A structural domain mutant of protein A, affinity chromatography medium prepared therefrom and application thereof

Abstract

The present invention relates to the field of biomedicine technology, and specifically to a domain mutant of protein A, an affinity chromatography medium prepared therefrom, and applications thereof. The present invention provides a domain mutant of protein A, which has excellent alkali resistance and high affinity for binding to Fc, and can be used as an affinity chromatography ligand for separating and purifying antibodies, Fc fusion proteins and other Fc-containing proteins. The affinity chromatography medium using the domain mutant of protein A of the present invention as a ligand has a small dynamic load attenuation during the circulation cleaning process of a high-concentration alkaline solution, and can still maintain a high load, which significantly improves the separation efficiency of the affinity chromatography medium, is conducive to reducing the separation and purification costs of antibodies, Fc fusion proteins and other Fc-containing proteins, improves their production efficiency, and is suitable for large-scale industrial production.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a domain mutant of protein A, an affinity chromatography medium prepared therefrom and applications thereof. Background Art

[0002] Biological products (pharmaceuticals) (such as therapeutic antibodies) are expensive and their quality is subject to strict scrutiny and regulation to ensure their effectiveness and safety. Therefore, low-cost and high-efficiency purification methods are particularly important for the life cycle of biological products (pharmaceuticals).

[0003] In recent years, antibody drugs have developed rapidly. In 2021, 70% of the top 10 drugs in global sales are antibody drugs. After years of technological accumulation, the expression of target proteins in the upstream fermentation of antibody drugs has become higher and higher, from 1-3g / L to 5-10g / L or even higher. The scale of production is also getting bigger and bigger, gradually increasing from several thousand liters to tens of thousands of liters. The increase in the expression and production scale of antibody drugs also poses greater challenges to downstream separation and purification. According to the current technical level, the maximum diameter of the chromatography column can reach 2 meters, and the maximum column volume is 500-600L. In order to match the upstream fermentation scale, the loading capacity of the chromatography medium needs to be increased as much as possible.

[0004] The separation and purification steps in the downstream production process of antibody drugs usually adopt the classic three-step or two-step chromatography method. The first step is usually the affinity chromatography of Staphylococcus aureus protein A. Staphylococcus aureus protein A (hereinafter referred to as protein A) is a bacterial protein that was discovered in the 1970s and can bind to the Fc end of antibodies of multiple species. Since its discovery, it has been studied as an important antibody purification chromatography ligand. It is currently the most commercially complete and widely used affinity ligand (J Chromatogr B Analyt Technol Biomed Life Sci. 2007 Mar 15; 848 (1): 40-7. doi: 10.1016 / j.jchromb.2006.09.030). Studies have found that multiple domains in the protein A ligand have unique specific adsorption capabilities for the Fc segment of antibodies, which can be used as specific capture of antibodies. A one-step process can remove most of the impurities in the cell fermentation broth, and the purity can reach more than 98%. With the development of antibody drugs, affinity chromatography media used for antibody capture have also achieved rapid development.

[0005] The selection of affinity chromatography media is closely related to the quality of the product. For example, the early protein A affinity chromatography ligand shed seriously and was not easy to clean. If these process-related impurities cannot be well removed in the subsequent process, there is a risk of causing adverse immune reactions, which in turn affects the quality and safety of the drug. Therefore, foreign filler manufacturers have long been committed to increasing the ligand stability, binding capacity and alkali tolerance of the medium. For example, the current mainstream protein A affinity chromatography media, such as Cytiva's MabSelect SuRe, are alkali-resistant chromatography media that can withstand 0.1-0.5M NaOH cleaning, thereby achieving efficient and low-cost removal of impurities such as protein precipitates, hydrophobic proteins, nucleic acids, endotoxins and viruses attached to the filler. At the same time, the ligand stability has also been enhanced, with ligand shedding <100ppm and a dynamic binding capacity of more than 30g / L. Cytival's latest MabSelect PrismA affinity chromatography medium can withstand 1M NaOH cleaning and has a dynamic binding capacity of more than 60g / L. At present, most domestic antibody drug manufacturers use imported protein A affinity chromatography media. These imported protein A affinity chromatography media are usually extremely expensive, accounting for about 85% of the total cost of chromatography media in the downstream purification process of antibody drugs, and these media still have room for performance improvement and improvement. For example, the base microspheres of MabSelect SuRe are highly cross-linked agarose, which has the advantages of excellent hydrophilicity and good biocompatibility, and is very suitable for the separation and purification of antibody biomacromolecules; the disadvantages are: first, the structure is relatively soft, the particle size distribution is wide, and there are problems such as poor column packing repeatability and low mechanical strength during actual use; second, Mabselect SuRe is generally cleaned with 0.1M NaOH in biopharmaceuticals, and its alkali resistance needs to be improved; third, the dynamic loading capacity of MabSelect SuRe is >30g / L, which is still a certain distance from the loading capacity required for large-scale high-expression antibody production. In addition, Prosep Ultra Plus from Millipore is also a widely used protein A affinity chromatography medium. The matrix is ​​a glass bead structure with controllable pores, which has good physical stability and high loading capacity. However, the glass bead structure has a fatal flaw, that is, it cannot withstand high-concentration NaOH cleaning, which limits its application in large-scale biological sample preparation.

