CN106977591A - A kind of method for isolating and purifying Recombinant Staphylococal Protein A - Google Patents
A kind of method for isolating and purifying Recombinant Staphylococal Protein A Download PDFInfo
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
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Abstract
The invention discloses a kind of method for isolating and purifying Recombinant Staphylococal Protein A; using the filler of two kinds of mixed mechanisms; two steps are chromatographed and the mode of ultrafiltration effectively removes a series of impurity such as endotoxin, coli somatic residual protein (HCP), Escherichia coli residual DNA, protein aggregate; using minimum step; obtain most pure target sample; save technology controlling and process cost, at the same have that stability is good, process control index clearly, the features such as be adapted to large-scale industrial production.Protein solution after purified, purity of protein is measured with efficient liquid phase and SDS PAGE electrophoresis more than 97%；Protein recovery is more than 75%；Endotoxin is reduced to below 1EU/mg；HCP and Escherichia coli residual DNA are below 5ppm, can be used to synthesize clinic staphylococcal protein A immunosorbent as raw material.
Description
Technical field：
The present invention relates to biomedicine technical field, and in particular to a kind of side for isolating and purifying Recombinant Staphylococal Protein A
Method.
Background technology：
Staphylococcal protein A (abbreviation albumin A or SPA) is a kind of protein of polypeptide single-stranded structure, contains 5 height classes
As immunoglobulin Fc section land, can specifically with human immunoglobulin (IgG) combine.SPA is coupled to agar
On sugar carrier, it is prepared into carrier-SPA compounds loading pillar and staphylococcal protein A immunoabsorbent column is made, human plasma passes through
During the chromatographic column, the IgG types pathogenic antibody in blood plasma will be fallen by specifically absorption, and some change because IgG type antibody quality and quantities
Disease significantly can be treated and alleviated with symptom caused by change.Staphylococcal protein A immunoabsorbent column extensively should
For the treatment of the diseases such as autoimmune disease, organ transplant and malignant tumour, wide market.
Widely used due to staphylococcal protein A, market demand increasingly increases.It is straight only from staphylococcus aureus
Extraction native protein is connect, does not simply fail to meet the market demand.Therefore people start to turn to the method next life of genetic engineering in recent years
Produce Recombinant Staphylococal Protein A.Current staphylococcal protein A is mainly expressed by genetic engineering bacterium-Escherichia coli, is led to
Chromatographic technique purifying is crossed, satisfactory product is finally given.Due to containing multiple protein impurity in the nutrient solution of Escherichia coli
And the non-protein impurity such as nucleic acid, lipid, the purifying to downstream brings baptism.Wherein endotoxin be influence it is maximum because
Element, the endotoxin harmful effect such as human body can be caused to generate heat, shiver, pharmaceuticals industry has higher to the endotoxin content of pharmaceutical products
It is required that, such as endotoxin content must assure that every kg body weight intake hourly is less than 5EU in injection medicament.But from
The problem that endotoxin is still industry is removed in albumen system, its main cause is that protein system is more complicated, and
There is strong active force between multiple proteins and endotoxin, both are combined together, it is difficult to separate, therefore there is presently no
Endotoxic pervasive rule is removed from protein system, correlative study is also all only applicable to a certain albumen system.Impurity place
Main albumen and host DNA influence also larger to body, and 100ppm coli somatic residual protein (HCP) just can cause trouble
The immune response of person, host DNA always is the weight of domestic and international drug administration department concern because there is special potential security risk
Point., it is necessary to set up circulation in vitro when staphylococcal protein A immunoabsorbent column carries out clinical treatment, belong to three class medicine equipments,
Risk is larger.Staphylococcal protein A directly affects the security of adsorption column as one of important raw and processed materials, its quality.So opening
Hair technique is easy, can amplify, the purification process for the staphylococcal protein A that purity is higher is particularly important.
