CN105315369A - Protein purification using cation exchange chromatography - Google Patents
Protein purification using cation exchange chromatography Download PDFInfo
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Abstract
The present invention provides a method for purifying pollutant-containing protein through cation exchange chromatography, wherein the composition, the concentration, the pH value and theconductivity of buffer liquids of sample loading, washing, elution and the like are selectively optimized to achieve good removal effects of HCP, DNA and other pollutants, excellent aggregate removal effect and high target protein yield. According to the present invention, the method has characteristics of simple operation step, simple operation and production cost reducing, and is suitable for large-scale industrial antibody separation and purification.
Description
Technical field
The invention belongs to protein purification field, be specifically related to utilize cation-exchange chromatography method purifying to contain the antibody of pollutent.
Background technology
Along with the development of biotechnology, the protein more and more comprising antibody is prepared on a large scale by bio-reactor (as engineering bacteria or engineering cell strain) or other modes, to obtain containing target protein matter culture, to carry out separation and purification be requisite step in its production process downstream technique, how to pass through the improvement to method of purifying protein, optimize protein purification condition, further increasing removes the efficiency of pollutent, and improving the purity of protein and yield etc. is Problems existing always in protein suitability for industrialized production.
It is generally import in host cell by the recombinant vectors containing goal gene that utilizing works bacterium or engineering cell strain express target protein matter method, carries out cell cultures under suitable conditions, thus obtains the expression of target protein matter.In the culture obtained except target protein, also comprise host cell proteins (Hostcellprotein, HCP), host cell DNA, RNA, nutrient media components, target protein variant and/or aggregate, target protein fragment and the toxin that may exist, virus, microorganism etc. at interior other pollutents etc.; Therefore the purer protein meeting application purpose could must be obtained by a series of purge process.
Ion exchange chromatography (IonExchangeChromatography is referred to as IEC) is a kind of purification process conventional in current biochemical field.Ion exchange chromatography take ion-exchanger as stationary phase, reaches a kind of chromatography method being separated object according to the component ion in moving phase and the counterion on exchanger by reversible exchange.So-called ion-exchange, refer to that a certain ion in solution and the another kind of ion be combined on carrier carry out the process of reversible exchange, the ion in the ionic bond in ie in solution to carrier on carrier is replaced.If carrier combines positively charged active group, then exchangeable anions, it is anionite; If carrier combines electronegative active group, then exchangeable cation, it is cationite.Under certain conditions, in mixture the different proteins charged character of institute and electric charge how much different, what have can be combined with specific ion exchanger, and what have can not be in conjunction with; In the protein that can be combined with ion-exchanger, the size of bonding force is also not necessarily identical, then adopts suitable elution requirement, effectively can be separated them.Ion exchange chromatography is widely used in the separation and purification of various biochemical substances as amino acid, albumen, carbohydrate, Nucleotide etc. at present.
Due to complicacy and the other factors impact of biological sample, general biomacromolecule and ion-exchanger in conjunction with situation meter more difficult to estimate, the particularly complicacy of protein structure, the number of its bonding force with ion-exchanger and the electric charge entrained by protein molecule is relevant, still has certain relation with other character such as bulk of molecule and charge placement of protein.Therefore, particularly be used as the antibody of medicine for different protein, optimized choice goes out suitable cation exchange chromatography technique, reduces production cost as far as possible and improves antibody yield, to obtain the antibody of higher degree, often need to be groped by a large amount of experiments.
Patent 200880119331.X discloses a kind of method by cation-exchange chromatography antibody purification, wherein comes to use high pH cleaning step to clear the pollution off before wash-out expects antibody at the elution buffer using specific conductivity to raise; By comprising at least two cleaning steps in cation exchange purification scheme, wherein at least the first is carried out at high pH (about pH6.8 or larger), can significantly improve purifying effect, achieve higher step productive rate.This patent used high pH cleaning step before being increased in antibody elution, and to clear the pollution off, but increasing of processing step can make the process time extend, and adds production cost.
