Development of optimized drug-like small molecule inhibitors of the SARS-CoV-2 3CL protease for treatment of COVID-19.

The SARS-CoV-2 3CL protease is a critical drug target for small molecule COVID-19 therapy, given its likely druggability and essentiality in the viral maturation and replication cycle. Based on the conservation of 3CL protease substrate binding pockets across coronaviruses and using screening, we identified four structurally distinct lead compounds that inhibit SARS-CoV-2 3CL protease. After evaluation of their binding specificity, cellular antiviral potency, metabolic stability, and water solubility, we prioritized the GC376 scaffold as being optimal for optimization. We identified multiple drug-like compounds with <10 nM potency for inhibiting SARS-CoV-2 3CL and the ability to block SARS-CoV-2 replication in human cells, obtained co-crystal structures of the 3CL protease in complex with these compounds, and determined that they have pan-coronavirus activity. We selected one compound, termed coronastat, as an optimized lead and characterized it in pharmacokinetic and safety studies in vivo. Coronastat represents a new candidate for a small molecule protease inhibitor for the treatment of SARS-CoV-2 infection for eliminating pandemics involving coronaviruses.

UPLC-MS assessment on the structural similarity of recombinant human erythropoietin (rhEPO) analogues from manufacturers in China for attribute monitoring.

Recombinant human erythropoietin (rhEPO) is a glycoprotein that acts as the main hormone involved in regulating red blood cell production to treat anemia caused by chronic kidney disease or chemotherapy. Since the expiration of the patent of the innovator epoetin alfa, numerous rhEPO products have emerged in global markets. As described here, multiple complementary analytical approaches are utilized for the extensive characterization of rhEPO molecules, and more importantly for the structural comparison of the rhEPO analogues on the Chinese market. The focus of this study is placed on the overall glycosylation profiling, O-glycan profiling, and N-glycan mapping by UPLC-MS with an aim to develop an effective analytical methodology to monitor the product quality attributes of rhEPO analogues. Two rhEPO analogues manufactured in China were analyzed to demonstrate the principle of the developed methods. Each rhEPO product showed a characteristic glycoform profile with respect to the distribution of sialic acids across multi-antennary structures, the occurrence of O-glycosylation, O-acetylation on sialic acids, and the extension of N-glycan antennae with N-acetyllactosamine units. The study demonstrates that UPLC-MS is an effective analytical tool to characterize and monitor the glycosylation profiles among rhEPO analogues in order to detect and account for the divergence between rhEPO products, as well as the presence of unusual or unexpected glycans.

Intact mAb LC-MS for drug concentration from pre-clinical studies: bioanalytical method performance and in-life samples.

Background: Antibody biotherapeutic measurement from pharmacokinetic studies has not been traditionally based on intact molecular mass as is the case for small molecules. However, recent advancements in protein capture and mass spectrometer technology have enabled intact mass detection and quantitation for dosed biotherapeutics. A bioanalytical method validation is part of the regulatory requirement for sample analysis to determine drug concentration from in-life study samples. Results/methodology: Here, an intact protein LC-MS assay is subjected to mock bioanalytical method validation, and unknown samples are compared between intact protein LC-MS and established bioanalytical assay formats: Ligand-binding assay and peptide LC-MS/MS. Discussion/conclusion: Results are presented from the intact and traditional bioanalytical method evaluations, where the in-life sample concentrations were comparable across method types with associated data analyses presented. Furthermore, for intact protein LC-MS, modification monitoring and evaluation of data processing parameters is demonstrated.

The Characterization of Drug-to-Antibody Ratio and Isoforms of ADCs Using LC and MS Technologies.

Hydrophobic interaction chromatography (HIC) and MS are leading techniques for the characterization of the critical quality attributes (CQA) of antibody-drug conjugates (ADCs). This includes the average drug-to-antibody ratio (DAR) and drug loading distribution. A workflow that effectively utilizes the synergy between chromatography and detection technologies has been developed and was assessed using cysteine-conjugated ADCs. The DAR of low, moderate and high drug-loaded ADC samples were calculated from the chromatographic peak areas using LC(HIC)/UV or the deconvoluted mass spectra using native LC(SEC)/MS. The results of DAR by both technologies produced comparable results. In addition, the 2D-LC/MS system has been evaluated in combination with HIC and reversed-phase chromatography for structural identification. Individual peaks from the 1st dimension of the HIC separation were isolated online and re-directed to the 2nd dimension reversed-phase column. ADC was detected as the sub-units by MS and the conjugation site was identified via a middle down approach.