[0006] In summary, in order to improve the production efficiency of biological products and reduce production costs, it is still necessary to further improve the performance of protein A affinity chromatography media and develop high-efficiency and low-cost protein A affinity chromatography media. Summary of the invention

[0007] The invention provides a domain mutant of protein A, an affinity chromatography medium prepared therefrom and application thereof.

[0008] The present invention genetically modifies the natural B domain and Z domain of protein A, and obtains, through screening, domain mutants of protein A with significantly improved alkali resistance, higher Fc affinity and dynamic binding capacity. The domain mutants are used to prepare protein A affinity chromatography ligands and affinity chromatography media, which can significantly improve the capacity and separation efficiency of affinity chromatography.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] In the first aspect, the present invention provides a domain mutant of protein A, which, compared with the native B domain or Z domain of protein A, contains mutations in which the amino acid at position 3 mutates to alanine, the amino acid at position 6 mutates to aspartic acid, the amino acid at position 7 mutates to an amino acid other than asparagine, glutamine, proline and cysteine, the amino acid at position 9 mutates to alanine, the amino acid at position 11 mutates to alanine, the amino acid at position 23 mutates to threonine, the amino acid at position 40 mutates to valine, and the amino acids at positions 42, 43 and 44 mutate to lysine, alanine and isoleucine, respectively.

[0011] The present invention has been experimentally verified to find that compared with reported protein A ligands and commonly used commercial protein A ligands, the protein A domain mutant containing the above mutation has obvious advantages in alkaline resistance, affinity, and dynamic binding capacity.

[0012] Specifically, compared with the native B domain or Z domain of protein A, the mutant contains mutations in which asparagine at position 3 is mutated to alanine, asparagine at position 6 is mutated to aspartic acid, lysine at position 7 is mutated to an amino acid other than asparagine, glutamine, proline and cysteine, glutamine at position 9 is mutated to alanine, asparagine at position 11 is mutated to alanine, asparagine at position 23 is mutated to threonine, glutamine at position 40 is mutated to valine, and alanine at position 42, asparagine at position 43 and leucine at position 44 are mutated to lysine, alanine and isoleucine, respectively.

[0013] Preferably, the amino acid other than asparagine, glutamine, proline and cysteine ​​is isoleucine.

[0014] Preferably, the mutations of the mutant compared to the native B domain or Z domain of protein A are: asparagine at position 3 mutates to alanine, asparagine at position 6 mutates to aspartic acid, lysine at position 7 mutates to isoleucine, glutamine at position 9 mutates to alanine, asparagine at position 11 mutates to alanine, asparagine at position 23 mutates to threonine, glutamine at position 40 mutates to valine, and alanine at position 42, asparagine at position 43 and leucine at position 44 mutate to lysine, alanine and isoleucine, respectively.

[0015] In the present invention, the amino acid sequence of the natural B domain of the protein A is shown as SEQ ID NO.1, and the amino acid sequence of the Z domain is shown as SEQ ID NO.2.

[0016] In some specific embodiments of the present invention, the amino acid sequence of the domain mutant of protein A is shown as SEQ ID NO.8.

[0017] In a second aspect, the present invention provides a fusion protein, wherein the fusion protein is any one of the following (1)-(3):

[0018] (1) The fusion protein is obtained by adding a protein tag to the C-terminus and / or N-terminus of the domain mutant of protein A described above;

[0019] (2) The fusion protein is obtained by adding a signal peptide to the C-terminus and / or N-terminus of the domain mutant of protein A described above;

[0020] (3) The fusion protein comprises an amino acid sequence formed by fusion of 2 to 8 of the domain mutants of protein A described above.

[0021] In the above (3), the fusion protein is obtained by connecting 2-8 domain mutants of the above-mentioned protein A in series. The domain mutants of each protein A can be directly connected or connected by a connecting peptide. The connecting peptide can be a flexible connecting peptide conventionally used in the art, and an exemplary connecting peptide can be a sequence rich in glycine and serine.

