Patent Application
20250000997
The present application is based on the application with CN application Ser. No. 202111021482.5 filled on Sep. 1, 2021, and claims its priority. The disclosure of the CN application is hereby incorporated herein in its entirety.
The present application relates to the field of medicinal chemistry, and specifically to a method for improving the product quality of an antibody-drug conjugate.
With the development of the field of antibody-drug conjugates, the development of production process conditions of purification and conjugation for biologically active drugs containing target antibodies is particularly important. The development and effective control of separation and purification processes for antibodies and a conjugation process for antibody-drug conjugates can ensure the product quality of drugs and are the key to the industrial production process of antibody-drug conjugates.
The design and parameter control of separation and purification process routes for antibodies directly affect the product quality and yield of antibody-drug conjugates. The existing purification process route of the antibody part of antibody-drug conjugates often requires including ultrafiltration and/or diafiltration steps to make the antibody meet the conjugation requirements. However, such steps increase the antibody purification steps and production costs, affecting the yields of the antibody and even the final conjugate.
On the other hand, the drug conjugation process has an important impact on the quality of antibody-drug conjugates. Currently, common conjugation methods for antibody-drug conjugates include: lysine conjugation, light and heavy chain reduced sulfide bond conjugation. and site-directed conjugation. Due to the diversity of conjugation sites and methods, for lysine conjugation, light and heavy chain reduced sulfide bond conjugation, or site-directed conjugation processes, the obtained antibody-drug conjugates are all accompanied by certain non-target products, and even occurrence of protein aggregation, affecting the pharmaceutical safety and effectiveness of the antibody-drug conjugates. The number of toxins attached to the antibody (drug-antibody ratio. DAR) determines the uniformity of the drug product. How to ensure product quality while controlling the DAR uniformity as much as possible is a difficulty in the production process of antibody-drug conjugate products. In the prior art, conjugation and purification processes of antibody-drug conjugates with DAR values between 3-4 are relatively common. Therefore, if purification and conjugation processes of antibody-drug conjugates with high DAR (for example, 6-8) can be developed, it is of great significance for improving product quality and reducing costs and safety risks.
The inventors of the present application have conducted a large number of experiments and repeated explorations on the upstream antibody purification process and antibody-drug conjugation process of antibody-drug conjugates, and improved the reaction conditions and process parameters of each step in the production process. The method results in antibody products that can meet the conjugation requirements, improves the product purity and quality of final antibody-drug conjugates, significantly reduces the content of related substances and potential medication risks, and achieves stable and reliable product quality, thus being suitable for industrial continuous scale-up production.
The present invention provides a method for improving the product quality of an antibody-drug conjugate, comprising:
wherein the structure of the drug to be conjugated is shown in formula (I):
D-[L1-(L2)m1-(L3)m2-(L4)m3-E]-G Formula (I)
wherein, each R1 and R2 are independently hydrogen, halogen, carboxylate, sulfonate, cyano, C1-6 alkyl, halogenated C1-6 alkyl, cyano substituted C1-6 alkyl (e.g., —CH2CN), C1-6 alkoxy, C2-10 alkenyl, or C2-10 alkynyl; Z1 is an amino acid or a peptide consisting of 2 to 10 amino acids; x1 and x2 each are individually 0, 1, 2, 3, 4, 5, or 6; and L1 is connected to D at position 1 and L1 is connected to L2 at position 2;
or absent; wherein, y: is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and L2 is connected to L1 at position 1 and L2 is connected to L3 at position 2;
wherein, each R4 is independently hydrogen, β is 0, 1 or 2, and E is connected to G at position 2 and E is connected to L4 at position 1;
In certain embodiments, the present invention provides a method for improving the product quality of an antibody-drug conjugate, wherein a 5-20 mM reducing agent is added to an antibody that is pretreated and mixed uniformly for a reduction reaction, and then a drug to be conjugated is added, and after it is stirred and rested, an acidic solution is added;
In certain embodiments, the antibody-drug conjugate in step (1) is obtained by the following method: a reducing agent solution with a concentration of 5-20 mM is added to an antibody that is pretreated, and then a drug to be conjugated is added to the reaction system, and after it is stirred and rested, an acidic solution is added to obtain the antibody-drug conjugate.
