CN105777896B - A kind of purification process at antibody acidity peak - Google Patents
A kind of purification process at antibody acidity peak Download PDFInfo
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- CN105777896B CN105777896B CN201610154004.4A CN201610154004A CN105777896B CN 105777896 B CN105777896 B CN 105777896B CN 201610154004 A CN201610154004 A CN 201610154004A CN 105777896 B CN105777896 B CN 105777896B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/165—Extraction; Separation; Purification by chromatography mixed-mode chromatography
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
- C07K16/241—Tumor Necrosis Factors
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Abstract
The present invention relates to a kind of purification process at antibody acidity peak, specifically, at 2~8 DEG C, comprise the steps of: that (1) uses MEP HyperCel filler, the elution to be changed from small to big using conductivity, target peak eluent is collected, the phenylbutyrate sodium of 5~10mmol/L is wherein contained in eluent；(2) the target peak eluent in step (1) is subjected to weak cation chromatography, uses butyrate as buffering eluting salt target peak.It carries out after purification, the removal to antibody acidity peak capable of being reinforced by the above method, it can be effectively by the control of acid peak within the scope of 11%-14%.
Description
Technical field
The present invention relates to antibody purification fields, and in particular to a kind of method of antibody acidity peak purifying.
Background technique
Monoclonal antibody after a variety of translations modification, will lead to charge heterogeneity, be embodied in during expression
Electricity point is different, generates a series of antibody of different isoelectric points.It analyzes, occurs successional by high-resolution ion exchange column
Peak, rather than single peak.Charge heterogeneity influences the stability and biological activity of monoclonal antibody, therefore in production,
Charge heterogeneity becomes Key Quality attribute, needs strict control.
Since charge heterogeneity difference is very small, it is difficult to chromatograph by a step and can antibody acidity peak be dropped to and can be connect
By in the range of.The ion-exchange packing of very high resolution is needed, then cooperates other chromatography means, such as affinity column
Protein A etc..Although Protein A chromatographic effect imitates the removals such as the other impurities such as HCP, DNA, light chain of monoclonal antibody
Fruit is best, but is not so good as high-resolution ion-exchange packing to the removal effect at acid peak.Cooperate high-resolution ion exchange
The SP Sepharose High Performance of filler GE company, effect does not comply with one's wishes, and cost is very expensive, GE
The ProteinA of company usually requires more than ten just in case rising, and service life is shorter, in addition, affine filler contains protein A component, uses
Albumin A, which can fall off, afterwards introduces new impurity, needs to establish the method and removal technique of detection albumin A.It is utilized using compounded mix
Conventional minimizing technology, due to being fully loaded loading, partial size is larger, and resolution ratio is not inherently high, removes the effect at acid peak not
Ideal, to the whole plus-pressure of removal of downstream impurity.
In addition, using the combination of other fillers, it is anti-in conventional method removal such as using cation exchange and anion exchange
The effect at body acidity peak is also not highly desirable.Currently, the content at control antibody acidity peak in medical purification art is one general
Time problem.
Summary of the invention
The present invention selects MEP filler and the weak cation conduct of pall company according to the deficiency of above-mentioned technical background herein
The content at acid peak is effectively reduced in means of purification, reduces the pressure of subsequent purification.
Specifically, the present invention provides a set of purifying process that may be adapted to antibody acidity peak, at 2~8 DEG C, comprising following
Step:
(1) the MEP HyperCel filler of Pall company is used, the elution to be changed from small to big using conductivity is collected
Target peak eluent wherein contains the phenylbutyrate sodium of 5~10mmol/L in eluent；
(2) target peak eluent described in step (1) is subjected to weak cation chromatography, butyrate is used to wash as buffer salt
De- target peak.
