CN103450328A - Method for purifying antibody by using high-density protein A coated magnetic beads - Google Patents
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Abstract
The invention discloses a method for purifying an antibody by using high-density protein A coated magnetic beads. The method comprises the following steps: adding magnetic beads and an expression product containing the antibody into a reaction vessel, applying a magnetic field to the outside or inside of the reaction vessel, and then, transferring out a supernatant; adding a washing buffer solution into the reaction vessel, and removing the magnetic field; repeatedly washing the magnetic beads for 2-4 times; adding an eluting buffer solution, removing the magnetic field, and incubating for 5-15 minutes at room temperature; enabling the magnetic beads to be attached to the wall through applying the magnetic field, transferring out the solution, and adjusting the pH value of the solution to be neutral, thereby obtaining a purified antibody. According to the method, antibody purification is carried out by using a magnetic bead affinity separation technology, so that the antibody capture speed is higher compared with that of the traditional antibody affinity chromatography; in addition, the purifying operation has no need of complicated chromatography systems, and samples have no need of clarifying treatment, so that the samples with relatively high viscosity can be directly subjected to purifying operation; moreover, the unique protein A coated magnetic beads are used, so that the antibody binding efficiency is increased greatly, and the shedding rate of a protein A ligand reaches a very low level.
Description
Technical field
The present invention relates to a kind of method of antibody purification, be specifically related to a kind of method of utilizing the coated magnetic beads for purifying antibody of high-density of albumin A.
Background technology
Monoclonal antibody is that the hybridoma that bone-marrow-derived lymphocyte and myeloma cell are hybridized formation produces, and the formed structural domain of its heavy chain and light chain can be identified and in conjunction with specific antigen.FDA in 1997 ratify the chimeric mAb medicine Rituxan Anti-CD20 antibody listing of first treatment lymphatic cancer, to 2007, the FDA approved more than 20 kind of therapeutic monoclonal antibodies medicine, approximately half is used for the treatment of cancer, has a plurality ofly become " the bomb medicine " that annual sales amount surpasses 1,000,000,000 dollars.The market growth of whole world monoclonal antibody drug is very swift and violent, more than 200 kind of monoclonal antibody is approximately arranged in the clinical experiment stage, accounts for more than 1/3rd of whole clinical biotechnology medicine sum, and wherein 1/4th in clinical three phases or wait for a green light.Monoclonal antibody medicine has become the theme of global field of biological pharmacy development.
The fast development antagonist purifying industry of monoclonal antibody medicine has proposed new challenge.At present, the antibody purification production of main flow mainly comprises the following steps: 1, fermented liquid clarification; 2, antibody capture; 3, antibody polishing purification; 4, the concentrated filter wash of antibody; 5, antibody filtration sterilization/preparation.In the antibody capture stage, mainly apply the protein A affinity chromatography technology and extract antibody from fermented supernatant fluid.Albumin A is a kind of albumen extracted from the staphylococcus aureus cytolemma, has 5 IgG binding domainss, can with the antibody Fc end, be combined specifically.Present stage, the most frequently used protein A affinity chromatography medium was to make affine filler by albumin A is fixed on to the sepharose surface by the method for chemical coupling, then was packed into chromatography column.The fermented supernatant fluid that will contain antibody pumps up and injects chromatography column, and antibody is caught by albumin A fixing on the chromatography filler, and captive antibody can dissociate from the affine filler of albumin A by the flushing of elute soln, reaches purifying and concentrated purpose.
Yet there is following shortcoming in above antibody purification process:
1, fermented soln need be clarified operation.Monoclonal antibody adopts the method for mammalian cell serum-free suspension culture to be prepared usually, has a large amount of cells in fermented liquid, easily chromatographic system is resulted in blockage.Therefore needed through centrifugal or filter operation before the chromatography operation, remove the cell in fermented liquid.Industrial large-scale clarification operation needs streaming whizzer or ultrafiltration system usually, and length consuming time, price is high and routine maintenance is complicated.