In certain embodiments, the method further includes the step of bacteria removal filtration.
In certain embodiments, the antibody is subjected to a bacteria removal filtration step prior to reduction reaction.
In certain embodiments, the reducing agent is tris (2-carboxyethyl) phosphine.
In certain embodiments, the reducing reaction includes treating the antibody with the reducing agent for 10 min-2 h, for example, 30 min-1 h.
In certain embodiments, the reduction reaction is treating the antibody with the reducing agent at room temperature: and/or, the reduction reaction is performed at 4-37° C., for example, 18-26° C. In certain embodiments, the column chromatography method is hydrophobic chromatography. Preferably, the hydrophobic chromatography includes steps of equilibration and elution.
In certain embodiments, the equilibrium step of the hydrophobic chromatography is using 20 mmol/L phosphate buffer-ammonium sulfate solution as an equilibrium solution under pH 6-7 conditions, wherein the conductivity is controlled at 50-80 mS/cm.
In certain embodiments, the equilibrium step of the hydrophobic chromatography is using 20 mmol/L phosphate buffer-ammonium sulfate solution as an equilibrium solution under pH 6-7 conditions, wherein the conductivity is controlled at 62-70 mS/cm.
In certain embodiments, the elution step of the hydrophobic chromatography is using the same solution as the equilibrium solution as an eluent under pH 6-7 conditions, wherein the conductivity is controlled at 22-30 mS/cm.
In certain embodiments, the chromatography column uses Butyl Sepharose High Performance packing.
In certain embodiments, the ultrafiltration and/or diafiltration concentration uses Pellicon 3 Cassette Biomax membrane.
In certain embodiments, a replacement solution used in the diafiltration is a 10 mmol/L histidine-histidine hydrochloride solution with a pH of 5.7-6.3.
In certain embodiments, the drug to be conjugated is added to the reaction system and stirred for 5-15 min, and then rested to react, preferably, rested for 1 h-12 h, such as 1 h-6 h, 1 h-4 h, 1 h-3 h, 1.5 h-3 h, or 1.5 h-2.5 h.
In certain embodiments, the stirring time is 5-15 min, the resting temperature is 4-37° C., for example, 18-26° C., and the resting time is 1.5-3 h.
In certain embodiments, the drug to be conjugated is a small molecular compound with a connecting arm. Preferably, the small molecular compound is selected from a DNA topoisomerase inhibitor, a tubulin inhibitor, or derivatives thereof.
In certain embodiments, the DNA topoisomerase inhibitor is a topoisomerase I inhibitor, and preferably, the topoisomerase I inhibitor is camptothecin, SN-38, irinotecan, topotecan, belotecan, rubitecan, or derivatives thereof.
In certain embodiments, the DNA topoisomerase inhibitor is a topoisomerase II inhibitor, and preferably, the topoisomerase II inhibitor is actinomycin D, doxorubicin, doxorubicin, docamicin, daunorubicin, mitoxantrone, podophyllotoxin, etoposide, or derivatives thereof.
In certain embodiments, the tubulin inhibitor is vinca alkaloid, vincristine, vinblastine, paclitaxel, docetaxel, cabazitaxel, or derivatives thereof.
In certain embodiments, the small molecular compound with a connecting arm is one selected from N-((S)-1-(((S)-1-((4-(((S)-2-((2R.3R)-3-((S)-1-((3R.4S,5S)-4-((S)-2-((S)-2-(dimethylamino)-3-methylbutylamido) -N,3-dimethylbutylamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropionylamino) -3-phenylpropionamido)methyl)phenyl)amino) -1-oxo-5-ureido-2-yl)amino)-3-methyl-1-oxobutan-2-yl) -6-(2-(methylsulfonyl) pyrimidin-5-yl)-5-hexyneamide;
In certain embodiments, the drug to be conjugated is a small molecular compound with a connecting arm, and the small molecular compound with a connecting arm is selected from the compounds shown in the following structures:
In certain embodiments, the antibody is Sacituzumab; the drug to be conjugated is a small molecular compound with a connecting arm, the structure of which is as follows:
In some embodiments, the structure of the antibody-drug conjugate is as follows:
In certain embodiments, the dosing concentration of the drug to be conjugated is 10-70 mM, preferably 50 mM.
