CN103254311A - Method for preparing antibody-maytansine alkaloid medicine conjugate - Google Patents
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Abstract
The invention relates to a method for preparing antibody-maytansine alkaloid medicine conjugate. The method comprises the following steps of replacing the antibody into a reaction buffer solution; dissolving a dual-function bridging agent-maytansine alkaloid with an organic solvent so as to prepare the mother liquor of maytansine alkaloid medicine; mixing the replaced antibody with the mother liquor of maytansine alkaloid medicine for coupled reaction for 1-4 hours at 20-30 DEG C; and carrying out anion exchange chromatography on the reaction liquid, conducting Sephadex TMG25 desalination chromatographic column purification on collected flow liquid, and collecting a first peak sample as the prepared antibody-maytansine alkaloid medicine conjugate. The prepared antibody-maytansine alkaloid medicine conjugate is proper in coupling rate, high in purity and low in endotoxin content.
Description
Technical field
The present invention relates to the preparation method of a kind of antibody-maytenin alkaloids medicament conjugate, belong to biological technical field.
Background technology
Along with the exploitation of target therapeutic agent, treatment for cancer has obtained obvious improvement.In recent years, cell receptor and antigen that the researchist utilizes the cancer cells surface selectivity to express had been developed many antibody drugs, and these antibody can carry out specificity with tumour cell is combined.On this basis, with cytotoxic molecule, carrying out chemistry such as bacterium with plant poison, radionuclide and some chemotherapeutic agent and antibody is connected, obtain antibody-drug conjugates, this antibody-like-drug conjugates is killed tumour cell be combined the cytotoxic activity of back release medicine or activation medicine with tumour cell.The specificity of antibody-drug conjugates can reduce Normocellular toxicity, has therefore improved the tolerance of medicine in patient's body.
Maytenin (Maytansine) at first is to be separated from East Africa shrub tingia Caulis Mayteni (Maytenus serrata) by people such as Kupchan.The maytenin alkaloid is the Cytotoxic medicine of a class height, and according to the study, its toxicity is eager to excel 100 to 1000 times than the cytotoxicity of traditional cancer chemotherapeutic drug such as methotrexate, daunorubicin etc., referring to US3896111.Afterwards, find that some microorganisms also can produce the maytenin alkaloid, as the C-3 ester of Ansamitocin Po (maytansinol) and Ansamitocin Po, referring to US4151042.
Maytenin is mitotic inhibitor, reports, with the L1210 cell that maytenin is handled, cause 67% the m period that is accumulated in, and not processed control cells indication range is between 3.2% to 5.8%.With studies show that of sea urchin egg and clam ovum, maytenin disturbs the formation of microtubule by the polymerization that suppresses tubulin, thereby suppresses mitotic division.Referring to Remillard, 189Science1002-1005(1975).
Because the alkaloidal height cytotoxicity of maytenin indicates that they have effect in the treatment cancer, but, the alkaloidal clinical trial of maytenin is also unsatisfactory, mainly is because their side effect.As if side effect to central nervous system and gastrointestinal symptoms causes the patient to refuse further treatment, and the maytenin alkaloid is relevant with peripheral neuropathy, this peripheral neuropathy may be (the Issel at207) of accumulation.
Can directed medicine be sent to the target cell site by preparation antibody-maytenin alkaloid conjugate, thereby reduce the toxicity to healthy tissues and cell, reduce the alkaloidal side effect of maytenin.
CN101087611A has reported with cutting conjugate that joint connects antibody and the preparation of maytenin alkaloid and being connected antibody and comparing with the conjugate that the maytenin alkaloid prepares with cutting joint, both have identical external and anti-tumor in vivo activity, but the former is showing significant reduction aspect plasma clearance and the toxicity.
US5208020 and US5416064 etc. disclose the method for preparing antibody-maytenin alkaloid conjugate, at first use bifunctional linking reagent and monoclonal antibody coupling, utilize Sephadex
TMG25 chromatographic column purifying connects the monoclonal antibody that goes up linking agent, will be connected with the monoclonal antibody of linking agent and the excessive chemical toxicant coupling that contains sulfydryl again, utilizes Sephadex again
TMG25 chromatographic column purifying.
CN101087611A also discloses the method that the mode identical with US5208020, US5416064 prepares antibody-maytenin alkaloid conjugate, namely earlier with bifunctional linking reagent and monoclonal antibody coupling, purifying obtains first mixture, again with the chemical toxicant coupling, carries out purifying and obtains conjugate.The antibody-drug conjugates purity of utilizing this mode to obtain is low and unstable.For example, the purity of the appropriate pearl of disclosed song-SMCC-DM1 conjugate has only about 95% among the CN101087611A embodiment, and the content of albumen aggressiveness thing has accounted for more than 4.2%, and there is the risk that causes higher immunogenicity in existing in of this superpolymer in the therapeutic process.
