High-Throughput Analyses of Therapeutic Antibodies Using High-Field Asymmetric Waveform Ion Mobility Spectrometry Combined with SampleStream and Intact Protein Mass Spectrometry.

Intact protein mass spectrometry (MS) coupled with liquid chromatography was applied to characterize the pharmacokinetics and stability profiles of therapeutic proteins. However, limitations from chromatography, including throughput and carryover, result in challenges with handling large sample numbers. Here, we combined intact protein MS with multiple front-end separations, including affinity capture, SampleStream, and high-field asymmetric waveform ion mobility spectrometry (FAIMS), to perform high-throughput and specific mass measurements of a multivalent antibody with one antigen-binding fragment (Fab) fused to an immunoglobulin G1 (IgG1) antibody. Generic affinity capture ensures the retention of both intact species 1Fab-IgG1 and the tentative degradation product IgG1. Subsequently, the analytes were directly loaded into SampleStream, where each injection occurs within ∼30 s. By separating ions prior to MS detection, FAIMS further offered improvement in signal-overnoise by ∼30% for denatured protein MS via employing compensation voltages that were optimized for different antibody species. When enhanced FAIMS transmission of 1Fab-IgG1 was employed, a qualified assay was established for spiked-in serum samples between 0.1 and 25 µg/mL, resulting in ∼10% accuracy bias and precision coefficient of variation. Selective FAIMS transmission of IgG1 as the degradation surrogate product enabled more sensitive detection of clipped species for intact 1Fab-IgG1 at 5 µg/mL in serum, generating an assay to measure 1Fab-IgG1 truncation between 2.5 and 50% with accuracy and precision below 20% bias and coefficient of variation. Our results revealed that the SampleStream-FAIMS-MS platform affords high throughput, selectivity, and sensitivity for characterizing therapeutic antibodies from complex biomatrices qualitatively and quantitatively.

Strategy to improve the quantitative LC-MS analysis of molecular ions resistant to gas-phase collision induced dissociation: application to disulfide-rich cyclic peptides.

Due to observed collision induced dissociation (CID) fragmentation inefficiency, developing sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for CID resistant compounds is especially challenging. As an alternative to traditional LC-MS/MS, we present here a methodology that preserves the intact analyte ion for quantification by selectively filtering ions while reducing chemical noise. Utilizing a quadrupole-Orbitrap MS, the target ion is selectively isolated while interfering matrix components undergo MS/MS fragmentation by CID, allowing noise-free detection of the analyte's surviving molecular ion. In this manner, CID affords additional selectivity during high resolution accurate mass analysis by elimination of isobaric interferences, a fundamentally different concept than the traditional approach of monitoring a target analyte's unique fragment following CID. This survivor-selected ion monitoring (survivor-SIM) approach has allowed sensitive and specific detection of disulfide-rich cyclic peptides extracted from plasma.

Bioactivation of 2-(alkylthio)-1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles.

Certain functional groups/structural motifs are known to generate chemically reactive metabolites that can covalently modify essential cellular macromolecules and, therefore, have the potential to disrupt biological function and elicit idiosyncratic adverse drug reactions. In this report, we describe the bioactivation of 5-substituted 2-(alkylthio)-1,3,4-thiadiazoles and 2-(alkylthio)-1,3-benzothiazoles, which can be added to the growing list of structural alerts. When 5-substituted 2-(methylthio)-1,3,4-thiadiazoles and 2-(methylthio)-1,3-benzothiazole were incubated with pooled human liver microsomes in the presence of NADPH and GSH, unusual GSH adducts were formed. Characterization of these GSH adducts by high-resolution mass spectrometry indicated the replacement of the methylthio- group by GSH, and NMR experiments ascertained the proposed structures. On the basis of the metabolic profile change in incubation samples with/without GSH, we proposed that the GSH adduct formation involved two steps: (1) enzymatic oxidation of the alkylthio- group to form sulfoxide and sulfone and (2) nucleophilic displacement of the formed sulfoxide and sulfone by GSH. The proposed mechanism was confirmed by the formation of the same GSH adduct from the incubation of synthetically prepared sulfoxide and sulfone compounds in buffer. We found the sulfur oxidation step was significantly inhibited (80-100%) by preincubation with 1-aminobenzotriazole but was much less affected by thermoinactivation (0-45%), suggesting that the sulfoxidation step is primarily catalyzed by cytochrome P450s and not by flavin monooxygenases. We also investigated the presence of this bioactivation pathway in more than a dozen compounds containing 2-(alkylthio)-1,3,4-thiadiazole and 2-(alkylthio)-1,3-benzothiazoles. The common GSH adduct formation pathway demonstrated by current studies raises a new structural alert and potential liability in drug safety when 2-alkylthio derivatives of 1,3-benzothiazoles and 1,3,4-thiadiazoles are incorporated in drug design.