[0022] In a third aspect, the present invention provides a recombinant protein A, wherein the recombinant protein A comprises the domain mutant of protein A described above.

[0023] Preferably, the B domain and / or Z domain of the recombinant protein A is a domain mutant of protein A described above.

[0024] In a fourth aspect, the present invention provides a nucleic acid molecule encoding the domain mutant of protein A or the fusion protein or the recombinant protein A described above.

[0025] For the nucleotide sequence of the above-mentioned nucleic acid molecule, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding these amino acid sequences according to the amino acid sequence and codon rules of the domain mutant of protein A or the fusion protein or the recombinant protein A described above. Due to the degeneracy of codons, the nucleotide sequence encoding the same amino acid sequence is not unique, and all nucleic acid molecules capable of encoding the domain mutant of protein A or the fusion protein or the recombinant protein A are within the protection scope of the present invention.

[0026] In some specific embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the domain mutant of protein A is shown in SEQ ID NO.19.

[0027] In a fifth aspect, the present invention provides a biological material comprising the above-mentioned nucleic acid molecule or expressing the above-mentioned domain mutant of protein A or the fusion protein or the recombinant protein A; the biological material is an expression cassette, a vector or a host cell.

[0028] The above-mentioned expression cassette can be obtained by connecting a transcription or translation regulatory element such as a promoter upstream of the nucleic acid molecule and / or connecting a transcription or translation regulatory element such as a terminator downstream thereof.

[0029] The above-mentioned vectors include, but are not limited to, plasmid vectors, phage vectors, virus vectors, artificial chromosome vectors, etc. The vector may be a cloning vector or an expression vector.

[0030] The host cells include microbial cells or animal cells. The microorganisms include but are not limited to Escherichia coli, yeast, etc. The animal cells include but are not limited to insect cells, mammalian cells, etc.

[0031] In a sixth aspect, the present invention provides an affinity chromatography ligand, which comprises one or more domain mutants of protein A as described above, or comprises one or more fusion proteins as described above, or comprises one or more recombinant protein A as described above.

[0032] The affinity chromatography ligand of the present invention can specifically bind to proteins containing Fc, such as immunoglobulins, Fc fusion proteins, etc., thereby achieving the separation of Fc-containing proteins such as immunoglobulins.

[0033] In a seventh aspect, the present invention provides an affinity chromatography medium, which comprises the domain mutant of protein A or the fusion protein or the recombinant protein A or the affinity chromatography ligand described above coupled to a solid support.

[0034] The present invention also provides a separation matrix, which comprises the domain mutant of protein A or the fusion protein or the recombinant protein A or the affinity chromatography ligand coupled to a solid support.

[0035] Based on the fact that the domain mutants, fusion proteins, and recombinant protein A of protein A described above have high alkali resistance, the above affinity chromatography medium or separation matrix can withstand high concentration alkaline solutions (such as 0.1-1M NaOH solutions) during cleaning. During the circulation cleaning process of the alkaline solution, the dynamic loading capacity of the affinity chromatography decays less and can still ensure a high loading capacity.

[0036] The solid phase support mentioned above can be any solid phase support known in the art for separation and purification of Fc-containing proteins.

[0037] The solid support may be a porous material or a non-porous material.

[0038] Exemplary solid phase supports include, but are not limited to, polysaccharides and other polyhydroxy-containing polymers or other synthetic polymers, wherein the polysaccharides include agarose, agar, dextran, starch, cellulose, pullulan, and the like.

[0039] The solid support may be in the shape of a microsphere.

[0040] Preferably, the coupling is via a thioether bond. The solid support is a polysaccharide.

[0041] The domain mutant or fusion protein of protein A and recombinant protein A of the present invention can be connected to the corresponding solid support through conventional coupling techniques via the thiol group, amino group, carboxyl group, etc. Commonly used coupling reagents include but are not limited to diepoxide, N-hydroxysuccinimide, etc.

[0042] In some specific embodiments of the present invention, the affinity chromatography medium is a porous microsphere medium, and preferably the solid support is agarose microspheres.

[0043] In some specific embodiments of the present invention, the affinity chromatography medium is obtained by activating agarose microspheres and coupling the affinity chromatography ligand. The affinity chromatography medium has a stable structure, moderate hardness and softness, and a particle size distribution within a certain range. Therefore, when used, the column has good repeatability, high mechanical strength, greatly improved flow rate, low cost, and is suitable for large-scale production.

[0044] In an eighth aspect, the present invention provides a chromatographic column, comprising the affinity chromatography medium described above.