Patent report at present on Recombinant Staphylococal Protein A purifying is very more, such as passes through Ni post affinitive layer purifications
(CN201010614100.5, CN201210096913.9, CN200810028707.8, CN200810028706.3,
CN200810028704.4, CN201010110754.4, CN201010110754.4)；With Sephacryl S200 sieve methods
Purify (CN03149982.1, CN200710085148.X, CN201010225393.8)；It is affine using IgG Fc fragments as aglucon
Chromatographic stuffing single step purification or coupled ion exchange purifying (CN201010227290.5, CN201210118134.4)
GSTrapFF posts affinity chromatography and Superdex 7510/300GL sieve methods purifying (CN201080013514.0).
The first chromatographic column used in CN200680034577.8 is anion-exchange column, HIC posts and ceramic hydroxyapatite column, the second layer
Analysis post is cation exchange column, or 1,2 posts are exchanged using order.These above-mentioned patents do not mention impurity in protein solution
Content.But for the isolating and purifying of albumin A, do not require nothing more than and reach higher purity, also strictly to control endotoxin, egg
The content of the white non-protein impurity such as impurity and nucleic acid, lipid.Patent CN103145813B refer to how to remove endotoxin, main
Will be using charcoal absorption-hydrophobic chromatography twice, because activated carbon is powdered, practical operation is inconvenient.
The content of the invention：
It is an object of the invention to provide a kind of method for isolating and purifying Recombinant Staphylococal Protein A, this method is easy to operate,
Effectively remove endotoxin, protein aggregate, coli somatic residual protein (HCP), Escherichia coli residual DNA etc. miscellaneous
Escherichia coli residual DNA and HCP amounts are below 5ppm in matter, the protein solution of purifying, and endotoxin is less than 1EU/mg, purity of protein
Up to more than 97%, it is adaptable to industrialized production, it can be used to synthesize clinic staphylococcal protein A immuno absorbence as raw material
Agent.
Term albumin A or Recombinant Staphylococal Protein A refer to total length or only containing wherein some or some knots in the present invention
The staphylococcal protein A gene in structure domain is through clone, conversion and obtained recombinant protein is expressed in Bacillus coli cells.
It is many that term purification refers to the raising purpose from composition or sample containing target protein and one or more impurity
The purity of peptide or protein matter or target protein.
Term immunoglobulin (Ig) refers to be connected by two identical light chains and two identical heavy chains by interchain disulfide bond
Four peptide chain structures connect.
Term " chromatographic stuffing ", " medium ", " filler ", " chromatography media " meaning are identical, refer to and are loaded in chromatographic column, utilize
The difference of each component physicochemical properties, multicomponent mixture is separated, to complete the material of chromatography process.
The present invention is achieved by the following technical programs：
A kind of method for isolating and purifying Recombinant Staphylococal Protein A, this method comprises the following steps：
1) thalline is pre-processed：Thalline containing staphylococcal protein A is added to Bis-tris buffer solutions, homogeneous is then used
Supernatant is collected by centrifugation after crusher machine, pH is to 2.0-4.0 for regulation, and supernatant is collected by centrifugation, then adjusts pH to 6.5-7.2, obtain
To protein liquid；
2) the first chromatographic column is handled：By step 1) obtained protein liquid loading is equipped with salt-resistant type to what is balanced with equilibrium liquid
In the chromatographic column of anion exchange filler, continuation is balanced with equilibrium liquid, is rinsed with flushing liquor, finally with elution grape ball
Mycoprotein A, collects eluting peak and obtains the first collection liquid；The equilibrium liquid Bis-tris containing 10-30mM, 0.5-2wt%TritonX-
100th, 0.1mol/L NaCl, pH6.5-7.2；The flushing liquor Bis-tris containing 10-30mM, 0.1mol/LNaCl, pH 6.5-
7.2；The eluent Bis-tris containing 10-30mM, 1mol/L NaCl, pH 6.5-7.2；
3) ultrafiltration displacement buffer solution：By step 2) obtained the first collection liquid be 5KD with the molecular weight that dams milipore filter bag
Ultrafiltration is replaced into pH 6.8-7.5, the Na containing 5-20mM2HPO4-NaH2PO4And the buffer solution of 0.1-0.3M proline, thus
To protein liquid；
4) the second chromatographic column is handled：By step 3) obtained protein solution loading is equipped with hydroxyl to what is balanced with equilibrium liquid
In the pillar of apatite chromatographic stuffing, continuation is balanced with equilibrium liquid, then with elution, is collected eluting peak and is obtained the second collection
Liquid；Na of the equilibrium liquid containing 5-20mM2HPO4-NaH2PO4And 0.1-0.3M proline, pH 6.8-7.5；The eluent contains
100mM Na2HPO4-NaH2PO4, pH 6.8-7.5；
5) ultrafiltration is replaced into preservation liquid：By step 4) obtained the second collection liquid be 5KD with the molecular weight that dams milipore filter
Bag ultrafiltration is replaced into normal saline solution, the staphylococcal protein A thus isolated and purified.
