CN114409801B

CN114409801B - Affinity chromatography medium based on recombinant protein A and preparation method and application thereof

Info

Publication number: CN114409801B
Application number: CN202111585634.4A
Authority: CN
Inventors: 吕小林; 杨克; 毛慧明
Original assignee: Suzhou Saifen Technology Co ltd
Current assignee: Suzhou Saifen Technology Co ltd
Priority date: 2021-12-22
Filing date: 2021-12-22
Publication date: 2023-04-18
Anticipated expiration: 2041-12-22
Also published as: CN114409801A

Abstract

The invention discloses an affinity chromatography medium based on recombinant protein A and a preparation method and application thereof, the invention recombines A or D structural domain of wild protein A and mutant Z structural domain to obtain chimera ZA or ZD, the mutation of ZA chimera relative to Z structural domain comprises: asp 53Glu, ala 54Ser, whereas mutations of the ZD chimera relative to the Z domain include: ala 42Thr, leu 44Val, ala 46Gly, asp 53Glu, ala 54Ser. The alkali resistance is further improved through the mutation without influencing the space structure of the whole structural domain, the His-Tag label and the flexible connecting arm are added on the basis of the mutant, the activity freedom degree of the chimera is ensured, the binding capacity of the chimera and the target molecule is improved, and the finally obtained affinity medium can obtain higher dynamic loading capacity and stronger alkali resistance.

Description

Affinity chromatography medium based on recombinant protein A and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an affinity chromatography medium based on recombinant protein A, and a preparation method and application thereof.

Background

In recent years, antibody therapy has been greatly advanced for the treatment of various cancers, rheumatoid arthritis, viral infections, and other diseases. To date, over 100 monoclonal antibody drugs have been approved by the FDA and there are also very abundant development lines, and thus, the demand for producing and purifying antibody drugs has increased. The purification of antibody drugs generally goes through a capture stage and a purification stage, wherein the capture stage uses an affinity chromatography medium formed by coupling a protein ligand capable of specifically binding to immunoglobulin to a solid phase carrier, such as a protein A ligand, a protein G ligand, a protein L ligand, etc.

Protein a is a membrane protein isolated from the cell wall of staphylococcus aureus and has a molecular weight of about 42kDa, and has been found to have strong binding force to the Fc region of immunoglobulin and weak binding force to the Fab region, and thus has been widely used for purification of immunoglobulin and Fc fusion proteins since the seventies of the last century. The wild-type protein A comprises 5 homologous structural domains, namely E, D, A, B and C, each structural domain comprises 3 antiparallel alpha helical structures (Helix) and two flexible connecting arms (Loop), and the arrangement sequence from the N end to the C end is Helix1-Loop1-Helix2-Loop2-Helix3 (Figure 1). Studies have shown that all 5 homeodomains of wild-type protein a can bind to immunoglobulins and Fc fusion proteins, with the B domain showing better solvent tolerance and temperature stability than the other domains. For immunoglobulin G (IgG), each domain exhibits strong affinity for the Fc region of IgG subclasses IgG1, igG2 and IgG4, and very weak affinity for IgG 3. The affinity effect of protein A on the Fc region is mainly the interaction between Helix1 and Helix2 and the Fc region, including hydrogen bond, hydrophobicity and van der Waals force, and Helix3 does not participate.

Protein a affinity chromatography media adsorb impurities such as host proteins, pigments, nucleic acids, endotoxins, and viruses during use, and require in-situ Cleaning (CIP) with NaOH to remove the impurities. The tolerance of the wild-type protein A to NaOH is not enough to support the long-term use of the medium, and mutation modification needs to be carried out on the wild-type protein A molecule to enhance the alkali resistance. In recent years, protein a produced by fermentation using e.coli cells (e.coli cells) has also made a great progress in alkali resistance, and in particular, Z domain obtained by modifying B domain is widely used. However, with the continuous progress of antibody expression technology, the expression level of antibody is increased, and the most effective method for improving the production efficiency without changing the existing equipment is to use high-loading affinity chromatography medium to increase the sample loading.

Therefore, how to obtain high dynamic loading and have higher and more stable alkali-resistant performance of the recombinant protein A and the affinity medium thereof will face more challenges.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a chimera of recombinant protein A.

The technical problem to be solved by the invention is to provide a recombinant protein A with high affinity to antibody and Fc fusion protein, wherein the recombinant protein A is formed by chimeric wild-type protein A structural domain and mutant Z structural domain, and has flexible connecting arm and cysteine at the C end of the sequence and high alkali resistance.

The technical problem to be solved by the invention is to provide a nucleic acid or a gene for coding the recombinant protein A.

The technical problem to be solved by the present invention is to provide an expression cassette, a recombinant vector or a cell.

The technical problem to be solved by the invention is to provide a method for preparing the recombinant protein A.

The technical problem to be solved by the invention is to provide an affinity chromatography medium.

The invention finally solves the technical problem of providing a method for purifying immunoglobulin or Fc fusion protein from fermentation liquor by using the affinity chromatography medium.

