CN101318991A - Method for syncretizing protein employing composite chromatography medium purification - Google Patents
Method for syncretizing protein employing composite chromatography medium purification Download PDFInfo
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Abstract
The invention discloses a method for adopting a compound chromatography medium to purify Fc fusion protein. The method comprises the following steps that: the ion-exchange hydrophobic compound chromatography medium is adopted to adjust the pH of a chromatography buffer system to be below the average pI of purification target protein and to adjust the chromatography buffer system to have high ionic strength simultaneously, utilizes proper chromatography conditions to ensure that the purification target protein exists in flow-through liquid; the flow-through liquid is collected, while polluting impurities (such as host cell protein, polymer, etc.) are adsorbed onto the compound chromatography medium, so as to improve the purity of the target protein. The method ensures that: the removal rate of final impurities reaches over 1 percent; the purity of the target protein reaches over 99 percent; the recovery rate of the target protein reaches over 90 percent. The method is compact in cohesion in realization process, can effectively remove the polluting impurities in one step, and is characterized in good stability, clear in process control index, suitable for mass production, etc.
Description
Technical field
The invention belongs to bioprotein purifying field, relate to the antibody class protein purification, particularly a kind of method that adopts composite chromatography medium purification Fc fused protein.This method is mainly used in animal cell large-scale and cultivates the treatment of producing Fc fused protein medicine production preparation.
Background technology
Antybody therapy has become the important means of current biotherapy tumour, popularity and infectious diseases.The antibody class medicine becomes a big class new therapeutic agent on the international drug market with specificity, validity and the security of its height
[1]
Animal cell large-scale is cultivated and is produced the main flow that the antibody class medicine has become current field of biological pharmacy.Clinical many antibody class medicine, particularly medicine for treatment need with higher dosage or long-term prescription, and the market requirement is bigger, so the scale operation of such bio-pharmaceuticals has relatively high expectations to processing quality, must meet the U.S. FDA standard.At present, the antibody purification of about 70-80% has used Protein A, Protein G affinity chromatography
[2,3], because this method can one step antibody purification (normally IgG antibody) almost completely from cell culture supernatant.
A-protein (Protein A) derives from the strain system of streptococcus aureus.Protein A is fixed on the agarose as affinity ligand, 5 and IgG Fc section bonded structural domain that it comprised.ProteinA also can be in conjunction with other immunoglobulin (Ig)s and as IgA, IgM.Protein A (rProteinA) natural and reorganization has special combination for the Fc section of immunoglobulin IgG.The Protein A of reorganization contains a C-terminal halfcystine after transforming, can be fixed on the agarose in single site, has increased its binding ability.The chromatographic behavior of ProteinA depends on this proteic source of species with the bonding strength of Fc, and dynamically binding ability then is decided by chromatogram environment and chromatographic condition.
Along with improving constantly for the requirement of therapeutic antibodies quality product such as FDA, SFDA, antibody behind the Protein A affinitive layer purification often still can not meet the demands, mainly show as the existence of some contaminative impurity, for example: the Protein A that comes off, host cell proteins (HCP), polymer (D/A) etc.
Because the part that Protein A affinity chromatography medium inevitably can take place to a certain degree when using repeatedly comes off, this may be because protease hydrolysis disintegrates down protein from matrix result.And the pollutents such as Protein A or its fragment that come off have still kept the avidity to IgG antibody, will form mixture like this and are difficult to remove from antibody purified, and this situation never allows in the therapeutic antibodies medicine, and it is necessary therefore removing
[4,5]Protein A is that bacterioprotein can cause the undesirable immune response reaction simultaneously, can activate the white corpuscle and the complement system of carrying Fc in vivo according to the mixture of reporting the formation of Protein A and IgG antibody and get immunne response, produces oxidation and anaphylaxis
[6]
People such as Balint point out and can separate this kind Protein A-IgG complex body and IgG monomer by gel-filtration
[7], but this method treatment capacity is few, is unsuitable for amplifying.
