Multidimensional Chromatographic Method for the Analysis of Antibody-Drug Conjugates

Abstract

The present disclosure relates to a method for analyzing antibody-drug conjugate complexes. In particular, the present disclosure relates to a sensitive multidimensional chromatography method for the extraction, detection and quantification of non-conjugated cytotoxic agents and associated linker molecules used in the preparation of cysteine-based antibody-drug conjugates.

Description

[0001] Citation of Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 215,339, filed September 8, 2015, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure relates to sensitive multidimensional chromatographic methods for the extraction, detection, and quantification of unconjugated cytotoxic agents and associated linker molecules used in the preparation of cysteine-based antibody-drug conjugates. Background Art

[0004] Antibody-drug conjugate (ADC) complexes represent a growing class of immunoconjugate therapies. ADC complexes are complex molecules consisting of monoclonal antibodies connected to biologically active, highly cytotoxic drugs via cleavable linkers (e.g., acid-labile linkers, protease-cleavable linkers, and disulfide linkers) or non-cleavable linkers. The conjugation of potent drugs to monoclonal antibodies enables targeted delivery of toxic payloads to the tumor surface while minimizing systemic toxic effects on healthy tissues, thus improving the therapeutic window of such modalities in cancer therapy.

[0005] Incomplete conjugation processes can produce free or non-conjugated drugs, drug-linkers, or drug-related impurities. In addition, degradation products may appear in the formulation and in vivo over time, thereby increasing the risk to the patient and reducing the efficacy of the ADC complex. Despite the purification steps implemented during the preparation process, trace levels of these free drug substances may also be present in the formulated ADC complex. For these reasons, it is necessary to characterize the residual free drug and associated products to ensure a safe and effective product.

[0006] Current methods for detecting trace free drug substances are ultimately hampered by several shortcomings, including low specificity and / or sensitivity in the detection, characterization, and quantification of trace free drug substances in impurity-free ADC complex samples. Therefore, there remains a need in the art for new detection methods that can be directly integrated into existing or novel workflows. Summary of the invention

[0007] The present disclosure relates to novel and useful multidimensional chromatographic methods for detecting and / or quantifying one or more unconjugated drugs (e.g., cytotoxic and anticancer compounds) and associated linker molecules used in the preparation of cysteine-based antibody-drug conjugate complexes.

[0008] Thus, provided herein is a method for analyzing an antibody-drug conjugate complex in a sample comprising the antibody-drug conjugate complex and an unconjugated drug compound. The method comprises the steps of exposing the sample to a first dimension having a mixed mode stationary phase, and exposing the sample to a second dimension having a hydrophobic stationary phase. The method provides separation of the antibody-drug conjugate complex and the unconjugated drug compound in the sample.

[0009] In some embodiments, the method further comprises a step of capturing unconjugated drug compound. In one embodiment, the capturing step is performed before the step of exposing the sample to the first dimension. Alternatively, the capturing step is performed between the step of exposing the sample to the first dimension and the step of exposing the sample to the second dimension.

[0010] Also provided herein is a method for analyzing an antibody-drug conjugate complex in a sample comprising an antibody-drug conjugate complex, an unconjugated drug compound, and an associated linker molecule. In some embodiments, the unconjugated drug compound is a free drug. In other embodiments, the unconjugated drug compound is connected to a reactive form of an associated linker molecule. In other embodiments, the unconjugated drug compound is connected to a quenched or inactivated form of a linker molecule. First, the sample is exposed to a first dimension separation, and then the sample is exposed to a second dimension separation. The method involves a step of trapping a portion of an unconjugated drug compound and / or an associated linker molecule with a stationary phase before the first dimension separation or between the first and second dimension separations.

[0011] The methods provided herein detect each of the antibody-drug conjugate complex, unconjugated drug compound, and optionally associated linker molecules in the samples of these methods. In some embodiments, each of the antibody-drug conjugate complex, unconjugated drug compound, and optionally associated linker molecules is detected in the samples of the method using mass spectrometry. In specific embodiments, the methods provided herein use mass spectrometry to establish the mass-to-charge ratio of each of the antibody-drug conjugate complex, unconjugated drug compound, and optionally associated linker molecules in the samples of these methods. In other embodiments, each of the antibody-drug conjugate complex, unconjugated drug compound, and optionally associated linker molecules is detected in the samples of the method using ultraviolet and / or visible spectroscopy. In other embodiments, each of the antibody-drug conjugate complex, unconjugated drug compound, and optionally associated linker molecules is detected in the samples of the method using fluorescence spectroscopy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 An exemplary embodiment of the method of the present invention is shown.

[0013] Figure 2 An exemplary embodiment of the method of the present invention is shown.

[0014] Figure 3 An exemplary embodiment of the method of the present invention is shown.

[0015] Figure 4 A to 4C describe antibody-fluorophore-conjugate (AFC) mimicking components. The drug component used in the preparation of non-toxic AFCs that mimic the chemical structure and linker material of brentuximab is based on A) a dansylsulfonamide ethylamine (DSEA) moiety attached to B) a maleimidocaproyl valine-citrulline linker material (Mal-linker-DSEA). The residual reactive mal-linker-DSEA is quenched with N-acetyl-cysteine ​​after the conjugation step, resulting in C) a quenched linker-fluorophore (NAc-linker-DSEA) adduct material.

[0016] Figure 5 Reference standard evaluation using a mixture of three reference standards that were baseline separated within an applied gradient (500) is shown. DSEA (510), NAc-linker-DSEA (520), and Mal-linker-DSEA (530) reference standards were separated using a superficially porous C18 RPLC column with acetonitrile (containing 0.1% FA v / v) as the organic mobile phase in a gradient from 5% to 50% in 10 min (dashed line). 540 represents the conditioning peak. The [M+H] peak of each component was measured using the optimized MS settings (see Materials and Methods found in the Examples). +1 and [M+2H] +2 The charge states are derived from the combined spectra of the collected SIRs.

[0017] Figure 6 The dynamic range of the assay is shown. Analysis of the reference standard was performed in triplicate. The most abundant [M+2H] +2 The peak areas from the SIRs by charge state generated calibration curves for the reference standards and were fitted with a general linear regression model. Using ICH guidelines, the MS quadrupole dynamic range for the mal-linker-DSEA reference standard was determined to be 1.35 pg to 688.5 pg, and the MS quadrupole dynamic range for the NAc-linker-DSEA reference standard was 1.65 pg to 854.5 pg.

[0018] Figure 7A to 7C depict schematic diagrams of the instrument configuration. A column manager housing two six-way two-position valves is constructed as shown in the schematic diagram to facilitate transfer of retained drug substances between the SPE (first) dimension (730) and the RPLC (second) dimension (760). The valve positions are numerically indicated as positions 700 and 705. Other components of the instrument include: QSM: quaternary solvent manager (710), AS: automatic sampler (720), ACD: on-column dilution (740); TUV: tunable ultraviolet detector (770), BSM: binary solvent manager (750), MS: mass spectrometer (780).

[0019] Figure 8 Method evaluation of SPE with spiked samples is described. DSEA, mal-linker-DSEA, and NAc-linker-DSEA were spiked into neat AFC samples for SPE optimization. The optimal SPE loading conditions for extraction of mal-linker-DSEA and NAc-linker-DSEA components from the spiked AFC samples were determined to be 18% acetonitrile, 2% FA. A step gradient to 36% acetonitrile, 2% FA was determined to be the optimal condition for eluting the bound drug component in a narrow peak centered at 13.5 min.

[0020] Fig. 9 A to 9B describe the evaluation of 2DLC configurations. Figure 7 2DLC configurations shown in A to 7B serve as proof of principle to successfully transfer NAc-linker-DSEA doped into AFC samples from A) SPE (1st dimension) to B) RPLC (2nd dimension).

[0021] Fig.10 A to 10B describe DSEA samples. A) Using Figure 7 Optimized 2DLC method shown, water blanks were performed before each DSEA injection to monitor carryover between DSEA injections. Overlay chromatograms of three water blanks indicate negligible mal-linker-DSEA carryover between runs and no observable NAc-linker-DSEA carryover. B) Mal-linker-DSEA and NAc-linker-DSEA drug components were detected in 10 μL injections (19.4 μg) of pure AFC sample. Overlays of these three runs indicate a high degree of assay reproducibility. DETAILED DESCRIPTION

[0022] Reference will now be made in detail to certain embodiments of the present invention, examples of which are shown in the accompanying drawings. Although the present invention will be described in conjunction with the enumerated embodiments, it should be understood that they are not intended to limit the present invention to these embodiments. On the contrary, the present invention is intended to encompass all alternatives, modifications and equivalents that may be included within the scope of the invention as defined by the appended claims. Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein may also be used to implement the present invention. The present invention is by no means limited to the methods and materials described.