Described step 1) thalline pretreatment be preferably：The thalline containing staphylococcal protein A is taken, according to quality:Body
Product=1kg:10L ratio adds the bacteria into pH=7.2, and concentration is 10-30mM Bis-tris buffer solutions, with high-pressure homogeneous
Crushed under machine 800bar pressure and be then centrifuged for collecting supernatant；With salt acid for adjusting pH to 2.0-4.0, supernatant is collected by centrifugation, so
PH is adjusted to 6.5-7.2 with NaOH afterwards, obtains protein liquid.Step 1) heated with broken substitution under high pressure homogenizer 800bar pressure
And the method for lysozyme crushes thalline, energy consumption is low, it is not necessary to complicated heating and heat-insulating device, can realize continuous operation and work
Industry is amplified.
Step 2) in salt-resistant type anion exchange filler be TOYOPEARL NH2- 750F salt-resistant type anion exchanges are filled out
Material, its particle diameter is 30-60 μm, and endotoxin is in pH>The negative electrical charge of part is carried in 3.1 solution, is incorporated into by charge effect
TOYOPEARL NH2In -750F anion exchange fillers, after the albumen loading of contaminated with endotoxins, with equilibrium liquid and flushing liquor
Remove part endotoxin, then using the method for gradient elution, first elute target protein, then with high-salt buffer and
The endotoxin removal that NaOH will be left on pillar.
Step 2), it is preferable that equilibrium liquid Bis-tris containing 20mM and 1%TritonX-100,0.1mol/LNaCl, pH
7.0；Flushing liquor Bis-tris containing 20mM, 0.1mol/LNaCl, pH 7.0；Eluent Bis-tris containing 20mM and 1mol/L
NaCl, pH7.0.
Step 4) hydroxyapatite chromatography packing material size be 40 μm, preferably CHT type I.Hydroxyapatite is current application
Widest inorganic chromatography filler, highly alkaline-resisting, biological safety is good, due to its unique separation mechanism, can carry out high-resolution
The separation of rate.
Step 4), it is preferable that equilibrium liquid Na containing 5mM2HPO4-NaH2PO4And 0.2M proline, pH 7.0；Eluent is
100mM Na2HPO4-NaH2PO4, pH 7.0.
Step 3), step 5) in the material of milipore filter bag be preferably polyether sulfone (PES).
Beneficial effects of the present invention are as follows：
1. the present invention is extremely stable in acid condition using albumin A, minimum pH 2.0 extreme condition is resistant to, is walked
It is rapid 1) in the pH of broken supernatant is reduced to after 2.0-4.0, can be by the miscellaneous egg of the Escherichia coli residual DNA wherein contained and part
In vain, cell fragment be co-precipitated, greatly reduce the turbidity of solution so that and anion-exchange column combination it is even closer, albumen
Yield is improved；The residual quantity of Escherichia coli residual DNA has been greatly reduced simultaneously, improves purity of protein；It it also avoid with deep
Deimpurity method is gone in layer filter filtering, greatly reduces production cost.