Summary of the invention
In order to few ion exchange chromatography step of being tried one's best by use carries out purifying, reduce production cost, and obtain the antibody of higher degree, the invention provides a kind of method that cation-exchange chromatography purifying contains the antibody of pollutent, it comprises the following steps:
A) loading: the solution containing antibody and pollutent is loaded on cationic exchange coloum,
B) clean: clean cation exchange material with cleaning buffer solution,
C) wash-out: with elution buffer from object antibody under wash-out cation exchange material.
The present invention, by being optimized by lot of experiments the composition of various damping fluid and concentration, pH, specific conductivity etc., reaches good pollutant removal and higher yield.
A preferred embodiment of the present invention, the specific conductivity of cleaning buffer solution is 5-9ms/cm, and preferred specific conductivity is 6-8ms/cm.
A preferred embodiment of the present invention, the pH of cleaning buffer solution is 5.5-5.9, and preferred pH is 5.7-5.9;
Buffer substance in cleaning buffer solution in the present invention is MES, citric acid, phosphoric acid, Citrate trianion, phosphoric acid salt or its two kinds and two or more mixtures; Containing the salt of two or more mixtures being selected from Repone K (KCl), sodium-chlor (NaCl), salt of wormwood, sodium acetate, potassium sulfate, sodium sulfate, Citrate trianion, phosphoric acid salt or these compositions in damping fluid.
In a preferred embodiment of the present invention, cleaning buffer solution is the solution containing 20-25mMMES, 40-50mMNaCl;
In the preferred embodiment of the invention, the specific conductivity of elution buffer is 9-14ms/cm, the preferred 9-10ms/cm of specific conductivity of elution buffer, more preferably 9.0-9.6ms/cm.
In another preferred embodiment of the present invention, the pH of elution buffer is 5.5-5.9, and preferred pH is 5.7-5.9.
Buffer substance in elution buffer in the present invention is MES, citric acid, phosphoric acid, Citrate trianion, phosphoric acid salt or its two kinds and two or more mixtures; Containing the salt of two or more mixtures being selected from Repone K (KCl), sodium-chlor (NaCl), salt of wormwood, sodium acetate, potassium sulfate, sodium sulfate, Citrate trianion, phosphoric acid salt or these compositions in damping fluid.
In another preferred embodiment of the present invention, elution buffer is the solution containing 20-25mMMES, 75-110mMNaCl, the preferred 80-95mMNaCl of NaCl concentration.
Utilizing the inventive method before carrying out cation-exchange chromatography, period or afterwards, other purification steps one or more can carried out to the antibody containing pollutent.Such as by carrying out cation-exchange chromatography again after the preliminary purification of Depth Filtration, protein A affinity chromatography, better antibody purification effect can be obtained.
Be in conjunction with the antibody of people VEGF (human vascular endothelial growth factor) or the antibody in conjunction with h CD20 with the antibody that cation-exchange chromatography purifying process of the present invention is purified.
The antibody in conjunction with people VEGF that can be used for purifying process of the present invention refer to comprise be combined with people VEGF polyclonal antibody, monoclonal antibody, there are the antibody fragment of binding specificity or the various modified forms etc. of antibody; The VEGF antibody that the present invention preferably can be used for purifying, for having the antibody of the light chain hypervariable region sequence shown in the heavy chain hypervariable region sequence shown in SEQIDNO:1 (CDRH1), SEQIDNO:2 (CDRH2), SEQIDNO:3 (CDRH3) and SEQIDNO:4 (CDRL1), SEQIDNO:5 (CDRL2), SEQIDNO:6 (CDRL3), is more preferably rhuMAb-VEGF (Bevacizumab).