[0023] definition

[0024] Unless otherwise indicated, the terms and phrases used herein are intended to have the following meanings:

[0025] As used herein, an "antibody drug conjugate" or "ADC" is a monoclonal antibody (mAb) attached to a biologically active drug by a chemical linker with a labile bond.

[0026] "Antibody" is used in the broadest sense herein and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments. Antibodies may be murine, human, humanized, chimeric, or derived from other species. Antibodies are proteins produced by the immune system that are capable of recognizing and binding to specific antigens. (Janeway et al. (2001) "Immunobiology", 5th edition, Garland Publishing, New York). Target antigens generally have many binding sites, also referred to as epitopes, that are recognized by the CDRs on multiple antibodies. Each antibody that specifically binds to a different epitope has a different structure. Thus, an antigen may have more than one corresponding antibody.

[0027] Antibodies also refer to full-length immunoglobulin molecules or immunologically active portions of full-length immunoglobulin molecules, i.e., molecules comprising an antigen binding site that immunospecifically binds to an antigen of a target of interest or a portion thereof, such targets including but not limited to one or more cancer cells that produce autoimmune antibodies associated with autoimmune diseases. The immunoglobulins disclosed herein may be any type (e.g., IgG, IgE, IgM, IgD, and IgA), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecules. Immunoglobulins may be derived from any species. However, in one aspect, immunoglobulins are human, mouse, or rabbit.

[0028] The term "linker unit" refers to the direct or indirect bond of an antibody to a drug. Attachment of the linker to the mAb can be achieved in a variety of ways, such as through surface lysines, reductive coupling with oxidized carbohydrates, and cysteine ​​residues released by reduction of interchain disulfide bonds. A variety of ADC bond systems are known in the art, including hydrazone-based, disulfide-based, and peptide-based bonds.

[0029] The term "active pharmaceutical ingredient" or "API" refers to the biologically active ingredient in a pharmaceutical drug. The terms "pharmaceutical drug," "drug," and "payload" are used interchangeably throughout and refer to any substance that is biologically active or detectably active, such as a therapeutic agent, a detectable marker, a binding agent, etc., as well as a prodrug that is metabolized to an active agent in vivo.

[0030] As used herein, the term "mass spectrometry" or "MS" refers to an analytical technique that identifies compounds by their mass. MS refers to a method of filtering, detecting, and measuring ions based on their mass-to-charge ratio, or "m / z." MS techniques generally include (1) ionizing compounds to form charged compounds; and (2) detecting the molecular weight of the charged compounds and calculating the mass-to-charge ratio. The compounds can be ionized and detected by any suitable device. A "mass spectrometer" generally includes an ion generator and an ion detector. Generally speaking, one or more molecules of interest are ionized, and the ions are then introduced into a mass spectrometer where, due to a combination of magnetic and electric fields, the ions follow a specific path in space that depends on mass ("m") and charge ("z").

[0031] As used herein, the term "ionization" or "ionizing" refers to the process of producing analyte ions having a net charge equal to one or more electron units. Negative ions are those having a net negative charge of one or more electron units, while positive ions are those having a net positive charge of one or more electron units.

[0032] As used herein, the term "chromatography" refers to a process in which a chemical mixture carried by a liquid or gas is separated into components due to the differential distribution of the chemical entities as they flow around or over a stationary liquid or solid phase.

[0033] As used herein, the term "liquid chromatography" or "LC" means the process of selective retardation of one or more components of a liquid solution as the fluid uniformly permeates through a column of particulate matter or through a capillary passage. The retardation is caused by the distribution of the components of the mixture between one or more stationary phases and the bulk fluid (i.e., mobile phase) as it flows relative to one or more stationary phases. Examples of "liquid chromatography" include reverse phase liquid chromatography (RPLC), high performance liquid chromatography (HPLC), ultra high performance liquid chromatography (UHPLC), turbulent flow liquid chromatography (TFLC) (sometimes referred to as high turbulence liquid chromatography (HTLC) or high throughput liquid chromatography).

[0034] As used herein, the term "high performance liquid chromatography" or "HPLC" (sometimes also referred to as "high pressure liquid chromatography") refers to liquid chromatography that enhances separation by forcing a mobile phase under pressure through a stationary phase, typically a densely packed column. As used herein, the term "ultra-high performance liquid chromatography" or "UHPLC" (sometimes referred to as "ultra-high pressure liquid chromatography") refers to HPLC that occurs at much higher pressures than conventional HPLC techniques.

[0035] The term "LC / MS" refers to a liquid chromatograph (LC) coupled to a mass spectrometer.

[0036] Method of the present invention

[0037] The present disclosure relates to novel and useful multidimensional chromatographic methods for detecting and / or quantifying one or more unconjugated drugs (e.g., cytotoxic and anticancer compounds) and associated linker molecules used in the preparation of cysteine-based antibody-drug conjugate complexes.

[0038] Therefore, if Figure 1 and Figure 2 As shown, methods (100, 200) for analyzing antibody-drug conjugate complexes in a sample comprising the antibody-drug conjugate complexes and unconjugated drug compounds are provided herein. These methods include the steps of exposing the sample to a first dimension (120, 220) having a mixed mode stationary phase, and exposing the sample to a second dimension (130, 230) having a hydrophobic stationary phase. These methods provide separation of the antibody-drug conjugate complexes and unconjugated drug compounds in the sample (140, 240).

[0039] In some embodiments, some of these methods further include a step of capturing unconjugated drug compounds. In one embodiment, the capturing step is performed before the step of exposing the sample to the first dimension. Alternatively, the capturing step is performed between the step of exposing the sample to the first dimension and the step of exposing the sample to the second dimension.

[0040] like Figure 3 As shown, the present invention also provides a method (300) for analyzing an antibody-drug conjugate complex in a sample including an antibody-drug conjugate complex, an unconjugated drug compound, and an associated linker molecule. In some embodiments, the unconjugated drug compound is a free drug. In other embodiments, the unconjugated drug compound is connected to a reactive form of an associated linker molecule. In other embodiments, the unconjugated drug compound is connected to a quenched or inactivated form of a linker molecule. The sample is first exposed to a first dimension separation (330), and then the sample is exposed to a second dimension separation (350). The method involves a step of capturing a portion of the unconjugated drug compound and / or the associated linker molecule with a stationary phase before the first dimension separation (320) or between the first dimension separation and the second dimension separation (340).

[0041] like Figure 1 As shown, the methods provided herein detect (150) each of the antibody-drug conjugate complex, the unconjugated drug compound, and the optionally associated linker molecule in the sample of these methods. In some embodiments, mass spectrometry is used to detect each of the antibody-drug conjugate complex, the unconjugated drug compound, and the optionally associated linker molecule in the sample of the method. Figure 2 As shown, in certain embodiments, the methods provided herein use mass spectrometry to establish the mass-to-charge ratio of each of the antibody-drug conjugate complex, the unconjugated drug compound, and the optionally associated linker molecule in the sample of these methods (250). In other embodiments, ultraviolet and / or visible spectroscopy is used to detect each of the antibody-drug conjugate complex, the unconjugated drug compound, and the optionally associated linker molecule in the sample of the method. In other embodiments, fluorescence spectroscopy is used to detect each of the antibody-drug conjugate complex, the unconjugated drug compound, and the optionally associated linker molecule in the sample of the method.

[0042] In some embodiments, antibody-fluorophore-conjugate (AFC) mimetics ( Figure 4 Such non-toxic AFCs can be used to mimic the chemical structure and linker materials of, for example, brentuximab, and thus the "drug" component can be based on the dansylsulfonamide ethylamine (DSEA) moiety ( Figure 4 A), which is attached to a maleimidocaproylvaline-citrulline linker substance (Mal-Linker-DSEA) ( Figure 4 B). Residual reactive mal-linker-DSEA can be quenched with N-acetyl-cysteine ​​after the conjugation step, resulting in a quenched linker-fluorophore (NAc-linker-DSEA) adduct species ( Figure 4 C).

[0043] The methods provided herein are particularly useful for quantifying low levels (eg, ng / mL) of unconjugated drug compounds in the sample.

[0044] The inventive method provides several advantages over the prior art. For example, the inventive method avoids the need for precipitated proteins and does not require sample preparation (e.g., does not require pre-concentration, buffer exchange or dilution). In addition, there are no residual proteins or biological matrix components that may affect detection and quantitative results. The disclosed method also extends the linear dynamic range, thereby expanding the executable scope of operational specifications.

