Methods for evaluating antibody-drug conjugates

Abstract

The present disclosure provides methods for assessing the DAR of ADC products that offer advantages over known methods: specifically, the disclosed methods can be used in high-throughput applications and / or can be used without requiring dilution of the ADC sample during the assessment. [Selection diagram] 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 herein by reference. [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-to-antibody ratio ("DAR"). The DAR is an important quality attribute of ADC products because it can affect the efficacy, safety, and / or stability of the product. Therefore, a reliable and high-throughput method for assessing the DAR of ADC products 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: specifically, the disclosed methods can be used in high-throughput applications and / or can be used without requiring dilution of the ADC sample during the assessment. DETAILED DESCRIPTION OF THE INVENTION

[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 describes the direct proportionality between the absorbance and concentration of a substance: A = εcl, where A is 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 an ADC product 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 for the conjugate at 280 nm and 252 nm. While UV-Vis methods are widely used in the art, they lack the throughput required for formulation screening studies. Furthermore, 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 for measuring DAR using size exclusion chromatography (e.g., UPLC) and gradient spectroscopy. These methods were characterized and compared to UV-Vis spectroscopy with respect to 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, the DAR is determined using size exclusion. In some embodiments, the methods disclosed herein comprise applying a sample containing an antibody-drug conjugate to a size-exclusion chromatographic matrix. In some embodiments, the methods disclosed herein comprise 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 DAR was assessed using the following UPLC-based methodology: [Table 1]

[0008] Data collected at 280 nm were integrated using Empower's Apex Track integration method with peak shoulder detection. The retention time integration range was molecule-dependent but 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 an "HMW" peak. Any peak eluting later than the "native peak" was classified as an "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 percent areas were reported as an indication of purity: % total HMW, % native (or main or monomer), and % total LMW. The total areas of all peaks were summed and used in 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 was molecule-dependent but typically within 3-9 minutes. The total area of ​​all peaks was summed and used in subsequent DAR calculations. However, in some embodiments, only the area of ​​the native peaks is used.

[0011] DAR is the total peak area at 280 nm (A of ADC) max ), and the total peak area at 252 nm (A of the drug max ) was determined from the general A of the drug conjugates used in ADCs. max Although, for example, known methods can be used to select the appropriate wavelength for a particular conjugate, the amount of drug bound to the antibody can be determined by the difference in the total peak area 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 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 antibody or drug at known concentrations.

number

[0014] Equation 2 does not require the extinction coefficient of the antibody at 252 nm to be determined, but does require the collection of UPLC data for a naked 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, where the chromatographic elution time is characteristic for a particular molecule. Additional methods include, for example, SEC-HPLC, reversed-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 entire ADC sample is applied to a size exclusion chromatography 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 DAR is determined by calculating the concentrations of antibody and drug in an ADC sample. 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 compounds 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 were adapted to measure the concentrations of antibody and drug in an 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 contact 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 antibody's absorbance maximum; measurements can be taken by repeatedly moving the probe relative to the sample; 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 antibody concentration. These steps can then be repeated using the drug's absorption maximum to determine the drug concentration. The DAR can be calculated from the determined drug concentration and antibody concentration.

[0019] In some embodiments, the ADC sample is not diluted before 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 As used herein, the term "antibody-drug conjugate" refers to a protein created by conjugating an antibody to a biologically active cytotoxic payload or drug. Antibody-drug conjugates (ADCs) are generally created 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 provides a non-exhaustive list of specific human polypeptide antigens targeted by known, available antibody agents, and identifies 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 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 pro-apoptotic, cytostatic, and / or cytotoxic, e.g., agents 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 disruptors (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 disclosure include 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, glemutamomab 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 drug substance or drug product manufacturing (e.g., before or after distribution to a fill / finish environment or facility), and before or after release of the drug substance or drug product for 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 (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; before packaging into individual dosage forms, such as syringes, pens, vials, or multi-dose vials; or before determining a batch is ready for commercial distribution prior to the generation of a Certificate of Test, Material Safety Data Sheet (MSDS), or Certificate of Analysis (CofA).

[0025] Evaluation by the methods described herein is useful in guiding, controlling, or implementing numerous activities or steps in the process of manufacturing, distributing, and monitoring ADC formulations, as well as providing for their safe and effective use. Thus, in some embodiments, a decision is made or a step is performed in response to the evaluation, e.g., 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., subjecting the preparation to a previous process step); 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 by, e.g., labeling or disabling, as indicated in step (c).

Claims

1. 1. A method for determining a drug-to-antibody ratio (DAR) in a sample containing an antibody-drug conjugate using size exclusion chromatography, comprising: applying the sample to a size exclusion chromatography matrix; detecting an absorbance response of the sample at a first wavelength of light (λ1), the first wavelength of light being a predetermined absorbance maximum of the antibody; detecting an absorbance response of the sample at a second wavelength of light (λ2), the second wavelength of light being at a predetermined absorbance maximum of the drug; determining a total absorbance of the sample at the first wavelength of light and determining a total absorbance of the sample at the second wavelength of light, wherein each total absorbance is calculated by integrating the peaks of the absorbance response over an elution time interval by summing the areas of a high molecular weight (HMW) peak, a low molecular weight (LMW) peak, and a main peak, wherein the main peak is the peak with the greatest height and area, the HMW peaks are any peaks that elute earlier than the main peak, and the LMW peaks are any peaks that elute later than the main peak; and, Calculating the DAR using the following equation (1): [Equation 1] During the ceremony, [Equation 2] is the extinction coefficient of the antibody at the first wavelength of light; [Equation 3] is the extinction coefficient of the antibody at the second wavelength of light; [Equation 4] is the extinction coefficient of the drug at the first wavelength of light; [Equation 5] is the extinction coefficient of the drug at the second wavelength of light; Total area λ1 is the total absorbance of the sample at the first wavelength of light; Total area λ2 is the total absorbance of the sample at the second wavelength of light; wherein the predetermined absorbance maximum of the antibody is the wavelength at which the antibody absorbance is greatest, and the predetermined absorbance maximum of the drug is the wavelength at which the drug absorbance is greatest.