[0045] The affinity chromatography medium is filled in the chromatographic column.

[0046] In a ninth aspect, the present invention provides the use of the domain mutant of protein A or the fusion protein or the recombinant protein A or the affinity chromatography ligand or the affinity chromatography medium or the chromatographic column described above in separating Fc-containing proteins or preparing Fc-containing proteins.

[0047] Preferably, the Fc-containing protein is an antibody or an Fc fusion protein.

[0048] In a tenth aspect, the present invention provides a method for separating Fc-containing proteins, the method comprising: contacting a sample containing Fc-containing proteins with the domain mutant of protein A or the fusion protein or the recombinant protein A or the affinity chromatography ligand or the affinity chromatography medium or the chromatographic column described above.

[0049] Preferably, the Fc-containing protein is an antibody or an Fc fusion protein.

[0050] Preferably, the method for separating Fc-containing proteins comprises: contacting a sample containing Fc-containing proteins with the domain mutant of protein A or the fusion protein or the recombinant protein A or the affinity chromatography ligand or the affinity chromatography medium or the chromatographic column described above, then washing with a washing solution, and then eluting the Fc-containing proteins with an eluent to collect the Fc-containing proteins.

[0051] The present invention has no particular limitation on the composition of the cleaning solution and the eluting solution, and the cleaning solution and the eluting solution commonly used in the field for separation and purification of Fc-containing proteins can be used.

[0052] The beneficial effects of the present invention include at least: the domain mutant of protein A provided by the present invention has excellent alkali resistance and high affinity for binding to Fc, and can be used as an affinity chromatography ligand for separating and purifying antibodies, Fc fusion proteins and other Fc-containing proteins; the protein A affinity chromatography medium using the domain mutant of protein A of the present invention as a ligand has a small dynamic load attenuation during the circulation cleaning process of a high-concentration alkaline solution and can still maintain a high load, which significantly improves the separation efficiency of the affinity chromatography medium, is beneficial to reducing the separation and purification costs of antibodies, Fc fusion proteins and other Fc-containing proteins, improves their production efficiency, and is suitable for large-scale industrial and widespread use. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0054] Figure 1 Schematic diagram of the rational design and large-scale screening optimization of the B and Z domain sequences of Protein A in Example 1 of the present invention.

[0055] Figure 2This is a schematic diagram of the structure of constructing the artificially synthesized mutant library into the pET-28a-aggregation peptide (AGG) expression vector in Example 1 of the present invention and the detection result of the precipitation formed by the mutant ProteinA-aggregation peptide AGG.

[0056] Figure 3 Schematic diagram of the screening scheme of the artificially synthesized mutant library in Example 1 of the present invention.

[0057] Figure 4 This is a schematic diagram of the structure of the pET28a(+) vector into which the mutant DNA sequence is inserted in Example 2 of the present invention, and a diagram showing the results of repeated screening of IgG binding ability of each mutant. In the right figure, numbers such as PA, 26, and 33 represent the screening numbers of different mutant strains, respectively.

[0058] Figure 5 The electrophoresis detection results of 10 candidate protein A variants (1-10) and the parent Z23T protein (N23T) in Example 2 of the present invention after purification by Ni-NTA.

[0059] Figure 6 The results are the affinity test results of each ligand and human IgG in Example 3 of the present invention.

[0060] Figure 7 This is a curve of the change rate of loading capacity versus the number of cycles in the CIP alkaline resistance test of the chromatography medium using mutant No. 5 (T_SEQAA-5) as a ligand using 0.5M NaOH in Example 5 of the present invention.

[0061] Figure 8 This is a curve of the change rate of loading capacity versus the number of cycles in the alkali resistance test of CIP using 1M NaOH using the chromatographic medium with mutant No. 5 (T_SEQAA-5) as the ligand in Example 5 of the present invention.

[0062] Fig. 9 This is a curve showing the change in dynamic binding capacity of the chromatography medium with mutant No. 5 (T_SEQAA-5) as a ligand as a function of the number of cycles in Example 5 of the present invention, in which the alkali resistance test was carried out using the monoclonal antibody culture supernatant as the loading liquid to simulate the antibody drug production process.

[0063] Fig.10 This is a curve showing the change in antibody recovery rate versus cycle number for the chromatography medium with mutant No. 5 (T_SEQAA-5) as the ligand in Example 5 of the present invention, in which the supernatant of the monoclonal antibody culture fluid was used as the loading liquid to simulate the antibody drug production process for alkaline resistance testing. DETAILED DESCRIPTION

[0064] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0065] Example 1 Rational design and large-scale screening optimization of alkali-resistant Protein A ligands

[0066] Aiming at the protein function optimization goal of improving alkali resistance and IgG binding ability, the present invention performs targeted optimization of Protein A affinity ligands through alkali-intolerant amino acid substitution, large-scale random mutation and AlphaFold2 structure alignment.