J. people such as Bang Najie proves and adopts ion-exchange can effectively remove the ProteinA that in use comes off
[4,5]The description of relevant antibody purification in its patent: adopt ion exchange chromatography, carry out the circulation peak of classification collection of ions exchange, Protein A-IgG complex body greatly reduces in the product that Elisa detection classification is collected.But this method has adopted fractional separation, and the product rate of recovery is caused bigger influence, and its rate of recovery is 70%-80%, and optimum only is more than 80%, and this is very uneconomic for the antibody product with high added value; And, only come off in this method and study at the Protein A of affinity chromatography medium in early stage comparatively, not representative.
It is worth noting that what adopt in the existing report is in the past affinity media, the Protein A problem that comes off is comparatively serious
[4,5], wherein can obtain in every milligram of antibody of Streamline recombinant protein A affinity media approximately or surpass 1000ng contaminative Protein A
[8]Therefore be necessary to adopt effective ways to remove.Yet, development along with technology, the affinity chromatography medium that is widely used in the antibody purification field at present is by Protein A Sepharose FF, media such as rProtein A Sepharose develop into ProSep-Va Ultra, Mabselect, Mabselect Xtra, media of new generation such as Mabselect SuRe, the Protein A problem of coming off of these novel affinity chromatography mediums has had very big improvement, studies show that in a large number that wherein Mabselect SuRe application potential is bigger
[9,10,11]
The Protein A part of MabSelect series is through genetic engineering modified, and the C end contains a halfcystine, forms the sulfide linkage of an orientation, has increased the effective combination to IgG simultaneously.Albumin A and gel have adopted brand-new technical process when crosslinked, the stability of medium and the surface-area of glue endoporus have been increased, therefore increased flow velocity and dynamic carrying capacity: at linear flow velocity is that 500cm/h and post bed height are under the condition of 20cm, the dynamic carrying capacity of MabSelect can reach more than the 30mg IgG/ml, surpass original A albumen medium several times, also obviously be better than other several albumin A link coupled media aspect the purifying characteristic.MabSelect Xtra and the Mabselect SuRe that release the end of the year 2004, carrying capacity has increased nearly 30% than common MabSelect again
[12]
At present, the amount of residual Protein A should be lower than 10ppm～12ppm in the U.S. FDA suggestion product
[13]People such as R.Hahn studies show that, the Protein A of Mabselect SuRe medium comes off lower with respect to other media (Mabselect Xtra, ProSep-Va Ultra), generally is in below the 3ppm, far below above standard
[11]And this studies show that be circulated throughout through 50 uses after, do not have trend of rising.This is because MabSelect is because by the epoxy covalent cross-linking, coming off of albumin A is at half above than other similar medium.This shows that carry out the Fc fusion rotein of purifying for media such as using Mabselect, the Protein A that comes off not is main contaminative impurity, and the Protein A that comes off can't influence the quality of product in suitable use age.
Therefore, the removal for other contaminative impurity (as: host cell proteins, polymer etc.) then seems more outstanding and important.
Host cell proteins (Host Cell Protein, HCP) be meant the protein ingredient that derives from host cell in the biological products, its main component comprises: host cell structural protein and transforming protein (being the promotes growth albumen of emiocytosis), the former danger is to cause the body anaphylaxis, and the latter is to cause cell transformation
[14]In theory, there are oncogenic possibility in the DNA of passage cell and albumen.Simultaneously, the cell residue albumen in the biological products is heterologous protein (anaphylactogen), might cause the anaphylaxis of body
[15]There are some researches show that the anaphylaxis of hamster kidney cell Vaccinum Encephalitidis Epidemicae may be relevant with hamster kidney cell protein content height in the vaccine
[16]
But the host cell proteins of different cells is different, just is same cell, and the HCP in the finished product also is different.At first, this mode with antibody expression is relevant, and is secondly relevant with the purification process that uses.People such as Rainer Hahn have compared the characteristic of different affinity medias, find MabSelectXtra, MabSelect SuRe, behind three kinds of affinity chromatography medium purifying of ProSep-vA Ultra in the antibody HCP residual each is variant
[11]
Chinese hamster ovary celI is used for the passage cell of the production of biological products such as antibody and recombinant protein by WHO and SFDA approval.This cytogenetics background is clear, and caryogram is stable, and no exogenous factor pollutes, and is fit to bio-reactor large scale culturing and production, has guaranteed the uniformity and the security of cell in enormous quantities.But the HCP that this cell produces still can influence the quality of product.Studies show that the HCP level after the affinity chromatography is generally between 200ppm～3000ppm
[17], studies show that more the HCP of 100ppm can cause patient's immune response
[18]" Chinese pharmacopoeia three ones (2005 editions) claims to the product of expressing cho cell: host cell residual protein (HCP) accounts for below 0.1% of total protein content.Therefore the removal of HCP is imperative in the purge process.