CN102596922A discloses a kind of new bifunctional linking reagent, namely incorporates hydrophilic PEG spacer at traditional bifunctional linking reagent it is modified to the wetting ability linker.The antibody-drug conjugates that utilizes this linking agent coupling to obtain can allow maximum 15 drug molecules of each antibody molecule coupling, thereby has improved joint efficiency.Simultaneously, also disclose a kind of method of new antibody-drug coupling, namely earlier bifunctional linking reagent has been connected with chemicals, it is being connected with monoclonal antibody.Utilize this coupling method to reduce the purification step in the coupling, improved product yield, also greatly reduce the heterogeneity of antibody-drug conjugates simultaneously.But the bifunctional linking reagent that utilizes the disclosed scheme synthesis hydrophilic of this patent document is a more loaded down with trivial details chemical reaction process, may limit the application of this kind bifunctional linking reagent.And the disclosed coupling method of CN102596922A synthesizes bent appropriate pearl-SMCC-DM1, utilize TSK-GEL size exclusion chromatography (moving phase contains 15% Virahol, can better conjugate monomer and aggressiveness be separated) analyze to find still to contain conjugate aggressiveness up to 4%.And, in order to obtain suitable coupling rate, the i.e. mol ratio of conjugate Chinese traditional medicine and antibody, generally need when reaction, add 6-10 times of overdose of medicine thing, for example, in the appropriate pearl of song-SMCC-DM1 preparation, need to drop into 7-9 times of overdose of medicine thing, generally between 7.5-8.5 times, cost is increased.
Summary of the invention
At the deficiencies in the prior art, the invention provides the coupling method that a kind of high coupling efficiency prepares antibody-maytenin alkaloid conjugate, the conjugate that this method prepares has suitable coupling rate, high purity and low endotoxin content.
Detailed technology scheme of the present invention is as follows:
The preparation method of a kind of antibody-CHROMATOGRAPHIC FRACTIONATION AND MASS alkaloidal drug conjugate may further comprise the steps:
(1) antibody displacement damping fluid
Utilize the mode of ultrafiltration and concentration or desalination chromatography to replace antibody stoste to reaction buffer, contain the tensio-active agent of 0.02%～0.1%v/v in the reaction buffer, be concentrated into antibody concentration at 20～30mg/ml; Antibody after must replacing;
The stoste of described antibody stoste monoclonal antibody;
(2) preparation maytenin alkaloids medicament mother liquor
Get the maytenin alkaloids medicament that is connected with bifunctional linking reagent, namely bifunctional linking reagent-maytenin alkaloid is used organic solvent dissolution, and preparation contains the mother liquor of 10mg/ml maytenin alkaloidal drug.
(3) linked reaction
Mol ratio in difunctional link agent-maytenin alkaloid and antibody is the ratio of 5～6:1, antibody after the displacement that obtains in (1) step is mixed with the maytenin alkaloid mother liquor that obtains during (2) goes on foot, carry out linked reaction, 20～30 ℃ of temperature of reaction, 1～4 hour reaction times;
(4) anion-exchange chromatography
Sample Zhiyin ion exchange column on the above-mentioned reaction solution is carried out purifying; At first use level pad balance anion chromatography column, go up sample then, the chromatography column carrying capacity is 30-50mg/ml, and last sample flow velocity is 75-100cm/h, collects stream and wears liquid, after last sample finishes, with level pad flushing anion chromatography post, finishes until collection again;
(5) desalination chromatography
The stream of above-mentioned anion-exchange chromatography collection is worn liquid carry out Sephadex
TMG25 desalination chromatography column purifying; Desalination damping fluid balance desalination chromatography column, the desalination damping fluid contains 0.02～0.1%(v/v) tensio-active agent, with sample on the collection liquid of anion-exchange chromatography to the desalination chromatography column, flow velocity is 2～5cm/h, collect first and go out the peak sample, be prepared antibody-maytenin alkaloids medicament conjugate.
Preferred according to the present invention, the described antibody stoste of step (1) is selected from Herceptin, Cetuximab or handkerchief Buddhist nun monoclonal antibody.
Preferred according to the present invention, the reaction buffer of the described displacement antibody of step (1) stoste is selected from sodium phosphate salt damping fluid, potassium phosphate salt damping fluid, Tris-Hcl damping fluid or Sodium phosphate dibasic-citrate buffer solution, pH of buffer 6.0～9.0.
Preferred according to the present invention, the mode of the described ultrafiltration and concentration of step (1) or desalination chromatography can adopt prior art, for example can carry out ultrafiltration and concentration by the Pellicon system, or adopts Sephadex
TMThe G25 gel chromatography column carries out the desalination chromatography.
Preferred according to the present invention, the described tensio-active agent of step (1) mainly refers to nonionic surface active agent, the smooth class of preferred fatty acid sorb and poly yamanashi esters, the wherein preferred span 40 of the smooth class of lipid acid sorb, sorbester p18; The preferred polysorbas20 of poly yamanashi esters, polysorbate40, polysorbate60 and tween 80, the consumption of tensio-active agent are 0.02%～0.1%(v/v).
Preferred according to the present invention, the described organic solvent of step (2) is selected from N,N-DIMETHYLACETAMIDE (DMA), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO) (DMSO) or ethanol (EtOH).