The technical scheme is as follows: to achieve the above technical object, the present invention provides a chimera of a recombinant protein a, which comprises a chimera ZA comprising α -helix structures helix1 and helix2 in the mutant Z domain and α -helix structure helix3 in the a domain of the wild-type protein a or a chimera ZD; the chimera ZD comprises alpha helical structures helix1 and helix2 in a mutant Z domain and alpha helical structure helix3 in a D domain of a wild-type protein A, the amino acid sequence of the chimera ZA is shown as SEQ ID NO.1, and the amino acid sequence of the chimera ZD is shown as SEQ ID NO. 2.

The chimera also comprises a flexible connecting arm and cysteine which are added at the C end of the chimera, and the amino acid sequence of the chimera is shown as SEQ ID NO.3 or SEQ ID NO. 4.

The invention provides a recombinant protein A, which comprises a plurality of chimeras, preferably, the recombinant protein A also comprises a flexible joint, an N-terminal His-Tag and a TEV proteolytic site; preferably, the recombinant protein a is composed of 5 repeated ZA domains or 5ZD domains.

Wherein, the amino acid sequence of the recombinant protein A is shown as SEQ ID NO.5 or SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO. 8.

Wherein the recombinant protein a is formed by tandem of two or more ZA or ZD chimeras whose amino acid mutations relative to the Z domain include: asp 53Glu, ala 54Ser, amino acid mutations of the ZD chimera relative to the Z domain including: ala 42Thr, leu 44Val, ala 46Gly, asp 53Glu, ala 54Ser.

The invention provides nucleic acid or gene, wherein the nucleic acid or gene codes the recombinant protein A, the nucleic acid or gene sequence coding the recombinant protein A of SEQ ID NO.7 is shown as SEQ ID NO.9, and the nucleic acid or gene sequence coding the recombinant protein A of SEQ ID NO.8 is shown as SEQ ID NO. 10.

The invention provides expression cassettes, recombinant vectors or cells containing said nucleic acids or genes.

The invention provides a method for preparing the recombinant protein A, which comprises the following steps: inserting the DNA sequence of the recombinant protein A into a pET derivative expression vector carrying a T7 promoter and a kanamycin resistance gene, and then transforming the constructed plasmid into a host cell for expression to obtain the recombinant protein A;

preferably, the host cell comprises prokaryotic cells, eukaryotic cells, yeast, insect cells, more preferably escherichia coli cells, and most preferably escherichia coli BL21 cell strain.

The invention provides an affinity chromatography medium, which contains the recombinant protein A.

Wherein the affinity chromatography medium is formed by coupling the recombinant protein A to a solid phase carrier through a proper spacing arm;

preferably, the sulfhydryl of the terminal cysteine of the recombinant protein A reacts with the activated functional group of the solid phase carrier to couple the recombinant protein A to the solid phase carrier;

preferably, the His-Tag label of the recombinant protein A is formed by cutting off with TEV enzyme and then coupling the cut His-Tag label to a solid phase carrier through a thioether bond;

preferably, a spacer arm is connected between the recombinant protein A and the solid phase carrier, and the spacer arm comprises, but is not limited to, epichlorohydrin, ethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, glycerol triglycidyl ether, 1,4-butanediol diglycidyl ether or 1,6-hexanediol diglycidyl ether;

preferably, the solid phase carrier comprises agarose microspheres, silica gel microspheres, glass beads, polymer microspheres and porous polymer microspheres;

preferably, the polymeric microspheres include polyethylene-divinylbenzene microspheres, polymethacrylate microspheres, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides, polyvinyl alcohols, polyhydroxyalkyl acrylates, and other vinyl monomer-containing polymers;

preferably, the solid support can be a membrane, chip, capillary, filter or other matrix material having a three-dimensional structure;

preferably, the solid support is a spherical medium with a particle size of <200 μm, preferably <100 μm, most preferably a particle size of 3-70 μm;

preferably, the spherical medium has a porous structure and an average pore diameter

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Preferably, the porous polymeric microspheres have a bilayer structure. One characteristic of the porous polymer solid phase carrier is a double-layer structure, such as Monomix Core 60 of Sedtz technology, in which the inner layer can be bonded with the recombinant protein A as an affinity layer, and the outer layer is modified with other chemical functional groups, and similarly, the outer layer can be bonded with the recombinant protein A as an affinity layer, and the inner layer is modified with other chemical functional groups.

The invention provides a method for purifying immunoglobulin or Fc fusion protein from fermentation liquor by using the affinity chromatography medium, which comprises but is not limited to the following steps: contacting the fermentation broth with an affinity chromatography medium, washing the medium with a buffer solution, and then reducing the pH to elute the immunoglobulin or Fc fusion protein.

Preferably, the method specifically comprises the following steps:

(1) An equilibrium chromatographic column; (2) loading; (3) post-balancing; (4) leaching 1; (5) leaching 2; (6) eluting; (7) cleaning; (8) CIP cleaning;

preferably, the sample loading amount in the step (2) is not higher than 80% of the dynamic loading amount, and the residence time is 5-8min;

preferably, the equilibrium solution adopted in the step (3) is 0.02-0.1mol/L phosphate or Tris-HCl buffer system, contains 0.1-0.2mol/L NaCl and has pH of 7-7.5;

preferably, the leacheate 1 adopted in the step (4) is 0.02-0.1mol/L phosphate or Tris-HCl buffer system containing 1-2mol/L NaCl and has the pH value of 7-7.5;

preferably, the leacheate 2 adopted in the step (5) is an acetic acid-sodium acetate buffer system with the pH value of 5-5.5 and the concentration of 0.02-0.1 mol/L;

preferably, the eluent in the step (6) is selected from a 0.02-0.1mol/L acetic acid-sodium acetate or glycine-hydrochloric acid or citric acid-sodium citrate buffer system with the pH value of 3-4;

preferably, the cleaning solution in the step (7) is 0.1-1mol/L acetic acid solution;

preferably, the CIP solution used in the CIP cleaning in the step (8) is selected from 0.1mol/L to 0.5mol/L NaOH.