Quantitative estimation of circulating metabolites without synthetic standards by ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry in combination with UV correction.

An early assessment of metabolite exposure in preclinical species can provide quantitative estimation on possible active or toxic metabolites. Frequently, synthetic metabolite standards are not available at the preclinical stage, precluding the quantitation of metabolites by means of calibration curves and quality control (QC) samples. We present here an approach to determine the extent of circulating metabolites using 'metabolite standards' generated by in vitro incubations in combination with the correction for mass spectrometry response based on UV response. The study was done by coupling ultra-high-performance liquid chromatography (UHPLC) to LTQ-Orbitrap high-resolution mass spectrometry, and the quantitation was based on full scan high-resolution accurate mass analysis in combination with retention time. First, we investigated the separation capacity of a 10.5 min UHPLC method and the quantitative capability of an LTQ-Orbitrap for full scan accurate mass quantitation by spiking chemical standards of buspirone and its six metabolites in blank plasma. Then we demonstrated the use of a UV correction approach to quantitatively estimate buspirone and its metabolites in plasma samples from a rat pharmacokinetics study. We compared the concentration versus time profiles of buspirone and its six metabolites in rat plasma samples obtained using three different approaches, including using UV correction, using individual standard curves for each metabolite prepared from the synthetic standard, and using a calibration curve of the parent compound buspirone. We demonstrated the estimated metabolite exposure of buspirone using this UV correction approach resulted in rank ordering of metabolite exposure within three-fold of the value obtained with metabolite standards, in contrast to eight-fold without UV correction. The approach presented in this paper provides a practical solution to an unmet bioanalytical need for quantitative information on metabolites without standards in preclinical in vivo studies.

Metabolite generation via microbial biotransformations with Actinomycetes: rapid screening for active strains and biosynthesis of important human metabolites of two development-stage compounds, 5-[(5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]non7-yl-methyl]-3-thiophenecarboxylic acid (BMS-587101) and dasatinib.

The enzymes present in many microbial strains are capable of carrying out a variety of biotransformations when presented with drug-like molecules. Although the enzymes responsible for the biotransformations are not well characterized, microbial strains can often be found that produce metabolites identical to those found in mammalian systems. However, traditional screening for microbial strains that produce metabolites of interest is done with many labor intensive steps that include multiple shake flasks and many manual manipulations, which hinder the application of these techniques in drug metabolite preparation. A 24-well microtiter plate screening system was developed for rapid screening of actinomycetes strains for their ability to selectively produce metabolites of interest. The utility of this system was first demonstrated with the well characterized cytochrome P450 substrate diclofenac. Subsequently, the use of this system allowed the rapid identification of several actinomycetes strains that were capable of converting two drug candidates under development, 5-[(5S,9R)-9-(4-cyanophenyl)-3-(3,5-dichlorophenyl)-1-methyl-2,4-dioxo-1,3,7-triazaspiro[4.4]non7-yl-methyl]-3-thiophenecarboxylic acid and N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1-piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, Sprycel, BMS-345825), to mammalian metabolites of interest. Milligram quantities of the metabolites were then prepared by scaling-up the microbial biotransformation reactions. These quantities were sufficient for initial characterization, such as testing for pharmacological activity and use as analytical standards, prior to the availability of authentic chemically synthesized compounds.