VEGF antibody of the present invention can be obtained by following methods: by heavy chain CDR region sequence Transplanted Human heavy chain subgroup III framework of the SEQIDNO:1-3 of synthesis, the light chain CDR region sequence of SEQIDNO:4-6 is transplanted on people's light chain κ subgroup 1 framework, obtain VEGF antibody VH and VL sequence, again VH and VL is cloned into respectively on the expression vector containing human IgG CH and constant region of light chain, by the expression vector cotransfection Chinese hamster ovary celI containing VEGF antibody heavy chain and light chain, cultivate under proper culture conditions, the VEGF antibody of expressing cho cell is secreted in the outer substratum of born of the same parents, collect containing VEGF antibody cell culture, after cell culture is carried out preliminary purification, obtain the mixture containing VEGF antibody and pollutent being used for carrying out further ion exchange chromatography.
The antibody in conjunction with h CD20 that can be used for purifying process of the present invention refer to comprise be combined with h CD20 polyclonal antibody, monoclonal antibody, there are the antibody fragment of binding specificity or the various modified forms etc. of antibody, the preferably available anti-CD20 antibodies of the present invention, for having the antibody of the light-chain variable sequence shown in the weight chain variabl area sequence shown in SEQIDNO:7 and SEQIDNO:8, is more preferably Rituximab (Rituximab).
Anti-CD20 antibodies of the present invention can be obtained by following methods: be cloned on the expression vector of human IgG CH and constant region of light chain respectively by the light-chain variable sequence of the weight chain variabl area sequence of the SEQIDNO:7 of synthesis and SEQIDNO:8, by the expression vector cotransfection Chinese hamster ovary celI containing anti-CD20 antibodies heavy chain and light chain, cultivate under proper culture conditions, the anti-CD20 antibodies of expressing cho cell is secreted in the outer substratum of born of the same parents, collect the cell culture containing anti-CD20 antibodies, after cell culture is carried out preliminary purification, obtain the mixture containing anti-CD20 antibodies and pollutent being used for carrying out further ion exchange chromatography.
In another embodiment of the present invention, before loading, balanced by cationic exchange coloum level pad, level pad can be same damping fluid with cleaning buffer solution.
Solution load solution containing antibody and pollutent before loading, by being optimized load solution pH, pH value ± 0.05 of selected level pad when load solution pH value is purifying.
The specific conductivity of load solution regulates according to the character of ion-exchange material, if adopt the not halophilic ion-exchange materials such as SP-HP, then need the specific conductivity≤3.5ms/cm of load solution, if adopt the halophilic fillers such as poros, the specific conductivity regulating load solution can not be needed.
In another embodiment of the present invention, exchange material used is the ion-exchange material that particle size diameter is not more than about 50 μm.In some preferred embodiments, cationic exchange coloum exchange material used is SP-HP (SP-SepharoseHP, HighPerformanceGE), CaptoSPImpRes, porosHS and porosXS (polystyrene resin; I.e. Vinylstyrene and cinnamic multipolymer), fractogel (polymethacrylate resin), the strong cation-exchanging resin resin etc. that the resolving power such as NuviaHRS are high.
The present invention, by being optimized by lot of experiments the composition of various damping fluid and concentration, pH, specific conductivity etc., reaches good pollutant removal and higher target protein yield.In wherein the selection of cleaning buffer solution specific conductivity being optimized, by improving specific conductivity, while removing pollutent equally, ensure that required antibody is still combined on exchange material, and can be same damping fluid with level pad, improve the simplicity of technological operation, saved production cost.Adopt a step cleaning step, obtain the removal efficiency of good HCP, DNA pollution thing, to the selection optimization of elution buffer pH value and specific conductivity, by the scope of control ph and specific conductivity, obtain excellent aggregate removal effect, higher antibody yield can be obtained again simultaneously.
PH value MettlerToledopH meter of the present invention is 24 DEG C of mensuration.
