Alkali-resistant Fc binding protein

Abstract

The invention relates to a Z structural domain of alkali-resistant Fc binding protein protein A and protein A containing the structural domain. The Z structural domain is an amino acid sequence as shown in SEQ ID NO: 6. The mutant protein obtained by the invention can be used as an affinity chromatography filler ligand, and has excellent alkali resistance and good high temperature resistance. When a chromatography medium bonded with the protein A is cleaned for 120 minutes by using 1M NaOH and is cleaned for 100 hours by using 0.5 M NaOH, the DBC is not obviously reduced, and 98% of the initial loading capacity can still be maintained; the residual DBC is kept at 83% or above of the initial value after the high-temperature sterilization at 121 DEG C is carried out for 30 minutes once, and the residual DBC can still be kept at 75% or above of the initial value after the high-temperature sterilization at 121 DEG C is carried out for 30 minutes twice, so that the method has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an alkali-resistant Fc binding protein protein A and use thereof as an affinity chromatography filler ligand in antibody or Fc fusion protein purification. Background Art

[0002] Staphylococcal protein A (SPA or protein A) is a protein on the surface of Staphylococcus aureus, which is covalently linked to the peptidoglycan of the cell wall. Since it can bind to the Fc segment of human and various mammalian IgG, the binding of protein A to the Fc segment of the antibody does not affect the antibody's adsorption activity to the antigen, so it is widely used in antibody affinity purification.

[0003] The wild-type protein A gene coding sequence consists of 1464 bases (excluding the 108 bases encoding the N-terminal signal peptide), encoding a total of 488 amino acids, with a molecular weight of about 42kD. Protein A does not contain cysteine, so there are no disulfide bonds and thiol groups inside the molecule. It is a single-chain polypeptide that is connected to the peptidoglycan of Staphylococcus aureus through a covalent bond. Protein A includes five highly homologous IgG binding domains, E, D, A, B, and C, which have about 65% to 90% homology between them. Each domain consists of about 58 amino acids and three α-helical bundles arranged in reverse parallel in a Z shape (helices H1, H2, and H3, respectively), and its three-dimensional structure is stabilized by a hydrophobic core.

[0004] Each domain has the ability to autonomously bind to the antibody IgG Fc fragment, but the application of this natural protein A ligand is limited due to its stability and elution conditions. Protein A is easily degraded by microorganisms and is sensitive to organic solvents and pH changes, which shortens the service life of the chromatographic column; after protein A affinity filler purifies IgG, it needs to be eluted in a lower pH environment, which will reduce the biological activity of IgG; during the affinity separation process, protein A will be released from the chromatographic column and needs to be further purified and removed.

[0005] Usually, the chromatography medium used in the first step of monoclonal antibody purification is the chromatography medium material coupled with protein A. Because it specifically binds to IgG, the purity and yield after one-step purification can usually reach more than 90%. However, this chromatography medium is expensive and not durable. It must be cleaned after each chromatography step to remove host proteins, host DNA and other strongly adsorbed impurities introduced by fermentation culture. The most effective cleaning method is 0.5M sodium hydroxide washing. However, when strong alkali is used for washing, protein A cross-linked to the chromatography medium material is prone to hydrolysis, denaturation or shedding. After multiple batches of alkali treatment, the protein loading capacity can be significantly reduced due to protein A shedding and protein denaturation and hydrolysis, which poses a great challenge to the robustness of the production process.

[0006] At present, there are many literature reports at home and abroad that some protein A with strong alkali resistance have been obtained by mutation modification of protein A. For example, GE healthcare pointed out in patent application US20200079878A1 that multiple protein A fragments with strong alkali resistance were obtained through mutation screening. After 300 washes with 0.5M NaOH, the antibody load can still be maintained at about 50%. Patent application CN104059133A performed mutation screening based on the B domain of protein A, and the obtained mutants were washed with 0.5M NaOH 100 times (about 25h), and the load did not change significantly.

[0007] In the antibody purification process, the cost of affinity chromatography fillers is relatively high. Compared with ion exchange fillers, reverse phase chromatography fillers and other chromatography fillers, the current protein A alkali resistance still has a lot of room for improvement. An affinity protein A molecule with better alkali resistance is still needed as a ligand for affinity chromatography. Summary of the invention

[0008] In view of the above technical status, the present invention provides an Fc binding protein A with better alkali resistance through mutation screening. The specific invention is as follows:

[0009] The present invention provides a Z domain of an alkaline-resistant Fc binding protein protein A, wherein the Z domain is an amino acid sequence shown in SEQ ID NO:6.

