Immune globulin binding protein and application thereof

Abstract

The invention discloses an immunoglobulin binding protein, the immunoglobulin binding protein comprises a mutant of a Z structural domain of protein A, the mutant of the Z structural domain has good affinity and thermal stability, and a chromatography medium obtained by using the recombinant Protein A protein has higher binding capacity and alkali stability.

Description

Technical Field

[0001] This application belongs to the field of biotechnology. Specifically, this invention relates to a novel recombinant staphylococcal protein A and its applications. Background Technology

[0002] Immunoglobulins (such as monoclonal or polyclonal antibodies) are currently a highly sought-after biopharmaceutical product in the research and manufacturing field. Since the first monoclonal antibody drug was launched in 1986, more than 100 monoclonal antibody drugs have been approved for marketing worldwide. Purification of immunoglobulins is a crucial step in obtaining these products.

[0003] Staphylococcal protein A (SPA), or simply protein A, is a protein found on the surface of Staphylococcus aureus. Its molecule contains five domains: E, D, A, B, and C. It can bind to the Fc fragment of human and various mammalian IgG, acting as an immunoglobulin-binding protein. In 1987, Nilsson et al. replaced the first alanine amino acid in the B domain of SPA with valine and the 29th amino acid with alanine, thus obtaining the Z domain. Protein A affinity chromatography media containing the Z domain exhibit improved alkali stability compared to those containing the B domain. Researchers have conducted extensive studies based on the Z domain, obtaining protein A mutants with further improved alkali stability.

[0004] Currently, various purification packing materials for protein A ligands are available on the market, such as those in the form of natural protein A (e.g., Protein A SEPHAROSE™, Cytiva) and some engineered protein A packing materials (e.g., prismA, Cytiva), but their performance varies considerably. For example, early protein A affinity chromatography ligands exhibited poor alkali resistance. Natural protein A and traditional modified forms (e.g., Z-domain) are prone to conformational denaturation or hydrolysis under strong alkaline conditions (e.g., 0.5–1 M NaOH), leading to ligand inactivation. After 48 hours of treatment with 0.5 M NaOH, the binding activity of natural protein A decreased to 53%. Although Z-domain mutants have shown some improvement, their long-term stability remains unsatisfactory. If these process-related impurities cannot be effectively removed in subsequent processes, there is a risk of triggering an immune response, posing a significant challenge to drug quality. Currently, research on improving the alkali resistance of Protein A is extensive; for example, Susanne Gilich et al. proposed that protein engineering can improve the stability of protein ligand domains under alkaline conditions. Currently, various research institutions are engineering the amino acids in each domain of protein ligands and using AI calculations to mutate amino acid sites in order to explore sequences that can improve the alkali resistance of protein ligands.

[0005] However, existing packing materials face the following core problems when undergoing alkali resistance modification: First, loading capacity is limited. The dynamic binding capacity (DBC) of traditional agarose-based packing materials is generally lower than 50 mg IgG / mL medium, restricting production efficiency. Second, ligand detachment is a major concern. Insufficient coupling stability leads to a shortened packing material lifespan and increases production costs. The ligand detachment rate of some products is as high as 100 ppm. Summary of the Invention

[0006] This invention relates to an immunoglobulin-binding protein that exhibits higher affinity and thermal stability compared to commercially available products. The affinity separation matrix prepared from it has higher binding capacity and alkali stability. Specifically, this invention provides an immunoglobulin-binding protein comprising a mutant of the Z domain of protein A, wherein the mutant of the Z domain is selected from SEQ ID No: 1 to 7.

[0007] Further, the immunoglobulin-binding protein comprises 2 to 8 repeating mutants of the Z domain, wherein the 4th and 6th amino acids of the mutant Z domain are proline; or the mutant Z domain is selected from SEQ ID No: 1 to 3.

[0008] Furthermore, the C-terminus and / or N-terminus of the immunoglobulin-binding protein may be further linked to a protein tag, preferably, the protein tag being one or more cysteine ​​residues, lysine residues, or histidine residues.

[0009] Furthermore, the immunoglobulin-binding protein is selected from SEQ ID NO:8 or SEQ ID NO:9.

[0010] Secondly, the present invention provides a nucleotide sequence encoding the aforementioned immunoglobulin-binding protein.

[0011] Thirdly, the present invention provides an expression vector comprising the nucleotide sequence described above.