2. the first step that the present invention is purified uses TOYOPEARL NH2- 750F salt-resistant type anion exchange fillers, this it is cloudy from
Son exchanges filler and has had hydrophobic effect and ion exchange, height salt tolerant, so step 1 concurrently) obtained protein liquid is without dilute
Release can direct loading, simplify purifying flow, albumen yield increases；While 30-60 μm of the particle diameter of filler, than traditional
90 μm of sepharose average grain diameters, particle diameter is smaller, and separating effect is better, can remove more foreign proteins, mitigates next step
Purify pressure.In addition, ion-exchange packing can effectively remove the endotoxin in the broken supernatant of Escherichia coli, endotoxin content
Below 100EU/mg is down to by 5000~10000EU/mg.
3. agarose is mainly connected to by protein amino in downstream synthetic adsorbent filler, on cellulose carrier.
Conventional ion exchange column uses Tris-HCl buffer solutions, a small amount of residual is had in whole protein solution, because of Tris (structural formulas
For：) molecular formula contains amino, so micro tris (mM) can also interfere with the conjunction of downstream adsorbent agent
Into causing absorption property to reduce.In the present invention the first chromatographic column use Bis-tris (structural formula for) buffer solution replaces conventional Tris-HCl buffer solutions to significantly improve albumin A and chromatography
The adhesion of post, increases the rate of recovery of albumen.Meanwhile, because Bis-tris molecular structures do not contain free amino, Bu Huiyu
Amino competition binding site on protein, so being connected to carrier synthetic proteins A adsorption stuffings on the albumen in downstream without influence.
4. the present invention is using hydroxyapatite as final step polishing purification, 40 μm of its average grain diameter can be further
The impurity such as dimer, endotoxin, micro Escherichia coli residual DNA, HCP are removed, product purity is improved.
5. because the reasons such as concentration, pH, temperature can cause the aggregation of protein in protein purification procedures, with class dimer or
The form of polymer is present, and is to influence the main cause of purity of protein.L-PROLINE is 18 kinds of ammonia of human body synthetic protein
One of base acid, can suppress the aggregation of protein, strengthen the stability of protein.Albumen can be promoted by adding a small amount of proline
Matter is in native conformation, albumin A is preferably separated with class dimer, by being finely separated off more protein impurities.
In summary, the present invention is simple to operate, using the filler of 2 kinds of mixed mechanisms, and the mode of two steps chromatography and ultrafiltration has
Eliminating endotoxin, coli somatic residual protein (HCP), Escherichia coli residual DNA, protein aggregate etc. one to effect is
Row impurity, using minimum step, obtains most pure target sample, saves technology controlling and process cost, at the same have stability it is good,
Process control index clearly, be adapted to large-scale industrial production the features such as.Protein solution after purified, with efficient liquid phase and
SDS-PAGE electrophoresis measures purity of protein more than 97%；Protein recovery is more than 75%；Endotoxin is reduced to below 1EU/mg；
HCP and Escherichia coli residual DNA are below 5ppm, can be used to synthesize the immune suction of clinic staphylococcal protein A as raw material
Attached dose, the large-scale production of annual production kilogram level can be achieved, while also the industrialized production for other protein purifications provides reference.
Brief description of the drawings：
Fig. 1 is the PAGE gel electrophoresis result for Recombinant Staphylococal Protein A；Wherein swimming lane 1 is low molecule amount egg
White marker, molecular size range is respectively 98kDa, 66.2kDa, 45kDa, 31kDa, 20kDa, 14.4kDa, restructuring from top to bottom
The molecular size range of staphylococcal protein A is about 20kDa；Swimming lane 2 is the whole protein solution that embodiment 1 is obtained；Swimming lane 3 is implementation
The whole protein solution that example 2 is obtained.