2, chromatography operates length consuming time, the instrument costliness.Because pressure and flow velocity that filler can bear are limited, so interior accessible sample size of unit time is limited to size and the filling property of chromatography column.Large-scale tomography devices and albumin A affinity purification filler are very expensive, and basic dependence on import, are the bottlenecks of antibody purification industry.
3, chromatographic system can not be processed high viscosity samples.Due to the pressure that is limited to filler and can bears, therefore full-bodied sample (as serum or high protein concentration sample) all needs through dilution and filters just to be applied to the chromatography operation, causes the amplification of sample volume and the dilution of target product.
4, coming off of albumin A part easily occurs in the protein A affinity chromatography filler in the process of repeatedly rinsing.Albumin A is to realize fixing by covalent coupling on agarose matrix.More advanced filler for realize the albumin A aglucon towards consistence reach the purpose that improves the aglucon utilization ratio, all adopt the directed fixation method of single-point, be albumin A with agarose between only by a covalent linkage, be connected, but the fixing method of this single-point easily comes off albumin A in chromatography column flushing process repeatedly.And albumin A is high sensitinogen, easily in human body, cause immune response, there are strict restriction in U.S. food and Drug Administration (FDA) to the residual quantity of albumin A aglucon in monoclonal antibody medicine.The albumin A obscission has also affected effective carrying capacity and the work-ing life of affine filler.
In recent years, along with the development of magnetic separation technique, the coated superparamagnetism magnetic bead of albumin A is applied to immunodetection and small-scale immunoaffinity purification field more and more.The coated magnetic beads for purifying technology of albumin A is without complicated tomography devices, not restriction of clarity to sample, only need simple magnetic absorption step easily to separate monoclonal antibody quickly from the monoclonal antibody expression product, efficiently solve the weak point of traditional chromatographic technique.But, because the antibodies efficiency of the coated magnetic bead of the albumin A occurred on the market at present still has very large gap with respect to traditional agarose filler, therefore still fail to be applied on a large scale antibody purification industry.
Summary of the invention
In order to overcome the shortcoming of above-mentioned existing antibody purification process, the object of the present invention is to provide a kind of method of utilizing the coated magnetic beads for purifying antibody of high-density of albumin A.
Purpose of the present invention is achieved through the following technical solutions:
A kind of method of utilizing the coated magnetic beads for purifying antibody of high-density of albumin A comprises the following steps:
(1) the coated magnetic bead of the high-density of albumin A, the expression product that contains antibody are joined in reaction vessel, stir evenly, under room temperature, hatch 5～15min, the magnetic bead state that is evenly distributed in solution; Magnetic bead and antibodies in this process;
(2) apply magnetic field in reaction vessel outside or inside, make magnetic bead move and be attached on wall of container (see figure 2) on (see figure 1) or magnet along field direction;
(3) migrate out supernatant liquor from reaction vessel; In this process, antibody is attached on wall of container with magnetic bead or, on magnet, all the other compositions are transferred out of reaction vessel;
(4) lavation buffer solution is added in reaction vessel, remove magnetic field, make magnetic bead again be dispersed in lavation buffer solution; Repeating step (2)～(3), complete a magnetic bead washing;
(5) repeating step (4) washing magnetic bead is 2～4 times, thoroughly washes away the non-specific adsorbent on magnetic bead;
(6) elution buffer is added in reaction vessel, remove magnetic field, magnetic bead is dispersed in lavation buffer solution again, under room temperature, hatch 5～15min; This operation is that the antibody elution on magnetic bead is got off;
(7) apply magnetic field and make magnetic bead adherent, the solution in the shift reaction container, the pH value of regulator solution is to neutral, obtains the antibody after purifying.
The coated magnetic bead of the high-density of the albumin A in step (1) is applied for a patent, and is Chinese patent application 201210107107.7(application number) the crosslinked magnetic bead that immobilization SL-PA fusion rotein is arranged that obtains of embodiment 1.
The described expression product that contains antibody of step (1) can be serum, cell expressing liquid or ascites.
The described magnetic field that applies in step (2) and (7), its magnetic flux is 1000～10000 Gausses, be 1-5min action time.
The phosphoric acid buffer that the described lavation buffer solution of step (4) contains 0.01～0.5M, the NaCl of 1～500mM, surplus is water, pH value 6～9.
