CN107405408B - Preparation method of antibody drug conjugate - Google Patents
Preparation method of antibody drug conjugate Download PDFInfo
- Publication number
- CN107405408B CN107405408B CN201680011806.8A CN201680011806A CN107405408B CN 107405408 B CN107405408 B CN 107405408B CN 201680011806 A CN201680011806 A CN 201680011806A CN 107405408 B CN107405408 B CN 107405408B
- Authority
- CN
- China
- Prior art keywords
- antibody
- reaction
- drug conjugate
- purification
- trastuzumab
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Peptides Or Proteins (AREA)
- Cell Biology (AREA)
Abstract
A method for preparing an antibody drug conjugate. In particular, it relates to the synthesis and purification steps of antibody drug conjugates, said purification steps comprising a size exclusion step.
Description
Technical Field
The invention relates to a preparation method of an antibody drug conjugate, in particular to a purification step of the preparation method of the antibody drug conjugate. More particularly, the invention relates to a preparation method of an antibody drug conjugate T-DM1 and P-mab-linker-MC-MMAF.
Background
Chemotherapy remains one of the most important anticancer modalities, including surgery, radiation therapy, and targeted therapies. Although the variety of highly effective cytotoxic drugs is large, the difference between tumor cells and normal cells is small, which limits the wide clinical application of these antitumor compounds due to toxic side effects. The specificity of the anti-tumor monoclonal antibody to the surface antigen of tumor cells, the antibody drug has become the front-line drug of anti-tumor therapy, but the curative effect is often not satisfactory when the antibody is used alone as the anti-tumor drug.
The Antibody Drug Conjugate (ADC) connects the monoclonal antibody or antibody fragment with the cytotoxic drug with biological activity through a stable chemical linker compound, fully utilizes the specificity of the antibody for combining the surface antigens of normal cells and tumor cells and the high efficiency of the cytotoxic drug, and simultaneously avoids the defects of low curative effect of the antibody and overlarge toxic and side effect of the antibody. This means that the antibody Drug conjugate binds tumor cells precisely and has a reduced effect on normal cells compared to conventional chemotherapeutic drugs (Mullard A, (2013) Nature Reviews Drug Discovery, 12: 329-332; DiJoseph JF, Armellono DC, (2004) Blood, 103: 1807-1814).
The preparation method of the antibody drug conjugate mainly comprises two steps of preparation and purification of the conjugate. The purification step can remove impurities such as small molecules, polymers and the like in the initial preparation liquid of the antibody drug conjugate, and the purity is improved.
Typically, the purification step will be performed by Ion Exchange Chromatography (Ion Exchange Chromatography abbreviated as IEC) or tangential flow ultrafiltration (TFF, ultrafiltration). Ion exchange chromatography is a chromatographic method in which an ion exchanger is used as a stationary phase and separation is performed according to the difference in the binding force between a component ion in a mobile phase and a counter ion on the exchanger during reversible exchange. When the ion exchange chromatography is adopted, the charge distribution of the antibody drug conjugate with different drug-loading rates is influenced due to different amounts of antibody molecule conjugate drugs in the antibody drug conjugate sample. Therefore, when an ion column is used for chromatographic separation, the charge distribution caused by drug loading can greatly influence the chromatographic result, such as the purity and the average drug loading of the final chromatographic product.
Tangential flow ultrafiltration is a form of filtration in which the flow direction of liquid is perpendicular to the filtration direction, and when tangential flow filtration is performed, the flow direction of the liquid to be filtered is parallel to the direction of the plane of the filtration membrane, and the liquid can pass through the membrane pores perpendicular to the surface of the membrane. The tangential flow ultrafiltration method can conveniently remove small molecular impurities in the system, but has the disadvantage that some impurities with relatively large molecular weight cannot be removed.