Fig. 2 is the HPLC collection of illustrative plates of Recombinant Staphylococal Protein A in embodiment 1.
Fig. 3 is the HPLC collection of illustrative plates of Recombinant Staphylococal Protein A in embodiment 2.
Embodiment：
Further illustrated the following is to the present invention, rather than limitation of the present invention.
Embodiment 1：
1) thalline is pre-processed：Take thalline (the gene work as expressed staphylococcal protein A that 1kg contains staphylococcal protein A
Journey bacterium Escherichia coli), 10L, pH=7.2 are added, concentration is 10mM Bis-tris buffer solution suspension thallines, then equal with high pressure
Matter machine is broken 2 times in pressure 800bar, and supernatant about 10L is collected by centrifugation.Resulting solution is added into salt acid for adjusting pH extremely
2.0, centrifugation, supernatant is adjusted to pH=6.5 with sodium hydroxide, obtains protein liquid.
2) the first chromatographic column is handled：It will be equipped with TOYOPEARL NH2The chromatographic column of -750F fillers Bis- containing 10mM
Tris and 0.5wt%TritonX-100,0.1mol/LNaCl, pH 6.5 equilibrium liquid balances 5 times of column volumes, and step 1 is obtained
To protein liquid loading to balanced with equilibrium liquid be equipped with TOYOPEARL NH2- 750F salt-resistant type anion exchange fillers
In chromatographic column, continuation balances 5 times of column volumes with equilibrium liquid, then with Bis-tris containing 10mM, 0.1mol/LNaCl, pH 6.5
Flushing liquor rinse 5 times of column volumes, finally with Bis-tris containing 10mM and 1mol/L NaCl, pH 6.5 elution mesh
Albumen staphylococcal protein A is marked, eluting peak is collected and obtains the first collection liquid.
3) ultrafiltration displacement buffer solution：By step 2) obtained the first collection liquid be 5KD with the molecular weight that dams PES milipore filters
Bag, isometric 5 times of volumes of ultrafiltration are replaced into pH 6.8, the Na containing 5mM2HPO4-NaH2PO4And the buffer solution of 0.1M proline,
Obtain protein liquid.
4) the second chromatographic column is handled：It will be equipped with Na of the pillar containing 5mM of hydroxyapatite chromatography filler2HPO4-
NaH2PO4And 0.1M proline, pH6.8 equilibrium liquid balances 5 times of column volumes, by step 3) obtained protein liquid loading has been to having used
In the pillar equipped with hydroxyapatite chromatography filler of equilibrium liquid balance, continuation balances 5 times of column volumes, Ran Houyong with equilibrium liquid
100mM Na2HPO4-NaH2PO4, the elutions of pH 6.8, collect eluting peak obtain the second collection liquid.
5) ultrafiltration is replaced into preservation liquid：By step 4) obtained the second collection liquid be 5KD with the molecular weight that dams milipore filter
Isometric 5 times of volumes of ultrafiltration are wrapped, normal saline solution is replaced into, thus obtains protein solution, the grape ball as isolated and purified
Mycoprotein A.
The measure of purity of protein and impurity in the protein solution of step 5：Purity of protein uses electrophoresis and high-efficient liquid phase color
Spectrometry is determined, as a result respectively referring to Fig. 1 and Fig. 2, and purity of protein is measured with RP chromatography for 97.1% (Fig. 2).Endotoxin contains
Amount is measured with gel method TAL.Coli somatic residual protein amount uses escherichia coli host residual protein
(E.coli P) ELISA kit is determined.Escherichia coli residual DNA residual quantity uses Quant-iTTM dsDNA
Kit is measured.It the results are shown in Table 1.
