CN110272496A - The method of the purity of bispecific antibody is improved using magnetic beads for purifying - Google Patents
The method of the purity of bispecific antibody is improved using magnetic beads for purifying Download PDFInfo
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- CN110272496A CN110272496A CN201910525890.0A CN201910525890A CN110272496A CN 110272496 A CN110272496 A CN 110272496A CN 201910525890 A CN201910525890 A CN 201910525890A CN 110272496 A CN110272496 A CN 110272496A
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- magnetic bead
- eluent
- bispecific antibody
- equilibration buffer
- cleaned
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/46—Hybrid immunoglobulins
- C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
Abstract
A kind of method that the present invention discloses purity that bispecific antibody is improved using magnetic beads for purifying, comprising steps of activated Protein A coating magnetic bead is mixed with the supernatant to be purified containing bispecific antibody, it is incubated for, after magnetic bead is sufficiently combined with bispecific antibody, supernatant is removed by Magneto separate；Magnetic bead first is cleaned with equilibration buffer, is cleaned 2~5 times；Magnetic bead is washed with water again；Magnetic bead after obtained cleaning is eluted with eluent, Magneto separate simultaneously collects eluent；Magnetic bead after separation is carried out elution 1~3 time with eluent again, is merged eluent, is obtained bispecific antibody after purification.Method of the invention is not necessarily to clarification activities, saves the time, and reduce device requirement, can be completed in a relatively short time the purifying of more high-throughput, more volume sample bispecific antibody.
Description
Technical field
The present invention relates to field of biotechnology, improve the pure of bispecific antibody using magnetic beads for purifying more particularly to a kind of
The method of degree.
Background technique
Monoclonal antibody is that bone-marrow-derived lymphocyte and myeloma cell hybridize the hybridoma to be formed and generate, light chain and heavy chain
Specific antigen can be identified and combine by being formed by structural domain.Ratify the chimeric list of first treatment lymph cancer to FDA in 1997
Since clonal antibody lists, there are many antibody drug is granted, multiple " bomb drugs " for surpassing 1,000,000,000 dollars as annual sales amount.
Bispecific antibody is to be generated on the basis of monoclonal antibody by manually engineering, can identify simultaneously two it is identical or different
The immunoglobulin of epitope, its this feature have significant superiority compared with traditional monoclonal antibody.For example, bispecific is anti-
Body combines two epitopes of a single receptor, can star the different suppression mechanisms for the target, to significantly mention
Rise the ability for combining single epitope.In addition, bispecific antibody can be in combination with two pathogenic intermediaries (as neutralized two trainings
Base hinders two receptors), two accesses are limited, collaboration or superposition are formed.In the case of other, while demarcating tumour cell
These two types of cells, active cell toxicity can be effectively coupled with the bispecific antibody of immunocyte effector.Catumaxomab
(Removab, Trion) is formal in 2009 to obtain EMA approval in Europe listing, for treating malignant ascite (abdominal metastas cancer
A kind of common complication of patients with terminal), become the bispecific antibody of first listing in the whole world.The preparation of bispecific antibody
Mainly there is double cross oncocyte method, chemical coupling, the methods of recombination, wherein recombination is current most commonly used skill
Art.
Antibody drug has become the theme of global field of biological pharmacy, and fast development isolates and purifies proposition to antibody
Higher requirement.The purification process of antibody is typically based on capture affinity chromatography, generally Protein A, followed by ion is handed over
Change/hydrophobic/mixed mode chromatographic step.Protein A is a kind of albumen extracted from Staphylococcus aureus cell membrane, tool
There are 5 IgG binding structural domains, it can be specifically in conjunction with antibody Fc end.Advantage using Protein A affinity purification is
Technique initial stage can obtain high purity product.Most common Protein A affinity chromatography medium is by by Protein at this stage
The method of A chemical coupling is fixed on Ago-Gel surface and affine filler is made, and is then packed into chromatographic column.
Although the advantage that bispecific antibody has monoclonal antibody incomparable, however, due to up to four specific polypeptide chains
Co-expression or be related to extended length polypeptide chain assembling, the recombinant production of IgG class bispecific antibody usually along with
Due to heavy chain homodimerization, the mispairing of heavy chain and light chain, the unbalanced expression of different chains and intermolecular mistake are distributed
Etc. the increase (byproduct or aggregation) for the impurity for leading to purpose species.Since some by-products are shown and the dual anti-height of purpose
Similitude, their removal proposes very high challenge to production technology.Therefore, a kind of efficient Protein A parent is found
And purification process, the purity of bispecific antibody can be improved in preliminary purification, be of great significance.