In-depth structural characterization of Kadcyla® (ado-trastuzumab emtansine) and its biosimilar candidate.

ASBTRACT The biopharmaceutical industry has become increasingly focused on developing biosimilars as less expensive therapeutic products. As a consequence, the regulatory approval of 2 antibody-drug conjugates (ADCs), Kadcyla® and Adcetris® has led to the development of biosimilar versions by companies located worldwide. Because of the increased complexity of ADC samples that results from the heterogeneity of conjugation, it is imperative that close attention be paid to the critical quality attributes (CQAs) that stem from the conjugation process during ADC biosimilar development process. A combination of physicochemical, immunological, and biological methods are warranted in order to demonstrate the identity, purity, concentration, and activity (potency or strength) of ADC samples. As described here, we performed extensive characterization of a lysine conjugated ADC, ado-trastuzumab emtansine, and compared its CQAs between the reference product (Kadcyla®) and a candidate biosimilar. Primary amino acid sequences, drug-to-antibody ratios (DARs), conjugation sites and site occupancy data were acquired and compared by LC/MS methods. Furthermore, thermal stability, free drug content, and impurities were analyzed to further determine the comparability of the 2 ADCs. Finally, biological activities were compared between Kadcyla® and biosimilar ADCs using a cytotoxic activity assay and a HER2 binding assay. The in-depth characterization helps to establish product CQAs, and is vital for ADC biosimilars development to ensure their comparability with the reference product, as well as product safety.

Reduction of metal adducts in oligonucleotide mass spectra in ion-pair reversed-phase chromatography/mass spectrometry analysis.

RATIONALE: Electrospray ionization mass spectrometry (ESI-MS)-based techniques commonly used in oligonucleotide analyses are known to be sensitive to alkali metal adduct formation. Adducts directly impact the sensitivity of MS-based analyses as the available charge is distributed across the parent peak and adduct(s). The current study systematically evaluated common liquid chromatography (LC) components in LC/ESI-MS configurations used in oligonucleotide analysis to identify metal adduct contributions from LC instrumentation. METHODS: A UPLC liquid chromatography system was configured with a single quadrupole MS detector (ACQUITY QDa, Waters Corp.) to monitor adduct formation in oligonucleotide separations. An ion-pairing mobile phase comprised of 15 mM triethylamine and 400 mM hexafluoro-2-propanol was used in conjunction with an oligonucleotide separation column (Waters OST BEH C18, 2.1 mm × 50 mm) for all separations. A 10-min method was used to provide statistical figures of merit and evaluate adduct formation over time. RESULTS: Trace alkali metal salts in the mobile phase and reagents were determined to be the main source of metal salt adducts in LC/ESI-MS-based configurations. Non-specific adsorption sites located throughout the fluidic path contribute to adduct formation in oligonucleotide analyses. Ion-pairing mobile phases prepared at neutral or slightly basic pH result in up to a 57% loss of spectral abundance to adduct formation in the current study. CONCLUSIONS: Implementation of a short low pH reconditioning step was observed to effectively displace trace metal salts non-specifically adsorbed to surfaces in the fluidic path and was able to maintain an average MS spectral abundance ≥94% with a high degree of repeatability (relative standard deviation (R.S.D.) 0.8%) over an extended time study. The proposed method offers the ability to rapidly regenerate adsorption sites with minimal impact on productivity while retaining assay sensitivity afforded by MS detection with reduced adduct formation. © 2016 The Authors. Rapid Communications in Mass Spectrometry Published by John Wiley & Sons Ltd.

A rapid on-line method for mass spectrometric confirmation of a cysteine-conjugated antibody-drug-conjugate structure using multidimensional chromatography.