In certain embodiments, the pH value of the tris (2-carboxyethyl) phosphine solution is 7.0-8.0, for example, 7.4-8.0.
In certain embodiments, the acidic solution is selected from citric acid or acetic acid.
In certain embodiments, the acidic solution adjusts the pH of the reaction system to acidity; preferably, the acidic solution adjusts the pH of the reaction system to 6.0-7.0; further preferably, the acidic solution adjusts the pH of the reaction system to 4.5-7.0.
In certain embodiments, the antibody pretreatment includes steps of adding a disodium edetate solution to the antibody and adjusting the pH with a Tris solution. Preferably, the concentration of the disodium edetate solution is 2-10 mM, and the concentration of the Tris solution is 1-3 M; further preferably, the concentration of the disodium edetate solution is 2-10 mM, and the concentration of the Tris solution is 2 M.
In certain embodiments, the antibody pretreatment includes steps of adding a disodium edetate solution to the antibody and adjusting the pH using a phosphate. Preferably, the phosphate is sodium phosphate; further preferably, the sodium phosphate is disodium hydrogen phosphate; preferably, the concentration of the disodium edetate solution is 2-10 mM, and the concentration of the disodium hydrogen phosphate solution is 0.2-2 M, preferably 1 M.
In certain embodiments, the antibody is an anti-Her2 antibody, an anti-EGFR antibody, an anti-PD-1 antibody, an anti-PD-LI antibody, or an anti-Trop-2 antibody.
In certain embodiments, the anti-Her2 antibody is Trastuzumab or Pertuzumab, the anti-EGFR antibody is Cetuximab or Nimotuzumab, the anti-PD-I monoclonal antibody is Nivolumab or Pembrolizumab, the anti-PD-LI monoclonal antibody is Atezolizumab or Durvalumab, and the anti-Trop-2 antibody is Sacituzumab or Datopotamab.
In certain embodiments, in the reduction reaction, the dosing concentration of the antibody is 10-25 g/L.
In certain embodiments, the regulation solution is selected from ammonium sulfate. In certain embodiments, the regulation solution is added to the antibody-drug conjugate prior to the hydrophobic chromatography step.
In certain embodiments, the regulation solution adjusts the antibody-drug conjugate
to a high conductivity: preferably, the regulation solution adjusts the conductivity of the antibody-drug conjugate to 62-70 mS/cm.
In certain embodiments, the antibody is further purified prior to pretreatment.
In certain embodiments, the purification includes steps of chromatography selected from affinity chromatography, anion exchange chromatography, and/or cation exchange chromatography, and filtration.
In certain embodiments, the purification sequentially includes steps of affinity chromatography, incubation, depth filtration, anion exchange chromatography, and cation exchange chromatography.
In certain embodiments, the purification does not include ultrafiltration, diafiltration and the like.
In certain embodiments, a packing of the affinity chromatography is selected from Cytiva Mabselect Sure LX, Cytiva MabSelect SuRe, Cytiva MabSelect PrismA, Merck Eshmuno A, and Tosoh AF rProteinA-HC 650F.
In certain embodiments, the affinity chromatography further includes steps of equilibration, washing and elution. Preferably, the equilibration step uses NaCl-phosphate buffer: preferably, the washing can be performed at a pH of 5.4-6.2 for one, two or multiple times; preferably, the washing solution is selected from phosphate buffer-NaCl and sodium acetate-acetic acid solution; preferably, the elution uses sodium acetate-acetate buffer.
In certain embodiments, the elution step of the affinity chromatography requires controlling the pH at 3.5-3.7.
In certain embodiments, the incubation includes incubation at room temperature and pH 3.6-3.8.
In certain embodiments, the depth filtration uses Millipore XOHC.