[0012] Fourthly, the present invention provides a host cell comprising the above-described expression vector, or having the above-described nucleotide sequence integrated into its genome.

[0013] Fifthly, the present invention provides an affinity chromatography ligand comprising one or more of the immunoglobulin-binding proteins described above.

[0014] In a sixth aspect, the present invention provides an affinity chromatography medium comprising the aforementioned immunoglobulin-binding protein or the aforementioned affinity chromatography ligand coupled to a solid support.

[0015] In a seventh aspect, the present invention provides the use of the above-mentioned affinity chromatography medium for the purification of antibodies or Fc fusion proteins, and for the preparation of therapeutic agents for treating clinical autoimmune diseases or other diseases related to Ig in the human body.

[0016] This invention utilizes protein computational simulation tools and combined with screening tests to obtain a specific recombinant Protein A protein. Testing revealed good affinity and thermostability, further validating its loading capacity as a chromatography medium of approximately 70 mg / ml, comparable to commercially available products. Regarding alkali resistance comparison, the dynamic binding capacity (DBC) remained stable after immersion in 1 mol / L NaOH solution for 48 hours. Measurements of actual samples immersed in 1 M NaOH for 48 hours showed antibody residue levels below 20 ppm, effectively ensuring the recyclability of the chromatography medium. Attached Figure Description

[0017] Figure 1 Schematic diagram of SPA-11M structure. Detailed Implementation

[0018] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0019] The terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and not to describe a specific order or sequence. It should be understood that such use of data can be interchanged where appropriate so that embodiments of this application can be implemented in orders other than those illustrated or described herein, and the objects distinguished by "first," "second," etc., are generally of the same class and the number of objects is not limited; for example, a first object can be one or more. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0020] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0021] The term "immunoglobulin-binding protein" can refer to a protein that can bind to the Fc fragment of an immunoglobulin. However, it does not preclude proteins that bind to the Fc fragment from binding to other regions, such as the Fab region of an immunoglobulin.

[0022] As used herein, unless otherwise stated, the terms "recombinant Protein A," "immunoglobulin-binding protein," "recombinant protein A," "SpA recombinant protein," and "recombinant protein A polymer" are used interchangeably and all refer to the immunoglobulin-binding protein of this invention obtained by modifying the amino acid sequence of wild-type SpA.

[0023] The mutant of the Z-domain described in this invention is the result of a specific mutation in the Z-domain sequence.

[0024] The Z-sequence of the structural domain is:

[0025] VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK

[0026] The immunoglobulin-binding proteins described in this invention, in addition to referring to proteins with the aforementioned antibody-binding recombinant protein A activity and having amino acid sequences such as SEQ ID NO. 1 to SEQ ID NO. 7, also include variant forms with sequences of SEQ ID NO. 1 to SEQ ID NO. 7 having the same or similar functions as the recombinant Protein A protein. These variant forms include (but are not limited to): deletions, insertions, and / or substitutions of several amino acids (typically 1-20, more preferably 1-10, 1-8, 1-5, 1-3, or 1-2), and the addition or deletion of one or more amino acids (typically 1-20, more preferably 1-10, or 1-5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not change the function of the protein. Similarly, adding one or more amino acids at the C-terminus and / or N-terminus generally does not change the function of the protein. In another preferred embodiment, one or more cysteine ​​residues, lysine residues, or histidine residues are added to the C-terminus of the immunoglobulin-binding protein.

[0027] Preferably, the 4th and 6th amino acids of the mutant Z domain are proline.

[0028] The inventors conducted in-depth analysis of the mutant Z-domain and discovered that mutating amino acids at positions 4 and 6 of the Z-domain to proline resulted in protein A with better binding capacity and base stability. The structure of the mutant Z-domain (SEQ ID No:1) is as follows. Figure 1 As shown.

[0029] Further preferably, the mutant of the Z-domain is selected from SEQ ID No: 1 to 3. More preferably, the mutant of the Z-domain is selected from SEQ ID No: 1 or 2.

[0030] Immunoglobulin-binding proteins may include 2 to 8 repeating mutants of the Z domain, preferably 4 to 8 repeating mutants of the Z domain, for example 4, 5, 6, 7 or 8, and may be 5 to 7 repeating mutants of the Z domain.