Each impurity determination result in the protein solution of the step 5 of table 1
| Measure project | As a result |
| Purity of protein | 97.1% |
| Protein recovery | 78% |
| Endotoxin | ＜ 1EU/mg |
| HCP | ＜ 2ppm |
| Escherichia coli residual DNA | ＜ 5ppm |
Embodiment 2
1) thalline is pre-processed：Take thalline (the gene work as expressed staphylococcal protein A that 1kg contains staphylococcal protein A
Journey bacterium Escherichia coli), 10L, pH=7.2 are added, concentration is 30mM Bis-tris buffer solution suspension thallines, then equal with high pressure
Matter machine is broken 2 times in pressure 800bar, and supernatant about 10L is collected by centrifugation.Resulting solution is added into salt acid for adjusting pH extremely
4.0, centrifugation, supernatant is adjusted to pH=7.2 with sodium hydroxide, obtains protein liquid.
2) the first chromatographic column is handled：It will be equipped with TOYOPEARL NH2The chromatographic column of -750F fillers (contains 20mM with equilibrium liquid
Bis-tris and 1wt%TritonX-100,0.1mol/LNaCl, pH 7.0) 5 times of column volumes of balance, the egg that step 1 is obtained
White liquor loading is equipped with TOYOPEARL NH to what is balanced with equilibrium liquid2The chromatographic column of -750F salt-resistant type anion exchange fillers
In, continuation balances 5 times of column volumes with equilibrium liquid, then with flushing liquor (Bis-tris containing 20mM, 0.1mol/LNaCl, pH
7.0) 5 times of column volumes are rinsed, finally target egg are eluted with eluent (Bis-tris containing 20mM and 1mol/L NaCl, pH 7.0)
White staphylococcus albumin A, collects eluting peak and obtains the first collection liquid.
3) ultrafiltration displacement buffer solution：By step 2) obtained the first collection liquid be 5KD with the molecular weight that dams PES milipore filters
Bag, isometric 5 times of volumes of ultrafiltration are replaced into pH 7.0, the Na containing 5mM2HPO4-NaH2PO4And the buffer solution of 0.2M proline,
Obtain protein liquid.
4) the second chromatographic column is handled：It will be equipped with the pillar equilibrium liquid (pH 7.0, containing 5mM of hydroxyapatite chromatography filler
Na2HPO4-NaH2PO4And 0.2M proline) 5 times of column volume of balance, by step 3) obtained protein liquid loading is to flat
Weigh in the pillar equipped with hydroxyapatite chromatography filler of liquid balance, continuation balances 5 times of column volumes with equilibrium liquid, then with elution
Liquid (100mM Na2HPO4-NaH2PO4, pH 7.0) and elution, collect the collection liquid of eluting peak second.
5) ultrafiltration is replaced into preservation liquid：By step 4) obtained the second collection liquid be 5KD with the molecular weight that dams milipore filter
Isometric 5 times of volumes of ultrafiltration are wrapped, normal saline solution is replaced into, thus obtains protein solution (Protein A solution), as separate pure
The staphylococcal protein A of change.
The detection method be the same as Example 1 of purity of protein and each impurity in the protein solution of step 5.As a result respectively referring to Fig. 1
And Fig. 2, purity of protein is measured with RP chromatography for 97.6% (Fig. 3).It the results are shown in Table 2.
Each impurity determination result in the protein solution of table 2
| Measure project | As a result |
| Purity of protein | 97.6% |
| Protein recovery | 76% |
| Endotoxin | ＜ 1EU/mg |
| HCP | ＜ 1ppm |
| Escherichia coli residual DNA | ＜ 1ppm |
Embodiment 3
Basic be the same as Example 1, difference is, step 1) thalline pretreatment：1kg is taken to contain staphylococcal protein A
Thalline, adds 10L, pH=7.2, and concentration is 20mM Bis-tris buffer solution suspension thallines, then with high pressure homogenizer in pressure
Power 800bar is broken 2 times, and supernatant about 10L is collected by centrifugation.Resulting solution is added into salt acid for adjusting pH to 3.0, centrifuged, on
Clear liquid is adjusted to pH=7.2 with sodium hydroxide, thus obtains protein solution.
