CN103254311B - Method for preparing antibody-maytansine alkaloid medicine conjugate - Google Patents
Method for preparing antibody-maytansine alkaloid medicine conjugate Download PDFInfo
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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, the treatment of cancer achieves significant progress.In recent years, researchist utilized cell receptor and the antigen of cancer cell surfaces selective expression, developed many antibody drugs, and these antibody can carry out specific binding with tumour cell.On this basis, by cytotoxic molecule, such as bacterium carries out chemistry with plant poison, radionuclide and some chemotherapeutic agent and antibody and is connected, obtain antibody-drug conjugates, this kind of antibody-drug conjugates discharges medicine or activates the cytotoxic activity of medicine after being combined with tumour cell, is killed by tumour cell.The specificity of antibody-drug conjugates can reduce Normocellular toxicity, therefore improves the tolerance of medicine in patient body.
Maytenin (Maytansine) is first separated from East Africa shrub tingia Caulis Mayteni (Maytenusserrata) by people such as Kupchan.Maytenin alkaloid is the medicine of a class high cell toxicity, according to the study, its toxicity than traditional cancer chemotherapeutic drug as the cytotoxicity of methotrexate, daunorubicin etc. is eager to excel 100 to 1000 times, see US3896111.Afterwards, find that some microorganisms also can produce maytenin alkaloid, as the C-3 ester of Ansamitocin Po (maytansinol) and Ansamitocin Po, see US4151042.
Maytenin is mitotic inhibitor, reports, with the L1210 cell of maytenin process, cause 67% be accumulated in m period, and not processed compared with control cells indication range is between 3.2% to 5.8%.Show with the research of sea urchin egg and clam ovum, maytenin disturbs the formation of microtubule by suppressing the polymerization of tubulin, thus suppresses mitotic division.See Remillard, 189Science1002-1005(1975).
Due to the alkaloidal high cell toxicity of maytenin, indicate that they have effect in Therapeutic cancer, but, the alkaloidal clinical trial of maytenin is also unsatisfactory, mainly due to their side effect.Cause patient to refuse further treatment to the side effect of central nervous system and gastrointestinal symptoms, and maytenin alkaloid seems relevant with peripheral neuropathy, this peripheral neuropathy may be accumulation (Issel at207).
Directed medicine can be sent to target cell site by Dispersal risk-maytenin alkaloid conjugate, thus reduce the toxicity of normal tissue and cell, reduce the alkaloidal side effect of maytenin.
CN101087611A reports and connects conjugate prepared by antibody and maytenin alkaloid with cutting joint and connect compared with conjugate prepared by antibody and maytenin alkaloid with cutting joint, both have identical in vitro and in vivo anti-tumor activity, but the former shows significant reduction in plasma clearance and toxicity.
US5208020 and US5416064 etc. disclose the method for Dispersal risk-maytenin alkaloid conjugate, first use bifunctional linking reagent and monoclonal antibody coupling, utilize Sephadex
tMg25 chromatography connects the monoclonal antibody of upper linking agent, then by being connected with the monoclonal antibody of linking agent and the excessive chemical toxicant coupling containing sulfydryl, again utilizes Sephadex
tMg25 chromatography.
CN101087611A also discloses the method for the mode Dispersal risk identical with US5208020, US5416064-maytenin alkaloid conjugate, namely first by bifunctional linking reagent and monoclonal antibody coupling, purifying obtains the first mixture, then with chemical toxicant coupling, carry out purifying obtain conjugate.The antibody-drug conjugates purity that profit obtains in this way is low and unstable.Such as, disclosed in CN101087611A embodiment, the purity of bent appropriate pearl-SMCC-DM1 conjugate only has about 95%, and the content of albumen aggressiveness thing has accounted for more than 4.2%, there is the risk causing higher immunogenicity in the existence of this superpolymer over the course for the treatment of.
CN102596922A discloses a kind of new bifunctional linking reagent, on traditional bifunctional linking reagent, is namely incorporated to hydrophilic PEG spacer is modified to hydrophilic linkers.The antibody-drug conjugates utilizing this linking agent coupling to obtain can allow maximum 15 drug molecules of each antibody molecule coupling, thus improves joint efficiency.Meanwhile, also disclose a kind of method of new antibody-drug conjugate, namely first bifunctional linking reagent is connected with chemicals, it is being connected with monoclonal antibody.Utilize this coupling method to decrease purification step in coupling, improve product yield, also greatly reduce the heterogeneity of antibody-drug conjugates simultaneously.But, utilize the bifunctional linking reagent of scheme synthesis hydrophilic disclosed in this patent document to be a more loaded down with trivial details chemical reaction process, the application of this kind of bifunctional linking reagent may be limited.And the bent appropriate pearl-SMCC-DM1 of coupling method synthesis disclosed in CN102596922A, TSK-GEL size exclusion chromatography (moving phase contains the Virahol of 15%, can better be separated with aggressiveness by conjugate monomer) analysis is utilized to find the conjugate aggressiveness still contained up to 4%.Further, in order to obtain suitable Conjugate ratio, i.e. the mol ratio of conjugate Chinese traditional medicine and antibody, general needs add 6-10 times of overdose of medicine thing when reacting, such as, in prepared by the appropriate pearl of song-SMCC-DM1, 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
For the deficiencies in the prior art, the invention provides a kind of coupling method of high coupling efficiency Dispersal risk-maytenin alkaloid conjugate, the conjugate that the method prepares has suitable Conjugate ratio, high purity and low endotoxin content.