Size exclusion chromatography, also known as Size Exclusion Chromatography (SEC) or gel chromatography, is a method of separating solutes using the relative relationship between the uniqueness of a porous gel stationary phase, the pore size of the gel pores, and the size of the coils of the macromolecule sample molecules. The separation mechanism mainly depends on the relationship between the pore size of the gel and the molecular size of the separated components, and has no direct relationship with the properties of the mobile phase; there is no interaction between the sample molecules and the stationary phase. The chromatographic stationary phase is a porous gel that allows only components with a diameter smaller than the pore size to enter, which pores are so large for solvent molecules that they can freely diffuse in and out. Macromolecules in the sample can not enter the gel pores and are completely excluded, and only can pass through the chromatographic column along the gaps among the porous gel particles, and are firstly eluted from the chromatographic column by the mobile phase; the medium-sized molecules can enter some appropriate pores in the gel, but cannot enter smaller micropores, are retained in the column, and elute from the column more slowly; the small molecules can enter most of holes in the gel, are more strongly retained in the column and are more slowly eluted; the molecules of the solvent that dissolve the sample, which have the smallest molecular weight, can enter all the pores of the gel and finally flow out of the column, thus achieving complete separation of samples with different molecular sizes. Size exclusion can distinguish between components of different molecular weights by their different retention times in the gel. Furthermore, the method does not require different charge distributions of the components. Therefore, size exclusion chromatography is not affected by the charge distribution of the molecule during purification.
T-DM1 is an antibody-drug conjugate (ADC) which is formed by coupling herceptin (trastuzumab) and a micromolecular microtubular drug DM 1. We have found that when T-DM1 is prepared by conventional methods, no product is obtained that meets both drug loading and SEC purity requirements and recovery.
CN101267841B discloses a process for the preparation of purified T-DM1, wherein both purification steps use tangential flow ultrafiltration. However, since tangential flow ultrafiltration does not remove some of the higher molecular weight impurities, the final product cannot be obtained stably with high purity using this method, and sometimes additional purification steps are required to obtain a product with acceptable purity, prolonging the work-up process.
WO2015113476 discloses a novel antibody-drug conjugate pertuzumab-propyl-1-thio-MC-MMAF (P-mab-linker-MC-MMAF) which is effective in inhibiting the growth of tumor cells. In the preparation of P-mab-linker-MC-MMAF, we have also found that purification by conventional methods does not yield a product that meets both drug loading and SEC purity and recovery.
Through research, the method can maintain the activity and improve the recovery rate while ensuring the drug-loading rate, so that the preparation efficiency of the antibody conjugate is effectively improved. The method is also suitable for preparing other antibody conjugates and achieves excellent effects.
Disclosure of Invention
In order to improve the preparation effect of ADC drugs, the invention provides an improved preparation method of an antibody drug conjugate, which comprises the steps of carrying out a coupling reaction of an antibody-linking agent and a cytotoxic drug to prepare the antibody drug conjugate and purifying the antibody drug conjugate, wherein the purification step comprises a molecular exclusion step.
Wherein the antibody linking agent is prepared by reacting a bifunctional crosslinking reagent with the antibody, thereby resulting in covalent linking of the linker molecule to the antibody, resulting in a modified antibody. The 'bifunctional crosslinking reagent' is preferably 4- (N-maleimidomethyl) cyclohexane-1-carboxylic acid succinimide ester (SMCC) and 3-acetylmercaptopropionaldehyde.
In a preferred embodiment of the invention, the concentration of the antibody stock solution is 5-15mg/mL, preferably 10 mg/mL.
In a preferred embodiment of the invention, the antibody linker is present at a concentration of 5-15mg/mL, preferably 10 mg/mL.
In a preferred embodiment of the present invention, the size exclusion step comprises using gel with separation molecular weight in the range of 5000-5000000, preferably 10000-600000 as packing material of the purification column.
In a preferred embodiment of the present invention, the gel filler with a separation molecular weight range of 10000-600000 comprises G200 gel. The molecular weight of the protein which can be separated by G200 gel is within the range of 10000-600000, and commercial products are Sephadex G200, Superdex G200 and SuperdexTM200pg, etc. Superdex is preferred in the present inventionTM200 pg. More meets the production purpose.
In a preferred embodiment of the present invention, in the method for preparing the antibody drug conjugate, the antibody is trastuzumab.
In a preferred embodiment of the present invention, in the method for preparing the antibody drug conjugate, the antibody drug conjugate is T-DM 1.
In a preferred embodiment of the present invention, in the method for preparing the antibody drug conjugate, the antibody is pertuzumab.