[0045] Dimension 1

[0046] The first dimension column should be able to separate or isolate the protein or antibody from the free drug component with sufficient resolution between the two species to allow the drug to be sent to a trap or second dimension column while directing the protein component to waste. This is performed using the physicochemical properties of the ADC. An example of mixed mode is the net charge difference between the antibody and the free drug molecule. Size exclusion chromatography or SEC takes advantage of the size difference between a large antibody and a small drug substance.

[0047] In one embodiment, the first dimension is a mixed mode anion exchanger. Thus, net positive proteins pass through the column under acidic conditions, while net neutral or basic drugs are adsorbed to the column stationary phase for subsequent elution.

[0048] In another embodiment, the first dimension is a mixed mode cation exchanger. Thus, positively charged proteins are retained while allowing the drug substance to pass through the injection gap to be captured by the trap or second dimension column.

[0049] In another embodiment, the first dimension is size exclusion chromatography. Thus, large antibodies flow to the waste in an isocratic manner and elute earlier, while small drug molecules elute later in or after the total inclusion peak due to secondary interactions with the stationary phase.

[0050] In one embodiment, the first dimension column is an online 2.1 mm x 20 mm, 30 micron solid phase extraction column ( MAX, purchased from Waters Technologies Corporation, Milford, MA). Thus, both trapping and first dimension separation occur within the column.

[0051] The second pillar

[0052] The second-dimension column should be designed to minimize the stress on the first dimension if the first-dimension column cannot withstand high pressures. For example, using A 50 mm, 2.0-mm column (available from Waters Scientific, Milford, MA) is used as an embodiment of the first dimension separation; the pressure of the first dimension column should not exceed 6,000 PSI. Factors that affect pressure are column length (shorter = lower pressure), particle size (larger particles = lower pressure), flow rate (lower flow = lower pressure), column inner diameter (larger inner diameter = lower pressure), and temperature (higher temperature = lower pressure). However, the balance of these parameters (considered to be short, large particle, wide bore columns) can lead to reduced chromatographic performance. Therefore, in one embodiment, the disclosed method uses a superficially porous small particle 50 mm Cortecs column. In some embodiments, the second dimension column is a 2.1 mm x 50 mm, 2.7 micron superficially porous C18 column ( C18, purchased from Waters Scientific, Milford, MA).

[0053] However, if not limited by the first dimension column, the second dimension column can have any size and stationary phase that exhibits sufficient retention to retain the drug substance after on-column dilution and result in acceptable separation of the drug components. Nevertheless, columns with too much retention can result in trace protein carryover, long elution times, and / or poor chromatography of hydrophobic drug substances.

[0054] Organic modifier and acid %

[0055] Hydrophobic active pharmaceutical ingredients (APIs) require optimization of the elution strength of organic solvents. Under low acid concentration conditions, more hydrophobic analytes may not elute completely, or may be too broad to elute in the defined window. Higher acid concentration conditions allow the drug substance to elute in an acceptable window.

[0056] In some embodiments, pure organic solvents or mixed compositions with increased elution strength are used to elute more hydrophobic drug substances from dimension 1. For drug components that are too hydrophilic or elute at relatively mild organic compositions, the initial organic composition at the time of injection can be reduced to retain these species, and a two-step gradient can be used to capture the less hydrophobic species with the more hydrophobic species.

[0057] In other embodiments, the acid % is adjusted at the time of injection so that a lower acid % is used at the time of injection to retain the more hydrophilic species and then raised to a 2% acid composition to elute all drug components. This can be accomplished using one or more additional solvent lines (e.g., pumps) on a quaternary solvent manager.

[0058] Exemplary solvents that can be used in the disclosed methods include, but are not limited to, water, acetonitrile, ammonium acetate, ammonium formate, methanol, ethanol, isopropanol, propanol, tetrahydrofuran, and combinations thereof. Exemplary acids that can be used in the disclosed methods include, but are not limited to, acetic acid, formic acid, difluoroacetic acid, and trifluoroacetic acid.

[0059] Transfer of drug components

[0060] The mechanism of transferring drug components to the second dimension of analysis can be performed using a trapping column or on-column dilution.

[0061] When using on-column dilution, the goal is to dilute the strong elution solvent composition % to promote adsorption of the eluted drug component onto the head of the 2nd dimension column. If the organic composition is too high, the drug can cause peak splitting or reduced chromatographic performance when adsorbed, or in the worst case, the drug passes through the column without being retained, resulting in an erroneous assessment of drug levels. Adjusting the flow rate of the 2nd dimension pump allows for appropriate dilution. An example would be: if the drug is eluted from the 1st dimension at 50% organic composition at a constant flow rate of 0.100ml / min and the desired organic composition for re-adsorption in the 2nd dimension column is 25%, the method would flow from the 2nd dimension pump at 0.100mL / min to effectively dilute the sample at a 1:1 ratio.

[0062] In some embodiments, a trap is used. Thus, the trap should have enough retention to retain the eluted drug substance and it is very likely that the on-column dilution must still be used. In addition, the trap should have equal or lower retention than the second dimension column. If the retention of the trap is greater than the second dimension column, one or more analytes will be eluted from the trap with a mobile phase composition that is not conducive to adsorption to the second dimension column, and the second dimension column will not increase additional separation benefits. The use of a trap can help reduce the pressure exposure on the first dimension column and allow the method to be performed with higher flow and shorter run time.

[0063] Connector and Drug Type

[0064] In one embodiment, the methods disclosed herein use ADC mimetics with fluorophore conjugates that exhibit lower hydrophobicity than ADCs containing commercial APIs, as demonstrated by Li et al. [J Chromatogr A. 2015; Vol. 1393, pp. 81-88]. For more hydrophobic drug substances, the organic percentage and acid concentration can be adjusted in a similar manner to more hydrophobic APIs, as discussed above. Similarly, if increased organic composition is required to elute the API component, the on-column dilution and retention of the second dimension can be adjusted accordingly.

[0065] The methods disclosed herein can be applied to non-cleavable linkers and / or pH labile linkers.

[0066] In one embodiment, the method of the present invention can be used to analyze cysteine-conjugated ADCs. Alternatively, the method of the present invention can be used to analyze lysine-conjugated ADCs. Cysteine-conjugated ADCs typically have relatively low drug loadings (e.g., 0 to 8 drugs). In contrast, lysine-conjugated ADCs typically have higher drug loadings (e.g., 1 to 10 or more drugs). Lysine-conjugated ADCs may exhibit a higher degree of hydrophobicity due to (1) increased drug loading and (2) the fact that interchain disulfide bonds remain intact. If the enhanced hydrophobicity causes the protein material to remain on the first dimension and then elute to the second dimension column, it may be appropriate to cleave the conjugated drug prior to analysis, at which point the cleaved material will generate a unique mass that can be distinguished from the free drug component. By cleaving the drug, the hydrophobicity of the antibody is reduced, thereby allowing it to pass through the designed first dimension column while retaining the cleaved drug and free drug. If the protein is still too hydrophobic, addition of a reducing agent can be used to reduce the interchain disulfides, thereby reducing the intact antibody to its corresponding subunits.

[0067] Detection Technology

[0068] The method of the present technology includes one or more detection steps. For example, some embodiments utilize mass spectrometry to detect antibody-drug conjugate complexes and unconjugated drug compounds in a sample. In some embodiments, mass spectrometry is used to establish the mass-to-charge ratio of each of the antibody-drug conjugate complexes and unconjugated drug compounds in the sample.

[0069] In certain other embodiments, spectroscopy is used as the preferred detection technique.In certain embodiments, ultraviolet (UV) and / or visible spectroscopy is used.

[0070] Mass spectrometry

[0071] A variety of mass spectrometry systems with high mass accuracy, high sensitivity and high separation are known in the art and can be used for the method of the present invention. The mass analyzer of this type of mass spectrometer includes but is not limited to quadrupole (Q), time of flight (TOF), ion trap, sector magnetic field or FT-ICR or a combination thereof. The ion source of a mass spectrometer should mainly produce sample molecular ions or quasi-molecular ions and some characterizable fragment ions. The example of this type of ion source includes an atmospheric pressure ionization source, such as electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI). ESI and MALDI are two of the most commonly used methods for ionizing proteins for mass spectrometry. ESI and APC1 are the most commonly used ion source technologies (Lee, M. "LC / MS Applications in Drug Development" (2002) J. Wiley & Sons, New York) of LC / MS.

[0072] Surface enhanced laser desorption ionization (SELDI) is an example of a surface-based ionization technique that realizes high-throughput mass spectrometry (U.S. Patent 6,020,208). Typically, SELDI is used to analyze complex mixtures of proteins and other biomolecules. SELDI uses chemically reactive surfaces (such as "protein chips") to interact with analytes (e.g., proteins) in solution. Such surfaces selectively interact with analytes and fix analytes thereon. Therefore, the analyte of the present invention can be partially purified on the chip, and then quickly analyzed in a mass spectrometer. By providing different reactive moieties at different sites on the substrate surface, throughput can be increased.