Detailed technology scheme of the present invention is as follows:
A preparation method for antibody-CHROMATOGRAPHIC FRACTIONATION AND MASS alkaloidal drug conjugate, comprises the following steps:
(1) antibody displacement damping fluid
Utilize the mode of ultrafiltration and concentration or desalination chromatography to replace antibody stoste in reaction buffer, the tensio-active agent containing 0.02% ~ 0.1%v/v in reaction buffer, is concentrated into antibody concentration at 20 ~ 30mg/ml; Antibody after must replacing;
The stoste of described antibody stoste monoclonal antibody;
(2) maytenin alkaloids medicament mother liquor is prepared
Get the maytenin alkaloids medicament being connected with bifunctional linking reagent, i.e. bifunctional linking reagent-maytenin alkaloid, with organic solvent dissolution, the mother liquor of preparation containing 10mg/ml maytenin alkaloidal drug.
(3) linked reaction
Be the ratio of 5 ~ 6:1 in the mol ratio of difunctional link agent-maytenin alkaloid and antibody, antibody after the displacement obtained in (1) step is mixed with the maytenin alkaloid mother liquor obtained in (2) step, carry out linked reaction, temperature of reaction 20 ~ 30 DEG C, 1 ~ 4 hour reaction times;
(4) anion-exchange chromatography
Above-mentioned reaction solution loading Zhiyin ion exchange column is carried out purifying; First use equilibration buffer anion chromatography post, then loading, chromatography column carrying capacity is 30-50mg/ml, and loading flow velocity is 75-100cm/h, collects stream and wears liquid, after loading, then use Equilibration buffer wash anion chromatography post, until collect complete;
(5) desalination chromatography
The stream that above-mentioned anion-exchange chromatography is collected is worn liquid and carries out Sephadex
tMg25 desalination column chromatography; Desalination damping fluid balance desalination chromatography column, desalination damping fluid contains 0.02 ~ 0.1%(v/v) tensio-active agent, by the collection liquid loading of anion-exchange chromatography to desalination chromatography column, flow velocity is 2 ~ 5cm/h, collect first peak sample, be prepared antibody-maytenin alkaloids medicament conjugate.
Preferred according to the present invention, the antibody stoste described in step (1) is selected from Herceptin, Cetuximab or Victibix.
Preferred according to the present invention, the reaction buffer of the displacement antibody stoste described in step (1) is selected from sodium phosphate buffer, 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 ultrafiltration and concentration described in step (1) or the mode of desalination chromatography can adopt prior art, such as, can carry out ultrafiltration and concentration by Pellicon system, or adopt Sephadex
tMg25 gel chromatography column carries out desalination chromatography.
Preferred according to the present invention, the tensio-active agent described in step (1) mainly refers to nonionic surface active agent, the smooth class of preferred fatty acid sorb and poly yamanashi esters, wherein the 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 is 0.02% ~ 0.1%(v/v).
Preferred according to the present invention, the organic solvent described in 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, bifunctional linking reagent described in step (3) is selected from N-bromosuccinimide base 4-(maleimidomehyl) cyclohexane carboxylate (SMCC), N-bromosuccinimide base 4-(maleimidomehyl) hexanaphthene-1-carboxyl-(6-amidocaproate) (LC-SMCC), all can purchased from Suzhou Hao Fan biotech company.
Preferred according to the present invention, the maytenin alkaloids medicament described in step (3) is synthesized by ansamitocin P-3.The method reference literature J Med.Chem.2006 of synthesis maytenin alkaloidal drug, the method for 49,4392-4408 is carried out.Ansamitocin P-3 is commercially available.Such as purchased from Yan Yu biological medium company.
Preferred according to the present invention, the method synthesis that the maytenin alkaloids medicament being connected with bifunctional linking reagent described in step (3) can provide with reference to [0438] section of CN102596922A.
