CN105777903A - Method for purifying adalimumab by aid of cation exchange chromatography - Google Patents
Method for purifying adalimumab by aid of cation exchange chromatography Download PDFInfo
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Abstract
The invention discloses a method for purifying adalimumab by the aid of cation exchange chromatography. The method has the advantages that pollutants in mixtures with antibodies and the pollutants are removed by urea and betaine surfactants which are added in CEX (cation exchange) multi-step elution procedures, polymers are separated from the antibodies, then hydrophobic interaction chromatography is carried out, and accordingly good pollutant separation effects can be realized; samples are easy to regulate in chromatography procedures, drastic change of pH (potential of hydrogen) values can be prevented, and the contents of polymers in ultimately purified products can be obviously lowered.
Description
Technical field
The present invention relates to monoclonal antibody preparation, be specifically related to a kind of cation exchange chromatography method of adalimumab.
Background technology
Adalimumab is the full human monoclonal antibody of the first approved Tumor necrosis factorα (TNF α) in the whole world.Adalimumab can stop TNF α to be combined with its cell surface receptor, thus blocking the biologic activity of TNF α, finally reducing inflammation and reacts and reduce osteoclast activation, reaches to control the purpose of also relief of symptoms sign.
Conventional purification methods is Chinese patent CN102257006A such as, disclose the method catching step separation and antibody purification by affinity chromatograph, ProteinA post is owing to selectivity is high and thing ability of depolluting is strong, often carried out antibody capture by first-selection, but it is expensive, the low pH of eluting is easily caused the shortcomings such as high molecular polymer is formed, ProteinA aglucon drops, and makes increasing research begin to focus on and solves existing issue by non-ProteinA technique.Chinese patent CN103998469A discloses and substitutes ProteinA post removal antibody pollutant with cation exchange column, hydrophobic interaction post and anion-exchange column.Wherein, there is the cationic fillers improving carrying capacity and can be used to process the feed liquid of fermentation post processing, but feed liquid is after being loaded in particular column, owing to pollutant load is higher, it is adsorbed onto chromatographic column generation competition and can affect polymer removal in chromatography, make troubles to removal polymeric in subsequent purification.Chinese patent CN102119168A discloses a kind of chromatographic step using non-ionic polymers and removes protein aggregate, the additive that strengthening is dissolved is used to carry out ion-exchange chromatography subsequently, but the additive concentration used is higher, it is used in antibody purification procedures, has the risk causing antibody deactivation.
Summary of the invention
It is an object of the invention to the defect for above-mentioned prior art, it is proposed to the purification process of a kind of adalimumab that can effectively remove the pollutant such as polymer and stablize sample.Applicant finds under study for action, and the cofactor (such as the low concentration denaturant such as carbamide and surfactant) added in renaturation process can suppress aggregation to generate by the extensibility of raising folding intermediate or stretching, extension peptide chain.And these cofactors are except removing polymer better, itself also has the effect of stabilization of antibodies higher structure, it is suppressed that polymeric formation.Therefore, chromatographic run introduces multistep and rinses and eluting, and in elution buffer, add suitable additive, it is possible to assist in removing the pollutant such as polymer and stable sample.According to above-mentioned discovery, applicant proposed following technical scheme:
Technical scheme provides a kind of method of purification adalimumab, carries out cation-exchange chromatography eluting, hydrophobic interaction chromatography eluting including to the antibody of Chinese hamster ovary celI fermentation expression.The eluent of cation-exchange chromatography is the buffer of pH5.5-6.5, carries out 2 eluting altogether, wherein containing 0.1-0.5mol/L carbamide in the eluent of first time cation-exchange chromatography；The eluent of second time cation-exchange chromatography contains the betaines surfactant of mass fraction 0.1%-1.0%；Described hydrophobic interaction chromatography eluting is divided into 2 step eluting, and wherein, the eluent of first step eluting is pH5.5-6.5,0.15-0.4mol/L (NH4)2SO445mmol/LPBS buffer, the eluent of second step eluting is the 45mmol/LPBS buffer of pH5.5-6.5.
According to the method that technique scheme provides, in some embodiments, described betaines surfactant is selected from dodecyl dihydroxy ethyl glycine betaine, octadecyl dihydroxy ethyl glycine betaine or cocamido propyl betaine.
The present invention introduces low urea and glycine betaine in the eluent of cation-exchange chromatography.Carbamide dissolves the effect (8M) of inclusion body when being generally used for renaturing inclusion bodies, and the carbamide of low concentration can make protein renaturation, is conducive to being folded into native conformation.Debita spissitudo surfactant can be combined with the hydrophobic position of adsorbent and albumen simultaneously, thus weakening the hydrophobic adsorption of protein, betaines zwitterionic surfactant, amphion group or zwitterion end moieties mixture are contained in surface, the solvation of charged amphion functional group and Hydrogen bonding interaction make zwitterionic compound surface form hydration layer, and this hydration layer surface formed based on electrostatic interaction can be adsorbed by virtual impedance nonspecific proteins.
HIC (hydrophobic interaction chromatography) is the chromatography method that protein is easily separated by a class according to the hydrophobic difference between chromatographic stuffing surface ligand and protein surface hydrophobic residue.The hydrophobic surface residue of protein can interact with the aglucon in HIC substrate, and bond strength can according to the hydrophobicity of surface institute exposed residue is different difference to some extent.Generally, hydrophobicity effect improves with ionic strength and strengthens, and therefore generally adopts " high salt is adsorbed, low eluting salt " in HIC process.Under suitable conditions, in conjunction with the trend eluting that is gradually increased according to hydrophobicity of protein.Polymer is usually the polymerization of multiple monomeric protein or relates to Denatured protein, and its hydrophobicity is more higher than monomer, thus HIC can be used for polymeric removal make its in elution process more late go out peak and and monomer separation.