[0073] Commercially available mass spectrometers can sample and record the entire mass spectrum simultaneously and at a frequency that allows acquisition of enough spectra for multiple components in a mixture to ensure that the mass spectral signal intensity or peak area is quantitatively representative. This will also ensure that the elution times observed for all masses are not modified or distorted by the mass analyzer, and this will help ensure that quantitative measurements are not affected by the need to measure the abundance of transient signals.

[0074] Spectroscopy

[0075] A variety of spectroscopic systems with high accuracy, high sensitivity and high resolution are known in the art and can be used in the methods of the present invention. Absorption spectroscopy refers to a spectroscopic technique that measures the absorption of radiation that varies with frequency or wavelength due to its interaction with a sample. The sample absorbs energy, i.e., photons, from the radiation field. The intensity of the absorption varies with frequency, and this variation is the absorption spectrum. Absorption spectroscopy is performed across the entire electromagnetic spectrum.

[0076] Absorption spectroscopy is used as an analytical chemistry tool to determine the presence of a specific substance in a sample, and in many cases to quantify the amount of substance present. Infrared and UV-visible spectroscopy are particularly common in analytical applications.

[0077] There are a variety of experimental methods available for measuring absorption spectra. The most common arrangement is to direct a generated radiation beam toward a sample and detect the intensity of the radiation that passes through the sample. The absorption can be calculated using the energy transmitted. The source, sample arrangement, and detection technique vary significantly depending on the frequency range and the purpose of the experiment.

[0078] The most direct method of absorption spectroscopy is to generate radiation with a source, measure a reference spectrum of that radiation with a detector, and then remeasure a sample spectrum after placing the material of interest between the source and the detector. The two measured spectra can then be combined to determine the absorption spectrum of the material. The sample spectrum alone is not sufficient to determine the absorption spectrum because it will be affected by the experimental conditions - the spectrum of the source, the absorption spectra of other materials between the source and the detector, and the wavelength-dependent properties of the detector. But the reference spectrum will be affected by these experimental conditions in the same way, and so the combination alone produces the absorption spectrum of the material.

[0079] A wide variety of radiation sources can be used to cover the electromagnetic spectrum. For spectroscopy, it is generally desirable that the source covers a wide range of wavelengths in order to measure a wide region of the absorption spectrum. Some sources inherently emit a wide spectrum. Examples of these sources include silicon carbide rods or other black body sources in the infrared region, mercury lamps in the visible and ultraviolet regions, and x-ray tubes. A recently developed new type of wide spectrum radiation source is synchrotron radiation, which covers all of these spectral regions. Other radiation sources generate narrow spectra, but the emission wavelength can be tuned to cover the spectral range. Examples of these radiation sources include klystrons in the microwave region and lasers that span the infrared, visible, and ultraviolet regions (but not all lasers have tunable wavelengths).

[0080] The detector used to measure the radiant power will also depend on the wavelength range of interest. Most detectors are sensitive over a fairly wide spectral range, and the sensor chosen will generally depend more on the sensitivity and noise requirements for a given measurement. Examples of common detectors in spectroscopy include heterodyne receivers in the microwave region, bolometers in the millimeter-wave and infrared regions, mercury cadmium telluride and other cooled semiconductor detectors in the infrared region, and photodiodes and photomultiplier tubes in the visible and ultraviolet regions.

[0081] UV / Visible spectroscopy

[0082] "UV / visible spectroscopy" refers to absorption spectroscopy or reflectance spectroscopy in the ultraviolet (UV) and / or visible regions of the electromagnetic spectrum. The wavelength of ultraviolet (UV) electromagnetic radiation can range from 100 nm (30 PHz) to 380 nm (750 THz), which is shorter than the wavelength of visible light but longer than the wavelength of X-rays. Visible light is a type of electromagnetic radiation that is visible to the human eye. The wavelength of visible electromagnetic radiation can range from about 390 nm (430 THz) to about 700 nm (770 THz).

[0083] The instrument used in UV-Vis spectroscopy is called a UV / Vis spectrophotometer. It measures the intensity of light that has passed through a sample (I) and compares that intensity to the intensity of the light before it passed through the sample (I o ) for comparison. Ratio I / I o It is called transmittance and is usually expressed as a percentage (%T). The absorbance A is based on the transmittance:

[0084] Fluorescence spectroscopy

[0085] "Fluorescence spectroscopy" refers to a type of electromagnetic spectroscopy that analyzes fluorescence from a sample. It involves using a beam of light (usually ultraviolet) to excite electrons in the molecules of certain compounds and cause those electrons to emit light; usually visible light, but not necessarily. A companion technique is absorption spectroscopy. In the special case of single-molecule fluorescence spectroscopy, the intensity fluctuations of the emitted light are measured by a single fluorophore or a pair of fluorophores.

[0086] There are two common types of instruments: (1) filter fluorometers that use filters to separate incident light and fluorescence; and (2) spectrofluorometers that use a diffraction grating monochromator to separate incident light and fluorescence. Both types use the following scheme: light from an excitation source passes through a filter or monochromator and is incident on a sample. A portion of the incident light is absorbed by the sample, and some of the molecules in the sample fluoresce. The fluorescence is emitted in all directions. Some of this fluorescence passes through a second filter or monochromator and reaches a detector, which is usually set at 90° to the incident beam to minimize the risk of transmitted or reflected incident light reaching the detector.

[0087] Various light sources can be used as excitation sources, including lasers, LEDs, and lamps; specifically xenon arc lamps and mercury vapor lamps. Lasers emit light with high irradiance only in a very narrow wavelength interval (usually at 0.01 nm), which makes an excitation monochromator or filter unnecessary. Mercury vapor lamps are line lamps, which means that they emit light close to the peak wavelength. In contrast, xenon arc lamps have a continuous emission spectrum with an intensity that is nearly constant from 300 nm to 800 nm, and have an irradiance sufficient to measure all the way down to just over 200 nm.

[0088] Optical filters and / or monochromators can be used in fluorometers. A monochromator transmits light of adjustable wavelengths with adjustable tolerances. The most common type of monochromator utilizes a diffraction grating, i.e., collimated light strikes the grating and is present at different angles depending on the wavelength. The monochromator can then be adjusted to select which wavelengths are transmitted. To allow anisotropy measurements, two polarization filters need to be added: one after the excitation monochromator or filter, and one before the emission monochromator or filter.

[0089] As mentioned above, fluorescence is most often measured at a 90° angle relative to the excitation light. This geometry is used instead of placing the sensor at a 180° angle in the path of the excitation light in order to avoid interference with the transmitted excitation light. There is no perfect monochromator and it will transmit some stray light, i.e. light with wavelengths other than the target wavelength. An ideal monochromator would transmit only light within a specified range and have a high wavelength-independent transmission. When measuring at a 90° angle, only the light scattered by the sample causes stray light. This results in a better signal-to-noise ratio when compared to the 180° geometry and reduces the detection limit by about 10,000 times. In addition, fluorescence can also be measured from the front, which is usually done for turbid or opaque samples.

[0090] Detectors can be single-channel or multi-channel. Single-channel detectors can only detect the intensity of one wavelength at a time, while multi-channel detectors detect the intensity of all wavelengths simultaneously, making emission monochromators or filters unnecessary. Different types of detectors have advantages and disadvantages.

[0091] The most common fluorometers with dual monochromators and continuous excitation light sources can record both excitation and fluorescence spectra. When measuring fluorescence spectra, the wavelength of the excitation light is kept constant, preferably at a wavelength of high absorption, and the emission monochromator scans the spectrum. For measuring excitation spectra, the wavelength passed through the emission filter or monochromator is kept constant and the excitation monochromator is scanned. Excitation spectra are generally the same as absorption spectra, because the fluorescence intensity is proportional to the absorption.

[0092] Incorporated by Reference

[0093] The contents of all references, patents, and published patent applications cited in this application, as well as the Figures, are hereby incorporated by reference in their entirety.

[0094] Example

[0095] Having described the invention, however, the invention may be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention in any way.

[0096] Example 1: Evaluation of pure antibody-drug using mass spectrometry detection for improved specificity and sensitivity Targeted Multidimensional Approach (SPE-RPLC / MS) for Trace Free Drug Substances in Conjugate (ADC) Samples

[0097] This example uses a SPE-RPLC / MS method that is specific, sensitive, and achieves method control in two dimensions. The method was evaluated using a clinically relevant valine-citrulline surrogate molecule based on brentuximab. It was found that the assay sensitivity when using MS detection was two orders of magnitude higher than that of UV-based detection, with a LOQ of 0.30 ng / mL. Free drug substances were present in impurity-free ADC surrogate samples at concentrations below 7.0 ng / mL (i.e., levels that could not be detected by UV alone). The 2DLC method provides high specificity and sensitivity when evaluating trace free drug substances and improves control of each dimension, enabling direct integration into existing or novel workflows.

