CN109369806A - The minimizing technology of cysteine variant in Su Jin monoclonal antibody product - Google Patents
The minimizing technology of cysteine variant in Su Jin monoclonal antibody product Download PDFInfo
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- CN109369806A CN109369806A CN201910029571.0A CN201910029571A CN109369806A CN 109369806 A CN109369806 A CN 109369806A CN 201910029571 A CN201910029571 A CN 201910029571A CN 109369806 A CN109369806 A CN 109369806A
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- 239000012514 monoclonal antibody product Substances 0.000 title claims abstract description 16
- 235000018417 cysteine Nutrition 0.000 title claims description 29
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 title claims description 28
- 238000005516 engineering process Methods 0.000 title description 6
- 238000000034 method Methods 0.000 claims abstract description 78
- 238000005571 anion exchange chromatography Methods 0.000 claims abstract description 15
- 210000004962 mammalian cell Anatomy 0.000 claims abstract description 12
- 238000005215 recombination Methods 0.000 claims abstract description 9
- 230000006798 recombination Effects 0.000 claims abstract description 9
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 37
- 239000000872 buffer Substances 0.000 claims description 25
- 239000000945 filler Substances 0.000 claims description 23
- 238000002360 preparation method Methods 0.000 claims description 18
- 239000012228 culture supernatant Substances 0.000 claims description 16
- 238000010828 elution Methods 0.000 claims description 16
- 239000000523 sample Substances 0.000 claims description 14
- 239000012488 sample solution Substances 0.000 claims description 11
- 238000002835 absorbance Methods 0.000 claims description 8
- 238000004113 cell culture Methods 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 7
- 230000008859 change Effects 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 5
- 229910003460 diamond Inorganic materials 0.000 claims description 4
- 239000010432 diamond Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 238000009790 rate-determining step (RDS) Methods 0.000 claims description 3
- 230000003139 buffering effect Effects 0.000 claims description 2
- -1 compound anion Chemical class 0.000 claims description 2
- 238000012544 monitoring process Methods 0.000 claims description 2
- 230000003111 delayed effect Effects 0.000 claims 1
- 238000011010 flushing procedure Methods 0.000 claims 1
- 230000008569 process Effects 0.000 abstract description 14
- 239000000047 product Substances 0.000 abstract description 9
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 abstract description 3
- 239000012071 phase Substances 0.000 description 48
- 239000006101 laboratory sample Substances 0.000 description 37
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 31
- 238000004587 chromatography analysis Methods 0.000 description 17
- 239000003814 drug Substances 0.000 description 8
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 6
- 229940010466 cosentyx Drugs 0.000 description 6
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 6
- 238000007833 oxidative deamination reaction Methods 0.000 description 6
- 239000002253 acid Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 239000012535 impurity Substances 0.000 description 5
- 102000015696 Interleukins Human genes 0.000 description 4
- 108010063738 Interleukins Proteins 0.000 description 4
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 229940047122 interleukins Drugs 0.000 description 4
- 229910021529 ammonia Inorganic materials 0.000 description 3
- 230000027455 binding Effects 0.000 description 3
- 150000001945 cysteines Chemical class 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 238000004255 ion exchange chromatography Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 1
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 201000004681 Psoriasis Diseases 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 238000013368 capillary electrophoresis sodium dodecyl sulfate analysis Methods 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N cystine group Chemical group C([C@@H](C(=O)O)N)SSC[C@@H](C(=O)O)N LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000014155 detection of activity Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000002086 displacement chromatography Methods 0.000 description 1
- 235000015177 dried meat Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 229960004540 secukinumab Drugs 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- 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
- C07K16/244—Interleukins [IL]
-
- 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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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention provides a kind of method using cysteine isomers in anion-exchange chromatography removal Su Jin monoclonal antibody product, this method includes that the Su Jin monoclonal antibody product is made to be subjected to anion-exchange chromatography, wherein the Su Jin monoclonal antibody product includes the Su Jin monoclonal antibody that mammalian cell recombination generates.Method of the invention can effectively remove cysteine isomers, and simple process is stablized, reproducible, and will not destroy the normal configuration of antibody；Compared with prior art products, the quality of Su Jin monoclonal antibody is also improved.