In some embodiments, the filler of weak cation chromatography is CM Sephadex C-25, TOYOPEARL CM-
650(S)、Uni CM-30S。
In the chromatography process of weak cation, the concentration of buffer its butyrate when being eluted often is 150~
250mmol/L。
In other embodiments, purifying process comprises the steps of:
(1) the MEP HyperCel filler for using Pall company after loading balances, first uses the 30mmol/L lemon of pH6.5
The elution of lemon phthalate buffer reuses the 50mmol/L citrate buffer elution of pH7.0, collects target peak eluent,
Containing the phenylbutyrate sodium of 5~10mmol/L in middle citrate buffer；
(2) target peak eluent described in step (1) is subjected to weak cation chromatography, uses pH7.0's after balance
200mmol/L butyric acid salt buffer elution target peak.
In some embodiments, butyrate is sodium butyrate, calcium butyrate, butyric acid potassium.
In some embodiments of the invention, antibody is TNF α antibody or its antigen-binding portion thereof, such as infliximab
(infliximab), adalimumab (adalimumab), Golimumab are TNF α class antibody.
The beneficial effects of the invention are that:
A. the technique has many advantages, such as to remove that acid peak impurity effect is good, and product recovery rate is high, sample can direct loading,
Pre-treatment work is less, simple for process only to be chromatographed by two steps, so that it may control acid peak in enclosing 11%~14%；
B. under cryogenic, by adding phenylbutyrate sodium and butyrate in eluent, effectively acid peak can be carried out
Separation, and have preferable control to turbidity, host cell etc..
Specific embodiment
As described below is the preferred embodiment of the present invention, and what the present invention was protected is not limited to following preferred implementation side
Formula.It should be pointed out that for those skilled in the art on the basis of the inventive concept, several deformations for making and
It improves, belongs to protection scope of the present invention.
Raw material used in embodiment or consumptive material can be prepared by commercial sources or known method.Embodiment
In the tomographic system used be GE company AKTA purifer, chromatographic column is Pall LRC15X080-200V01 column.Embodiment party
The chromatography matrix MEP-HyperCel used in case is a compounded mix of Pall company, is had hydrophobic and two kinds of ion exchange
Mechanism, filler are made of 4 mercaptoethyl pyridine coupler rigid fiber elements.CM Sephadex C-25 (CM sephadex C-25)
For weak cation exchange mechanism, filler is carboxymethyl cross-link dextran, and TOYOPEARL CM-650 (S) is the production of TOSOH company,
Filler is carboxymethyl cross-linked poly-methyl methacrylate, and Uni CM-30S is weak cation exchange mechanism, and filler is that carboxymethyl crosslinking is poly-
Acrylate.
It is the fermentation liquid of the adalimumab (adalimumab) of expressing cho cell used in the embodiment of the present invention, it will
Chromatogram purification experiment is carried out after the feed liquid centrifugal filtration that fermentor gets off.Following embodiment of the present invention is to use two step centrifugal process,
Centrifugation 4000g/min for the first time, is centrifuged 10min, Chinese hamster ovary celI and bulky grain is centrifuged off, and collects supernatant；Second of use
8000g/min removes cell fragment and fine particle, collects supernatant.By the feed liquid plate-type filtering after centrifugation, by nephelometer
Detection feed liquid turbidity is 6NTU, and following experiment is used for after meeting the requirements.
Embodiment 1
Compounded mix chromatography
A. prepare: feeding liquid (antibody concentration 2.18mg/ml) 400ml uses the tomographic system AKTA of GE company
Purifer selects Pall LRC15X080-200V01 column, includes 25ml MEP HyperCel filler.Sample and pillar are always
It is maintained at 4 DEG C.
