Immunoassay methods

Abstract

Immunoassay method. Protein A variants, particularly protein A variants having high alkaline resistance and high IgG binding affinity, are disclosed. Applications of the protein A variants are also disclosed.

Description

[0001] This application is a divisional application of Chinese patent application No. 202111572524.4, filed on December 21, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to protein A variants, particularly protein A variants with high alkali resistance. The invention also relates to the application of these protein A variants. Background Technology

[0003] In recent years, antibody drugs have developed rapidly. In 2020, half of the top 10 best-selling drugs globally were antibody drugs. The purification step in antibody drug production typically employs a classic three-step or two-step chromatography method. Staphylococcus aureus protein A, discovered in the 1970s, is a bacterial protein capable of binding to the Fc terminus of antibodies from multiple species. Since its discovery, it has been studied as an important ligand for antibody purification chromatography and is currently one of the most commercially viable and widely used affinity ligands. Research has found that multiple regions on the protein A ligand possess unique specific adsorption capabilities for the antibody Fc fragment, which can be used for specific antibody capture. A single-step process can remove most impurities from cell fermentation broth, achieving a purity of over 98%. With the development of antibody drugs, affinity chromatography media used for antibody capture have also experienced rapid advancements.

[0004] The choice of affinity chromatography media is closely related to product quality. For example, early protein A affinity chromatography suffered from severe ligand detachment, making it difficult to clean. 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. Therefore, foreign packing material manufacturers have long been committed to increasing the ligand stability, binding capacity, and alkali tolerance of the media. For example, current mainstream protein A affinity chromatography media, such as Cytiva's MabSelect SuRe, are alkali-resistant media, capable of withstanding 0.1-0.5M NaOH washing, thus efficiently and cost-effectively removing protein precipitates, hydrophobic proteins, nucleic acids, endotoxins, and viruses attached to the packing material. Ligand stability has also been enhanced, with ligand detachment <100ppm. Dynamic binding capacity reaches over 30g / L. Currently, most domestic antibody drug manufacturers use imported protein A affinity chromatography media. These imported protein A affinity chromatography media are typically expensive, accounting for approximately 85% of the total cost of chromatography media in downstream purification processes for antibody drugs, and they also have various shortcomings that can be improved. For example, MabSelect Sure's microspheres are based on highly cross-linked agarose, which has the advantages of excellent hydrophilicity and good biocompatibility, making it very suitable for the separation and purification of antibody-like biomolecules; however, its disadvantages are: firstly, its structure is relatively soft and its particle size distribution is relatively wide, resulting in poor column packing reproducibility and low mechanical strength in practical use; secondly, MabSelect Sure is generally washed with 0.1M NaOH in biopharmaceuticals, and its alkali resistance is still insufficient; thirdly, MabSelect Sure's dynamic loading capacity is >30g / L, which is still somewhat insufficient for the loading capacity required for large-scale high-expression antibody production. On the other hand, Millipore's Prosep UltraPlus is also a widely used protein A affinity chromatography medium, with a matrix of glass beads with controllable pores, good physical stability, and a high loading capacity. However, the glass bead structure has a fatal flaw: it cannot withstand high concentrations of NaOH for cleaning, which limits its application in large-scale biological samples.

[0005] Because of the high price and various shortcomings of imported fillers, it is necessary to develop domestically produced, high-efficiency, low-cost protein A affinity fillers. Summary of the Invention

[0006] This article provides an immunoassay method, including the use of a alkali-resistant protein A variant, said alkali-resistant protein A variant comprising...

[0007] 1) The amino acid sequence shown in any one of SEQ ID NO: 1-3 or 5-10; or

[0008] 2) An amino acid sequence that has at least 90% sequence identity with any of the amino acid sequences shown in SEQ ID NO: 1-3, 5-10.

[0009] In some embodiments, the KD value of the alkali-resistant protein A variant binding to the IgG antibody is no higher than 1 × 10⁻⁶. -6 M, not higher than 1×10 -7 M or not higher than 3×10 -8 M.