Description
Technical field
The present invention relates to antibody preparation technical field of purification, specifically, the present invention relates in Su Jin monoclonal antibody or its product
The minimizing technology of cysteine variant.
Background technique
Interleukins (IL-17) monoclonal antibody is a kind of monoclonal antibody in conjunction with IL-17, and Su Jin monoclonal antibody (AIN457) is made
For one of IL-17 monoclonal antibody, it has been approved for the treatment of the diseases such as psoriasis and ankylosing spondylitis, has had and significantly controls
Therapeutic effect and application prospect.
However, Su Jin monoclonal antibody at the 97th of light chain variable region there are free cysteine, on the free cysteine
Free sulfhydryl groups chemical property is very active, can chemically react with a large amount of substance, such as with present in cultivating system half
Cystine reacts (i.e. cysteine modification), and the cysteine variant of Su Jin monoclonal antibody is consequently formed, causes antibody activity
Loss.
Chinese patent application discloses CN107001460A and proposes a kind of this half Guang ammonia of selective reduction Su Jin monoclonal antibody
The method for being acidified variant, this method restores masked cysteine residues using reducing agent, to keep maximum Su Jin
Monoclonal antibody antigen-binding activity.The preferred cysteine of the patent application is as reducing agent, by the half Guang ammonia that certain mol proportion is added
Acid, and the dissolved oxygen in reaction process, pH and temperature are adjusted, pass through the pH etc. of adjusting reaction system again after reacting a period of time
Mode terminates reaction, obtains complete antibody.
But the method for the patent application has significant defect when being applied to and commercially producing.Compared to classics
The step of monoclonal antibody-purified method, the more redox of this method are incubated for, the step for need strict control condition,
Therefore the complexity for increasing technique, is also unfavorable for being mass produced.Furthermore the cysteine being added can not only go back original antibody
Cysteine variant, can also destroy the normal disulfide bond of antibody, it is possible to cause antibody fragments to generate, reduce antibody
Purity and activity increase process risk.In addition, the step for required unconventional antibody purification equipment, reagent, consumptive material it is also big
The cost of technique is increased greatly.
Therefore, for the cysteine variant in Su Jin monoclonal antibody or its product, this field, which still has, removes it
The needs of method.
Summary of the invention
In view of the deficiencies of the prior art, solve above-mentioned technical problem, the purpose of the present invention is to provide it is a kind of using yin from
The method of the cysteine variant of sub- displacement chromatography technology removal Su Jin monoclonal antibody, this method utilize Su Jin monoclonal antibody and its half Guang
Propylhomoserin variant removes the cysteine variant in physical and chemical qualitative natural differences, separation.Non- the passing through of this method
The mode of reaction is learned to change the structure of antibody, therefore the normal configuration of antibody will not be destroyed, simple process is stablized, repeatability
It is good.
As used herein, Su Jin monoclonal antibody (secukinumab) the of the invention cis- dried meat ammonia in complementary determining region of light chain CDR3
There are free cysteines after acid, correspond to light chain variable region, which is located at the 97th.For example, Novartis Co., Ltd
The AIN457 of production.
As used herein, the cysteine variant of Su Jin monoclonal antibody of the invention refers to, in being somebody's turn to do for normal monoclonal antibody molecule
Sulfydryl on free cysteine residues reacts with the sulfydryl of the free cysteine of another non-monoclonal antibody molecule itself,
Disulfide bond is formed, the normal monoclonal antibody molecular weight+119Da or+238Da(of the ratio ultimately generated depends on free half on two light chains
Whether cystine residue all reacts) cysteine variant.