People such as Andreas Stein adopt directly antibody purification from nutrient solution of a kind of cation exchange medium, and the HCP amount is reduced to 80ppm from the 155000ppm of fermented liquid in its product, and decontamination factor reaches 1800 times
[19], obtained good effect.But as the first step preliminary purification antibody from fermented liquid, restricted factor is more in its amplification process.
J. people such as Bang Najie proves and adopts ion-exchange can effectively remove the Protein A that in use comes off
[4,5], obtained good effect.But its removal for HCP in the product is not reported.
At present, fewer at the document that HCP further removes, especially the research of removing at HCP in the product after the affinity chromatography is reported still less.
Except removing Protein A and HCP, another issues of purification that relates to Fc fusion rotein (but can expand to any other type biological medicine albumen) is homogeneous dimer and higher polymeric formation.To form mixture (mainly also can form based on avidity even natural protein) opposite with a-protein, idiopathic or form polymer by salt, when being made solvent to expose near the hydrophobic region on surface to cause Fc fusion or analogous protein seen by the driving of chemical quality law by pH inductive at least a portion branch protein denaturation.Therefore, form on the contrary with mixture based on avidity, non-specific cohesion is driven by the solvent repulsive interaction mainly, is similar to the crystal growth behavior in this
[4,5], still polymer soluble increases in time at first, and then protein precipitates from solution.After medicine gave, the contaminative polymkeric substance of low per-cent also may cause deleterious immune response
[20]
Up to now, can remove the most frequently used volume-exclusion chromatography of method (SEC) of polymkeric substance reliably.Yet SEC is the bottleneck of purifying, compares with other chromatographic techniques, and it needs a large amount of treatment times, and material expensive and load capacity are low.It is reported
[21], SEC can effectively detect the existence of polymkeric substance, and can carry out quantitative examination to it, but also uses as just detection means, can't be applied to product preparation.
On the other hand, the purity of target product is no doubt important in purge process, but purification efficiency is also extremely important.Purification efficiency is mainly reflected in the rate of recovery and media processes ability two aspects.
Though people such as Andreas Stein have obtained the better products quality, ion exchange chromatography itself requires high, so sample preparation is comparatively complicated, will certainly cause the product unnecessary loss
[19]J. people such as Bang Najie faces same problem.In their method sample must through dialysis and or method such as ultrafiltration handle, just can carry out next step purifying
[4,5]This has relatively high expectations to factors such as time, equipment, environment, personnel, is not easy to satisfy the demand of large-scale production.Simultaneously, the mode that they have adopted classification to collect is come antibody purification, is prerequisite (its rate of recovery is only up to more than 85%) to sacrifice the rate of recovery when obtaining high-purity product like this
[4,5]
The processing power of medium is determined jointly by carrying capacity and flow velocity.For the mass-producing purifying, higher carrying capacity and flow velocity are more suitable.The dynamic carrying capacity of Ion Exchange Medium that people such as Andreas Stein and J. Bang Najie adopt is generally the 50mgIgG/ml medium, and flow velocity is generally 300cm/h～500cm/h
[22]And its carrying capacity is subjected to the sample preparation effect and effects of operation conditions is bigger, is not suitable for scale operation
According to the data-searching that the applicant carried out, retrieve have below with reference to document relevant with the present invention:
1. rice power, Tang Hao etc., a kind of method of efficient fast purifying fragment antibody, 2004, Chinese invention patent, the patent No.: ZL200410026022.1;
2.Deborah K.Follman，Robert L.Fahrner.Factorial screening ofantibody purification processes using three chromatography stepswithout protein A.Journal of Chromatography A，1024(2004)79-85。