[0067] The specific optimization and screening technical solution includes the following steps:

[0068] Step 1: The B and Z domain sequences of Protein A were used as the initial sequences for rational design and large-scale screening optimization, respectively. Referring to the literature (Computational and Structural Biotechnology Journal 19(2021)3372–3383, doi:10.1016 / j.csbj.2021.05.048), the amino acids K7, Q10, F13, Y14, L17, E24, R27, N28, I31, and K35 that bind to IgG remained unchanged ( Figure 1 , red mark), between alpha helix 1a and alpha helix 2a, between alpha helix 2a and alpha helix 3a, and amino acids P20, P38, and P57 after alpha helix 3a remain unchanged ( Figure 1 ), while the alkali-intolerant amino acid residue N was mutated ( Figure 1 , yellow mark), and an artificial synthetic mutation library was designed based on this.

[0069] Step 2: The synthetic mutant library was constructed into the pET-28a-aggregation peptide (AGG) expression vector. The mutant ProteinA-aggregation peptide AGG formed can form a precipitate (see Figure 2 ) was adsorbed on a 24-well plate to facilitate subsequent treatment with NaOH. The mutant ProteinA-aggregation peptide AGG after alkali treatment was further tested for IgG binding activity.

[0070] Step 3: The mutant ProteinA-aggregation peptide AGG after alkali treatment was tested for IgG binding activity by ELISA. The alkali-resistant Z (N23T) was used as a positive control to screen the sequences in the library. The specific screening scheme is as follows Figure 3 shown.

[0071] Step 4: Rescreening and verification. The clones with positive signals screened out were sequenced, and the expression vector was reconstructed with at least 4 replicates, and then expression verification was performed. Four replicates of alkali-resistant Z (N23T) were used as positive controls.

[0072] The B and Z domain sequences of Protein A (initial sequences) are as follows:

[0073] >SEQAA_B domain(SEQ ID NO.1):

[0074]

[0075] >SEQAA_Z domain(SEQ ID NO.2):

[0076]

[0077] The sequence of alkali-resistant Z(N23T) is as follows:

[0078] >SEQAA_Z(N23T)(SEQ ID NO.3):

[0079]

[0080] The sequences of the ten target proteins (domain mutants of Protein A) with the best performance such as alkaline resistance obtained through preliminary screening are as follows:

[0081] >T_SEQAA-1 (SEQ ID NO.4):

[0082]

[0083] >T_SEQAA-2 (SEQ ID NO.5):

[0084]

[0085] >T_SEQAA-3 (SEQ ID NO.6):

[0086]

[0087] >T_SEQAA-4 (SEQ ID NO.7):

[0088]

[0089] >T_SEQAA-5 (SEQ ID NO. 8):

[0090]

[0091] >T_SEQAA-6 (SEQ ID NO.9):

[0092]

[0093] >T_SEQAA-7 (SEQ ID NO.10):

[0094]

[0095] >T_SEQAA-8 (SEQ ID NO.11):

[0096]

[0097] >T_SEQAA-9 (SEQ ID NO. 12):

[0098]

[0099] >T_SEQAA-10 (SEQ ID NO. 13):

[0100]

[0101] Example 2 Expression and purification of candidate protein A ligands

[0102] The ten amino acid sequences T_SEQAA-1 to T_SEQAA-10 were optimized using the dominant codons of E. coli to obtain the following ten DNA sequences T-SEQDNA-1 to T-SEQDNA-10. These DNA sequences can be repeated 2-8 times to form tandem proteins. This embodiment uses a 6-repeat sequence (the sequences of T-SEQDNA-1 to T-SEQDNA-10 are repeated 6 times to form tandem proteins). The ten DNA sequences were inserted into the pET28a(+) vector using BamHI and EcoRI at the 5' and 3' ends ( Figure 4 ), and then transformed into E. coli BL21 competent cells by heat shock transformation method, inoculated on LB plate and cultured overnight, picked a single colony in 3mL LB medium, and cultured in a shaking incubator at 37℃ for 18 hours. 3mL bacterial solution was further expanded and cultured to 30mL (OD 600<0.4). Take out 20mL of bacterial solution and add glycerol to 30% (v / v) for cryopreservation. Add the remaining 10mL to 1L LB medium (1:100) and expand the culture until OD 600 =0.6-0.8 (about 2 hours), add IPTG to a final concentration of 0.5mM, cool to 16°C overnight to induce protein expression. After 24 hours of overnight culture, collect the bacteria by centrifugation. Resuspend the cell pellet in lysis buffer (50mM NaHPO4, pH=8, 0.3NaCl, Dnase I, Protease inhibitor). After ultrasonic disruption of the bacteria, add MgCl2 to a final concentration of 75mM, stir at 4°C for 3 hours, and collect the supernatant by centrifugation. 10 candidate protein A variants and the parent Z23T protein were purified with Ni-NTA with a purity of more than 95%. The results are as follows Figure 5 As shown. After analysis, except for variant #4 with a slightly lower expression level, there was no significant difference in expression between the other mutants and the parent. The affinity of the obtained protein A variant protein to human IgG1 antibody was tested using Fortebio Octet. The results are shown in Table 1. Among them, mutants 5 (T_SEQAA-5) and 10 (T_SEQAA-10) had better comprehensive performance and were evaluated later.

