 20 below. The experimental results show that high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) can all ensure a relatively high recovery rate.

[0187] Table 19. Standard Curve of MabSelect Prism A

[0188]

[0189] Table 20. Detection Results of MabSelect Prism A at High, Medium, and Low Concentrations

[0190]

[0191]

[0192] Prepare the standard curve of Eshmuno A (see Table 21 below) and perform repeated detections on Eshmuno A at high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) respectively. The results are shown in Table 22 below. The experimental results show that high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) can all ensure a relatively high recovery rate.

[0193] Table 21. Standard Curve of Eshmuno A

[0194]

[0195] Table 22. Detection Results of Eshmuno A at High, Medium, and Low Concentrations

[0196]

[0197]

[0198] Prepare the standard curve of Amsphere A3 (see Table 23 below) and repeat the detection of Amsphere A3 at high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) respectively. The results are shown in Table 24 below. The experimental results show that high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) can all ensure a high recovery rate.

[0199] Table 23. Standard Curve of Amsphere A3

[0200]

[0201] Table 24. Detection Results of Amsphere A3 at High, Medium, and Low Concentrations

[0202]

[0203] Prepare the standard curve of rProtein A ligand (see Table 25 below) and repeat the detection of rProtein A at high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) respectively. The results are shown in Table 26 below. The experimental results show that high concentration (600 pg / mL), medium concentration (200 pg / mL), and low concentration (60 pg / mL) can all ensure a high recovery rate.

[0204] Table 25. Standard Curve of rProtein A

[0205]

[0206]

[0207] Table 26. Detection Results of rProtein A at High, Medium, and Low Concentrations

[0208]

[0209] Those skilled in the art will further recognize that the present invention can be implemented in other specific forms without departing from its spirit or central characteristics. Since the foregoing description of the present invention has only disclosed its exemplary embodiments, it should be understood that other variations are considered to be within the scope of the present invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Instead, reference should be made to the appended claims to indicate the scope and content of the present invention.

Claims

1. A method for detecting protein A in a sample, the method comprises: adding the sample and an anti-protein A antibody conjugated with a label to a plate coated with Fc protein, and incubating to form a complex of Fc protein, protein A in the sample, and the anti-protein A antibody, and determining the content of protein A in the sample by detecting the signal generated by the label.

2. The method of claim 1, wherein the sample and the anti-protein A antibody conjugated with the label are added to the plate coated with Fc protein and then incubated together.

3. The method of claim 1, wherein the sample is added to the plate coated with Fc protein and a first incubation is carried out, and then the anti-protein A antibody conjugated with the label is added and a second incubation is carried out.

4. The method according to any one of claims 1-3, wherein the incubation time for each time is 30 minutes to 2 hours, such as 1 hour to 2 hours.

5. The method according to any one of claims 1-4, wherein the Fc protein can specifically bind to protein A and comprises the Fc region of human immunoglobulin or its fragment or variant.

6. The method of claim 5, wherein the Fc protein is the Fc region of human IgG1 or its fragment.

7. The method according to any one of claims 1-6, wherein the method further comprises pre-coating the plate by incubating the Fc protein with the plate.

8. The method according to any one of claims 1-7, wherein the anti-protein A antibody is selected from anti-protein A antibodies of chicken origin, anti-protein A antibodies of rabbit origin, and anti-protein A antibodies of human origin.

9. The method of claim 8, wherein the anti-protein A antibody is a monoclonal antibody or a polyclonal antibody.

10. The method according to any one of claims 1-9, wherein the label is selected from HRP, alkaline phosphatase, fluorescent label, dye, and biotin.

11. The method according to any one of claims 1-10, wherein the sample is an elution sample of a target protein (such as an antibody) from protein A affinity chromatography.

12. The method according to any one of claims 1-11, wherein the sample is pretreated before being added to the plate, and the pretreatment is selected from acid treatment, dilution treatment, and heat treatment.

13. The method of claim 12, wherein the pretreatment comprises heating the sample at about 95-100 °C for about 20 minutes, then cooling to room temperature and centrifuging, and harvesting the supernatant as the sample to be added to the plate.

14. The method according to any one of claims 1-13, which further comprises adding a chromogenic substrate to make the label generate a detectable signal, then terminating the reaction (such as by adding a chromogenic termination solution) and measuring the absorbance value.

15. The method according to any one of claims 1-14, which further comprises preparing a series of concentration solutions of protein A standards as a reference to draw a standard curve.

16. The method according to any one of claims 1-15, wherein the plate is a microplate such as a multi-well plate, preferably having a high adsorption capacity.

17. A kit, which comprises Fc protein and an anti-protein A antibody, and the anti-protein A antibody is optionally conjugated with a label.

18. The kit of claim 17, which comprises a plate coated with Fc protein.

19. The kit of claim 17 or 18, wherein the Fc protein comprises an Fc region or a fragment or variant thereof derived from a human immunoglobulin, for example, the Fc protein is a human IgG1 Fc region or a fragment thereof.

20. The kit of any one of claims 17-19, wherein the label is selected from HRP, alkaline phosphatase, a fluorescent label, a dye, and biotin.

21. The kit of any one of claims 17-20, which further comprises one or more of a washing buffer, a blocking buffer, a chromogenic solution, a chromogenic termination solution, and a carbonate-bicarbonate (CBS) buffer.

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