Numeral in the present invention is approximation, no matter whether uses the wording such as " about " or " about ".The numerical value of numeral likely there will be the difference such as 1%, 2%, 5%, 7%, 8%, 10%.Whenever disclosing one and having N value digital, any have N+/-1%, N+/-2%, N+/-3%, N+/-5%, N+/-7%, the numeral of N+/-8% or N+/-10% value can be specifically disclosed, and wherein " +/-" refers to and add deduct, and the scope between N-10% to N+10% is also disclosed.Such as, for " eluent of first time eluting is containing 0.2mol/L carbamide ", then there is 0.2mol/L+/-1%, 0.2mol/L+/-2%, 0.2mol/L+/-3%, 0.2mol/L+/-5%, 0.2mol/L+/-7%, the value of 0.2mol/L+/-8% and 0.2mol/L+/-10% is by simultaneously open, simultaneously, concentration range between 0.2mol/L-10% to 0.2mol/L+10% falls within scope of disclosure, that is 0.18-0.22mol/L and value between, all comprising in scope at the eluent of first time eluting.
The definition " antibody " of the present invention refers to any immunoglobulin, complex or its pieces.This term includes but not limited to monoclonal or the polyclonal antibody of IgA, IgD, IgE, IgG and IgM, including nature or by the form of the genetic engineering modifications such as people source, chimeric, synthesis, restructuring, hybridization, sudden change.In embodiments of the present invention, antibody can be the monoclonal antibody IgG in full people source.
The definition " monoclonal antibody " of the present invention or " monoclonal antibody " refer to the antibody for a certain specific antigen determinant synthesized by single effect B cell.Its difference according to developmental stage, it is possible to be mouse, mosaic, humanization and full human monoclonal antibody.In embodiments of the present invention, monoclonal antibody can be full human monoclonal antibody, the variable region of its antibody and Dou Shiren source, constant region.
The definition " adalimumab " of the present invention or " A Da wood monoclonal antibody " are the full human monoclonal antibodies of a kind of Tumor necrosis factorα (TNF α).Adalimumab can stop TNF α to be combined with its cell surface receptor, thus blocking the biologic activity of TNF α.In embodiments of the present invention, adalimumab can be through the form that Chinese hamster ovary celI fermentation obtains.Wherein, " monoclonal antibody " or " monoclonal antibody " follows aforementioned definitions.
The definition " high molecular gathering thing " of the present invention or " HWM " refer to the aggregated forms of multiple protein monomers, are generally pentamer and higher level aggregation.One or more peaks that this outflow part is generally viewed as in SEC-HPLC chromatogram before main peak.
The definition " Chinese hamster ovary celI " of the present invention refers to mammalian Chinese hamster ovary cell, and it is used to express the maximum also the most successful class cell of foreign protein, is conventional mammalian host cell.When the recombinant expression carrier of encoding antibody genes is introduced in Chinese hamster ovary celI, it can be made to express by suitable CMC model host cell or secretory antibody is in culture medium, obtain the mixture containing target antibody.In embodiments of the present invention, " Chinese hamster ovary celI " can be used to express the Chinese hamster ovary cell of adalimumab.
Term " pollutant " refers to the material different from desired antibody products.Pollutant include but not limited to: host cell material, such as host cell proteins (HCP)；Expect the variant of antibody, fragment, aggregation or derivant；Cell culture media component.
Term " cleaning buffer solution " is in this article for referring to after loading compositions and flow through before eluting proteins of interest matter the buffer of chromatographic material.Cleaning buffer solution can be used for closing chromatographic material and removes one or more pollutant, and substantially eluting does not expect antibody products.According to the preferred embodiment invented herein, use " the first cleaning buffer solution " and " the second cleaning buffer solution ".
Term " elution buffer " is for from solid phase eluting antibody interested.In this article, elution buffer has relative to the second lower pH of cleaning buffer solution so that expectation antibody products's eluting in chromatography media.
Term " ion exchange " or " ion-exchange chromatography " refer to that utilizing ion-exchanger is fixing phase, carries out, according to the difference of electrostatic interactions between some solute and ion-exchanger, the chromatography method that solute separates.Different in kind according to ion-exchanger, can be classified as anionite and cationite.Anion is had exchange capacity by the former, and active group is alkalescence；Cation is had exchange interaction by the latter, and active group is acid.Having the pH range size of ion-exchange capacity according to it, ion-exchanger has again strong, weak dividing: the pH value range that strong ion-exchanger has ion exchange is big, and rate of ionization is affected less by pH；The pH value range of weak ion-exchanger ion exchange is little, and rate of ionization is affected greatly by pH." cation chromatography " or " cation-exchange chromatography " refer both to the chromatography that utilizes cationite to carry out in embodiments of the present invention.In above-mentioned steps, owing to the isoelectric point, IP of antibody is higher, positively charged in pH is lower than the buffer of isoelectric point, IP, cationite can combine strong and weak different by cationes different from solution and is easily separated by target product.Cationite of the present invention typically has containing sulfonic group (SO3 2-), sulfopropyl (SP), the strong cation exchanger of the group such as phosphonate group (P) and containing carboxymethyl (CM), carboxyl (COO-) weak cation exchanger of plasma cation exchange groups；It is preferably containing CM or COO-Weak cation exchanger Deng group, it is more preferred to, for ToyopearlCM or FractogelCOO-。
Term " polymer " (D/A) can be understood as the Non-covalent binding of same antibody, by the molecule of plural antibodies.Described antibody can be made up of the homogenizing of single-chain antibody covalent bond (such as disulfide bond) or heterogeneous a plurality of polypeptide.The polymer water soluble solution of the present invention.Such as, dimer is the non-specific binding of two IgG molecules.Polymeric formation is closely related with natural antibody is folding and antibody structure factors influencing deformation.Such as, high salt forms polymer with extreme pH induction of antibodies degeneration.