The amino acid that the described elution buffer of step (6) contains 0.01～4M, pH value 1.9～5; Described amino acid is more than one in glycine, Methionin or arginine.
The described adjusting of step (7) pH value is to regulate with Tutofusin tris (Tris) solution of 1M.
Magnetic bead repeating step (4) after use, wash after the pH value is neutrality and can be recycled and reused for antibody purification.
Mechanism of the present invention is:
The magnetic variation with temperature of magneticsubstance and changing, when low temperature, the magnetic of material is difficult to be changed; And, when temperature raises, material will become " paramagnetic material ", its magnetic is easy to change with magnetic field on every side.If temperature further improves, or the granularity of magnetic-particle is when very little, even if at normal temperatures, the polarity of magnet also presents randomness, is difficult to the magnetic property that keeps stable, and this phenomenon is exactly the superparamagnetic effect.The superparamagnetism magnetic bead is externally assembled rapidly under the effect in magnetic field, after magnetic field is withdrawn, can again disperse and without remanent magnetism.The present invention utilize this characteristic of magnetic bead make its as a kind of novel separation and purification matrix the separating and purifying technology for biologically active substance.
The present invention is based on the method for utilizing SLP that functional protein is directed fixing, obtain the superparamagnetism magnetic bead (seeing patent 201210107107.7 embodiment 1) with immobilization albumin A aglucon.Utilize the efficient specific binding of this magnetic bead antagonist, capture antibody from serum or antibody expression product, then utilize magnetic separator that magnetic bead-antibody complex is separated from mixed solution, through the repeatedly washing to the magnetic bead mixture, remove the non-specific adsorbent of magnetic bead surfaces, finally with elution buffer, antibody and magnetic bead dissociated and be discharged in elution buffer, reaching the purpose of antibody purification.
The present invention has following advantage and effect with respect to prior art:
1, the present invention uses the affine isolation technique of magnetic bead to carry out antibody purification, than traditional antibody affinity chromatography technology, has antibody capture speed (<15 minutes) faster; In addition, purification process is without complicated chromatographic system, and sample is without carrying out clarifying treatment, can directly to viscosity, larger sample carries out purification process.
2, with the affine isolation technique of existing magnetic bead, compare, the present invention is due to the coated magnetic bead of the albumin A that has used unique SLP immobilization technology to prepare, antibodies efficiency significantly improves, it is extremely low-level that the expulsion rate of albumin A aglucon reaches, and makes the application of magnetic bead isolation technique in monoclonal antibody purifying in enormous quantities become possibility.
3, purification process of the present invention has been saved the fermented liquid clarification steps.Mode capture antibody from sample that the present invention adopts magnetic bead and sample directly to mix to hatch, clarity and viscosity to sample do not have particular requirement, the step of sample clarification filtration in traditional antibody chromatography purifying process be can save, corresponding production time and device requirement saved.
4, purification process of the present invention has solved traditional antibody purification chromatography technique length consuming time, the problem of instrument costliness.Purification process of the present invention is without expensive import tomography devices, only need simple magnetic field (by permanent magnet or electromagnet provide all can) can carry out separation and the collection of magnetic bead.Because magnetic bead has nano level diameter, therefore can provide and catch greatly area, can reach the saturated adsorption with antibody in 15 minutes.
5, purification process of the present invention has solved the low problem of the affine magnetic bead carrying capacity of existing antibody.The present invention adopts that to take the coated magnetic bead of the albumin A with nanometer grade diameter prepared by SLP functional protein immobilization technology be raw material, there is the antibodies efficiency that is several times as much as general albumin A magnetic bead product, every milligram of magnetic bead antibodies amount, up to 250 μ g, makes the extensive use of magnetic beads for purifying technology more economical more efficient.
The accompanying drawing explanation
Fig. 1 utilizes the magnetic field of reaction vessel outside to carry out the schematic diagram of magnetic bead solid-liquid separation.
Fig. 2 utilizes the magnetic field of reaction vessel interior to carry out the schematic diagram of magnetic bead solid-liquid separation.
Fig. 3 is the SDS-PAGE detected result figure of each step products in the purification process of embodiment 1; Wherein, the standard control of human IgG-humanized IgG, the human serum before the S-purifying, the human serum after the F-purifying, the reacted twice washing supernatant of W1, W2-magnetic bead and human serum, E1-eluted product.
Fig. 4 is the western blot the result figure of embodiment 1 magnetic beads for purifying product, and left figure is the SDS-PAGE electrophorogram, and right figure is corresponding Western blot fluorescence developing figure; Wherein, M-protein standard molecular weight, 1-human serum sample, the eluted product of 2～5-magnetic bead purifying from human serum, 6-humanized IgG standard substance.
Fig. 5 is the human IgG1's that in embodiment 2, two kinds of purification process obtain SDS-PAGE detected result figure; Wherein, M-protein standard molecular weight, 1～2-SL-PA magnetic beads for purifying product, 3-affinitive layer purification product.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited to this.
Utilize the SL-PA magnetic bead to extract humanized IgG from human serum, comprise the following steps:
SL-PA magnetic bead preparation method is shown in Chinese patent application " a kind of method that SLP of utilization is directed fixing by functional protein ", and (number of patent application: 201210107107.7) embodiment 1.
(1) get SL-PA magnetic bead 100mg, add in the 5ml centrifuge tube, then (pH value 7.5, contain 50mM phosphoric acid buffer and 150mM NaCl to add the 5ml lavation buffer solution; Lower same), vibrate resuspended, be positioned over 2min on magnetic separator, suck supernatant liquor after solid-liquid separation.Repeat this step once;
(2) magnetic bead and 3ml human serum after washing (purchased from the special bio tech ltd of Guangzhou stamen) (being the front human serum S of purifying) are mixed, incubated at room 10min, during frequently shake to keep magnetic bead to mix with the even of serum;
(3) centrifuge tube is positioned over to 2min on magnetic separator, sucks supernatant liquor (F) after solid-liquid separation;
(4) add the 5ml lavation buffer solution, vibrate resuspended, be positioned over 2min on magnetic separator, suck supernatant liquor (W1) after solid-liquid separation.Repeat this step 2 time (washing for the second time supernatant liquor is W2);
(5) add 3ml elution buffer (containing the 0.1M glycine, pH value 2.5), mix, incubated at room 10min, during frequently shake to keep magnetic bead to mix with the even of solution;
(6) centrifuge tube is positioned over to 2min on magnetic separator, collects supernatant liquor after solid-liquid separation, this supernatant liquor is the purified product that contains human IgG.In supernatant liquor, add 100 μ l1M Tris solution (pH8.0) regulator solution pH values to neutral at once, be eluted product E1;
(7) purified product of step (6) is dialysed or ultrafiltration with ultrapure water, collected dialysis or ultrafiltration product, with vacuum freeze drier, made lyophilized powder.Purified product can be used for further polishing purification operation.
The purification effect of humanized IgG:
(1) the polyacrylamide gel electrophoresis detected result of each step products of magnetic beads for purifying is shown in as Fig. 3.
As can be seen from Figure 3, the purified product electrophoretic band (E1) of SL-PA magnetic bead from human serum is consistent with the electrophoretic band of humanized IgG standard substance, and purity is consistent with standard substance.By measuring human IgG total amount in 280nm place absorbance value calculating eluted product, be 23.14mg, every milligram of magnetic bead is 231.4 μ g in conjunction with the amount of human IgG.The whole purifying flow process 40min that is less than consuming time, sample serum is without being diluted and being filtered, and purification process is without chromatographic system.
And adopt traditional antibody affinity chromatography method to process the serum sample of equal volume, need to be by 5～10 times of serum stoste dilutions, through 0.22 μ m aperture membrane filtration, by chromatographic system, sample is pressurizeed, make sample flow into albumin A affinity purification chromatography column, human IgG in sample is combined with the albumin A aglucon, and then remove unconjugated foreign protein through rinsing chromatography column, finally with elution buffer, in connection with the antibody elution on chromatography column, obtain the antibody after purifying, whole process is consuming time surpasses 3 hours, needs expensive chromatographic system and filtration unit.
(2) checking of purified product: the mouse anti human IgG mono-clonal monomer (purchased from American AB cam company) of take is primary antibodie, take goat anti-mouse igg-HRP(purchased from Wuhan doctor's moral company) as two anti-, carry out protein immunoblotting detection (Western blot), whether checking SL-PA magnetic beads for purifying product is human IgG, and result is as Fig. 4.
As can be seen from Figure 4, the eluted product of magnetic bead purifying from human serum has identical immunoblotting colour developing result with the humanized IgG standard substance, proves that this purified product is humanized IgG.
(3) the albumin A determination of residual amount in purified product
Get the human IgG product of SL-PA magnetic bead purifying from human serum, and with albumin A affinity purification chromatography column (HiTrap MabSelect1ml; U.S. GE lifesciences company) from human serum, the human IgG product of purifying is control group, by albumin A ELISA detection kit (Protein A ELISA Kit, U.S. RepliGen company), detects albumin A residual quantity in product.Result is as following table:
| The purifying mode | Albumin A content (ppm) |
| SL-PA magnetic beads for purifying method | 12.4 |
| Protein A-sepharose affinity chromatography | 16.7 |
The HiTrap MabSelect protein A affinity chromatography post of U.S. GE company is the chromatography media with minimum albumin A expulsion rate of generally acknowledging now, has application the most widely in antibody purification industry.Through the enzyme linked immunological test, the purified product of SL-PA magnetic bead has the albumin A residual quantity lower than HiTrap MabSelect, meets U.S. food and Drug Administration (FDA) limits value (<20ppm) about albumin A residual quantity in antibody drug.
Extract humanized IgG 1 monoclonal antibody from the expressing cho cell supernatant liquor, comprise the following steps:
Get the cho cell expressing recombinant system (purchased from Guangzhou Yuansheng Pharmaceutical Technology Co., Ltd.) of containing humanized IgG 1 gene, through the serum-free suspension culture, recombinaant CHO cell system expresses humanized IgG 1 and is secreted in culture supernatant, and expression amount is 1.15mg/ml.Get the nutrient solution 20ml of filtered, mix with 100mg SL-PA magnetic bead, purification step is with embodiment 1 step 1～7.
Result: successfully extract the 21.62mg human IgG1 from the recombinaant CHO cell culture supernatant, the purifying yield is 96%, and product purity is 94%, 37 minutes consuming time of purification process.
Use the antibody affinity chromatography method to carry out purification process (chromatographic system: AKTA purifer10, U.S. GE lifescience company to the equal volume same sample; Albumin A affinity purification post: HiTrap MabSelect, 1ml, U.S. GE lifesciences company).The nutrient solution sample, through centrifugal, is got supernatant liquor, then through 0.22 μ m membrane filtration once.After filtering, sample, through the chromatographic system purifying, obtains human IgG1 19.75mg, and the purifying yield is 86%, and product purity is 95%, 3 hours consuming time of purification process.
Fig. 5 is shown in by human IgG1's polyacrylamide gel electrophoresis (SDS-PAGE) collection of illustrative plates that two kinds of purification process obtain.
Extract Rituximab (Rituximab) monoclonal antibody from the Chinese hamster ovary celI mixed solution, comprise the following steps:
Get Rituximab (purchased from U.S. Roche Group) 20mg, mix with 20ml Chinese hamster ovary celI nutrient solution, obtain the Chinese hamster ovary celI mixed solution that contains Rituximab.Use 100mg SL-PA magnetic bead from then in mixed solution, to extract Rituximab, step is with embodiment 1 step 1～7.As a result, successfully extract the 19.49mg Rituximab from mixed solution, the purifying yield is 97.45%, purity 95%.
Extract A Damu (Adalimumab) monoclonal antibody from the Chinese hamster ovary celI mixed solution, comprise the following steps:
Get Rituximab (purchased from German Vetter Pharma-Fertigung GmbH&Co.KG) 20mg, mix with 20ml Chinese hamster ovary celI nutrient solution, obtain the Chinese hamster ovary celI mixed solution that contains adalimumab.Use 100mg SL-PA magnetic bead from then in mixed solution, to extract adalimumab, step is with embodiment 1 step 1～7.As a result, successfully extract the 18.59mg adalimumab from mixed solution, the purifying yield is 93%, purity 94%.
Extract Ying Fulixi (Infliximab) monoclonal antibody from the Chinese hamster ovary celI mixed solution, comprise the following steps:
Get infliximab (purchased from Johnson Co.) 20mg, mix with 20ml Chinese hamster ovary celI nutrient solution, obtain the Chinese hamster ovary celI mixed solution that contains infliximab.Use 100mg SL-PA magnetic bead from then in mixed solution, to extract infliximab, step is with embodiment 1 step 1～7.As a result, successfully extract the 19.02mg infliximab from mixed solution, the purifying yield is 95.1%, purity 92%.
Extract (Cetuximab) monoclonal antibody of western appropriate former times from the Chinese hamster ovary celI mixed solution, comprise the following steps:
Get Cetuximab (purchased from Merck KGaA Lyons drugmaker) 20mg, mix with 20ml Chinese hamster ovary celI nutrient solution, obtain the Chinese hamster ovary celI mixed solution that contains Cetuximab.Use 100mg SL-PA magnetic bead from then in mixed solution, to extract Cetuximab, step is with embodiment 1 step 1～7.As a result, successfully extract the 19.65mg Cetuximab from mixed solution, the purifying yield is 98.25%, purity 97%.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (4)
1. the method for the coated magnetic beads for purifying antibody of the high-density of utilizing albumin A is characterized in that comprising the following steps:
(1) expression product that the high-density of albumin A is coated with to magnetic bead, contains antibody joins in reaction vessel, stirs evenly, and under room temperature, hatches 5～15min;
(2) apply magnetic field in reaction vessel outside or inside;
(3) migrate out supernatant liquor from reaction vessel;
(4) lavation buffer solution is added in reaction vessel, remove magnetic field; Repeating step (2)～(3) once;
(5) repeating step (4) washing magnetic bead is 2～4 times;
(6) elution buffer is added in reaction vessel, remove magnetic field, under room temperature, hatch 5～15min;
(7) apply magnetic field and make magnetic bead adherent, the solution in the shift reaction container, the pH value of regulator solution is to neutral, obtains the antibody after purifying;
The phosphoric acid buffer that the described lavation buffer solution of step (4) contains 0.01～0.5M, the NaCl of 1～500mM, surplus is water, pH value 6～9;
The amino acid that the described elution buffer of step (6) contains 0.01～4M, pH value 1.9～5.
2. the method for the coated magnetic beads for purifying antibody of the high-density of utilizing albumin A according to claim 1, it is characterized in that: the contained amino acid of the described elution buffer of step (6) is more than one in glycine, Methionin or arginine.
3. the method for the coated magnetic beads for purifying antibody of the high-density of utilizing albumin A according to claim 1, it is characterized in that: the described expression product that contains antibody of step (1) is serum, cell expressing liquid or ascites.
4. the method for the coated magnetic beads for purifying antibody of the high-density of utilizing albumin A according to claim 1 is characterized in that: the described magnetic field that applies in step (2) and (7), and its magnetic flux is 1000～10000 Gausses, be 1-5min action time.
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| CN110177617A (en) * | 2017-01-04 | 2019-08-27 | 南京金斯瑞生物科技有限公司 | The alkaline-resisting albumin A magnetic bead of high carrying capacity and its application method |
| CN108314701A (en) * | 2018-04-09 | 2018-07-24 | 青岛汉德森生物科技有限公司 | A method of utilizing magnetic bead extraction and antibody purification |
| CN110272496A (en) * | 2019-06-18 | 2019-09-24 | 上海药明生物技术有限公司 | The method of the purity of bispecific antibody is improved using magnetic beads for purifying |
| CN111484975A (en) * | 2020-04-29 | 2020-08-04 | 上海轩锋生物科技有限公司 | Preparation method of human T cell CD3/CD28 activated magnetic beads |
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