2. 10. The method of claim 1, further comprising calculating an extinction coefficient of the antibody at a first wavelength of light or a second wavelength of light based on the absorbance of a solution of the antibody of known concentration at the first wavelength of light or the second wavelength of light, respectively.

3. 10. The method of claim 1, further comprising calculating an extinction coefficient of the drug at the first wavelength of light or the second wavelength of light based on the absorbance of a solution of the drug of known concentration at the first wavelength of light or the second wavelength of light, respectively.

4. 10. The method of claim 1, wherein the size exclusion chromatography comprises ultra performance liquid chromatography (UPLC), reverse phase (RP) UPLC, or high performance liquid chromatography (HPLC).

5. 10. The method of claim 1, wherein the sample comprises 1 μg / μL to 500 μg / μL of antibody-drug-conjugate (ADC).

6. The method of claim 1, wherein the antibody is an anti-tumor antibody.

7. 10. The method of claim 1, wherein the drug is a pro-apoptotic agent, a cytostatic agent, or a cytotoxic agent.

8. The method of claim 1 , wherein integrating the peaks of the absorbance response comprises detecting a peak shoulder of each peak of the absorbance response.

9. 2. The method of claim 1, wherein the time interval is 3 to 9 minutes.

10. 1. A method for determining a drug-to-antibody ratio (DAR) in a first sample containing an antibody-drug conjugate using size exclusion chromatography, comprising: measuring the total absorbance of a first sample containing the antibody-drug conjugate; applying the first sample containing the antibody-drug conjugate to a size exclusion chromatography matrix; detecting an absorbance response of the first sample at a first wavelength of light (λ1), the first wavelength of light being a predetermined absorbance maximum of the antibody; detecting an absorbance response of the first sample at a second wavelength of light (λ2), the second wavelength of light being at a predetermined absorbance maximum of the drug; measuring the total absorbance by determining a total absorbance of the first sample at the first wavelength of light and a total absorbance of the first sample at a second wavelength of light, wherein each total absorbance is calculated by integrating the peaks of the absorbance response over an elution time interval by summing the areas of a high molecular weight (HMW) peak, a low molecular weight (LMW) peak, and a main peak, wherein the main peak is the peak with the greatest height and area, the HMW peaks are any peaks that elute earlier than the main peak, and the LMW peaks are any peaks that elute later than the main peak; measuring the total absorbance of a second sample containing the antibody, applying the second sample containing the antibody to a size exclusion chromatography matrix; detecting the absorbance of a second sample containing the antibody at the first wavelength of light (λ1); detecting the absorbance of a second sample containing the antibody at the second wavelength of light (λ2); measuring the total absorbance by determining a total absorbance of a second sample comprising the antibody at the first wavelength of light and a total absorbance of a second sample comprising the antibody at a second wavelength of light, wherein each total absorbance is calculated by integrating the peaks of the absorbance response over an elution time interval by summing the areas of a high molecular weight (HMW) peak, a low molecular weight (LMW) peak, and a main peak; and Calculating the DAR using the following equation (2): [Equation 6] During the ceremony, [Equation 7] is the extinction coefficient of the antibody at the first wavelength of light; [Equation 8] is the extinction coefficient of the drug at the first wavelength of light; [Equation 9] is the extinction coefficient of the antibody at the second wavelength of light; [Equation 10] is the total absorbance of the second sample containing the antibody at the first wavelength of light; [0011] is the total absorbance of the second sample containing the antibody at the second wavelength of light; [0012] is the total absorbance of the first sample containing the antibody-drug conjugate at the first wavelength of light; [0013] is the total absorbance of the first sample containing the antibody-drug conjugate at the second wavelength of light; wherein the predetermined absorbance maximum of the antibody is the wavelength at which the antibody has a maximum absorbance, and the predetermined absorbance maximum of the drug is the wavelength at which the drug has a maximum absorbance.

11. 11. The method of claim 10, further comprising calculating an extinction coefficient of the antibody at the first wavelength of light or the second wavelength of light based on the absorbance of a solution of the antibody of known concentration at the first wavelength of light or the second wavelength of light, respectively.

12. 11. The method of claim 10, further comprising calculating an extinction coefficient of the drug at the first wavelength of light or the second wavelength of light based on the absorbance of a solution of the drug of known concentration at the first wavelength of light or the second wavelength of light, respectively.

13. 11. The method of claim 10, wherein the size exclusion chromatography comprises ultra performance liquid chromatography (UPLC), reverse phase (RP) UPLC, or high performance liquid chromatography (HPLC).

14. 11. The method of claim 10, wherein the first sample comprises 1 μg / μL to 500 μg / μL of antibody-drug-conjugate (ADC).

15. The method of claim 10, wherein the antibody is an anti-tumor antibody.

16. 11. The method of claim 10, wherein the drug is a pro-apoptotic agent, a cytostatic agent, or a cytotoxic agent.

17. The method of claim 10 , wherein integrating the peaks of the absorbance response comprises detecting a peak shoulder of each peak of the absorbance response.

18. 11. The method of claim 10, wherein the time interval is between 3 and 9 minutes.

Images