In certain embodiments, a packing of the anion exchange chromatography is selected from Cytiva Q Sepharose Fast Flow, Thermo POROS 50HQ, Thermo POROS XQ, and Bio-Rad Nuvia HP-Q
In certain embodiments, the pH of an operating system of the anion exchange chromatography is 5.5-6.5, preferably 5.8-6.2.
In certain embodiments, a packing of the cation exchange chromatography is selected from Tosoh Gigacap S 650M, Merck Eshmuno CPX, Thermo POROS 50HS, Thermo POROS XS, and Bio-Rad Nuvia HP-S.
In certain embodiments, the pH of an operating system of the cation exchange chromatography is 5.5-6.5, preferably 5.9-6.1.
In certain embodiments, the purification further includes steps of virus removal filtration and bacteria removal filtration.
The method of the present invention at least has the following beneficial effects:
The technical solutions in the embodiments of the present invention are clearly and fully described below with reference to the accompanying drawings in the embodiments of the present invention. It is clear that the described embodiments are merely some embodiments of the present invention rather than all the embodiments of the present invention. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, and applications or uses thereof. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention. Unless otherwise specified, the reagents used in the following examples are commercially available products, and the solutions can be prepared using conventional techniques in the art.
An affinity chromatography column (packing Mabselect Sure LX) was equilibrated with 20 mM phosphate buffer at pH 7.2 and 150 mM NaCl solution. The solution containing Sacituzumab antibody was loaded with a loading of 42.7 g/L. After completion, it was washed with at least 3 column volumes of 20 mM PB at pH 6.0 and 1M NaCl solution, and then washed a second time with at least 3 column volumes of 20 mM sodium acetate-acetic acid buffer at pH 5.5. After washing, it was eluted with 20 mM sodium acetate-acetic acid solution at pH 3.57.
The collected eluate was incubated at room temperature using citric acid to adjust to pH 3.7. After inactivation for 2 h, the resulting sample was neutralized with 2 M Tris. The above sample was clarified through depth filtration, with a loading of 1366 g/m2 and Millipore XOHC as a membrane material. The treated sample was first subjected to anion exchange chromatography with a loading of 108.5 g/L, Cytiva Q Sepharose Fast Flow as packing, a loading conductivity of 2.68 mS/cm, and a pH of 6.09. The flow-through peak was collected after loading. The flow-through sample was chromatographed with a cationic column packed with Gigacap S 650M, a loading of 49.2 g/L, an elution pH of 6.02, and 20 mM PB+105 mM sodium chloride solution as eluent. The product was collected and used in the next step. The collected product was prefiltered and then subjected to virus removal filtration and bacteria removal filtration using Millipore Viresolve Pro to obtain the final pretreated antibody.
An affinity chromatography column (packing Mabselect Sure LX) was equilibrated with 20 mM phosphate buffer at pH 7.2 and 150 mM NaCl solution. The solution containing Sacituzumab antibody was loaded with a loading of 41.9 g/L. After completion, it was washed with at least 3 column volumes of 20 mM PB at pH 6.0 and 1M NaCl solution, and then washed a second time with at least 3 column volumes of 20 mM sodium acetate-acetic acid buffer at pH 5.5. After washing, it was eluted with 20 mM sodium acetate-acetic acid solution at pH 3.62.
The collected eluate was incubated at room temperature using citric acid to adjust to pH 3.69. After inactivation for 2 h, the resulting sample was neutralized with 2 M Tris solution. The above sample was clarified through depth filtration, with a loading of 1394.9 g/m2 and Millipore XOHC as a membrane material. The treated sample was first subjected to anion exchange chromatography with a loading of 107.7 g/L, GE Q Sepharose Fast Flow as packing, a loading conductivity of 2.71 mS/cm, and a pH of 6.07. The flow-through peak was collected after loading. The flow-through sample was chromatographed with a cationic column packed with Gigacap S 650M, a loading of 48.6 g/L, an elution pH of 6.03, and 20 mM PB+105 mM sodium chloride solution as eluent. The product was collected and used in the next step. The collected product was prefiltered and then subjected to virus removal filtration and bacteria removal filtration using Millipore Viresolve Pro to obtain the final pretreated antibody.