Technical scheme is as follows.
On the one hand, the present invention provides a kind of method for removing cysteine variant in Su Jin monoclonal antibody product, institute
The method of stating includes that the Su Jin monoclonal antibody product is made to be subjected to anion-exchange chromatography.
Wherein, the Su Jin monoclonal antibody product includes the Su Jin monoclonal antibody that mammalian cell recombination generates；Preferably, the Soviet Union
Golden monoclonal antibody product is the culture supernatant for the mammalian cell that recombination generates Su Jin monoclonal antibody, or is the culture supernatant
Purified part.
Wherein, the anion-exchange chromatography is carried out using compound anion-exchange chromatography filler；Preferably, the yin
Ion-exchange chromatography uses the linear gradient elution of mobile phase A and Mobile phase B, and wherein mobile phase A is 5-50 mM Tris-HCl
Buffer, pH 8.9, Mobile phase B are 10-100 mM Tris-HCl buffer, and pH 7.1, linear gradient elution includes: 70-0%
Mobile phase A and 30%-100% Mobile phase B elute at least 20 column volumes (CV), wherein 100% Mobile phase B elution at least 8
A CV.
Specifically, it the described method comprises the following steps:
1) column is filled with anion-exchange chromatography filler, using 5-50 mM Tris-HCl buffer, pH 8.9 balances chromatographic column extremely
Baseline is flat；
2) by the sample solution tune pH value containing the Su Jin monoclonal antibody to 8.5 ± 0.1, loading is buffered using 5-50 mM Tris-HCl
Liquid, it is flat to baseline that pH 8.9 balances the chromatographic column；
3) linear gradient elution is carried out with mobile phase A and Mobile phase B, wherein mobile phase A is 5-50 mM Tris-HCl buffering
Liquid, pH 8.9, Mobile phase B are 10-100 mM Tris-HCl buffer, pH 7.1, by the absorbance at monitoring A280 in egg
Elutriated fraction is collected after white appearance.
In the step 1) of the method for the present invention, it is preferable that the anion-exchange chromatography filler is the friendship of compound anion
Change chromatographic stuffing, such as the Capto of GE companyTMAdhere, Bo Gelong MMA of adhere ImpRes filler, GE.According to this hair
The Capto of GE company can be selected in bright specific embodimentTMAdhere ImpRes filler or Bo Gelong company
Bestarose Diamond MMA filler；Remaining any compound anion-exchange chromatography filler can reach identical experiment
Effect.
Preferably, the Tris-HCl buffer is 25 mM Tris-HCl buffers, pH 8.9.
In the step 2) of the method for the present invention, it is preferable that the sample solution includes to be generated by mammalian cell recombination
Su Jin monoclonal antibody or its product.For example, the sample solution is the culture supernatant for the mammalian cell that recombination generates Su Jin monoclonal antibody,
Or the purified part for the culture supernatant.Specific embodiment according to the present invention, the sample solution is by including
The method of following steps obtains: making mammalian cell expression Su Jin monoclonal antibody, harvests cell culture supernatant, Protein A parent
And chromatographic purifying.
Preferably, applied sample amount is not higher than 60 mg or not higher than filler described in Su Jin monoclonal antibody/mL described in 50 mg.
Preferably, the chromatographic column to baseline is balanced using the Tris-HCl buffer in step 1) to put down.
In the step 3) of the method for the present invention, it is preferable that the mobile phase A is 25 mM Tris-HCl buffers, pH
8.9, the Mobile phase B is 50 mM Tris-HCl buffers, pH 7.1.
Preferably, the linear gradient elution includes: with the mobile phase A of 70-0% and the Mobile phase B of 30%-100% elution 20
A CV, wherein 100% Mobile phase B elutes 8 CV.
Preferably, in the method for the invention, rate-determining steps 2 respectively) in sample solution and step 3) mobile phase flow velocity,
So that the retention time of first eluting peak is greater than 3 minutes.