Preferred according to the present invention, the described bifunctional linking reagent of step (3) is selected from N-succsinic acid imide 4-(maleimide methyl) cyclohexane carboxylate (SMCC), N-succsinic acid imide 4-(maleimide methyl) hexanaphthene-1-carboxyl-(6-amido capronate) (LC-SMCC), all can be available from Suzhou sky sail biotech company.
Preferred according to the present invention, the described maytenin alkaloids medicament of step (3) is synthetic by ansamitocin P-3.The method reference literature J Med.Chem.2006 of synthetic maytenin alkaloidal drug, the method for 49,4392-4408 is carried out.Ansamitocin P-3 is commercially available.For example available from beautiful territory biological medium company.
Preferred according to the present invention, the method that the described maytenin alkaloids medicament that is connected with bifunctional linking reagent of step (3) can provide with reference to [0438] section of CN102596922A is synthetic.
Preferred according to the present invention, the chromatography media of the described anion-exchange chromatography of step (4) is selected from Q Sepharose, DEAE Sepharose, Capto Q, Capto DEAE, Capto Adhere, preferably uses Capto Adhere.
Preferred according to the present invention, the used level pad of the described anion-exchange chromatography of step (4) can be identical with the used reaction buffer of step (1), also can select different damping fluids; The used level pad of anion-exchange chromatography can be selected from sodium phosphate salt damping fluid, potassium phosphate salt damping fluid, Tris-Hcl damping fluid or Sodium phosphate dibasic-citrate buffer solution, still needs to add 0.02%～0.1%(v/v) tensio-active agent in the damping fluid.Described tensio-active agent is identical with the tensio-active agent of step (1).
Preferred according to the present invention, the desalination damping fluid that the described desalination chromatography of step (5) uses is selected citrate buffer, acetate buffer, succsinic acid damping fluid or phosphate buffered saline buffer, contains 0.02%～0.1%(v/v) tensio-active agent in the damping fluid.Described tensio-active agent is identical with the tensio-active agent of step (1).
In the present invention, the described anion-exchange chromatography of step (4) adopts and passes pattern, and namely the required antibody-drug conjugates monomer flow that obtains is worn, and the antibody-drug conjugates aggressiveness is attracted on the anion chromatography post; In order to improve the effect of anion-exchange chromatography purifying, the present invention has selected lower linear rate of flow, is generally 75～100cm/h, chromatography carrying capacity 30～50mg/ml.
In the present invention, the described desalination chromatography of step (5) is Sephadex
TMG25 chromatographic column purifying for effectively unreacted maytenin alkaloids medicament, organic solvent etc. and antibody-maytenin alkaloidal drug conjugate being separated, has been selected lower linear rate of flow, is generally 2～5cm/h.
" antibody " described in the inventive method can be any immunoglobulin (Ig), any immunoglobulin fragment such as Fab, F(ab ')
2, dsFv, sFv, double antibody and three chain antibodies, perhaps immunoglobulin chimeric body, they can be combined by the antigen on cell surface.Antibody can be polyclonal antibody or monoclonal antibody, and optimal selection is monoclonal antibody.Monoclonal antibody refers to the antibody molecule of homogeneous, and it is specific to specific antigen.General by the generation of bone-marrow-derived lymphocyte mono-clonal.Preferred antibody is Humanized monoclonal antibodies in present method, such as comprising huN901, huMy9-6, huB4, huC242, Herceptin, handkerchief Buddhist nun monoclonal antibody and Rituximab etc.
The cytotoxic agent that uses in the inventive method is the maytenin alkaloid, and being selected from the DM1(chemical name is N
2 '-Tuo acetyl-N
2 '-3-sulfydryl-1 oxopropyl)-and maytenin), the DM4(chemical name is N
2 '-Tuo acetyl-N
2 '-4-methyl-4 sulfydryl-1 oxo amyl group)-maytenin).Cytotoxic agent is optional other suitable chemical toxicity medicines also, can cause necrocytosis, lure necrocytosis or reduce any compound of cell survival, suitable cytotoxic agent is selected from taxanes, CC-1065 and CC-1065 analogue or dolastatin and analogue etc.Preferred described maytenin alkaloid is DM1 or DM4.
The inventive method, preferred antibody-CHROMATOGRAPHIC FRACTIONATION AND MASS alkaloidal drug conjugate is: bent appropriate pearl-SMCC-DM1, Pa Ni-SMCC-DM1, western appropriate former times-SMCC-DM1.