The steps can be optimized according to different sample types.

Wherein SEQ ID NO.1 of the invention shows an amino acid sequence of a Z-domain and A-domain chimera ZA;

wherein SEQ ID NO.2 shows the amino acid sequence of the Z-domain and D-domain chimera ZD;

wherein SEQ ID NO.3 shows an amino acid sequence in which a flexible connecting arm and cysteine are added to the C-terminal of the ZA chimera;

wherein SEQ ID NO.4 shows an amino acid sequence of adding a flexible connecting arm and cysteine to the C-terminal of the ZD chimera;

wherein SEQ ID NO.5 shows an amino acid sequence of 5ZA chimeras with added flexible connecting arms to form recombinant protein A (5 ZA) and semi-leucine (5 ZA-cys) added at the C terminal;

wherein, SEQ ID NO.6 shows an amino acid sequence that 5ZD chimeras with added flexible connecting arms form a recombinant protein A (5 ZD) and a cysteine (5 ZD-cys) is added at the C-terminal;

wherein, SEQ ID NO.7 shows that His-Tag label and TEV restriction enzyme cutting site amino acid sequence are added at the N end of the recombinant protein A (5 ZA-cys);

wherein SEQ ID NO.8 shows an amino acid sequence of adding a His-Tag label and a TEV enzyme cutting site at the N end of the recombinant protein A (5 ZD-cys).

Has the beneficial effects that: compared with the prior art, the invention has the following advantages: the invention recombines A or D structure domain of wild protein A and mutant Z structure domain to obtain chimera ZA or ZD, the mutation of ZA chimera relative to Z structure domain includes: asp 53Glu, ala 54Ser, whereas mutations of the ZD chimera relative to the Z domain include: ala 42Thr, leu 44Val, ala 46Gly, asp 53Glu, ala 54Ser, on the basis of the mutation, the invention also adds a flexible connecting arm with the length of 10 amino acids at the C end, the flexibility of the polypeptide chain of the flexible connecting arm enables the recombinant protein A to be more spherical and more compact in volume, the influence of steric hindrance when the recombinant protein A is combined with an antibody is reduced, meanwhile, the flexible connecting arm also ensures that the recombinant protein A has sufficient freedom of movement after being coupled to a solid phase carrier, and further shows high dynamic loading, meanwhile, the mutation further improves the alkali resistance of the recombinant protein A and does not influence the spatial structure of helix1 and helix2 in the recombinant protein A structure domain, and the finally obtained affinity medium can obtain higher dynamic loading and stronger alkali resistance.

Drawings

FIG. 1 is a graph showing the results of SDS-page analysis of an intermediate and a purified product of recombinant protein A;

FIG. 2 is a diagram of a recombinant protein A (SEQ ID NO. 7) affinity chromatography medium for purifying monoclonal antibodies;

FIG. 3 shows the results of alkali resistance tests of affinity chromatography media prepared from recombinant protein A expressed according to the sequences of SEQ ID NO.7 and SEQ ID NO. 8.

Detailed Description

The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.

Wild type protein A

The antibody or fusion protein has 5 structural domains which are arranged in the sequence of E, D, A, B, C from the N end to the C end, each structural domain can be combined with an antibody or fusion protein containing an Fc region, the specific acting force is the interaction between Helix1 and Helix2 and the Fc region in each structural domain, including hydrogen bond, hydrophobicity and van der Waals force, and Helix3 does not participate in the interaction, so that whether the spatial structure of Helix1 and Helix2 is reserved by mutational modification on protein A or not to a certain extent can influence the affinity of the protein A and the Fc region. The Z domain obtained by mutating the B domain has good alkali resistance and high affinity to the Fc region, and an affinity chromatography medium prepared from recombinant protein A (consisting of 4Z domains, one cysteine added at the C terminal and labeled as 4Z-cys) is widely used for the production of antibody drugs in the last 20 years.

The amino acid sequence of the Z domain is shown in SEQ ID NO.11.

Recombinant protein A

The recombinant protein A provided by the invention is composed of any structure and chimeric structure of a Z structural domain and E, D, A, B, C, for example, the Z structural domain and the A structural domain form a chimeric ZA or the Z structural domain and the D structural domain form a chimeric ZD, amino acid sequences are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, and the amino acid sequences are respectively shown as Helix1 and Helix2 sequences of the Z structural domain, so that the recombinant protein A has high affinity to an Fc region, and the change of the Helix3 sequence further improves alkali resistance without influencing the space structure of the whole structural domain.