An affinity chromatography column (packing Mabselect Sure LX) was equilibrated with 20 mM phosphate buffer at pH 7.2 and 150 mM NaCl solution. The solution containing Sacituzumab antibody was loaded with a loading of 43.2 g/L. After completion, it was washed with at least 3 column volumes of 20 mM PB at pH 6.0 and 1M NaCl solution, and then washed a second time with at least 3 column volumes of 20 mM sodium acetate-acetic acid buffer at pH 5.5. After washing, it was eluted with 20 mM sodium acetate-acetic acid solution at pH 3.58.
The collected eluate was incubated at room temperature using citric acid to adjust to pH 3.66. After inactivation for 2 h, the resulting sample was neutralized with 2 M Tris solution. The above sample was clarified through depth filtration, with a loading of 1447.8 g/m2 and Millipore XOHC as a membrane material. The treated sample was first subjected to anion exchange chromatography with a loading of 116 g/L, GE Q Sepharose Fast Flow as packing, a loading conductivity of 2.69 mS/cm, and a pH of 6.06. The flow-through peak was collected after loading. The flow-through sample was chromatographed with a cationic column packed with Gigacap S 650M, a loading of 52.8 g/L, an elution pH of 6.0, and 20 mM PB+105 mM sodium chloride solution as eluent. The product was collected and used in the next step. The collected product was prefiltered and then subjected to virus removal filtration and bacteria removal filtration using Millipore Viresolve Pro to obtain the final pretreated antibody.
Samples collected in each step of the above three batches were tested for IEC purity using ion exchange chromatography and for SEC purity using SEC-HPLC method. The results are shown in the table below.
It can be seen from Table 1 that the three batches of antibodies produced by the process of the present invention all meet the requirements in terms of the purity indicators and can be used for the subsequent preparation of antibody-drug conjugates.
Samples collected in each step of the above three batches were tested for DNA residue and HCP residue. Residual DNA was tested using fluorescence quantitative PCR and residual HCP was tested using ELISA. The results are shown in the table below.
It can be seen from Table 2 that the three batches of antibodies produced by the process of the present invention all meet the requirements in terms of DNA residue and HCP residue and can be used for the subsequent preparation of antibody-drug conjugates.
Sacituzumab antibody was diluted with 0.25 mL of a solution (pH 7.6) containing 20 mM PB, 150 mM NaCl and 5 mM disodium edetate to adjust the pH to 7.4, 10 mM TCEP was added as a reducing agent for conjugation of a drug to be conjugated and the Sacituzumab antibody. The two was mixed uniformly and rested at room temperature for 2 h. After completion, cysteine was added to terminate the reaction. Finally, the buffer was replaced using G-25 gel column to obtain the conjugated product.
In this example, the drugs to be conjugated as shown in the following structures were respectively conjugated with Sacituzumab to obtain antibody-drug conjugates:
5
A 5 mM disodium edetate regulation solution was added to a slowly stirred pretreated antibody to be conjugated (prepared according to the method of Example 1), and 2M Tris was used to adjust the pH concentration to alkaline. 10 mM TCEP solution was added to the antibody solution and mixed evenly with stirring. After reacting for 35 min at 23.3°° C., a 50 mM solution of a drug to be conjugated was added to the reaction system, mixed evenly with stirring, and rested at room temperature. After reacting for 150 min, 1 M citric acid was used to adjust the pH to acidic (pH 6.0-7.0) to obtain the target product. Herein, the structural formula of the drug to be conjugated is
Ammonium sulfate regulation solution was added into the target product, and then hydrophobic column chromatography flowthrough was performed to collect a product, where the hydrophobic chromatography column packing was Butyl Sepharose High Performance, the equilibrium solution was a 20 mmol/L phosphate buffer-ammonium sulfate solution at pH 6.59, and conductivity was 66.51 mS/cm. The same eluent as the equilibrium solution was selected for elution, where conductivity was 26.93 mS/cm. The product of hydrophobic chromatography was further concentrated by ultrafiltration to obtain an antibody-drug conjugate.
