CN108101982A - A kind of purification process of monoclonal antibody - Google Patents
A kind of purification process of monoclonal antibody Download PDFInfo
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- CN108101982A CN108101982A CN201810058773.3A CN201810058773A CN108101982A CN 108101982 A CN108101982 A CN 108101982A CN 201810058773 A CN201810058773 A CN 201810058773A CN 108101982 A CN108101982 A CN 108101982A
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Abstract
The invention discloses a kind of purification process of monoclonal antibody, and including low pH virus incubations, chromatography pre-treatment, chromatography process, the chromatography pre-treatment is included the following steps using the method for two steps readjustment pH：Monoclonal antibody after low pH virus incubations is adjusted into liquid with pH and adjusts pH for the first time, filtering adjusts liquid with pH and adjusts pH for the second time, obtains the monoclonal antibody after readjustment pH.The present invention has the residual quantity for reducing the impurity such as endotoxin, aggregation, host cell proteins, and the advantages of improving the purity of monoclonal antibody and increase the service life of anion chromatography filler, and technological operation is simple, can put into industrialized production.It is understood according to experiment, the peak area percentage of the monoclonal antibody after present invention chromatography is up to 94.38%, and therefore, the present invention has good purification effect, can separate monoclonal antibody well with impurity.
Description
Technical field
The present invention relates to biopharmaceutical technologies, and in particular to a kind of purification process of monoclonal antibody.
Background technology
The technique research and development of contemporary antibody drug be based on genetic engineering, cell engineering and Fermentation Engineering basis, in
State's Hamster Qvary (CHO) cell has that genetic background is clear, possesses and turns over as main mammalian cell expression host cell
The features such as translating post-processing ability becomes the standard industrial cell line of protein production, is equally also suitable for antibody protein
Expression.With the development of cell suspension cultures technology so that large-scale culture is possibly realized, and the application of serum free medium is then
So that protein purification technique is more easy, everything has all greatly accelerated the industrialization process of antibody drug, to prepare inexpensive matter
Excellent antibody drug is laid a good foundation.
It is flourishing by nearly 30 years on the basis of Koehler and Milstein since prepared by first monoclonal antibody
Development, monoclonal antibody technology of preparing are made that huge contribution in terms of life science and medical practice, it has also become
One of modern biotechnology pillar industry.For monoclonal antibody drug because of its targeting specific, toxic side effect is low, treatment it is efficient
Property etc. clear superiorities antitumor immune response disease, organ-graft refection and virus infection etc. fields have important application.
Antibody drug manufacturing process is pure from the cell line structure in research and development period, medium optimization, upstream, cell culture, downstream
Any one link for changing this five aspects of preparation to the end all has antibody drug critically important influence, wherein again pure with downstream
Chemical industry skill is the most complicated cumbersome, and downstream process contains multiple steps, and currently used is affinity chromatography, ion chromatography and hydrophobic
Chromatograph the method being used in series.Although at present all using this pattern, have can for the variation of tiny parameter in each technique
It can cause huge variation.In the chromatography technique of downstream in addition to the impurity such as HCP to be removed, residual DNA and dimer polymer,
The virus that may be also introduced to upstream is removed.
In antibody drug production process, affinity chromatography is commonly used in the acquisition phase of antibody, i.e. first purifying process step
Suddenly, Selective Separation goes out required antibody from fermented feed liquid, then with ion-exchange chromatography and/or hydrophobic chromatography and/or mixing
Pattern chromatographs, and carrys out the antibody that further consummate affinity chromatography elutes.For general technology, what affinity chromatography afforded
Sample would generally carry out low pH virus incubations, and all the low pH samples being incubated can be handled before chromatographing in next step,
To reach the operating condition of different chromatography method needs.
The processing of sample before traditional anion chromatography, typically a step pH, i.e., the sample being directly incubated low pH
Adjust back the pH scopes needed for anion chromatography loading.But to may result in antibody unstable for this method, generation precipitation so as to
The loss of sample is caused, it is in addition undesirable to the impurity removal effect of antibody, increase the amount of anion chromatography adsorbing contaminant, to layer
Analysis filler damages, and shortens its service life.
