Method for evaluating antibody-medicine composite body

Abstract

To provide a method for evaluating the DAR of an ADC product that provides advantages over known methods.SOLUTION: Specifically, the method of the present disclosure can be used for a high-throughput application and / or can be used without requiring dilution of an ADC sample when the evaluation is being made.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 556,153, filed September 8, 2017, the entire contents of which are incorporated by reference herein. [Background technology]

[0002] Antibody-drug conjugates (ADCs) are a new class of drug molecules. Their ability to seek out specific targets and deliver potent drugs makes them an attractive option for developing target-based therapeutics. ADCs are generated by chemically linking potent drug molecules to monoclonal antibodies via selected chemical linkers. The average number of drug molecules bound to a monoclonal antibody is referred to as the drug / antibody ratio ("DAR"). DAR is an important quality attribute of ADC products, as it can affect the efficacy, safety, and / or stability of the product. Therefore, a method to assess the DAR of ADC products in a reliable and high-throughput manner is desirable. Summary of the Invention [Means for solving the problem]

[0003] The present disclosure provides methods for assessing the DAR of ADC products that offer advantages over known methods, in particular, the methods of the present disclosure can be used in high throughput applications and / or can be used without the need for dilution of the ADC sample while performing the assessment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0004] UV-Vis and Beer-Lambert Law Traditionally, DAR has been measured using UV-Vis spectroscopy (see, e.g., Chen, Methods Mol. Biol. 1045:267-73 (2013)). The basis of this analysis is Lambert's law, which relates the direct proportionality between the absorbance and concentration of a substance: A = εcl, where A is the absorbance, ε is the extinction coefficient (a physical constant of a substance), l is the optical path length through a cell containing the analyte, and c is the concentration.

[0005] The DAR measurement of ADC products using UV-Vis spectroscopy utilizes the difference between the absorption maximum of the antibody (e.g., 280 nm) and the absorption maximum of the drug (e.g., 252 nm). For example, the average DAR can be calculated using the difference in absorbance measured at 280 nm and 252 nm for the conjugate. Although UV-Vis methods are widely used in the art, they lack the throughput required for formulation screening studies. Also, they cannot be used without sample dilution, which introduces errors due to sample dilution.

[0006] Thus, the present disclosure is based, at least in part, on alternative methods of measuring DAR using size exclusion chromatography (e.g., UPLC) and gradient spectroscopy. These methods were characterized and compared to UV-Vis spectroscopy in terms of reproducibility, precision, and sensitivity. The data generated supports the use of UPLC-based DAR methods, which overcome the throughput limitations of traditional UV-Vis methods. Furthermore, the gradient spectroscopy-based methods can be used to analyze ADC samples without sample dilution.

[0007] UPLC-based methods In some embodiments, size exclusion is used to determine the DAR. In some embodiments, the methods disclosed herein include applying a sample containing an antibody-drug conjugate to a size-exclusion chromatographic matrix. In some embodiments, the methods disclosed herein include applying a sample containing an antibody-drug conjugate to a size-exclusion chromatographic matrix and allowing it to migrate. In some embodiments, the total amount of the ADC sample is applied to the size-exclusion matrix for analysis. For example, the following UPLC-based methodology was used to evaluate the DAR. [Table 1]

[0008] Data collected at 280 nm were integrated using Empower's Apex Track integration method with peak shoulder detection. Retention time integration ranges depended on the molecule but were typically within 3-9 minutes. The peak with the greatest height and area was classified as the "native", "main", or "monomer" peak. Any peak eluting earlier than the "native" peak was classified as a "HMW" peak. Any peak eluting later than the "native peak" was classified as a "LMW" peak.

[0009] The relative percentages of each species were calculated from the ratio of the area of ​​each individual peak to the total area of ​​all peaks. The following relative percentage areas were reported as an indication of purity: % total HMW, % native (or main or monomeric), and % total LMW. The total areas of all peaks were summed and used in the subsequent DAR calculations. However, in some embodiments, only the areas of the native peaks are used.

[0010] Data collected at 252 nm were integrated using Empower's Apex Track integration method with peak shoulder detection. The retention time integration range is molecule dependent but is typically within 3-9 minutes. The total areas of all peaks were summed and used in the subsequent DAR calculations. However, in some embodiments, only the areas of the native peaks are used.