Charge variant native mass spectrometry benefits mass precision and dynamic range of monoclonal antibody intact mass analysis.

The preponderance and diversity of charge variants in therapeutic monoclonal antibodies has implications for antibody efficacy and degradation. Understanding the extent and impact of minor antibody variants is of great interest, and it is also a critical regulatory requirement. Traditionally, a combination of approaches is used to characterize antibody charge heterogeneity, including ion exchange chromatography and independent mass spectrometric variant site mapping after proteolytic digestion. Here, we describe charge variant native mass spectrometry (CVMS), an integrated native ion exchange mass spectrometry-based charge variant analytical approach that delivers detailed molecular information in a single, semi-automated analysis. We utilized pure volatile salt mobile phases over a pH gradient that effectively separated variants based on minimal differences in isoelectric point. Characterization of variants such as deamidation, which are traditionally unattainable by intact mass due to their minimal molecular weight differences, were measured unambiguously by mass and retention time to allow confident MS1 identification. We demonstrate that efficient chromatographic separation allows introduction of the purified forms of the charge variant isoforms into the Orbitrap mass spectrometer. Our CVMS method allows confident assignment of intact monoclonal antibody isoforms of similar mass and relative abundance measurements across three orders of magnitude dynamic range.

An automated high throughput liquid chromatography-mass spectrometry process to assess the metabolic stability of drug candidates.

An automated high throughput process, termed the MetFast assay, is described to assess in vitro the general microsomal cytochrome P450 beta-nicotinamide adenine dinucleotide phosphate-mediated first-pass metabolic stability of potential drug candidates as a utility for pharmaceutical profiling. Utilizing robotic protocols with a multiprobe liquid handler, compounds are incubated with liver microsomes from different species. Samples are then analyzed by in-line liquid chromatography (LC)-mass spectrometry (MS) to determine the amount of compound remaining after a certain time, which allows calculation of metabolism rates. To quantitatively assess large numbers of structurally diverse compounds by LC-MS, a strategy based on an iterative two-step process was devised. Initially compounds are qualitatively analyzed by LC-ultraviolet (UV)/MS (step 1) to determine purity (UV detection) and structural integrity (MS detection). This step ensures that only correct and verified compounds with sufficient purity are being assayed to obtain reproducible high data quality. In addition, all necessary information is gathered to automatically generate specific quantitative methods for the subsequent bioanalytical analysis of metabolic stability samples by LC-UV/MS (step 2). In-house-developed, highly flexible and sophisticated data management software, termed SmartReport, is utilized for automated qualitative and quantitative LC-MS analysis set-up, data processing, and results reporting. The integration of key aspects, inherent "universal" collision-induced dissociation settings of ion trap mass spectrometers for tandem mass spectrometric scan functions utilized for compound-specific and sensitive quantitative MS methods, generic fast-LC conditions, generic MS instrument settings, and the functionality of SmartReport software resulted in an analytical process that routinely provides reproducible high-quality metabolic stability data on structurally diverse compounds. Described here is the setup of the MetFast assay, and metabolic stability data from assay validation compounds are given.

A semi-automated method for the integrated evaluation of half-life and metabolic soft spots of discovery compounds.