[0010] The present invention also provides an alkaline-resistant protein A containing the Z domain; as one of the embodiments, the protein A comprises 2 to 8 repeated Z domains; in the present invention, as one of the embodiments, the protein A comprises 3 to 6 repeated Z domains; in the present invention, as one of the embodiments, the protein A comprises 4-6 repeated Z domains, for example, 4, 5, or 6 repeated Z domains.

[0011] In the present invention, as one of the embodiments, the C-terminus of protein A is further connected to one or more cysteines, preferably one cysteine.

[0012] In the present invention, as one of the embodiments, the protein A has an amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:17.

[0013] Accordingly, the present invention also claims to protect the nucleotide sequence of the Z domain or protein A. According to the amino acid sequence provided by the present invention, those skilled in the art can easily obtain the corresponding nucleotide sequence and optimize the nucleotide sequence according to the codon characteristics of different organisms.

[0014] Accordingly, the present invention also claims to protect an expression vector comprising the nucleotide sequence.

[0015] Accordingly, the present invention also claims protection for a host cell comprising the expression vector or a host cell having the nucleotide sequence of the aforementioned Z domain or protein A integrated into its genome.

[0016] By inserting the nucleotide sequence into a suitable expression vector and further transfecting a host cell, a recombinant expression strain for expressing the protein A can be obtained for the production of the protein A; the expression vector is preferably the Escherichia coli expression vector pET-29a(+), and the host cell is preferably an Escherichia coli BL21(DE3) cell.

[0017] The present invention also provides an affinity adsorption medium, wherein the aforementioned protein A is coupled to a solid phase carrier. As one embodiment, the solid phase carrier is selected from natural or modified polymers, such as agarose, dextran, etc.; or synthetic polymers, such as polymethacrylate, polystyrene-divinylbenzene, etc.; or inorganic matrix carriers, such as ceramics, silica, etc. As one embodiment, the solid phase carrier is in the form of microspheres, magnetic beads, filter membranes, chips, or capillaries.

[0018] The present invention also provides the use of the aforementioned affinity adsorption medium for purifying antibodies or Fc fusion proteins and for preparing therapeutic agents for treating clinical autoimmune diseases or other diseases related to Ig in the human body; the clinical autoimmune diseases or other diseases related to Ig in the human body include systemic lupus erythematosus / lupus nephritis, anti-glomerular basement membrane antibody disease, anti-neutrophil cytoplasmic antibody-associated vasculitis, renal transplant rejection, membranoproliferative nephritis, allergic purpura nephritis, rheumatoid arthritis, Sjögren's syndrome, scleroderma, dermatomyositis, pyogenic infection, dilated cardiomyopathy, etc.

[0019] The present invention also provides the use of the aforementioned protein A as an affinity chromatography filler ligand in the purification of antibodies or Fc fusion proteins. As an exemplary illustration, the affinity chromatography filler ligand of the protein A described in the present invention can be cleaned and disinfected with sterile sodium hydroxide or saturated sodium carbonate or saturated sodium bicarbonate solution to extend the service life of the adsorption column.

[0020] Technical effects:

[0021] 1. The protein A mutant protein obtained by the present invention can be used as an affinity chromatography filler ligand with excellent alkali resistance. Among them, the (2953)-4 and (2953)-6 chromatography media have no significant decrease in DBC after cumulative cleaning with 1M NaOH for 120 minutes and 0.5M NaOH for 100 hours, and can still maintain 98% of the initial load, and the chromatography yield can be maintained at more than 90%; the two chromatography media are immersed in 1M NaOH solution for 100 hours, and the load retention rate is still more than 80%. The alkali resistance is significantly better than the alkali-resistant protein A disclosed in the prior art US20200079878A1.

[0022] 2. The protein A mutant protein obtained in the present invention has good high temperature resistance. After the chromatography media (2953)-4 and (2953)-6 were sterilized at 121°C for 30 min once, the remaining DBC remained above 83% of the initial value. After being sterilized at 121°C for 30 min twice, the remaining DBC could still be maintained above 75% of the initial value. DETAILED DESCRIPTION

[0023] The following examples are used to further illustrate the present invention, but are not intended to limit the effective scope of the present invention in any way.

