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.

Identification of hnRNP-A1 as a pharmacodynamic biomarker of type I PRMT inhibition in blood and tumor tissues.

Arginine methylation has been recognized as a post-translational modification with pleiotropic effects that span from regulation of transcription to metabolic processes that contribute to aberrant cell proliferation and tumorigenesis. This has brought significant attention to the development of therapeutic strategies aimed at blocking the activity of protein arginine methyltransferases (PRMTs), which catalyze the formation of various methylated arginine products on a wide variety of cellular substrates. GSK3368715 is a small molecule inhibitor of type I PRMTs currently in clinical development. Here, we evaluate the effect of type I PRMT inhibition on arginine methylation in normal human peripheral blood mononuclear cells and utilize a broad proteomic approach to identify type I PRMT substrates. This work identified heterogenous nuclear ribonucleoprotein A1 (hnRNP-A1) as a pharmacodynamic biomarker of type I PRMT inhibition. Utilizing targeted mass spectrometry (MS), methods were developed to detect and quantitate changes in methylation of specific arginine residues on hnRNP-A1. This resulted in the development and validation of novel MS and immune assays useful for the assessment of GSK3368715 induced pharmacodynamic effects in blood and tumors that can be applied to GSK3368715 clinical trials.

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.

Antibody Subunit LC-MS Analysis for Pharmacokinetic and Biotransformation Determination from In-Life Studies for Complex Biotherapeutics.

Complex biotherapeutics present challenges from drug discovery, screening, and development perspectives. While monoclonal antibody drugs are not monitored for metabolites in the same manner as small molecules, biotherapeutics such as fusion proteins, antibody-drug conjugates, or bispecific antibodies may undergo biotransformation (such as clipping, deamidation, or oxidation) in vivo, resulting in catabolites that can have a direct impact on drug safety or efficacy. Here antibody subunit LC-MS is utilized for evaluation of two classes of complex biotherapeutics: an antibody-drug conjugate and a mAb-fusion biotherapeutic. Pharmacokinetic concentration, biotransformation, and DAR data are collectively presented using the subunit LC-MS approach for the two molecules, and the methods shared in detail can be applied to any humanized IgG1 mAb biotherapeutic for preclinical study support. Overall, the data generated from antibody LC-MS analyses can provide key information in early phase development and deliver multiple study end points with a single data set.

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.

Application of high-resolution MS for development of peptide and large-molecule drug candidates.

BACKGROUND: For quantitative bioanalysis utilizing MS, the instrument of choice is typically a triple quadruple mass spectrometer. However, advances in high-resolution MS have allowed sensitivity and dynamic ranges to approach that of triple quadrupole instruments. RESULTS: A matrix-free protein digest, a digested therapeutic protein and the intact peptide therapeutic liraglutide were each analyzed on high-resolution and triple quadrupole mass spectrometers with data compared. Samples from a mouse PK study with liraglutide were analyzed using the two different instruments, and equivalent PK exposure data were demonstrated. CONCLUSION: High-resolution and triple quadrupole mass spectrometers can generate data resulting in identical PK parameters from an in-life sample set, thus giving confidence in either technique in support of biotherapeutic PK exposure studies.

Integrating internal and external bioanalytical support to deliver a diversified pharmaceutical portfolio.

The portfolios of pharmaceutical companies have diversified substantially over recent years in recognition that monotherapies and/or small molecules are less suitable for modulating many complex disease etiologies. Furthermore, there has been increased pressure on drug-development budgets over this same period. This has placed new challenges in the path of bioanalytical scientists, both within the industry and with contract research organizations (CROs). Large pharmaceutical, biotechnology and small-medium healthcare enterprises have had to make important decisions on what internal capabilities they wish to retain and where CROs offers a significant strategic benefit to their business model. Our journey has involved asking where we believe an internal bioanalytical facility offers the greatest benefit to progressing drug candidates through the drug-development cycle and where externalization can help free up internal resources, adding flexibility to our organization in order to deal with the inevitable peaks and troughs in workload.

The Gyrolab™ immunoassay system: a platform for automated bioanalysis and rapid sample turnaround.

BACKGROUND: The Gyrolab™ workstation benefits from fully automated transfer of reagents and samples originating from a storage microplate onto a compact disc containing solid-phase microstructures composed of a 15 nl streptavidin-derivitized bead bed. RESULTS: This paper describes the development, full validation and use of the method in a regulated environment to measure a humanized bispecific monoclonal antibody-domain antibody (GSK-A) molecule using the Gyrolab immunoassay system in cynomolgus nonhuman primate plasma ranging from 5 to 250 µg/ml. The method was subsequently used in support of the TK portion of a regulated preclinical study in monkeys. CONCLUSION: The Gyrolab immunoassay system proved to be a viable alternative to traditional immunoassays and was used to support a regulated preclinical TK study. The speed of analysis that the Gyrolab provides was beneficial in meeting timelines to complete this project as multiple assays and repeat sample analysis could be completed in the same day.