" specific conductivity " herein refers to the ability of solution conduction current.The specific conductivity of solution can be changed, as can be raised the specific conductivity of elution buffer by the salt concn increased in elution buffer by the salt concn changed in solution.The salt that may be used for raising specific conductivity includes but not limited to two or more mixtures of Repone K (KCl), sodium-chlor (NaCl), salt of wormwood, sodium acetate, potassium sulfate, sodium sulfate, Citrate trianion, phosphoric acid salt or these salt.
In the present invention conductivity value with MettlerToledo conductivity meter 24 DEG C of mensuration, measuring method be: probe is put into solution, direct reading.
For purifying containing the protein of pollutent, it can be the protein obtained by chemosynthesis or other synthetic method, or takes from the protein of natural origin, also can be the protein obtained by the cultivation of engineering bacteria or engineering cell strain.
Antibody described herein comprises polyclonal antibody, monoclonal antibody, multi-specificity antibody or has the antibody fragment of binding specificity or the various modified forms etc. of antibody.
It is generally import in host cell by the recombinant vectors containing goal gene that utilizing works bacterium or engineering cell strain express target protein matter method, carries out cell cultures under suitable conditions, thus obtains the expression of target protein matter.The host cell being suitable for cloning or expressing goal gene is prokaryotic organism, yeast cell or higher eucaryotic cells.At present for antibody drug, major part is prepared in the mode of zooblast large scale culturing.Conventional animal cell line includes but not limited to chicken embryo, porcine kidney cell, HEKC, hamster kidney cell, monkey-kidney cells, hamster kidney cell (BHK), Chinese hamster ovary cell CHO), human cervical carcinoma cell (HELA), human pneumonocyte, human liver cell, mouse mammary tumor, people's liver tumor cell etc.Wherein Chinese hamster ovary celI is current most widely used antibody expression systems.
Carry out cell cultures by seeding cells into bio-reactor, target protein is expressed, and the protein of expression can be secreted in cell culture medium, or is accumulated in cell.For being accumulated in intracellular protein, first need collecting cell to carry out cracking to cell, then by such as centrifugal, filter or ultrafiltration removing host cell and lysis produce fragment.For the protein be secreted in substratum, then directly can be contained the supernatant liquor of target protein by filtration or centrifugal acquisition.
The preferred embodiment of the invention carries out purifying to the antibody produced by animal cell culture.Preferably by the vector introduction Chinese hamster ovary (CHO) containing goal gene, by cell cultures, the culture collected containing object antibody carries out purifying.A series of pollutent can be produced, as Chinese hamster ovary protein (CHOP) by Chinese hamster ovary celI cultivation; The variant of cell DNA, nutrient media components, antibody and/or aggregate, antibody fragment and the toxin that may exist, virus, microorganism etc. are at interior other pollutents etc.Obtain containing object antibody and other pollutents cell culture fluid after, before carrying out cation-exchange chromatography, period or afterwards, other purification steps one or more can be carried out to the antibody containing pollutent.
People VEGF refers to human vascular endothelial growth factor, and (vascularpermeabilityfactor, is called for short: VPF), can induction of vascular is newborn in vivo to be also called vascular permeability factor in early days.The antibody in conjunction with people VEGF that can be used for purifying process of the present invention refer to comprise be combined with people VEGF polyclonal antibody, monoclonal antibody, there are the antibody fragment of binding specificity or the various modified forms etc. of antibody.
CD20 antigen is the surface membrane protein being positioned at bone-marrow-derived lymphocyte, it is a kind of B cell differentiation antigen, CD20 is expressed in nearly all normal B cells and malignant B cell, expresses, and do not express in hemopoietic stem cell, plasma cell and other healthy tissuess in its B cell lymphoma more than 95%.The antibody in conjunction with h CD20 that can be used for purifying process of the present invention refer to comprise be combined with the CD20 on human B lymphocyte surface polyclonal antibody, monoclonal antibody, there are the antibody fragment of binding specificity or the various modified forms etc. of antibody.