Chimeras ZA or ZD

The chimera ZA or ZD provided by the invention is additionally provided with a flexible connecting arm (composed of 10 amino acids, the tail end is alanine) at the C end, and the chimera ZA or ZD has the function of pulling the space distance between the chimera and the solid phase carrier open and improving the binding capacity with target molecules. Furthermore, the C-terminal is added with cysteine, so that the chimera can be coupled with a solid phase carrier in a single-point bonding mode, the moving freedom degree of the chimera is ensured, the binding capacity of the chimera and a target molecule is improved, and the sequences are shown as SEQ ID NO.3 and SEQ ID NO. 4.

Recombinant protein A

The invention provides a recombinant protein A consisting of 5 chimeras ZA or ZD in series, and the C end of each chimera is added with a flexible connecting arm to increase the space distance between the chimeras, the sequence of the recombinant protein 5ZA-cys or 5ZD-cys consisting of the five chimeras is shown as SEQ ID NO.5 and SEQ ID NO.6, and the tail end cysteine is used for coupling with a solid phase carrier.

The invention provides a recombinant protein A with an increased His-Tag sequence and a TEV enzyme digestion recognition site sequence at the N end, and the sequences are shown as SEQ ID NO.7 and SEQ ID NO. 8. The recombinant protein A can be purified by using an immobilized metal affinity chromatography medium (such as IMAC-Ni), and then the His-Tag label is cut off by using TEV enzyme to obtain the recombinant protein A.

Example 1 Gene Synthesis and plasmid preparation

1. Gene synthesis

The gene encoding recombinant protein A (SEQ ID NO.7 and SEQ ID NO. 8) was assembled from 50 double-stranded DNAs having 25 to 30 base pairs as described in the references (Krebs, et al Synthesis of a gene for sensing rhodopsin I and matter functional expression in Halobacterium halobium. Proc Natl Acad. Sci U S A.1993, 90 (8): 3486-3490), i.e., 25 pairs of complementary DNA oligonucleotides were prepared by a DNA synthesizer, followed by phosphorylation, annealing of DNA oligonucleotides, and ligation of 25 double-stranded DNAs to produce the entire synthetic gene. The gene sequence of the recombinant protein A coded by the SEQ ID NO.7 is shown as SEQ ID NO.9, and the gene sequence of the recombinant protein A coded by the SEQ ID NO.8 is shown as SEQ ID NO. 10.

2. Preparation of expression vector

The synthetic recombinant protein A coding genes of SEQ ID NO.9 and SEQ ID NO.10 were inserted into pET-16b vector through Nde I and Xba I sites, and after confirming the sequence in the plasmid, the plasmid was transferred into E.coli host cell BL21 (DE 3).

EXAMPLE 2 production and purification of recombinant protein A (recombinant protein A encoded by the amino acid sequence SEQ ID NO. 7)

Production of recombinant protein A was carried out in a 10L fermenter, induction was carried out using LB medium with 200mM IPTG, and whether expression was successful was confirmed by SDS-PAGE. After expression, the cells were harvested by centrifugation, resuspended in 1X PBS solution, then disrupted with a high pressure homogenizer (18000 psi), and then centrifuged to remove cell debris. After clarification and filtration, the recombinant protein A is captured by a Ni affinity chromatographic column, the chromatographic medium is Polar MC60-Ni Excel (Sepax PN #: 270660800), the specification of the chromatographic column is 10cm (ID) × 37cm (L), after the loading is finished, 1 XPBS solution with 2 times of column volume is used for leaching impurities, and the target protein is eluted by 0.1M phosphate buffer solution +0.2M imidazole at pH 7.5. The collected eluate was subjected to ultrafiltration to remove imidazole, and then a TEV enzyme was added to cleave off the His-Tag, and the results of SDS-page analysis of the intermediate and purified product of recombinant protein A are shown in FIG. 1. After removing the TEV enzyme and His-Tag, the recombinant protein A solution was concentrated to 50g/L for storage. The average yield was 3g/L.

Example 3 coupling of recombinant protein A to polymethacrylate microspheres

Activating hydrophilic polymethacrylate microspheres: 2L Monomix MC45-SEC (45 μm,

sepax PN #:280145950 Loading the base spheres into a 10L reaction kettle, adding 2L 1.5M NaOH, uniformly stirring, reacting at 45 ℃ and 150rpm for 1.5h, adding 2L 1, 4-butanediol diglycidyl ether, continuously reacting for 1.5h, and washing the base spheres with ethanol and water respectively after the reaction is finished to obtain the activated base spheres.

Coupling of recombinant protein a produced in example 2 to the activation spheres: 2L of the activated spheres were charged into a 10L reactor, then 1L of 0.5M sodium carbonate solution was added and stirred to disperse the activated spheres uniformly, then 1L of recombinant protein A solution (1 XPBS solution containing 40g of recombinant protein A in reduced state) was added, and finally 426g of Na was added ₂ SO ₄ And stirring uniformly, reacting for 16h at 30 ℃ and 150rpm, adding 10g of thioglycerol after the reaction is finished, and continuing to react for 3h, and washing a filler by using 10L 50mM phosphate buffer solution and pH7.4 after the reaction is finished to finally obtain the recombinant protein A affinity chromatography medium. The dynamic loading of the affinity chromatography medium for the recombinant protein A is tested to be 55mg/mL by hIgG, and the binding time is 5min.