B. balance: equilibration buffer is 150mmol/LPBS (pH7.3), and holding flow velocity is 4.5ml/min.
C. loading: every milliliter of filler applied sample amount is 35mg antibody, loading volume about 400ml.
D. it rinses: going to rinse with the equilibration buffer of 150mmol/LPBS (pH7.3) steady to baseline.
E. it elutes: eluting in two steps, elution buffer one are as follows: 30mmol/L citrate+10mmol/L phenylbutyrate sodium
(pH6.5), a peak is eluted to give up for acid peak impurity.After ready to balance, with two: 50mmol/L citrate of elution buffer
+ 10mmol/L phenylbutyrate sodium (pH7.0) is eluted, and a tall and big peak is separated, and is collected second peak, is about obtained
40ml。
Detect the method bibliography " the charge heterogeneity analysis that ion-exchange chromatography fights VEGFR2 monoclonal antibody " at acid peak
In detection method, purpose antibody is detected using high-resolution ion exchange column.
The yield of calculating antibody is 93.54%, and the content for detecting acid peak is 20.71%.
Weak cation GE CM Sephadex C-25 chromatography
A. prepare: being diluted with water, about 1.2 times of water is added, with fourth acid for adjusting pH to 7.0, the feed liquid after dilution is used not
Rust steel plate type filter Millipore crosses 0.20 μm of filter membrane, obtains 90ml sample (antibody concentration 9.10mg/ml).Sample
4 DEG C are remained at pillar.Using the tomographic system AKTA purifer of GE company, Pall LRC15X080- is selected
200V01 column includes CM Sephadex C-25 (Capto SP ImpRes) filler of 27ml GE company.
B. balance: equilibration buffer is 60mmol/LPBS (pH7.0), and holding flow velocity is 4ml/min.
C. loading: every milliliter of filler applied sample amount is 30mg antibody, loading volume about 90ml.
D. it rinses: going to rinse with the dcq buffer liquid of 50mmol/L butyric acid salt buffer (pH7.0) steady to baseline.
E. it elutes: being eluted with 200mmol/L butyric acid salt buffer (pH7.0), elute a peak, which is carried out
Fractional Collections, the peak before half peak height are not collected, start to collect after half peak height, and collection starts to stop to 200mAu.
Detection method is same as above, and the yield of calculating antibody is 75.31%, and the content for detecting acid peak is 13.72%.
Embodiment 2
Compounded mix chromatography
A. prepare: feeding liquid (antibody concentration 2.5mg/ml) 350ml uses the tomographic system AKTA of GE company
Purifer selects Pall LRC15X080-200V01 column, includes 25ml MEP HyperCel filler.Sample and pillar are always
It is maintained at 4 DEG C.
B. balance: equilibration buffer is 150mmol/LPBS (pH7.3), and holding flow velocity is 4.2ml/min.
C. loading: every milliliter of filler applied sample amount is 35mg antibody, loading volume about 350ml.
D. it rinses: going to rinse with the equilibration buffer of 150mmol/LPBS (pH7.3) steady to baseline.
E. it elutes: eluting in two steps, elution buffer one are as follows: 30mmol/L citrate+8mmol/L phenylbutyrate sodium
(pH6.5), a peak is eluted to give up for acid peak impurity.After ready to balance, with two: 50mmol/L citrate of elution buffer
+ 8mmol/L phenylbutyrate sodium (pH7.0) is eluted, and a tall and big peak is separated, and is collected second peak, is about obtained
38ml。
Detect the method bibliography " the charge heterogeneity analysis that ion-exchange chromatography fights VEGFR2 monoclonal antibody " at acid peak
In detection method, purpose antibody is detected using high-resolution ion exchange column.
The rate of recovery of calculating antibody reaches 91.37%, and the content through detecting acid peak reaches 19.72%.
Weak cation GE CM Sephadex C-25 chromatography
A. pre-treatment: being diluted with water, and about 1.2 times of water is added, and with fourth acid for adjusting pH to 7.0, the feed liquid after dilution is used
Stainless steel plate type filter Millipore crosses 0.20 μm of filter membrane, obtains 90ml sample (antibody concentration 8.75mg/ml).Make
With the tomographic system AKTA purifer of GE company, Pall LRC15X080-200V01 column is selected, 27ml GE company is included
CM Sephadex C-25 (Capto SP ImpRes) filler.Sample and pillar remain at 4 DEG C.