[0011] DAR is the total peak area at 280 nm (A max ), and the total peak area at 252 nm (A max ) was determined from the typical A of drug conjugates used in ADCs. max However, the appropriate wavelengths can be selected for a particular conjugate, for example, using known methods. The amount of drug bound to the antibody can be determined by the difference in the total peak areas at these two wavelengths, using naked antibody as a reference standard, if applicable.

[0012] The following two equations (derived from the Beer-Lambert law) were examined to demonstrate consistency:

number

[0013] Equation 1 does not require the use of a naked antibody reference standard. However, it does require a systematic determination of the extinction coefficient (ε) for both the antibody and drug at 252 nm. The extinction coefficient at a given wavelength can be easily calculated from the Beer-Lambert law by measuring the absorbance at a given wavelength using solutions of either the antibody or drug of known concentration.

number

[0014] Equation 2 does not require the extinction coefficient of the antibody at 252 nm to be determined, but does require collection of UPLC data for a bare antibody reference standard.

[0015] Although UPLC is exemplified, other size exclusion chromatography techniques can be used in the methods described herein. Size exclusion chromatography generally refers to the separation of molecules by size, and the chromatographic elution time is characteristic for a particular molecule. Additional methods include, for example, SEC-HPLC, reverse phase (RP) HPLC, and RP-UPLC.

[0016] In some embodiments, the ADC sample is not diluted prior to analysis by size exclusion chromatography (e.g., HPLC or UPLC). In some embodiments, the total amount of the ADC sample is applied to a size exclusion chromatographic matrix, so no dilution is necessary prior to analysis of the ADC sample by size exclusion chromatography. In some embodiments, samples containing about 1 μg / μL to about 500 μg / μL of ADC are analyzed.

[0017] Gradient spectroscopy-based methods In some embodiments, the concentrations of the antibody and drug in the ADC sample are calculated to determine the DAR. For example, gradient spectroscopy is a known method for determining the absorbance of a solution at various path lengths. The absorbance values ​​at various path lengths can then be used to calculate the concentration of the compound in the solution based on the Beer-Lambert law. Methods and systems using gradient spectroscopy are known (see, e.g., U.S. Publication No. 20120130649) and are commercially available (see, e.g., SoloVPE (C Technologies, Inc., Bridgewater, NJ)). Such methods and systems have been adapted to measure the concentrations of the antibody and drug in the ADC formulation, from which the DAR was determined.

[0018] For example, an ADC sample can be placed in a container; a probe can be moved relative to the container to bring the probe into contact with the bottom of the container; the probe can be moved relative to the container according to a predetermined increment that fills a preselected optical path length through the solution, moving the probe from the bottom of the container through the sample; the absorbance can be read at the absorbance maximum of the antibody; the probe can be moved repeatedly relative to the sample to perform measurements; a regression line can be generated from the absorbance and optical path length to obtain the slope of the regression line; and the slope of the regression line can be divided by the extinction coefficient of the antibody to determine the concentration of the antibody. These steps can then be repeated to determine the concentration of the drug using the absorption maximum of the drug. The DAR can be calculated from the determined drug concentration and antibody concentration.

[0019] In some embodiments, the ADC sample is not diluted prior to analysis by gradient spectroscopy. In some embodiments, a sample containing about 0.1 μg / μL to about 500 μg / μL of ADC is analyzed.

[0020] Antibody-drug conjugates The term "antibody-drug conjugate" as used herein refers to a protein that is produced by binding an antibody to a biologically active cytotoxic payload or drug. Antibody-drug conjugates (ADCs) are generally produced by chemical modification / coupling reactions known to those skilled in the art. Any antibody-drug conjugate can be analyzed using the methods described herein.

[0021] In some embodiments, the antibody-drug conjugate is an anti-tumor antibody (see, e.g., Adler et al., Hematol. Oncol. Clin. North Am. 26:447-81 (2012); Li et al., Drug Discov. Ther. 7:178-84 (2013); Scott et al., Cancer Immun. 12:14 (2012); and Sliwkowski et al., Science 341:1192-1198 (2013)). Table 1 is a non-exhaustive list of specific human polypeptide antigens targeted by known available antibody agents and indicates the specific cancer indications for which the antibody agents are proposed to be useful. Any of the antibodies in Table 1 can be used in antibody-drug conjugates to be evaluated using the methods of the present disclosure. [Table 2-1] [Table 2-2] [Table 2-3]