EXAMPLE 4 coupling of recombinant protein A to agarose microspheres

Activating the agarose microspheres: 2L of Agarosix-65 (65 μm, sepax PN #: 250165990) base sphere is put into a 10L reaction kettle, added with 2L 1.5M NaOH and stirred evenly, then reacted for 1.5h under the condition of 45 ℃ and 150rpm, then added with 2L 1, 4-butanediol diglycidyl ether and continuously reacted for 1.5h, and after the reaction is finished, the base sphere is washed by ethanol and water respectively to obtain the activated base sphere.

Coupling of recombinant protein a (using recombinant protein a encoded by sequence SEQ ID No.8, produced as in example 2) to the activation knob: 2L of activated basic spheres are put into a 10L reaction kettle, then 1L0.5M sodium carbonate solution is added and stirred to ensure that the activated basic spheres are uniformly dispersed, then 1L of recombinant protein A solution (1X PBS solution containing 33g of recombinant protein A in a reduction state) is added, and finally 426g of Na is added ₂ SO ₄ And stirring uniformly, reacting for 16h at 30 ℃ and 150rpm, adding 10g of thioglycerol after the reaction is finished, and continuing to react for 3h, and washing a filler by using 10L 50mM phosphate buffer solution and pH7.4 after the reaction is finished to finally obtain the recombinant protein A affinity chromatography medium.The dynamic loading of the affinity chromatography medium for the recombinant protein A is 65mg/mL by using hIgG, and the binding time is 5min.

EXAMPLE 5 purification of monoclonal antibodies (CHO cell broth) Using protein A affinity chromatography media

The protein a affinity chromatography medium prepared in example 4 was packed into a column (column volume 5.13 mL) of 6.6mm (ID) × 150mm (L) for monoclonal antibody purification from the fermentation broth, as shown in fig. 2, which is a UV signal overlay during 80 consecutive purifications, with very high consistency during the purification process, and thus the protein a affinity chromatography medium prepared in example 4 had very stable performance.

The purification method is optimized according to the specification, and comprises the following specific steps:

1) Equilibrating 5CV of the chromatographic column by using equilibration solution (0.02 mol/L sodium phosphate buffer solution +0.15mol/L sodium chloride aqueous solution, pH 7.3-7.5) for 5min of residence time;

2) Carrying out deep filtration on the fermentation liquor, and then loading, wherein the concentration of the monoclonal antibody is 6g/L, the loading amount is 42.5mL, and the residence time is 5min;

3) Reequilibrating the chromatographic column for 3CV with equilibration solution (0.02 mol/L sodium phosphate buffer solution +0.15mol/L sodium chloride aqueous solution, pH 7.3-7.5) for 5min;

4) Eluting the chromatographic column 3CV with eluent 1 (0.05 mol/L sodium acetate buffer solution + 1mol/L sodium chloride aqueous solution, pH 7.3-7.5) for 5min;

5) Eluting the chromatographic column 3CV with eluent 2 (0.05 mol/L sodium acetate buffer solution, pH 5.4-5.6) for 5min;

6) Washing the chromatography column with eluent (0.05 mol/L sodium acetate buffer solution, pH 3.3-3.5) for 5CV, collecting UV280nm peak (increasing 0.1 AU-decreasing 0.1 AU), and standing for 5min;

7) Cleaning the chromatographic column with 3CV of cleaning solution (1 mol/L acetic acid water solution) for 5min;

8) CIP 3CV is carried out by using 0.1M NaOH solution, and the residence time is 5min;

EXAMPLE 6 study of the alkali resistance of protein A affinity chromatography Medium

The recombinant protein a affinity chromatography media prepared in example 3 and example 4 were packed into chromatography columns (column volume 1.03 mL) of 6.6mm (ID) × 30mm (L) respectively, and alkali resistance of the recombinant protein a affinity chromatography media was studied by testing dynamic loading after using 0.5M NaOH CIP chromatography column, as shown in fig. 3, dynamic loading of the recombinant protein a affinity chromatography media was maintained at 80% of the initial value after continuous 120 times of 0.5M NaOH CIP, which was very useful.

Sequence listing

<110> Suzhou Sai Ke Tech technology GmbH

<120> affinity chromatography medium based on recombinant protein A, preparation method and application thereof

<160> 11

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<210> 1

<211> 58

<212> PRT

<213> ZA Chimera(Artificial Sequence)

<400> 1

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55

<210> 2

<211> 58

<212> PRT

<213> ZD Chimera(Artificial Sequence)

<400> 2

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala

35 40 45

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

50 55

<210> 3

<211> 69

<212> PRT

<213> ZA Chimara with a flexible linker, and terminal cysteine(Artificial Sequence)

<400> 3

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp

50 55 60

Ala Lys Phe Ala Cys

65

<210> 4

<211> 69

<212> PRT

<213> ZD Chimera with a flexible linker, and terminal cysteine(Artificial Sequence)

<400> 4

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala

35 40 45

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp

50 55 60

Ala Lys Phe Ala Cys

65

<210> 5

<211> 341

<212> PRT

<213> Multimer of 5 ZA Chimara with flexible linkers, and one terminal cysteine(Artificial Sequence)