Preferred according to the present invention, the chromatography media of the anion-exchange chromatography described in 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 reaction buffer that the level pad used of the anion-exchange chromatography described in step (4) can be used with step (1) is identical, also can select different damping fluids; Anion-exchange chromatography level pad used can be selected from sodium phosphate buffer, potassium phosphate salt damping fluid, Tris-Hcl damping fluid or Sodium phosphate dibasic-citrate buffer solution, still needs interpolation 0.02% ~ 0.1%(v/v in damping fluid) tensio-active agent.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 desalination chromatography described in step (5) uses selects citrate buffer, acetate buffer, succinate buffer or phosphate buffered saline buffer, containing 0.02% ~ 0.1%(v/v in damping fluid) tensio-active agent.Described tensio-active agent is identical with the tensio-active agent of step (1).
In the present invention, the anion-exchange chromatography described in step (4) adopts and passes pattern, and the antibody-drug conjugates monomer flow obtained is namely worn, and antibody-drug conjugates aggressiveness is attracted on anion chromatography post; In order to improve the effect of anion-exchange chromatography purifying, the present invention have selected lower linear rate of flow, is generally 75 ~ 100cm/h, chromatography carrying capacity 30 ~ 50mg/ml.
In the present invention, the desalination chromatography described in step (5) is Sephadex
tMg25 chromatography, for effectively unreacted maytenin alkaloids medicament, organic solvent etc. and antibody-maytenin alkaloidal drug conjugate being separated, have selected lower linear rate of flow, being generally 2 ~ 5cm/h.
" antibody " described in the inventive method can be any immunoglobulin (Ig), and any immunoglobulin fragment is as Fab, F(ab ')
2, dsFv, sFv, double antibody and three chain antibodies, or immunoglobulin chimera, 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.Generally produced by bone-marrow-derived lymphocyte mono-clonal.In present method, preferred antibody is Humanized monoclonal antibodies, such as comprises huN901, huMy9-6, huB4, huC242, Herceptin, Victibix and Rituximab etc.
The cytotoxic agent used in the inventive method is maytenin alkaloid, and being selected from DM1(chemical name is N
2 '-Tuo acetyl-N
2 '-3-sulfydryl-1 oxopropyl)-maytenin), DM4(chemical name is N
2 '-Tuo acetyl-N
2 '-4-methyl-4 sulfydryl-1 oxopentyl)-maytenin).Cytotoxic agent is other suitable chemotoxic drug optional also, can cause necrocytosis, lure any compound of necrocytosis or minimizing 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, preferred technical scheme one, the preparation method of bent appropriate pearl-SMCC-DM1 conjugate, step is as follows:
(1) Sephadex is utilized
tMg25 chromatography desalination chromatography scheme displacement Herceptin stoste is to reaction buffer, and reaction buffer consists of 0.1M sodium phosphate salt, pH7.5, containing the polysorbas20 of 0.05%v/v; Concentrated antibody concentration, to 20mg/ml, obtains the appropriate pearl antibody of song of displacement;
(2) take SMCC-DM1, fully dissolve the SMCC-DM1 mother liquor of preparation 10mg/ml with N,N-DIMETHYLACETAMIDE (DMA);
(3) be 5 ~ 5.5:1 by the mol ratio of SMCC-DM1 and antibody, add the SMCC-DM1 mother liquor that step (2) is prepared in the Herceptin prepared by step (1), start linked reaction, temperature of reaction is 25 DEG C, and the reaction times is 1.5h;
(4) by above-mentioned reaction solution loading Capto Adhere anion-exchange chromatography post.Chromatography column first uses corresponding reaction buffer in step (1) to balance, and then by step (3) reaction solution loading after completion of the reaction, flow velocity is 75cm/h, carrying capacity is 35mg/ml, collects stream and wears sample, after loading, rinse with corresponding reaction buffer, complete to collecting;
(5) the above-mentioned collection liquid through anion-exchange chromatography is carried out Sephadex respectively
tMg25 desalination chromatography.Desalination chromatography column is first by desalination damping fluid balance, and flow velocity is 3cm/h.Desalination damping fluid used is 20mM succsinic acid, 0.05% polysorbas20, pH5.5.After loading, with same wash buffer, collect peak sample according to UV280 ultraviolet absorption value, be the appropriate pearl of the song-SMCC-DM1 conjugate of preparation.
The preparation method of optimal technical scheme two, Pa Ni-SMCC-DM1 conjugate of the present invention, as described in Example 3.
Optimal technical scheme three of the present invention, the preparation method of western appropriate former times-SMCC-DM1 conjugate, as described in Example 4.
In monoclonal antibody-purified process, the general stream of the anion-exchange chromatography pattern of wearing that adopts removes monoclonal antibody aggressiveness, is attracted on anionic exchange medium because monoclonal antibody aggressiveness is generally easy with more multi-charge than monomer.The conjugate aggressiveness produced in linked reaction is also removed by anion-exchange chromatography.In addition, anion-exchange chromatography also has removes endotoxic effect, reduces hidden danger during clinical application.