In a preferred embodiment of the present invention, in the method for preparing the antibody drug conjugate, the antibody drug conjugate is P-mab-linker-MC-MMAF.
In a preferred embodiment of the invention, the method of purification of the antibody linker may use small molecule filtration, preferably tangential flow ultrafiltration. In a preferred embodiment of the invention, the ultrafiltration membrane has a pore size of 10-50kD, preferably 30 kD.
The preparation and purification steps of the antibody-linking agent may also be included before the antibody-linking agent is subjected to a conjugation reaction with the cytotoxic drug.
The invention purifies the antibody-drug conjugate by a molecular exclusion method, can effectively remove polymers in the antibody, hardly influences the drug loading capacity of a sample, and improves the purity of the conjugate drug while ensuring the activity. The method has the advantages that the method can remove the polymer generated in the coupling process and the unreacted micromolecule toxin in the system by a one-step method to obtain the high-purity coupling substance, reduces the purification steps, improves the purification efficiency and is beneficial to industrial production.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described herein. In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below.
When tradenames are used in the present invention, applicants intend to include the formulation of the tradename product, the non-patent and active pharmaceutical portions of the tradename product.
Unless stated to the contrary, terms used in the specification and claims have the following meanings.
The term "cytotoxic drug" refers to a chemical molecule within a tumor cell that is strong enough to disrupt its normal growth. Cytotoxic drugs can kill tumor cells in principle at a high enough concentration, but due to lack of specificity, while killing tumor cells, they can also cause apoptosis of normal cells, resulting in serious side effects. In embodiments of the invention, non-limiting examples thereof include tubulin inhibitors, DNA alkylating agents, tyrosine kinase inhibitors, topoisomerase inhibitors, or DNA synthesis inhibitors.
The term "antibody" refers to any form of antibody that exhibits a desired biological activity. Thus, it is used in the broadest sense and specifically includes, but is not limited to, full length antibodies, antibody binding fragments or derivatives. Sources of antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, engineered antibodies (e.g., bispecific antibodies).
The term "antibody stock solution" refers to an antibody solution formed by storing an antibody obtained after purification in an appropriate buffer system conventionally used by those skilled in the art. Such as the acetic acid-sodium acetate buffer system or Phosphate Buffer System (PBS) used in the present invention.
The linker (SMCC) of the present invention may be commercially available or may be prepared and purified according to the references simple Synthesis of Reagents Containing a Terminal macromolecular Ligand Linked to an Active Ester, Synthesis 1991(10), 819. quadrature. 821.
The antibody-linker in the present invention refers to the antibody and SMCC obtained after the coupling reaction, such as trastuzumab-linker in step 2 of example 1. The reaction substrate equivalence ratio of the coupling reaction described herein, i.e., SMCC: the antibody range is 2-10: 1; in the present invention, 8: 1 is preferred. The reaction time is 1 to 5 hours, preferably 2 hours.
The antibody-linking agent and cytotoxic drug undergoing a coupling reaction described in the present invention means that the antibody-linking agent and cytotoxic drug undergo a C-S coupling reaction. The reaction substrate equivalence ratio for the coupling reaction described herein, i.e., L-DM 1: the range of antibody-linker is 2-15: 1; in the present invention, 6: 1 is preferred. The reaction solution used for the coupling reaction is Phosphate Buffered Saline (PBS) containing EDTA, and the pH is 5.5-7.5, preferably 6.5. The reaction time is 2 to 18 hours, preferably 4 hours. The reaction temperature is 4-37 deg.C, preferably 25 deg.C. The reaction solution also contains one or more solvents selected from acetonitrile, ethanol, DMSO, DMA and DMF, and the content of organic solvent is 2-20%, preferably 5-10%; in the present invention, 10% ethanol and 2% acetonitrile are preferred. Reference is also made to the description of US 2005/0169933 a 1.
The G200 gel in the invention represents the type of the gel, the G value represents the magnitude of the crosslinking degree, the figure represents 10 times of the water absorption capacity of the swelling glue per gram of dry glue, for example, the Superdex G200 can absorb 20 grams of water per gram of dry glue. Sephadex is a trade name for dextran gel (dextran gel); superdex is a trade name for a complex gel composed of agarose and dextran, which is a modified form of Sephadex. SuperdexTM200pg, a commercially available preparative grade gel model.