[0024] Example

[0025] Example 1: Screening of Fc binding protein-Staphylococcus aureus protein A mutants

[0026] Taking the mutant G8 in patent application CN202211411171.4 (amino acid sequence: VDAKFDKEAQEAFYEILHLPNLTEEQRNAFIRSLKLDPSVSGTALAEAKKLNDAQAPK) as the starting molecule, random mutations and screening were performed on the E11, H18, R32, S33, L36, D37, V40, G42, T43, A44, K50, and L51 sites. The amino acid sequence of the screened protein A mutant is shown in Table 1, where the positive control C2 molecule is the Z domain of the alkali-resistant mutant disclosed in SEQ ID NO: 20 in GE's US2020079878A1.

[0027] Table 1 Amino acid sequences of protein A mutants

[0028]

[0029]

[0030] C2 tetramer, referred to as (C2)-4, SEQ ID NO: 9

[0031] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

[0032] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

[0033] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK

[0034] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IQSLKDDPSV SKAILAEAKK LNDAQAPK 2938 tetramer, referred to as (2938)-4, SEQ ID NO: 10

[0035] VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPK

[0036] VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPK

[0037] VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPK

[0038] VDAKFDKEAQ KAFYEILKLP NLTEEQRNAF IQKLKDEPSQ SRAILAEAKR YNDAQAPK 2950 tetramer, referred to as (2950)-4, SEQ ID NO: 11

[0039] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAALAEAKK LNDAQAPK

[0040] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAALAEAKK LNDAQAPK

[0041] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAALAEAKK LNDAQAPK

[0042] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAALAEAKK LNDAQAPK 2951 Tetramer, abbreviated as (2951)-4, SEQ ID NO:12

[0043] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKAALAEAKK LNDAQAPK

[0044] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKAALAEAKK LNDAQAPK

[0045] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKAALAEAKK LNDAQAPK

[0046] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKAALAEAKK LNDAQAPK 2952 Tetramer, abbreviated as (2952)-4, SEQ ID NO:13

[0047] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGTILAEAKK LNDAQAPK

[0048] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGTILAEAKK LNDAQAPK

[0049] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGTILAEAKK LNDAQAPK

[0050] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGTILAEAKK LNDAQAPK 2953 four tandem repeats, abbreviated as (2953)-4, SEQ ID NO:14

[0051] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK

[0052] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK

[0053] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK

[0054] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK 2954 four tandem repeats, abbreviated as (2954)-4, SEQ ID NO:15

[0055] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKAILAEAKK LNDAQAPK

[0056] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKAILAEAKK LNDAQAPK

[0057] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKAILAEAKK LNDAQAPK

[0058] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKAILAEAKK LNDAQAPK 2955 four tandem repeats, abbreviated as (2955)-4, SEQ ID NO:16

[0059] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKALLAEAKK LNDAQAPK

[0060] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKALLAEAKK LNDAQAPK

[0061] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKALLAEAKK LNDAQAPK

[0062] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SKALLAEAKK LNDAQAPK 2953 hexamer, abbreviated as (2953)-6, SEQ ID NO: 17

[0063] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK

[0064] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK

[0065] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK

[0066] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK

[0067] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK

[0068] VDAKFDKEAQ EAFYEILHLP NLTEEQRNAF IRSLKLDPSV SGAILAEAKK LNDAQAPK

[0069] Example 2: Expression and purification of protein A mutant protein

[0070] 1. Construction of protein A recombinant expression strain

[0071] The Protein A recombinant vector plasmid was artificially synthesized by the DNA full sequence synthesis method (synthesized by Nanjing GenScript Biotechnology Co., Ltd.). A cysteine ​​was introduced into the C-terminus of (C2)-4, (2938)-4, (2950)-4, (2951)-4, (2952)-4, (2953)-4, (2954)-4, (2955)-4, and (2953)-6 in Example 1, and an OmpA signal peptide (amino acid sequence: MKKTAIAIAVALAGFATVAQA) and a DsbA connecting peptide (amino acid sequence: AQYEDGKQYTGT) were inserted into the N-terminus. The nucleotide sequence was obtained by reverse translation of the amino acid sequence, and the optimized nucleotide sequence was obtained according to the codon degeneracy and the codon preference of Escherichia coli. An NdeI restriction site was added to the N-terminus of the gene sequence and an XhoI restriction site was added to the C-terminus. The residues were ligated to the Escherichia coli expression vector pET-29a(+) to obtain a protein A mutant recombinant plasmid and a positive control (C2)-4 recombinant plasmid.