The feature of technical solution of the present invention: namely adopt the damping fluid adding tensio-active agent in antibody displacement damping fluid step, like this in follow-up linked reaction system, the wetting ability of medicine can be increased, improve the efficiency of linked reaction, thus reduce the feed ratio of linked reaction.Meanwhile, the damping fluid adding tensio-active agent in following purification steps also can effectively reduce antibody-drug conjugates produces conjugate aggressiveness probability because of drugs hydrophobility.In antibody purification procedures, add a step anion-exchange chromatography step, compare with the coupling process not increasing anion-exchange chromatography, the purity of finished product can be improved, reduce endotoxin content in product, and on Conjugate ratio and total recovery almost without impact.
Excellent results of the present invention is:
1, in the reaction buffer and purification buffer afterwards of Dispersal risk-maytenin alkaloid conjugate, with the addition of tensio-active agent, thus make Dispersal risk-maytenin alkaloid conjugate have higher efficiency, for the appropriate pearl of song-SMCC-DM1, linked reaction is carried out under equal proportion, the feed ratio adding the linked reaction of tensio-active agent with do not add compared with surface-active feed ratio, feed ratio can be reduced to 1:6 by 1:7.5, substantially increase the efficiency of linked reaction, reduce cost.
2, the antibody of preparation-maytenin alkaloid conjugate is made to have higher purity and lower endotoxin content by increase by step anion-exchange chromatography.For the appropriate pearl of song-SMCC-DM1, SEC-HPLC purity reaches more than 98%, and the antibody-drug conjugates aggressiveness of formation is less than 2%; Product endotoxin content is reduced to 0.2EU/mg by the 0.8EU/mg of prior art, and in antibody-drug conjugates, the level of free drug is less than 2%(relative to total medicine).
Embodiment
The measuring method of Conjugate ratio: measure Conjugate ratio and refer to the quantity measuring and each monoclonal antibody molecule is connected with maytenin alkaloids medicament, i.e. the ratio of maytenin alkaloids medicament mole number and monoclonal antibody mole number.Ultraviolet spectrophotometry and mass spectrometry two kinds of methods can be adopted to measure.Ultraviolet spectrophotometry: by measuring the antibody-ultraviolet absorption value of maytenin alkaloids conjugate at 252nM and 280nM place, and undertaken calculating Conjugate ratio by langbobier law; Mass spectrometry: antagonist-maytenin alkaloid conjugate carries out ESI-MS analysis, can obtain the peak type figure connected without number medicine, carry out calculating Conjugate ratio according to the area at each peak.The Conjugate ratio obtained by two kinds of methods has good consistence.
Conjugate concentration determination: adopt ultraviolet spectrophotometry, determine the ultraviolet absorption value at 252nM and 280nM place, the concentration of antibody and medicine can be calculated by langbobier law; Conjugate concentration described in the present invention is monoclonal antibody concentration and the summation being connected to the maytenin alkaloidal drug on monoclonal antibody.
SEC-HPLC analyzes: utilize GLK3000 efficient molecular sieve post antagonist-maytenin alkaloidal drug to analyze, moving phase selects 20mM PB, 15% Virahol, pH7.0.The ratio shared by conjugate aggressiveness and conjugate monomer is calculated according to area normalization method.
Endotoxin content detects: carry out with reference to " Chinese Pharmacopoeia 2010 editions three " annex XII E bacterial endotoxins test.
Further illustrate the present invention by the following examples, but, should be appreciated that these embodiments are only used for the use specifically described more in detail, and should not be understood as limiting the present invention in any form.In this article, unless otherwise indicated, agents useful for same is commercially available prod.
The maytenin alkaloids medicament (i.e. bifunctional linking reagent-maytenin alkaloid) being connected with bifunctional linking reagent in embodiment is SMCC-DM1.SMCC is purchased from Suzhou Hao Fan biotech company, and DM1 is by ansamitocin P-3 reference literature JMed.Chem.2006, and the method for 49,4392-4408 carries out synthesizing, and wherein ansamitocin P-3 is purchased from Yan Yu biological medium company.The method that [0438] section of SMCC-DM1 reference CN102596922A provides is synthesized.
Product in embodiment adopts the phraseology of " antibody-bifunctional linking reagent-maytenin alkaloid ".
Embodiment 1, prepare bent appropriate pearl-SMCC-DM1 conjugate, checking adds the impact on coupling efficiency after tensio-active agent.