Anion-exchange chromatography mainly removes bacterial endotoxin, host protein and DNA impurity etc., and conventional method is to adopt
With (flow-through) mode is flowed through, antibody is directly by pillar, and pollutant is absorbed.
The presence of impurity can cause drug effect to reduce in antibody drug, serious that human body can be caused dead.Such as：Contain endogenous toxic material
Element can cause a variety of allergy after entering human body by the drug of contaminated with endotoxins, such as：High fever, even shock, death.It is many
Antibody including IgG and IgM, can form aggregation, and the presence of aggregation can generate the drug effect different with major product, can cause
The formation of resistance to the action of a drug antibody.Remaining host cell proteins generate corresponding antibody it is possible that stimulating body that immune response occurs,
Human body can equally be damaged.Therefore, for antibody drug in purification process, the removal effect of impurity directly affects product matter
Amount.
The content of the invention
Technical problems based on background technology, the present invention propose a kind of purification process of monoclonal antibody, pass through
The method of the present invention can reduce the impurity residual of monoclonal antibody, improve the purity of monoclonal antibody product.
The present invention proposes a kind of purification process of monoclonal antibody, including low pH virus incubations, chromatography pre-treatment, chromatography
Process, the chromatography pre-treatment are included the following steps using the method for two steps readjustment pH：By the monoclonal after low pH virus incubations
Antibody adjusts liquid with pH and adjusts pH for the first time, and filtering adjusts liquid with pH and adjusts pH for the second time, obtains the monoclonal after readjustment pH and resists
Body.
Preferably, the value that the first time adjusts pH is 4.00-7.00；Preferably, the value of second of adjusting pH is
8.00-9.50。
Preferably, described be filtered into is filtered with the fiber filter film in 0.2-0.5 μm of aperture.
Preferably, the pH adjusts liquid and delays selected from Tris-HCl buffer solutions, phosphate buffer, Glycine-NaOH
One kind in fliud flushing, sodium chloride-sodium hydrate buffer solution.
Preferably, the pH adjusts liquid and selects Tris-HCl buffer solutions.
Preferably, the pH adjusts the concentration of liquid as 0.01-1.0mol/L, and the pH value that pH adjusts liquid is 6.00-11.00.
Preferably, the chromatography is that the monoclonal antibody after readjustment pH is carried out anion-exchange chromatography, including following step
Suddenly：
S1, pre-equilibration is carried out to anion chromatography column with pre-equilibration buffer solution, then with level pad to anion layer
Analysis column is balanced；
S2, the monoclonal antibody after readjustment pH is added in the anion chromatography column balanced, collects what 40mAU started
Effluent A；Then chromatographic column is cleaned with leacheate, collects the effluent B before 40mAU；
S3, chromatography media is eluted with eluent, with pure water and 20% alcohol flushing chromatographic column；
S4, effluent A and effluent B are mixed to get to after anion chromatography monoclonal antibody to get to list after purification
Clonal antibody.
Preferably, the pre-equilibration buffer solution and eluent are Tris-HCl buffer solutions with high salt.
Preferably, the level pad and leacheate are less salt Tris-HCl buffer solutions.
Preferably, the pH value of the Tris-HCl buffer solutions with high salt is 8.00-10.00.
Preferably, the Tris-HCl buffer solutions with high salt include 600-1000mmol/L NaCl and 10-100mmol/L
Tris。
Preferably, the pH value of the less salt Tris-HCl buffer solutions is 8.00-10.00.
Preferably, the less salt Tris-HCl buffer solutions include 10-100mmol/L NaCl and 10-100mmol/L
Tris。
Preferably, the chromatography media is selected from Ago-Gel class (Sepharose/Capto), polystyrene type
(SOURCE) one kind in, aglucon are selected from one kind in quaternary ammonium group (Q), diethyl amino ethyl group (DEAE).