3.Jorg Thommes，Andrea Bader，Markus Halfar，etal.Isolation ofmonoclonal antibodies from cell containing hybridoma broth using aprotein A coated adsorbent in expanded beds.Journal of ChromatographyA，752(1996)11l-122。
4.J. Bang Najie, R.P. cloth clarke, M.R. Davis etc. are used for the affinity-plus ion exchange-chromatography of antibody purification, and 2005, PCT patent application, application number: 200580035236.8;
5.J. Bang Najie, A. Pu Lunta comes antibody purification with albumin A and ion exchange chromatography, 2004, and PCT patent, application number: 20048009347.7;
6.Balint，et al.Tumoricidal response following perfusion overimmobilized protein′A：identification of immunoglobulin oligomers inserum after perfusion and their partial characterization，Cancer Res，44(1984)734。
7.Das，et al，Separation of complexes containing protein A and IgGor Fc gamma Fragments by high-performance liquid chromatography，Analyt.Biochem，145(1985)27-36。
8.Ingenmar Johansson，IGG separation medium Amersham，2002，USAPatent(US6399750)；
9.R.Hahn，R.Schlegel，A.Jungbauer，Comparison of protein A affinitysorbents，J.Chromatogr.B 790(2003)35-51；
10.R.Hahn，P.Bauerhansl，K.Shimahara，C.Wizniewski，A.Tscheliessnig，A.Jungbauer，Comparison of protein A affinity sorbentsII.Mass transfer properties.Journal of Chromatography.A 1093(2005)98-110；
11.Rainer Hahn，Kazumichi Shimahara，Franz Steindl，Alois Jungbauer，Comparison of protein A affinity sorbents III.Life time study，Journalof Chromatography A.1102(2006)224-231；
12.MabSelect Xtra Datafile Amersham Biosciences 11-0011-57；
13.The Gold Sheet，2004，38，9；
14.Tanja Wolter and Andreas Richter，Assays for controllingHost-cell impurities in Biopharmaceuticals，BioProcess technical；
15. the Hou Ji cutting edge of a knife or a sword, Cheng Yaqin, different methods is measured the comparison and analysis of Chinese hamster ovary cell albumen residual quantity in the genetically engineered drug, Chinese biochemical drug magazine, 23 (2002) 168-170;
16. Xu grows into forest, the detection of residual cells matrix among the Tang Qiaoying, former generation hamster kidney cell Vaccinum Encephalitidis Epidemicae, and the Chinese biological goods are learned magazine, 14 (2001) 48-50;
17.R.L.Fahrner，H.L.Knudsen，C.D.Basey，W.Galan，D.Feuerhelm，Vanderlaan，G.S.Blank，Biotechnol.Genet.Eng.Rev.18(2001)301；
18.Konrad，M.Immunogenicity of proteins administered to humansfor therapeutic purposes.Trends Biotechnol.7(1989)175；
19.Andreas Stein，Ander Kiesewetter，Cation exchange chromatographyin antibody purification pH screening for optimised binding and HCPremoval，Journal of Chromatography B，848(2007)151-158；
20. appoint jump bright, Cheng Yaqin, Ni Daoming, the quality control of intravenous injection human normal immunoglobulin, the Chinese biological goods are learned magazine, 18 (2005) 73;
21.S.Gibert，N.Bakalara，X.Santarelli，Three step chromatographicprocedure for the production of a His-tag recombinant kinesinoverexpressed in E.coli，Journal of Chromatography B，737(2007)143-150；
22.Q Sepharose FF.Datafile GE Healthcare.17-0510-10；
23.Chiti，et al.Rationalization of the effects of mutations onprotein aggregation rate，Nature，424(2004)805-808；
Summary of the invention
Defective or deficiency at above-mentioned prior art existence, the objective of the invention is to, a kind of method that adopts composite chromatography medium purification Fc fusion rotein is provided, through groping for a long time and a large amount of experimental results show that, this method can reduce the level of contaminative impurity in the product, improve the purity of target product, effectively guaranteed product quality.
In order to realize above-mentioned task, the present invention takes following technical solution:
A kind of method that adopts composite chromatography medium purification Fc fusion rotein, it is characterized in that the hydrophobic composite chromatography medium of this method selection ion-exchange, the pH that regulates the chromatography buffering system is lower than the proteic average pI of purification of target, regulate this chromatography buffering system simultaneously and have higher ionic strength, utilize suitable chromatography condition, making purification of target albumen be present in stream wears in the liquid, contaminative impurity then is adsorbed onto on the composite chromatography medium, collect described stream and wear liquid, thereby purification of target albumen is effectively removed the impurity of Fc fusion rotein.