<400> 5

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp

50 55 60

Ala Lys Phe Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala

65 70 75 80

Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn

85 90 95

Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu

100 105 110

Leu Ala Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala

115 120 125

Pro Lys Val Asp Ala Lys Phe Ala Val Asp Asn Lys Phe Asn Lys Glu

130 135 140

Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu

145 150 155 160

Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln

165 170 175

Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala

180 185 190

Pro Lys Gln Ala Pro Lys Val Asp Ala Lys Phe Ala Val Asp Asn Lys

195 200 205

Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro

210 215 220

Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp

225 230 235 240

Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn

245 250 255

Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp Ala Lys Phe Ala

260 265 270

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

275 280 285

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

290 295 300

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

305 310 315 320

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp

325 330 335

Ala Lys Phe Ala Cys

340

<210> 6

<211> 341

<212> PRT

<213> Multimer of 5 ZD Chimara with flexible linkers, and one terminal cysteine(Artificial Sequence)

<400> 6

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala

35 40 45

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp

50 55 60

Ala Lys Phe Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala

65 70 75 80

Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn

85 90 95

Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val

100 105 110

Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala

115 120 125

Pro Lys Val Asp Ala Lys Phe Ala Val Asp Asn Lys Phe Asn Lys Glu

130 135 140

Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu

145 150 155 160

Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln

165 170 175

Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala

180 185 190

Pro Lys Gln Ala Pro Lys Val Asp Ala Lys Phe Ala Val Asp Asn Lys

195 200 205

Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro

210 215 220

Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp

225 230 235 240

Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn

245 250 255

Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp Ala Lys Phe Ala

260 265 270

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

275 280 285

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

290 295 300

Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala

305 310 315 320

Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp

325 330 335

Ala Lys Phe Ala Cys

340

<210> 7

<211> 367

<212> PRT

<213> Multimer of 5 ZA chimera with flexible linkers, one terminal cysteine and N-terminus HIStag and TEV proteolysis site.(Artificial Sequence)

<400> 7

Met Lys His His His His His His Pro Met Ser Asp Tyr Asp Ile Pro

1 5 10 15

Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala Val Asp Asn Lys Phe Asn

20 25 30

Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu

35 40 45

Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro

50 55 60

Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Glu Ser

65 70 75 80

Gln Ala Pro Lys Gln Ala Pro Lys Val Asp Ala Lys Phe Ala Val Asp

85 90 95

Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His

100 105 110

Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu

115 120 125

Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys

130 135 140

Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp Ala Lys

145 150 155 160

Phe Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr

165 170 175

Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe

180 185 190

Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala

195 200 205

Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys

210 215 220

Val Asp Ala Lys Phe Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln

225 230 235 240

Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln

245 250 255

Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala

260 265 270

Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

275 280 285

Gln Ala Pro Lys Val Asp Ala Lys Phe Ala Val Asp Asn Lys Phe Asn

290 295 300

Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu

305 310 315 320

Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro

325 330 335

Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala Lys Lys Leu Asn Glu Ser

340 345 350

Gln Ala Pro Lys Gln Ala Pro Lys Val Asp Ala Lys Phe Ala Cys

355 360 365

<210> 8

<211> 367

<212> PRT

<213> Multimers of 5 ZD chimera with flexible linkers, one terminal cysteine and N-terminus HIStag and TEV proteolysis site.(Artificial Sequence)

<400> 8

Met Lys His His His His His His Pro Met Ser Asp Tyr Asp Ile Pro

1 5 10 15

Thr Thr Glu Asn Leu Tyr Phe Gln Gly Ala Val Asp Asn Lys Phe Asn

20 25 30

Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu

35 40 45

Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro

50 55 60

Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser

65 70 75 80

Gln Ala Pro Lys Gln Ala Pro Lys Val Asp Ala Lys Phe Ala Val Asp

85 90 95

Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His

100 105 110

Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu

115 120 125

Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys

130 135 140

Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys Val Asp Ala Lys

145 150 155 160

Phe Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr

165 170 175

Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe

180 185 190

Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr Asn Val Leu Gly

195 200 205

Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys Gln Ala Pro Lys

210 215 220

Val Asp Ala Lys Phe Ala Val Asp Asn Lys Phe Asn Lys Glu Gln Gln

225 230 235 240

Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu Asn Glu Glu Gln

245 250 255

Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro Ser Gln Ser Thr

260 265 270

Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser Gln Ala Pro Lys

275 280 285

Gln Ala Pro Lys Val Asp Ala Lys Phe Ala Val Asp Asn Lys Phe Asn

290 295 300

Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile Leu His Leu Pro Asn Leu

305 310 315 320

Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln Ser Leu Lys Asp Asp Pro

325 330 335

Ser Gln Ser Thr Asn Val Leu Gly Glu Ala Lys Lys Leu Asn Glu Ser

340 345 350

Gln Ala Pro Lys Gln Ala Pro Lys Val Asp Ala Lys Phe Ala Cys

355 360 365

<210> 9

<211> 1101

<212> DNA

<213> recombinant protein A (Artificial Sequence)