[0098] result :

[0099] It is preferred to select such an ADC for this embodiment, which provides the widest applicability of the proposed method and exhibits a pH-stable linker molecule. For ease of use and handling, such an ADC is also needed, which exhibits negligible or no cytotoxicity while being very close to the properties of commercial ADCs. To this end, an antibody-fluorophore-conjugate (AFC) as described previously [Wagner-Rousset, E. et al., mAbs, 2014, Vol. 6, No. 1, pp. 173-184] was used for this study. The key aspect in the design of AFC is that it mimics the clinically relevant ADC brentuximab Physiological and chemical properties of the drug [Pro, B. et al., Journal of Clinical Onocology, 2012, Vol. 30, No. 18, pp. 2190-2196; Francisco, JA et al., Blood, 2003, Vol. 102, No. 4, pp. 1485-1465], by using non-cytotoxic drug mimetics to minimize cytotoxicity and have minimal impact on the integrity of the original ADC. Monomethyl auristatin E (MMAE) was replaced with dansyl sulfonamide (DSEA, Figure 4 A), while retaining the maleimidocaproyl valine-citrulline linker (mal-linker-DSEA, Figure 4 B), thus successfully meeting these criteria. Wagner-Rousett and colleagues conducted an in-depth study of the manufactured AFC, establishing that the integrity of the ADC alternative was intact and well suited for ADC development without the risk of exposure to cytotoxic agents. [mAbs, 2014, Vol. 6, No. 1, pp. 173-184.] As part of the conjugation process, N-acetyl-cysteine ​​(NAc-linker-DSEA, Figure 4 C) to quench the reactive maleimide containing the valine-citrulline linker that did not undergo conjugation and remove it via SEC purification. The residual drug substance was assessed using a multidimensional approach that combined an online SPE mixed-mode anion exchange column ( MAX, Waters) and Superficially Porous High Resolution C 18 Column (Cortecs C 18 , Waters Corporation) with online detection using both a tunable UV (ACQUITY TUV, Waters Corporation) and a single quadrupole MS detector (ACQUITY QDa, Waters Corporation).

[0100] Reference Standards

[0101] Reference standards consisting of DSEA, linker-DSEA, and NAc-linker-DSEA were characterized using an ACQUITY H-Class Bio with 2D technology (Waters Corp.) in 1DLC configuration (see Materials and Methods). 18 Column (Cortecs C 18 , Waters Corporation) was used to perform a 10 min reverse phase (RP) gradient to evaluate the suitability of the reference standards for the proposed method. For each standard, [M+1H] +1 and [M+2H] +2 Selected ion monitoring (SIR) of charge state. Figure 5 As shown, the mixture of these three reference standards is the baseline separated within the applied gradient (500). Interestingly, compared with the organic mobile phase (MP) compositions of 42% and 46% for NAc-linker-DSEA and mal-linker-DSEA standards, respectively, the hydrophobicity of the DSEA (510) drug mimetic is significantly weaker than the mal-linker-DSEA (530) and NAc-linker-DSEA (520) standards eluted at 24% MP composition. The relative retention times of NAc-linker-DSEA and mal-linker-DSEA are similar to the RPLC results observed by Li et al. [J Chromatogr A, 2015, Vol. 1393, pp. 81-88] using the same valine-citrulline linker. In contrast, the hydrophobicity of the free drug mimetic (DSEA) is weaker.

[0102] Reference standard calibration curve

[0103] Adopt and Figure 5Serial dilutions of mal-linker-DSEA and NAc-linker-DSEA standards were analyzed in triplicate using the same instrument and column configuration using a 10 min RP gradient. A tunable UV detector set to an absorbance wavelength of 280 nm and the [M+2H] +2 Data acquisition was performed using a single quadrupole MS detector with SIR of charge states. Figure 6 ) and regression analysis of TUV chromatograms integrate, plot and analyze the collected data (data not shown). Use regulatory guidelines [Green, JM Analytical Chemistry, 1996, Vol. 68, No. 9, pp. 305A-309A] to assess the suitability of the assay for accuracy (<20% RSD at LOQ, otherwise <15%) and precision (<20% relative error (RE) at LOQ, otherwise <15%) to determine the dynamic range of the method.

[0104] Ordinary linear regression was determined to accurately model the data. The dynamic range of the single quadrupole (Table 1) was determined to span 2.5 orders of magnitude for the mal-linker-DSEA standard (0.27 ng / mL to 137.70 ng / mL) and 2.5 orders of magnitude for the NAc-linker-DSEA standard (0.33 ng / mL to 170.90 ng / mL), with LOQs of 0.27 ng / mL (1.35 pg on column; SNR D 9.56) and 0.33 ng / mL (1.65 pg on column; SNR D 9.97), respectively. These dynamic range results combined with good agreement with the fitted data at lower concentrations ( Figure 6 , inset) indicates that a detection limit of 0.10 ng / mL (0.5 pg on column) should be achievable with the proposed assay. In practice, an undesirable level of data processing is required at lower concentrations to achieve adequate integration, hampering accurate analysis. Nevertheless, the ability to detect target compounds at a nominal 0.3 ng / mL (1.5 pg on column) demonstrates the ability of these methods to detect pharmaceutical compounds with a high degree of sensitivity.

[0105] The linear dynamic range of the TUV measurement (Table 1) was determined to span 2 orders of magnitude for mal-linker-DSEA (34.42 ng / mL to 4,406.25 ng / mL) and NAc-linker-DSEA (85.45 ng / mL to 5,468.75 ng / mL) standards, with LOQs determined to be 34 ng / mL (0.17 ng on column) and 85 ng / mL (0.43 ng on column), respectively. The UV measurement was not evaluated at higher concentrations to extend the dynamic range because higher concentrations would exceed current regulatory recommendations for tolerable impurity levels. The lower sensitivity (or higher limit of detection) of the TUV measurement was expected and supports studies to construct online MS detection to improve sensitivity. The incorporation of mass detection expanded the sensitivity of the proposed method by 2 orders of magnitude beyond conventional UV-based detection and provided a 150-fold improvement in sensitivity for free drug substance compared to a previously published method using a SEC-RPLC / UV configuration for similar compounds, with a LOQ of 0.30 ng / mL (1.5 pg on column). [Li Y et al., J Chromatogr A 2015;1393,81-88.]

[0106]

[0107]

[0108] Table 1: Assay suitability. Standards were analyzed in triplicate and accuracy was assessed using ICH guidelines. (<20% R.SD at LOQ, otherwise <15%) and precision (<20% relative error (RE) at LOQ, otherwise <15%). Extending the dynamic range by 2 orders of magnitude using a quadrupole MS detector configured in tandem with an LC-TUV optical detector .

[0109] Solid Phase Extraction Optimization

[0110] Method development for multidimensional methods was performed independently for each dimension prior to coupling the column for AFC analysis as an efficient means of performing diagnostic runs. Optimization of the SPE method was performed using a 2.1 mm × 20 mm, 30 um SPE column (Oasis MAX, Waters Corporation) to evaluate the transfer of mal-linker-DSEA and NAc-linker-DSEA from the first dimension (SPE column) to the second dimension (analysis of C 18 To this end, Figure 7 The 2D LC configuration shown in A to 7C is used with a stainless steel union in place of the 2nd column with left and right valve groups in position 2.

[0111] Table 2 shows the column manager event table. The extracted drug substance was transferred in the 5.50 elution window using on-column dilution with both valves in position 2 to refocus the eluted drug substance at the head of the analytical column. There was a 0.6 second interval in position 2,1 between the transfers to purge the fluid path.

[0112] Column Manager: Event Table

[0113] Time (min) event action initial Left valve Position 1 initial Right valve Position 1 12.00 Left valve Position 2 12.01 Right valve Position 2 17.50 Right valve Position 1 17.51 Left valve Position 1

[0114] Table 2: Figure 7 Column Manager Event Table for Instruments A to 7C .

[0115] A small aliquot of the diluted trastuzumab sample used for column conditioning was spiked with mal-linker-DSEA and NAc-linker-DSEA reference standards. The spiked trastuzumab sample was injected onto the SPE column with screening conditions based on the RPLC results of the reference standards. The initial and elution MP compositions were adjusted in an iterative manner until no observable ions associated with the reference standards were detected between the 5min to 12min and 15min to 20min portions of the MS chromatogram. The presence of ions between 5min to 12min would indicate poor retention of the reference standard, while the detection of ions between 15min to 20min would indicate poor recovery of the reference standard. The optimized compositions were determined to be 18% and 36% organic solvent for the loading and elution conditions, respectively.