Detailed Description
The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
The experimental method of the present invention, in which the specific conditions are not specified, is generally carried out under the conventional conditions or the conditions recommended by the manufacturers of the raw materials or the commercial products. Reagents of specific sources are not indicated, and conventional reagents are purchased in the market.
Example 1: synthesis and purification of antibody drug conjugate T-DM1
Step one, synthetic process route of L-DM1
Reference is made to semi synthetic Maytansine assays for the Targeted Treatment of Cancer, J.Med.chem.2006, 49, 4392-.
Step two, synthetic process route of T-DM1
10.0 g of stock antibody of monoclonal antibody Trastuzumab (Trastuzumab, antibody disclosed in US6165464A) (Trastuzumab was diluted to a final antibody concentration of 10mg/mL with 50mM phosphate buffer, pH 6.5) and 181 mg of antibody in a buffer (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate, as well as 10% ethanol and 5% acetonitrile at 25 deg.C4- (N-Maleimidomethyl) cyclohexane-1-carboxylic acid succinimidyl ester(connecting agent SMCC, Shanghai Honghenkeke science and technology limited) in a reaction table for 2 hours, to generate a reaction solution of Trastuzumab-MCC; the Trastuzumab-MCC reaction solution obtained by the reaction was subjected to equal-volume ultrafiltration (ultrafiltration membrane using 30kd of a 30kd cellulose membrane as a ultrafiltration membrane) at 25 ℃ in 10 volumes using 50mM NaCl, 2mM EDTA and 50mM potassium phosphate, and a buffer solution (pH 6.5) containing 10% ethanol and 5% acetonitrileWrap), to remove unreacted SMCC and other small molecule impurities in the reaction solution, and finally performing equal-volume ultrafiltration with 5 volumes of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate buffer (pH 6.5) to obtain a 50mM NaCl, 2mM EDTA and 50mM potassium phosphate buffer solution (pH 6.5) of Trastuzumab-MCC.
The concentration of the obtained Trastuzumab-MCC in a buffer solution (pH 6.5) of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate was adjusted to 10mg/mL by replacing the buffer solution (pH 6.5) with 50mM NaCl, 2mM EDTA and 50mM potassium phosphate and 10% ethanol and 2% acetonitrile, and 300mgL-DM1 was added to the mixture, followed by reaction. Carrying out reaction on a reaction shaking bed for 4 hours to generate a reaction solution of Trastuzumab-MCC-DM1, and taking a sample to carry out drug loading test and SEC-HPLC purity test, wherein the SEC-HPLC result is 94.59%; the filler for the reaction liquid of Trastuzumab-MCC-DM1 generated by the reaction is SuperdexTMA 200pg size exclusion column was used to purify the sample to remove aggregates and other small impurities from the sample, recover the monomers, and sample for drug load testing and SEC-HPLC purity testing, with a SEC-HPLC result of 99.11%. The drug loading was calculated from the samples taken during the purification (UV method) and the results are shown in Table 1.
TABLE 1 calculation of drug loading from purification process samples (UV method)
| Sample (I) | Before purification | After purification |
| Capacity of carrying capacity | 3.40 | 3.34 |
Attached: the method for measuring the drug load of Trastuzumab-MCC-DM1 comprises the following steps:
the method for measuring the drug loading of Trastuzumab-MCC-DM1 comprises an ultraviolet-visible spectrophotometry method and an LC/MS method.
The principle of UV-visible spectrophotometry is based on The effect of drug and monoclonal antibody on total absorbance at 280nm (The absorbance at 280nm of The drug and The antibody conjugate to The total absorbance (A)280nm) The calculation process is as follows:
A280nm＝ε1bc1+ε2bc2 (1)
wherein:
ε1: extinction coefficient of drug at 280 nm;
ε2: extinction coefficient of monoclonal antibody at 280 nm;
c1: the concentration of the drug;
c2: the concentration of monoclonal antibody;
b: a detector path length (detector path length);
the same equation can be obtained for the total absorbance value of the sample at 252 nm:
A252nm＝ε3bc3+ε4bc4 (2)
the drug loading can be calculated by combining two equations (1) and (2) with the extinction coefficient and concentration data of the monoclonal antibody and the drug at two detection wavelengths. In the process, the final loading of the stock solution is measured and calculated by adopting an ultraviolet-visible spectrophotometry method.