Summary of the invention
Technical problem to be solved by the present invention lies in provide a kind of purification process of bispecific antibody, improve double special
The purity of heterogenetic antibody, compared to traditional chromatographic column have many advantages, such as to save the time, it is easy to operate, reduce device requirement.
In order to solve the above technical problems, the present invention provides a kind of purity for improving bispecific antibody using magnetic beads for purifying
Method, comprising steps of
(1) it combines: activated Protein A coating magnetic bead is mixed with the supernatant to be purified containing bispecific antibody
It closes, is incubated for, after magnetic bead is sufficiently combined with bispecific antibody, supernatant is removed by Magneto separate, the magnetic bead in conjunction with after；
(2) clean: the magnetic bead first obtained with equilibration buffer cleaning step (1), the equilibration buffer for 0.05~
0.5M Tris buffer, pH 6.7~7.3 are cleaned 2~5 times；Magnetic bead is washed with water again；
(3) it elutes: the magnetic bead after cleaning that step (2) obtains is eluted with eluent, the eluent is 0.05
~0.5M glycine solution, pH 2.5~3.5, Magneto separate simultaneously collect eluent；Magnetic bead after separation is washed with eluent again
It is 1~3 time de-, merge eluent, obtains bispecific antibody after purification.
Preferably, the equilibration buffer is 0.06~0.4M Tris buffer；Preferably, the equilibration buffer is
0.07~0.3M Tris buffer；Preferably, the equilibration buffer is 0.08~0.2M Tris buffer；Preferably, institute
Stating equilibration buffer is 0.09~0.1M Tris buffer；Preferably, the equilibration buffer is 0.1M Tris buffer.
Preferably, the pH value of the equilibration buffer is pH 6.8~7.2；Preferably, the pH value of the equilibration buffer
For pH 6.9~7.1；Preferably, the pH value of the equilibration buffer is pH 7.0.
Preferably, it in the step (2), is cleaned 3~4 times with equilibration buffer；Preferably, in the step (2), with flat
Weighing apparatus buffer solution for cleaning 3 times.
Preferably, the eluent is 0.06~0.4M glycine solution；Preferably, the eluent is 0.07~0.3M
Glycine solution；Preferably, the eluent is 0.08~0.2M glycine solution；Preferably, the eluent be 0.09~
0.1M glycine solution；Preferably, the eluent is 0.1M glycine solution.
Preferably, the pH value of the eluent is pH 2.6~3.4；Preferably, the pH value of the eluent is pH 2.7
~3.3；Preferably, the pH value of the eluent is pH 2.8~3.2；Preferably, the pH value of the eluent be pH 2.9~
3.1；Preferably, the pH value of the eluent is pH 3.0.
Preferably, in the step (3), with eluent co-elute 2~3 times；Preferably, in the step (3), with elution
Liquid co-elute 3 times.
Preferably, the Protein A coating magnetic bead is AmMag Protein A magnetic bead.
Specifically, in the step (1), the activation method of magnetic bead are as follows: first pass through Magneto separate and remove the solution for storing magnetic bead
It removes, obtains magnetic bead；Then magnetic bead is cleaned with NaOH solution；Magnetic bead is cleaned with equilibration buffer again, cleans 2~5 times, obtains the magnetic of activation
Pearl.
Specifically, in the step (1), incubation time is 0.5~for 24 hours.Preferably, incubation time is 1~4 hour.It is excellent
Choosing, incubation time is 2~3 hours.
Specifically, the magnetic bead after completing elution is first cleaned with NaOH solution in the step (3), then wash with water, then
20% ethyl alcohol is added, magnetic bead is saved in 4 DEG C.
In method of the invention, Protein A coating magnetic bead refers to that using super paramagnetic microsphere as matrix, surface is by covalently connecting
The mode connect, the Protein A of alkali resistance in coating.Protein A is coated with magnetic bead tolerance 0.1M NaOH cleaning, is suitable for needing
Control the purifying of endotoxin sample.Super paramagnetic microsphere paramagnetism with super strength, can assemble rapidly in magnetic field, leave magnetic
It can aid in magnetic bead again after uniformly to scatter.Secondly, there is the suitable and lesser partial size of difference, for example diameter is
30-100 μm, it ensure that sufficiently strong magnetic responsiveness is not again easily settled.Again, there are surface active groups abundant, in order to
To be coupled with biochemical substances, and realized and the separation of sample to be tested under the action of external magnetic field.Compared with traditional separation method,
Magnetic bead is used for the separation of biological sample complex component, can be realized separation and enrichment while progress, effectively improve point
From speed and bioaccumulation efficiency, while also greatly promote the sensitivity of analysis detection.