[0010] In some implementations, the IgG antibody is an IgG1 molecule.

[0011] In some embodiments, the amino acid sequence of the alkali-resistant protein A variant is shown in SEQ ID NO: 1.

[0012] In some embodiments, the amino acid sequence of the alkali-resistant protein A variant is shown in SEQ ID NO: 3.

[0013] In some embodiments, the amino acid sequence of the alkali-resistant protein A variant is shown in SEQ ID NO: 10.

[0014] In some embodiments, the alkali-resistant protein A variant is in the form of a fusion protein, the fusion protein comprising two, three, four, five or more of the alkali-resistant protein A variants connected in tandem.

[0015] In some implementations, the alkali-resistant protein A variant is conjugated with a tracer.

[0016] In some embodiments, the tracer is selected from fluorescein, enzymes, colloidal gold, and ferritin. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the structure of protein A. S represents the signal sequence, E, D, B, A, and C are five IgG binding domains, and X and M sequences are related to cell wall attachment. The diagram also shows the Z domain, a mutated version of the B domain, which includes the G29A mutation compared to the B domain.

[0018] Figure 2 This is a flowchart of the protein A variant targeted optimization method of the present invention.

[0019] Figure 3 This is an identity matrix diagram between the target sequence of the protein A variant obtained in this invention and the initial sequence.

[0020] Figure 4 Map of the pET28a(+) vector used for expression of protein A variant.

[0021] Figure 5This is an electrophoresis image of the purified protein variant A.

[0022] Figure 6 The results of measuring the dynamic loading of protein A variant of the present invention are shown. Detailed Implementation

[0023] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0024] Protein A (SpA) is a cell wall protein derived from Staphylococcus aureus. Natural protein A contains five highly homologous domains: E, D, A, B, and C domains. Figure 1 Each domain can bind to the Fc fragment of immunoglobulin G (IgG) in humans or other mammals such as mice, pigs, dogs, and cattle. This binding usually does not affect the ability of the Fab fragment in the IgG molecule to bind specifically to the antigen. Natural protein A, after being conjugated to microspheres (such as agarose) or otherwise immobilized, is widely used for antibody purification. Because the binding strength of the five domains of natural protein A to the same antibody molecule varies slightly, elution conditions vary, often requiring elution of the purified product under different pH or denaturant concentrations. This leads to complex procedures or inactivation of the antibody molecule due to its inability to withstand such conditions. Therefore, researchers have used genetic engineering techniques to modify the protein A gene, using only one or a few specific domains in tandem to obtain recombinant protein A with desired binding affinity or other properties (e.g., alkali resistance).

[0025] A "domain" refers to a spatially distinct and relatively independent regional structure within a larger protein molecule. For smaller protein molecules, a domain is often equivalent to its tertiary structure. Domains can have specific functions, such as ligand binding (e.g., the B domain of protein A binding to IgG) or enzymatic activity. In this article, unless otherwise stated, a domain may also refer to a polypeptide fragment or the protein itself, which comprises only one domain.

[0026] "Protein A variant" generally refers to a protein or protein domain that has IgG binding ability but differs from the natural protein A or its specific domains in its amino acid sequence. For example, it may include one or more amino acid mutations (e.g., substitution, deletion, insertion, etc.) relative to the natural protein A. In this paper, "Protein A variant" refers to a target protein (or polypeptide fragment) obtained by the inventors through targeted optimization based on protein A and other proteins. It is significantly different from the natural protein A or its domains in its amino acid sequence, for example, the amino acid sequence identity is no higher than 85% after alignment. In addition, it is alkali-resistant and may or may not have IgG binding ability.

[0027] "Alkali-resistant protein A variant" refers to a protein A variant that can withstand alkali treatment. Here, "alkali-resistant" means that the protein A variant retains its ability to bind IgG molecules after exposure to alkaline solutions (e.g., 0.1, 0.2, 0.3, 0.4, or 0.5 M NaOH solutions) for a period of time or a certain number of times. Preferably, the alkali-resistant protein A variant retains more than 50%, 60%, 70%, 80%, or even 90% of its affinity for binding IgG molecules after 10 (e.g., 50, 100, 150, or even 180) exposures to alkaline solutions (e.g., 0.1 or 0.5 M NaOH solutions).