Example 2: synthesis of antibody drug conjugate P-mab-linker-MC-MMAF
The method comprises the steps of taking a stock solution (100 mM acetic acid-sodium acetate buffer solution with pH 4.3-4.5) of pertuzumab (P-mAb, an antibody disclosed in US7041292B1), concentrating the stock solution to a concentration of about 10.0mg/mL to obtain 200mL (13.5mmol) of the acetic acid-sodium acetate buffer solution of the P-mAb, dissolving 3-acetylmercaptopropionaldehyde (14.3mg, 0.108mmol) in 20mL of acetonitrile, dropwise adding the dissolved solution to the buffer solution, dissolving sodium cyanoborohydride (173mg, 2.7mmol) in 10mL of water, dropwise adding the dissolved solution to the reaction solution, and stirring the reaction solution at 25 +/-2 ℃ for 2-3 hours. After the reaction, 5 times of volume of buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA, 50mM potassium phosphate and 10% acetonitrile is used for equal-volume ultrafiltration to remove other unreacted small molecular impurities in the reaction solution, and finally, 5 times of volume of buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate is used for equal-volume ultrafiltration to obtain 200mL of pertuzumab-ethyl propanethiol solution with the concentration of about 10 mg/mL.
To the pertuzumab-propanethiol ethyl ester solution (prepared in the first step), 4.0mL of 2M hydroxylamine hydrochloride was added, and after 1 hour of reaction with stirring at 25 ℃, the reaction solution was subjected to equal-volume ultrafiltration using 5 volumes of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate buffer (pH 6.5) to obtain 200mL of pertuzumab-propanethiol solution with a concentration of 10 mg/mL.
Dissolving a compound MC-MMAF (the compound is disclosed in WO2005081711, 125mg and 13.5mmol) in 20mL of acetonitrile, adding the solution into a pertuzumab-propanethiol solution (prepared in the second step), stirring at 25 ℃ for 4 hours for reaction to obtain an ADC sample of the P-mab-linker-MC-MMAF, and performing complete molecular weight test and SEC-HPLC purity test on the sample, wherein the SEC-HPLC result is 94.70% in detail and the drug loading is 2.1. The filler for the ADC sample reaction liquid of the obtained P-mab-linker-MC-MMAF is SuperdexTMA 200pg size exclusion column was run to purify the sample to remove aggregates and other small molecule residues from the sample, and a purified sample was obtained, and samples were run for complete molecular weight testing and SEC-HPLC purity testing, with a SEC-HPLC result of 99.39% and a drug load of 2.08.
Attached: testing complete molecular weight experimental conditions of a monoclonal antibody sample P-mab-linker-MC-MMAF:
chromatographic conditions are as follows:
a chromatographic column: poroshell 300 SB-C85 um 2.1.1 x 75mm
Mobile phase: a: 0.1% HCOOH/H2O B：0.1％HCOOH/ACN
Gradient:
| RT(min) | 0 | 5 | 8 | 12 | 12.1 | 15 |
| B％ | 5 | 5 | 95 | 95 | 5 | 5 |
flow rate: 0.4mL/min
Column temperature: 75 deg.C
Mass spectrum conditions:
mass spectrum: agilent 6530Q-TOF LC-MS
Mode (2): 1GHz
Capillary voltage: 3500V
Temperature of desolventizing gas: 325 deg.C
Gas flow rate: 10L/min
Fragment voltage: 200V
MS acquisition range: 500m/z-5000m/z
Example 3: synthesis and purification of antibody drug conjugate T-DM1
50.0 g of monoclonal antibody Trastuzumab (Trastuzumab final concentration: 10mg/mL) was reacted with 905 mg of succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate in a buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate, and 10% ethanol and 5% acetonitrile at 25 ℃ for 2 hours on a reaction shaker to give a reaction solution of Trastuzumab-MCC; the reaction solution of Trastuzumab-MCC obtained by the reaction was subjected to equal-volume ultrafiltration at 25 ℃ using 10 volumes of a buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate and 10% ethanol and 5% acetonitrile to remove unreacted SMCC and other small-molecule impurities in the reaction solution, and finally subjected to equal-volume ultrafiltration using 5 volumes of a buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate to obtain a buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate.