According to the present invention, optimized technical scheme one, the preparation method of bent appropriate pearl-SMCC-DM1 conjugate, step is as follows:
(1) utilizes Sephadex
TMG25 chromatographic column purifying desalination chromatography mode is replaced Herceptin stoste to reaction buffer, and reaction buffer consists of the 0.1M sodium phosphate salt, and pH7.5 contains the polysorbas20 of 0.05%v/v; Concentrated antibody concentration makes the bent appropriate pearl antibody of displacement to 20mg/ml;
(2) take by weighing SMCC-DM1, fully dissolve the SMCC-DM1 mother liquor of preparation 10mg/ml with N,N-DIMETHYLACETAMIDE (DMA);
(3) mol ratio by SMCC-DM1 and antibody is 5～5.5:1, adds the SMCC-DM1 mother liquor of step (2) preparation in the prepared Herceptin of step (1), the beginning linked reaction, and temperature of reaction is 25 ℃, the reaction times is 1.5h;
(4) with sample Capto Adhere anion-exchange chromatography post on the above-mentioned reaction solution.Chromatography column carries out balance with corresponding reaction buffer in the step (1) earlier, sample on the reaction solution after then step (3) reaction being finished, and flow velocity is 75cm/h, carrying capacity is 35mg/ml, collects stream and wears sample, after last sample finishes, with corresponding reaction buffer flushing, finish to collecting;
(5) above-mentioned collection liquid through anion-exchange chromatography is carried out Sephadex respectively
TMG25 desalination chromatography.With desalination damping fluid balance, flow velocity is 3cm/h to the desalination chromatography column earlier.Used desalination damping fluid is the 20mM succsinic acid, 0.05% polysorbas20, pH5.5.After last sample finishes, with same damping fluid flushing, collect out the peak sample according to the UV280 ultraviolet absorption value, be the bent appropriate pearl-SMCC-DM1 conjugate of preparation.
Optimal technical scheme two of the present invention, the preparation method of Pa Ni-SMCC-DM1 conjugate is as described in embodiment 3.
Optimal technical scheme three of the present invention, the preparation method of western appropriate former times-SMCC-DM1 conjugate is as described in embodiment 4.
In the monoclonal antibody purge process, generally adopt the stream of the anion-exchange chromatography pattern of wearing to remove the monoclonal antibody aggressiveness, because the monoclonal antibody aggressiveness generally has more multi-charge than monomer and is attracted on the anionic exchange medium easily.The conjugate aggressiveness that produces in the linked reaction also can be removed by anion-exchange chromatography.In addition, anion-exchange chromatography also has the endotoxic effect of removal, the hidden danger when having reduced clinical application.
The characteristics of technical solution of the present invention: namely adopt the damping fluid that adds tensio-active agent in antibody displacement damping fluid step, like this in follow-up linked reaction system, can increase the wetting ability of medicine, improve the efficient of linked reaction, thereby reduce the feed ratio of linked reaction.Simultaneously, the damping fluid of adding tensio-active agent also can effectively reduce antibody-drug conjugates produces the conjugate aggressiveness because of the medicine hydrophobicity probability in the subsequent purification step.In the antibody purification step, increased by a step anion-exchange chromatography step, compare with the coupling process that does not increase anion-exchange chromatography, can improve the purity of finished product, reduced endotoxin content in the product, and coupling rate and total recovery have not almost been had influence.
Excellent results of the present invention is:
1, in the preparation antibody-reaction buffer of maytenin alkaloid conjugate and purifying damping fluid afterwards, added tensio-active agent, thereby make preparation antibody-maytenin alkaloid conjugate have higher efficient, be example with the appropriate pearl-SMCC-DM1 of song, with carrying out linked reaction under the equal proportion, add tensio-active agent linked reaction feed ratio with do not add surface-active feed ratio and compare, feed ratio can be reduced to 1:6 by 1:7.5, improve the efficient of linked reaction greatly, reduced cost.
2, make the antibody-maytenin alkaloid conjugate of preparation have higher purity and lower endotoxin content by increasing by a step anion-exchange chromatography.Be example with the appropriate pearl-SMCC-DM1 of song, SEC-HPLC purity reaches more than 98%, and the antibody-drug conjugates aggressiveness of formation is less than 2%; The product endotoxin content is reduced to 0.2EU/mg by the 0.8EU/mg of prior art, in the antibody-drug conjugates level of free medicine less than 2%(with respect to total medicine).
Embodiment
The measuring method of coupling rate: measure the coupling rate and refer to measure the quantity that is connected with the maytenin alkaloids medicament on each monoclonal antibody molecule, the i.e. ratio of maytenin alkaloids medicament mole number and monoclonal antibody mole number.Can adopt two kinds of methods of ultraviolet spectrophotometry and mass spectrometry to measure.Ultraviolet spectrophotometry: by measuring antibody-maytenin alkaloids conjugate at the ultraviolet absorption value at 252nM and 280nM place, and calculate the coupling rate by langbobier law; Mass spectrometry: antagonist-maytenin alkaloid conjugate carries out ESI-MS to be analyzed, and can obtain to connect the peak type figure without the number medicine, calculates the coupling rate according to the area at each peak.The coupling rate that obtains by two kinds of methods has good consistence.
Conjugate concentration determination: adopt ultraviolet spectrophotometry, determine the ultraviolet absorption value at 252nM and 280nM place, can calculate antibody and drug concentrations by langbobier law; Conjugate concentration described in the present invention is monoclonal antibody concentration and the summation that is connected to the maytenin alkaloidal drug on the monoclonal antibody.