Cysteine-conjugated antibody-drug conjugates (ADCs) are manufactured using controlled partial reduction and conjugation chemistry with drug payloads that typically occur in intervals of 0, 2, 4, 6, and 8. Control of heterogeneity is of particular importance to the quality of ADC product because drug loading and distribution can affect the safety and efficacy of the ADC. Liquid chromatography ultra-violet (LC-UV)-based methods can be used to acquire the drug distribution profiles of cysteine-conjugated ADCs when analyzed using hydrophobic interaction chromatography (HIC). However, alternative analysis techniques are often required for structural identification when conjugated drugs do not possess discrete ultra-violet absorbance properties for precise assessment of the drug-to-antibody ratio (DAR). In this study, multidimensional chromatography was used as an efficient method for combining non-compatible techniques, such as HIC, with analysis by mass spectrometry (LC/LC/QTOF-MS) for rapid on-line structural elucidation of species observed in HIC distribution profiles of cysteine-conjugated ADCs. The methodology was tested using an IgG1 mAb modified by cysteine conjugation with a non-toxic drug mimic. Structural elucidation of peaks observed in the HIC analysis (1(st) dimension) were successfully identified based on their unique sub-unit masses via mass spectrometry techniques once dissociation occurred under denaturing reversed phase conditions (2(nd) dimension). Upon identification, the DAR values were determined to be 2.83, 4.44, and 5.97 for 3 drug load levels (low-, medium-, and high-loaded ADC batches), respectively, based on relative abundance from the LC-UV data. This work demonstrates that multidimensional chromatography coupled with MS, provides an efficient approach for on-line biotherapeutic characterization to ensure ADC product quality.

In vivo isotopically labeled atherosclerotic aorta plaques in ApoE KO mice and molecular profiling by matrix-assisted laser desorption/ionization mass spectrometric imaging.

RATIONALE: The ability to quantify rates of formation, regression and/or remodeling of atherosclerotic plaque should facilitate a better understanding of the pathogenesis and management of cardiovascular disease. In the current study, we coupled a stable isotope labeled tracer protocol with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) to examine spatial and temporal lipid dynamics in atherosclerotic plaque. METHODS: To promote plaque formation in the aorta region, ApoE KO mice were fed a high cholesterol diet (0.15% cholesterol) and orally dosed with (2,2,3,4,4,6-d(6))-cholesterol over several weeks. Tissue sections of ~10 µm thickness were analyzed by MALDI-MSI using matrix deposition by either chemical sublimation or acoustic droplet ejection. RESULTS: MALDI-MSI yielded distinct spatial distribution information for a variety of lipid classes including specific lysophosphatidylcholines typically associated with atherosclerosis-related tissue damage such as phospholipase 2 (Lp-PLA(2)) that mediate chemotactic responses to inflammation (e.g. LPC 16:0, LPC 18:0 and LPC 18:1) as well as free cholesterol and cholesteryl esters that contribute to atheroma formation. MALDI mass spectra acquired from aorta tissue sections clearly distinguished non-esterified and esterified versions of (2,2,3,4,4,6-d(6))-cholesterol within aortic plaque regions and showed distinct spatial accumulation of the cholesterol tracer. CONCLUSIONS: The ability to couple stable isotope based protocols with MALDI-MSI enables a novel strategy to characterize the effects of therapeutic treatments on atherosclerotic plaque formation, regression and potential remodeling of the complex lipid components with high chemical specificity and spatiotemporal information.

Distribution of chloroquine in ocular tissue of pigmented rat using matrix-assisted laser desorption/ionization imaging quadrupole time-of-flight tandem mass spectrometry.