Cationite for cation-exchange chromatography refers to that stationary phase dielectric surface is combined with anionic group, the anionic group of this solid phase is combined with positively charged counterion (gegenion), protein positively charged in this counterion and solution carries out reversibility exchange.According to the ionization different in kind of active group, ion-exchanger has dividing of strongly-acid, strong basicity, slightly acidic and weakly alkaline.Available ion-exchange material comprises ion exchange resin, ion-exchange cellulose, ion exchange sephadex, sepharose, polyacrylamide gel and silica gel etc.
Multiple commercial cation exchange material is had to can be used for the present invention at present, be included in SP650S or SP650M of immobilized sulfopropyl (SP) (as TosohBiosciences) on methacrylate ester), immobilized sulfopropyl (the SP) (SP-SEPHAROSEHIGHPERFORMANCETM (SP-HP) of such as GEHealthcare on agarose, SP-SEPHAROSEFASTFLOWTM or SP-SEPHAROSEFASTFLOWXLTM), FRACTOGEL-SEHICAPTM, FRACTOGEL-SO3TM, with FRACTOPREPTM (EMDMerck), immobilized alkylsulfonyl (the S-SEPHAROSEFASTFLOWTM of such as GEHealthcare on agarose, the SUPERSpTM of CaptoSPImpRes and TosohBiosciences), immobilization sulfopropyl (Poros50HS or PorosXS as AppliedBiosystems) on the styrene-divinylbenzene that bag is crosslinked, with immobilized sulphur isobutyl-(FractogelEMDSO3 as Merck-Millipore) on the methacrylic ester of polymerization feeler, immobilized sulfo group (SCeramicHyperD as PallCorporation) on the ceramic bead that hydrogel is full of, and immobilized sulfo group (NuviaHS as Bio-rad) on polymkeric substance.The preferred cation exchange material of the present invention comprises SP-HP (SP-SepharoseHP, HighPerformanceGE), CaptoSPImpRes, porosHS and porosXS (polystyrene resin; I.e. Vinylstyrene and cinnamic multipolymer), fractogel (polymethacrylate resin), NuviaHRS etc.
Mixture containing antibody and pollutent is after cation-exchange chromatography technique purifying of the present invention, pollutant removal is remarkable, wherein host cell proteins matter has higher removal efficiency, in certain embodiments, host cell proteins matter content even content lower than 20ppm, even lower than 10ppm; The content of DNA is lower than 1pg/mgMab; The Protein A content <10ppm leached; The purifying of antibody is carried out through present invention process, aggregate separating effect highly significant, after purifying, aggregate content is lower than 5%, and aggregate content is even lower than 1% in certain embodiments, in the antibody-solutions of purifying process purifying of the present invention, substantially do not detect the composition such as substratum, toxin.
Accompanying drawing explanation
The content of principal pollutant after accompanying drawing 1:5 different batches VEGF antibody sample cation displacement chromatography
Below by specific embodiment, the present invention is illustrated, but any restriction is not formed to the present invention.
Specific embodiment
Embodiment 1, preparation containing the mixture of VEGF antibody and pollutent
By on VEGF antibody heavy chain CDR region sequence Transplanted Human heavy chain subgroup III framework of the SEQIDNO:1-3 of synthesis, the light chain CDR region sequence of SEQIDNO:4-6 is transplanted on people's light chain κ subgroup 1 framework, obtain VEGF antibody VH and VL sequence, again VH and VL is cloned into respectively on the expression vector containing human IgG CH and constant region of light chain of structure, by the expression vector cotransfection Chinese hamster ovary celI containing VEGF antibody heavy chain and light chain, cultivate under proper culture conditions, the VEGF antibody of expressing cho cell is secreted in the outer substratum of born of the same parents, collect containing VEGF antibody cell culture, after cell culture is carried out preliminary purification, obtain the mixture containing VEGF antibody and pollutent being used for carrying out further ion exchange chromatography.