SEC-HPLC analyzes: utilize GLK3000 efficient molecular sieve post antagonist-maytenin alkaloidal drug to analyze, moving phase is selected 20mM PB for use, 15% Virahol, pH7.0.Calculate conjugate aggressiveness and the shared ratio of conjugate monomer according to area normalization method.
Endotoxin content detects: carry out with reference to " 2010 editions three ones of Chinese Pharmacopoeias " appendix XII E bacterial endotoxins test.
Further specify the present invention by the following examples, still, should be appreciated that these embodiment are only used for the more detailed usefulness that specifically describes, and it should be interpreted as for limiting the present invention in any form.In this article, unless otherwise indicated, agents useful for same is the commercially available prod.
The maytenin alkaloids medicament that is connected with bifunctional linking reagent among the embodiment (being bifunctional linking reagent-maytenin alkaloid) is SMCC-DM1.SMCC is available from Suzhou sky sail biotech company, and DM1 is that the method for 49,4392-4408 is synthesized by ansamitocin P-3 reference literature JMed.Chem.2006, and wherein ansamitocin P-3 is available from beautiful territory biological medium company.SMCC-DM1 is synthetic with reference to [0438] section method that provides of CN102596922A.
Product among the embodiment adopts the phraseology of " antibody-bifunctional linking reagent-maytenin alkaloid ".
Embodiment 1, preparation bent appropriate pearl-SMCC-DM1 conjugate, checking add behind the tensio-active agent influence to coupling efficiency.
The preparation method of bent appropriate pearl-SMCC-DM1 conjugate, step is as follows:
(1) antibody displacement damping fluid
Utilize desalination chromatography (Sephadex
TMG25) mode is replaced Herceptin stoste to reaction buffer A1(0.1M sodium phosphate salt, pH7.5) in, and concentrated antibody concentration prepares bent appropriate pearl antibody A 1 to 20mg/ml;
Utilize desalination chromatography (Sephadex
TMG25) mode is replaced Herceptin stoste to reaction buffer A2(0.1M sodium phosphate salt, pH7.5,0.05%v/v polysorbas20) in, and concentrated antibody concentration prepares bent appropriate pearl antibody A 2 to 20mg/ml;
(2) preparation SMCC-DM1 mother liquor
Take by weighing SMCC-DM1, fully dissolve the SMCC-DM1 mother liquor of preparation 10mg/ml with N,N-DIMETHYLACETAMIDE (DMA);
(3) linked reaction
The SMCC-DM1 mother liquor that in the prepared Herceptin of step (1), adds step (2) preparation, the beginning linked reaction, temperature of reaction is 25 ℃, the reaction times is 1.5h; Reaction is divided into three groups and carries out:
A: use reaction buffer A1 and bent appropriate pearl antibody A 1, add the SMCC-DM1 mother liquor of 7.5 times of excess molar ratio in the reaction;
B: use reaction buffer A2 and bent appropriate pearl antibody A 2, add the SMCC-DM1 mother liquor of 7.5 times of excess molar ratio in the reaction;
C: use reaction buffer A2 and bent appropriate pearl antibody A 2, add the SMCC-DM1 mother liquor of 5.5 times of excess molar ratio in the reaction;
(4) anion-exchange chromatography
Respectively with sample Capto Adhere anion-exchange chromatography post on above-mentioned a, b, the c three group reaction liquid.Chromatography column carries out balance with corresponding reaction buffer in the step (1) earlier, sample on the reaction solution after then step (3) reaction being finished, and flow velocity is 75cm/h, carrying capacity is 35mg/ml, collects stream and wears sample, after last sample finishes, with corresponding reaction buffer flushing, finish to collecting;
(5) desalination chromatography
Above-mentioned three groups of collection liquid through anion-exchange chromatography are carried out Sephadex respectively
TMG25 desalination chromatography.With desalination damping fluid balance, flow velocity is 3cm/h to the desalination chromatography column earlier.The used desalination damping fluid of a group sample is the 20mM succsinic acid, pH5.5, and b group and the used damping fluid of c group sample are the 20mM succsinic acid, 0.05%v/v polysorbas20, pH5.5.After last sample finished, each group with corresponding damping fluid flushing, was collected out the peak sample according to the UV280 ultraviolet absorption value respectively, is the bent appropriate pearl-SMCC-DM1 conjugate of preparation.
The bent appropriate pearl-SMCC-DM1 conjugate that obtains is utilized concentration and the coupling rate of ultraviolet spectrophotometry (measuring conjugate respectively in the absorbancy of 252nm and 280nm) counting yield, also use simultaneously the coupling rate of mass spectrometry counting yield, and compare, data see Table 1.