The definition " loading " of the present invention refers to and is joined in chromatographic column by sample to be separated, so as to the operation contacted with chromatographic stuffing.In embodiments of the present invention, loading can be that the adalimumab fermentation liquid after suitably processing is joined the operation in CEX or HIC chromatographic column.
The definition "or" that the present invention uses represents alternative, if appropriate, it is possible to they combined, say, that term "or" includes each listed independent alternative and their combination.Such as, " betaines surfactant is selected from dodecyl dihydroxy ethyl glycine betaine, octadecyl dihydroxy ethyl glycine betaine or cocamido propyl betaine " represents in some embodiments, betaines surfactant can be the one among dodecyl dihydroxy ethyl glycine betaine, octadecyl dihydroxy ethyl glycine betaine, cocamido propyl betaine, it is also possible to be its more than one combination.
Accompanying drawing explanation
Fig. 1 is the thin layer chromatography figure that in embodiment 1, ToyopearlCM650M catches antibody.
Fig. 2 is PhenylSepharoseFF polishing purification chromatography ultraviolet detection figure in embodiment 1.
Fig. 3 is the SEC-HPLC size exclusion chromatography ultraviolet detection figure of CEX purified product in embodiment 1 and comparative example 1.
Fig. 4 is the SEC-HPLC size exclusion chromatography ultraviolet detection figure of HIC purified product in embodiment 1 and comparative example 1.
Detailed description of the invention
The specific embodiment of the present invention comprises the steps:
1) bacterium solution separation is carried out by after Chinese hamster ovary celI fermentation expression；
2) by fermentation liquid dilute with water, pH to 6.00 ± 0.10 is regulated；
3) cation exchange column is crossed, first time eluting is carried out with pH5.5-6.5, buffer containing 0.1-0.5mol/L carbamide, then carry out second time eluting with the buffer of pH5.5-6.5, betaines surfactant containing mass fraction 0.1%-1.0%, collect elution fraction；
4) component collected is passed through hydrophobic interaction chromatography eluting, collect elution fraction.
In some embodiments, described betaines surfactant is selected from dodecyl dihydroxy ethyl glycine betaine, octadecyl dihydroxy ethyl glycine betaine or cocamido propyl betaine.
In some embodiments, step 1) described in be separated into and fermentation liquid is carried out 4000-8000g be centrifuged, then supernatant is carried out 6000-8000g and is centrifuged.In embodiments of the present invention, described being centrifuged carries out on SIGMA6K15 centrifuge, and centrifugation time is advisable with 10-15min.Centrifugal effect is to separate cell and fermentation liquid and remove cell debris.
In some embodiments, step 2) described in adjustment pH regulate to electrical conductivity 7.0 ± 0.20mS/cm with citric acid solution.In some embodiments, step 2) described in adjustment pH before, fermentation liquid is filtered with 0.45 μm of filter membrane, takes supernatant.
In some embodiments, step 3) ToyopearlCM650M that filler is Tosoh company of cationic exchange chromatography, volume containing the sample≤60g protein/L filler.Can using 20mmol/LPBS, 10mmol/L sodium chloride, pH6.0 balances filler, after fully loaded good antibody, is rinsed with equilibrating buffer.In some embodiments, adopt Fractional Collections, rise to the 1% of total height from 280nm ultraviolet absorption value and start to collect, until rising to the 80% of peak is first paragraph；Then collecting, until it is second segment that absorption value is reduced to the 10% of summit, wherein second segment gleanings is cation purified.
In some embodiments, step 4) PhenylSepharoseFF that filler is GEHealthcare of hydrophobic interaction chromatography, volume containing the sample 40mg/mL filler.20-50mmol/LPBS and the 1mol/L (NH of pH5.5-6.5 can be used4)2SO4Mixed liquor balance filler, after fully loaded good antibody, is rinsed with equilibrating buffer, after eluting, regenerates with 1mol/LNaOH.
According to the method that technique scheme provides, in some embodiments, step 3) described in the 20mmol/LPBS buffer containing 0.2mol/L carbamide that eluent is pH6.0 of first time eluting, in the embodiment that some are more excellent, described buffer is 20mmol/LPBS buffer.
In some embodiments, step 3) described in the eluent of second time eluting be 20-60mmol/L citrate buffer, in the embodiment that some are more excellent, step 3) described in second time eluting eluent in containing 150-200mmol/L sodium chloride.In the embodiment that some are more excellent, step 3) described in the eluent of second time eluting be 45mmol/L citrate buffer；Step 3) described in second time eluting eluent in ethanol containing 180mmol/L sodium chloride, the betaines surfactant of 1% mass parts and 2% parts by volume.
Above-mentioned citrate buffer can also be disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, Acetate-acetate buffer solution, or disodium hydrogen phosphate-potassium phosphate buffer.Some preferred embodiment in, it is sodium citrate-citric acid buffer.
According to the method that preceding solution provides, in some embodiments, step 4) described in hydrophobic interaction chromatography eluting be divided into 2 step eluting, wherein, the eluent of first step eluting is pH5.5-6.5,0.15-0.4mol/L (NH4)2SO445mmol/LPBS buffer, the eluent of second step eluting is the 45mmol/LPBS buffer of pH5.5-6.5.In more excellent embodiment, step 4) described in hydrophobic interaction chromatography eluting be divided into 2 step eluting, wherein, the eluent of first step eluting is pH6.3,0.2mol/L (NH4)2SO445mmol/LPBS buffer, the eluent of second step eluting is the 45mmol/LPBS buffer of pH6.3.