[0072] Take 1 μl of 0.08 ng / μl recombinant plasmid and add it to 50 μl BL21 (DE3) competent cells. After ice bathing for 30 minutes, heat shock at 42°C for 60 seconds. After ice bathing again for 3 minutes, add 500 μl of antibiotic-free LB liquid culture medium in the clean bench and resume culture at 37°C and 150 rpm for 1 hour.

[0073] Take 100 μl of the bacterial solution after recovery culture and apply it to LB solid medium containing 50 μg / ml Kan, place it in a 37°C incubator, and invert and culture overnight. Pick a single clone from the overnight culture plate and inoculate it into 50 mL LB liquid medium containing 50 μg / ml Kan. Pick two single clones for each strain and culture them in a shake flask at 37°C and 220 rpm. Perform colony PCR verification at the same time, using EasyTaq as the DNA enzyme and T7 as the amplification primer (the nucleotide sequence of the T7 promoter primer is TAATACGACTCACTATAGGG; the nucleotide sequence of the T7 terminator primer is GCTAGTTATTGCTCAGCGG) to verify the target gene.

[0074] The PCR amplification products were subjected to agarose gel electrophoresis: the agarose concentration was 1%, 8 μl of sample was added to the loading well, and DL2000 Marker was used as a control, 1×TAE buffer, voltage 120V, current 108mA, and electrophoresis for 30 minutes. After the electrophoresis, the gel was placed in ethidium bromide solution for staining for 10 minutes, and the electrophoresis results were observed. It was found that the size of the four-concatemer band was about 1000bp, and the size of the six-concatemer band was about 1500bp, which was consistent with expectations.

[0075] Take 1 ml of the protein A recombinant expression strain cultured to a certain concentration and inoculate it into a secondary 50 mL LB liquid medium containing 50 μg / ml Kan. Make two parallels for each single clone and culture in a 37°C 220 rpm shake flask. Take the remaining bacterial liquid and mix it with 60% sterile glycerol, dispense 1 ml into 5 1.5 mL sterile centrifuge tubes, and store in an ultra-low temperature refrigerator.

[0076] 2. Shake flask screening of protein A recombinant expression strains

[0077] The positive control molecule (C2)-4 strain and protein A mutant expression strain were taken out from the ultra-low temperature refrigerator, 100 μl was inoculated into 50 mL LB liquid culture medium containing 50 μg / ml Kan, and cultured in a shaking flask at 37°C and 220 rpm. When the OD of the bacterial liquid grew to 0.6-0.8, IPTG induction was performed, the induction concentration was 0.5 mM, the induction temperature was 28°C, and the induction time was 16 h.

[0078] 4 ml of the induced bacterial solution at 0 h and 16 h were taken and centrifuged at 12000 g for 5 min. The fermentation supernatant was identified by SDS-PAGE electrophoresis. The results showed that the positive control molecule (C2)-4 strain and the protein A recombinant expression strain had obvious expression of the target protein in the 16 h fermentation supernatant.

[0079] 3. Expression of protein A recombinant expression strain in 5L fermenter

[0080] Take out the stored positive control molecule (C2)-4 strain and protein A mutant expression strain from the ultra-low temperature refrigerator for primary seed liquid inoculation, take 1 ml of bacterial liquid and inoculate it in 50 mL LB anti-resistant medium, shake the flask at 37°C and 220 rpm until the bacterial liquid OD is between 0.8 and 3. For secondary seed liquid inoculation, take 1 ml of primary seed liquid and inoculate it in 100 mL LB anti-resistant medium, shake the flask at 37°C and 220 rpm until the bacterial liquid OD is between 0.8 and 3, and pour the secondary bacterial liquid into the inoculation bottle. Inoculate the bacterial liquid in the inoculation bottle into a 5L fermentation tank, control the growth temperature at 35°C, and the induction temperature at 28°C for batch fermentation.