The present invention can effectively remove the contaminative impurity in the product after the affinity chromatography, has improved the end product quality, and its purity is reached more than 99%.The hydrophobic composite chromatography medium of the ion-exchange of Cai Yonging is the medium of a kind of high carrying capacity, high flow rate through facts have proved simultaneously, is more suitable for industrial production.To sum up, it is tight that technology of the present invention is connected, and a step is effectively removed contaminative impurity, has characteristics such as good stability, clear and definite, the suitable large-scale production of process control index.
Description of drawings
Fig. 1 is a Capto adhere chromatography color atlas;
Fig. 2 is Superdex-200 color atlas (a MabSelect SuRe elutriant);
Fig. 3 is Superdex-200 color atlas (Capto adhere stream is worn liquid);
Fig. 4 is SE-HPLC color atlas (Capto adhere stream is worn liquid);
The present invention is described in further detail below in conjunction with embodiment that accompanying drawing and contriver provide.
Embodiment
The method of employing composite chromatography medium purification Fc fusion rotein of the present invention, adopt the hydrophobic composite chromatography medium of ion-exchange, the pH that regulates the chromatography buffering system is lower than the proteic average pI of purification of target, regulate this chromatography buffering system simultaneously and have higher ionic strength, by effective chromatography condition, making purification of target albumen be present in stream wears in the liquid, contaminative impurity (as host cell proteins, polymer etc.) then is adsorbed onto on the composite chromatography medium, one step collected this stream and wears liquid, thereby purification of target albumen is effectively removed the impurity of Fc fusion rotein.
Specifically may further comprise the steps:
1) selects composite chromatography medium for use
The medium of composite chromatography medium has been compound ion exchange and hydrophobic interaction, wherein ion exchange is the anionresin effect, preferred reinforcing yin essence ion exchange, the wherein preferred strong hydrophobic interaction of hydrophobic interaction;
2) the Fc fusion rotein of selection behind Protein A affinity chromatography purifying, Protein A is natural protein A or its functional derivatives;
3) regulate sample to be purified (containing target protein) pH than the low 0.5-2.5 pH unit of the average pI of described target protein, regulating its ionic strength simultaneously is 10ms/cm～50ms/cm, is higher than the used ionic strength of conventional ion exchang medium (0ms/cm～5ms/cm);
Should notice that this pI refers to the pI of actual cut and antibody purification, be not the pI that only estimates from aminoacid sequence.The polypeptide backbone of many antibody purified molecules has been modified in the reality, for example glycosylation.Described modification can be to add electrically charged part, thereby changes the pI of this molecule.Point focusing (IEF) such as pass through and measure the pI of product antibody, find the especially small unhomogeneity of monoclonal antibody protein translation post-treatment formation, and the product antibody of glycosylation form causes wideer pI scope, all these antibody form in the IEF gel and are similar to band in blocks but not single band, and most of at least products have specific (pI) numerical value.Therefore, according to the present invention, " average pI " is meant that pI is in the common pI of antibody product molecule in the above-mentioned pI preferable range, its prerequisite distributed number of glycosylation form (antibody) that has been this rationally fair representative of hypothesis.
4) the pH value of chromatography buffering system is made as the low 0.5-2.5 pH unit of average pI than described target protein, simultaneously the ionic strength of buffering system is made as 10ms/cm～50ms/cm, is higher than the used ionic strength of conventional ion exchang medium (0ms/cm～5ms/cm);
5) the chromatography flow velocity is made as 100cm/h～600cm/h; Make target protein be present in stream and wear in the liquid, contaminative impurity (as host cell proteins, polymer etc.) then is adsorbed onto on the composite chromatography medium.
6) all streams of step collection are worn the target protein in the liquid, detect collected stream and wear liquid, wherein the purity of protein monomer is at least 99%, removes the contaminative impurity (as host cell proteins, polymer form albumen) more than 1% at least, and the rate of recovery of target protein is at least more than 90%.
Described Fc fusion rotein is the Fc fusion rotein that animal cell expression is produced, and promptly contains the fusion rotein or the IgG antibody-like albumen of antibody Fc structure; Wherein, the fusion rotein that contains the antibody Fc structure is the species albumen that ProteinA is had higher affinity, and wherein IgG antibody-like albumen can be mouse source property, or humanized chimeric antibody, CDR-grafted antibody and complete humanized engineered antibody.