[0031] Protein variants include: homologous sequences, conserved variants, allelic variants, natural mutants, proteins encoded by DNA that hybridizes with the DNA of antibody-binding recombinant protein A under high or low severity conditions, and proteins obtained using antiserum of said antibody-binding recombinant protein A.

[0032] The immunoglobulin-binding protein of the present invention can be chemically synthesized or produced from a prokaryotic or eukaryotic host using recombinant technology. The host can be any cell known to those skilled in the art capable of expressing the recombinant protein (e.g., bacterial, yeast, higher plant, insect, and mammalian cells). In another preferred embodiment, the host is *Escherichia coli* BL21(DE3).

[0033] The present invention also provides a nucleotide sequence encoding the immunoglobulin-binding protein. Accordingly, the present invention includes protein DNA sequences that can be used to produce mutants by expression in a recombinant host using existing techniques. (Based on codon degeneracy, the present invention includes all nucleotide sequences that can encode the recombinant protein A-mer.)

[0034] The full-length nucleotide sequence or fragment thereof of the immunoglobulin-binding protein of the present invention can be synthesized by chemical synthesis, PCR amplification, or recombinant synthesis. In another preferred embodiment, the nucleotide sequence is obtained by chemical synthesis.

[0035] This invention relates to a recombinant expression vector for expressing an immunoglobulin-binding protein, the expression vector comprising a nucleotide sequence encoding the immunoglobulin-binding protein and a vector sequence. The term "recombinant expression vector" refers to bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors well known to those skilled in the art. In short, any plasmid and vector that can stably replicate within a host can be used in this invention. In another preferred embodiment, the expression vector comprises one or more selective marker genes to provide phenotypic characteristics for selecting transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green fluorescent protein (GFP), or kanamycin or ampicillin resistance for *E. coli*. In another preferred embodiment, the expression vector is selected from the pGEX or pET series vectors. In another preferred embodiment, the expression vector is the pET-28a vector.

[0036] The present invention also provides a host for expressing the immunoglobulin-binding protein of the present invention, wherein the expression host comprises the recombinant expression vector of the present invention. The host can be any expression cell known to those skilled in the art, and can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. In another preferred embodiment, the host is *Escherichia coli* BL21(DE3).

[0037] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0038] The obtained transformants can be cultured using conventional methods to express the immunoglobulin-binding protein of the present invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth, and once the host cells have grown to an appropriate cell density, they are induced to express the target protein using suitable methods (such as temperature adjustment or chemical induction).

[0039] The immunoglobulin-binding proteins described in this invention can be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, their physical, chemical, and other properties can be utilized to separate and purify the target proteins using various separation methods. These separation and purification methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, protein precipitation (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0040] The present invention provides an affinity chromatography ligand comprising one or more immunoglobulin-binding proteins obtained in this invention.

[0041] This invention provides an affinity chromatography medium comprising an affinity chromatography ligand coupled to a solid-phase support. The solid-phase support of this invention can be any solid-phase support known in the art for the separation and purification of Fc-containing proteins. For example, the solid-phase support can be a porous or non-porous material. Exemplary solid-phase supports include, but are not limited to, polymers containing multiple hydroxyl groups, such as polysaccharides, or other synthetic polymers. The polysaccharides include agarose, agar, dextran, starch, cellulose, pullulan, etc. Preferably, the solid-phase support is in the form of microspheres. Preferably, the coupling is via a thioether bond. The solid-phase support is a polysaccharide.

[0042] The method by which the immunoglobulin-binding protein of the present invention attaches as a ligand to the affinity medium during affinity chromatography can be a carrier binding method, a physical adsorption method, a cross-linking method, or an embedding method. In another preferred embodiment, the method by which the protein is linked to the carrier is a cross-linking method. In another preferred embodiment, the protein and the carrier are cross-linked together via thioether bonds.

[0043] The immunoglobulin-binding protein of the present invention can be used to prepare affinity media for purifying antibodies. Therefore, the present invention provides an affinity chromatography medium that can be used for purifying antibodies or Fc fusion proteins, and for preparing therapeutic agents for treating clinical autoimmune diseases or other diseases related to Ig (immunoglobulin) in the human body. This includes an affinity medium and the immunoglobulin-binding protein of the present invention attached to said affinity medium.

[0044] The specific embodiments and comparative examples of the present invention are listed below, but the present invention is not limited to the following examples.