[0081] 4. Purification of fermentation samples of protein A recombinant expression strains

[0082] The 16h fermentation sample in the 5L fermenter was centrifuged, 325ml of the fermentation supernatant was taken, 75ml of ammonium sulfate solution was added to make the final concentration of ammonium sulfate 1.0M, and the pH was adjusted to 7.0 after mixing as the sample to be loaded for hydrophobic chromatography. A 40mL chromatography column was used for hydrophobic chromatography, Uniphenyl 50M was used as the hydrophobic chromatography filler, A solution (20mM Na2HPO4·12H2O+1.0M(NH4)2SO4+10mM EDTA-2Na, pH7.0) was the balance solution, and B solution (10mM Na2HPO4·12H2O+10mMEDTA-2Na, pH7.0) was the eluent. The specification of the chromatography column is 1.6*40cm. 100% A solution is used to flush 3 column volumes. The flow rate is controlled at 3 ml / min to balance the column. 400 ml of sample is loaded. After loading, 100% A solution is used to flush 3 column volumes to rebalance the column. 100% B solution is used to flush 3 column volumes to elute the purified protein, and the elution peak is collected.

[0083] The sample collected by hydrophobic chromatography was washed with ion chromatography medium DEAE Sepharose FF, solution A (20mM PBS, 20mMNaCl, pH7.0) was used as the balancing solution, solution B (20mM PBS, 0.5M NaCl, pH7.0) was used as the eluting solution, the chromatography column specification was 1.6*40cm, 100% solution A was used to wash 3 column volumes, the flow rate was controlled at 3ml / min to balance the column, the hydrophobic collection solution was loaded, and after the loading was completed, 100% solution A was used to wash 3 column volumes to rebalance the column, and solutions A and B were used for gradient elution to collect the protein elution peak. After electrophoresis detection, the purity of the target protein was higher than 90%.

[0084] Example 3: Preliminary evaluation of alkali resistance of purified protein A mutant samples

[0085] The purified samples of fermentation supernatants of protein A mutants (2938)-4, (2950)-4, (2951)-4, (2952)-4, (2953)-4, (2954)-4, (2955)-4, and (2953)-6 collected in Example 2 and the purified sample of positive control (C2)-4 were each divided into two portions. 6 M NaOH was added to one portion to make the final concentration of NaOH 0.5 M, and 6 M NaOH was added to make the final concentration of NaOH 1.0 M. After standing for 48 hours, the pH was adjusted to 7.0. The samples treated with alkali for 48 hours were analyzed by SDS-PAGE. From the changes in protein concentration, the fermentation yield and alkali resistance of (C2)-4, (2952)-4, (2953)-4, and (2953)-6 were better, while the fermentation yield and alkali resistance of (2950)-4 and (2951)-4 were poor. Therefore, (2950)-4 and (2951)-4 were not evaluated for the next round of dynamic binding capacity.

[0086] Table 2 Fermentation yield of protein A mutants and results of 48h alkali treatment

[0087] Mutants Fermentation yield Protein concentration after 48h treatment with 0.5M NaOH Protein concentration after 48h treatment with 1.0M NaOH (C2)-4 +++++ +++++ +++++ (2938)-4 + +++ +++ (2950)-4 + + + (2951)-4 + + + (2952)-4 +++++ +++++ +++++ (2953)-4 +++ +++++ +++++ (2954)-4 + +++ +++ (2955)-4 + +++ +++ (2953)-6 +++++ +++++ +++++

[0088] Note: Fermentation yield: "+" indicates extremely low yield, "+++" indicates relatively high yield, and "+++++" indicates high yield;

[0089] Main peak concentration after alkali treatment for 48h: “+” indicates poor alkali resistance, “+++” indicates good alkali resistance, and “+++++” indicates good alkali resistance.

[0090] Example 4: Alkali resistance test of protein A affinity chromatography medium

[0091] The collected protein A mutants (2938)-4, (2952)-4, (2953)-4, (2954)-4, (2955)-4 purified samples and the positive control (C2)-4 purified sample were lyophilized to obtain lyophilized powder, dissolved in 20mM pH8.0 PBS solution, and reacted with epoxy-activated high-rigidity agarose gel (average particle size 75μm). Each 1ml of activated agarose gel was reacted with 10mg of protein A. The temperature was controlled at 37°C and the reaction was carried out for 10h to couple the carboxyl-terminal cysteine ​​of protein A with the activated agarose gel to obtain 6 affinity chromatography media bonded with different protein A.