[0028] The “Z domain” is a modified B domain of natural protein A that also possesses IgG binding ability. The Z domain includes the G29A mutation relative to domain B. This mutation increases the stability of the Z domain against certain chemical reagents (e.g., CNBr, hydroxylamine, etc.) and enhances its ability to bind to IgG. For more information on the Z domain, see the description by Nilsson B et al. (A Synthetic IgG-Binding Domain Based on Staphylococcal Protein A. Protein Eng. 1987, 1(2): 107–113, which is incorporated herein by reference in its entirety.)

[0029] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also known as "sequence consistency") refers to the degree of similarity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Generally, sequence alignment is performed and gaps (if any) are introduced before calculating the percentage of similarity between two amino acid or nucleotide sequences. If, at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be identical or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, sequence consistency is obtained by dividing the number of matching positions by the total number of positions in the alignment window. In other algorithms, the number of gaps and / or gap length are also taken into account. For the purposes of this invention, the publicly available alignment software BLAST (available at ncbi.nlm.nih.gov) can be used to obtain the optimal sequence alignment and calculate the sequence consistency between two amino acid or nucleotide sequences using default settings.

[0030] In some embodiments, the protein A variants provided herein may include an amino acid sequence that has at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) with any of the sequences shown in SEQ ID NO: 1-3, 5-10.

[0031] In some embodiments, the encoding DNA sequence of the protein A variant provided herein may include a nucleotide sequence that has at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) with any of the sequences shown in SEQ ID NO: 11-13, 15-20.

[0032] Those skilled in the art will understand that, based on the specific amino acid sequences provided herein, variants of the protein A variant provided herein can be obtained by substituting (e.g., conserved substitutions), deleting, adding, and verifying or screening the binding affinity and / or alkali resistance of the resulting product to IgG. These variants should also be included within the scope of this invention. Similarly, based on the specific nucleotide sequences provided herein, those skilled in the art can obtain other nucleotide sequences that are functionally substantially identical to the nucleotide sequences provided herein by substituting (e.g., synonymous mutations), deleting, adding, and verifying or screening their expression of the target product and the binding affinity and / or alkali resistance of the target product to IgG. These other sequences should also be included within the scope of this invention.

[0033] The protein A variants provided herein can be used as components of other proteins. In one embodiment, a fusion protein comprising one or more protein A variants (e.g., 1, 2, 3, 4, 5, 6, or even more) is provided herein. The protein A variants in this fusion protein are linked in tandem by a linker molecule (e.g., a short amino acid sequence). Preferably, the linker molecule is also alkali-resistant, such as that described, for example, in PCT Publication WO03080655. In another embodiment, a fusion protein comprising one or more protein A variants (e.g., 1, 2, 3, 4, 5, 6, or even more) and at least one other domain also capable of IgG binding is provided herein. This other domain is, for example, domains E, D, A, B, and C of protein A, or the “Z domain” described above. Additionally, it is contemplated that the protein A variants provided herein may be linked to a signal peptide or a readily purifiable tag, such as a histidine tag, a Strep II tag, etc. Based on the amino acid and nucleotide sequences of the protein A variants provided herein, those skilled in the art can readily obtain these fusion proteins using genetic engineering techniques.

[0034] The protein A variants provided herein generally possess IgG (e.g., IgG1) binding ability (except for #4), and some protein A variants (#1, #3, and #10) even have higher IgG binding affinity than the Z domain used as a control (see Example 2). Accordingly, when these protein A variants are used to isolate IgG from IgG-containing cellular components (such as cell culture supernatants), they can have higher dynamic loading or purification capabilities.

[0035] Meanwhile, some of the protein A variants (#1, #3, and #10) provided in this article exhibit good alkali resistance, with protein A variant #10 showing even better alkali resistance than Mabselect SuRe (see Example 4). These protein A variants can withstand cleaning with NaOH up to 0.5M, meeting the requirements for repeated sterilization and pyrogen removal of equipment in pharmaceutical processes.

[0036] In addition, the codon-optimized protein A variant coding sequences (#1, #2, #3 and #10) provided in this paper can increase the expression level of protein A variants in host cells (such as Escherichia coli BL21), for example, by 30% higher than the expression level of the coding sequence for the Z domain used as a control.

[0037] As mentioned above, the protein A variant (and fusion proteins including this protein A variant) provided herein can be used for IgG purification. Additionally, in some cases, it can be used to remove IgG molecules from certain samples (e.g., blood) to obtain IgG-free samples. It is also anticipated that the protein A variant (and fusion proteins including this protein A variant) provided herein can be used for immunoassays; for example, the protein A variant can be labeled with tracers (such as fluorescein, enzymes, colloidal gold, ferritin) to replace secondary antibodies for antigen detection.

[0038] The present invention will be further described below through specific embodiments.

[0039] Example 1: Targeted Optimization Method for Alkali-Resistant Protein A Variant

[0040] To optimize protein function by improving alkali resistance and binding ability to IgG, this invention uses bioinformatics and convolutional neural networks to obtain a targeted optimized protein A variant.

[0041] The specific technical solution of this method may include the following steps (see...) Figure 2 ):

[0042] Step 1: Use the E, D, C, B, and A domains and the Z domain of protein A as the initial sequences for directional optimization, and obtain the crystal structures of the corresponding sequences from the RCSB-PDB database. The structure of the A domain is obtained by homology modeling.

[0043] Step 2: Perform cross-alignment of the initial sequence and structural superposition cluster analysis on the amino acid residues.

[0044] Step 3: Based on the sequence identity and amino acid residue structure matching results, the sequence is divided into backbone region and variable region. Backbone region is regarded as conserved sequence and variable region is regarded as fragmented sequence. Then, the variable region sequence is split into three types of short sequences containing single amino acids, double amino acids and triple amino acids.

[0045] The basic amino acids in the backbone region sequence include:

[0046] Q / AFY / L / PNL / QRN / FIQSL / DPS / S / L / EA / KLN / QAPK.

[0047] The basic amino acids in the variable region sequence include:

[0048] QAVI / QDA / NA / NKQ / FH / ND / KE / DEA / QH / SNA / EQ / IV / HN / LM / TN / EA / EDA / AG / KR / DH / VQ / LAKT / EN / ILV / GAS / KQ / DE / AS.

[0049] Step 4: By using random sequence growth and combination, short sequences from the fragment region are assembled on the basis of the backbone region to establish a potential sequence library containing random sequences. The sequence length in this library is 58 amino acids, and the sequences have identity with the E, D, C, B, and A domains and the Z domain of protein A under certain conditions (50% < identity < 85%).

[0050] Step 5: Obtain Protein A sequence with alkali resistance and IgG binding ability through literature review and calculate the primary structure molecular descriptor. After normalization, use it as the training set to pre-train a convolutional neural network containing input layer, hidden layer and output layer. The hidden layer contains 4 convolutional layers and 2 pooling layers, and the output layer is a dense connection layer.

[0051] Step 6: Calculate the primary structure molecular descriptors for the sequences in the potential sequence library and normalize them. Use the model trained in the previous step to make predictions. Select the top 10 sequences with both high alkali resistance and high IgG binding ability based on the prediction values ​​for further experimental verification.

[0052] The target sequence obtained by this method is as follows:

[0053] >T_SEQAA-1(SEQ ID NO: 1)

[0054] IDNKFNEEQQAAFYEVLHMPNLNAEQRNGFIQSLKDDPSQSTNLLAEAQKLNEAQAPK

[0055] >T_SEQAA-2(SEQ ID NO: 2)

[0056] QDNQFNKEQQNAFYQILHLPNLNAEQRNAFIQSLRHDPSQSLNLLGEAQKLNDSQAPK

[0057] >T_SEQAA-3(SEQ ID NO: 3)

[0058] AQNKFDKEQQNAFYQILHMPNLTADQRNGFIQSLKDDPSQSANVLAEAQKLNDAQAPK

[0059] >T_SEQAA-4(SEQ ID NO: 4)

[0060] AQNKHNKEHQNAFYQILHLPNLNEEQRNGFIQSLKDDPSVSANILGEAKKLNESQAPK

[0061] >T_SEQAA-5(SEQ ID NO: 5)

[0062] QQNKHDEAQQSAFYEVLHMPNLTEEQRNGFIQSLKDDPSQSLELLGEAQKLNDSQAPK

[0063] >T_SEQAA-6(SEQ ID NO: 6)

[0064] AAAQFNEEQQNAFYEILHMPNLTEAQRNAFIQSLKDDPSQSTNVLGEAQKLNDSQAPK

[0065] >T_SEQAA-7(SEQ ID NO: 7)

[0066] QDNKFDEDQQSAFYQILHMPNLTEDQRNGFIQSLKHDPSVSANLLSEAQKLNESQAPK

[0067] >T_SEQAA-8(SEQ ID NO: 8)

[0068] QDANFDKAHQSAFYEVLHLPNLNEEQRNAFIQSLKDDPSQSKNVLAEAQKLNDAQAPK

[0069] >T_SEQAA-9(SEQ ID NO: 9)

[0070] IDNKFNKAQQNAFYEVLNMPNLTAAQRNGFIQSLRDDPSVSTELLGEAKKLNESQAPK

[0071] >T_SEQAA-10(SEQ ID NO: 10)

[0072] QDNQHDEAQQAAFYEILNLPNLNEEQRNGFIQSLRHDPSQSAEILSEAKKLNESQAPK

[0073] The initial sequence for this method is as follows:

[0074] >SEQAA_Parental(Z domain) (SEQ ID NO: 21)

[0075] VDNKFNKEQQNAFYEILHLPNLNEEQRNAFIQSLKDDPSQSANLLAEAKKLNDAQA PK

[0076] The identity matrix between the target sequence and the initial sequence obtained by this method is as follows: Figure 3 As shown.

[0077] Example 2 Expression and purification of candidate protein A variant

[0078] The ten amino acid sequences from T_SEQAA-1 to T_SEQAA-10 and the Z domain sequence were optimized using the dominant codons of E. coli to obtain the corresponding DNA coding sequences as follows.

[0079] T-SEQDNA-1 (SEQ ID NO: 11)

[0080] ATTGATAACAAATTTAACGAAGAACAGCAGGCGGCGTTTTATGAAGTGCTGCAT

[0081] ATGCCGAACCTGAACGCGGAACAGCGTAACGGCTTTATTCAGAGCCTGAAAGAT

[0082] GATCCGAGCCAGAGCACCAACCTGCTGGCGGAAGCGCAGAAACTGAACGAAGC

[0083] GCAGGCGCCGAAA

[0084] T_SEQDNA-2(SEQ ID NO: 12)

[0085] CAGGATAACCAGTTTAACAAAGAACAGCAGAACGCGTTTTATCAGATTCTGCAT

[0086] CTGCCGAACCTGAACGCGGAACAGCGTAACGCGTTTATTCAGAGCCTGCGTCAT

[0087] GATCCGAGCCAGAGCCTGAACCTGCTGGGCGAAGCGCAGAAACTGAACGATAG

[0088] CCAGGCGCCGAAA

[0089] T_SEQDNA-3 (SEQ ID NO: 13)

[0090] GCGCAGAACAAATTTGATAAAGAACAGCAGAACGCGTTTTATCAGATTCTGCAT

[0091] ATGCCGAACCTGACCGCGGATCAGCGTAACGGCTTTATTCAGAGCCTGAAAGAT

[0092] GATCCGAGCCAGAGCGCGAACGTGCTGGCGGAAGCGCAGAAACTGAACGATGC

[0093] GCAGGCGCCGAAA

[0094] T_SEQDNA-4(SEQ ID NO:14)

[0095] GCGCAGAACAAACATAACAAAGAACATCAGAACGCGTTTTATCAGATTCTGCAT

[0096] CTGCCGAACCTGAACGAAGAACAGCGTAACGGCTTTATTCAGAGCCTGAAAGAT

[0097] GATCCGAGCGTGAGCGCGAACATTCTGGGCGAAGCGAAAAAACTGAACGAAAG

[0098] CCAGGCGCCGAAA

[0099] T_SEQDNA-5(SEQ ID NO:15)

[0100] CAGCAGAACAAACATGATGAAGCGCAGCAGAGCGCGTTTTATGAAGTGCTGCAT

[0101] ATGCCGAACCTGACCGAAGAACAGCGTAACGGCTTTATTCAGAGCCTGAAAGAT

[0102] GATCCGAGCCAGAGCCTGGAACTGCTGGGCGAAGCGCAGAAACTGAACGATAG

[0103] CCAGGCGCCGAAA

[0104] T_SEQDNA-6(SEQ ID NO:16)

[0105] GCGGCGGCGCAGTTTAACGAAGAACAGCAGAACGCGTTTTATGAAATTCTGCAT

[0106] ATGCCGAACCTGACCGAAGCGCAGCGTAACGCGTTTATTCAGAGCCTGAAAGAT

[0107] GATCCGAGCCAGAGCACCAACGTGCTGGGCGAAGCGCAGAAACTGAACGATAG

[0108] CCAGGCGCCGAAA

[0109] T_SEQDNA-7(SEQ ID NO:17)

[0110] CAGGATAACAAATTTGATGAAGATCAGCAGAGCGCGTTTTATCAGATTCTGCAT

[0111] ATGCCGAACCTGACCGAAGATCAGCGTAACGGCTTTATTCAGAGCCTGAAACAT

[0112] GATCCGAGCGTGAGCGCGAACCTGCTGAGCGAAGCGCAGAAACTGAACGAAAG

[0113] CCAGGCGCCGAAA

[0114] T_SEQDNA-8(SEQ ID NO:18)

[0115] CAGGATGCGAACTTTGATAAAGCGCATCAGAGCGCGTTTTATGAAGTGCTGCAT

[0116] CTGCCGAACCTGAACGAAGAACAGCGTAACGCGTTTATTCAGAGCCTGAAAGAT

[0117] GATCCGAGCCAGAGCAAAAACGTGCTGGCGGAAGCGCAGAAACTGAACGATGC

[0118] GCAGGCGCCGAAA

[0119] T_SEQDNA-9(SEQ ID NO:19)

[0120] ATTGATAACAAATTTAACAAAGCGCAGCAGAACGCGTTTTATGAAGTGCTGAAC

[0121] ATGCCGAACCTGACCGCGGCGCAGCGTAACGGCTTTATTCAGAGCCTGCGTGAT

[0122] GATCCGAGCGTGAGCACCGAACTGCTGGGCGAAGCGAAAAAAACTGAACGAAAG

[0123] CCAGGCGCCGAAA

[0124] T_SEQDNA-10 (SEQ ID NO: 20)

[0125] CAGGATAACCAGCATGATGAAGCGCAGCAGGCGGCGTTTTATGAAATTCTGAAC

[0126] CTGCCGAACCTGAACGAAGAACAGCGTAACGGCTTTATTCAGAGCCTGCGTCAT

[0127] GATCCGAGCCAGAGCGCGGAAATTCTGAGCGAAGCGAAAAAAACTGAACGAAAG

[0128] CCAGGCGCCGAAA

[0129] SEQ DNA-Parental (Z domain) (SEQ ID NO: 22)

[0130] GTGGATAACAAATTTAACAAAGAACAGCAGAACGCGTTTTATGAAATTCTGCAT

[0131] CTGCCGAACCTGAACGAAGAACAGCGTAACGCGTTTATTCAGAGCCTGAAAGAT

[0132] GATCCGAGCCAGAGCGCGAACCTGCTGGCGGAAGCGAAAAAAACTGAACGATGC

[0133] GCAGGCGCCGAAA

[0134] The 10 DNA sequences mentioned above, along with the Z domain DNA sequence, were inserted into the pET28a(+) vector using BamHI and EcoRI at the 5' and 3' ends, respectively. Figure 4The cells were then transformed into BL21 expression bacteria using heat shock with competent bacteria. After inoculation onto LB agar plates and overnight culture, single colonies were picked and cultured in 3 mL LB medium at 37°C on a shaker for 18 hours. The 3 mL bacterial culture was further expanded to 20 mL (OD600 < 0.4). 10 mL of the bacterial culture was taken out and glycerol was added to 30% (v / v) for cryopreservation. The remaining 10 mL was added to 1 L LB medium (1:100) and cultured to OD600 = 0.6-0.8 (for about 2 hours). IPTG was added to a final concentration of 0.5 mM, and the culture was cooled to 16°C overnight to induce protein expression. After 24 hours of overnight culture, the cells were collected by centrifugation. The cell pellet was resuspended in lysis buffer (50 mM NaHPO4, pH = 8, 0.3 NaCl, DNase I, Proteinase inhibitor). After sonication to lyse the cells, MgCl2 was added to a final concentration of 75 mM and stirred at 4°C for 3 hours. The supernatant was collected by centrifugation. Ten candidate protein A variants and the control Z domain were purified using Ni-NTA, achieving a purity of over 95%. SDS-PAGE electrophoresis results are shown below. Figure 5 As shown, these protein variants and the Z domain are approximately 7 kDa in size. Data analysis revealed that variants #1 (corresponding to T_SEQAA-1, and so on), #2, #3, and #10 showed expression levels more than 30% higher than the DNA encoding the Z domain, while #4, #5, #6, #7, #8, and #9 showed lower expression levels than the DNA encoding the Z domain. The affinity of the obtained protein A variant and the Z domain for human IgG1 antibody was tested using Fortebio Octet, and the results are shown in Table 1.

[0135] Table 1. Affinity test results of protein A variant and Z domain control bound to IgG1.

[0136] Protein A variant number Kon(1 / Ms) Koff(1 / s) KD(M) Z 1.4E5 3.6E-3 2.6E-8 T_SEQAA-1 1E5 2.2E-3 2.2E-8 T_SEQAA-2 1.3E4 1.1E-2 8.4E-7 T_SEQAA-3 1.2E5 1.3E-3 1.1E-8 T_SEQAA-4 ND ND N / A T_SEQAA-5 1.8E4 3.3E-2 1.8E-6 T_SEQAA-6 1.2E4 5.6E-3 2.1E-7 T_SEQAA-7 3.8E3 6.6E-4 1.7E-7 T_SEQAA-8 9.5E3 1.5E-3 1.6E-7 T_SEQAA-9 8E2 2.8E-4 3.5E-7 T_SEQAA-10 2.1E5 4E-3 1.9E-8

[0137] ND: Not detected N / A: Not applicable

[0138] As shown in Table 1, except for #4, the other 9 protein A variants all have detectable binding affinity for IgG1 antibody. Among them, #1, #3, and #10 have even better binding affinity to IgG1 antibody than the control Z domain.

[0139] Example 3: Coupling of candidate protein A variant and microspheres

[0140] In this embodiment, agarose microspheres with high uniformity in particle size and good mechanical strength were selected as the scaffold to couple candidate protein A variants (1#, 3# and 10#) with similar affinity to the Z domain.

[0141] The steps for coupling are briefly as follows.

[0142] Step 1: Measure 5 mL of epoxy-activated microspheres and wash them twice with 10 mL of 0.1 M PB, pH 8.6 buffer.