[0116] like Figure 8 As shown, use of these conditions allows protein to flow to the waste, where the retained reference standard elutes in a relatively narrow window using minimal amounts of organic MP, thereby improving on-column dilution efficiency.

[0117] To verify this, the stainless steel union was replaced with a second-dimensional porous C 18 RP column and the initial valve state was set to position 1. A fresh aliquot of the diluted trastuzumab sample was spiked with only the NAc-linker-DSEA reference standard and injected using the optimized conditions as described for the AFC analysis (see Materials and Methods) and timed valve changes as indicated in Table 1. Fig. 9 As shown in A, the protein eluted in the void during the initial injection, allowing the spiked NAc-linker-DSEA to be successfully transferred and refocused on the second dimension RPLC column, which was then eluted using a 10 min gradient ( Fig. 9 B).

[0118] Recovery efficiency evaluation

[0119] In the case of NAc-joint-DSEA reference standards successfully transferred using the proposed 2DLC configuration, the natural extension is to test the recovery efficiency of the method. Four samples of NAc-joint-DSEA were prepared at a nominal concentration of 2ng / mL, 17ng / mL, 74ng / mL, and 146ng / mL, and these four concentrations represent four points spaced apart in the entire dynamic range of the method as determined by regression analysis of quadrupole MS data. Three injections were repeated in 1DLC and 2DLC instrument configurations. The peak areas of these two instrument configurations were compared to evaluate the recovery. As shown in Table 3, the recovery was determined to be within the recommended guidelines [Green, JM Analytical Chemistry, 1996, Vol. 68, No. 9, pp. 305A-309A. ], wherein the accuracy (RSD) of the two configurations was less than 5% and the precision (RE) was within 3%. These results indicate that the SPE column effectively extracted the NAc-linker-DSEA sample from the eluent, with no observable loss of the standard in the void space. In addition, the area agreement between the 1D and 2D configurations indicates that the transfer efficiency between the columns is maintained, with no losses from the stainless steel tee used for on-column dilution. The combined recovery results indicate that the proposed multidimensional method of coupling SPE-RPLC is sufficiently robust and fit for purpose, with the NAc-linker-DSEA standard being confirmed to be recovered over the entire dynamic range established.

[0120]

[0121] Table 3: 2DLC recovery evaluation. Nac-linker- DSEA reference standard, and use Figure 7 The 2DLC configuration shown was used to evaluate the recovery efficiency. The same system with a union was used The same separation was performed in 1DLC configuration, replacing the SPE column (1st dimension) as a reference. Comparison of peak areas in three replicate injections indicates that sample recoveries are identical between 1DLC and 2DLC configurations. .

[0122] AFC Sample Analysis

[0123] AFC samples at a concentration of 1.94 mg / mL were used as received without additional preparation, and Figure 7 The 2DLC configuration shown evaluates the presence of mal-linker-DSEA and NAc-linker-DSEA. A 10 μL injection of the pure AFC sample was analyzed in triplicate using the same method as described above (see "Materials and Methods"). The same separation was performed using a water blank before each sample run to evaluate carryover. Fig.10 As shown in A, the overlay of three replicate SIR spectra of the blank injections shows no observable NAc-linker-DSEA species remaining at 22 min and negligible (<5% by area) residual mal-linker-DSEA species eluted at 23 min, indicating that the method is reproducible and can be performed over multiple runs, thereby extending column usage. Fig.10As shown in Table 4, the presence of NAc-linker-DSEA and mal-linker-DSEA in the AFC samples was confirmed, with the two species reproducibly eluting at 22 min and 23 min, respectively. The method accuracy was confirmed after a closer inspection of the data shown in Table 4, where the RSDs were determined to be less than 3% for both mal-linker-DSEA and NAc-linker-DSEA drug components. Using the MS-based calibration curve ( Figure 6 ), the concentrations of the free drug component were determined to be 7.19 ng / mL and 3.82 ng / mL for the NAc-linker-DSEA and mal-linker-DSEA components, respectively. Optical detection alone is not able to detect drug substance at these levels in samples of moderate concentration and injection volume, thus emphasizing the utility of 2DLC methods such as this one equipped with MS.

[0124]

[0125] Table 4: AFC sample results. As recommended by the ICH guidelines, within the dynamic range of the assay ( Figure 6 ) The concentrations of NAc-linker-DSEA and mal-linker-DSEA were determined to be 7.19 ng / mL and 3.82ng / mL .

[0126] discuss :

[0127] Traditional methods for detecting, characterizing, and quantifying trace free drug substances have included assays based on ELISA and RPLC techniques with varying degrees of success. [Stephan, JP, KR Kozak, and WL Wong Bioanalysis, 2011, Vol. 3, No. 6, pp. 677-700; Kozak, KR et al., Bioconjug Chem, 2013, Vol. 24, No. 5, pp. 772-779.] By combining multidimensional techniques to reduce sample preparation steps, this has provided analysts with a more effective method for evaluating trace drug substances with improved sensitivity. Selecting orthogonal column chemistries for multidimensional assays can be a challenging prospect because eluents and specifications can affect assay specificity, efficiency, and sensitivity. [Fleming, MS et al., Analytical Biochemistry, 2005, Vol. 340, No. 2, pp. 272-278; Li, Y. et al., J Chromatogr A, 2015, Vol. 1393, pp. 81-88; He, Y. et al., J Chromatogr A, 2012, Vol. 1262, pp. 122-129. ] The use of SPE-based techniques for targeted removal of APIs from biological matrices has long been used in the pharmaceutical industry. For this reason, SPE techniques have become increasingly common in the characterization and routine testing of biopharmaceuticals. [Souverain, S., S. Rudaz and JL Euthey, Journal of Chromatography B, 2004, Vol. 801, No. 2, pp. 141-156. ] The flexibility of the method with offline and online options combined with the ability to use volatile solvents makes SPE technology ideal for multidimensional methods with MS detection. Unique mixed-mode OASIS chemistries facilitate the ability to separate complex mixtures based on different physicochemical properties, and this is ideal when considering the unique properties of ADCs composed of a hydrophilic substrate conjugated to a hydrophobic drug. [Wakankar, A. et al., mAbs, 2011, Vol. 3, No. 2, pp. 161-172.] The current study has addressed the challenges associated with residual free drug analysis by developing a specific and sensitive MS-compatible multidimensional method coupled to SPE-RPLC chemistry.

[0128] Targeting free or non-conjugated hydrophobic drug substances for selective extraction is achieved by using hydrophobic SPE resins interspersed with anion exchange functional groups. Conceptually, net positive substrates such as mAbs in solutions with a pH below their pI pass through positively charged SPE materials, including mAb substances attached with drugs. In contrast, free drugs and associated products are adsorbed to the mixed-mode surface of the SPE ligand for downstream analysis. The current study successfully demonstrated this method by capturing and eluting clinically relevant valine-citrulline-based surrogate molecules and their N-acetyl-cysteine ​​quenched products from impurity-free ADC simulation samples using a SPE-RPLC / MS method. Similar hydrophobic properties observed in surrogate molecules and established literature support the feasibility of this method in clinical practice. [Li, Y. et al., J ChromatogrA, 2015, Vol. 1393, pp. 81-88. ]

[0129] The success of the proposed method relies on several key aspects that work in a synergistic manner. The implementation of the SPE column as the first dimension (which has retention for the API material) makes it easy for the analyst to tune the specificity of the method for different substrates or drug candidates. In contrast, first-dimensional separations such as SEC do not provide the same degree of specificity or selectivity. In addition, the mixed-mode SPE column effectively removes protein substrates and reduces residues due to the repulsion of like charges, thereby allowing the column to be reused. Due to the strong hydrophobicity of the captured drug substances, the organic percentage when they are eluted can reduce the performance of the second-dimensional RP chromatography. In order to overcome this challenge, on-column dilution is combined to effectively retain the drug product at the head of the second-dimensional column in the first-dimensional eluent. [Hurwitz E Fau-Levy, R. et al., Cancer Research, 1975, Vol. 35, pp. 1175-1181; Souverain, S., S. Rudaz and JL Euthey Journal of Chromatography B, 2004, Vol. 801, No. 2, pp. 141-156.] The degree to which the eluent needs to be diluted will depend on the organic strength required to elute the drug product from the first dimension, but is easily adjusted using the proposed configuration. The improved second dimension separation efficiency by increasing the on-column dilution combined with online MS detection resulted in a 125-fold increase in the sensitivity of the assay for mal-linker-DSEA and a 250-fold increase in the sensitivity of the assay for NAc-linker-DSEA drug substance when compared to UV detection, with a nominal LOQ of 0.3 ng / mL (Table 4).