[0092] The prepared protein A-bonded affinity chromatography medium was loaded onto the column, and Romosozumab monoclonal antibody fermentation broth (prepared with reference to Example 1 of CN103435698B, with a monoclonal antibody protein content of 2.18 mg / ml) was used as the loading liquid; the chromatography column was washed with 0.5 M NaOH solution for 15 min before use, and balanced with a balance solution (2 mM NaH2PO4·2H2O+8 mM Na2HPO4·12H2O+0.14 M NaCl) until the baseline was stable.

[0093] Dynamic binding capacity test: Inject samples at different retention times until the penetration absorbance reaches 10% UVBypass, and record the injection volume. The injection volume*sample content is DBC (dynamic binding capacity). After the loading is completed, use 0.5M NaOH to wash for 15 minutes. Repeat this process for one batch; DBC after washing / DBC without washing = retention rate of loading capacity.

[0094] Table 3 Trends in dynamic binding capacity of each protein A mutant

[0095]

[0096] As shown in Table 3, the affinity chromatography medium bonded with the (2953)-4 mutant protein can maintain a 97% loading retention rate after 350 washes and a 81% loading retention rate after 450 washes, and its alkali resistance is significantly better than that of the affinity chromatography medium bonded with the (C2)-4 protein.

[0097] Example 5: Alkali resistance test of protein A affinity chromatography medium at high concentration

[0098] The purified protein A mutants (2953)-4 and (2953)-6 were freeze-dried to obtain 16 g and 12 g of freeze-dried powder, respectively, which were dissolved in 20 mM pH 8.0 PBS solution and reacted with epoxy-activated high-rigidity agarose gel (average particle size 75 μm). Each 1 ml of activated agarose gel was reacted with 20 mg of protein A. The temperature was controlled at 37°C and the reaction was carried out for 10 hours to couple the carboxyl-terminal cysteine ​​of protein A with the activated agarose gel. Two affinity chromatography media bonded with different ligands were obtained, totaling 715 ml of (2953)-4 and 415 ml of (2953)-6.

[0099] Two affinity chromatography media (2953)-4 and (2953)-6 were loaded into columns with a column size of 0.5×20 cm, and Romosozumab monoclonal antibody solution (prepared with reference to Example 1 of CN103435698B, with a monoclonal antibody protein content of 2.18 mg / ml) was used as the loading solution; the chromatography column was cleaned with 0.5M NaOH solution for 15 min before use, and equilibrated with a balance solution (2mM NaH2PO4·2H2O+8mM Na2HPO4·12H2O+0.14M NaCl) until the baseline was stable.

[0100] Dynamic binding capacity test: Perform two dynamic binding capacity tests after every 50 batches of cycles. Method: Inject the sample at a retention time of 4 minutes until the penetration absorbance reaches 10% UV Bypass, and record the injection volume. The injection volume*sample content is DBC (dynamic binding capacity). After loading, use eluent (0.1 M HAc, pH 3.9) for elution.

[0101] Cyclic batch: The chromatography medium was dynamically flushed with 0.5 M NaOH, and every 15 min was recorded as one cycle, that is, one batch was used.

[0102] Normal use batch: After the chromatography column is balanced, 80% of the maximum dynamic binding capacity tested is injected, the sample retention time is 4 minutes, the target protein is eluted with eluent, and the residual Protein A in the collected sample and the chromatography yield are detected.

[0103] After each cycle of 50 batches, two normal batch experiments were performed.

[0104] CIP method: After each injection, wash with 0.5M NaOH for 15 minutes; after every 50 batches, wash with 1M NaOH for 15 minutes for deep cleaning; DBC after washing / DBC without washing = loading retention rate.

[0105] Table 4 Dynamic binding capacity variation trend of two affinity chromatography media in alkaline resistance test

[0106]

[0107] Table 5 Statistics of normal batch chromatography yields of two affinity chromatography media

[0108]

[0109] The above results show that the DBC of (2953)-4 and (2953)-6 chromatographic media did not decrease significantly after cumulative cleaning with 1M NaOH for 120 minutes and 0.5M NaOH for 100 hours, and the initial loading capacity was still maintained at 98%, and the chromatographic yield could be maintained above 90%.