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 replaces Herceptin stoste to reaction buffer A1(0.1M sodium phosphate salt, pH7.5) in, and concentrated antibody concentration is to 20mg/ml, prepares bent appropriate pearl antibody A 1;
Utilize desalination chromatography (Sephadex
tMg25) mode replaces Herceptin stoste to reaction buffer A2(0.1M sodium phosphate salt, pH7.5,0.05%v/v polysorbas20) in, and concentrated antibody concentration is to 20mg/ml, prepares bent appropriate pearl antibody A 2;
(2) SMCC-DM1 mother liquor is prepared
Take SMCC-DM1, fully dissolve the SMCC-DM1 mother liquor of preparation 10mg/ml with N,N-DIMETHYLACETAMIDE (DMA);
(3) linked reaction
In the Herceptin prepared by step (1), add the SMCC-DM1 mother liquor that step (2) is prepared, start linked reaction, temperature of reaction is 25 DEG C, and the reaction times is 1.5h; Reaction is divided into three groups to carry out:
A: use reaction buffer A1 and the appropriate pearl antibody A 1 of song, add the SMCC-DM1 mother liquor of 7.5 times of excess molar ratio in reaction;
B: use reaction buffer A2 and the appropriate pearl antibody A 2 of song, add the SMCC-DM1 mother liquor of 7.5 times of excess molar ratio in reaction;
C: use reaction buffer A2 and the appropriate pearl antibody A 2 of song, add the SMCC-DM1 mother liquor of 5.5 times of excess molar ratio in reaction;
(4) anion-exchange chromatography
Respectively by above-mentioned a, b, c tri-group reaction liquid loading Capto Adhere anion-exchange chromatography post.Chromatography column first uses corresponding reaction buffer in step (1) to balance, and then by step (3) reaction solution loading after completion of the reaction, flow velocity is 75cm/h, carrying capacity is 35mg/ml, collects stream and wears sample, after loading, rinse with corresponding reaction buffer, complete to collecting;
(5) desalination chromatography
Above-mentioned three groups of collection liquid through anion-exchange chromatography are carried out Sephadex respectively
tMg25 desalination chromatography.Desalination chromatography column is first by desalination damping fluid balance, and flow velocity is 3cm/h.A group sample desalination damping fluid used is 20mM succsinic acid, and pH5.5, b group and c group sample damping fluid used are 20mM succsinic acid, 0.05%v/v polysorbas20, pH5.5.After loading, use corresponding wash buffer respectively for each group, collect peak sample according to UV280 ultraviolet absorption value, be the appropriate pearl of the song-SMCC-DM1 conjugate of preparation.
The appropriate pearl of the song-SMCC-DM1 conjugate of acquisition is utilized concentration and the Conjugate ratio of ultraviolet spectrophotometry (measuring the absorbancy of conjugate at 252nm and 280nm respectively) counting yield, also use the Conjugate ratio of mass spectrometry counting yield simultaneously, and contrast, data are in table 1.
Table 1 adds the impact of tensio-active agent on coupling efficiency
| Sample | Product concentration (mg/ml) | Yield | Conjugate ratio (ultraviolet method) | Conjugate ratio (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, effectively coupling efficiency can be improved when adding a certain amount of tensio-active agent (polysorbas20 as 0.05%) in buffer solution system.Identical molar feed ratio can obtain higher Conjugate ratio, can obtain the antibody-drug conjugates of about 3.5 Conjugate ratio when reduction molar feed ratio is 5.5 simultaneously.
Embodiment 2, in coupling process, add content and endotoxin content that anion-exchange chromatography can reduce conjugate aggressiveness.
The preparation of bent appropriate pearl-SMCC-DM1 conjugate, step is as follows:
(1) antibody displacement damping fluid: first utilize desalination chromatography (Sephadex
tMg25) mode replaces bent appropriate pearl antibody stoste in reaction buffer (0.1M sodium phosphate salt, pH7.5,0.05%v/v polysorbas20), and concentrated antibody concentration is to 20mg/ml;
(2) SMCC-DM1 mother liquor is prepared: take the SMCC-DM1 with Herceptin mol ratio 5.5 times in step (1), fully dissolve the SMCC-DM1 mother liquor of preparation 10mg/ml with N,N-DIMETHYLACETAMIDE (DMA);
(3) linked reaction:
In the Herceptin prepared by step (1), add the SMCC-DM1 mother liquor that step (2) is prepared, start linked reaction, temperature of reaction is 25 DEG C, and the reaction times is 1.5h;
Reaction solution is divided into d, e two groups, d group only carries out desalination chromatography, and e group first carries out anion-exchange chromatography, then carries out desalination chromatography;
(4) anion-exchange chromatography
By above-mentioned e group reaction liquid loading Capto Adhere anion-exchange chromatography post.Chromatography column first uses the reaction buffer in step (1) to balance, then the corresponding reaction solution of loading, and flow velocity is 75cm/h, and carrying capacity is 35mg/ml, collects stream and wears sample, after loading, rinses with reaction buffer, complete to collecting;
(5) desalination chromatography
The above-mentioned collection liquid of e group anion-exchange chromatography and the reaction solution of d group are carried out Sephadex respectively
tMg25 desalination chromatography.Desalination chromatography column is first by desalination damping fluid balance, and flow velocity is 3cm/h, and damping fluid used is 20mM succsinic acid, 0.05%v/v polysorbas20, pH5.5.Loading after balance, continues to rinse with damping fluid, collects peak sample, be the appropriate pearl of the song-SMCC-DM1 conjugate of preparation according to UV280 absorption value.