[0103] The specific sequences of the ten DNA sequences T-SEQDNA-1 to T-SEQDNA-10 described above are as follows:

[0104] SEQDNA-Parental(Z23T)(SEQ ID NO.14):

[0105]

[0106] T-SEQDNA-1 (SEQ ID NO.15):

[0107]

[0108] T_SEQDNA-2 (SEQ ID NO.16):

[0109]

[0110] T_SEQDNA-3 (SEQ ID NO.17):

[0111]

[0112] T_SEQDNA-4 (SEQ ID NO.18):

[0113]

[0114] T_SEQDNA-5 (SEQ ID NO.19):

[0115]

[0116] T_SEQDNA-6 (SEQ ID NO.20):

[0117]

[0118] T_SEQDNA-7 (SEQ ID NO.21):

[0119]

[0120] T_SEQDNA-8 (SEQ ID NO.22):

[0121]

[0122] T_SEQDNA-9 (SEQ ID NO.23):

[0123]

[0124] T_SEQDNA-10 (SEQ ID NO.24):

[0125]

[0126] Table 1

[0127]

[0128]

[0129] Example 3 Comparison of affinity and alkali resistance with commercial ligands

[0130] In order to further study the affinity and alkaline resistance of the protein A affinity ligands screened in Examples 1 and 2, this example compares the affinity and alkaline resistance of the commercial MaXtar APRA ligand and its optimized variants, Protein A ligand 1 and Protein A ligand 2 provided by a domestic supplier, S ligand and P ligand from a foreign company C, and protein A mutants No. 5 (T_SEQAA-5) and No. 10 (T_SEQAA-10) screened in Examples 1 and 2. The sample numbers of each ligand are shown in Table 2.

[0131] Table 2

[0132] Sample No. Corresponding ligand #1 Domestic supplier ProteinA ligand 1 #2 Domestic supplier ProteinA ligand 2 #3 S ligand from foreign company C #4 MaXtar APRA ligand optimized variants #5 The #10 mutant screened by the present invention #6 The #5 mutant screened by the present invention #7 P ligand from foreign company C

[0133] 1. Biacore analysis of different ligand affinities

[0134] The affinity between the above ligands and human IgG was determined using Biacore. The specific results are as follows: Figure 6 And as shown in Table 3.

[0135] Table 3

[0136] Sample No. ka(1 / Ms) kd(1 / s) KD(M) #1 3.85E+05 1.62E-04 4.21E-10 #2 3.16E+05 2.51E-04 7.95E-10 #3 1.61E+05 1.04E-05 6.43E-11 #4 2.56E+05 1.15E-04 4.47E-10 #5 1.10E+06 8.81E-04 7.99E-10 #6 5.92E+05 3.06E-04 5.16E-10 #7 6.10E+05 5.96E-05 9.78E-11

[0137] 2. Biacore analysis of alkali resistance of different ligands

[0138] After multiple cycles of NaOH washing, the differences in the ability of the ligands to capture IgG were compared under PBS solution conditions. The ligand was fixed on the Biacore chip, the mobile phase was IgG solution, and IgG was loaded in PBS until the highest point (about 180ms). The value of the highest point of IgG Load in the first cycle was 100%, and the value of the highest point of IgG Load after different numbers of alkaline washing cycles was divided by the value of the highest point of the first cycle to compare the alkali resistance of different ligands. The results showed that #6 and #7 had the best alkali resistance, and #6 (T_SEQAA-5) was used as a candidate ligand for subsequent research.