[0110] Example 6: Alkali resistance test of protein A affinity chromatography medium soaked in 1M NaOH

[0111] According to the Protein A affinity chromatography medium preparation method described in Example 5, two affinity chromatography media (2953)-4 and (2953)-6 bonded with different ligands were obtained and loaded into columns with a column size of 0.5×20 cm.

[0112] Alkaline solution soaking method: 1M NaOH flows through the column loaded with Protein A affinity chromatography medium until the conductivity and pH of the effluent are the same as those of the 1M NaOH solution. Place at room temperature for 12 to 19 hours as one alkali solution soaking cycle. After the soaking, immediately rinse the column with purified water until the pH of the effluent is neutral.

[0113] Dynamic binding capacity test: After each alkaline solution soaking cycle, the sample was injected according to the sample retention time of 6 minutes, and the maximum dynamic binding capacity of the filler was tested using the Romosozumab single antigen solution according to the method described in Example 5.

[0114] Table 6 Dynamic binding capacity of two affinity chromatography media after immersion in 1M NaOH

[0115]

[0116] The data in the above table show that the loading retention rate of the two fillers is still above 80% after being immersed in 1M NaOH solution for 100 hours, showing excellent alkali resistance.

[0117] Example 7: Performance test of protein A affinity chromatography medium to resist high temperature sterilization at 121°C

[0118] Affinity chromatography media (2953)-4 and (2953)-6 were prepared according to the method described in Example 5, and the high temperature sterilization performance of the two chromatography media was tested. The specific method is as follows.

[0119] The prepared affinity chromatography media (2953)-4 and (2953)-6 were loaded into columns, respectively, with a column size of 0.5×20 cm. Before the first use, the chromatography column was cleaned with a 0.5M NaOH aqueous solution for 15 minutes, and balanced with an equilibrium solution (2mM NaH2PO4·2H2O+8mMNa2HPO4·12H2O+0.14M NaCl) until the baseline was stable, and the dynamic binding capacity test of the chromatography medium was performed according to the method described in Example 5. After the test, the chromatography medium was taken out and sterilized with high temperature and wet heat (condition: 121°C, 30min), and the chromatography medium sterilized at 121°C for 30min was re-loaded into the column and then tested for dynamic binding capacity, and the above sterilization operation was repeated once. The experiment was sterilized twice, and the dynamic binding capacity was tested after each time, and the remaining DBC (%) = DBC after sterilization / initial DBC.

[0120] Table 7 DBC data statistics of two chromatography media before and after high temperature sterilization

[0121]

[0122]

[0123] The data in the above table show that after sterilization once, the remaining DBC of the two chromatography media (2953)-4 and (2953)-6 remains above 83% of the initial value. After sterilization twice, the remaining DBC can still be maintained above 75% of the initial value, indicating that both chromatography media have good high temperature resistance.

Claims

1. A Z domain of an alkaline-resistant Fc binding protein protein A, characterized in that: The Z domain is the amino acid sequence shown in SEQ ID NO:

6.

2. Protein A comprising the Z domain according to claim 1, characterized in that: Protein A includes 2 to 8, preferably 3 to 6, and most preferably 4 to 6 repeating Z domains.

3. Protein A according to claim 2, characterized in that The C-terminus of protein A is further linked to one or more cysteines, preferably one cysteine.

4. Protein A according to claim 2, characterized in that The protein A is the amino acid sequence shown in SEQ ID NO:14 or SEQ ID NO:

17.

5. A nucleotide sequence encoding the Z domain of claim 1 or the protein A of any one of claims 2 to 4.

6. An expression vector comprising the nucleotide sequence according to claim 5.

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

8. An affinity adsorption medium, characterized in that The protein A described in any one of claims 2 to 4 is coupled to the solid phase carrier of the affinity adsorption medium.

9. The affinity adsorption medium according to claim 8, characterized in that The solid phase carrier is selected from natural or modified polymers, preferably agarose or dextran; or synthetic polymers, preferably polymethacrylate or polystyrene-divinylbenzene; or inorganic matrix carriers, preferably ceramics or silica.

10. Use of the affinity adsorption medium according to claim 8 for purifying antibodies or Fc fusion proteins, and for preparing therapeutic agents for treating clinical autoimmune diseases or other diseases related to Ig in the human body.

11. Use of the protein A according to claims 2-4 as an affinity chromatography filler ligand in the purification of antibodies or Fc fusion proteins.

Images