Table 1 adds tensio-active agent to the influence of coupling efficiency
| Sample | Product concentration (mg/ml) | Yield | Coupling rate (ultraviolet method) | Coupling rate (mass spectroscopy) |
| Bent appropriate pearl-SMCC-DM1a | 6.50 | 89.5% | 3.21 | 3.19 |
| Bent appropriate pearl-SMCC-DM1b | 6.23 | 90.05% | 4.30 | 4.37 |
| Bent appropriate pearl-SMCC-DM1c | 6.36 | 87.6% | 3.62 | 3.66 |
As can be seen from Table 1, in buffer solution system, add certain amount of surfactant (polysorbas20 as 0.05%) and can effectively improve coupling efficiency.Identical molar feed ratio can obtain higher coupling rate, reduces molar feed ratio simultaneously and be can obtain the antibody-drug conjugates of 3.5 left and right sides coupling rates at 5.5 o'clock.
Embodiment 2, in coupling process, add content and endotoxin content that anion-exchange chromatography can reduce the conjugate aggressiveness.
The preparation of bent appropriate pearl-SMCC-DM1 conjugate, step is as follows:
(1) antibody displacement damping fluid: at first utilize desalination chromatography (Sephadex
TMG25) mode is replaced bent appropriate pearl antibody stoste to reaction buffer (0.1M sodium phosphate salt, pH7.5,0.05%v/v polysorbas20), and concentrated antibody concentration is to 20mg/ml;
(2) preparation SMCC-DM1 mother liquor: take by weighing with step (1) in the SMCC-DM1 of 5.5 times of Herceptin mol ratios, with the abundant SMCC-DM1 mother liquor of dissolving preparation 10mg/ml of N,N-DIMETHYLACETAMIDE (DMA);
(3) linked reaction:
The SMCC-DM1 mother liquor that in the prepared Herceptin of step (1), adds step (2) preparation, the beginning linked reaction, temperature of reaction is 25 ℃, the reaction times is 1.5h;
Reaction solution is divided into two groups of d, e, and the d group is only carried out the desalination chromatography, and the e group is carried out anion-exchange chromatography earlier, carries out the desalination chromatography again;
(4) anion-exchange chromatography
With sample Capto Adhere anion-exchange chromatography post on the above-mentioned e group reaction liquid.Chromatography column carries out balance with the reaction buffer in the step (1) earlier, goes up the corresponding reaction solution of sample then, and flow velocity is 75cm/h, and carrying capacity is 35mg/ml, collects stream and wears sample, after last sample finishes, with the reaction buffer flushing, finishes to collecting;
(5) desalination chromatography
The collection liquid of above-mentioned e group anion-exchange chromatography and the reaction solution of d group are carried out Sephadex respectively
TMG25 desalination chromatography.With desalination damping fluid balance, flow velocity is 3cm/h to the desalination chromatography column earlier, and used damping fluid is the 20mM succsinic acid, 0.05%v/v polysorbas20, pH5.5.Go up sample after balance finishes, continue flushing with damping fluid, collect out the peak sample according to the UV280 absorption value, be the bent appropriate pearl-SMCC-DM1 conjugate of preparation.
The bent appropriate pearl-SMCC-DM1 conjugate that obtains is utilized concentration and the coupling rate of ultraviolet spectrophotometry (measuring conjugate respectively in the absorbancy of 252nm and 280nm) counting yield, utilize the purity of SEC-HPLC testing product simultaneously, utilize endotoxin content in the limulus reagent test testing product.
Table 2 anion-exchange chromatography is to the influence of conjugate
| Sample | Product concentration (mg/ml) | Yield | SEC-HPLC aggressiveness % | SEC-HPLC monomer % | Intracellular toxin |
| Bent appropriate pearl-SMCC-DM1d | 9.32 | 93.5% | 4.16 | 95.76 | 0.8EU/mg |
| Bent appropriate pearl-SMCC-DM1e | 6.16 | 88.9% | 1.23 | 98.70 | 0.2EU/mg |
As can be seen from Table 2, the coupling reaction solution is being carried out in the process of purifying, increasing by a step anion-exchange chromatography, can effectively reduce the content of conjugate aggressiveness, improving the purity of conjugate, also can further reduce endotoxic content in the product simultaneously.With respect to the coupling process that does not increase anion-exchange chromatography, the total recovery value reduces and about 5%.
Embodiment 3, preparation Pa Ni-SMCC-DM1 conjugate, step is as follows:
(1) antibody displacement damping fluid
At first utilize ultrafiltration and concentration (pellicon system) mode replace handkerchief Buddhist nun monoclonal antibody stoste to reaction buffer (20mM potassium phosphate salt damping fluid, the 0.02%v/v polysorbas20, pH6.0) in, and concentrated antibody concentration is to 30mg/ml.
(2) preparation SMCC-DM1 mother liquor
Take by weighing with step (1) in the SMCC-DM1 of 5.0 times of handkerchief Buddhist nun monoclonal antibody mol ratios, with the abundant SMCC-DM1 mother liquor of dissolving preparation 10mg/ml of N,N-DIMETHYLACETAMIDE (DMA).