The appropriate pearl of the song-SMCC-DM1 conjugate of acquisition is utilized concentration and the Conjugate ratio of ultraviolet spectrophotometry (measuring the absorbancy of conjugate at 252nm and 280nm respectively) counting yield, utilize the purity of SEC-HPLC testing product simultaneously, utilize endotoxin content in limulus reagent test testing product.
Table 2 anion-exchange chromatography is on the impact 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, coupling reaction solution is being carried out in the process of purifying, increasing by a step anion-exchange chromatography, the content of conjugate aggressiveness can be effectively reduced, improving the purity of conjugate, also can reduce endotoxic content in product further simultaneously.Relative to the coupling process not increasing anion-exchange chromatography, total recovery value reduces and about 5%.
Embodiment 3, preparation Pa Ni-SMCC-DM1 conjugate, step is as follows:
(1) antibody displacement damping fluid
First utilize ultrafiltration and concentration (pellicon system) mode to replace Victibix stoste in (20mM potassium phosphate salt damping fluid, 0.02%v/v polysorbas20, pH6.0) in reaction buffer, and concentrated antibody concentration is to 30mg/ml.
(2) SMCC-DM1 mother liquor is prepared
Take the SMCC-DM1 with Victibix mol ratio 5.0 times in step (1), fully dissolve the SMCC-DM1 mother liquor of preparation 10mg/ml with N,N-DIMETHYLACETAMIDE (DMA).
(3) linked reaction
In the Victibix prepared by step (1), add the SMCC-DM1 mother liquor that step (2) is prepared, start linked reaction, temperature of reaction is 25 DEG C, and the reaction times is 2.0h;
(4) anion-exchange chromatography
By above-mentioned reaction solution loading Capto Adhere anion-exchange chromatography post.Chromatography column first uses the reaction buffer in step (1) to balance, then loading reaction solution, and flow velocity is 75cm/h, and carrying capacity is 45mg/ml, collects stream and wears sample, after loading, rinses with reaction buffer, complete to collecting;
(5) desalination chromatography
The collection liquid of above-mentioned anion-exchange chromatography is carried out Sephadex
tMg25 desalination chromatography.Desalination chromatography column is first by desalination damping fluid balance, and flow velocity is 3.5cm/h.Desalination damping fluid used is 20mM sodium phosphate salt, 0.02%v/v polysorbas20, pH7.2.After loading, use desalination wash buffer, collect peak sample according to UV280 ultraviolet absorption value, be the Pa Ni-SMCC-DM1 conjugate of preparation.
Pa Ni-SMCC-DM1 the conjugate of acquisition is utilized concentration and the Conjugate ratio of ultraviolet spectrophotometry (measuring the absorbancy of conjugate at 252nm and 280nm respectively) counting yield, utilize the purity of SEC-HPLC testing product simultaneously, utilize endotoxin content in limulus reagent test testing product.
The character of table 3 Pa Ni-SMCC-DM1 conjugate product
Embodiment 4, prepare western appropriate former times-SMCC-DM1 conjugate
(1) antibody displacement damping fluid
First ultrafiltration and concentration (pellicon system) or desalination chromatography (Sephadex is utilized
tMg25) mode replaces Cetuximab stoste in (20mM Sodium phosphate dibasic-10mM citric acid, 0.1%v/v tween 80, pH8.0) in reaction buffer, and concentrated antibody concentration is to 20mg/ml;
(2) SMCC-DM1 mother liquor is prepared
Take the SMCC-DM1 with Cetuximab mol ratio 6.0 times in step (1), fully dissolve the SMCC-DM1 mother liquor of preparation 10mg/ml with N,N-DIMETHYLACETAMIDE (DMA);
(3) linked reaction
In the Cetuximab prepared by step (1), add the SMCC-DM1 mother liquor that step (2) is prepared, start linked reaction, temperature of reaction is 22.5 DEG C, and the reaction times is 4.0h;
(4) anion-exchange chromatography
By above-mentioned reaction solution loading Capto Adhere anion-exchange chromatography post.Chromatography column first balances with reaction buffer, then loading reaction solution, and flow velocity is 75cm/h, and carrying capacity is 40mg/ml, collects stream and wears sample, after loading, rinses with reaction buffer, complete to collecting;
(5) desalination chromatography
The collection liquid of above-mentioned anion-exchange chromatography is carried out Sephadex
tMg25 desalination chromatography.Desalination chromatography column is first by desalination damping fluid balance, and flow velocity is 3cm/h.Desalination damping fluid used is 20mM citric acid-sodium citrate, containing 0.1%v/v tween 80, and pH5.0.After loading, use desalination wash buffer, collect peak sample according to UV280 ultraviolet absorption value, be the western appropriate former times-SMCC-DM1 conjugate of preparation.