In the embodiment of the present invention, water used is deionized water.
The present invention provides 4 specific embodiments and 1 comparative example altogether.Embodiment 1 shows when low applied sample amount, with the addition of in cation chromatographic step and contribute to polymer pollutant removal and the impact on purification effect of the sample stable additive under more excellent elution requirement.Comparative example 1 is then the purification result of not doping eluting under embodiment 1 the same terms.It follows that purification 3 batch samples, wherein embodiment 2 is the purification result increasing quantity of sample handling under preferably purification condition；Embodiment 3 and 4 is increase on the basis of quantity of sample handling respectively, changes the antibody purification result of purifying process parameter.
The following stated is the preferred embodiment of the present invention, and what the present invention protected is not limited to following preferred embodiment.It should be pointed out that, for a person skilled in the art on the basis that these innovation and creation are conceived, the some deformation made and improvement, broadly fall into protection scope of the present invention.Raw material used in embodiment or consumptive material all can pass through to be either commercially available.
Embodiment 1
One) fermentation liquid obtains
The Chinese hamster ovary celI (Chinese hamster ovary cell) expressing full people source adalimumab is cultivated and expressed adalimumab, the antibody of expression is centrifuged, SIGMA6K15 centrifuge carries out the centrifugal 10-15min of 4000-8000g, separate cell and fermentation liquid, supernatant is carried out second time centrifugal, the centrifugal 10-15min of 6000-8000g removes cell debris, and supernatant is proceeded to next step.
Filter: the supernatant after two times centrifugal is filtered by 0.45 μm of filter membrane, can directly go up cation seperation column after the feed adjustment filtered.
Two) cation exchange column (CEX) is crossed
1) sample regulates: the feed liquid after clarification is diluted 1 times, and with 0.5M Fructus Citri Limoniae acid for adjusting pH to 6.0, now volume is about 210ml；
2) balance: use the tomographic system AKTApurifer of GE company, selects XK16/20 post, includes 20mlToyopearlCM650M filler.Maintenance flow velocity is 5mL/min, with 20mMPBS, 10mM sodium chloride, the equilibration buffer of pH6.0.Spend about 50ml balance liquid；
3) loading: maintenance flow velocity is 5mL/min, starts loading；Loading volume is about 200ml；The applied sample amount of antibody is about 600mg；With 20mMPBS, 10mM sodium chloride, the wash buffer of pH6.0 is steady to baseline.Spend about 50ml.
4) eluting 1: with 20mMPBS, 0.2M carbamide, the level pad of pH6.0 goes flushing steady to baseline.Collect a peak (first step eluting peak), be mainly pollutant after testing, spend buffer and be about 70ml；
5) eluting 2: with 45mM citrate buffer+180mMNaCl+0.5% dodecyl dihydroxy ethyl glycine betaine+2% ethanol, pH6.3, carry out eluting, collect two parts eluting peak, Part I reaches the 1% of peak from ultraviolet 280nm absorption to start to collect, stop (second step eluting collects 1) to 80% maximum absorption value and start second segment collection (second step eluting collects 2), stop at 10% place of summit to A280.Part I eluting peak is pollutant peak after testing, peak for the purpose of Part II.Part II eluting spends about 70ml buffer, and eluting peak collected volume is about 45ml.
6) regeneration, preservation: with 1M sodium chloride cleaning and regeneration.Reduce electrical conductivity to zero with deionized water, add 20% ethanol and preserve.
Three) hydrophobic interaction post (HIC) is crossed
1) sample regulates: the eluting of cation exchange column is collected peak 2M (NH4)2SO4Regulate to 1M (NH4)2SO4, volume is about 95ml.
2) balance: use the tomographic system AKTApurifer of GE company, selects the XK16/20 post of GE, includes 20mlPhenylSepharoseFF filler.Keeping flow velocity 2mL/min, level pad is 45mMPBS, 1M (NH4)2SO4, pH6.3, spend about 45ml balance liquid.
3) loading: maintenance flow velocity is 2mL/min, starts loading.Loading volume 95ml.
4) rinse: with the about 30ml of Equilibration buffer wash.
5) eluting 1: flow velocity 2ml/min, with 45mMPBS, 0.2M (NH4)2SO4, pH6.3, collects eluting peak, spends elution buffer and be about 45ml；
6) eluting 2: keeping flow velocity 2ml/min, with 45mMPBS, pH6.3 eluting, eluting peak is pollutant peak after testing, spends elution buffer and is about 25ml.
7) regeneration, preservation: carry out column regeneration, flow velocity 2ml/min with 1MNaOH.Reduce electrical conductivity to zero with deionized water, add 20% ethanol and preserve.
Four) result detection
Being detected by the purified antibody collected, project includes the response rate, polymer.Detection method and condition are as follows:
Antibody concentration is measured by PorosA (A albumen) HPLC.Application of samples dilution, to reach the reading in calibration range.ShimadzuHPLC system is configured with PorosAImmunoDetection sensor cartridge (AppliedBiosystems, FosterCity, CA).Post maintains ambient temperature.System was run with 2mL/ minute.Automatic sampler temperature of tray is set to 4 DEG C.Absorbance is monitored at 280nm.Buffer A is 40mMPBS and 400mMNaCl.Buffer B is 50mM potassium dihydrogen phosphate and 150mM sodium chloride.Injected sample and use 0-100% buffer B eluting adalimumab.