B. balance: equilibration buffer is 60mmol/LPBS (pH7.0), and holding flow velocity is 4ml/min.
C. loading: every milliliter of filler applied sample amount is 29mg antibody, loading volume about 90ml.
D. it rinses: going to rinse with the dcq buffer liquid of 50mmol/L butyric acid salt buffer (pH7.0) steady to baseline.
E. it elutes: being eluted with 200mmol/L butyric acid salt buffer (pH7.0), elute a peak, which is carried out
Fractional Collections, the peak before half peak height are not collected, start to collect after half peak height, and collection starts to stop to 200mAu.
Detection method is same as above, and the yield of calculating antibody is 75.31%, and the content for detecting acid peak is 12.44%.
Embodiment 3
Compounded mix chromatography
A. prepare: feeding liquid 380ml, antibody concentration 2.24mg/ml use the tomographic system AKTA of GE company
Purifer selects Pall LRC15X080-200V01 column, includes 25ml MEP filler.Sample and pillar remain at 4 DEG C.
B. balance: equilibration buffer is 150mmol/LPBS (pH7.3), and holding flow velocity is 4.5ml/min.
C. loading: every milliliter of filler applied sample amount is 34mg antibody, loading volume about 380ml.
D. it rinses: going to rinse with the equilibration buffer of 150mmol/LPBS (pH7.3) steady to baseline.
E. it elutes: eluting in two steps, elution buffer one are as follows: 30mmol/L citrate+5mmol/L phenylbutyrate sodium
(pH6.5), a peak is eluted to give up for acid peak impurity.After ready to balance, with two: 50mmol/L citrate of elution buffer
+ 5mmol/L phenylbutyrate sodium (pH7.0) is eluted, and a tall and big peak is separated, and is collected second peak, is about obtained
40ml。
Detect the method bibliography " the charge heterogeneity analysis that ion-exchange chromatography fights VEGFR2 monoclonal antibody " at acid peak
In detection method, purpose antibody is detected using high-resolution ion exchange column.
The yield of calculating antibody is 92.88%, and the content for detecting acid peak is 19.04%.
Weak cation GE CM Sephadex C-25 chromatography
A. prepare: being diluted with water, about 1.2 times of water is added, with fourth acid for adjusting pH to 7.0, the feed liquid after dilution is used not
Rust steel plate type filter Millipore crosses 0.20 μm of filter membrane, obtains 88ml sample (antibody concentration 8.90mg/ml).It uses
The tomographic system AKTA purifer of GE company selects Pall LRC15X080-200V01 column, includes the CM of 27mlGE company
Sephadex C-25 filler.Sample and pillar remain at 4 DEG C.
B. balance: equilibration buffer is 60mmol/LPBS (pH7.0), and holding flow velocity is 4ml/min.
C. loading: every milliliter of filler applied sample amount is 29mg antibody, loading volume about 88ml.
D. it rinses: going to rinse with the dcq buffer liquid of 50mmol/L butyric acid salt buffer (pH7.0) steady to baseline.
E. it elutes: being eluted with 200mmol/L butyric acid salt buffer (pH7.0), elute a peak, which is carried out
Fractional Collections, the peak before half peak height are not collected, start to collect after half peak height, and collection starts to stop to 200mAu.
Detection method is same as above, and the yield of calculating antibody is 73.84%, and the content for detecting acid peak is 11.89%.
Claims (3)
1. a kind of purification process of antibody, which is characterized in that at 2~8 DEG C comprising the steps of:
(1) it after using MEP HyperCel filler, loading to balance, is first washed using the 30mmol/L citrate buffer of pH6.5
It is de-, the 50mmol/L citrate buffer elution of pH7.0 is reused, collects target peak eluent, wherein Citrate buffer
Containing the phenylbutyrate sodium of 5~10mmol/L in liquid；
(2) target peak eluent described in step (1) is subjected to weak cation chromatography, the 200mmol/L of pH7.0 is used after balance
Butyric acid salt buffer elution target peak；
Wherein the antibody is adalimumab.