Comparison of the quantification of a therapeutic protein using nominal and accurate mass MS/MS.

BACKGROUND: The quantification of proteins and peptides in in vivo samples is a critical part of supporting the drug development process for biotherapeutics. LC-MS/MS using tandem quadrupole mass spectrometers is well established as the technology of choice for the quantification of small-molecule drugs and their metabolites in biological fluid. The application of accurate mass MS for quantification in a DMPK environment has attracted considerable interest in recent years. MATERIALS & METHODS: In this article we describe and compare the application of LC-high-resolution MS and LC-selected reaction monitoring (SRM) for the quantification of a therapeutics proteins. RESULTS: The accurate mass instrumentation showed acceptable linearity and sensitivity to quantify the protein therapeutic to the level of 10 ng/ml. The accurate mass instrument was operated in accurate mass SRM using high resolution (SRM-HR), the assay was demonstrated to be linear over three orders of magnitude. By narrowing the mass window from 100 mDa to 40 mDa and then to 20 mDa the assay specificity was significantly improved, hence increasing the S/N and improving the assay sensitivity. CONCLUSION: The high-resolution instrument was demonstrated to be reproducible over the course of the assay. The accurate mass method sensitivity was determined to be within one order of magnitude of that obtained with a tandem quadrupole MS/MS assay.

Absolute quantification of a therapeutic domain antibody using ultra-performance liquid chromatography-mass spectrometry and immunoassay.

BACKGROUND: Domain antibodies (dAbs; ∼10-15 kDa) are made up of the variable heavy chain or the variable light chain of the antibody structure, and retain binding capability. dAbs have proved difficult to detect in plasma using immunoassay without specific antibodies raised against the dAb. RESULTS: A sensitive and selective UPLC-MS/MS method for the absolute quantification of a dAb in monkey plasma was developed (range: 1 to 500 ng/ml) without the need for a specific capture antibody. This method was used to analyze pharmacokinetic studies early on in drug development. Furthermore, an immunoassay was developed and the pharmacokinetic samples were reanalyzed. CONCLUSION: The two assays show good correlation (r(2) = 0.92), giving confidence in using either method for quantification of the dAb.

Identification of proteins adducted by lipid peroxidation products in plasma and modifications of apolipoprotein A1 with a novel biotinylated phospholipid probe.

Reactive electrophiles generated by lipid peroxidation are thought to contribute to cardiovascular disease and other oxidative stress-related pathologies by covalently modifying proteins and affecting critical protein functions. The difficulty of capturing and analyzing the relatively small fraction of modified proteins complicates identification of the protein targets of lipid electrophiles. We recently synthesized a biotin-modified linoleoylglycerylphosphatidylcholine probe called PLPBSO ( Tallman et al. Chem. Res. Toxicol. 2007, 20, 227-234 ), which forms typical linoleate oxidation products and covalent adducts with model peptides and proteins. Supplementation of human plasma with PLPBSO followed by free radical oxidation resulted in covalent adduction of PLPBSO to plasma proteins, which were isolated with streptavidin and identified by liquid chromatography-tandem mass spectrometry (LC-MS-MS). Among the most highly modified proteins was apolipoprotein A1 (ApoA1), which is the core component of high density lipoprotein (HDL). ApoA1 phospholipid adduct sites were mapped by LC-MS-MS of tryptic peptides following mild base hydrolysis to release esterified phospholipid adducts. Several carboxylated adducts formed from phospholipid-esterified 9,12-dioxo-10( E)-dodecenoic acid (KODA), 9-hydroxy, 12-oxo-10( E)-dodecenoic acid (HODA), 7-oxoheptanoic acid, 8-oxooctanoic acid, and 9-oxononanoic acid were identified. Free radical oxidations of isolated HDL also generated adducts with 4-hydroxynonenal (HNE) and other noncarboxylated electrophiles, but these were only sporadically identified in the PLPBSO-adducted ApoA1, suggesting a low stoichiometry of modification in the phospholipid-adducted protein. Both phospholipid electrophiles and HNE adducted His162, which resides in an ApoA1 domain involved in the activation of Lecithin-cholesterol acyltransferase and maturation of the HDL particle. ApoA1 lipid electrophile adducts may affect protein functions and provide useful biomarkers for oxidative stress.

Development of a highly sensitive and selective UPLC/MS/MS method for the simultaneous determination of testosterone and 5alpha-dihydrotestosterone in human serum to support testosterone replacement therapy for hypogonadism.