[0022] In some embodiments, the antibody-drug conjugate comprises one or more drugs that are proapoptotic, cytostatic, and / or cytotoxic, e.g., agents that are specifically available for and / or recommended for use in the treatment of one or more diseases, disorders, or conditions associated with unwanted cell proliferation. In many embodiments, the drug is a chemotherapeutic agent useful in the treatment of cancer. In some embodiments, the chemotherapeutic agent may be or include one or more alkylating agents, one or more anthracyclines, one or more cytoskeletal disrupting agents (e.g., microtubule targeting agents such as taxanes, maytansine, and their analogs), one or more epothilones, one or more histone deacetylase inhibitors (HDACs), one or more topoisomerase inhibitors (e.g., inhibitors of topoisomerase I and / or topoisomerase II), one or more kinase inhibitors, one or more nucleotide analogs or nucleotide precursor analogs, one or more peptide antibiotics, one or more platinum-based agents, one or more retinoids, one or more vinca alkaloids, and / or one or more analogs of one or more of the following (i.e., those that share related antiproliferative activity): In certain embodiments, the chemotherapeutic agent is actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea , idarubicin, imatinib, irinotecan, maytansine and / or its analogs (e.g., DMI), mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoids, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, valrubicin, vinblastine, vincristine, vindesine, vinorelbine, and combinations thereof.

[0023] In some embodiments, the antibody-drug conjugates evaluated using the methods of the present disclosure are hLL1-doxorubicin, hRS7-SN-38, hMN-14-SN-38, hLL2-SN-38, hA20-SN-38, hPAM4-SN-38, hLL1-SN-38, hRS7-Pro-2-P-Dox, hMN-14-Pro-2-P-Dox, hLL2-Pro-2-P-Dox, hA20-Pro-2-P-Dox, hPAM4-Pro-2-P-Dox, hLL1-Pro-2-P-Dox, P4 / D 10-doxorubicin, gemtuzumab ozogamicin, brentuximab vedotin, trastuzumab emtansine, inotuzumab ozogamicin, glemtamomab vedotin, SAR3419, SAR566658, BIIB015, BT062, CMC-544, SAR3419, CDX-011, SGN-75, SGN-CD19A, AMG-172, AMG-595, BAY-94-9343, ASG-5ME, ASG-22ME, ASG-16M8F, MDX-1203, MLN-0264, anti-PSMA ADC, RG-7450, RG-7458, RG-7593, RG-7596, RG-7598, RG-7599, RG-7600, RG-7636, ABT-414, IMGN-853, IMGN-529, IMGN-901, borsetuzumab mafodotin, or lorvotuzumab mertansine (see, e.g., Sassoon et al., Methods Mol. Biol. 1045:1-27 (2013); Bouchard et al., Bioorganic Med. Chem. Lett. 24:5357-5363 (2014)).

[0024] Purpose The disclosed methods have a variety of applications, including quality control at different stages in the manufacture of a drug substance or drug product, analysis of ADC preparations before and / or after completion of the manufacture of a drug substance or drug product (e.g., before or after distribution to a fill / finish environment or facility), before or after release of the drug substance or drug product into commerce (e.g., before distribution to a pharmacy, caregiver, patient, or other consumer). In some cases, the ADC formulation is a drug substance (active pharmaceutical ingredient or "API") or a drug product (an API formulated for use in a subject, such as a human patient). In some cases, the ADC formulation is from a stage of manufacture or use prior to distribution to a caregiver or other consumer; prior to packaging into individual dosage forms, such as syringes, pens, vials, or multi-dose vials; prior to the generation of a Certificate of Test, Material Safety Data Sheet (MSDS), or Certificate of Analysis (CofA), prior to determining that a batch is suitable for commercial distribution.

[0025] The evaluation of the methods described herein is useful in guiding, controlling, or performing numerous activities or steps in the process of manufacturing, distributing, and monitoring, as well as providing for the safe and effective use of, ADC formulations. Thus, in some embodiments, decisions are made or steps are performed in response to the evaluation, for example, depending on whether a criterion (e.g., a particular DAR, mean DAR, and / or DAR range) is met. The methods described herein can include: (a) determining whether the ADC formulation can be processed into a drug substance or drug product; (b) determining whether the ADC formulation can be reprocessed (e.g., the preparation can be subjected to a previous process step again); and / or (c) determining that the ADC formulation is not suitable for processing into a drug substance or drug product. In some embodiments, the methods include: a processing operation as indicated in step (a), reprocessing as indicated in step (b), or rendering the formulation unsuitable for commercial distribution, e.g., by labeling or disabling, as indicated in step (c).