[0143] Step 2: Add 3 mg of candidate protein A variant per milliliter of packing material;

[0144] Step 3: React at 30℃±1℃ for 1 hour;

[0145] Step 4: Wash the gel twice with 20ml of water each time, collect the washing solution and measure the protein concentration (OD280), and calculate the content of protein A variant on the coupled gel.

[0146] Step 5: Rinse the gel with 20ml of water, then rinse 5 times.

[0147] Step 6: Wash twice with 5ml of 20% ethanol;

[0148] Step 7: Store 3 mL of 20% ethanol at 2-8℃.

[0149] Example 4: Determination of Alkali Resistance and Dynamic Loading of Affinity Chromatography Media

[0150] Pack a 1 mL Hitrip column with the protein A affinity chromatography medium prepared above and wash with equilibration buffer. Using Mabselect SuRe (Cytiva) as a reference, test the alkali resistance and dynamic binding capacity of the affinity medium conjugated with the test mutant. Before use, the AKTA purifier protein purification instrument was rinsed and equilibrated with purified water, and the baseline was zeroed after equilibration. 20 mM PBS, 150 mM NaCl, pH 7.0 buffer and 50 mM Gly, pH 3.0 buffer were used as the equilibration and elution phases, respectively, and the system pump was equilibrated with the equilibration phase. Connect the column to the AKTA purifier, taking care to prevent air bubbles from entering during connection. Run the pump at a controlled flow rate during connection, thoroughly wet the connector with the mobile phase, and fill the top of the column with the mobile phase before connecting. Wash the column sequentially with 10 CV each of 50 mM Gly, pH 3.0 and 20 mM PBS, 150 mM NaCl, pH 7.0 until baseline equilibration. The antibody-cell culture supernatant (~1 mg / ml) was initially injected at a rate of 0.25 mL / min, approximately 50 mL per chromatography column. The antibody was eluted with the elution phase, and the dynamic binding capacity of the column was calculated by analyzing the total amount of antibody eluted. Subsequently, the column was subjected to CIP with 0.1 M NaOH and 0.5 M NaOH, respectively. The above steps were repeated for 180 cycles. Changes in dynamic binding capacity were detected and analyzed. Results are shown below. Figure 6As shown, all three variants (#1, #3, and #10) exhibited superior alkali resistance, withstanding multiple CIP treatments with NaOH concentrations up to 0.5 M. They maintained over 80% of their initial loading after 100 cycles and showed no rapid decline in dynamic loading after 180 cycles. In particular, variant #10 demonstrated a higher dynamic loading than Mabselect SuRe throughout the testing process, regardless of whether it was treated with 0.1 M or 0.5 M NaOH.

[0151] The Mabselect SuRe control currently exhibits excellent affinity for IgG and alkali resistance, while the novel protein A variant provided by this invention is similar to or even better in these aspects, offering users more options. Considering that protein A variants #1, #2, #3, and #10 have a significant advantage in expression levels compared to the wild-type Z domain (more than 30% higher), expressing these protein A variants using the encoding DNA sequences provided herein also offers a cost advantage.

Claims

1. An immunoassay method for non-diagnostic or therapeutic purposes, comprising immunoassay using a alkali-resistant protein A variant or its fusion protein form in place of a second antibody, wherein the amino acid sequence of the alkali-resistant protein A variant is as shown in SEQ ID NO: 1, 3 or 10, and the KD value of the alkali-resistant protein A variant binding to IgG antibody is not higher than 1 × 10⁻⁶. -6 M, not higher than 1×10 -7 M or not higher than 3×10 -8 M, the IgG antibody is an IgG1 molecule.

2. The method of claim 1, wherein the fusion protein comprises 2, 3, 4, 5 or more of the alkali-stable protein A variants linked in tandem.

3. The method of claim 1, wherein the alkali-stable protein A variant is coupled to a tracer.

4. The method of claim 3, wherein the tracer is selected from the group consisting of fluorescein, an enzyme, colloidal gold, and ferritin.

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