Embodiment 2 contains the preparation of the mixture of anti-CD20 antibodies and pollutent
The light-chain variable sequence of the weight chain variabl area sequence of the SEQIDNO:7 of synthesis and SEQIDNO:8 is cloned on the expression vector containing human IgG CH and constant region of light chain of structure respectively, by the expression vector cotransfection Chinese hamster ovary celI containing anti-CD20 antibodies heavy chain and light chain, cultivate under proper culture conditions, the anti-CD20 antibodies of expressing cho cell is secreted in the outer substratum of born of the same parents, collect the cell culture containing anti-CD20 antibodies, after cell culture is carried out preliminary purification, obtain the mixture containing anti-CD20 antibodies and pollutent being used for carrying out further ion exchange chromatography.
Embodiment 3: the purifying of VEGF antibody
Experimental technique and result: the chromatography column adopting post height of bed position 20cm, is packed into post by Zeo-karb SP-HP; With the chromatography column that equilibration buffer installs; After column equilibration, the solution containing VEGF antibody and pollutent obtained by embodiment 1 is slowly added along chromatography column tube wall; Chromatography column is cleaned with cleaning buffer solution; Then carry out antibody elution with elution buffer, collect elutriant.Each process parameter that the present embodiment adopts sees the following form 1.
Table 1: the purifying process condition that the present embodiment adopts:
Table 2: purified antibodies monomer principal pollutant content
After cation-exchange chromatography purification step, in gained antibody-solutions, antibody monomer and each major impurity content are in table 2, and experimental result shows, the pollutent in VEGF antibody mixture is removed significantly, the yield of antibody is also higher, and does not detect the composition of substratum.
In addition, by the optimization of processing parameter, in experiment, level pad and cleaning buffer solution can the same damping fluids of choice for use, can reduce process costs further.
Design the impact on pollutant removal and protein yield such as different concns, pH value and the conductance etc. of investigating each step of purifying process solution used respectively by experiment, filter out for the preferred concentration range of VEGF antibody cation-exchange chromatography purifying process, pH value and specific conductivity (see table 3), under purifying process preferred concentration range of the present invention, pH value and specific conductivity condition, in antibody-solutions after purified, host cell proteins matter content even content lower than 20ppm, even lower than 10ppm; The content of DNA is lower than 1pg/mgMab; The Protein A content <10ppm leached; Aggregate separating effect highly significant, after purifying, aggregate content is lower than 5%, and aggregate content is even lower than 1% in certain embodiments,
The preferred concentration range of table 3, VEGF antibody cation-exchange chromatography purifying process, pH value and specific conductivity
Embodiment 4: the purifying of anti-CD20 antibodies
Experimental technique and result: the chromatography column adopting height of bed position 10cm, is packed into post by Zeo-karb (POROSHS50); With the chromatography column that equilibration buffer installs; After column equilibration, the solution containing anti-CD20 antibodies and pollutent obtained by embodiment 2 is slowly added along chromatography column tube wall; Chromatography column is cleaned with cleaning buffer solution; Then carry out antibody elution with elution buffer, collect elutriant.The processing parameter that the present embodiment adopts sees the following form 4.
Table 4: purifying process condition:
Table 5: purified antibodies monomer and principal pollutant content
After cation-exchange chromatography purification step, in gained antibody-solutions, antibody monomer and each major impurity content are in table 5, and experimental result shows, the pollutent in anti-CD20 antibodies mixture is removed significantly, the yield of antibody is also higher, and does not detect the composition of substratum.