<400> 9

atgaaacatc atcatcatca tcatccgatg agcgattatg atattccgac caccgaaaac 60

ctgtattttc agggcgcggt ggataacaaa tttaacaaag aacagcagaa cgcgttttat 120

gaaattctgc atctgccgaa cctgaacgaa gaacagcgca acgcgtttat tcagagcctg 180

aaagatgatc cgagccagag cgcgaacctg ctggcggaag cgaaaaaact gaacgaaagc 240

caggcgccga aacaggcgcc gaaagtggat gcgaaatttg cggtggataa caaatttaac 300

aaagaacagc agaacgcgtt ttatgaaatt ctgcatctgc cgaacctgaa cgaagaacag 360

cgcaacgcgt ttattcagag cctgaaagat gatccgagcc agagcgcgaa cctgctggcg 420

gaagcgaaaa aactgaacga aagccaggcg ccgaaacagg cgccgaaagt ggatgcgaaa 480

tttgcggtgg ataacaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 540

ctgccgaacc tgaacgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 600

agccagagcg cgaacctgct ggcggaagcg aaaaaactga acgaaagcca ggcgccgaaa 660

caggcgccga aagtggatgc gaaatttgcg gtggataaca aatttaacaa agaacagcag 720

aacgcgtttt atgaaattct gcatctgccg aacctgaacg aagaacagcg caacgcgttt 780

attcagagcc tgaaagatga tccgagccag agcgcgaacc tgctggcgga agcgaaaaaa 840

ctgaacgaaa gccaggcgcc gaaacaggcg ccgaaagtgg atgcgaaatt tgcggtggat 900

aacaaattta acaaagaaca gcagaacgcg ttttatgaaa ttctgcatct gccgaacctg 960

aacgaagaac agcgcaacgc gtttattcag agcctgaaag atgatccgag ccagagcgcg 1020

aacctgctgg cggaagcgaa aaaactgaac gaaagccagg cgccgaaaca ggcgccgaaa 1080

gtggatgcga aatttgcgtg c 1101

<210> 10

<211> 1101

<212> DNA

<213> recombinant protein A (Artificial Sequence)

<400> 10

atgaaacatc atcatcatca tcatccgatg agcgattatg atattccgac caccgaaaac 60

ctgtattttc agggcgcggt ggataacaaa tttaacaaag aacagcagaa cgcgttttat 120

gaaattctgc atctgccgaa cctgaacgaa gaacagcgca acgcgtttat tcagagcctg 180

aaagatgatc cgagccagag caccaacgtg ctgggcgaag cgaaaaaact gaacgaaagc 240

caggcgccga aacaggcgcc gaaagtggat gcgaaatttg cggtggataa caaatttaac 300

aaagaacagc agaacgcgtt ttatgaaatt ctgcatctgc cgaacctgaa cgaagaacag 360

cgcaacgcgt ttattcagag cctgaaagat gatccgagcc agagcaccaa cgtgctgggc 420

gaagcgaaaa aactgaacga aagccaggcg ccgaaacagg cgccgaaagt ggatgcgaaa 480

tttgcggtgg ataacaaatt taacaaagaa cagcagaacg cgttttatga aattctgcat 540

ctgccgaacc tgaacgaaga acagcgcaac gcgtttattc agagcctgaa agatgatccg 600

agccagagca ccaacgtgct gggcgaagcg aaaaaactga acgaaagcca ggcgccgaaa 660

caggcgccga aagtggatgc gaaatttgcg gtggataaca aatttaacaa agaacagcag 720

aacgcgtttt atgaaattct gcatctgccg aacctgaacg aagaacagcg caacgcgttt 780

attcagagcc tgaaagatga tccgagccag agcaccaacg tgctgggcga agcgaaaaaa 840

ctgaacgaaa gccaggcgcc gaaacaggcg ccgaaagtgg atgcgaaatt tgcggtggat 900

aacaaattta acaaagaaca gcagaacgcg ttttatgaaa ttctgcatct gccgaacctg 960

aacgaagaac agcgcaacgc gtttattcag agcctgaaag atgatccgag ccagagcacc 1020

aacgtgctgg gcgaagcgaa aaaactgaac gaaagccagg cgccgaaaca ggcgccgaaa 1080

gtggatgcga aatttgcgtg c 1101

<210> 11

<211> 58

<212> PRT

<213> Z Domain (Artificial Sequence)

<400> 11

Val Asp Asn Lys Phe Asn Lys Glu Gln Gln Asn Ala Phe Tyr Glu Ile

1 5 10 15

Leu His Leu Pro Asn Leu Asn Glu Glu Gln Arg Asn Ala Phe Ile Gln

20 25 30

Ser Leu Lys Asp Asp Pro Ser Gln Ser Ala Asn Leu Leu Ala Glu Ala

35 40 45

Lys Lys Leu Asn Asp Ala Gln Ala Pro Lys

50 55

Claims

1. A recombinant protein a comprising a chimera ZA or a chimera ZD comprising the alpha helical structures helix1 and helix2 in the mutant Z domain and the alpha helical structure helix3 in the a domain of wild-type protein a; the chimera ZD comprises alpha helical structures helix1 and helix2 in a mutant Z domain and alpha helical structure helix3 in a D domain of a wild-type protein A, the chimera ZA has an amino acid sequence shown in SEQ ID NO.1, the chimera ZD has an amino acid sequence shown in SEQ ID NO.2, the recombinant protein A consists of 5 repeated ZA domains or 5ZD domains, and the amino acid sequence of the recombinant protein A is shown in SEQ ID NO.5 or SEQ ID NO.6 or SEQ ID NO.7 or SEQ ID NO. 8.