[0045] Example 1 Screening of recombinant protein A

[0046] Based on the analysis of the Z-sequence of protein A domain (VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQAPK) using protein computational simulation tools (such as deep learning models), and considering the structural and functional characteristics of the protein, protein structure simulation was performed based on the collected protein sequence data. Functional features such as active and binding sites were extracted, and suitable mutation and retention regions were selected for virtual mutation screening. Screening included pH stability prediction and affinity change prediction. Combining the results of the virtual screening, the structure of protein A was analyzed, including the degrees of freedom of the surface peptide chain, amino acid residue interactions at subunit interfaces, and interactions between amino acid residues in the active pocket and the substrate, to identify potential directed evolutionary hotspots. The obtained mutation sites will be used to construct a mutation library, and the impact of mutation sites on the performance of protein A will be screened and evaluated. Finally, 25 sequences were selected from the 2520 mutation library. Structural simulation was performed on these 25 sequences, and 19 sequences were selected based on base stability and affinity prediction. Structural simulations were performed on the 19 selected sequences, and 7 sequences were found to have better predicted base stability and affinity than the control. After multiple rounds of selection, the sequences shown in Table 1 were obtained.

[0047] Table 1

[0048]

[0049] Example 2 Affinity of Recombinant Protein A

[0050] The affinity between recombinant protein A obtained in Example 1 and antibody IgG was determined using biomembrane interference (BLI) technology. The specific procedure is as follows:

[0051] Sensor immobilization: The recombinant protein A variant was dissolved in 1xPBS buffer at a concentration of 50-100 μg / mL and immobilized on the surface of the anti-HIS biosensor by amino coupling method, with a target immobilization amount of approximately 1.0 nm.

[0052] Analyte preparation: Antibody IgG was serially diluted with PBST running buffer (1X PBS, pH 7.4, containing 0.02% Tween-20) to prepare at least 5 concentration gradients.

[0053] Binding and dissociation: The solidified sensor was sequentially immersed in IgG samples of different concentrations and oscillated at 1000 rpm at 25°C for 300 seconds to monitor the binding process; then the sensor was transferred to the running buffer and the dissociation process was monitored for 300 seconds.

[0054] Sensor regeneration: After each measurement, immerse the sensor in a 10 mM glycine-HCl (pH 1.5) solution for 10 seconds to completely remove the bound antibody and ensure that the sensor can be reused.

[0055] Data Analysis: Using the instrument's software, a 1:1 global fitting of the binding and dissociation curves was performed to obtain the binding rate constant (Ka), dissociation rate constant (Kd), and affinity constant (K). D ).

[0056] The reaction conditions were as follows: different protein A samples were added with 1M NaOH to a final concentration, and the reaction was terminated after 1 hour at room temperature. Samples without alkali treatment and samples treated with 1M NaOH were then analyzed. The results are detailed in Table 2.

[0057] Table 2

[0058]

[0059] Note: NA indicates exceeding the detection limit; in most cases, it represents K. D The value is extremely low, meaning the antibody has very high affinity.

[0060] A commercially available protein A sequence with good alkali resistance and high loading capacity was selected as the positive control group. The specific sequence is as follows:

[0061] VDAKFDKEAQEAFYEILHLPNLTEEQRNAFIQSLKDDPSVSKAILAEAKKLNDAQAPK (SEQ ID No:10)

[0062] Affinity constant K D The affinity is the most direct indicator; the smaller the value, the stronger the affinity. The affinity of the protein A domain variant obtained in this invention is comparable to, or even better than, that of commercially available protein A with good alkali resistance and high loading capacity, and it maintains good affinity even after alkali treatment.

[0063] Example 3: Base stability of recombinant protein A

[0064] The stability of protein A mutants under alkaline conditions was detected using Nanotemper (DSF), and the stability of each protein mutant was evaluated by isothermal denaturation experiments.

[0065] Reaction conditions: Protein samples were treated with 1 M NaOH (detection concentration was 2.5 mg / ml), reacted at 32℃ for 48 h, and data were detected by Isothermal Scan every 5 min. The detection results are detailed in Table 3.