In pharmacology and toxicology, localization of the distribution of a drug molecule in its target tissue provides very important in vivo biological information. Traditionally, this has been examined using autoradiography (ARG). However, there are significant limitations in this application. One is the synthesis and use of radiolabeled compounds, the other is that the image generated expresses an undifferentiated mixture of the parent drug and/or its metabolites. The objective of the study was to define the specific distribution of the parent drug in rat ocular tissue containing melanin (e.g. the retina) using non-labeled chloroquine by MALDI Imaging tandem mass spectrometry (MS/MS). After single oral administration (at 20 mg/kg) of chloroquine, sections (10 µm) of rat eye tissue were prepared at 24 h. The MS system used was a quadrupole time-of flight (Q-TOF) tandem mass spectrometer (MALDI Synapt™, Waters, Milford, MA, USA). Tissue sections were sprayed with CHCA (α-cyano-4-hydroxycinnamic acid, 5 mg/mL) in 80% acetonitrile (ACN) containing 5% formic acid (FA) using either a manual sprayer (airbrush) or an automated sprayer (TM-Sprayer™, HTX Technologies, Carrboro, NC, USA). Chloroquine was readily detected in the MS/MS mode by monitoring one of its major fragment ions (m/z 247.10) and imaged through the rat eye tissue. The image of the specific distribution within the retina in the rat eye tissue was confirmed, and found to be similar to autoradiograms after oral administration of (14)C-chloroquine reported previously.

Collision cross-section determination and tandem mass spectrometric analysis of isomeric carotenoids using electrospray ion mobility time-of-flight mass spectrometry.

Carotenoids are natural pigments with provitamin A and antioxidant activities. Biosynthesized in plants as their all-trans isomers, carotenoids isomerize in solution and in humans to multiple cis isomers which can have different bioavailabilities and functions. Since separation and characterization of isomeric carotenoids using high-pressure liquid chromatography (HPLC) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) is time-consuming, the potential for ion mobility mass spectrometry (IM-MS) to resolve and characterize carotenoid isomers rapidly without chromatography was investigated using traveling-wave ion mobility spectrometry on a quadrupole time-of-flight mass spectrometer. The all-trans isomers of lycopene and ß-carotene were separated by several milliseconds from the cis-isomers which were detected as partially overlapping peaks. The collision cross-section values of these carotenoid isomers were determined using IM-MS to be 180 and 236 Å(2) for cis-lycopene and all-trans-lycopene, and 181 and 225 Å(2) for cis-ß-carotene and all-trans-ß-carotene, respectively. Collision-induced dissociation MS/MS of ion mobility-resolved isomers indicated that cis and all-trans carotenoid isomers can be distinguished by their fragmentation patterns. Previous MS/MS studies of cis- and all trans-carotenoids had suggested that they produced identical tandem mass spectra, but this appears to have been the result of isomerization during ionization. Introduction of specific cis or trans isomers by infusion or HPLC resulted in cis/trans isomerization in the ion source during electrospray, and the relative levels of cis carotenoids forming in the ion source compared to the all-trans isomers were temperature dependent.

Improving de novo sequencing of peptides using a charged tag and C-terminal digestion.

An improved method for peptide de novo sequencing by MALDI mass spectrometry is presented. The method couples a charge derivatization reaction with C-terminal digestion to modify tryptic peptides. The charge derivatization attaches a fixed charge group onto the N-termini of peptides, and the enzymatic digestion after the derivatization step removes C-terminal basic amino acid residues such as arginine and lysine. The fragmentation of the modified peptide(s) under low-energy CID conditions (MALDI Q-TOF mass spectrometer) yields a simplified yet complete ion series of the peptide sequence. The validity of the method is demonstrated by the results from several model protein digests, where peptide sequences were correctly deduced either manually or through an automated sequencing program.

Analysis of human plasma proteome by 2DE- and 2D nanoLC-based mass spectrometry.

We compared the 2DE coupled to MALDI-TOF-MS and ESI-MS/MS analysis (2DE-MS) and the on-line 2D nanoLC, followed by nanoESI-MS/MS analysis (2DLC-MS), for the separation and identification of proteins in high abundance protein-depleted human plasma. Identification of proteins in the plasma by the two methods demonstrated that the majority of the identified protein set was unique to each method. Therefore, if a comprehensive coverage of the proteome identification is desired, it is ideal to apply both methods. The 2DE-MS method is amenable to protein spot-based quantitation, whereas the 2DLC-MS method may provide an advantage of the high throughput application.