The intermediates in each step of the above preparation were collected, and tested for purity using SEC-HPLC method, DAR using RP-HPLC method, free toxin using RP-HPLC method, and bacterial endotoxin according to the general rule 1143 of Chinese Pharmacopoeia, 2015 edition—Gel method. The results are shown in the table below.
As can be seen from Table 3, the antibody-drug conjugate produced by the conjugation process of the present invention has high purity, uniform DAR value, and effective control of free toxin and endotoxin contents, which significantly improves the product quality of the prepared antibody-drug conjugate.
A 5 mM disodium edetate regulation solution was added to a slowly stirred
pretreated antibody to be conjugated (prepared according to the method of Example 1), and 2M Tris was used to adjust the pH concentration to alkaline. 10 mM TCEP solution was added to the antibody solution and mixed evenly with stirring. After reacting for 32 min at 23.5° C., a 50 mM solution of a drug to be conjugated was added to the reaction system, mixed evenly with stirring, and rested at room temperature. After reacting for 2.5 h, 1 M citric acid was used to adjust the pH to acidic (pH 6.0-7.0) to obtain the target product. Herein, the structural formula of the drug to be conjugated is
Ammonium sulfate regulation solution was added into the target product, and then hydrophobic column chromatography flowthrough was performed to collect a product, where the hydrophobic chromatography column packing was Butyl Sepharose High Performance (GE), the equilibrium solution was a 20 mmol/L phosphate buffer-ammonium sulfate solution at pH 6.59, and conductivity was 66.69 mS/cm. The eluent was the same as the equilibrium solution and conductivity was 27.16 mS/cm. The product of hydrophobic chromatography was further concentrated by ultrafiltration to obtain an antibody-drug conjugate.
The intermediates in each step of the above preparation were tested for purity, DAR, free toxin and bacterial endotoxin (the test method was the same as in Example 3), and the results are as shown in the table below.
As can be seen from Table 4, the antibody-drug conjugate produced by the conjugation process of the present invention has high purity, uniform DAR value, and effective control of free toxin and endotoxin contents, which significantly improves the product quality of the prepared antibody-drug conjugate.
A 5 mM disodium edetate regulation solution was added to a slowly stirred pretreated antibody to be conjugated (prepared according to the method of Example 1), and 2 M Tris was used to adjust the pH concentration to alkaline. 10 mM TCEP solution was added to the antibody solution and mixed evenly with stirring. After reacting for 35 min at 22.3° C., a 50 mM solution of a drug to be conjugated was added to the reaction system, mixed evenly with stirring, and rested at room temperature. After reacting for 153 min, 1 M citric acid was used to adjust the pH to acidic (pH 6.0-7.0) to obtain the target product. Herein, the structural formula of the drug to be conjugated is
Ammonium sulfate regulation solution was added into the target product, and then hydrophobic column chromatography flowthrough was performed to collect a product, where the hydrophobic chromatography column packing was Butyl Sepharose High Performance (GE), the equilibrium solution was a 20 mmol/L phosphate buffer-ammonium sulfate solution at pH 6.51, and conductivity was 66.66 mS/cm. The eluent was the same as the equilibrium solution and conductivity was 26.98 mS/cm. The product of hydrophobic chromatography was further concentrated by ultrafiltration to obtain an antibody-drug conjugate.
The intermediates in each step of the above preparation were tested for purity, DAR, free toxin and bacterial endotoxin (the test method was the same as in Example 3), and the results are as shown in the table below.
As can be seen from Table 5, the antibody-drug conjugate produced by the conjugation process of the present invention has high purity, uniform DAR value, and effective control of free toxin and endotoxin contents, which significantly improves the product quality of the prepared antibody-drug conjugate.
The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention. In addition, the technical solutions between the various embodiments can be combined to each other, but this must be based on the ability of ordinary skilled in the art to achieve. When a combination of technical solutions conflicts or cannot be achieved, it should be considered that this combination of technical solutions does not exist, nor is it within the scope of protection claimed by the present invention.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111021482.5 | Sep 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/113869 | 8/22/2022 | WO |