The concentration of the obtained Trastuzumab-MCC in a buffer solution (pH 6.5) of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate was adjusted to 10mg/mL by replacing the buffer solution with 50mM NaCl, 2mM EDTA and 50mM potassium phosphate and 10% ethanol and 2% acetonitrile in Trastuzumab-MCC in a buffer solution (pH 6.5), and 1500mgL-DM1 in ethanol was added to the mixture to react with the above reaction system. The reaction is carried out for 4 hours on a reaction shaker to obtain a reaction solution of Trastuzumab-MCC-DM1, and the purity and the drug loading capacity of the reaction solution are tested. SEC-HPLC purity was 95.25% with drug loading of 3.43.
The filler for the obtained Trastuzumab-MCC-DM1 reaction liquid is SuperdexTMA200 pg size exclusion column was used to purify the sample to remove aggregates and residual other small molecule material from the sample. Finally, purity test and drug load test are carried out, the SEC-HPLC purity is 99.26%, and the drug load is 3.40. The sample recovery was 95%.
TABLE 2 drug loading calculation from purification process samples (UV method)
| Sample (I) | Before purification | After purification |
| Capacity of carrying capacity | 3.43 | 3.40 |
Comparative example 1:
in a buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate, as well as 10% ethanol and 5% acetonitrile, 10.0 g of monoclonal antibody Trastuzumab (Trastuzumab final concentration of 10mg/ml) was reacted with 180 mg of succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate on a reaction shaker at 25 ℃ for 2 hours to give a reaction solution of Trastuzumab-MCC; after the reaction, the reaction solution of Trastuzumab-MCC obtained by the reaction was desalted and purified by Sephadex G25 gel column (elution phase: buffer solution containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate at pH 6.5) to remove the unreacted SMCC and other small molecule impurities in the reaction solution, and finally a buffer solution of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate (pH 6.5) of Trastuzumab-MCC was obtained.
The obtained Trastuzumab-MCC was replaced with a buffer solution (pH 6.5) of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate in a buffer solution (pH 6.5) of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate and a buffer solution (pH 6.5) of 10% ethanol and 2% acetonitrile in Trastuzumab-MCC, and the concentration was adjusted to 10mg/ml, and then 300mg of an ethanol solution of L-DM1 was added to the mixture to react with the above reaction system. Carrying out reaction on a reaction shaker for 4 hours to generate a reaction solution of Trastuzumab-MCC-DM 1; after the reaction, the reaction solution of Trastuzumab-MCC-DM1 obtained by the reaction was desalted and purified by Sephadex G25 gel column (elution phase: buffer solution containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate at pH 6.5) to remove unreacted L-DM1 and other small molecule impurities in the reaction solution, and finally a buffer solution of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate (pH 6.5) of Trastuzumab-MCC-DM1 was obtained. Finally, purity test and drug load test are carried out, the SEC-HPLC purity is 94.45%, and the drug load is 3.45%.
Comparative example 2:
in a buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate, as well as 10% ethanol and 5% acetonitrile, 10.0 g of monoclonal antibody Trastuzumab (Trastuzumab final concentration of 10mg/ml) was reacted with 181 mg of succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate on a reaction shaker at 25 ℃ for 2 hours to give a reaction solution of Trastuzumab-MCC; the reaction solution of Trastuzumab-MCC obtained by the reaction is subjected to equal-volume ultrafiltration (the ultrafiltration membrane is a 30kd cellulose membrane) at 25 ℃ by using 5 times of the volume of a buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate and 10% ethanol and 5% acetonitrile to remove unreacted SMCC and other small molecule impurities in the reaction solution, and finally subjected to equal-volume ultrafiltration by using 5 times of the volume of a buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate to obtain a buffer solution (pH 6.5) containing 50mM NaCl, 2mM EDTA and 50mM potassium phosphate of Trastuzumab-MCC.