Embodiment 4
Basic be the same as Example 1, difference is, step 2) in the first chromatographic column handle：Equilibrium liquid Bis- containing 30mM
Tris and 2wt%TritonX-100,0.1mol/LNaCl, pH7.2；Flushing liquor Bis-tris containing 30mM, 0.1mol/
LNaCl, pH7.2 eluent Bis-tris containing 30mM and 1mol/L NaCl, pH 7.2.
Embodiment 5
Basic be the same as Example 2, difference is, step 3) in ultrafiltration displacement buffer solution be pH 7.2, containing 10mM's
Na2HPO4-NaH2PO4And the buffer solution of 0.2M proline.Step 4) the second chromatographic column processing in equilibrium liquid containing 10mM's
Na2HPO4-NaH2PO4And 0.2M proline, pH is 7.2；Eluent Na containing 100mM2HPO4-NaH2PO4, pH 7.2.
Embodiment 6
Basic be the same as Example 2, difference is, step 3) in ultrafiltration displacement buffer solution be the Na containing 20mM2HPO4-
NaH2PO4And 0.3M proline, pH7.5 buffer solution.Step 4) the second chromatographic column processing in Na of the equilibrium liquid containing 20mM2HPO4-
NaH2PO4And 0.3M proline, pH7.5；Eluent Na containing 100mM2HPO4-NaH2PO4, pH 7.5.
Embodiment 7：Protein A activity is identified
The Recombinant Staphylococal Protein A obtained after purification is connected on agarose carrier, plasma adsorption experiment is carried out, surveyed
Surely the adsorbent synthesized evaluates the activity of albumen with this to the adsorption effect of IgG types antibody in human plasma.
Using sodium periodate oxidation, the Protein A solution that embodiment 1,2 is obtained is coupled to Sepharose 6FF respectively
On.Specific method is as described in patent 201010512304.8.The step of by simulating clinically immuno absorbence, in vitro to blood plasma
Interior immunoglobulin absorption property test, evaluates its absorption property, and then will be seen that its Clinical practice value, and specific steps are such as
Shown in patent 201010512304.8, it is the immunosorbent of aglucon to people to measure Recombinant Staphylococal Protein A in embodiment 1,2
IgG adsorbance is respectively 34mg/g, 37mg/g filler in blood plasma, and contrast anion-exchange chromatography with the conditions of uses tris-
The albumen of HCl systems purifying, the absorption property of its synthetic filling is respectively 30mg/g, 33mg/g filler, and average adsorption performance is carried
It is high by 13% and 12%.Absorption property is higher, under identical treatment time or cycle-index, and the rate of descent of pathogenic antibody is higher,
It is more helpful to lapsing to for disease, so the absorption property for improving 10%-15% is also significantly.
To the protein solution in embodiment 3,4,5,6 according to above-mentioned detection method respectively to purity of protein, HCP residual quantities,
Escherichia coli residual DNA residual quantity, absorption property etc. are detected that testing result meets the requirements, i.e., purity of protein is more than
97%；Protein recovery is more than 75%；Absorption property is more than 33mg/g；Endotoxin is reduced to below 1EU/mg；HCP and DNA are equal
Less than 5ppm.