Sample antibody-containing is added in magnetic bead, a bit of time is incubated on shaking table, makes antibody by method of the invention
It is sufficiently combined with magnetic bead.After antibody is integrated on magnetic bead, antibody and Beads enrichment, then benefit can be made by acidic elution solution
Magnetic bead is adsorbed in tube wall with magnetic frame, facilitates the taking-up of sample.Magneto separate eliminates the needs of centrifugation, reduces to greatest extent
The loss of sample.
Protein A coating magnetic bead is applied to the affinity purification of bispecific antibody by the present invention, is also had the advantage that
(1) bispecific antibody expression supernatant is not necessarily to clarification activities, saves the time, and reduce device requirement.
(2) traditional chromatographic column is compared, method of the invention can be completed in a relatively short time more high-throughput, more substantially
The purifying of the bispecific antibody of product sample.Purification process is simple, does not need expensive tomography devices.
(3) in method of the invention, the contact due to antibody expression supernatant with magnetic bead is more uniform, it is not easy to generation office
The excessive problem of portion's concentration, sample elution are more complete.
(4) the bispecific antibody purity of magnetic beads for purifying is generally higher than the sample that traditional chromatographic column method obtains, reduce into
The demand of the more purifications such as row SEC, IEX or CHT, improves sample recovery rate.
The present invention is applied to the purifying of bispecific antibody using the coated Superparamagnetic beads of Protein A, to replace
Existing affinity purification operation.Protein A is coated with magnetic beads for purifying technology without complicated tomography devices, to the clarity of sample
There is no limit, it is only necessary to simple magnetic absorption step conveniently and efficiently can isolate antibody from antibody expression product, have
Effect solves the shortcoming of traditional chromatographic technique.But the antibody combination energy of the coating magnetic bead of the Protein A due to listing at present
Power still has biggish gap for traditional agarose plugs, and comparatively the service life of Protein A coating magnetic bead also compares
It is shorter, therefore, still fail to be applied to antibody purification industry on a large scale.The present invention is by adjusting sample incubation time, certain journey
ProteinA coating magnetic bead effect in conjunction with antibody is improved on degree.
WuXiBody is medicine open-birth object bispecific antibody technology platform with independent intellectual property rights, which exists
Commercialized operation is realized both at home and abroad, is best one of the bispecific antibody platform of industry.The platform allows any list
Anti- sequence facilitates the anti-of the various format difference chemical valences of building to bispecific structure, unique configuration flexibility is assembled into
Body, to meet the biological property demand of disparity items.Method provided by the invention is particularly suitable for WuXiBody bispecific
Antibody purification.
Detailed description of the invention
Fig. 1 is the process for using figure that Protein A is coated with magnetic bead.
Fig. 2 is monoclonal antibody sample SDS-PAGE testing result figure in embodiment 1
Specific embodiment
Clear, complete description will be carried out to the present invention/invention technical solution below, it is clear that described embodiment
It is the present invention/invention a part of the embodiment, instead of all the embodiments.Based on the embodiment in the present invention/invention, ability
Domain those of ordinary skill every other embodiment obtained without making creative work, belongs to this hair
The range of bright/invention protection.
Embodiment 1.AmMag Protein A magnetic bead is applied to monoclonal antibody-purified and control endotoxin
Prepare AmMag Protein A magnetic bead first: (1) being removed 20% ethyl alcohol for storing magnetic bead by Magneto separate；(2)
0.1M NaOH is added and cleans magnetic bead, is incubated for after 0.5h and NaOH is removed by Magneto separate；(3) 0.1M Tris is added, pH7.0 is flat
Weigh magnetic bead, and repeated washing 3 times；(4) 0.1M Tris, the pH7.0 mixing of three times magnetic bead volume is added, the magnetic bead activated is outstanding
Supernatant liquid.
The bead suspension activated in right amount is added in 20ml monoclonal antibody expression supernatant, 2h is incubated on shaking table, is made
Magnetic bead is sufficiently combined with antibody, removes supernatant by Magneto separate afterwards.Magnetic bead is cleaned, is repeated 3 times with 0.1M Tris, pH7.0, then
Use H2O cleans magnetic bead.1.5ml 0.1M Glycine, pH 3.0 is added to be eluted, is incubated for 10 minutes, will be washed after Magneto separate
De- liquid is sucked out, and repeats elution three times.
Sample Purification on Single terminates to clean magnetic bead: (1) 0.1M NaOH cleaning magnetic bead is added, removes NaOH by Magneto separate；(2)
H is added2O cleans magnetic bead, removes H by Magneto separate2O；(3) 20% ethyl alcohol is added, magnetic bead is stored in 4 DEG C.
Table 1 is Sample Purification on Single result: the purity of step purifying is greater than 98%, and level of endotoxin is below 2EU/mg.
Table 1: monoclonal antibody Sample Purification on Single statistical data
The present invention handles AmMag Protein A magnetic bead with 0.1M NaOH, can control the endotoxin water of sample well
It is flat.
Embodiment 2.AmMag Protein A magnetic bead and conventional filler are applied to the purifying of WuXiBody bispecific antibody
Compare
Magnetic bead is cleaned and activated with example 1 first.Prepare traditional GE MabSelect SuRe chromatographic column simultaneously,
First with 0.5M NaOH rinse 0.5h, then use 0.1M Tris, pH7.0 balance after can loading.Bispecific antibody is expressed
Final proof product are equally divided into two parts, while being purified.AmMag Protein A magnetic bead is washed using 0.1M Glycine pH3.0
De-, GE MabSelect SuRe chromatographic column is eluted using 0.1M Glycine pH3.5.
Table 2 is that the purification result of four tests compares: the bispecific antibody purity of AmMag magnetic beads for purifying is totally all higher than
The resulting sample of MabSelect SuRe column chromatography.
Table 2: dual anti-Sample Purification on Single statistical data
Embodiment 3.AmMag Protein A is applied to the purifying of the bispecific antibody sample of large volume
Bispecific antibody structure is complex, especially in the case that sample is easy to produce poly, uses GE
MabSelect SuRe chromatographic column is purified it is possible that difficult the phenomenon that eluting.5 sample of BsAb uses GE MabSelect
When SuRe chromatographic column is purified, product, change elution are hardly obtained when eluting using conventional 0.1M Glycine pH3.5
Condition uses 0.1M Glycine, 0.2M Arginine, and the antibody purity that pH3.0 is afforded is lower.It attempts to use AmMag magnetic
Although yield is still lower after purification for pearl, product purity is higher, therefore amplifies volume and produced.First by the same example of magnetic bead
1 is cleaned and is activated.In order to keep sample more abundant in conjunction with magnetic bead, the antibody expression supernatant of 2L is divided into 10 parts, is added
Enter appropriate magnetic bead and be placed on shaking table to be incubated for 3h.It is cleaned and is eluted with example 1 afterwards, the purifying data such as table that finally obtained
3:
Table 3: the dual anti-Sample Purification on Single statistical data of large volume
| Sample | Amount(mg) | Purity |
| BsAb 5 | 29.82 | 95.95% |
In conclusion the various embodiments described above are only the present invention/invention preferred embodiment, not to limit this hair
The protection scope of bright/invention, all any modifications within the present invention/invention spirit and principle, made, change equivalent replacement
Into etc., it should all be included in the present invention/invention protection scope.
Claims (11)
1. a kind of method for the purity for improving bispecific antibody using magnetic beads for purifying, which is characterized in that comprising steps of
(1) it combines: activated Protein A coating magnetic bead is mixed with the supernatant to be purified containing bispecific antibody,
It is incubated for, after magnetic bead is sufficiently combined with bispecific antibody, supernatant is removed by Magneto separate, the magnetic bead in conjunction with after；
(2) clean: the magnetic bead first obtained with equilibration buffer cleaning step (1), the equilibration buffer are 0.05~0.5M
Tris buffer, pH 6.7~7.3 are cleaned 2~5 times；Magnetic bead is washed with water again；
(3) it eluting: the magnetic bead after cleaning that step (2) obtains is eluted with eluent, the eluent is 0.05~
0.5M glycine solution, pH 2.5~3.5, Magneto separate simultaneously collect eluent；Magnetic bead after separation carries out elution 1 with eluent again
~3 times, merges eluent, obtain bispecific antibody after purification.
2. the method as described in claim 1, which is characterized in that the equilibration buffer is 0.06~0.4M Tris buffer；
Preferably, the equilibration buffer is 0.07~0.3M Tris buffer；Preferably, the equilibration buffer be 0.08~
0.2M Tris buffer；Preferably, the equilibration buffer is 0.09~0.1M Tris buffer；Preferably, the balance
Buffer is 0.1M Tris buffer.
3. the method as described in claim 1, which is characterized in that the pH value of the equilibration buffer is pH 6.8~7.2；It is preferred that
, the pH value of the equilibration buffer is pH 6.9~7.1；Preferably, the pH value of the equilibration buffer is pH 7.0.
4. the method as described in claim 1, which is characterized in that in the step (2), cleaned 3~4 times with equilibration buffer；
Preferably, it in the step (2), is cleaned 3 times with equilibration buffer.
5. the method as described in claim 1, which is characterized in that the eluent is 0.06~0.4M glycine solution；It is preferred that
, the eluent is 0.07~0.3M glycine solution；Preferably, the eluent is 0.08~0.2M glycine solution；
Preferably, the eluent is 0.09~0.1M glycine solution；Preferably, the eluent is 0.1M glycine solution.
6. the method as described in claim 1, which is characterized in that the pH value of the eluent is pH 2.6~3.4；Preferably,
The pH value of the eluent is pH 2.7~3.3；Preferably, the pH value of the eluent is pH 2.8~3.2；Preferably, institute
The pH value for stating eluent is pH 2.9~3.1；Preferably, the pH value of the eluent is pH3.0.
7. the method as described in claim 1, which is characterized in that in the step (3), with eluent co-elute 1~3 time；It is excellent
Choosing, in the step (3), with eluent co-elute 3 times.
8. the method as described in claim 1, the Protein A coating magnetic bead is AmMag Protein A magnetic bead.
9. the method as described in claim 1, in the step (1), the activation method of magnetic bead are as follows: first passing through Magneto separate will store
The solution of magnetic bead removes, and obtains magnetic bead；Then magnetic bead is cleaned with sodium hydroxide solution；Magnetic bead, cleaning 2 are cleaned with equilibration buffer again
~5 times, obtain the magnetic bead of activation.
10. the method as described in claim 1, in the step (1), incubation time is 0.5~for 24 hours.Preferably, incubation time
It is 1~4 hour.Preferably, incubation time is 2~3 hours.
11. the method as described in claim 1, in the step (3), the magnetic bead after completing elution is first cleaned with NaOH solution,
Then it washes with water, adds 20% ethyl alcohol, magnetic bead is saved in 4 DEG C.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2021208839A1 (en) * | 2020-04-13 | 2021-10-21 | Wuxi Biologics (Shanghai) Co., Ltd. | Purification of bispeciifc antibodies |
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| CN103450328A (en) * | 2012-09-20 | 2013-12-18 | 海狸纳米科技（苏州）有限公司 | Method for purifying antibody by using high-density protein A coated magnetic beads |
| US20160264685A1 (en) * | 2015-03-13 | 2016-09-15 | Novimmune Sa | Methods of purifying bispecific antibodies |
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| WO2021208839A1 (en) * | 2020-04-13 | 2021-10-21 | Wuxi Biologics (Shanghai) Co., Ltd. | Purification of bispeciifc antibodies |
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Effective date of registration: 20191230 Address after: 200131 A location of 301 parts of 2 building, 299 Fu Tai Road, Pudong New Area Free Trade Zone, Shanghai Applicant after: WUXI BIOLOGICS (SHANGHAI) Co.,Ltd. Applicant after: Wuxi Yaoming coupling Biotechnology Co.,Ltd. Applicant after: Wuxi Yaoming Biotechnology Co.,Ltd. Address before: 200131 A location of 301 parts of 2 building, 299 Fu Tai Road, Pudong New Area Free Trade Zone, Shanghai Applicant before: WUXI BIOLOGICS (SHANGHAI) Co.,Ltd. |
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Address after: 200131 Shanghai Pudong New Area pilot Free Trade Zone Applicant after: WUXI BIOLOGICS (SHANGHAI) Co.,Ltd. Applicant after: Wuxi Yaoming United Biotechnology Co.,Ltd. Applicant after: Wuxi Yaoming Biotechnology Co.,Ltd. Address before: 200131 Shanghai Pudong New Area pilot Free Trade Zone Applicant before: WUXI BIOLOGICS (SHANGHAI) Co.,Ltd. Applicant before: Wuxi Yaoming coupling Biotechnology Co.,Ltd. Applicant before: Wuxi Yaoming Biotechnology Co.,Ltd. |
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Application publication date: 20190924 |