Specific embodiment according to the present invention, which comprises
1) with CaptoTMAdhere ImpRes filler or Bestarose Diamond MMA filler fill column, use 25 mM
Tris-HCl buffer, it is flat to baseline that pH 8.9 balances chromatographic column；
2) by the sample solution tune pH 8.5 ± 0.1 containing the Su Jin monoclonal antibody, to revive not higher than 60 mg or not higher than described in 50 mg
Filler loading described in golden monoclonal antibody/mL, reuses 25 mM Tris-HCl buffers, and pH 8.9 balances the chromatographic column to baseline
It is flat；
3) linear gradient elution is carried out with mobile phase A and Mobile phase B, wherein mobile phase A is 25 mM Tris-HCl buffers,
PH 8.9, Mobile phase B are 50 mM Tris-HCl buffers, pH 7.1, with the flowing of the mobile phase A of 70-0% and 30%-100%
Phase B elutes 20 CV, wherein 100% Mobile phase B elutes 8 CV, the absorbance at A280 is during which monitored, after albumen appearance
Collect elutriated fraction.
Wherein, rate-determining steps 2 respectively) in sample solution and step 3) mobile phase flow velocity so that first eluting peak
Retention time is greater than 3 minutes.
On the other hand, the present invention provides a kind of preparation method of Su Jin monoclonal antibody, which comprises makes mammalian cell
Su Jin monoclonal antibody is expressed, then cell culture supernatant is being harvested and is optionally carrying out cell culture supernatant after purification, making thin
Born of the same parents' culture supernatant or its purified part carry out of the invention for removing cysteine variant in Su Jin monoclonal antibody product
Method.
There is document to prove, the Su Jin monoclonal antibody product of Su Jin monoclonal antibody, particularly the recombination generation of Su Jin monoclonal antibody mammalian cell
In cysteine variant acidic variant (Acidic Variant) is shown as in CEX charge heterogeneity map, can
It analyzes it to be characterized by CEX charge heterogeneity map and whether there is and its relative amount.Therefore, the present inventor is logical
PH gradient CEX analysis method is crossed, the Su Jin monoclonal antibody experiment using before and after technique redox described in CN107001460A patent
Sample and Su Jin single antigen grind the medicine (Cosentyx of Novartis Co., Ltd's production®) head to head compared, it is determined that Su Jin monoclonal antibody
Synergy of the cysteine variant on pH gradient CEX map.Based on this, Su Jin monoclonal antibody system of the invention is developed
The minimizing technology of cysteine variant in product.
Compared with prior art, method of the invention carries out Su Jin monoclonal antibody or its product using anion-exchange chromatography pure
Change, specially removes the cysteine variant of Su Jin monoclonal antibody.By carrying out conductance to Poros HS, Capto S ImpAct
Gradient elution technique is groped, and is carried out pH technique to Poros HS, Poros XS, Capto MMC ImpRes and is groped, passes through
Cysteine variant chromatographic peak situation of change before and after the chromatography on pH gradient CEX map in Su Jin monoclonal antibody product is detected,
It demonstrates and anion-exchange chromatography is carried out using compound anion-exchange chromatography filler, it is different cysteine can be effectively removed
Structure body, or even be completely removed.Method and process simple and stable of the invention, it is reproducible, and the normal of antibody will not be destroyed
Structure；Compared with prior art products, the quality of Su Jin monoclonal antibody is also improved.
Detailed description of the invention
Hereinafter, carrying out the embodiment that the present invention will be described in detail in conjunction with attached drawing, in which:
Fig. 1 shows the CEX map of reference preparation in embodiment 1, the front and back Redox laboratory sample.
Fig. 2 shows the CEX map of reference preparation in embodiment 2, chromatography front and back laboratory sample.
Fig. 3 shows the CEX map of reference preparation in embodiment 3, chromatography front and back laboratory sample.