2. the method according to claim 1, wherein the filler of weak cation chromatography is CM Sephadex
C-25、TOYOPEARL CM-650S、Uni CM-30S。
3. the method according to claim 1, wherein the butyrate is sodium butyrate, calcium butyrate, indolebutyric acid
Potassium.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2012149197A2 (en) | 2011-04-27 | 2012-11-01 | Abbott Laboratories | Methods for controlling the galactosylation profile of recombinantly-expressed proteins |
| US9067990B2 (en) | 2013-03-14 | 2015-06-30 | Abbvie, Inc. | Protein purification using displacement chromatography |
| US9150645B2 (en) | 2012-04-20 | 2015-10-06 | Abbvie, Inc. | Cell culture methods to reduce acidic species |
| US9181572B2 (en) | 2012-04-20 | 2015-11-10 | Abbvie, Inc. | Methods to modulate lysine variant distribution |
| US9512214B2 (en) | 2012-09-02 | 2016-12-06 | Abbvie, Inc. | Methods to control protein heterogeneity |
| WO2014151878A2 (en) | 2013-03-14 | 2014-09-25 | Abbvie Inc. | Methods for modulating protein glycosylation profiles of recombinant protein therapeutics using monosaccharides and oligosacharides |
| US9017687B1 (en) | 2013-10-18 | 2015-04-28 | Abbvie, Inc. | Low acidic species compositions and methods for producing and using the same using displacement chromatography |
| EP3052640A2 (en) | 2013-10-04 | 2016-08-10 | AbbVie Inc. | Use of metal ions for modulation of protein glycosylation profiles of recombinant proteins |
| US9181337B2 (en) | 2013-10-18 | 2015-11-10 | Abbvie, Inc. | Modulated lysine variant species compositions and methods for producing and using the same |
| US9085618B2 (en) | 2013-10-18 | 2015-07-21 | Abbvie, Inc. | Low acidic species compositions and methods for producing and using the same |
| WO2015073884A2 (en) | 2013-11-15 | 2015-05-21 | Abbvie, Inc. | Glycoengineered binding protein compositions |
| WO2018045587A1 (en) * | 2016-09-12 | 2018-03-15 | 广东东阳光药业有限公司 | Purification method for anti-vegf type monoclonal antibody |
| CN109022496A (en) * | 2018-07-03 | 2018-12-18 | 北京化工大学 | Promote enzymatic hydrolysis buffer and its application of the production of fiber acetone-butanol alcohol fermentation |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101218247A (en) * | 2005-04-11 | 2008-07-09 | 米德列斯公司 | Protein purification using HCIC and ion exchange chromatography |
| CN107629124A (en) * | 2010-12-28 | 2018-01-26 | 中外制药株式会社 | The cultural method of zooblast |
-
2016
- 2016-03-17 CN CN201610154004.4A patent/CN105777896B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101218247A (en) * | 2005-04-11 | 2008-07-09 | 米德列斯公司 | Protein purification using HCIC and ion exchange chromatography |
| CN107629124A (en) * | 2010-12-28 | 2018-01-26 | 中外制药株式会社 | The cultural method of zooblast |
Non-Patent Citations (4)
| Title |
|---|
| 单克隆抗体电荷异构体分离方法优化;程洪杰 等;《中国医药生物技术》;20141231;第9卷(第5期);385-389 |
| 新型疏水性电荷诱导配基设计及层析分离抗体研究;童红飞;《中国博士学位论文全文数据库 工程科技I辑》;20140815;B015-2 |
| 疏水性电荷诱导层析——原理、性能及其应用;夏海锋 等;《化工进展》;20101231;第29卷(第10期);1951-1956 |
| 疏水性电荷诱导层析纯化免疫球蛋白IgY;童红飞等;《化工学报》;20110630;第62卷(第6期);1574-1580 |
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