Claims (7)
1. a kind of method for isolating and purifying Recombinant Staphylococal Protein A, it is characterised in that this method comprises the following steps：
1) thalline is pre-processed：Thalline containing staphylococcal protein A is added to Bis-tris buffer solutions, it is then broken with homogenizer
Supernatant is collected by centrifugation after broken, pH is to 2.0-4.0 for regulation, and supernatant is collected by centrifugation, then adjusts pH to 6.5-7.2, obtain egg
White liquor；
2) the first chromatographic column is handled：By step 1) obtained protein liquid loading to balanced with equilibrium liquid equipped with salt-resistant type it is cloudy from
Son is exchanged in the chromatographic column of filler, and continuation is balanced with equilibrium liquid, is rinsed with flushing liquor, finally with elution staphylococcus egg
White A, collects eluting peak and obtains the first collection liquid；The equilibrium liquid Bis-tris containing 10-30mM, 0.5-2wt%TritonX-100,
0.1mol/L NaCl, pH6.5-7.2；The flushing liquor Bis-tris containing 10-30mM, 0.1mol/LNaCl, pH 6.5-7.2；
The eluent Bis-tris containing 10-30mM, 1mol/L NaCl, pH 6.5-7.2；
3) ultrafiltration displacement buffer solution：By step 2) obtained the first collection liquid be 5KD with the molecular weight that dams milipore filter bag ultrafiltration
PH 6.8-7.5 are replaced into, the Na containing 5-20mM2HPO4-NaH2PO4And the buffer solution of 0.1-0.3M proline, thus obtain egg
White liquor；
4) the second chromatographic column is handled：By step 3) obtained protein solution loading is equipped with hydroxy-apatite to what is balanced with equilibrium liquid
In the pillar of stone chromatographic stuffing, continuation is balanced with equilibrium liquid, then with elution, is collected eluting peak and is obtained the second collection liquid；
Na of the equilibrium liquid containing 5-20mM2HPO4-NaH2PO4And 0.1-0.3M proline, pH 6.8-7.5；The eluent contains
100mM Na2HPO4-NaH2PO4, pH 6.8-7.5；
5) ultrafiltration is replaced into preservation liquid：By step 4) obtained the second collection liquid be 5KD with the molecular weight that dams milipore filter Bao Chao
Filter is replaced into normal saline solution, the staphylococcal protein A thus isolated and purified.
2. the method according to claim 1 for isolating and purifying Recombinant Staphylococal Protein A, it is characterised in that step 2) in
Salt-resistant type anion exchange filler be TOYOPEARL NH2- 750F salt-resistant type anion exchange fillers, its particle diameter is 30-60 μ
m。
3. the method according to claim 1 or 2 for isolating and purifying Recombinant Staphylococal Protein A, it is characterised in that described
Thalline is pre-processed：The thalline containing staphylococcal protein A is taken, according to quality:Volume=1kg:10L ratio adds thalline
To pH=7.2, concentration is 10-30mM Bis-tris buffer solutions, is then centrifuged for being crushed under high pressure homogenizer 800bar pressure
Collect supernatant；With salt acid for adjusting pH to 2.0-4.0, supernatant is collected by centrifugation, then adjusts pH to 6.5-7.2 with NaOH, obtains
To protein liquid.
4. the method according to claim 1 or 2 for isolating and purifying Recombinant Staphylococal Protein A, it is characterised in that step 2)
Middle equilibrium liquid Bis-tris containing 20mM and 1%TritonX-100,0.1mol/LNaCl, pH 7.0；Flushing liquor Bis- containing 20mM
Tris, 0.1mol/LNaCl, pH 7.0；Eluent Bis-tris containing 20mM and 1mol/L NaCl, pH7.0.
5. the method according to claim 1 or 2 for isolating and purifying Recombinant Staphylococal Protein A, it is characterised in that step 4)
Hydroxyapatite chromatography packing material size be 40 μm.
6. the method according to claim 1 or 2 for isolating and purifying Recombinant Staphylococal Protein A, it is characterised in that step 4)
Equilibrium liquid Na containing 5mM2HPO4-NaH2PO4And 0.2M proline, pH 7.0；Eluent is 100mM Na2HPO4-NaH2PO4,
pH 7.0。
7. the method according to claim 1 or 2 for isolating and purifying Recombinant Staphylococal Protein A, it is characterised in that step
3), step 5) in milipore filter bag material be polyether sulfone.
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