Specific embodiment
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Medicine as used in the following examples
Material raw material, reagent material etc. are commercially available products unless otherwise specified.
The laboratory sample of Su Jin monoclonal antibody used in embodiment is using the CHO(China storehouse using the building of DNA recombinant technique
Mouse ovary) cell expression Su Jin monoclonal antibody.Cell culture process is common monoclonal antibody culture process, is included in shaking flask and expands training
Support, be then seeded into 2 L(laboratory samples 1) or 20 L(laboratory samples 2) cultivate 14 days in stream plus bioreactor after harvest
Then cell culture supernatant obtains the sample of the monoclonal antibody containing Su Jin by a step Protein A affinitive layer purification.
Embodiment 1The characterization of chromatographic peak of the cysteine variant of Su Jin monoclonal antibody on pH gradient CEX map
Using oxidative deamination process described in patent application publication CN107001460A (Redox) to laboratory sample 1 into
Row selective reduction, the laboratory sample after obtaining Redox.
Then the laboratory sample before and after Redox is analyzed using pH gradient CEX, while medicine is ground with the original of Su Jin monoclonal antibody
(it is purchased from Novartis Co., Ltd, Cosentyx®, lot number S0029) and it is used as reference preparation, it carries out head to head comparing analysis.
PH gradient CEX method is as follows:
Liquid phase systems: Waters Acquity Arc
Chromatographic column: 4 × 250mm of Thermo ProPac WCX-10
Mobile phase A: 4 mM Piperazine, 4 mM Imidazole, 4 mM Tris, pH 5.0
Mobile phase B: 4 mM Piperazine, 4 mM Imidazole, 4 mM Tris
Mobile phase C:500 mM NaCl
Column temperature: 40 DEG C
Flow velocity: 1.0 ml/min
Sample treatment: with Su Jin monoclonal antibody protein meter, three kinds of samples are diluted to 1 mg/ml or so using 0.1 × PBS, so
The CpB enzyme of 1:20 afterwards: the ratio (mass ratio) of protein content is added CpB enzyme in sample, and 37 DEG C are incubated for 30 minutes, then directly
Sample introduction, about 50 μ g of sample volume.
Linear gradient elution is carried out with mobile phase A, Mobile phase B and mobile phase C, with 55% ~ 0% mobile phase A, 43.6%
~ 96.8% Mobile phase B and 1.4% ~ 3.2% mobile phase C elute 38 minutes.
The CEX map of acquisition is shown in Fig. 1.
As seen from Figure 1, acid peak (peak on the main peak left side, arrow the are signified) chromatographic peak profile of laboratory sample and guarantor after Redox
Stay the time consistent with reference preparation.Therefore, can confirm after Redox, the cysteine variant of laboratory sample is gone back
It originally was normal monoclonal antibody molecule.The CEX map of laboratory sample before and after Redox is compared, it is thus identified that cysteine becomes
Synergy of the allosome on the chromatogram of pH gradient CEX method.
Embodiment 2Using the removal of the cysteine variant of the Su Jin monoclonal antibody of the method for the present invention
With CaptoTMAdhere ImpRes filler (being purchased from GE company) dress column, is balanced using 25 mM Tris-HCl, pH 8.9
Chromatographic column is flat to baseline.
Laboratory sample 2 is adjusted into pH 8.5 ± 0.1, to be not higher than 60 mg antibody/mL filler loading, reuses 25 mM
It is flat to baseline that Tris-HCl, pH 8.9 balances chromatographic column.
Linear gradient elution is carried out with mobile phase A and Mobile phase B, wherein mobile phase A is 25mM Tris-HCl, pH 8.9,
Mobile phase B is 50 mM Tris-HCl, pH 7.1, elutes 20 CV with the mobile phase A of 70-0% and the Mobile phase B of 30%-100%,
Wherein 100% Mobile phase B elutes 8 CV, during which monitors the absorbance at A280, and the absorbance at A280 is greater than 100mAu
Start to collect elutriated fraction, is less than 100mAu and stops collecting.Laboratory sample after being chromatographed.