Claims

1. 1. A method for measuring a drug / antibody ratio (DAR) in a sample containing an antibody-drug conjugate (ADC), comprising the steps of: placing said sample in a container; contacting a probe with the bottom of said container; The probe is moved from the bottom of the container through the sample in predetermined increments, resulting in a preselected path length through the solution. detecting absorbance of the sample at the first wavelength of light (λ1), wherein the first wavelength is a predetermined absorbance maximum of an antibody; repeating the steps of moving the probe relative to the sample and taking measurements at a first wavelength; generating a regression line from the absorbance at the first wavelength and the path length, and obtaining a slope of the regression line; determining the concentration of the antibody by dividing the slope of the regression line by the extinction coefficient of the antibody at the first wavelength; detecting the absorbance of the sample at a second wavelength (λ), wherein the second wavelength is a predetermined absorbance maximum of the drug; repeating the steps of moving the probe relative to the sample and taking measurements at a second wavelength; generating a regression line from the absorbance at the second wavelength and the path length, and obtaining a slope of the regression line; determining the concentration of the drug by dividing the slope of the regression line by the extinction coefficient of the drug at the second wavelength; and Calculate the DAR using the determined drug concentration and the determined antibody concentration The method comprising:

2. 10. The method of claim 1, wherein the sample comprises 0.1 μg / μL to 500 μg / μL of ADC.

3. The method of claim 1 or 2, wherein the antibody is an anti-tumor antibody.

4. The method of any one of claims 1 to 3, wherein the drug is a pro-apoptotic, cytostatic or cytotoxic agent.

5. The method of any one of claims 1 to 3, wherein the drug comprises a chemotherapeutic agent.

6. 6. The method of claim 5, wherein the chemotherapeutic agent comprises a microtubule targeting agent, an epothilone, a histone deacetylase inhibitor, a topoisomerase inhibitor, a kinase inhibitor, a nucleotide analog, a nucleotide precursor analog, a peptide antibiotic, a platinum-based agent, a retinoid, or a vinca alkaloid.

7. 6. The method of claim 5, wherein the chemotherapeutic agent comprises actinomycin, all-trans retinoic acid, auristatin, azacitidine, azathioprine, bleomycin, bortezomib, carboplatin, capecitabine, cisplatin, chlorambucil, cyclophosphamide, curcumin, cytarabine, daunorubicin, docetaxel, doxifluridine, doxorubicin, epirubicin, epothilone, etoposide, fluorouracil, gemcitabine, hydroxyurea, idarubicin, imatinib, irinotecan, maytansine, mechlorethamine, mercaptopurine, methotrexate, mitoxantrone, maytansinoid, oxaliplatin, paclitaxel, pemetrexed, teniposide, thioguanine, topotecan, barbican, vinblastine, vincristine, vindesine, or vinorelbine.

8. The antibody is selected from the group consisting of adecatumumab, adotrastuzumab emtansine, alemtuzumab, anatumomab, apolizumab, bavituximab, bevacizumab, bectumomab, blinatumomab, brentuximab vedotin, cantuzumab, catumaxomab, cetuximab, daratumumab, denosumab, edrocolomab, epitumomab, etaracizumab, farletuzumab, gemtuzumab osagamicin, and glembatumumab.

8. The method of any one of claims 1 to 7, comprising ribozyme, ipilimumab, ibiritumomab, labetuzumab, lorvotuzumab, lucatumumab, mapatumumab, milatuzumab, minletumomab, nimotuzumab, opinutuzumab, ofatumumab, oregovomab, panitumumab, pemtumomab, pertuzumab, rituximab, sibrotuzumab, tositumomab, trastuzumab, veltuzumab, or volociximab.

9. Based on the DAR, the following: (i) formulating the composition into a drug substance or drug product; (ii) reprocessing the composition; or (iii) rendering the composition unusable for commercial release. The method according to any one of claims 1 to 8, comprising:

10. The method of claim 9, wherein the step of rendering the composition unusable for commercial release includes marking the composition or destroying the composition.