A highly sensitive and selective quantitative method to accurately determine testosterone (Te) and 5alpha-dihydrotestosterone (DHT) in human serum is crucial to the success of Te replacement therapy for hypogonadism. To this end we have developed and validated a semi-automated and relatively high-throughput method in a 96-well plate format using ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLC/MS/MS) for the simultaneous determination of Te and DHT in human serum. Te and DHT along with the internal standards [(2)H(3)]-Te and [(2)H(3)]-DHT were extracted from 300 microL of human serum by liquid-liquid extraction using methyl tertiary-butyl ether (MTBE), followed by derivatization with 2,3-pyridinedicarboxylic anhydride and solid-phase extraction for sample clean up. A novel chemical derivatization approach using 2,3-pyridinedicarboxylic anhydride was employed to achieve the MS sensitivity and selectivity required for DHT. Baseline separation of Te and DHT derivatives from endogenous steroid derivatives was achieved using UPLC technology on a C18 stationary-phase column with 1.7 microm particle size. The validity of using double charcoal-stripped female human serum as surrogate matrix for preparation of calibration standards was demonstrated through standard addition experiments. The method was validated over the concentration ranges of 0.2-40 ng/mL for Te and 0.01-2 ng/mL for DHT. The validation and study sample analysis results show that the method is rugged, precise, accurate, and well suited to support pharmacokinetic studies where approximately 300 samples can be extracted and analyzed in 1 day.

Phospholipid-protein adducts of lipid peroxidation: synthesis and study of new biotinylated phosphatidylcholines.

Oxidative stress gives rise to a number of electrophilic aldehydes from membrane phospholipids, and these compounds have been linked to pathophysiologic events associated with the progression of cardiovascular disease. A headgroup biotinylated phosphatidylcholine (PC) has been prepared, and its oxidation chemistry has been studied. Biotin or biotin-sulfoxide groups were attached to PC at the ammonium headgroup via a di-ethylene glycol link. The modified phospholipids have calorimetric and colloidal properties similar to those of the parent. The oxidation of PLPBSO (the biotin-sulfoxide analogue of 1-palmitoyl-2-linoleoylglycerylphosphatidylcholine, PLPC) was studied under a variety of conditions. PLPBSO, like PLPC, undergoes oxidation to give electrophiles that adduct to small model peptides as well as to isolated proteins such as human serum albumin. PLPBSO incorporates into human blood plasma, and treatment of the plasma with water soluble free radical initiators gives rise to a number of biotinylated plasma proteins that can be isolated via (strept)avidin affinity. Isolated peptide or protein-lipid adducts can be identified by proteomics analyses, and studies on model peptides show that phospholipid-protein adduction sites can be identified by known algorithms. Biotinylated lipids such as PLPBSO and modern proteomics tools would appear to provide a new approach to exploring the chemistry and biology of membrane peroxidation associated with oxidative stress.

Covalent adduction of human serum albumin by 4-hydroxy-2-nonenal: kinetic analysis of competing alkylation reactions.

The electrophilic lipid oxidation product 4-hydroxy-2-nonenal (HNE) reacts with proteins to form covalent adducts, and this damage has been implicated in pathologies associated with oxidative stress. HNE adduction of blood proteins, such as human serum albumin (HSA), yields adducts that may serve as markers of oxidative stress in vivo. We used liquid chromatography-tandem mass spectrometry (LC-MS-MS) and the P-Mod algorithm to map the sites of 10 adducts formed by reaction of HNE with HSA in vitro. The detected adducts included Michael adducts formed at histidine and lysine residues. The selectivity of HNE in competing adduction reactions was evaluated by analysis of kinetics for HNE Michael adduction at six targeted HSA histidine residues. Reaction kinetics were analyzed by selected reaction monitoring in LC-MS-MS using stable isotope tagging with phenyl isocyanate. Rate constants ranged over 4 orders of magnitude, with the order of reactivity being H242 > H510 > H67 > H367 > H247 approximately K233. The most reactive target, H242, is located in a fatty acid- and drug binding cavity in subdomain IIa of HSA and appears to be a hot-spot for HNE modification. Analysis of adduction kinetics together with HSA structure and target residue pK(a) values suggest that location in the hydrophobic binding cavity and low predicted pK(a) of H242 account for its high reactivity toward HNE. H242 adducts may be preferred products of adduction by lipophilic electrophiles and may comprise a family of biomarkers for oxidative stress.

The neurotoxin 2'-NH2-MPTP degenerates serotonin axons and evokes increases in hippocampal BDNF.