Described Fc fusion rotein is the monomeric protein of non-cohesion basically.
Described Fc fusion rotein is the Fc fusion rotein by the mammalian cell culture expression, or the antibody class albumen of Chinese hamster ovary celI culture expression.The pI of Fc fusion rotein is at least 6.5 or be higher than 6.5.
Condensation product of the present invention is interpreted as the non-covalent combination of same protein, and described protein can be made of the homogeneous or heterogeneous many polypeptide of single chain protein matter or covalent attachment (for example passing through disulfide-bonded).The same with its monomer of deriving, condensation product water soluble solution of the present invention.For example, the dimer condensation product is the non-specific binding of two IgG molecules.The formation of condensation product is closely related with the deformation effect (factor) of and quaternary structure of protein folding to natural protein.For example, heating can cause cohesion with the sex change of pH inductive protein folding.Therefore, given proteic cohesion rate has high degree of specificity, depends on the attack energy stability that each protein folding is specific
[23]
The composite chromatography medium that the present invention adopts is the medium of a kind of compound ion exchange interaction and hydrophobic interaction, experiment showed, that through the applicant it is a kind of high carrying capacity, high flow rate medium.Its dynamic carrying capacity can reach 75-300mg IgG/ml medium, and flow velocity can reach 600cm/h, and the ion-exchange chromatography media that adopts with respect to prior art is more suitable for large-scale production.
The pH of damping fluid is made as the pI that is different from product albumen to be purified or average pI, and described pH makes product albumen have surface charge, when contact or when being immersed in the described damping fluid, causes ion exclusion between the charged groups of product albumen and complex media.
The ionic strength of damping fluid makes product albumen have different hydrophobic propertys with contaminative impurity, when contacting or being immersed in the described damping fluid, cause hydrophobic repulsion between the hydrophobic grouping of product albumen and complex media, hydrophobic absorption between the hydrophobic grouping of contaminative impurity and complex media.
Below be the embodiment that the contriver provides, need to prove, the invention is not restricted to these embodiment.
The applicant adopts Chinese hamster ovary celI (TGLA) the excretory monoclonal antibody of transformation to carry out the purifying antibody preparation, and this cell is through genetic engineering modified Chinese hamster ovary celI, can express the monoclonal antibody of anti-CD20 related antigen by stability and high efficiency.
Use the mechanical agitation type bio-reactor, large scale and high density suspension culture TGLA cell; Clarification filtration cell culture fluid at first, adopt the IgG antibody in the MabSelect SuRe affinity chromatography one step absorption mass cell nutrient solution again, then regulate the pH and the ionic strength of affinity elution liquid, by complex media Capto adhere, obtain complete standard compliant mono-clonal IgG work in-process then.
Technology of the present invention is obtained antibody with the continuous purification process, and its technological process is mainly divided following a few step:
1, enchylema clarification: corporeal things such as the cell of removal 100% and cell debris.
2, MabSelect SuRe affinity media chromatography: preliminary purification and concentrated target product.
3, Capto adhere composite chromatography medium: obtain the high purity target product.
Experimental example:
1. enchylema clarification
Adopt 0.22 μ m capsule formula filter (PALL company), silicone tube, peristaltic pump (Baoding Lange pump company limited), constant current speed is filtered the Chinese hamster ovary celI culture supernatant, removes cell and cell debris.
2.MabSelect SuRe affinity chromatography
Utilize AKTA purifier-100 (GE Healthcare) chromatographic system, 20ml MabSelect SuRe (GE Healthcare) affinity chromatography medium is loaded in XK16/20 (GE Healthcare) chromatography column.With the abundant balance affinity column of level pad (20mM PB+150mM NaCl pH 6.0-7.0), treat that the uv-absorbing of 280nm gets back to baseline, and electricity is led, pH keeps beginning when stablizing to go up sample, not conjugated protein with the level pad flushing again, the uv-absorbing for the treatment of 280nm gets back to baseline and electricity is led, pH keep stable after, with the direct wash-out of elution buffer (100mM citrate buffer solution, pH 3.0-3.5), collect elutriant.
