Systematic optimization of targeted and multiplexed MS-based screening workflows for protein biomarkers.

Background: The capability of targeted MS-based methods to simultaneously measure multiple analytes with high selectivity and sensitivity greatly facilitates the discovery and quantitation of novel biomarkers. However, the complexity of biological samples is a major bottleneck that requires extensive sample preparation. Results: This paper reports a generic workflow to optimize surrogate peptide-based protein biomarker screening for seven human proteins in a multiplexed manner without the need for any specific affinity reagents. Each step of the sample processing and LC-MS methods is systematically assessed and optimized for better analytical performance. Conclusion: The established method is used for the screening of multiple myeloma patient samples to determine which proteins could be robustly measured and serve as potential biomarkers of the disease.

Experimental solubility profiling of marketed CNS drugs, exploring solubility limit of CNS discovery candidate.

We determined the experimental solubility of CNS marketed drugs. Of the 98 drugs measured, greater than 90% had solubility >10 µM in pH 7.4 buffer. Only seven drugs had solubility <10 µM. Using these data, we established a solubility criterion to support CNS discovery. The implication of poor solubility with potential safety concerns and undesirable side effects are discussed.

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.

Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones.

Aim: Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. Results: The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4 kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. Conclusion: HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.

Enantioselective Synthesis and Profiling of Two Novel Diazabicyclooctanone ß-Lactamase Inhibitors.

The enantioselective synthesis of two novel cyclopropane-fused diazabicyclooctanones is reported here. Starting from butadiene monoxide, the key enone intermediate 7 was prepared in six steps. Subsequent stereoselective introduction of the cyclopropane group and further transformation led to compounds 1a and 1b as their corresponding sodium salt. The great disparity regarding their hydrolytic stability was rationalized by the steric interaction between the cyclopropyl methylene and urea carbonyl. These two novel ß-lactamase inhibitors were active against class A, C, and D enzymes.

A high throughput dried DMSO LogD lipophilicity measurement based on 96-well shake-flask and atmospheric pressure photoionization mass spectrometry detection.

A rapid throughput octanol-water lipophilicity measurement based on 96-well shake-flask and LC/UV/APPI/MS is described. The method utilizes central liquid storage where compounds are stored as 10 mM solutions in dimethyl sulfoxide (DMSO). The DMSO is subsequently removed to generate solid like material used for LogD measurement. The removal of DMSO minimizes the concern for potential DMSO cosolvent effect on the measured value. Sample preparation is automated using a liquid handling workstation with 96-well pipetter. Both octanol and buffer phases are quantified using state of the art ultra-high pressure HPLC coupled with a superficially diffused core reversed-phase column and an atmospheric pressure photo ionization mass spectrometer. The throughput of the method is two days for a batch of 96 compounds. The method has been validated using 72 literature compounds with diverse ionization and LogD values ranging from -2 to +6. The observed coefficient of determination r(2) is 0.9973.

Application of a Dried-DMSO rapid throughput 24-h equilibrium solubility in advancing discovery candidates.

A rapid throughput equilibrium solubility measurement is described. The method utilizes central liquid storage where compounds are stored as 10mM solution in dimethyl sulfoxide (DMSO). The DMSO is subsequently removed to generate solid like material used for solubility measurement. A full range of available technologies is used including automated liquid handling, automated data collection using both HPLC/UV and LC/MS/MS. The method is fully validated and has been used to measure solubility for over 20,000 compounds across all phases of drug discovery. A detailed discussion on data interpretation and comparison to traditional solubility measurement using solid material is presented. An in-house solubility predictive model has been developed from the vast data set and has been employed successfully as part of compound design resulting in over 30% reduction in the number of poorly soluble compound synthesized.