1-Methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH2-MPTP) causes long-term depletions in cortical and hippocampal serotonin (5-HT) and norepinephrine (NE) that are accompanied by acute elevations in glial fibrillary acidic protein (GFAP) and argyrophilia. To further investigate the hypothesis that these changes are reflective of serotonergic and noradrenergic axonal degeneration, 2'-NH2-MPTP was administered to mice and innervation densities were determined immunocytochemically. Regional responses of the neurotrophin, brain-derived neurotrophic factor (BDNF), to putative damage were also assessed. Three days after 2'-NH2-MPTP, 5-HT axons exhibited a beaded, tortuous appearance indicative of ongoing degeneration. At 21 days, numbers of serotonin axons were significantly decreased, with the greatest axonal losses occurring in cortex and hippocampus. Serotonin axons in the amygdala were contrastingly spared long-term damage, as were 5-HT and NE cell bodies in the brain stem. BDNF protein levels were selectively increased in the hippocampus 3 days post-dose and returned to normal 21 days later. These results, in conjunction with previous findings, demonstrate that 2'-NH2-MPTP causes degeneration of serotonergic axons innervating the cortex and hippocampus on par with depletions in neurotransmitter levels. Moreover, damage to the hippocampus, a brain region important for learning and memory, and the modulation of anxiety and stress responsiveness, results in a transitory increase in BDNF.

Exploring the relationship between serotonin and brain-derived neurotrophic factor: analysis of BDNF protein and extraneuronal 5-HT in mice with reduced serotonin transporter or BDNF expression.

Serotonin (5-HT) has been proposed to promote neuronal plasticity during the treatment of mood and anxiety disorders and following neurodegenerative insult by altering the expression of critical genes including brain-derived neurotrophic factor (BDNF). In this study, mice with constitutive reductions in the serotonin transporter (SERT) or BDNF were investigated to further assess the functional relationship between serotonin neurotransmission and BDNF expression. Using a modified extraction procedure and a commercial enzyme-linked immunosorbant assay, 50% decreases in BDNF protein in hippocampus, frontal cortex and brain stem were confirmed in 4-month-old mice lacking one copy of the BDNF gene (BDNF(+/-)). By contrast, 4-month-old male and female mice with partial (SERT(+/-)) or complete (SERT(-/-)) reductions in SERT expression showed no differences in BDNF protein levels compared to SERT(+/+) mice, although male SERT knockout mice of all genotypes had higher BDNF levels in hippocampus, frontal cortex, and brain stem than female animals. Microdialysis also was performed in BDNF(+/-) mice. In addition to other phenotypic aspects suggestive of altered serotonin neurotransmission, BDNF(+/-) mice show accelerated age-related degeneration of 5-HT forebrain innervation. Nevertheless, extracellular 5-HT levels determined by zero net flux microdialysis were similar between BDNF(+/+) and BDNF(+/-) mice in striatum and frontal cortex at 8-12 months of age. These data illustrate that a 50% decrease in BDNF does not appear to be sufficient to cause measurable changes in basal extracellular 5-HT concentrations and, furthermore, that constitutive reductions in SERT expression are not associated with altered BDNF protein levels at the ages and in the brain regions examined in this study.

Late onset loss of hippocampal 5-HT and NE is accompanied by increases in BDNF protein expression in mice co-expressing mutant APP and PS1.

Transgenic mice expressing both mutant amyloid precursor protein (APPswe) and presenilin-1 (PS1DeltaE9) develop amyloid deposits as early as 4 months of age and preliminary evidence suggests that this may be associated with degenerative changes in serotonin axons innervating the dentate gyrus of the hippocampus. In the present investigation, which focused on further delineating the effects of amyloid deposition on hippocampal neurochemistry, decreases in serotonin neurotransmitter levels (-25%) were discovered to be present at 18 months in APP+/PS1+ mice, while norepinephrine was reduced in the hippocampus of 12- (-30%) and 18-month-old (-45%) APP+/PS1+ double mutants. In addition, brain-derived neurotrophic factor (BDNF) protein levels were investigated since changes in BDNF are reported to occur in AD, and BDNF has been shown to have trophic effects on serotonin and norepinephrine neurons. In doubly, but not singly mutant mice, hippocampal BDNF levels were increased at 12 (+70%) and 18 months (+170%). Furthermore, in a different model of serotonergic and noradrenergic degeneration, BDNF protein levels were similarly increased in response to depletions in hippocampal serotonin and norepinephrine caused by the chemical neurotoxin 1-methyl-4-(2'-aminophenyl)-1,2,3,6-tetrahydropyridine (2'-NH2-MPTP). These findings show that early amyloid deposition in mice expressing mutant human APP and PS-1 is associated with a progressive loss of serotonin and norepinephrine neurotransmitter levels in the hippocampus later in life. Furthermore, BDNF protein levels are increased in APP+/PS1+ and 2'-NH2-MPTP-treated mice, possibly as a compensatory response to serotonergic and noradrenergic neurodegeneration in a brain region important for learning and memory.