Perform size exclusion chromatography (SEC)-HPLC for the polymeric analysis of sample.Application of samples dilution, to reach the reading in standard curve.ShimadzuHPLC system is configured with SuperoseTM610/300GL post (GEHealthcare).Post maintains ambient temperature.System was run with 0.5mL/ minute.Automatic sampler temperature of tray is set to 4 DEG C.Absorbance is monitored at 280nm.Buffer A is 20mM disodium hydrogen phosphate/150mM sodium chloride.The degree such as injected sample and use 100% buffer A run adalimumab.
Testing result is in Table 1.In embodiment 1, the AKTA tomographic map of CEX and HIC is shown in Fig. 1 and Fig. 2 respectively.In figure, dotted line represents electrical conductivity, it is achieved represent the ultraviolet absorption value under 280nm.In Fig. 1, the peak before and after 100min is that eluting 1 goes out peak, and the peak before and after 120min is that eluting 2 goes out peak.In Fig. 2, the peak of about 95min is that eluting 1 goes out peak, and the peak of about 110min is eluting 2 goes out peak.The SEC-HPLC figure of CEX and HIC target collection thing is shown in accompanying drawing 3 and accompanying drawing 4 respectively.Wherein, the peak of 12.5-15.5min is heavy polymer (i.e. polymer, HMW), and the peak gone out in 15.5-18min is main peak (MP), is low molecular weight substance (LMW) after 18min.
Fig. 1 shows and adopts the ToyopearlCM650M AKTA method carrying out CEX purification.In figure shown in UV peak, during the Part I of elution step 1 after introduction of the sample and elution step 2 is collected, it is mainly pollutant peak, and collects in peak at the Part II of elution step 2 and be mainly target product.In Fig. 3, SEC-HPLC figure is visible, and after eliminating the pollutant such as substantial amounts of polymer, the required antibody of collection, it will how general 2% than reference examples 1 (not doping) for the polymeric removal amount of target contaminant.Illustrate that such additive combination is good to polymeric removal effect.
Fig. 2 shows the AKTA method adopting the PhenylSepharoseFF required antibody that CEX is collected to carry out further HIC purification.As it can be seen, press the collection method described in eluting 1 step, what collect is required pure antibody compound, and what eluting 2 obtained is mainly pollutant.
Embodiment 2
One) fermentation liquid obtains
The Chinese hamster ovary celI (Chinese hamster ovary cell) expressing full people source adalimumab is cultivated and expressed adalimumab, the antibody of expression is centrifuged, SIGMA6K15 centrifuge carries out the centrifugal 10-15min of 4000-8000g, separate cell and fermentation liquid, supernatant is carried out second time centrifugal, the centrifugal 10-15min of 6000-8000g removes cell debris, and supernatant is proceeded to next step.
Filter: the supernatant after two times centrifugal is filtered by 0.45 μm of filter membrane, can directly go up cation seperation column after the feed adjustment filtered.
Two) cation exchange column (CEX) is crossed
1) sample regulates: the feed liquid after clarification is diluted 1 times, and with 0.5M Fructus Citri Limoniae acid for adjusting pH to 6.0, now volume is about 400ml；
2) balance: use the tomographic system AKTApurifer of GE company, selects XK16/20 post, includes 20mlToyopearlCM650M filler.Maintenance flow velocity is 5mL/min, with 20mMPBS, 10mM sodium chloride, the equilibration buffer of pH6.0.Spend about 60ml balance liquid；
3) loading: maintenance flow velocity is 5mL/min, starts loading；Loading volume is about 400ml；The applied sample amount of antibody is about 1200mg；
4) rinse: with 20mMPBS, 10mM sodium chloride, the wash buffer of pH6.0 is steady to baseline.Spend about 50ml.
5) eluting 1: with 20mMPBS, 0.2M carbamide, the level pad of pH6.0 goes flushing steady to baseline.Collect a peak, be mainly pollutant after testing, spend buffer and be about 60ml；
6) eluting 2: with 45mM citrate buffer+180mMNaCl+0.5% dodecyl dihydroxy ethyl glycine betaine+2% ethanol, pH6.3, carry out eluting, collect two parts eluting peak, Part I reaches the 1% of peak from ultraviolet 280nm absorption to start to collect, stop starting second segment to 80% maximum absorption value to collect, stop at 10% place of summit to A280.Part I eluting peak is pollutant peak after testing, peak for the purpose of Part II.Part II eluting spends about 70ml buffer, and eluting peak-to-peak collected volume is about 70ml.
7) regeneration, preservation: with 1M sodium chloride cleaning and regeneration.Reduce conductance to zero with deionized water, add 20% ethanol and preserve.
Three) hydrophobic interaction post (HIC) is crossed
1) sample regulates: the eluting of cation exchange column is collected peak 2M (NH4)2SO4Regulate to 1M (NH4)2SO4, volume is about 140ml.
2) balance: use the tomographic system AKTApurifer of GE company, selects the XK16/20 post of GE, includes 20mlPhenylSepharoseFF filler.Keeping flow velocity 2mL/min, level pad is 45mMPBS, 1M (NH4)2SO4, pH6.3, spend about 50ml balance liquid.
3) loading: maintenance flow velocity is 2mL/min, starts loading.Loading volume 140ml.
4) rinse: with the about 30ml of Equilibration buffer wash.
5) eluting 1: flow velocity 2ml/min, with 45mMPBS, 0.2M (NH4)2SO4, pH6.3, collects eluting peak, spends elution buffer and be about 45ml；
6) eluting 2: keeping flow velocity 2ml/min, with 45mMPBS, pH6.3 eluting, eluting peak is pollutant peak after testing, spends elution buffer and is about 25ml.