The western appropriate former times-SMCC-DM1 conjugate obtained is utilized concentration and the Conjugate ratio of ultraviolet spectrophotometry (measuring the absorbancy of conjugate at 252nm and 280nm respectively) counting yield, utilize the purity of SEC-HPLC testing product simultaneously, utilize endotoxin content in limulus reagent test testing product.
The character of the western appropriate former times-SMCC-DM1 conjugate product of table 4
Claims (8)
1. a preparation method for antibody-CHROMATOGRAPHIC FRACTIONATION AND MASS alkaloidal drug conjugate, comprises the following steps:
(1) antibody displacement damping fluid
Utilize the mode of ultrafiltration and concentration or desalination chromatography to replace antibody stoste in reaction buffer, the tensio-active agent containing 0.02% ~ 0.1% v/v in reaction buffer, is concentrated into antibody concentration at 20 ~ 30mg/mL; Antibody after must replacing;
Described antibody stoste is the stoste of monoclonal antibody; Described tensio-active agent is span 40, sorbester p18, polysorbas20, polysorbate40, polysorbate60 or tween 80;
(2) maytenin alkaloids medicament mother liquor is prepared
Get the maytenin alkaloids medicament being connected with bifunctional linking reagent, i.e. bifunctional linking reagent-maytenin alkaloid, with organic solvent dissolution, the mother liquor of preparation containing 10mg/mL maytenin alkaloidal drug;
Described maytenin alkaloid is DM1 or DM4;
(3) linked reaction
Be the ratio of 5 ~ 6:1 in the mol ratio of bifunctional linking reagent-maytenin alkaloid and antibody, antibody after the displacement obtained in (1) step is mixed with the maytenin alkaloid mother liquor obtained in (2) step, carry out linked reaction, temperature of reaction 20 ~ 30 DEG C, 1 ~ 4 hour reaction times;
(4) anion-exchange chromatography
Above-mentioned reaction solution loading Zhiyin ion exchange column is carried out purifying; First use equilibration buffer anion chromatography post, then loading, chromatography column carrying capacity is 30-50mg/mL, and loading flow velocity is 75-100cm/h, collects stream and wears liquid, after loading, then use Equilibration buffer wash anion chromatography post, until collect complete;
The chromatography media of described anion-exchange chromatography is selected from Q Sepharose, DEAE Sepharose, Capto Q, Capto DEAE or Capto Adhere; The described anion-exchange chromatography level pad used reaction buffer used with step (1) is identical;
(5) desalination chromatography
The stream that above-mentioned anion-exchange chromatography is collected is worn liquid and carries out Sephadex
tMg25 desalination column chromatography; Desalination damping fluid balance desalination chromatography column, desalination damping fluid contains the tensio-active agent of 0.02 ~ 0.1% v/v, and the described tensio-active agent tensio-active agent used with step (1) is identical; By the collection liquid loading of anion-exchange chromatography to desalination chromatography column, flow velocity is 2 ~ 5cm/h, collects first peak sample, is prepared antibody-maytenin alkaloids medicament conjugate.
2. preparation method as claimed in claim 1, is characterized in that the antibody stoste described in step (1) is selected from Herceptin, Cetuximab or Victibix.
3. preparation method as claimed in claim 1, it is characterized in that the reaction buffer of the displacement antibody stoste described in step (1) is selected from sodium phosphate buffer, 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 ultrafiltration and concentration described in step (1) carries out ultrafiltration and concentration by Pellicon system, and described desalination chromatography adopts Sephadex
tMg25 gel chromatography column carries out desalination chromatography.
5. preparation method as claimed in claim 1, is characterized in that the organic solvent described in step (2) is selected from N,N-DIMETHYLACETAMIDE, dimethyl formamide, dimethyl sulfoxide (DMSO) or ethanol.
6. preparation method as claimed in claim 1, is characterized in that the bifunctional linking reagent described in step (3) is selected from N-bromosuccinimide base 4-(maleimidomehyl) cyclohexane carboxylate (SMCC), N-bromosuccinimide base 4-(maleimidomehyl) hexanaphthene-1-carboxyl-(6-amidocaproate) (LC-SMCC).
7. preparation method as claimed in claim 1, it is characterized in that the desalination damping fluid that the desalination chromatography described in step (5) uses selects citrate buffer, acetate buffer, succinate buffer or phosphate buffered saline buffer, the tensio-active agent containing 0.02% ~ 0.1%v/v in damping fluid.