2. The recombinant protein A as claimed in claim 1, wherein the chimera ZA or the chimera ZD further comprises a flexible linking arm and a cysteine added at the C-terminal end, the amino acid sequence of the chimera ZA is shown as SEQ ID NO.3, and the amino acid sequence of the chimera ZD is shown as SEQ ID NO. 4.

3. A nucleic acid encoding the recombinant protein a of claim 1.

4. An expression cassette, recombinant vector or cell comprising the nucleic acid of claim 3.

5. A method for preparing the recombinant protein a of claim 1, comprising the steps of: inserting the DNA sequence of the recombinant protein A into a pET derived expression vector carrying a T7 promoter and a kanamycin resistance gene, and then transforming the constructed plasmid into a host cell for expression to obtain the recombinant protein A.

6. The method of claim 5 for preparing the recombinant protein A of claim 1, wherein the host cell comprises a prokaryotic cell, a eukaryotic cell, a yeast, or an insect cell.

7. The method according to claim 5, wherein the host cell is an E.coli cell.

8. The method according to claim 5, wherein the host cell is E.coli BL21 cell line.

9. An affinity chromatography media comprising recombinant protein a of claim 1.

10. The affinity chromatography media of claim 9, wherein the affinity chromatography media is formed by coupling recombinant protein a to a solid support via a suitable spacer.

11. The affinity chromatography media of claim 10, wherein the thiol group of the cysteine at the terminus of recombinant protein a reacts with the activating functional group of the solid support to couple recombinant protein a to the solid support.

12. The affinity chromatography media of claim 11, wherein a spacer arm is coupled between the recombinant protein a and the solid support, the spacer arm comprising one or more of epichlorohydrin, ethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, glycerol triglycidyl ether, 1,4-butanediol diglycidyl ether, or 1,6-hexanediol diglycidyl ether.

13. The affinity chromatography media of claim 10, wherein the solid support comprises one or more of agarose microspheres, silica gel microspheres, glass beads, polymeric microspheres, or porous polymeric microspheres.

14. The affinity chromatography media of claim 13, wherein the polymeric microspheres comprise one or more of polyethylene-divinylbenzene microspheres, polymethacrylate microspheres, polyhydroxyalkyl methacrylates, polyacrylamides, polymethacrylamides, polyvinyl alcohols, polyhydroxyalkyl acrylates, and other polymers containing vinyl monomers.

15. The affinity chromatography media of claim 10, wherein the solid support comprises one or more of a membrane, a chip, a capillary, a filter, or other matrix material having a three-dimensional structure.

16. The affinity chromatography media of claim 10, wherein the solid support is a spherical media with a particle size <200 μ ι η.

17. The affinity chromatography media of claim 10, wherein the solid support is a spherical media with a particle size <100 μ ι η.

18. The affinity chromatography media of claim 10, wherein the solid support is a spherical media having a particle size of 3-7 μm.

19. The affinity chromatography media of claim 16, wherein the spherical media has a porous structure and an average pore diameter >300 a.

20. The affinity chromatography media of claim 16, wherein the spherical media has a porous structure and an average pore diameter >500 a.

21. The affinity chromatography media of claim 16, wherein the spherical media has a porous structure and an average pore diameter is >1000 a but <3000 a.

22. The affinity chromatography media of claim 13, wherein the porous polymeric microspheres have a bilayer structure.

23. A method for purifying immunoglobulin or Fc fusion protein from a fermentation broth by using the affinity chromatography medium of any one of claims 9 to 22, comprising the steps of: contacting the fermentation liquor with an affinity chromatography medium, washing the medium by using a buffer solution, and then reducing the pH value to elute the immunoglobulin or Fc fusion protein, wherein the pH value of the buffer solution is 2.0-4.0.

24. The method of claim 23, wherein the buffer has a pH of 2.5 to 4.0.

25. The method of claim 23, wherein the buffer has a pH of 3.0 to 3.5.

26. The method according to claim 23, comprising in particular the steps of: (1) an equilibrium chromatographic column; (2) loading; (3) post-balancing; (4) leaching 1; (5) leaching 2; (6) eluting; (7) cleaning; and (8) CIP cleaning.

27. The method of claim 26, wherein the loading in step (2) is no more than 80% of the dynamic loading and the residence time is 5-8 min.

28. The method of claim 26, wherein the equilibration solution used in step (3) is 0.02-0.1mol/L phosphate or Tris-HCl buffer system containing 0.1-0.2mol/L NaCl and having a pH of 7-7.5.

29. The method according to claim 26, wherein the eluent 1 used in step (4) is selected from 0.02-0.1mol/L phosphate or Tris-HCl buffer system containing 1-2mol/L NaCl and having a pH of 7-7.5.

30. The method as claimed in claim 26, wherein the leacheate 2 used in the step (5) is a 0.02 to 0.1mol/L acetic acid-sodium acetate buffer system with a pH of 5 to 5.5.

31. The method as claimed in claim 26, wherein the eluent in step (6) is selected from 0.02-0.1mol/L acetic acid-sodium acetate or glycine-hydrochloric acid or citric acid-sodium citrate buffer system with pH3-4.

32. The method as claimed in claim 26, wherein the washing solution in step (7) is 0.1-1mol/L acetic acid solution.

33. The method as claimed in claim 26, wherein the CIP solution used in the CIP cleaning in step (8) is 0.1mol/L to 0.5mol/L NaOH.