[0066] The stability of each protein mutant was assessed by isothermal denaturation experiments under a constant temperature of 32℃. The first denaturation time of the mutants SPA-2M, SPA-3M, SPA-4M, and SPA-5M was earlier than that of the control, while the first denaturation time of SPA-8M, SPA-9M, and SPA-11M was later. The first denaturation time directly reflects the kinetic stability of the protein against unfolding. Therefore, the above results indicate that the conformational stability of the mutants SPA-8M, SPA-9M, and SPA-11M was substantially improved compared to the control. On the other hand, the second denaturation time of all tested mutants was basically the same as that of the control group.

[0067] In summary, the protein A mutants of the present invention exhibit good development potential after alkali treatment: all mutants retain the excellent property of being non-aggregating, among which mutants SPA-8M, SPA-9M, and SPA-11M significantly improve their inherent alkali resistance while maintaining a low aggregation tendency, making them candidate molecules with outstanding advantages.

[0068] Table 3

[0069]

[0070] (The positive control group is the same as in Example 2)

[0071] Example 4: Expression and purification of recombinant protein A

[0072] The mutant obtained in Example 1 can be repeated 2 to 8 times to form a tandem protein. This example uses a 6-repetition sequence. Details are as follows:

[0073] 1. Construct six repeat sequences for the amino acid sequences of the SPA-9M and SPA-11M domains, as follows:

[0074] VDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNDAQAPKVDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNDAQAPKVDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNDAQAPKVD LPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNDAQAPKVDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNDAQAPKVDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNDAQAPKC (SEQ ID No:9, 6 repeat sequences of SPA-9M, referred to as SPA-9M-6)

[0075] VDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNAEQAPKVDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNAEQAPKVDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNAEQAPKVD LPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNAEQAPKVDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNAEQAPKVDLPFPKEAQAAFYEILHLPNLTEEQRNAFIQSLKDDPTVAAAVLAEARRLNAEQAPKC (SEQ ID No:8, 6 repeat sequences of SPA-11M, referred to as SPA-11M-6)

[0076] The amino acid sequences (SPA-9M-6 and SPA-11M-6) were translated into nucleotide sequences and the gene synthesis was outsourced to a gene synthesis company.

[0077] 4. Protein Expression and Purification

[0078] Recombinant plasmids were transformed into host cells to induce the expression of SPA-9M-6 and SPA-11M-6 proteins. After collecting the lysates, the His-tagged SPA-9M-6 and SPA-11M-6 proteins were purified using nickel column affinity chromatography. Purity was assessed by SDS-PAGE electrophoresis. The proteins were then concentrated to 20 mg / ml using tangential flow filtration (TFF) dialysis.

[0079] Example 5: Alkali Resistance Test of Protein A Affinity Chromatography Medium

[0080] The purified recombinant Protein A obtained in Example 4 was coupled to a solid support, as follows:

[0081] Activate the epoxy groups on the surface of agarose gel spheres by adjusting reaction conditions (e.g., pH 9-10, temperature 25°C). Add the purified recombinant Protein A solution to the activated agarose gel spheres and stir overnight to complete coupling. Wash away unbound ligands and block the remaining active sites.

[0082] Efficacy validation: Determination of the binding capacity, alkali resistance, and residual IgG1 levels of Protein A affinity chromatography medium.

[0083] 1. Combining load and alkali resistance testing methods

[0084] ①Preparation before the experiment: Prepare the buffer solution required for the experiment, and clean and balance the instrument system;

[0085] ②10% breakthrough assay of 3 mg / mL IgG1 standard;

[0086] ③ Equilibrate the chromatography column: Connect the chromatography column to the protein chromatography system and equilibrate the chromatography column with buffer for at least 5 CV (23.5 ml) until the UV 280 nm baseline is stable and close to 0 mAu (±5 mAU).

[0087] ④10% penetration test;

[0088] ⑤ Calculate the IgG1 binding load and IgG1 recovery rate according to the formulas; Packing material load = recovered antibody amount / packing material loading volume; Recovery rate = recovered antibody amount / C 上样浓度 *V 上样体积*90%

[0089] ⑥ After washing the column with the detected binding loading with 1 mol / L NaOH solution until the conductivity value is balanced, remove the column and store it in a refrigerator at 2-8℃ for 48 h. Then, detect the binding loading and recovery rate according to the above steps. After the detection is completed, continue to wash the column with 1 mol / L NaOH solution until the conductivity value is balanced. Then, remove the column and store it in a refrigerator at 2-8℃ for 72 h. Repeat the above operation.