7) regeneration, preservation: carry out column regeneration, flow velocity 2ml/min with 1MNaOH.Reduce conductance to zero with deionized water, add 20% ethanol and preserve.
Four) result detection
Being detected by the purified antibody collected, project includes the response rate, polymer.Detection method and condition are with embodiment 1.Result is in Table 1.
Embodiment 3
One) fermentation liquid obtains
The Chinese hamster ovary celI (Chinese hamster ovary cell) expressing full people source adalimumab is cultivated and expressed adalimumab, the antibody of expression is centrifuged, SIGMA6K15 centrifuge carries out the centrifugal 10-15min of 4000-8000g, separate cell and fermentation liquid, supernatant is carried out second time centrifugal, the centrifugal 10-15min of 6000-8000g removes cell debris, and supernatant is proceeded to next step.
Filter: the supernatant after two times centrifugal is filtered by 0.45 μm of filter membrane, can directly go up cation seperation column after the feed adjustment filtered.
Two) cation exchange column (CEX) is crossed
1) sample regulates: the feed liquid after clarification is diluted 1 times, and with 0.5M Fructus Citri Limoniae acid for adjusting pH to 6.0, now volume is about 400ml；
2) balance: use the tomographic system AKTApurifer of GE company, selects XK16/20 post, includes 20mlToyopearlCM650M filler.Maintenance flow velocity is 5mL/min, with 20mMPBS, 10mM sodium chloride, the equilibration buffer of pH6.0.Spend about 60ml balance liquid；
3) loading: maintenance flow velocity is 5mL/min, starts loading；Loading volume is about 400ml；The applied sample amount of antibody is about 1200mg；
4) rinse: with 20mMPBS, 10mM sodium chloride, the wash buffer of pH6.0 is steady to baseline.Spend about 50ml.
5) eluting 1: with 20mMPBS, 0.5M carbamide, the level pad of pH5.5 goes flushing steady to baseline.Collect a peak, be mainly pollutant after testing, spend buffer and be about 60ml；
6) eluting 2: with 45mM citrate buffer+200mMNaCl+0.1% dodecyl dihydroxy ethyl glycine betaine+2% ethanol, pH5.5, carry out eluting, collect two parts eluting peak, Part I reaches the 1% of peak from ultraviolet 280nm absorption to start to collect, stop starting second segment to 80% maximum absorption value to collect, stop at 10% place of summit to A280.Part I eluting peak is pollutant peak after testing, peak for the purpose of Part II.Part II eluting peak-to-peak collected volume is about 70ml.
7) regeneration, preservation: with 1M sodium chloride cleaning and regeneration.Reduce electrical conductivity to zero with deionized water, add 20% ethanol and preserve.
Three) hydrophobic interaction post (HIC) is crossed
1) sample regulates: the eluting of cation exchange column is collected peak 2M (NH4)2SO4Regulate to 1M (NH4)2SO4, volume is about 140ml.
2) balance: use the tomographic system AKTApurifer of GE company, selects the XK16/20 post of GE, includes 20mlPhenylSepharoseFF filler.Keeping flow velocity 2mL/min, level pad is 45mMPBS, 1M (NH4)2SO4, pH5.5, spend about 50ml balance liquid.
3) loading: maintenance flow velocity is 2mL/min, starts loading.Loading volume 140ml.
4) rinse: with the about 30ml of Equilibration buffer wash.
5) eluting 1: flow velocity 2ml/min, with 45mMPBS, 0.2M (NH4)2SO4, pH6.0, collects eluting peak, spends elution buffer and be about 60ml；
6) eluting 2: keeping flow velocity 2ml/min, with 45mMPBS, pH6.0 eluting, eluting peak is pollutant peak after testing, spends elution buffer and is about 40ml.
7) regeneration, preservation: carry out column regeneration, flow velocity 2ml/min with 1MNaOH.Reduce conductance to zero with deionized water, add 20% ethanol and preserve.
Four) result detection
Being detected by the purified antibody collected, project includes the response rate, polymer.Detection method and condition are shown in embodiment 1.Result is in Table 1.
Embodiment 4
One) fermentation liquid obtains
The Chinese hamster ovary celI (Chinese hamster ovary cell) expressing full people source adalimumab is cultivated and expressed adalimumab, the antibody of expression is centrifuged, SIGMA6K15 centrifuge carries out the centrifugal 10-15min of 4000-8000g, separate cell and fermentation liquid, supernatant is carried out second time centrifugal, the centrifugal 10-15min of 6000-8000g removes cell debris, and supernatant is proceeded to next step.
Filter: the supernatant after two times centrifugal is filtered by 0.45 μm of filter membrane, can directly go up cation seperation column after the feed adjustment filtered.
Two) cation exchange column (CEX) is crossed
1) sample regulates: the feed liquid after clarification is diluted 1 times, and with 0.5M Fructus Citri Limoniae acid for adjusting pH to 6.0, now volume is about 400ml；
2) balance: use the tomographic system AKTApurifer of GE company, selects XK16/20 post, includes 20mlToyopearlCM650M filler.Maintenance flow velocity is 5mL/min, with 20mMPBS, 10mM sodium chloride, the equilibration buffer of pH6.0.Spend about 60ml balance liquid；
3) loading: maintenance flow velocity is 5mL/min, starts loading；Loading volume is about 400ml；The applied sample amount of antibody is about 1200mg；
4) rinse: with 20mMPBS, 10mM sodium chloride, the wash buffer of pH6.0 is steady to baseline.Spend about 50ml.