[0130] Compared to the SEC-RPLC / UV method described by Li et al. using a similar, but different drug substance, the MS-based method in the current study demonstrated a sensitivity improvement of more than 150-fold. [Li, Y. et al., J Chromatogr A, 2015, Vol. 1393, pp. 81-88.] In addition to the improved sensitivity, the ability to effectively recover trace levels of drug substance across a wide dynamic range makes the proposed method particularly suitable for evaluating free drug substance throughout the ADC product life cycle, including development, formulation, and clinical trials.

[0131] As potentially more potent ADC drug candidates are identified [Thorson, JS et al., Current Pharmaceutical Design, 2000, Vol. 6, No. 18, pp. 1841-1879; Clardy, J. and C. Walsh Nature, 2004, Vol. 432, No. 7019, pp. 829-837], efforts to expand the therapeutic window will require assays with improved sensitivity in assessing and characterizing residual free drug species to ensure product safety and efficacy. [Wakankar, A. et al., mAbs, 2011, Vol. 3, No. 2, pp. 161-172]. ]The utility of multidimensional methods in biopharmaceutical characterization is becoming increasingly apparent [Fleming, MS et al., Analytical Biochemistry, 2005, Vol. 340, No. 2, pp. 272-278; Li, Y. et al., J Chromatogr A, 2015, Vol. 1393, pp. 81-88; He, Y. et al., J Chromatogr A, 2012, Vol. 1262, pp. 122-129; Li, Y. et al., Analytical Chemistry, 2014, Vol. 86, No. 10, pp. 5150-5157; Stoll, DR et al., Analytical Chemistry, 2015, Vol. 87, No. 16, pp. 8307-8315; Zhang, K. et al., Journal of Separation Science, 2013, Vol. 36, No. 18, pp. 2986-2992. ]. In this study, the SPE-RPLC / MS multidimensional approach coupled with on-column dilution was demonstrated to be an effective means to circumvent lengthy sample preparation steps while achieving control over each dimension; resulting in a method with specificity and sensitivity for free drug analysis that can be easily adapted to existing workflows. Future work will include evaluation of the proposed method for extraction of free drug substances in formulations and biological matrices using multiple panels of clinically relevant ADCs.

[0132]

[0133] Table 4: Assay suitability. Standards were analyzed in triplicate and accuracy was assessed using ICH guidelines (<20% RSD at LOQ, otherwise <15%) and precision (<20% relative error (RE) at LOQ, otherwise <15%). The dynamic range (grey highlighted) was increased using a quadrupole MS detector in a tandem configuration with an LC-TUV optical detector. Expanded by 2 orders of magnitude .

[0134] Materials and Methods :

[0135] Unless otherwise stated, chemicals and reagents were purchased from Sigma Aldrich. Mass spectrometry grade solvents were used for mobile phase and sample preparation.

[0136] Antibody and linker-payload preparation and purification

[0137] The AFC is based on the conjugation of dansylsulfonamide ethylamine (DSEAEA)-linker maleimide to the interchain cysteines of trastuzumab, which is used as a reference antibody. The trastuzumab used in this study is in a form and formulation approved by the European Medicines Agency (21 mg / mL). The linker-fluorophore payload is designed to mimic the linker-drug most commonly used in ADC clinical trials. The synthesis is briefly reported in the supplementary information by Wagner-Rousset et al.

[46] . It consists of maleimide-hexanoic acid dansylsulfonamide ethylamine (mc-DSEA, structure 5) with a valine-citrulline linker that simulates the cytotoxic agent. Figure 4 A) and the linker was conjugated to the mAb via the maleimide function through reduced interchain cysteines.

[0138] The mild reduction of trastuzumab and the coupling of DSEA-linker were performed as previously described. [Sun, MM et al., Bioconjug Chem, 2005, Vol. 16, No. 5, pp. 1282-1290.] In brief, trastuzumab was reduced for 2 h at 37 ° C in 10 mM borate pH 8.4 buffer containing 150 mM NaCl and 2 mM EDTA with 2.75 equivalents of TCEP. The concentration of free thiol groups was determined using Ellman's reagent with L-cysteine ​​as a standard, and about 5 thiol groups were usually generated per antibody. In order to achieve a DAR of 4, the partially reduced trastuzumab was then alkylated for 1 h at room temperature with 2 equivalents of DSEA-linker per thiol group in the same buffer. Any unreacted DSEA-linker was quenched using N-acetyl-cysteine ​​(1.5 equivalents / DSEA-linker). AFC was purified by size exclusion chromatography on a Superdex 200pg column (GE Life Sciences) and eluted by an AKTA Avant Biochromatography System (GE Life Sciences) using a 25mM histidine pH 6.5 buffer containing 150mM NaCl. AFC was characterized by most methods used for hinge region cysteine ​​ADCs (nr / rSDS-PAGE, SEC, HIC, native MS, LC-MS (IdeS / Red)) (average DAR = 4.0) and gave a profile similar to those reported for brentuximab. [Wagner-Rousset, E. et al., mAbs, 2014, Vol. 6, No. 1, pp. 173-184; Debaene, F. et al., Analytical Chemistry, 2014, Vol. 86, No. 21, pp. 10674-10683. ]The prepared AFC sample was used as pure substance at a concentration of 1.94 mg / mL.

[0139] Chromatography

[0140] An ACQUITY H-Class Bio (Waters Corporation) equipped with a commercially available 2D technology configuration was used for these experiments. Figure 7 A is a schematic diagram of the instrument setup showing the column, pump and tubing configuration for 2DLC with on-column dilution in place for 2D loading. Transfer of analytes retained on the SPE column (1st dimension) is performed by programmed valve events using the column manager control interface ( Figure 7B). Valve switches were staggered with 0.01 min delays to purge the column dilution fluid path before and after analyte transfer. A tunable UV detector (ACQUITY TUV, Waters Corporation) equipped with a 5 mm titanium flow cell was incorporated after the second dimension column to assess the optical detection limit of the separated analytes. The A detector was sampled at 20 Hz at 280 nm. max A 1DLC experiment with the appropriate column and mobile phase (MP) in the 1D column position and quaternary solvent manager (QSM) reservoir was performed using the same system by physically swapping the fluid path connections after the column in two dimensions and leaving the 2D binary solvent manager (BSM) pump idle with both valves in position 1.

[0141] Column Conditioning

[0142] Prior to sample running, the samples were concentrated in MS-grade HO containing 0.1% FA v / v. 2 The diluted sample of trastuzumab (2 mg / mL) prepared in O was adjusted to a 2.1 mm × 20 mm, 30 μm SPE column ( MAX, Waters Corporation). Column conditioning was performed with the chromatographic system in 1DLC configuration at a flow rate of 0.300 mL / min, with the column temperature set at 30°C. The QSM reservoir was prepared as MPA:H 2 O, 2% FA v / v, MP B: acetonitrile, 2% FA v / v. Separate a 2uL injection of the conditioning sample by performing a 10 min gradient from 0% MP B to 95% MP B until the baseline linear response is stable. Condition the second dimension column in a similar manner. Prior to the actual sample run, use ACN 0.1% FA v / v:H 2 A dilution mixture of the reference standard (0.5 ng / mL) prepared in 0.1% FA v / v was adjusted to a 2.1 mm × 50 mm, 2.7 um surface porous C 18 Column (Cortecs C 18 , Waters Corporation). The mobile phase was prepared as MP A:H 2 O, 0.1% FA v / v, MP B: acetonitrile, 0.1% FA v / v. A 5 uL injection of the diluted reference mixture was separated using a 10 min gradient from 5% MP B to 50% MP B at a flow rate of 0.300 ml / min and a column temperature of 40°C. The injections were repeated until the retention times and detector responses of the individual substances were stable. After conditioning both columns, the system was reconditioned in the 2DLC configuration for AFC analysis.

[0143] Calibration Standards

[0144] The stock reference standard ( Figure 4 ) was dissolved in pure DMSO and prepared at concentrations of 4 μg / mL, 2.82 μg / mL, and 3.5 μg / mL for DSEA, mal-linker-DSEA, and NAc-linker-DSEA, respectively. The stock solution was vortexed, briefly centrifuged, and divided into 50 uL aliquots and stored at -80°C before use. ACN 0.1% FA v / v:H was used at 50:50 2 Prepare initial calibration standard solutions by diluting each stock reference solution at a ratio of 1:5. Use 50:50 ACN 0.1% FA v / v:H 2 O 0.1% FA v / v, and sequential 1:1 serial dilutions of the initial calibration standard solutions of mal-linker-DSEA and NAc-linker-DSEA were performed. The reference standards were evaluated using the chromatography system in 1DLC configuration, with the QSM reservoir prepared as MPA:H 2 O, 0.1% FA v / v, MP B: acetonitrile, 0.1% FA v / v, MP C and MP D: acetonitrile. 5.0uL of each standard was loaded onto a 2.1mm×50mm, 2.7um surface porous C 18 Column (Cortecs C 18 , Waters) where the MP composition was kept constant for 1.0 min at 5% MP B at a flow rate of 0.300 ml / min and a column temperature of 40°C. The reference standard was eluted using a 10 min gradient from 5% MP B to 50% MP B. Column conditioning was performed using a fast 1.0 min gradient to increase the organic composition to 80% MP B, followed by a 1.0 min gradient to reach the initial condition (5% MP B) and kept constant for 2 min.