[0139] Example 4 Coupling of candidate protein A ligands and microspheres

[0140] In this example, agarose microspheres with high particle size uniformity and good mechanical strength are used as the matrix to couple the candidate protein A variants with affinity similar to the Z(N23T) control protein. The coupling specifically includes the following steps:

[0141] Step 1: Take 5 mL of epoxy-activated microspheres and wash them twice with 10 mL of 0.1 M PB, pH 8.6 buffer;

[0142] Step 2: Add 3 mg of candidate protein A per ml of filler;

[0143] Step 3: react at 30℃±1℃ for 1 hour;

[0144] Step 4: Wash the gel with 20 mL of water each time, wash twice in total, collect the washing solution and measure the protein concentration (OD 280 ), calculate the protein A content on coupling;

[0145] Step 5: Wash the gel with 20 mL of water and then wash it 5 times;

[0146] Step 6: Wash twice with 5 mL of 20% ethanol;

[0147] Step 7: 3 mL of 20% ethanol, store at 2-8°C.

[0148] Example 5 Determination of Dynamic Binding Capacity and Alkali Resistance of Affinity Chromatography Media

[0149] 1. Dynamic binding capacity determination

[0150] The affinity chromatography medium prepared by the above method with the candidate No. 5 (T_SEQAA-5) protein A ligand was loaded into the Hitrip 4.7mL chromatography column and rinsed with the equilibrium solution 20mM PBS (20mM PB + 150mM NaCl pH 7.0). The AKTA purifier protein purifier was rinsed and balanced with purified water before use, and the baseline was zeroed after equilibrium. 20mM PBS (20mM PB + 150mM NaCl pH7.0) buffer and 50mM Gly-HCl (pH 3.0) buffer were used as the equilibrium phase and elution phase during the experiment, respectively, and the system pump was balanced with the equilibrium phase. The small column was connected to the AKTA purifier, and attention was paid to prevent bubbles from entering during the connection process. During the connection, the pump was operated at a certain flow rate, the joint part was fully wetted with the mobile phase, and the upper end of the small column was also filled with the mobile phase, and then connected. The column was washed with 50mM Gly-HCl (pH 3.0) and 20mM PBS (20mM PB + 150mM NaCl pH 7.0) for 10CV each until the baseline was stable and returned to zero. Then, the prepared human IgG1 (3mg / mL) was injected at a flow rate of 0.77mL / min (retention time 6min) until the ultraviolet absorption value (280nm, flow cell with 2mm optical path) reached the maximum, and the dynamic binding capacity (QB) of the chromatographic medium at 10% flow-through was calculated based on the injection volume corresponding to 10% of the maximum absorption value. 10％ ), the calculation formula is as follows:

[0151]

[0152] Where, C0 = the concentration of injected human IgG1; V C = column volume (4.7 mL); V 10％ =10% maximum absorption value, i.e., injection volume for 10% flow-through; V0 = dead volume (can be confirmed by the peak volume of IgG subtypes that do not bind to the medium, such as IgG3).

[0153] For the convenience of testing and calculation, in the actual dynamic binding capacity test, the injection volume of the 1% acetone peak was taken as an approximate value for calculation in V0.

[0154] The best performing protein A ligand No. 5 (T_SEQAA-5) in the above test was subjected to a dynamic binding capacity test with the chromatography medium corresponding to the P ligand from the foreign company C.

[0155] The dynamic binding capacity calculation results are as follows:

[0156] Protein A ligand No. 5 (T_SEQAA-5):

[0157] P ligand of foreign company C:

[0158] 2. Determination of alkali resistance after coupling

[0159] (1) The affinity chromatography medium prepared by the above method with candidate mutant No. 5 (T_SEQAA-5) was refilled into a Hitrip 4.7mL chromatography column and rinsed with equilibration solution 20mM PBS (20mM PB + 150mM NaCl, pH 7.0). The AKTApurifier protein purifier was rinsed and equilibrated with purified water before use, and the baseline was returned to zero after equilibration. 20mM PBS (20mM PB + 150mM NaCl, pH 7.0) buffer and 50mM Gly-HCl (pH 3.0) buffer were used as the equilibrium phase and elution phase during the experiment, respectively, and the system pump was equilibrated with the equilibrium phase. The small column was connected to the AKTA purifier, and attention was paid to prevent bubbles from entering during the connection process. During the connection, the pump was operated at a certain flow rate, the joint part was fully wetted with the mobile phase, and the upper end of the small column was also filled with the mobile phase, and then connected. The column was washed with 20mM PBS (20mM PB + 150mM NaC, pH 7.0) for 5CV and 50mM Gly-HCl (pH 3.0) for 5CV at a flow rate of 1mL / min, followed by CIP with 0.5M NaOH (contact time 15min), and the above steps were repeated for 180 cycles. The dynamic binding capacity was measured every 10 cycles. The curve of the change rate of the binding capacity and the number of cycles is shown in Figure 2. Figure 7 shown.