5) eluting 1: with 50mMPBS, 0.1M carbamide, the level pad of pH6.5 goes flushing steady to baseline.Collect a peak, be mainly pollutant after testing, spend buffer and be about 60ml；
6) eluting 2: with 20mM citrate buffer+150mMNaCl+1.0% dodecyl dihydroxy ethyl glycine betaine+2% ethanol, pH6.5, carry out eluting, collect two parts eluting peak, Part I reaches the 1% of peak from ultraviolet 280nm absorption to start to collect, stop starting second segment to 80% maximum absorption value to collect, stop at 10% place of summit to A280.Part I eluting peak is pollutant peak after testing, peak for the purpose of Part II.Part II eluting peak-to-peak collected volume is about 70ml.
7) regeneration, preservation: with 1M sodium chloride cleaning and regeneration.Reduce electrical conductivity to zero with deionized water, add 20% ethanol and preserve.
Three) hydrophobic interaction post (HIC) is crossed
1) sample regulates: the eluting of cation exchange column is collected peak 2M (NH4)2SO4Regulate to 1M (NH4)2SO4, volume is about 140ml.
2) balance: use the tomographic system AKTApurifer of GE company, selects the XK16/20 post of GE, includes 20mlPhenylSepharoseFF filler.Keeping flow velocity 2mL/min, level pad is 45mMPBS, 1M (NH4)2SO4, pH6.5, spend about 50ml balance liquid.
3) loading: maintenance flow velocity is 2mL/min, starts loading.Loading volume 140ml.
4) rinse: with the about 30ml of Equilibration buffer wash.
5) eluting 1: flow velocity 2ml/min, with 45mMPBS, 0.2M (NH4)2SO4, pH6.5, collects eluting peak, spends elution buffer and be about 60ml；
6) eluting 2: keeping flow velocity 2ml/min, with 45mMPBS, pH6.0 eluting, eluting peak is pollutant peak after testing, spends elution buffer and is about 40ml.
7) regeneration, preservation: carry out column regeneration, flow velocity 2ml/min with 1MNaOH.Reduce electrical conductivity to zero with deionized water, add 20% ethanol and preserve.
Four) result detection
Being detected by the purified antibody collected, project includes the response rate, polymer.Detection method and condition are with embodiment 1.Result is in Table 1.
Comparative example 1
One) fermentation liquid obtains
The Chinese hamster ovary celI (Chinese hamster ovary cell) expressing full people source adalimumab is cultivated and expressed adalimumab, the antibody of expression is centrifuged, SIGMA6K15 centrifuge carries out the centrifugal 10-15min of 4000-8000g, separate cell and fermentation liquid, supernatant is carried out second time centrifugal, the centrifugal 10-15min of 6000-8000g removes cell debris, and supernatant is proceeded to next step.
Filter: the supernatant after two times centrifugal is filtered by 0.45 μm of filter membrane, can directly go up cation seperation column after the feed adjustment filtered.
Two) cation exchange column (CEX) is crossed
1) sample regulates: the feed liquid after clarification is diluted 1 times, and with 0.5M Fructus Citri Limoniae acid for adjusting pH to 6.0, now volume is about 210ml；
2) balance: use the tomographic system AKTApurifer of GE company, selects XK16/20 post, includes 20mlToyopearlCM650M filler.Maintenance flow velocity is 5mL/min, with 20mMPBS, 10mM sodium chloride, the equilibration buffer of pH6.0.Spend about 60ml balance liquid；
3) loading: maintenance flow velocity is 5mL/min, starts loading；Loading volume is about 200ml；The applied sample amount of antibody is about 600mg；
4) rinse: with 20mMPBS, 10mM sodium chloride, the wash buffer of pH6.0 is steady to baseline.Spend about 50ml.
5) eluting: with 45mM citrate buffer+250mMNaCl, pH6.3, carry out eluting, collect two parts eluting peak, Part I reaches the 1% of peak from ultraviolet 280nm absorption to start to collect, stop starting second segment to 80% maximum absorption value to collect, stop at 10% place of summit to A280.Part I eluting peak is pollutant peak after testing, peak for the purpose of Part II.Part II eluting peak collected volume is about 45ml.
6) regeneration: with 1M sodium chloride cleaning and regeneration.
7) preserve: reduce electrical conductivity to zero with deionized water, add 20% Alcohol Protection pillar.
Three) hydrophobic interaction post (HIC) is crossed
1) sample regulates: the eluting of cation exchange column is collected peak 2M (NH4)2SO4Regulate to 1M (NH4)2SO4, volume is about 95ml.
2) balance: use the tomographic system AKTApurifer of GE company, selects the XK16/20 post of GE, includes 20mlPhenylSepharoseFF filler.Keeping flow velocity 2mL/min, level pad is 45mMPBS, 1M (NH4)2SO4, pH6.3, spend about 50ml balance liquid.
3) loading: maintenance flow velocity is 2mL/min, starts loading.Loading volume 95ml.
4) rinse: with Equilibration buffer wash, spend about 30ml.
5) eluting 1: flow velocity 2ml/min, with 45mMPBS, 0.2M (NH4)2SO4, pH6.3, collects eluting peak, spends elution buffer and be about 45ml；
6) eluting 2: keeping flow velocity 2ml/min, with 45mMPBS, pH6.3 eluting, eluting peak is pollutant peak after testing, spends elution buffer and is about 25ml.
7) regeneration, preservation: carry out column regeneration, flow velocity 2ml/min with 1MNaOH.Reduce electrical conductivity to zero with deionized water, add 20% ethanol and preserve.
Four) result detection
Being detected by the purified antibody collected, project includes the response rate, polymer.Detection method and condition are with embodiment 1.Result is in Table 1.