Then it is analyzed using laboratory sample of the pH gradient CEX to chromatography front and back, while medicine is ground with the original of Su Jin monoclonal antibody
(it is purchased from Novartis Co., Ltd, Cosentyx®, lot number S0029) and it is used as reference preparation, it carries out head to head comparing analysis.
PH gradient CEX method and sample treatment etc. are carried out referring to described in embodiment 1.
It the results are shown in Table 1 and Fig. 2.Alkaline peak is the peak on the right side of main peak.
The CEX result of laboratory sample after 1. reference preparation of table, laboratory sample and chromatography
Table 1 and Fig. 2's goes out as the result is shown, from map and numerically, after laboratory sample is handled by method of the invention,
Cysteine isomers is completely removed, and the whole content at acid peak has very significant decline；From the point of view of main peak content,
Reference preparation is also significantly better than by the processed sample quality of method of the invention.
Embodiment 3Using the removal of the cysteine variant of the Su Jin monoclonal antibody of the method for the present invention
With Bestarose Diamond MMA filler (being purchased from Bo Gelong company) dress column, 25 mM Tris-HCl, pH 8.9 are used
It is flat to baseline to balance chromatographic column.
Laboratory sample 2 is adjusted into pH 8.5 ± 0.1, to be not higher than 50 mg antibody/mL filler loading, reuses 25 mM
It is flat to baseline that Tris-HCl, pH 8.9 balances chromatographic column.
Linear gradient elution is carried out with mobile phase A and Mobile phase B, wherein mobile phase A is 25mM Tris-HCl, pH 8.9,
Mobile phase B is 50 mM Tris-HCl, pH 7.1, elutes 20 CV with the mobile phase A of 70-0% and the Mobile phase B of 30%-100%,
Wherein 100% Mobile phase B elutes 8 CV, during which monitors the absorbance at A280, and the absorbance at A280 is greater than 100mAu
Start to collect elutriated fraction, is less than 100mAu and stops collecting.Laboratory sample after being chromatographed.
Then it is analyzed using laboratory sample of the pH gradient CEX to chromatography front and back, while medicine is ground with the original of Su Jin monoclonal antibody
(it is purchased from Novartis Co., Ltd, Cosentyx®, lot number SJ280) and it is used as reference preparation, it carries out head to head comparing analysis.
PH gradient CEX method and sample treatment etc. are carried out referring to described in embodiment 1.
It the results are shown in Table 2 and Fig. 3.Alkaline peak is the peak on the right side of main peak.
The CEX result of laboratory sample after 2. reference preparation of table, laboratory sample and chromatography
Table 2 and Fig. 3's goes out as the result is shown, and from map and numerically, laboratory sample uses the compound yin of same type of other brands
After ion chromatography filler, other conditions and method same treatment of the invention, cysteine isomers is completely removed, and
The whole content at acid peak has very significant decline；Also, the result of the result and embodiment 2 is without significant difference.
Embodiment 4Using the detection of activity and other original properties that the Su Jin monoclonal antibody that the method for the present invention obtains retains
Chromatography method of the invention is carried out described in reference implementation example 2 to laboratory sample 2.
Then, purity is carried out to the laboratory sample 2 after chromatography and impurity, structure and modification, antigen-binding activity etc. is crucial
The analysis of qualitative attribute (CQA), while medicine is ground (purchased from Novartis Co., Ltd, Cosentyx with the original of Su Jin monoclonal antibody®, lot number S0029
And SJ280) it is used as reference preparation, it is compared.Testing result is shown in Table 3.
Table 3
By the data in upper table it is found that the antigen-binding activity of the laboratory sample after chromatography, structure and modification and reference preparation one
It causes, and purity and impurity are better than reference preparation.Therefore, it is known that laboratory sample still remains its due purity, structure after chromatography
And activity.