BACKGROUND: An integrated method that provides rates of both parent disappearance and metabolite formation was developed. RESULTS: Buspirone, mirtazapine and verapamil were used as model compounds in developing the method. Incubations were carried out on a robotic platform. Qualitative analysis of metabolites in 30 µM samples was conducted by data-dependent HPLC-MS/MS on a high-resolution instrument. Quantitative analysis of the parent compound and metabolites in 0.5 µM samples was conducted by full-scan MS(2) with product ion extraction using an ion trap mass spectrometer. Data generated for the compounds included half-life and intrinsic clearance of the parent molecule, characterization of metabolites and relative rates of metabolite formation. A correction factor was used to convert MS responses of metabolites in 0.5 µM samples to UV areas in order to compare relative metabolite concentrations. CONCLUSION: The approach allows for the investigation of a set of six compounds simultaneously, with a turnaround time of 1 week or less.

On the inter-instrument and the inter-laboratory transferability of a tandem mass spectral reference library. 3. Focus on ion trap and upfront CID.

Mass spectral libraries represent versatile tools for the identification of small bioorganic molecules. Libraries based on electron impact spectra are rated robust and transferable. Tandem mass spectral libraries are often considered to work properly only on the instrument that has been used to build the library. An exception from that rule is the 'Wiley Registry of Tandem Mass Spectral Data, MSforID'. In various studies with data sets from different kinds of tandem mass spectrometric instruments, the outstanding sensitivity and robustness of this tandem mass spectral library search approach was demonstrated. The instrumental platforms tested, however, mainly included various tandem-in-space instruments. Herein, the results of a multicenter study with a focus on upfront and tandem-in-time fragmentation are presented. Five laboratories participated and provided fragment ion mass spectra from the following types of mass spectrometers: time-of-flight (TOF), quadrupole-hexapole-TOF, linear ion trap (LIT), 3-D ion trap and LIT-Orbitrap. A total number of 1231 fragment ion mass spectra were collected from 20 test compounds (amiloride, buphenin, cinchocaine, cyclizine, desipramine, dihydroergotamine, dyxirazine, dosulepin, ergotamine, ethambutol, etofylline, mefruside, metoclopramide, phenazone, phentermine, phenytoin, sulfamethoxazole, sulfamoxole, sulthiame and tetracycline) on seven electrospray ionization instruments using 18 different instrumental configurations for fragmentation. For 1222 spectra (99.3%), the correct compound was retrieved as the best matching compound. Classified matches (matches with 'relative average match probability' >40.0) were obtained for 1207 spectra (98.1%). This high percentage of correct identifications clearly supports the hypothesis that the tandem mass spectral library approach tested is a robust and universal identification tool.

Novel MS solutions inspired by MIST.

To improve patient safety and to help avoid costly late-stage failures, the pharmaceutical industry, along with the US FDA and International Committee on Harmonization (ICH), recommends the identification of differences in drug metabolism between animals used in nonclinical safety assessments and humans as early as possible during the drug-development process. LC-MS is the technique of choice for detection and characterization of metabolites, however, the widely different LC-MS response observed for a new chemical entity (NCE) and its structurally related metabolites limits the direct use of LC-MS responses for quantitative determination of NCEs and metabolites. While no method provides completely accurate universal response, UV, corona charged aerosol detection (CAD), radioactivity, NMR and low-flow (< 20 µl/min) nanospray approaches provide opportunities to quantify metabolites in the absence of reference standards or radiolabeled material with enough precision to meet the needs of early clinical development.

Creation and comparison of MS/MS spectral libraries using quadrupole ion trap and triple-quadrupole mass spectrometers.

Searchable libraries of MS/MS spectra, obtained using liquid chromatography/tandem mass spectrometry (LC/MS/MS) with data-dependent scan mode switching on both quadrupole ion trap and triple-quadrupole mass spectrometers in conjunction with electrospray ionization, are presented. The effects on library search scores of changing the parameters for producing collision-induced dissociation (CID) on both instrument types are systematically evaluated. These observations serve as a basis for determining a universal set of conditions for building MS/MS libraries. A group of 19 closely related steroids was used. The ability to obtain library-searchable spectra at low concentrations is demonstrated for the analysis of a sample of progesterone spiked with hydroxyprogesterone impurities at 0.1 and 0.01%.