Design the impact on pollutant removal and protein yield such as different concns, pH value and the conductance etc. of investigating each step of purifying process solution used respectively by experiment, filter out for the preferred concentration range of anti-CD20 antibodies cation-exchange chromatography purifying process, pH value and specific conductivity (see table 6).Under purifying process preferred concentration range of the present invention, pH value and specific conductivity condition, in the antibody-solutions after purified, host cell proteins matter content even content lower than 20ppm, even lower than 10ppm; The content of DNA is lower than 1pg/mgMab; The Protein A content <10ppm leached; Aggregate separating effect highly significant, after purifying, aggregate content is lower than 5%, and aggregate content is even lower than 1% in certain embodiments,
The preferred volumetric molar concentration scope of table 6, anti-CD20 antibodies cation-exchange chromatography purifying process, pH value and specific conductivity
Embodiment 5: the stability of present invention process
Experimental technique: the chromatography column adopting height of bed position 20cm, is packed into post by Zeo-karb SP-HP; With the chromatography column that equilibration buffer installs; After column equilibration, the solution containing VEGF antibody and pollutent obtained by embodiment 1 is slowly added along chromatography column tube wall; Chromatography column is cleaned with cleaning buffer solution; Then carry out antibody elution with elution buffer, collect elutriant.The each processing step parameter of the present embodiment sees the following form 7, has been carried out the purifying of 5 batches of laboratory samples by this processing step.
Table 7: purifying process parameter
Experimental result (see accompanying drawing 1) shows, 5 batch samples are respectively through after cation-exchange chromatography purification step, each all obtaining is removed effectively, present invention process not only goes effective, and it is easy and simple to handle, between different batches, removal effect is consistent, and purifying process of the present invention has good stability.
Embodiment 6: purifying process of the present invention compares with 200880119331.X patent purifying process
Experimental technique: the chromatography column adopting height of bed position 20cm, is packed into post by Zeo-karb SP-HP; Adopt the purifying process condition of table 8 respectively, carry out the purifying of the solution of VEGF antibody and pollutent, often kind of purifying process repeats 5 times respectively and carries out purification experiment, after collecting elutriant respectively, measure the content in the antibody-solutions obtained through patent 200880119331.X technique and present invention process purifying, compare effect and the antibody yield of patent 200880119331.X processing step and purification process of the present invention removal.
Table 8: process for purification of antibodies compares
Table 9: antibody monomer and principal pollutant comparision contents
Note: in table 9, numeric representation is: mean+SD (
)
Experimental result (see table 9), analyze through T inspection statistics, after present invention process purifying, in gained antibody-solutions, the content of HCP, DNA and lower-molecular substance is compared with 200880119331.X patent technique purification result, P value is all greater than 0.05, and therefore purifying process of the present invention has the removal effect of same HCP, DNA and lower-molecular substance preferably; Antibody monomer content and aggregate content and 200880119331.X patent technique purification result are through T check analysis, and P value <0.01, has pole significant difference; Therefore purifying process of the present invention has higher aggregate pollutant removal, thus obtains the object antibody monomer of more high-content.
To sum up experimental result shows, purifying process of the present invention is compared with 200880119331.X patent technique, present invention process step decreases the step using high ph-values cleaning buffer solution to wash before wash-out expects antibody, reach the removal effect of the pollutents such as good HCP, DNA equally, and there is the removal efficiency of more excellent aggregate.Operation steps of the present invention is simple, easy and simple to handle, reduce production cost, and level pad and cleaning buffer solution can adopt same damping fluid, can reduce costs further, is applicable to the antibody separation and purification of heavy industrialization.
Claims (12)
1. utilize a method for cation-exchange chromatography antibody purification, comprise the steps:
A) loading: the solution containing antibody and pollutent is loaded on cationic exchange coloum,
B) clean: clean cation exchange material with cleaning buffer solution,
C) wash-out: with elution buffer from object antibody under wash-out cation exchange material.
2. method according to claim 1, wherein the specific conductivity of cleaning buffer solution is 5-9ms/cm, preferred 6-8ms/cm.
3. method according to claim 2, wherein the pH of cleaning buffer solution is 5.5-5.9, and preferred pH is 5.7-5.9.