From table 1 and Fig. 4, polymer is had good removal effect by HIC, after wherein reference examples carries out drainage column, polymer was reduced to 0.895% from 2.727%, in embodiment 1, Content of polymer is reduced to 0.143% from 0.778%, Content of polymer is relatively low, and the latter reaches the crude drug requirement to these pollutant.
Table 1 testing result
By the result of embodiment 1 and comparative example 1 it is known that in the elution process caught of CEX the use of additive cause that in embodiment 1, Content of polymer significantly reduces than content in not additivated comparative example 1.And in follow-up HIC purification, embodiment 1, owing to employing additive at acquisition phase, causes that in the purified product finally given, Content of polymer is relatively low, meets the crude drug requirement to this pollutant load；And the CEX purification of samples in comparative example 1 continues through HIC chromatography, Content of polymer is not reduced to the level of crude drug requirement.The result of embodiment 2-4 shows, the response rate of three batches is all more than 91%, and by adding additive in first step chromatography, in purified product, polymeric content is relatively low, by second step HIC chromatography, is finally attained by crude drug standard.
Claims (12)
1. the cation exchange chromatography method of an adalimumab, cation-exchange chromatography eluting, hydrophobic interaction chromatography eluting is carried out including to the antibody of Chinese hamster ovary celI fermentation expression, it is characterized in that, the eluent of cation-exchange chromatography is the buffer of pH5.5-6.5, carry out 2 eluting altogether, wherein containing 0.1-0.5mol/L carbamide in the eluent of first time cation-exchange chromatography；The eluent of second time cation-exchange chromatography contains the betaines surfactant of mass fraction 0.1%-1.0%；Described hydrophobic interaction chromatography eluting is divided into 2 step eluting, and wherein, the eluent of first step eluting is pH5.5-6.5,0.15-0.4mol/L (NH4)2SO4PBS, the eluent of second step eluting is the PBS of pH5.5-6.5.
2. method according to claim 1, it is characterised in that described betaines surfactant is selected from dodecyl dihydroxy ethyl glycine betaine, octadecyl dihydroxy ethyl glycine betaine or cocamido propyl betaine.
3. method according to claim 1, it is characterised in that comprise the following steps:
1) bacterium solution after Chinese hamster ovary celI fermentation expression is separated；
2) by fermentation liquid dilute with water, pH to 6.00 ± 0.10 is regulated；
3) cation exchange column is crossed, first time eluting is carried out with pH5.5-6.5, buffer containing 0.1-0.5mol/L carbamide, then carry out second time eluting with the buffer of pH5.5-6.5, betaines surfactant containing mass fraction 0.1%-1.0%, collect elution fraction；
4) component collected is passed through hydrophobic interaction chromatography eluting, collect elution fraction.
4. method according to claim 3, it is characterised in that described betaines surfactant is selected from dodecyl dihydroxy ethyl glycine betaine, octadecyl dihydroxy ethyl glycine betaine or cocamido propyl betaine.
5. method according to claim 3, it is characterised in that step 1) described in be separated into and fermentation liquid is carried out 4000-8000g be centrifuged, then supernatant is carried out 6000-8000g and is centrifuged.
6. method according to claim 3, it is characterised in that step 2) described in fermentation liquid dilute with water be diluted to electrical conductivity 7.0 ± 0.20mS/cm, regulate pH be with citric acid solution regulate to 6.00 ± 0.10.
7. method according to claim 3, it is characterised in that step 2) regulate before pH, fermentation liquid is filtered, takes supernatant.
8. the method according to any one of claim 3-7, it is characterised in that step 3) described in the 20mmol/LPBS buffer containing 0.2mol/L carbamide that eluent is pH6.0 of first time eluting.
9. the method according to any one of claim 3-7, it is characterised in that step 3) described in the eluent of second time eluting be 20-60mmol/L citrate buffer；Step 3) described in second time eluting eluent in containing 150-200mmol/L sodium chloride.
10. method according to claim 9, it is characterised in that step 3) described in the eluent of second time eluting be 45mmol/L citrate buffer；Step 3) described in second time eluting eluent in ethanol containing 180mmol/L sodium chloride, the betaines surfactant of 1% mass parts and 2% parts by volume.
11. according to the method described in any one of claim 9 or 10, it is characterized in that, described citrate buffer is selected from sodium citrate-citric acid buffer, disodium hydrogen phosphate-phosphate sodium dihydrogen buffer solution, Acetate-acetate buffer solution or disodium hydrogen phosphate-potassium phosphate buffer.
12. method according to claim 11, it is characterised in that described citrate buffer is sodium citrate-citric acid buffer.
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| CN113563469A (en) * | 2020-04-28 | 2021-10-29 | 江苏中新医药有限公司 | Method for purifying adalimumab with high recovery rate |
| CN116272921A (en) * | 2023-02-15 | 2023-06-23 | 青岛盛瀚色谱技术有限公司 | Monodisperse weakly acidic cation chromatographic packing and preparation method and application thereof |
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| CN103998469A (en) * | 2011-12-15 | 2014-08-20 | 韩华石油化学株式会社 | A method of antibody purification |
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| CN113563469A (en) * | 2020-04-28 | 2021-10-29 | 江苏中新医药有限公司 | Method for purifying adalimumab with high recovery rate |
| CN116272921A (en) * | 2023-02-15 | 2023-06-23 | 青岛盛瀚色谱技术有限公司 | Monodisperse weakly acidic cation chromatographic packing and preparation method and application thereof |
| CN116272921B (en) * | 2023-02-15 | 2024-05-17 | 青岛盛瀚色谱技术有限公司 | Monodisperse weakly acidic cation chromatographic packing and preparation method and application thereof |
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