3.Capto adhere composite chromatography medium
The affinity chromatography elutriant is adjusted to pH 6-7 with 1M Tris-HCl, regulates ionic strength to 20ms/cm～50ms/cm with NaCl.
Utilize AKTA purifier-100 (GE Healthcare) chromatographic system, 20ml Capto adhere (GE Healthcare) composite chromatography medium is loaded in XK16/20 (GE Healthcare) chromatography column.With level pad (100mM citric acid-Tris damping fluid/160-320mM NaCl, pH 6.0-7.0) abundant balance Capto adhere composite chromatography post, treat that the uv-absorbing of 280nm gets back to baseline, and electricity is led, pH keeps beginning when stablizing to go up sample, not conjugated protein with the level pad flushing again, the uv-absorbing for the treatment of 280nm gets back to baseline and electricity is led, pH keep stable after, with the direct wash-out of elution buffer (100mM acetate).Collect stream and wear liquid, be target protein.
4.ELISA measure HCP
The HCP detection kit of this law employing Cygnus company (Immunoenzymetric Assay for theMeasurement of Chinese Hamster Ovary Host Cell Proteins, F015, CygnusTechnologies).Reference reagent box specification sheets, the concrete operations step is as follows:
1) in each centrifuge tube, adds standard substance (0-75ng/ml), reference substance, the test sample of 200ul respectively; 2) add the anti-CHO HCP antibody of the alkali phosphatase enzyme mark of 400ul in each centrifuge tube respectively; 3) build centrifuge tube, mixing, incubated at room 2 hours; 4) in 96 orifice plate bars, shift the above mixed solution that has reacted good, every hole 200ul; 5) build lath, and put into the plastics bag of sealing, 180rpm rotates and hatched 2 hours under the room temperature; 6) liquid in the drying lath adds scavenging solution 350ul, dries again, repeats 4 times; 7) developer of adding 200ul in every hole; 8) build lath, hatch 90min; 9) 405/492nm value of reading.
5. gel permeation chromatography (SEC) is measured D/A
Utilize AKTA purifier-100 (GE Healthcare) chromatographic system, with level pad (20mMPB+150mM NaCl, pH 7.2) abundant balance Superdex-200 post, the uv-absorbing for the treatment of 280nm gets back to baseline and electricity is led, pH keeps beginning to go up sample when stablize, washes 10 column volumes with level pad again.
6.SE-HPLC mensuration product purity
Utilize the Waters HPLC system of automatization, with level pad (100mM PB+100mMNa
2SO
4+ 0.05%NaN
3, pH 6.7) abundant balance TSKgel G3000SWXL post (10 column volumes), the uv-absorbing for the treatment of 280nm gets back to baseline and electricity is led, pH keeps beginning to go up sample when stablize, washes 10 column volumes with level pad again.
The result:
1.ELISA measure HCP
Table 1:Elisa measures HCP result in the Capto adhere chromatography process
| Sample number into spectrum | 1# | 2# | 3# |
| HCP concentration (ng/mL) | 368 | 26 | 235 |
| Sample concentration (mg/mL) | 0.6685 | 0.3492 | 0.4901 |
| HCP content (%) | 0.0005 | 0.00007 | 0.00048 |
| HCP content (ppm) | 551 | 74.4 | 480 |
Annotate: 1#MabSelect SuRe elution peak; 2#Capto adhere stream is worn the peak; 3#Capto adhere elution peak.
Table 1 shows that after Capto adhere handled, most of HCP all combined with Capto adhere medium, and gathers in the crops (see figure 1) in wash-out.Through 1# sample and 2# samples contg calculating complex media Captoadhere clearance is 86.5%.After the processing, the HCP residual quantity is 0.00007% in the product, much smaller than pharmacopeia regulation 0.1%, and is lower than 100ppm, can not cause possible immune response
[18]As seen, Captoadhere has the effect of good removal HCP.This step rate of recovery is 95.7% (result is not listed as) as calculated simultaneously.
2.Superdex-200 measure D/A
Table 2Superdex-200 measures D/A result in the Capto adhere chromatography process
| Sample number into spectrum | 1# | 2# | 3# |
| D/A content (%) | 3.46 | 0.25 | 54.73 |
Annotate: 1#MabSelect SuRe elutriant; 2#Capto adhere stream is worn; 3#Capto adhere wash-out.