4. method according to claim 3, buffer substance wherein in cleaning buffer solution is MES, citric acid, phosphoric acid, Citrate trianion, phosphoric acid salt or its two or more mixture, containing the salt of two or more mixtures being selected from Repone K, sodium-chlor, salt of wormwood, sodium acetate, potassium sulfate, sodium sulfate, Citrate trianion, phosphoric acid salt or these compositions in damping fluid; Preferred cleaning buffer solution is the solution containing 20-25mMMES, 40-50mMNaCl.
5. method according to claim 1, wherein the specific conductivity of elution buffer is 9-14ms/cm, preferred 9-10ms/cm, more preferably 9.0-9.6ms/cm.
6. method according to claim 5, wherein the pH of elution buffer is 5.5-5.9, and preferred pH is 5.7-5.9.
7. method according to claim 6, wherein in elution buffer, buffer substance is MES, citric acid, phosphoric acid, Citrate trianion, phosphoric acid salt or its two or more mixture, containing the salt of two or more mixtures being selected from Repone K, sodium-chlor, salt of wormwood, sodium acetate, potassium sulfate, sodium sulfate, Citrate trianion, phosphoric acid salt or these compositions in damping fluid; Preferred elution buffer is the solution containing 20-25mMMES, 75-110mMNaCl.
8. method according to claim 1, wherein said antibody is the antibody in conjunction with people VEGF or the antibody in conjunction with h CD20.
9. the method according to claim 1-6, balanced cationic exchange coloum level pad before loading.
10. method according to claim 9, level pad can be same damping fluid with cleaning buffer solution.。
11. methods according to claim 10, the pH of load solution is pH ± 0.05 of selected level pad.
12. methods according to claim 1-6, the exchange material that wherein cationic exchange coloum is used is the ion-exchange material that particle size diameter is not more than about 50 μm; Preferred ion exchange column exchange material used is the strong cation exchange material that the resolving power such as SP-HP, CaptoSPImpRes, porosHS, porosXS, fractogel, NuviaHRS are high.
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| CN106380519A (en) * | 2016-10-17 | 2017-02-08 | 深圳万乐药业有限公司 | Purification method of monoclonal antibodies |
| CN106380519B (en) * | 2016-10-17 | 2019-11-01 | 深圳万乐药业有限公司 | A kind of purification process of monoclonal antibody |
| CN111051495A (en) * | 2017-08-21 | 2020-04-21 | 凸版印刷株式会社 | Primary culture method |
| CN110272491A (en) * | 2018-03-13 | 2019-09-24 | 江苏恒瑞医药股份有限公司 | A kind of purifying process of anti-PD-1 antibody |
| CN110272491B (en) * | 2018-03-13 | 2023-01-24 | 江苏恒瑞医药股份有限公司 | Purification process of anti-PD-1 antibody |
| CN108467428A (en) * | 2018-03-26 | 2018-08-31 | 江苏中新医药有限公司 | A method of removing N-terminal truncation and abnormal variation body in rhNGF |
| CN109336970A (en) * | 2018-11-09 | 2019-02-15 | 杭州奕安济世生物药业有限公司 | The method of cation exchange chromatography antibody purification |
| CN114014906A (en) * | 2020-06-24 | 2022-02-08 | 信达生物制药(苏州)有限公司 | Method for purifying hydrophobic protein by using cation exchange chromatography |
| CN114014906B (en) * | 2020-06-24 | 2024-01-12 | 夏尔巴生物技术(苏州)有限公司 | Method for purifying hydrophobic protein by cation exchange chromatography |
| CN113150123A (en) * | 2021-05-27 | 2021-07-23 | 北京昭衍生物技术有限公司 | Method for preparing and separating protein charge isomers |
| CN114106086A (en) * | 2021-12-20 | 2022-03-01 | 苏州创胜医药集团有限公司 | Method for inactivating virus in chromatography process and application thereof |
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