8. preparation method as claimed in claim 1, is characterized in that step is as follows:
(1) Sephadex is utilized
tMg25 chromatography desalination chromatography scheme displacement Herceptin stoste is to reaction buffer, and reaction buffer consists of 0.1 M sodium phosphate salt, pH7.5, containing the polysorbas20 of 0.05% v/v; Concentrated antibody concentration, to 20mg/mL, obtains the appropriate pearl antibody of song of displacement;
(2) take SMCC-DM1, fully dissolve the SMCC-DM1 mother liquor of preparation 10mg/mL with N,N-DIMETHYLACETAMIDE;
(3) be 5 ~ 5.5:1 by the mol ratio of SMCC-DM1 and antibody, add the SMCC-DM1 mother liquor that step (2) is prepared in the Herceptin prepared by step (1), start linked reaction, temperature of reaction is 25 DEG C, and the reaction times is 1.5h;
(4) by above-mentioned reaction solution loading Capto Adhere anion-exchange chromatography post; Chromatography column first uses corresponding reaction buffer in step (1) to balance, and then by step (3) reaction solution loading after completion of the reaction, flow velocity is 75cm/h, carrying capacity is 35mg/mL, collects stream and wears sample, after loading, rinse with corresponding reaction buffer, complete to collecting;
(5) the above-mentioned collection liquid through anion-exchange chromatography is carried out Sephadex
tMg25 desalination chromatography; Desalination chromatography column is first by desalination damping fluid balance, and flow velocity is 3cm/h; Desalination damping fluid used is 20 mM succsinic acids, the polysorbas20 of 0.05% v/v, pH5.5; After loading, with same wash buffer, collect peak sample according to UV280 ultraviolet absorption value, be the appropriate pearl of the song-SMCC-DM1 conjugate of preparation.
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| MY183572A (en) | 2013-03-15 | 2021-02-26 | Regeneron Pharma | Biologically active molecules, conjugates thereof, and therapeutic uses |
| US20170319709A1 (en) * | 2013-07-05 | 2017-11-09 | Formation Biologics Inc. | Egfr antibody conjugates |
| CA2921412A1 (en) * | 2013-08-26 | 2015-03-05 | Regeneron Pharmaceuticals, Inc. | Pharmaceutical compositions comprising macrolide diastereomers, methods of their synthesis and therapeutic uses |
| CN103483357B (en) * | 2013-10-12 | 2015-11-18 | 齐鲁制药有限公司 | Intermediate new crystal of a kind of antibody-maytenin conjugate and preparation method thereof |
| CN106606784B (en) * | 2015-10-19 | 2020-01-21 | 泰州迈博太科药业有限公司 | Pre-antibody conjugate drug for target expression of EGFR (epidermal growth factor receptor) tumor cells and application thereof |
| CN114478801A (en) | 2016-01-25 | 2022-05-13 | 里珍纳龙药品有限公司 | Maytansinoid derivatives, conjugates thereof, and methods of use |
| CN105963711A (en) * | 2016-06-20 | 2016-09-28 | 杭州皓阳生物技术有限公司 | Anti-human CD20 monoclonal antibody-DM1 conjugate and preparation method thereof |
| CN111317754B (en) * | 2018-12-13 | 2022-02-18 | 泰州医药城国科化物生物医药科技有限公司 | Preparation method and application of asiatic moonseed total alkali |
| CN112691190A (en) * | 2019-10-23 | 2021-04-23 | 东曜药业有限公司 | Antibody-high-activity cytotoxic small molecule drug conjugate drug, preparation and application thereof |
| JP2024509700A (en) * | 2021-02-09 | 2024-03-05 | ジーンクアンタム ヘルスケア （スーチョウ） シーオー．， エルティーディー． | Conjugation device and conjugate production method |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5208020A (en) * | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
| CN101087611A (en) * | 2003-10-10 | 2007-12-12 | 伊缪诺金公司 | Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates, and methods of making said conjugates |
| CN102596922A (en) * | 2009-10-06 | 2012-07-18 | 免疫基因公司 | Potent conjugates and hydrophilic linkers |
-
2013
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5208020A (en) * | 1989-10-25 | 1993-05-04 | Immunogen Inc. | Cytotoxic agents comprising maytansinoids and their therapeutic use |
| CN101087611A (en) * | 2003-10-10 | 2007-12-12 | 伊缪诺金公司 | Method of targeting specific cell populations using cell-binding agent maytansinoid conjugates linked via a non-cleavable linker, said conjugates, and methods of making said conjugates |
| CN102596922A (en) * | 2009-10-06 | 2012-07-18 | 免疫基因公司 | Potent conjugates and hydrophilic linkers |
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