D/A content before and after the contrast Capto adhere purifying is shown and Fig. 2 by table 2, Fig. 3 as seen, behind Capto adhere antibody purification under the condition of the present invention, its D/A content is reduced to 0.25% by 3.46%, clearance is 92.8%.As seen, Capto adhere composite chromatography medium can be removed D/A effectively, has effectively guaranteed quality product.
3.SE-HPLC mensuration product purity
Product purity after SE-HPLC measures process Capto adhere purifying is calculated as 99.72% (see figure 4) by area integral.
Claims (9)
1. method that adopts composite chromatography medium purification Fc fusion rotein, it is characterized in that this method adopts the hydrophobic composite chromatography medium of ion-exchange, the pH that regulates the chromatography buffering system is lower than the proteic average pI of purification of target, regulate this chromatography buffering system simultaneously and have higher ionic strength, utilize suitable chromatography condition, make purification of target albumen be present in stream and wear in the liquid, collect this stream and wear liquid, contaminative impurity then is adsorbed onto on the composite chromatography medium.
2. the method for claim 1 is characterized in that, this method may further comprise the steps at least:
1) medium of to adopt composite chromatography medium, this composite chromatography medium be compound ion exchange of chromatography buffering system and hydrophobic interaction, wherein ion exchange is anionresin effect or reinforcing yin essence ion exchange, wherein hydrophobic interaction is strong hydrophobic interaction;
2) select Fc fusion rotein behind Protein A affinity chromatography purifying for use, Protein A is natural protein A or its functional derivatives;
3) regulate sample pH value to be purified than low 0.5 the pH unit～2.5 pH unit of the average pI of described target protein, regulating its ionic strength simultaneously is 10ms/cm～50ms/cm, is higher than the used ionic strength of conventional ion exchang medium;
4) the pH value of chromatography buffering system is than low 0.5 pH～2.5 pH unit of average pI of described target protein, and the ionic strength of buffering system is made as 10ms/cm～50ms/cm simultaneously, is higher than the used ionic strength of conventional ion exchang medium;
5) chromatography is made as 100cm/h～600cm/h with flow velocity, makes target protein be present in stream and wears in the liquid, and contaminative impurity is combined on the composite chromatography medium;
6) collect all streams and wear target protein in the liquid, collected stream is worn in the liquid, and stream wears that the purity of protein monomer is at least 99% in the liquid, and the rate of recovery of target protein is at least more than 90%.
3. method as claimed in claim 2 is characterized in that, described Fc fusion rotein is the Fc fusion rotein that animal cell expression is produced, and promptly contains the fusion rotein or the IgG antibody-like albumen of antibody Fc structure; Wherein, the fusion rotein that contains the antibody Fc structure is the species albumen that Protein A is had higher affinity, and wherein IgG antibody-like albumen can be mouse source property, or humanized chimeric antibody, CDR-grafted antibody and complete humanized engineered antibody.
4. method as claimed in claim 2 is characterized in that, described Fc fusion rotein is the monomeric protein of non-cohesion basically.
5. method as claimed in claim 2 is characterized in that, described Fc fusion rotein is the Fc fusion rotein by the mammalian cell culture expression, or the antibody class albumen of Chinese hamster ovary celI culture expression.
6. method as claimed in claim 2 is characterized in that, the pI of described Fc fusion rotein is at least 6.5 or be higher than 6.5.
7. method as claimed in claim 2, it is characterized in that, the pH of described damping fluid is made as pI or the average pI that is different from product albumen to be purified, described pH makes target protein have surface charge, when contact or when being immersed in the described damping fluid, cause ion exclusion between the charged groups of target protein and complex media.
8. method as claimed in claim 2, it is characterized in that, the ionic strength of described damping fluid makes target protein have different hydrophobic propertys with contaminative impurity, when contacting or being immersed in the described damping fluid, the hydrophobic grouping of target protein and complex media produces hydrophobic repulsion, and the hydrophobic grouping of contaminative impurity and complex media produces hydrophilic absorption.
9. method as claimed in claim 2 is characterized in that, the stream of described collection wears that the purity of protein monomer is at least 99% in the liquid, and this purity collects to realize by a step, removes the contaminative impurity more than at least 1%.
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