Preferably, the chromatography media is selected from Q-Sepharose FF, SOURCE-Q, Capto Q, DEAE-Sepharose
One kind in FF, Q-Big-Beds.
Preferably, the chromatography media selects Q-Sepharose FF.
Compared with prior art, beneficial effects of the present invention are as follows：
1) pre-treatment of present invention chromatography is the method using two steps readjustment pH to the monoclonal antibody after low pH virus incubations,
The impurity such as endotoxin, aggregation and host cell proteins can be effectively eliminated, improve the purity of antibody, and are increased anti-
The stability of body and the service life of anion chromatography column.
2) present invention can reduce antibody loss, improve the purity of monoclonal antibody after chromatography, can meet simultaneously high-purity
Spend the preparation of the monoclonal antibody of high-recovery.
Description of the drawings
Fig. 1 is the SEC-UPLC chromatograms of monoclonal antibody after low pH incubations in the embodiment of the present invention 1；
Fig. 2 is the SEC-UPLC chromatograms of monoclonal antibody after anion chromatography in the embodiment of the present invention 1.
Specific embodiment
In the following, technical scheme is described in detail by specific embodiment.
Embodiment 1
A kind of purification process of monoclonal antibody, it is specific to wrap including low pH virus incubations, chromatography pre-treatment, chromatography process
Include following steps：
Chromatograph pre-treatment：By the anti-CD52 monoclonal antibodies after low pH virus incubations with Tris-HCl buffer solutions adjust pH to
7.00, it is filtered with the fiber filter film in 0.2 μm of aperture, then pH to 9.50 is adjusted with Tris-HCl buffer solutions, it is adjusted back
Monoclonal antibody after pH；
Chromatography：S1, pre-equilibration is carried out to anion chromatography column with Tris-HCl buffer solutions with high salt, is delayed with less salt Tris-HCl
Fliud flushing is balanced anion chromatography column；
In S2, the anion chromatography column for having balanced the monoclonal antibody addition after readjustment pH, the stream that 40mAU starts is collected
Go out object A；With less salt Tris-HCl buffer solution for cleaning chromatographic columns, the effluent B before 40mAU is collected；
S3, chromatography media is eluted with Tris-HCl buffer solutions with high salt, anion chromatography medium is carried out with highly basic
Cleaning, rinses step anion chromatography medium with WFI water, anion chromatography medium is preserved with 20% ethyl alcohol；
S4, the effluent A being collected into and effluent B are mixed to get to after anion chromatography monoclonal antibody to get to pure
Anti- CD52 monoclonal antibodies after change；
Wherein, the concentration of anti-CD52 monoclonal antibodies is 6.88mg/mL；The actual carrying capacity of chromatographic stuffing is 80mg/mL.
Embodiment 2
A kind of purification process of monoclonal antibody, it is specific to wrap including low pH virus incubations, chromatography pre-treatment, chromatography process
Include following steps：
Chromatograph pre-treatment：By the anti-CD52 monoclonal antibodies of low pH virus incubations with Tris-HCl buffer solutions adjust pH to
4.00, it is filtered with the fiber filter film in 0.5 μm of aperture, then pH to 8.00 is adjusted with Tris-HCl buffer solutions, it is adjusted back
Monoclonal antibody after pH；
Chromatography：S1, pre-equilibration is carried out to anion chromatography column with Tris-HCl buffer solutions with high salt, is delayed with less salt Tris-HCl
Fliud flushing is balanced anion chromatography column；
In S2, the anion chromatography column for having balanced the monoclonal antibody addition after readjustment pH, the stream that 40mAU starts is collected
Go out object A；With less salt Tris-HCl buffer solution for cleaning chromatographic columns, the effluent B before 40mAU is collected；
S3, chromatography media is eluted with Tris-HCl buffer solutions with high salt, anion chromatography medium is carried out with highly basic
Cleaning, rinses step anion chromatography medium with WFI water, anion chromatography medium is preserved with 20% ethyl alcohol；
S4, the effluent A being collected into and effluent B are mixed to get to after anion chromatography monoclonal antibody to get to pure
Anti- CD52 monoclonal antibodies after change；
Wherein, the concentration of anti-CD52 monoclonal antibodies is 6.88mg/mL；The actual carrying capacity of chromatographic stuffing is 120mg/mL.
Comparative example 1
Anti- CD52 monoclonal antibodies after low pH virus incubations Tris-HCl buffer solutions are adjusted into pH to 9.50, with 0.2 μ
The fiber filter film in m apertures is filtered, and obtains the monoclonal antibody after readjustment pH；Wherein, anti-CD52 monoclonal antibodies is dense
It spends for 6.88mg/mL.
Test example 1
Respectively to the monoclonal antibody after the readjustment pH that is obtained in embodiment 1, the list after the readjustment pH that obtains in comparative example 1
Clonal antibody carries out impurity residues detection, wherein, the indices being related to use following detection method：Endotoxin is tried using horseshoe crab
Agent method detects, and aggregation and monoclonal antibody are detected using Size Exclusion High Performance liquid chromatography, and host cell proteins use enzyme
Join immunoabsorption detection.Testing result is shown in Table 1.
Table 1
| Project | Comparative example 1 | Embodiment 1 |
| Monoclonal antibody total amount (mg) | 1860 | 1980 |
| Endotoxin residual quantity (EU/mg) | 1.06 | 0.65 |
| Aggregation content (%) | 25.37 | 15.46 |
| Host cell proteins amount (ppm) | 1.08 | 0.60 |
As can be seen from Table 1, it is using the method for two steps readjustment pH, each impurity that pre-treatment is chromatographed in the embodiment of the present invention 1
Residual quantity than in comparative example 1 use a step readjustment pH methods impurity residual quantity it is low, meanwhile, also reduced in embodiment 1
The loss amount of monoclonal antibody, therefore, the present invention have apparent purification effect to monoclonal antibody.
Test example 2
Monoclonal antibody after being incubated to low pH in embodiment 1 carries out SEC-UPLC detections, obtains Fig. 1；To in embodiment 1
Monoclonal antibody carries out SEC-UPLC detections after obtained anion chromatography, obtains Fig. 2.
Fig. 1 is the SEC-UPLC chromatograms of the monoclonal antibody after low pH incubations in embodiment 1；Abscissa is albumen appearance
Time, 5 be target protein, i.e. monoclonal antibody, and in 16.5min appearances, ordinate represents A280 ultraviolet absorption values for mAU,
In, the peak area percentage of monoclonal antibody, glycoprotein polyprotein precursor and segment is respectively 51.25%, 45.5%, 3.25%.
Fig. 2 is the SEC-UPLC chromatograms of monoclonal antibody after anion chromatography in embodiment 1；Abscissa is albumen appearance
Time, 3 be target protein, i.e. monoclonal antibody, and in 16.5min appearances, ordinate represents A280 ultraviolet absorption values for mAU,
In, the peak area percentage of monoclonal antibody, glycoprotein polyprotein precursor and segment is respectively 94.38%, 5.43%, 0.19%.
According to Fig. 1, Fig. 2, it can be seen that：The peak area percentage of monoclonal antibody after present invention chromatography is up to
94.38%, it was demonstrated that the present invention can significantly improve the purity of monoclonal antibody, reduce impurity residual quantity, monoclonal antibody purification.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art in the technical scope disclosed by the present invention, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (10)
1. a kind of purification process of monoclonal antibody, including low pH virus incubations, chromatography pre-treatment, chromatography process, feature exists
In the chromatography pre-treatment is included the following steps using the method for two steps readjustment pH：Monoclonal after low pH virus incubations is resisted
Body adjusts liquid with pH and adjusts pH for the first time, and filtering adjusts liquid with pH and adjusts pH for the second time, obtains the monoclonal after readjustment pH and resists
Body.
2. the purification process of monoclonal antibody according to claim 1, which is characterized in that the value that first time adjusts pH is
4.00-7.00；Preferably, described second value for adjusting pH is 8.00-9.50.
3. the purification process of monoclonal antibody according to claim 1 or claim 2, which is characterized in that described be filtered into uses 0.2-0.5
The fiber filter film in μm aperture is filtered.
4. the purification process of monoclonal antibody according to claim 1, which is characterized in that the pH adjusts liquid and is selected from Tris-
One kind in HCl buffer solutions, phosphate buffer, Glycine-NaOH buffer solution, sodium chloride-sodium hydrate buffer solution；It is excellent
Selection of land, the pH adjust liquid and select Tris-HCl buffer solutions.
5. according to the purification process of the monoclonal antibody of claim 1 or 4, which is characterized in that the pH adjusts the concentration of liquid
For 0.01-1.0mol/L, the pH value that pH adjusts liquid is 6.00-11.00.
6. the purification process of monoclonal antibody according to claim 1, which is characterized in that the chromatography is will be after readjustment pH
Monoclonal antibody carries out anion-exchange chromatography, comprises the following steps：
S1, pre-equilibration is carried out to anion chromatography column with pre-equilibration buffer solution, then with level pad to anion chromatography column
It is balanced；
S2, the monoclonal antibody after readjustment pH is added in the anion chromatography column balanced, collects the outflow that 40mAU starts
Object A；Then chromatographic column is cleaned with leacheate, collects the effluent B before 40mAU；
S3, chromatography media is eluted with eluent, with pure water and 20% alcohol flushing chromatographic column；
S4, effluent A and effluent B are mixed to get to after anion chromatography monoclonal antibody to get to monoclonal after purification
Antibody.
7. the purification process of monoclonal antibody according to claim 6, which is characterized in that the pre-equilibration buffer solution and elution
Liquid is Tris-HCl buffer solutions with high salt；Preferably, the level pad and leacheate are less salt Tris-HCl buffer solutions.
8. the purification process of monoclonal antibody according to claim 7, which is characterized in that the Tris-HCl buffer solutions with high salt
PH value be 8.00-10.00；Preferably, the Tris-HCl buffer solutions with high salt include 600-1000mmol/L NaCl and 10-
100mmol/L Tris。
9. the purification process of monoclonal antibody according to claim 7, which is characterized in that the less salt Tris-HCl buffer solutions
PH value be 8.00-10.00；Preferably, the less salt Tris-HCl buffer solutions include 10-100mmol/L NaCl and 10-
100mmol/L Tris。
10. the purification process of monoclonal antibody according to claim 6, which is characterized in that the chromatography media is selected from agar
One kind in sugared gel-like, polystyrene type, the one kind of aglucon in quaternary ammonium group, diethyl amino ethyl group；Preferably, the chromatography
The one kind of medium in Q-Sepharose FF, SOURCE-Q, Capto Q, DEAE-Sepharose FF, Q-Big-Beds；
Preferably, the chromatography media selects Q-Sepharose FF.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114133446A (en) * | 2021-12-31 | 2022-03-04 | 方坦思(上海)生物医药有限公司 | Method for purifying monoclonal antibody |
| CN114181300A (en) * | 2021-12-20 | 2022-03-15 | 方坦思(上海)生物医药有限公司 | Preparation method of high-purity monoclonal antibody |
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| CN105017418A (en) * | 2014-03-27 | 2015-11-04 | 上海药明康德新药开发有限公司 | Monoclonal antibody purification process |
| CN106380507A (en) * | 2016-11-04 | 2017-02-08 | 郑州伊美诺生物技术有限公司 | Purification process of monoclonal antibodies |
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2018
- 2018-01-22 CN CN201810058773.3A patent/CN108101982A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105017418A (en) * | 2014-03-27 | 2015-11-04 | 上海药明康德新药开发有限公司 | Monoclonal antibody purification process |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114181300A (en) * | 2021-12-20 | 2022-03-15 | 方坦思(上海)生物医药有限公司 | Preparation method of high-purity monoclonal antibody |
| CN114133446A (en) * | 2021-12-31 | 2022-03-04 | 方坦思(上海)生物医药有限公司 | Method for purifying monoclonal antibody |
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