(3) linked reaction
The SMCC-DM1 mother liquor that in the prepared handkerchief Buddhist nun monoclonal antibody of step (1), adds step (2) preparation, the beginning linked reaction, temperature of reaction is 25 ℃, the reaction times is 2.0h;
(4) anion-exchange chromatography
With sample Capto Adhere anion-exchange chromatography post on the above-mentioned reaction solution.Chromatography column carries out balance with the reaction buffer in the step (1) earlier, goes up the sample reaction solution then, and flow velocity is 75cm/h, and carrying capacity is 45mg/ml, collects stream and wears sample, after last sample finishes, with the reaction buffer flushing, finishes to collecting;
(5) desalination chromatography
The collection liquid of above-mentioned anion-exchange chromatography is carried out Sephadex
TMG25 desalination chromatography.With desalination damping fluid balance, flow velocity is 3.5cm/h to the desalination chromatography column earlier.The used damping fluid of desalination is the 20mM sodium phosphate salt, 0.02%v/v polysorbas20, pH7.2.After last sample finishes, with the flushing of desalination damping fluid, collect out the peak sample according to the UV280 ultraviolet absorption value, be the Pa Ni-SMCC-DM1 conjugate of preparation.
Pa Ni-SMCC-DM1 the conjugate that obtains is utilized concentration and the coupling rate of ultraviolet spectrophotometry (measuring conjugate respectively in the absorbancy of 252nm and 280nm) counting yield, utilize the purity of SEC-HPLC testing product simultaneously, utilize endotoxin content in the limulus reagent test testing product.
The character of table 3 Pa Ni-SMCC-DM1 conjugate product
Embodiment 4, preparation western appropriate former times-SMCC-DM1 conjugate
(1) antibody displacement damping fluid
At first utilize ultrafiltration and concentration (pellicon system) or desalination chromatography (Sephadex
TMG25) mode replace Cetuximab stoste to reaction buffer (20mM Sodium phosphate dibasic-10mM citric acid, the 0.1%v/v tween 80, pH8.0) in, and concentrated antibody concentration is to 20mg/ml;
(2) preparation SMCC-DM1 mother liquor
Take by weighing with step (1) in the SMCC-DM1 of 6.0 times of Cetuximab mol ratios, with the abundant SMCC-DM1 mother liquor of dissolving preparation 10mg/ml of N,N-DIMETHYLACETAMIDE (DMA);
(3) linked reaction
The SMCC-DM1 mother liquor that in the prepared Cetuximab of step (1), adds step (2) preparation, the beginning linked reaction, temperature of reaction is 22.5 ℃, the reaction times is 4.0h;
(4) anion-exchange chromatography
With sample Capto Adhere anion-exchange chromatography post on the above-mentioned reaction solution.Chromatography column carries out balance with reaction buffer earlier, goes up the sample reaction solution then, and flow velocity is 75cm/h, and carrying capacity is 40mg/ml, collects stream and wears sample, after last sample finishes, with the reaction buffer flushing, finishes to collecting;
(5) desalination chromatography
The collection liquid of above-mentioned anion-exchange chromatography is carried out Sephadex
TMG25 desalination chromatography.With desalination damping fluid balance, flow velocity is 3cm/h to the desalination chromatography column earlier.The used damping fluid of desalination is the 20mM citric acid-sodium citrate, contains the 0.1%v/v tween 80, pH5.0.After last sample finishes, with the flushing of desalination damping fluid, collect out the peak sample according to the UV280 ultraviolet absorption value, be the western appropriate former times-SMCC-DM1 conjugate of preparation.
The western appropriate former times-SMCC-DM1 conjugate that obtains is utilized concentration and the coupling rate of ultraviolet spectrophotometry (measuring conjugate respectively in the absorbancy of 252nm and 280nm) counting yield, utilize the purity of SEC-HPLC testing product simultaneously, utilize endotoxin content in the limulus reagent test testing product.
The character of table 4 western appropriate former times-SMCC-DM1 conjugate product
Claims (10)
1. the preparation method of antibody-CHROMATOGRAPHIC FRACTIONATION AND MASS alkaloidal drug conjugate may further comprise the steps:
(1) antibody displacement damping fluid
Utilize the mode of ultrafiltration and concentration or desalination chromatography to replace antibody stoste to reaction buffer, contain the tensio-active agent of 0.02%～0.1%v/v in the reaction buffer, be concentrated into antibody concentration at 20～30mg/ml; Antibody after must replacing;
The stoste of described antibody stoste monoclonal antibody;
(2) preparation maytenin alkaloids medicament mother liquor
Get the maytenin alkaloids medicament that is connected with bifunctional linking reagent, namely bifunctional linking reagent-maytenin alkaloid is used organic solvent dissolution, and preparation contains the mother liquor of 10mg/ml maytenin alkaloidal drug;
Preferably, described maytenin alkaloid is DM1 or DM4;
(3) linked reaction
Mol ratio in difunctional link agent-maytenin alkaloid and antibody is the ratio of 5～6:1, antibody after the displacement that obtains in (1) step is mixed with the maytenin alkaloid mother liquor that obtains during (2) goes on foot, carry out linked reaction, 20～30 ℃ of temperature of reaction, 1～4 hour reaction times;
(4) anion-exchange chromatography
Sample Zhiyin ion exchange column on the above-mentioned reaction solution is carried out purifying; At first use level pad balance anion chromatography column, go up sample then, the chromatography column carrying capacity is 30-50mg/ml, and last sample flow velocity is 75-100cm/h, collects stream and wears liquid, after last sample finishes, with level pad flushing anion chromatography post, finishes until collection again;
(5) desalination chromatography
The stream of above-mentioned anion-exchange chromatography collection is worn liquid carry out Sephadex
TMG25 desalination chromatography column purifying; Desalination damping fluid balance desalination chromatography column, the desalination damping fluid contains the tensio-active agent of 0.02～0.1%v/v, with sample on the collection liquid of anion-exchange chromatography to the desalination chromatography column, flow velocity is 2～5cm/h, collect first and go out the peak sample, be prepared antibody-maytenin alkaloids medicament conjugate.
2. preparation method as claimed in claim 1 is characterized in that the described antibody stoste of step (1) is selected from Herceptin, Cetuximab or handkerchief Buddhist nun monoclonal antibody.
3. preparation method as claimed in claim 1, the reaction buffer that it is characterized in that the described displacement antibody of step (1) stoste is selected from sodium phosphate salt damping fluid, potassium phosphate salt damping fluid, Tris-Hcl damping fluid or Sodium phosphate dibasic-citrate buffer solution, pH of buffer 6.0～9.0.
4. preparation method as claimed in claim 1 is characterized in that the mode of the described ultrafiltration and concentration of step (1) or desalination chromatography can adopt prior art, for example can carry out ultrafiltration and concentration by the Pellicon system, or adopts Sephadex
TMThe G25 gel chromatography column carries out the desalination chromatography.
5. preparation method as claimed in claim 1 is characterized in that the described tensio-active agent of step (1) is span 40, sorbester p18, polysorbas20, polysorbate40, polysorbate60 and tween 80.
6. preparation method as claimed in claim 1 is characterized in that the described organic solvent of step (2) is selected from N,N-DIMETHYLACETAMIDE (DMA), dimethyl formamide (DMF), dimethyl sulfoxide (DMSO) (DMSO) or ethanol (EtOH).
7. preparation method as claimed in claim 1 is characterized in that the described bifunctional linking reagent of step (3) is selected from N-succsinic acid imide 4-(maleimide methyl) cyclohexane carboxylate (SMCC), N-succsinic acid imide 4-(maleimide methyl) hexanaphthene-1-carboxyl-(6-amido capronate) is (LC-SMCC).
8. preparation method as claimed in claim 1 is characterized in that the chromatography media of the described anion-exchange chromatography of step (4) is selected from Q Sepharose, DEAE Sepharose, Capto Q, Capto DEAE or Capto Adhere;
Preferably, the used reaction buffer of the used level pad of the described anion-exchange chromatography of step (4) and step (1) is identical.
9. preparation method as claimed in claim 1, it is characterized in that desalination damping fluid selection citrate buffer, acetate buffer, succsinic acid damping fluid or phosphate buffered saline buffer that the described desalination chromatography of step (5) uses, contain the tensio-active agent of 0.02%～0.1%v/v in the damping fluid; Described tensio-active agent is identical with the tensio-active agent of step (1).
10. preparation method as claimed in claim 1 is characterized in that step is as follows:
(1) utilizes Sephadex
TMG25 chromatographic column purifying desalination chromatography mode is replaced Herceptin stoste to reaction buffer, and reaction buffer consists of the 0.1M sodium phosphate salt, and pH7.5 contains the polysorbas20 of 0.05%v/v; Concentrated antibody concentration makes the bent appropriate pearl antibody of displacement to 20mg/ml;
(2) take by weighing SMCC-DM1, fully dissolve the SMCC-DM1 mother liquor of preparation 10mg/ml with N,N-DIMETHYLACETAMIDE (DMA);
(3) mol ratio by SMCC-DM1 and antibody is 5～5.5:1, adds the SMCC-DM1 mother liquor of step (2) preparation in the prepared Herceptin of step (1), the beginning linked reaction, and temperature of reaction is 25 ℃, the reaction times is 1.5h;
(4) with sample Capto Adhere anion-exchange chromatography post on the above-mentioned reaction solution.Chromatography column carries out balance with corresponding reaction buffer in the step (1) earlier, sample on the reaction solution after then step (3) reaction being finished, and flow velocity is 75cm/h, carrying capacity is 35mg/ml, collects stream and wears sample, after last sample finishes, with corresponding reaction buffer flushing, finish to collecting;
(5) above-mentioned collection liquid through anion-exchange chromatography is carried out Sephadex respectively
TMG25 desalination chromatography.With desalination damping fluid balance, flow velocity is 3cm/h to the desalination chromatography column earlier.Used desalination damping fluid is the 20mM succsinic acid, 0.05% polysorbas20, pH5.5; After last sample finishes, with same damping fluid flushing, collect out the peak sample according to the UV280 ultraviolet absorption value, be the bent appropriate pearl-SMCC-DM1 conjugate of preparation.
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