The concentration of the obtained Trastuzumab-MCC in a buffer solution (pH 6.5) of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate was adjusted to 10mg/ml by replacing the buffer solution (pH 6.5) with 50mM NaCl, 2mM EDTA and 50mM potassium phosphate and 10% ethanol and 2% acetonitrile, and 300mgL-DM1 was added to the mixture, followed by reaction. The reaction was carried out for 4 hours on a reaction shaker to produce a reaction solution of Trastuzumab-MCC-DM1, and isovolumetric ultrafiltration was carried out at 25 ℃ using 5 volumes of a buffer solution (pH 6.5) of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate and 10% ethanol and 2% acetonitrile (ultrafiltration membrane package using 30kd of a cellulose membrane) to remove unreacted L-DM1 and other small molecule impurities in the reaction solution, and isovolumetric ultrafiltration was finally carried out using 5 volumes of a buffer solution (pH 6.5) of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate to obtain a buffer solution (pH 6.5) of 50mM NaCl, 2mM EDTA and 50mM potassium phosphate of Trastuzumab-MCC-DM1(T-DM 1). Finally, purity test and drug load test were carried out, and the SEC-HPLC purity was 94.61%, and the drug load was 3.51.
Claims (7)
1. A method for preparing antibody drug conjugate comprises the preparation and purification of antibody-linking agent, wherein the purification method of the antibody-linking agent is ultrafiltration; the antibody-linking agent and the cytotoxic drug are subjected to coupling reaction to prepare an antibody drug conjugate, and the purification step of the antibody drug conjugate is also included, wherein the purification step comprises a molecular exclusion step;
the molecular exclusion step comprises the steps of using gel with the separation molecular weight range of 5000-5000000 as a filler of a purification column;
the aperture of the ultrafiltration membrane is 10-50 KD;
the antibody drug conjugate is trastuzumab-DM 1 or pertuzumab-propyl-1-sulfur-MC-MMAF.
2. The method of claim 1, wherein the step of size exclusion comprises using a gel with a separation molecular weight range of 10000-600000 as a packing material for a purification column.
3. The method of claim 2, wherein the size exclusion step comprises using a G200 gel as packing material for the purification column.
4. The method of claim 3 wherein the G200 gel is SuperdexTM 200pg。
5. The method of claim 1, wherein the ultrafiltration membrane has a pore size of 30 KD.
6. The method of preparing an antibody-drug conjugate of any of claims 1 to 5, wherein the concentration of antibody-linking agent in the conjugation reaction is 5 to 15 mg/mL.
7. The method of claim 6 wherein the antibody-linking agent is present at a concentration of 10 mg/mL.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510962987X | 2015-12-21 | ||
| CN201510962987 | 2015-12-21 | ||
| PCT/CN2016/109807 WO2017107817A1 (en) | 2015-12-21 | 2016-12-14 | Preparation method for antibody drug conjugate |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107405408A CN107405408A (en) | 2017-11-28 |
| CN107405408B true CN107405408B (en) | 2021-07-02 |
Family
ID=59089112
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201680011806.8A Active CN107405408B (en) | 2015-12-21 | 2016-12-14 | Preparation method of antibody drug conjugate |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN107405408B (en) |
| WO (1) | WO2017107817A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112206327A (en) * | 2019-07-12 | 2021-01-12 | 上海药明生物技术有限公司 | Preparation of antibody coupling drug and high-throughput screening method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1993146A (en) * | 2004-06-01 | 2007-07-04 | 健泰科生物技术公司 | Antibody-drug conjugates and methods |
| CN103068406A (en) * | 2010-06-08 | 2013-04-24 | 基因泰克公司 | Cysteine engineered antibodies and conjugates |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1992005200A1 (en) * | 1990-09-18 | 1992-04-02 | Chemex Pharmaceuticals, Inc. | Xenobiotic-antibody conjugates |
| SI2019104T1 (en) * | 2007-07-19 | 2013-12-31 | Sanofi | Cytotoxic agents comprising new tomaymycin derivatives and their therapeutic use |
| MX338932B (en) * | 2010-03-12 | 2016-05-06 | Immunogen Inc | Cd37-binding molecules and immunoconjugates thereof. |