Embodiment 5Compared to the detection of the technical effect of the optimization of oxidative deamination process described in CN107001460A patent.
According to described in embodiment 1, oxidative deamination process described in patent application publication CN107001460A is used
(Redox) selective reduction is carried out to laboratory sample 1, the laboratory sample 1 after obtaining Redox.
According to described in embodiment 2, Image processing is carried out to laboratory sample 2 using method of the invention, after being chromatographed
Laboratory sample 2.
The non-reduced electrophoretic analysis of CE-SDS is carried out to the laboratory sample 2 of laboratory sample 1 and chromatography front and back before and after Redox,
Medicine is ground (purchased from Novartis Co., Ltd, Cosentyx with the original of Su Jin monoclonal antibody simultaneously®, lot number S0029) and it is used as reference preparation, compared
To analysis.Testing result is shown in Table 4.
Table 4
As the data of table 4 it is found that after being handled using oxidative deamination process described in CN107001460A patent laboratory sample 1,
The low molecular weight impurities total amount of laboratory sample 1 has significant increase；And after using chromatography technique to handle laboratory sample 2, experiment
Before the low molecular weight impurities total amount of sample 2 is relatively handled, do not change significantly.
It follows that low molecular weight impurities will not be generated using chromatography process sample, and the attribute is monoclonal antibody medicine
Key Quality attribute, therefore this is chromatography technique of the invention with respect to oxidative deamination process described in CN107001460A patent
A big advantage.
In addition, oxidative deamination process described in CN107001460A patent is compared to classical monoclonal antibody chromatographic purifying
Method, operation is more complicated, needs tighter control condition, and it also requires special process equipment, therefore increase technique
Complexity, improve process costs, be also unfavorable for being mass produced.This is also chromatography technique of the invention with respect to Redox's
Another big advantage.
Therefore, the present invention eliminates the cysteine variant of Su Jin monoclonal antibody by simple and easy method.Compared to existing
There is technology, the chromatography method that the present invention uses reduces the complexity of technique, saved the operating time, and can promote product
Quality.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and as long as it does not depart from the spirit of the invention, should belong to the model of appended claims of the present invention
It encloses.
Claims (15)
1. a kind of method for removing cysteine variant in Su Jin monoclonal antibody product, the method includes making the Soviet Union
Golden monoclonal antibody product is subjected to anion-exchange chromatography, wherein the Su Jin monoclonal antibody product includes that mammalian cell recombination generates
Su Jin monoclonal antibody.
2. the method according to claim 1, wherein the Su Jin monoclonal antibody product is that recombination generates Su Jin monoclonal antibody
The culture supernatant of mammalian cell, or the purified part for the culture supernatant.
3. the method according to claim 1, wherein the anion-exchange chromatography is handed over using compound anion
Change chromatographic stuffing progress.
4. according to the method described in claim 3, it is characterized in that, the anion-exchange chromatography uses mobile phase A and flowing
The linear gradient elution of phase B, wherein mobile phase A is 5-50 mM Tris-HCl buffer, pH 8.9, Mobile phase B 10-100
MM Tris-HCl buffer, pH 7.1, linear gradient elution include: that the mobile phase A of 70-0% and the Mobile phase B of 30%-100% are washed
De- at least 20 column volumes (CV), wherein 100% Mobile phase B elutes at least eight CV.
5. the method according to claim 1, wherein the described method includes:
1) column is filled with compound anion-exchange chromatography filler, uses 5-50 mM Tris-HCl buffer, 8.9 balance layer of pH
It is flat to baseline to analyse column；
2) by the sample solution tune pH value containing the Su Jin monoclonal antibody to 8.5 ± 0.1, loading is buffered using 5-50 mM Tris-HCl
Liquid, it is flat to baseline that pH 8.9 balances the chromatographic column；
3) linear gradient elution is carried out with mobile phase A and Mobile phase B, wherein mobile phase A is 5-50 mM Tris-HCl buffering
Liquid, pH 8.9, Mobile phase B are 10-100 mM Tris-HCl buffer, pH 7.1, by the absorbance at monitoring A280 in egg
Elutriated fraction is collected after white appearance.