[0090] 2. Detection of residual IgG1 in the packing material after in-situ washing (CIP) was performed using enzyme-linked immunosorbent assay (ELISA). The specific steps are as follows:

[0091] ① Sample dilution: Take CIP effluent samples and determine the dilution gradient (400-16000X) based on the residual amount in the samples. At the same time, perform spike experiments and ensure that the spike recovery rate is within the range (70-130% / 80%-120%).

[0092] ② Sample lysis: The diluted sample is lysed, which can be done by acid hydrolysis or boiling, so that the residual IgG1 antibody can be exposed to its detection epitope;

[0093] ③ Immunoassay: Incubate and wash the sample obtained in step ② according to the kit instructions. The ELISA kit uses an antibody that specifically recognizes the IgG Fc fragment for detection;

[0094] ④ Color development and determination: After adding the color development substrate and stopping the reaction, use an ELISA reader to measure the absorbance value and calculate the residual concentration of IgG1 in the sample according to the standard curve.

[0095] The method described in this invention can be implemented using a commonly used IgG quantitative ELISA kit in the field, and its detection performance needs to be verified by spiked recovery experiments.

[0096] 3. The test results are shown in Table 4.

[0097] Table 4. Effect data of different recombinant Protein A proteins as ligands

[0098]

[0099] Note: DBC change rate = (DBC after alkali soaking - DBC before alkali soaking) / DBC before alkali soaking

[0100] MabSelect PrismA packing material is from Cytiva.

[0101] Loading capacity test: The loading capacity of the chromatographic packing was determined when 10% of the IgG1 antibody broke through the column. SPA-9M-6 and SPA-11M-6 were packing materials obtained by coupling to a packing carrier. MabSelect PrismA was a commercially available packing material. The loading capacity of multiple batches of packing materials was tested under the same conditions. The results showed that SPA-11M-6 was comparable to MabSelect PrismA in terms of loading capacity.

[0102] Alkali resistance of fillers: After soaking in 1M NaOH for 48 hours, both SPA-9M-6 and SPA-11M-6 maintained good alkali resistance, with an impact of less than 5% compared to before treatment.

[0103] IgG Residue Testing of Packing Material: Protein A affinity chromatography packing materials are relatively expensive and are often reused in practical applications. Detecting the residual amount of IgG1 is the sole criterion for whether the packing material can be recycled. After full-load testing with 1M NaOH CIP treatment, the residual levels in SPA-9M-6 and SPA-11M-6 packing materials remained below 20ppm, which is better than MabSelect PrismA.

[0104] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. An immunoglobulin-binding protein, characterized in that, The immunoglobulin-binding protein includes a mutant of the Z domain of protein A, wherein the mutant of the Z domain is selected from SEQ ID No: 1 to 7.

2. The immunoglobulin-binding protein according to claim 1, characterized in that, The immunoglobulin-binding protein comprises 2 to 8 repeating mutants of the Z domain; wherein the 4th and 6th amino acids of the mutant Z domain are proline; or the mutant Z domain is selected from SEQ ID No: 1 to 3.

3. The immunoglobulin-binding protein according to claim 2, characterized in that, The C-terminus and / or N-terminus of the mutant Z-domain can be further linked to a protein tag, preferably, the protein tag being one or more cysteine ​​residues, lysine residues, or histidine residues.

4. The immunoglobulin-binding protein according to claim 3, characterized in that, The immunoglobulin-binding protein is selected from SEQ ID NO:8 or SEQ ID NO:

9.

5. The nucleotide sequence encoding the immunoglobulin-binding protein according to any one of claims 1 to 4.

6. An expression vector comprising the nucleotide sequence as described in claim 5.

7. A host cell, characterized in that, The host cell comprises the expression vector of claim 6, or its genome integrates the nucleotide sequence of claim 5.

8. An affinity chromatography ligand, characterized in that, The affinity chromatography ligand comprises one or more immunoglobulin-binding proteins as described in any one of claims 1 to 4.

9. An affinity chromatography medium, characterized in that, The medium comprises the immunoglobulin-binding protein of any one of claims 1 to 4 or the affinity chromatography ligand of claim 8, coupled to the solid support.

10. The affinity chromatography medium of claim 9 is used for the purification of antibodies or Fc fusion proteins, and for the preparation of therapeutic agents for treating clinical autoimmune diseases or other Ig-related diseases in the human body.

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