| WO2013049410A1 (en) * | 2011-09-29 | 2013-04-04 | Seattle Genetics, Inc. | Intact mass determination of protein conjugated agent compounds |
| RU2015116485A (en) * | 2012-11-07 | 2016-12-27 | Пфайзер Инк. | Interleukin-13 alpha 2 receptor antibodies and antibody drug conjugates |
| MX2016009332A (en) * | 2014-01-29 | 2016-12-20 | Shanghai hengrui pharmaceutical co ltd | Ligand-cytotoxicity drug conjugate, preparation method therefor, and uses thereof. |
-
2016
- 2016-12-14 CN CN201680011806.8A patent/CN107405408B/en active Active
- 2016-12-14 WO PCT/CN2016/109807 patent/WO2017107817A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1993146A (en) * | 2004-06-01 | 2007-07-04 | 健泰科生物技术公司 | Antibody-drug conjugates and methods |
| CN103068406A (en) * | 2010-06-08 | 2013-04-24 | 基因泰克公司 | Cysteine engineered antibodies and conjugates |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2017107817A1 (en) | 2017-06-29 |
| CN107405408A (en) | 2017-11-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| ES2539232T3 (en) | Methods to reduce the level of one or more impurities in a sample during protein purification | |
| KR101149969B1 (en) | Antibody purification | |
| Lata et al. | Synthesis and application of cephalexin imprinted polymer for solid phase extraction in milk | |
| BRPI0518216B1 (en) | chromatography binder, methods of preparing a separation matrix, and separating one or more antibodies from one or more other compounds into a liquid sample, separation matrix, and disposable chromatography column. | |
| Elzahhar et al. | Bioconjugation in drug delivery: Practical perspectives and future perceptions | |
| EP3048109A1 (en) | Novel antibody purification method and antibody obtained therefrom, and novel antibody purification method using cation exchanger and antibody obtained therefrom | |
| JP4890453B2 (en) | Separation matrix and purification method | |
| Matsuda | Current approaches for the purification of antibody–drug conjugates | |
| US9452415B2 (en) | Sorbent comprising on its surface a cationic or protonizable aliphatic residue for the purification of organic molecules | |
| Cai et al. | Preparation of monodisperse, restricted-access, media-molecularly imprinted polymers using bi-functional monomers for solid-phase extraction of sarafloxacin from complex samples | |
| TW200418868A (en) | Sugar chain asparagine derivatives, sugar chain asparagine and sugar chain and manufacture thereof | |
| Chi et al. | Preparation of phenyl-boronic acid polymer monolith by initiator-free ring-opening polymerization for microextraction of sulfamethoxazole and trimethoprim from animal-originated foodstuffs | |
| CN105669857B (en) | A kind of quaternized chitosan oligosaccharide modifier of ES-2 peptide and the preparation method and application thereof | |
| Zhang et al. | Synthesis of a silica-bonded bovine serum albumin s-triazine chiral stationary phase for high-performance liquid chromatographic resolution of enantiomers | |
| CN107405408B (en) | Preparation method of antibody drug conjugate | |
| ES2545069T3 (en) | Procedure for selective binding of a substrate to sorbents by at least bivalent binding | |
| Omran et al. | Validation and Application of Molecularly Imprinted Polymers for SPE/UPLC–MS/MS Detection of Gemifloxacin Mesylate | |
| JP2007535658A (en) | Immunoglobulin purification | |
| Wang et al. | Antibody purification using affinity chromatography: A case study with a monoclonal antibody to ractopamine | |
| Carapito et al. | Multimodal ionic liquid-based chromatographic supports for an effective RNA purification | |
| TW200927254A (en) | Antibody production method | |
| Santos et al. | Plasmid purification by using a new naphthalene tripodal support | |
| Mi et al. | Protein adsorption on core‐shell resins for flow‐through purifications: Effect of protein molecular size, shape, and salt concentration | |
| EP2755757A1 (en) | Sorbent comprising on its surface an aromatic ring system having an anionic or deprotonizable group for the purification of organic molecules | |
| Ferreira et al. | Affinity analysis and application of dipeptides derived from l-tyrosine in plasmid purification |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |