CN109748955A - A kind of affinity chromatography medium and application suitable for antibody purification - Google Patents

Abstract

The invention discloses a kind of affinity chromatography medium suitable for antibody purification and applications.Affinity chromatography medium provided by the invention includes solid phase carrier and amino acid sequence recombinant protein A as shown in SEQ ID NO:1 with solid phase carrier coupling.Affinity chromatography medium provided by the invention has protein binding high specificity, and affinity is high, is very suitable for isolating and purifying for antibody.

Description

A kind of affinity chromatography medium and application suitable for antibody purification

Technical field

The invention belongs to field of biotechnology, a kind of affinity chromatography medium and application are related in particular to.

Background technique

Staphylococcal protein A (Staphylococcus aureus protein A, SPA, albumin A) is golden yellow grape Pneumoniae cells wall GAP-associated protein GAP is single chain polypeptide structure, is had and immunoglobulin in the mankind and other mammalians point The ability of the Fc section specific binding of son, primary attachment IgG and the immune complex containing IgG.1940, Vevwey had found at certain In a little staphylococcus aureuses, containing a kind of substance, in double diffusion test, it can be formed and be precipitated with normal human serum.1959 Year, Jensen has also discovered similar phenomenon, is named as Staphylococal Protein A.Nineteen sixty, Grov was named as protein staphylococcus A, abbreviation SPA albumin A, Lofkvist in 1963 etc. have separated Staphylococal Protein A, and prove that it is a kind of protein.It is inhaled since SPA has The affinity column of the characteristic of attached albumen, albumin A preparation has become the affine of the widely applied antibody purification of field of biotechnology Column, usually when preparing protein A affinity chromatography medium, used Epichlorohydrin activation agarose is all that reaction is situated between with water Matter, the solubility of epoxychloropropane in water is about 6.58% or so, therefore the reaction is a complicated oil (epoxy chloropropionate Alkane) Gu-water-(gel) three-phase reaction system, reaction rate are slower；Epoxidation process epoxy group pole under alkaline condition simultaneously Hydrolytic side reactions easily occur, need to carry out fully optimized and strict control to reaction condition that higher activation efficiency could be obtained. Shi Qinghong etc. effectively eliminates epoxychloropropane to the phase boundary in agarose activation process by being introduced into DMSO in reaction medium Face makes epoxy base density effectively improve 100 μm of ol/mL or so, but reaction medium is water, the still more serious [history of hydrolysis Clear flood etc..Epichlorohydrin activation Ago-Gel process intensification and performance evaluation [J] process engineering journal, 2007,7 (4): 743-746.】。

Currently, with biotechnology industry, the especially fast development of designative species, on numerous antibody drugs are approved City, for example, Imclone company exploitation a kind of mouse/people be fitted into monoclonal anti EGFR antibody (Erbitux, Cetuximab, Cetuximab), by the combination of competitive antagonism ligand and EGFR, block the combination of receptor and ligand after And inhibit the activation of ligand-mediated EGFR tyrosine kinase, to make in the stroma cell in tumour cell or tumor microenvironment A variety of cell processes adjusted by EGFR signal path are blocked, to achieve the purpose that treat tumour, have been used for controlling at present Treat the malignant diseases such as colorectal cancer, head and neck neoplasm.

With the listing of lot of antibodies drug, purified technology of protein is increasingly paid attention to by everybody, albumin A affinity column The market demand increases sharply, and requires the affinity of albumin A affinity column, purification efficiency etc. higher and higher.But contain in use When the affinity chromatography technology of albumin A, there are affinity it is low, purification efficiency is low the problems such as, therefore apply the affinity chromatography technology When, a kind of improved affinity chromatography medium is needed, purification effect is improved.

Summary of the invention

The present inventor develops a kind of novel affinity chromatography medium by gene technology means by numerous studies.Specifically , the present invention provides:

1, a kind of recombinant protein A, which is characterized in that the amino acid sequence of the recombinant protein A such as SEQ ID NO:1 institute Show.

2, a kind of affinity chromatography medium, which is characterized in that the affinity chromatography medium includes recombination described in claim 1 Albumin A and solid phase carrier, the solid phase carrier and recombinant protein A are coupled.

3, affinity chromatography medium described in above-mentioned 2, which is characterized in that the solid phase carrier be Ago-Gel, glucan, Cellulose, silica gel or hydroxyl high molecular polymer.

4, affinity chromatography medium described in above-mentioned 3, which is characterized in that the solid phase carrier is that agarose or glucan are solidifying Glue.

5, a kind of method for preparing any affinity chromatography medium of above-mentioned 1-4, the described method comprises the following steps:

(1) synthesis A gene of recombined protein monomer is designed by chemically synthesized method, 5 ' hold Ala-Asp to be mutated into Asn- Val is connected with forming AclI restriction enzyme site between each A gene of recombined protein monomer with AclI restriction enzyme site, first recombinant protein The NcoI restriction enzyme site being connected with expression vector, 6 His and EK restriction enzyme sites, the last one recombination is added in A gene monomer front end The BamH I restriction enzyme site for stopping codon TAA and being connected with expression vector pET32a is added in protein A gene monomer end；

(2) by the A gene of recombined protein monomer digestion of step (1) be followed by into carrier pET32a corresponding restriction enzyme site it Between, then with AclI endonuclease digestion, screen the transformant with amicillin resistance, extracted through plasmid, digestion screening obtains The expression vector pET32a-P of staphylococcal protein A containing 6 protein A gene monomers；

(3) the expression vector pET32a-P of step (2) is converted into e. coli bl21 DE3 with CaCl2 method, screened, digestion, Obtain the sub- BL21DE3/pET32a-P of recombinant conversion；

(4) the bacterial strain BL21DE3/pET32a-P of step (3) is subjected to cultivation and fermentation, makes its highly effective expressing recombinant protein A, Thallus is regathered, separated, purify to obtain recombinant protein A product；

(5) it is reacted in the medium with epoxychloropropane, sodium hydroxide with agarose or sephadex, reaction product The recombinant protein A obtained with step (4) reacts 15-25 hours at a temperature of 10-28 DEG C, and cleaning is drained again after having reacted, and obtains Recombinant protein A gel.

6, preparation method described in above-mentioned 5, which is characterized in that the Cross-linked Agar that step (5) Ago-Gel is 5% Sugared gel.

7, the preparation method as described in above-mentioned 6, which is characterized in that in the step (5), epoxychloropropane, sodium hydroxide It is reacted in DMSO medium with Ago-Gel.

8, any affinity chromatography medium of above-mentioned 2-4 is applied in protein separation.

9, application described in above-mentioned 8, which is characterized in that the protein is antibody.

10, application described in above-mentioned 9, which is characterized in that the antibody is anti-egfr antibodies.

It is of the invention the experimental results showed that, affinity chromatography medium of the invention has protein binding high specificity, affinity Height, the high advantage of purification efficiency, compared with commercially available SP- Ago-Gel FF, Dynamic Adsorption carrying capacity is significantly improved.

Specific embodiment

Further illustrate that the present invention, following embodiments should not be construed as limiting the invention with reference to embodiments.With In lower embodiment, anti-egfr antibodies (method referring to disclosed in China Patent No. 201010148069.0 is prepared) are other Experiment with material is commercially available by commercial channel.

The preparation of 1 Recombinant Staphylococal Protein A of embodiment

1, Recombinant Staphylococal Protein A gene monomer is constructed

Pass through chemically synthesized method, design synthesis one repeated fragment (repeated fragment sequence of staphylococcal protein A gene Shown in the 46-207 nucleotide as shown in SEQ ID NO:2) sequence monomers comprising length be 162bp repetition piece The NcoI restriction enzyme site being connected with expression vector is added in section and front end, 6 His (subtract in conjunction with nickel column for affinity chromatography Few purification step), EK restriction enzyme site (for cutting His and DDDDK, forming correct Protein A) and AclI digestion position Point (AACGTT, for being connected into multiple repeated fragments).In addition, further including terminator codon TAA, and clone is restricted with BamH I The total 9bp of restriction endonuclease connector.Labeled as " monomer 1 ".

In addition, design synthesis one repeated fragment of staphylococcal protein A gene (repeats piece by chemically synthesized method Duan Xulie is as shown in the 46-207 nucleotide of SEQ ID NO:2) sequence monomers, sequence includes length for 162bp weight Multiple segment and ends A clI restriction enzyme site.Labeled as " monomer 2 ".

2, the expression vector of Recombinant Staphylococal Protein A gene is constructed

With the monomer 1 obtained of restricted type restriction endonuclease NcoI and BamH I digestion embodiment 1, then and through NcoI and BamH The carrier pET32a connection of I digestion converts Escherichia coli, screens the transformant with amicillin resistance, extracts through plasmid, Prove that staphylococcal protein A gene monomer has been cloned into pET32a after digestion identification, to successfully construct a staphylococcus The pET32a carrier of protein A gene monomer.

3, the building of the pET32a-P carrier of the gene monomer containing staphylococcal protein A

By the monomer 2 obtained of above-mentioned steps 1 with AclI digestion, respective segments are recycled, then and constructed by above-mentioned steps 2 The pET32a carrier connection that grape globulin A gene monomer is recombinated containing one, convert Escherichia coli, screening has ammonia benzyl green The transformant of chloramphenicol resistance, is extracted through plasmid, and digestion screening obtains the staphylococcal protein A containing 6 protein A gene monomers Expression vector is labeled as " pET32a-P ", and through detecting, pET32a-P expression vector includes the gene sequence as shown in SEQ ID No:2 Column.

4, the coli strain of building expression Recombinant Staphylococal Protein A

Use CaCl₂The pET32a-P obtained of above-mentioned steps 3 is converted BL21DE3 by method, in the LB containing ampicillin Transformant is screened on plate, obtains recombinant conversion containing pET32a-P through plasmids detection and restriction analysis, is indicated and is “BL21DE3/pET32a-P”。

5, the production and purifying of Recombinant Staphylococal Protein A

(1) cultivation and fermentation of strain

Picking colibacillus engineering BL21DE3/pET32a-P, is inoculated in LB culture medium, 1~2%V/V of inoculum concentration, In 35 DEG C of overnight incubations, above-mentioned cultured seed culture medium is aseptically inoculated in fermentation training by l:8-1:4 by next day It supports on base, 35 DEG C of fermentations reach 0.4~0.7 to O.D600, are warming up to 48 DEG C of inductions, and bacterium is received in centrifugation after 3~5 hours.

It takes a small amount of cell to add 2 × sample-loading buffer, carries out PAGE gel electrophoresis detection, as the result is shown staphylococcus egg White A has induced soluble-expression.

(2) purifying of Recombinant Staphylococal Protein A

The thallus that step (1) is collected is suspended with phosphate buffer (0.2M, pH7.0-8.0 contain 0.1M NaCl), Ultrasonication, 4 DEG C of centrifugations, collects supernatant, obtains crude extract.

Crude extract is used into SDS-PAGE electrophoresis detection, most of staphylococcal protein A is slightly mentioned as the result is shown, is remained in The amount of thallus is atomic: crude extract being carried out 5200 molecular sieve purification of Sephacryl, collecting characteristic peak (the second eluting peak) is The Recombinant Staphylococal Protein A (being named as " Protein A 1 ") of purifying, through detecting, 1 amino acid sequence of Protein A is such as Shown in SEQ ID No:1.

Embodiment 2ELISA method detects Recombinant Staphylococal Protein A and antibody binding activity

The Protein A 1 and antibody binding activity prepared using ELISA method detection above-described embodiment 1, detailed process is such as Under:

1) it is coated with: the 1 sample 200u1 of every hole coating Protein A in 96 orifice plates, 30 DEG C, 1.5h；

2) close: every hole is closed with 1% BSA of 200u1, and 30 DEG C, 1.5h；

3) add primary antibody: every hole is added about 200ug human IgG antibody (being purchased from Mlbio company), and 38 DEG C, 1h；

4) add secondary antibody: about 200u1 is added in every hole, and 1:1000 Horseradish Peroxidase Conjugates (are purchased from Mlbio company), 30 DEG C, 1.5h；

5) it develops the color.

Same method detects commercially available Protein A (purchased from ancient biology) and antibody binding activity.

ELISA testing result: for Protein A 1 of the invention in conjunction with human IgG antibody, every hole is coated with the Portugal 0.5-1.2ng Grape pneumococcal proteins A can be obtained apparent detection signal, and commercially available Protein A needs every hole to be coated with 8.0-12.5ng grape ball Apparent detection signal is just obtained when mycoprotein A.It can be seen that Protein A1 of the invention has stronger antibody binding activity.

The preparation of 3 Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium of embodiment

Ago-Gel affinity chromatography medium is prepared using the Protein A1 of embodiment 1, comprising the following steps:

1, epoxychloropropane, sodium hydroxide and agarose (5% cross-linked agarose gel) activation of Ago-Gel: are used It is reacted in dimethyl sulfoxide (DMSO, be purchased from toray group) medium, it is small in 45-60 DEG C of at a temperature of reaction 2-3 When, the Ago-Gel of activation is obtained to draining after neutral wash with distilled water after having reacted；

2, the chemical coupling of Recombinant Staphylococal Protein A and the Ago-Gel of activation: the activation that above-mentioned steps 1 are obtained Ago-Gel reacts 15-25 hours with Protein A1 prepared by embodiment 1 at a temperature of 10-28 DEG C, cleaning after having reacted It drains, obtains staphylococcal protein A Ago-Gel affinity chromatography medium.

The Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium ligand density of preparation is about 5.3mg, absorption carries Measure 10~60mg/ml, 70-180 μm of the granular size of affinity media, maximum flow rate 350cm/h, pH range 2-11, storage temperature 3-10 DEG C, preservation 20% ethyl alcohol of liquid.

4 affinity chromatography medium of embodiment isolates and purifies antibody

Divided from antibody culture solution using the Recombinant Staphylococal Protein A affinity chromatography medium prepared in above-described embodiment 3 From antibody purification (preparing monoclonal antibody against EGFR according to method described in 201010148069.0), detailed process is as follows:

1, Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium isolates and purifies antibody

1) the Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium dress column that, prepared by above-described embodiment 3,1.7 × 20cm, bed volume 15ml；

2) 5-10 bed volume, is balanced with buffer solution A (PBS solution of pH7.4, the same below), l.5ml/min flow velocity is；

3) 2m1 monoclonal antibody against EGFR culture solution, is diluted to 30m1 with buffer solution A, 0.45 μm of membrane filtration, on Sample, flow velocity 1.5ml/min；

4) 5-10 bed volume, is washed again with buffer solution A, l.5ml/min flow velocity is；

5), with buffer solution B, (the 30mM citrate buffer solution of pH5.0, preparation method are as follows: citric acid 2.5g adds water 950m1 is adjusted to pH4.0 with 5M NaOH, adds water to 1000ml) elution, flow velocity is l.5ml/min, to collect eluting peak；

6) 10 bed volumes, are washed with stream of pure water, then wash 10 bed volumes, flow velocity 2.5ml/ with 20% ethanol stream Min, pillar are placed in 4-8 DEG C of environment and save；

7) monoclonal antibody against EGFR isolated and purified and reference substance, are subjected to SDS-PAGE electrophoretic analysis simultaneously.

(remarks: should be to buffer C (the 0.5M acetate buffer solution of pH4.0, preparation side after the every use several times of pillar Method is as follows: 0.5M acetum is adjusted to pH4.0 with solid NaOH) stream washes 1 time, so that more firm albumen removal will be adsorbed.)

SDS-PAGE electrophoresis result: Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium of the invention is primary Purify the monoclonal antibody against EGFR purity about 98.8% obtained.

2, commercially available SP- Ago-Gel FF isolates and purifies antibody

In addition, using identical method, using commercially available SP- Ago-Gel FF (being purchased from Bo Erxi scientific & technical corporation) from culture Antibody (preparing monoclonal antibody against EGFR according to method described in 201010148069.0) is isolated and purified in liquid.SDS-PAGE electricity Swimming is the results show that once purify the monoclonal antibody against EGFR purity about 88% of acquisition using MabSelectSure.

The experimental results showed that.Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium and a step of the invention is just The monoclonal antibody against EGFR that purity is greater than 98% can be obtained, purification effect is substantially better than commercial product.

Detection of 6 affinity chromatography medium of embodiment to the Dynamic Adsorption carrying capacity of antibody

Affinity chromatography medium of the invention and commercially available SP- Ago-Gel FF (being purchased from Bo Erxi scientific & technical corporation) are dynamic to antibody State absorption carrying capacity compares, and steps are as follows:

1,5-10 bed volume is balanced with buffer solution A (PBS solution of pH7.4), l.5ml/min flow velocity is；

2,2mg/ml concentration monoclonal antibody against EGFR loading, flow velocity are l.5ml/min, until 10% stopping when penetrating；

3, according to sample concentration, loading volume and column volume calculate 10% penetrate when each gel Dynamic Adsorption carrying capacity.

Experimental result: Recombinant Staphylococal Protein A Ago-Gel of the invention is about to the Dynamic Adsorption carrying capacity of antibody 50mg/ml, the Dynamic Adsorption carrying capacity about 30mg/ml of commercially available SP- Ago-Gel FF.

The experimental results showed that affinity chromatography medium of the invention the Dynamic Adsorption carrying capacity of antibody is apparently higher than it is commercially available The Dynamic Adsorption carrying capacity of SP- Ago-Gel FF has better purification effect.

Sequence table

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Asp Ala Pro Ser Leu Trp Ala Ile Ser Leu Ser Tyr Gln Ala Trp Arg

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Asn Phe Ala Ile Ser Asn Val Ala Ser Leu Trp Ser Asn Asn Lys Thr

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Trp Ser Thr Arg Ser Tyr Ile Lys Leu Trp Leu Asn Glu Ala Ala Arg

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Asn Phe Phe Leu Glu Thr Leu Lys Asp Ala Pro Ser Leu Trp Ala Ile

100 105 110

Ser Leu Ser Tyr Gln Ala Trp Arg Asn Phe Ala Ile Ser Asn Val Ala

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Ser Leu Trp Ser Asn Asn Lys Thr Trp Ser Thr Arg Ser Tyr Ile Lys

130 135 140

Leu Trp Leu Asn Glu Ala Ala Arg Asn Phe Phe Leu Glu Thr Leu Lys

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Asp Ala Pro Ser Leu Trp Ala Ile Ser Leu Ser Tyr Gln Ala Trp Arg

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Asn Phe Ala Ile Ser Asn Val Ala Ser Leu Trp Ser Asn Asn Lys Thr

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Claims (10)

1. a kind of recombinant protein A, which is characterized in that the amino acid sequence of the albumin A is as shown in SEQ ID NO:1.

2. a kind of affinity chromatography medium, which is characterized in that the affinity chromatography medium includes recombination described in the claims 1 Albumin A and solid phase carrier, the solid phase carrier and recombinant protein A are coupled.

3. affinity chromatography medium according to claim 2, which is characterized in that the solid phase carrier is Ago-Gel, Portugal Glycan, cellulose, silica gel or hydroxyl high molecular polymer.

4. affinity chromatography medium according to claim 3, which is characterized in that the solid phase carrier is agarose or glucan Gel.

5. a kind of method for preparing any affinity chromatography medium of claim 1-4, the described method comprises the following steps:

(1) it is designed by chemically synthesized method and synthesizes A gene of recombined protein monomer, 5 ' end Ala-Asp is mutated into Asn-Val, To form AclI restriction enzyme site, it is connected between each A gene of recombined protein monomer with AclI restriction enzyme site, first recombinant protein A base Because the NcoI restriction enzyme site being connected with expression vector, 6 His and EK restriction enzyme sites, the last one recombination egg is added in monomer front end The BamH I restriction enzyme site for stopping codon TAA and being connected with expression vector pET32a is added in white A gene monomer end；

(2) the A gene of recombined protein monomer digestion of step (1) is followed by between the corresponding restriction enzyme site of carrier pET32a, then With AclI endonuclease digestion, the transformant with amicillin resistance is screened, is extracted through plasmid, digestion screening, which obtains, contains 6 The expression vector pET32a-P of the staphylococcal protein A of a protein A gene monomer；

(3) the expression vector pET32a-P of step (2) is converted into e. coli bl21 DE3 with CaCl2 method, screening, digestion obtain The sub- BL21DE3/pET32a-P of recombinant conversion；

(4) the bacterial strain BL21DE3/pET32a-P of step (3) is subjected to cultivation and fermentation, makes its highly effective expressing recombinant protein A, then receive Collect thallus, is separated, purifies to obtain recombinant protein A product；

(5) it is reacted in the medium with epoxychloropropane, sodium hydroxide with agarose or sephadex, reaction product and step Suddenly the recombinant protein A that (4) obtain reacts 15-25 hours at a temperature of 10-28 DEG C, and cleaning is drained again after having reacted, and is recombinated Protein A Sepharose.

6. preparation method according to claim 5, which is characterized in that the crosslinking that step (5) Ago-Gel is 5% Ago-Gel.

7. preparation method according to claim 6, which is characterized in that in the step (5), epoxychloropropane, hydroxide Sodium is reacted in DMSO medium with Ago-Gel.

8. according to application of any affinity chromatography medium of claim 2-4 in protein separation.

9. application according to claim 8, which is characterized in that the protein is antibody.

10. application according to claim 9, which is characterized in that the antibody is anti-egfr antibodies.