[0160] The specific data are shown in Table 4.

[0161] Table 4

[0162] Cycle times Dynamic load change rate % <![CDATA[Dynamic binding capacity QB 10％ (mg / mL)]]> 0 100 78.8 10 100.6 79.3 20 99.5 78.4 30 97.8 77.1 40 99.2 78.2 50 99.1 78.1 60 98.7 77.8 70 96.6 76.1 80 95.2 75 90 98.4 77.5 100 97.0 76.4 110 98.9 77.9 120 100.0 78.8 130 95.6 75.3 140 96.3 75.9 150 96.7 76.2 160 97.5 76.8 170 98.4 77.5 180 97.0 76.4

[0163] From the above data, it can be seen that within 180 cycles, the dynamic loading capacity of protein A ligand No. 5 (T_SEQAA-5) decayed very little, and there was no sudden decline inflection point. The overall performance was stable, indicating that mutant No. 5 (T_SEQAA-5) as a ligand-coupled chromatographic medium has good tolerance to 0.5M NaOH.

[0164] The above method was repeated with 1M NaOH as the CIP solution. The initial loading was 100%. The curve of loading change rate and cycle number is shown in Figure 8 shown.

[0165] The specific data are shown in Table 5.

[0166] Table 5

[0167]

[0168]

[0169] From the above data, it can be seen that within 180 cycles, the dynamic binding capacity did not decrease significantly, and there was a slightly obvious downward trend after the 130th cycle, indicating that mutant No. 5 (T_SEQAA-5) as a ligand-coupled chromatography medium also has good tolerance to 1MNaOH.

[0170] (2) The affinity chromatography medium prepared using candidate mutant No. 5 (T_SEQAA-5) as the ligand was refilled into the Hitrip 4.7 mL chromatography column, and the instrument was connected and the equilibrated chromatography column was flushed according to the above method. The monoclonal antibody culture supernatant (antibody concentration of about 4 mg / mL) was used as the sample according to the steps in Table 6 and the QB 10％ 80% of the sample was used as the sample amount to simulate the actual production process for alkali resistance experiment (Table 6).

[0171] Table 6

[0172]

[0173] The dynamic binding capacity and antibody recovery rate were tested every 11 cycles, and their changes with the number of cycles were as follows: Fig. 9 and Fig.10 shown.

[0174] From the above data, it can be seen that the monoclonal antibody culture supernatant was used as the loading liquid to simulate the antibody drug production process for alkaline resistance testing. Within 181 cycles, the dynamic binding capacity of the antibody was basically maintained above 70 mg / mL, and the recovery rate was >92%. The loading capacity and recovery rate decayed relatively steadily throughout the process, with good alkali resistance and stable performance, which can well meet the production needs of biological drugs.

[0175] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A domain mutant of protein A, characterized in that: The amino acid sequence of the mutant is shown in SEQ ID NO.

8.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the domain mutant of protein A according to claim 1.

3. Biomaterial, characterized in that The biological material comprises the nucleic acid molecule of claim 2 or expresses the domain mutant of protein A of claim 1; The biological material is an expression cassette, a vector or a host cell.

4. An affinity chromatography ligand, characterized in that The affinity chromatography ligand comprises one or more domain mutants of the protein A according to claim 1.

5. An affinity chromatography medium, characterized in that The medium comprises the domain mutant of protein A according to claim 1 or the affinity chromatography ligand according to claim 4 coupled to a solid support.

6. The affinity chromatography medium according to claim 5, characterized in that The coupling is via thioether bond coupling, and / or the solid support is a polysaccharide.

7. A chromatographic column, characterized in that The chromatographic column comprises the affinity chromatography medium according to claim 5 or 6.

8. Use of the domain mutant of protein A according to claim 1, the affinity chromatography ligand according to claim 4, the affinity chromatography medium according to claim 5 or 6, or the chromatographic column according to claim 7 in separating or preparing Fc-containing proteins.

9. The use according to claim 8, characterized in that: The Fc-containing protein is an antibody or an Fc fusion protein.

10. A method for separating a protein containing Fc, characterized in that: The method comprises: contacting a sample containing an Fc-containing protein with the domain mutant of protein A according to claim 1 or the affinity chromatography ligand according to claim 4 or the affinity chromatography medium according to claim 5 or 6 or the chromatographic column according to claim 7.

11. The method according to claim 10, characterized in that The Fc-containing protein is an antibody or an Fc fusion protein.

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