[0145] SPE Optimization

[0146] A 2.1 mm x 20 um, 30 um SPE column ( The HPLC column was optimized to increase the MS response during acquisition. The liquid chromatograph was configured using two six-port two-position valves housed in a column manager (ACQUITY Column Manager, Waters Corporation). Figure 7 2DLC configuration shown. For optimization purposes, a stainless steel union was used instead of the 2nd dimension column and both valves were set to initial position 2. The 1st dimension QSM reservoir was prepared as MP A:H 2O, 2% FA v / v, MP B: acetonitrile, 2% FA v / v, MP C and MP D: acetonitrile. The second dimension BSM reservoir was prepared as MP A: H 2 O, 0.1% FA v / v, MP B: acetonitrile, 0.1% FA v / v. The 2nd dimension BSM was programmed to flow at a rate of 0.300 mL / min with an MP composition of 60% MP B (2nd dimension MP reservoir) when both columns were online to replicate the back pressure encountered by the 1st dimension column. The doped trastuzumab sample was injected into the SPE column at a flow rate of 0.100 ml / min with an initial MP composition of 23% MP B (1st dimension MP reservoir). The gradient was then gradually increased to 54% MP B to elute the retained reference standard. The initial and elution MP compositions were adjusted in an iterative manner until no observable ions associated with the reference standard were detected between the 5min to 12min and 15min to 20min portions of the MS spectrum. The optimized composition was determined to be 18% MP B for loading conditions and 36% MP B for elution conditions. Once optimized, the stainless steel union was replaced with the 2nd dimension superficially porous C18 column and the initial valve state was set to position 1. Proof of principle was performed using a fresh aliquot of the diluted trastuzumab sample spiked with a small amount of NAc-linker-DSEA reference standard and injected using the optimized conditions described in the AFC sample analysis.

[0147] AFC sample analysis (1st dimension)

[0148] AFC samples were analyzed in the first dimension using an optimized two-step gradient. The QSM reservoir was prepared as MP A: HO containing 2% FA v / v. 2 O, MP B: acetonitrile containing 2% FA v / v, MP C and MP D: acetonitrile. The neat AFC sample was injected at a volume of 10.0 uL using isocratic conditions set at 18% MP B, flow rate 0.100 ml / min, and column temperature 30°C. After 9 min, the MP composition was gradually increased to 36% MP B and kept constant for 8 min to elute the bound analytes. The transfer of the analytes to the 2nd dimension RPLC column was achieved by programmed valve events, where the left and right valves were switched to position 2 between the 12.00 min and 17.50 min marks of the gradient to combine the fluid paths of the 1st and 2nd dimension columns. The MP composition was increased to 90% MP B from 17.0 min to 17.5 min using a sharp 0.50 min gradient and kept constant for an additional 2.50 min. The sawtooth gradient from 90% MP B to 18% MP B was cycled 3 times to repair the Oasis MAX SPE column, with the final cycle returning to the initial start-up conditions.

[0149] AFC sample analysis (2nd dimension)

[0150] BSM reservoirs were prepared as MP A: HO containing 0.1% formic acid v / v 2 O, MP B: acetonitrile containing 0.1% formic acid v / v. As part of the 2DLC method, the 2nd dimension MP composition was set to 0% MP B at the time of injection and kept constant at a flow rate of 0.300 ml / min until the 17.50 min mark. On-column dilution was performed via a stainless steel splitter (Vicci Valco) with a 1:4 dilution (0.1 mL / min 1st dimension pump: 0.3 mL / min 2nd dimension pump) while the 1st and 2nd dimension fluid paths were combined between 12.00 min and 17.50 min of the method. After 17.50 min, the MP composition was increased to 25% MP B and held for 1 min. A 5.55 min gradient from 25% MP B to 50% MP B was performed and held constant for an additional 0.44 min. After the separation gradient was performed, the MP composition was increased to 90% MP B in 1 min, followed by two 1 min sawtooth gradients from 90% MP B to 5% MP B to condition the RPLC column, with the final cycle returning to the initial starting conditions.

[0151] Recycling Assessment

[0152] Evaluation of SPE column recovery efficiency was performed using a NAc-linker-DSEA reference standard. 2 Four samples were prepared at concentrations of 1.7 ng / mL, 16.2 ng / mL, 71.9 ng / mL, and 144.6 ng / mL in 0.1% FA v / v to span the dynamic range based on the NAc-linker-DSEA calibration curve ( Figure 6 ). Using the 2DLC system configuration described previously for AFC sample analysis, three injections were performed for the four reference standard samples. The system was then reconfigured to 1DLC mode, and the QSM reservoir was changed to MP A: H containing 0.1% FA v / v. 2 MP O, MP B: acetonitrile containing 0.1% FA v / v, MP C and MP D: acetonitrile. The same reference samples were directly injected into a 2.1 mm × 50 mm, 2.7 μm surface porous C 18The column was run with the MP composition held constant for 1.0 min at 5% MP B at a flow rate of 0.300 ml / min and a column temperature of 40°C. The reference standard was eluted using a 10 min gradient from 5% MP B to 50% MP B. Column conditioning was performed using a rapid 1.0 min gradient to increase the organic composition to 80% MP B, followed by a 1.0 min gradient to reach the initial condition of 5% MP B and held constant for 2 min. The recovery efficiency was determined by comparing the peak areas in the 1DLC and 2DLC system configurations.

[0153] MS Settings

[0154] MS analysis after TUV detection was performed using a single quadrupole mass spectrometer (ACQUITY QDa, Waters Corporation) Figure 7 ). represents the [M+1H] of DSEA, mal-linker-DSEA and NAc-linker-DSEA +1 and [M+2H] +2 The SIR of DSEA was collected in the positive polarity covering the mass-to-charge range of 30m / z to 1,250m / z. In addition to other charge states, the confirmation fragment (718.4m / z) of mal-joint-DSEA was also collected and optimized for MS. MS data were collected in the whole separation defined in the chromatography part, wherein the fluid passed through the MS capillary continuously. The adjustable instrument was set as follows: capillary voltage 0.8kV, sample cone 2.0V, source temperature 400°C. The data from MS analysis were processed in the chromatographic data system MassLynx. The corresponding SIR of DSEA, mal-joint-DSEA and NAc-joint-DSEA samples were summed and the window size of 5 scans and 1 iteration was applied to smooth the average value, and then integrated.

[0155] Other embodiments of the present invention may be found as disclosed in Birdsall et al., mAbs, 2016, Vol. 8, No. 2, pp. 305-317, which is incorporated herein by reference.

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Claims

1. A method for analyzing an antibody-drug conjugate complex, the method comprising: (i) providing a sample comprising an antibody-drug conjugate complex, an unconjugated drug compound, and a linker molecule; (ii) exposing the sample to a solid phase extraction column having a mixed mode stationary phase; (iii) diluting the elution solvent from the solid phase extraction column; (iv) exposing the sample to a reverse phase chromatography column having a hydrophobic stationary phase; (v) capturing a portion of the unconjugated drug compound and / or linker molecule with a stationary phase before the solid phase extraction column or between the solid phase extraction column and the reverse phase chromatography column; (vi) separating the antibody-drug conjugate complex, the unconjugated drug compound, and the linker molecule in the sample; (vii) detecting each of the antibody-drug conjugate complex, the unconjugated drug compound, and the linker molecule in the sample using mass spectrometry; as well as (viii) quantifying the amount of unconjugated drug compound in the sample.

2. The method of claim 1, wherein the mass spectrometry is used to establish the mass-to-charge ratio of each of the antibody-drug conjugate complex, the unconjugated drug compound, and the linker molecule in the sample.

3. The method according to claim 1, further comprising the step of adjusting the acid concentration so that the unconjugated drug compound is retained in the solid phase extraction column or the reverse phase chromatography column.

4. The method of claim 1, wherein the mixed mode stationary phase comprises a hydrophobic solid phase extraction resin interspersed with anion exchange functional groups.

5. The method of claim 1, wherein the linker molecule comprises a non-cleavable linker, a pH-labile linker, or a combination thereof.

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