6. according to the method described in claim 5, it is characterized in that, the Tris-HCl buffer is 25 mM in step 1)
Tris-HCl buffer, pH 8.9.
7. according to the method described in claim 5, it is characterized in that, the sample solution is that recombination generates Su Jin in step 2)
The culture supernatant of the mammalian cell of monoclonal antibody, or the purified part for the culture supernatant.
8. according to the method described in claim 5, it is characterized in that, applied sample amount is to revive not higher than described in 60 mg in step 2)
Filler described in golden monoclonal antibody/mL.
9. according to the method described in claim 5, it is characterized in that, applied sample amount is to revive not higher than described in 50 mg in step 2)
Filler described in golden monoclonal antibody/mL.
10. according to the method described in claim 5, it is characterized in that, being delayed in step 2) using the Tris-HCl in step 1)
It is flat to baseline that fliud flushing balances the chromatographic column.
11. according to the method described in claim 5, it is characterized in that, the mobile phase A is 25 mM Tris- in step 3)
HCl buffer, pH 8.9, the Mobile phase B are 50 mM Tris-HCl buffers, pH 7.1.
12. according to the method for claim 11, which is characterized in that the linear gradient elution includes: the flowing with 70-0%
The Mobile phase B of phase A and 30%-100% elute 20 CV, wherein 100% Mobile phase B elutes 8 CV.
13. according to the method described in claim 5, it is characterized in that, respectively rate-determining steps 2) described in sample solution and step 3)
Described in mobile phase flow velocity so that the retention time of first eluting peak be greater than 3 minutes.
14. method according to any one of claim 1 to 13, which is characterized in that the described method includes:
1) with CaptoTMAdhere ImpRes filler or Bestarose Diamond MMA filler fill column, use 25 mM
Tris-HCl buffer, it is flat to baseline that pH 8.9 balances chromatographic column；
2) by the sample solution tune pH 8.5 ± 0.1 containing the Su Jin monoclonal antibody, to revive not higher than 60 mg or not higher than described in 50 mg
Filler loading described in golden monoclonal antibody/mL, reuses 25 mM Tris-HCl buffers, and pH 8.9 balances the chromatographic column to baseline
It is flat；
3) linear gradient elution is carried out with mobile phase A and Mobile phase B, wherein mobile phase A is 25 mM Tris-HCl buffers,
PH 8.9, Mobile phase B are 50 mM Tris-HCl buffers, pH 7.1, with the flowing of the mobile phase A of 70-0% and 30%-100%
Phase B elutes 20 CV, wherein 100% Mobile phase B elutes 8 CV, the absorbance at A280 is during which monitored, after albumen appearance
Collect elutriated fraction.
15. a kind of preparation method of Su Jin monoclonal antibody, the preparation method includes making mammalian cell expression Su Jin monoclonal antibody, is received
Obtain cell culture supernatant and optionally carry out cell culture supernatant purifying, then make the cell culture supernatant or its
Purified part is subjected to method described in any one of claims 1 to 14.
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| WO2022180644A1 (en) * | 2021-02-25 | 2022-09-01 | Dr. Reddy's Laboratories Limited | Thiol variants and analytical methods thereof |
| CN114085291A (en) * | 2022-01-19 | 2022-02-25 | 迈威(上海)生物科技股份有限公司 | Method for reducing or eliminating CEX acidic peak of recombinant protein |
| CN114085291B (en) * | 2022-01-19 | 2022-04-12 | 迈威(上海)生物科技股份有限公司 | Method for reducing or eliminating CEX acidic peak of recombinant protein |
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